Buzzards Bay Action Committee · Buzzards Bay (now the Buzzards Bay Coalition) and the Buzzards Bay Action Committee (BBAC). The Buzzards Bay Coalition (BBC) is an educational and

Buzzards Bay Action Committee

Illicit Connection Detection and Stormwater Quality Monitoring

in the Buzzards Bay Watershed

Quality Assurance Project Plan

Jeff Osuch, Stormwater Collaborative Project Manager


Joseph Costa, Executive Director

Buzzards Bay National Estuary Program

Prepared by

Bernadette Taber, Stormwater Specialist

Buzzards Bay National Estuary Program

Prepared for

EPA New England Regional Laboratory

11 Technology Drive

North Chelmsford, MA 01863-2431

Date: March 16, 2016


Illicit Connection Detection and Stormwater Quality Monitoring

in the Buzzards Bay Watershed

Quality Assurance Project Plan

Approvals Signature:

________________________________________________________Date: ___________________

Stormwater Collaborative Project Manager

Jeff Osuch, BBAC

Fairhaven Town Hall

40 Center Street

Fairhaven, MA 02719

_______________________________________________________Date: ___________________

Project Quality Assurance Officer

Joseph E. Costa PhD

Executive Director, Buzzards Bay NEP

________________________________________________________Date: ___________________

EPA Project Manager/Officer

Ann Rodney, US EPA, Region 1

5 Post Office Sq. Suite 100 Boston MA 02109-3192

Tel. 617 918 1538 Email: [email protected]

_________________________________________________________Date: ___________________

EPA Quality Assurance Officer

Steve DiMettei, EPA New England Quality Assurance Unit

11 Technology Drive North Chelmsford MA 01863

Tel. 617 918 8369 Email: [email protected]

_________________________________________________________Date: ___________________

3

Table of Contents

1.3 Distribution List ...............................................................................................................................................5

1.4 Project Organization.........................................................................................................................................7

1.5 Problem Definition/Background ......................................................................................................................9

1.6 Project/Task Description and Schedule............................................................................................................9

1.7 Data Quality Objectives (DQOs) ...................................................................................................................17

1.8 Special Training Requirements/Certification .................................................................................................17

1.9 Documentation and Records ..........................................................................................................................17

2.0 Data Generation and Acquisition .......................................................................................................................21

2.1 Sampling Process Design ...............................................................................................................................21

2.2 Sampling Methods .........................................................................................................................................22

2.3 Sample Handling and Custody .......................................................................................................................25

2.4 Analytical Methods and Quality Control .......................................................................................................25

2.5 "In the field" and "In House" Field Measurements/Quality Control ..............................................................25

2.6 Instrument Testing, Inspection, and Maintenance..........................................................................................26

2.7 Instrument Calibration and Frequency ...........................................................................................................26

2.8 Inspection/Acceptance for Supplies and Consumables ..................................................................................26

2.9 Non-Direct Measurements .............................................................................................................................27

2.10 Data Management ........................................................................................................................................27

3.0 Assessment and Oversight ..................................................................................................................................27

3.1 Assessments and Response Actions ...............................................................................................................27

3.2 Reports ...........................................................................................................................................................28

4.0 Data Validation and Usability ............................................................................................................................28

4.1 Data Review, Verification, and Validation ....................................................................................................28

4.2 Verification and Validation Methods .............................................................................................................28

4

Tables

Table 1. Project Organization ...............................................................................................................................9 Table 2. Monitoring Parameters .........................................................................................................................11 Table 3. Project Schedule ...................................................................................................................................18 Table 4. Laboratory Tests - Bacteria, New Bedford Health Lab and Barnstable Co. Health and Environment 18 Table 5. "In the field" Field Tests .......................................................................................................................19 Table 6. "In house" Field Tests ...........................................................................................................................19 Table 7. Other Laboratory Tests (future optional tests), Barnstable County Health and Environment ..............20 Table 8. Monitoring Events by Municipality .....................................................................................................20 Table 9. Project SOPs .........................................................................................................................................23 Table 10. Water Quality Parameters and Indicators .............................................................................................23 Table 11. Supply Inspection Requirements and Acceptance Criteria...................................................................27

Figures

Figure 1. Organizational Chart...............................................................................................................................8 Figure 2. Bacteria-impacted waters in Buzzards Bay ..........................................................................................10 Figure 3. Dartmouth Monitoring Sites .................................................................................................................12 Figure 4. Fairhaven Monitoring Sites ..................................................................................................................13 Figure 5. Acushnet Monitoring Sites ...................................................................................................................14 Figure 6. Mattapoisett Monitoring Sites ..............................................................................................................15 Figure 7. Wareham Monitoring Sites ...................................................................................................................16 Figure 8. Sample Identification Label ..................................................................................................................24 Figure 9. Laboratory Sample Identification - Field Code Designation ................................................................24

Appendix

A - Laboratory SOPs and QAPs

B - Water Quality Grab Sample Collection

C - Field Water Quality Measurements

D - Wet and Dry Weather Field Data Sampling Sheet

5

1.3 Distribution List

Buzzards Bay Stormwater Collaborative Jeff Osuch

Fairhaven Town Hall

40 Center Street

Fairhaven, MA 02719

Buzzards Bay National Estuary Program Joseph Costa, Executive Director

2870 Cranberry Highway

East Wareham, MA 02538

Tel. 508 291-3625 x11

Email: [email protected]

Buzzards Bay Coalition Mark Rasmussen, President

114 Front Street

New Bedford, MA 02740

Tel. 508 999-6363


Town of Wareham David Menard, Municipal Maintenance Director

Town Barn , 95 Charge Pond Road

Wareham, MA 02571

Tel: 508 295-5300


Town of Dartmouth Mike O'Reilly, Conservation Agent

Dartmouth Town Hall, Room 119

400 Slocum Road

Dartmouth, MA 02747

Tel: 508-910-1822


Town of Fairhaven Mark Rees, Town Administrator

40 Center Street

Fairhaven, MA 02719

Tel: 508 979-4023, Ext. 2


Town of Mattapoisett Elizabeth Leidhold, Conservation Agent

Town Hall 16 Main Street P.O. Box 435

Mattapoisett, MA 02739

Tel: 508 758-4100 ext. 219,


Town of Acushnet Merilee Kelly

Acushnet Town Hall

122 Main Street

Acushnet, MA 02743

Tel: 508 998-0202


6

Office of MA Coastal Zone Management David Janik

2870 Cranberry Highway

East Wareham, MA. 02538

508 291-3625 x20


U.S. Environmental Protection Agency SteveDiMattei

New England Quality Assurance Unit

11 Technology Drive North Chelmsford MA 01863

Tel. 617 918-8369


U.S. Environmental Protection Agency Ann Rodney, EPA Project Officer

5 Post Office Square-Suite 100

Boston, MA 02109-3912

Tel: 617-918-1538


Barnstable Co. Dept of Health & Environment Gongmin Lei, Laboratory Director

P.O. Box 427 / 3195 Main Street

Barnstable, MA 02630

Tel: 508-375-6616


New Bedford Health Laboratory Leslie Aubut, Laboratory Director

1000 Rodney French Boulevard

New Bedford, MA 02744

Tel: 508- 991-6199


7

1.4 Project Organization

The Buzzards Bay Action Committee began in 1987 as an offshoot of the Buzzards Bay Project (renamed in 1996

to the Buzzards Bay National Estuary Program). The Buzzards Bay Project was established in 1985 and joined the

U.S. Environmental Protection Agency's National Estuary Program in 1987. As part of its structure, the Buzzards

Bay NEP had a "Citizen Advisory Committee" (CAC) that assisted with pollution source identification and

developing management options to protect and restore Buzzards Bay. The CAC established itself as a separate

entity in 1987 and eventually became two independent, not-for-profit organizations, one being the Coalition for

Buzzards Bay (now the Buzzards Bay Coalition) and the Buzzards Bay Action Committee (BBAC). The

Buzzards Bay Coalition (BBC) is an educational and outreach citizen-based group, whereas the BBAC is

comprised of municipal officials, and has become more involved with state, local, and federal legislative and

regulatory issues. Both organizations are represented on the Buzzards Bay NEP's Steering Committee. In 1997

and 2005, the BBAC signed Memorandum of Understandings (MOUs) with the Buzzards Bay NEP and the BBC

to improve collaboration, cooperation, and coordination of actions among the three organizations in their shared

goals to protect and restore water quality in Buzzards Bay and its surrounding watershed.

In 2015, the BBAC applied for and received a grant through U.S. Environmental Protection Agency's (EPA)

Healthy Communities Grant Program. The goal of this funding program is to combine available resources and

best identify competitive projects that will achieve measurable environmental and public health results in

communities across New England. BBAC's funding application was submitted under the Target Program Area of

Southeast New England Program for Coastal Watershed Restoration (SNEP), which focuses on restoring

connectivity, rebuilding natural hydrology, reducing stormwater inputs, supporting the return of fish and wildlife,

promoting fishable and swimmable uses of waterways, incorporating low-impact development, and integrating

innovative technologies and policies into restoration projects. Under this program, projects that managed the

impact of nutrients were given special attention.

As part of the Healthy Communities grant application, BBAC established a Stormwater Collaborative consisting

of itself, plus five Buzzards Bay municipalities (Dartmouth, Fairhaven, Acushnet, Mattapoisett, and Wareham).

By working together, the collaborative with assistance from the project partners (Buzzards Bay NEP and BBC)

look to combine resources in a cost-effective way to meet local goals of improving water quality and managing

stormwater systems. This collaborative will work to advance previous mapping efforts of stormwater networks by

the Buzzards Bay NEP and to monitor stormwater discharges located within nutrient- and pathogen-impaired

surface waters in Buzzards Bay. This Quality Assurance Project Plan will encompass water quality monitoring

parameters as described under EPA's the Healthy Communities grant application, plus any future (optional

parameters) expansion of the monitoring program.

Project Organization:

(1) The Buzzards Bay Action Committee's Stormwater Collaborative (BBAC-SC) will serve as the overall Lead

Organization for this project as per the organization structure of Figure 1. The BBAC-SC, with assistance and

support from the Buzzards Bay National Estuary Program (Buzzards Bay NEP), will provide general project

guidance and ensure that work performed meets the prescribed project scope. In addition, BBAC will hire the

Project Manager to oversee the project.

(2) Stormwater Collaborative Project Manager (Project Manager) will be the responsible official for overseeing

the overall project and budget. The Project Manager will work with the Stormwater Specialist (to be hired by

BBAC), the Technical Project Advisor, the Stormdrain Mapping Coordinator and the QA Project Officer in

reviewing/updating existing stormdrain mapping data, selecting sampling locations, choosing water quality

parameters and updating project partners regarding monitoring results.

(3) Stormwater Specialist (to be hired by BBAC) will be responsible for coordinating the stormdrain monitoring

program and stormdrain data collection. The monitoring duties include coordinating sample drop off with the

laboratories; planning, oversight and coordination with the field sampling crew (Municipal Public Works staff

and/or interns); analysis of "In house" (in the BBAC office) field samples and ensuring that the monitoring

samples are properly documented, preserved, handled, and delivered to the appropriate laboratory. The stormdrain

8

mapping duties include working with the Stormdrain Mapping Coordinator to determine the stormdrain data

(discharge point location including pipe type, size, and condition and roadcut condition.) to be collected.

(4) Stormdrain Mapping Coordinator, (5) Technical Project Advisor, and (6) Project QA Officer (Buzzards Bay

NEP) will provide guidance with regards to the locations of the initial monitoring stations and sampling data

collection. In addition, the Buzzards Bay NEP will provide data QA, analysis, and interpretation and will work

with the Project Manager and Stormwater Specialist to coordinate data collection for the Buzzards Bay

Stormdrain Mapping Atlas.

(7) Municipal Public Work Directors and Field Sampling Teams

The Field Sampling Team will be assigned by the Municipal Public Works Directors to pre-determined sampling

stations during wet- and dry- weather sampling events. Each sampling team will consist of the Stormwater

Specialist (or designee) and 1-2 staff from the municipal public works department. During sampling events,

members of the Field Sampling Team are responsible for assisting with pre-event planning, grab sample

collection, collection of field measurements, accurate completion of field data sheets and equipment worksheets.

If needed, team members might also deliver samples and field data sheets to the appropriate analytical laboratory.

All field personnel involved in the project will undergo training prior to field sampling and a record of training

will be documented and filed in the BBAC-SC's office.

In addition the Field Sampling Teams will be responsible for collecting and documenting (GPS, photographs, or

written as appropriate) stormdrain data as requested by the Stormdrain Mapping Coordinator.

Granting Agency

EPA Quality Assurance

Officer

Steve DiMattei

U.S. EPA Program Manager

Ann Rodney

EPA GIS Coordinator

Alexandra Dichter

Grant Recipient

(1) The Buzzards Bay Action Committee's

Stormwater Collaborative (BBAC-SC)

(2) Jeff Osuch,

Stormwater Collaborative Project Manager

(3)First name Last name,

BBAC Stormwater Specialist

Partners

Buzzards Bay

Coalition

(6) Buzzards Bay NEP

Project QA Officer, Joseph E. Costa, PhD

Acushnet Dartmouth Fairhaven Mattapoisett Dartmouth

(5) Technical

Project Advisor

(TPA):

Bernadette Taber,

Buzzards Bay NEP

(4) Stormdrain

Mapping

Coordinator (SMC):

Kevin Bartsch,

Buzzards Bay NEP

Figure 1. Organizational Chart

9

Table 1. Project Organization

Function Organization

Stormwater Collaborative Project Manager (Project Manager) Jeff Osuch, BBAC

Stormwater Network Mapping Coordinator (SMC) Kevin Bartsch, BBNEP

Stormwater Monitoring Technical Project Advisor (TPA) Bernadette Taber, BBNEP

Stormwater Specialist (SS) To be hired by BBAC

Project QA Officer Joseph Costa, BBNEP

EPA Project Officer Ann Rodney, US EPA

EPA QA Officer Steve DiMattei, US EPA

Nutrient Laboratory Barnstable Co. Health and Environment

Lab, Gongmin Lei

Biological Laboratories Barnstable Co. Health Lab, Gongmin Lei

New Bedford Health Lab, Leslie Aubut

1.5 Problem Definition/Background

In Buzzards Bay, there are roughly 6,000 acres of shellfish growing areas indefinitely closed to shellfishing,

another 6,000 acres closed after 0.2 inches or more of rain, and roughly 3,000 acres seasonally closed (Figure 2).

These closures are the result of bacterial contamination and are often largely the result of stormwater discharges.

Some of these stormwater discharges also contribute nutrients to these embayments, many of which are nitrogen

impaired and require a TMDL. Many of these discharges of concern are not regulated under existing stormwater

permits and regulations. The elimination of illicit discharges to stormwater networks and the treatment of

stormwater discharges conveying non-point sources of pollution will help reduce these impairments. These

actions can only be taken if problem discharges can be identified and prioritized.

The identification of problem discharges and the development of solutions must begin with monitoring

stormwater discharges and mapping stormwater networks for nutrients and bacteria. The BBAC, through its

establishment of the Buzzards Bay Stormwater Collaborative has already begun the process. The Collaborative

has applied for and received funding through EPA's Healthy Communities Grant Program. This grant will allow

the five municipalities within the Collaborative to begin testing stormwater discharges in coastal waters that are

closed to shellfishing resulting from high bacterial levels. In addition, the stormdrain network associated with

each discharge will be investigated using EPA New England Bacterial Source Tracking Protocol (Draft-January

2012) for the presence of illicit discharges.

1.6 Project/Task Description and Schedule

For the purposes of this field sampling program, the project scope has been defined as stormwater networks

discharging within 100 feet of either a shellfish closed embayment and/or bacteria impaired waters

(Massachusetts 2012 Integrated Waters List, Category 4 and 5) and are located within the municipalities

participating in the Stormwater Collaborative (Dartmouth, Fairhaven, Acushnet, Mattapoisett, and Wareham, see

Figure 3 to Figure 7).

Under EPA's Healthy Communities Grant funding, the sampling program will be conducted between April 2016

and November 2017 (with hopes of expanding the program with future funding). It will consist of three rounds of

wet weather sampling and four rounds of dry weather sampling and will include an illicit discharge detection

investigation. Since several of the outfalls are located close to the oceanfront, samples will be taken during storms

10

Figure 2. Bacteria-impacted waters in Buzzards Bay

11

that coincide with low tides for each embayment. Wet weather monitoring shall be conducted during the first

flush of stormwater runoff (less than 1 inch of rainfall) and must be preceded by 3 days (72 hours) without

rainfall. The first set of monitoring events will consist of one dry and one wet weather sample at each discharge

point. Once the analytical data from each monitoring event is received from the laboratories, the Project Manager,

Project QA Officer, and TPA will discuss the results with the Stormwater Specialist and determine the location of

the next monitoring location; either to remain at the discharge pipe for further analysis or proceed up the

stormdrain network to narrow down potential sources of contamination. Follow-up investigations may include dry

weather and/or wet weather sampling.

In the event of a prolonged drought, the BBAC will adapt the program to focus on dry weather flows and finding

illicit connections to the stormdrain systems. This would involve a more systematic evaluation of manhole

junctions and testing of dry weather flows. In addition, the BBAC may have the opportunity to expand the

monitoring target periods and/or extend the grant duration grant duration if needed.

Project Scope and Schedule

Table 2 shows a summary of contaminants that will be evaluated through this effort. Each round of sampling will

include confirmation of sampling locations (GPS), tidal data, antecedent precipitation, and compilation of all field

tests and field observations. Once the analytical data is received from the laboratories, the Project Manager, QA

Officer, and TPA will review the data and make recommendations for future monitoring locations. Final decisions

regarding locations will be made in consultation with the municipal DPW Director or designee

The assessment of outfalls and scope of work described above includes the following tasks, and according to the

schedule in Table 3:

Task 1: Water Quality Sampling Program Design

Task 2: Quality Assurance Project Plan (QAPP)

Task 3: Hiring of Staff

Task 4: Implementation of Sampling Program

Task 5: Reporting and Analysis

Table 2. Monitoring Parameters

Field Test Kits Laboratory Analysis

Surfactants (detergents as MBAS) Enterococci

Ammonia as Nitrogen Fecal Coliform

Temperature

Conductivity Optional Laboratory Analysis (future funding)

Nitrates as Nitrogen Nitrates as Nitrogen

Chlorine (future funding) Orthophosphate

Total Kjeldahl Nitrogen

Observations Oil and Grease

Color

Odor

Sewage/Septic System Waste products

(toilet paper, sanitary products, etc))

12

Figure 3. Dartmouth Monitoring Sites

13

Figure 4. Fairhaven Monitoring Sites

14

Figure 5. Acushnet Monitoring Sites

15

Figure 6. Mattapoisett Monitoring Sites

16

Figure 7. Wareham Monitoring Sites

17

1.7 Data Quality Objectives (DQOs)

Monitoring data collected by the Buzzards Bay Stormwater Collaborative's Field Sampling Teams (the Field

Teams) will meet the following water quality objectives:

The Field Teams will:

collect water quality data to characterize bacterial loading in areas closed to shellfishing and/or areas that

are impaired for bacteria (monitoring sites as depicted in Figure 3through Figure 7).

collect water quality data to identify illicit connections in the areas closed to shellfishing and/or areas that

are impaired for bacteria (monitoring sites as depicted in Figure 3 through Figure 7) that may be primary

contributors to pollutants in Buzzards Bay.

The methods and approaches utilized by the Collaborative are aimed at meeting the required levels of precision,

accuracy, field blank cleanliness, and detection limits. The minimum performance criteria for the bacteria

sampling is given in Table 4, for field tests sampling in Table 5 and Table 6 and the optional future tests in Table

7, below.

1.8 Special Training Requirements/Certification

This sampling program consists of standard field sampling techniques, field analyses, laboratory analyses, and

data validation techniques. All field personnel on the Field Sampling Teams will be required to complete a brief

training program to ensure all field sampling protocol is followed as specified in this Plan, as described in Section

B of this Plan. The Project Manager or Stormwater Specialist will maintain a signup sheet to include the name of

the trainee, date of training, and contact information

1.9 Documentation and Records

Field data will be recorded on standardized field data sheets (Appendix D) either on-site ("in the field") or at the

BBAC office ("In house").

The selected analytical laboratories are certified by MassDEP to perform all analytical procedures that will be

required during the completion of this field sampling program. All laboratory personnel are to have had training

in accordance with the procedures outlined in their QAPs. A summary of the laboratory SOPs for the required

analyses are provided in Appendix A.

The samples requiring laboratory analysis are picked up by the Stormwater Specialist (or designee) and driven to

the appropriate laboratory. Prior to releasing the samples, the onsite DPW staff member (or designee) will sign

and date the Chain of Custody (COC) on the bottom of the field data sheets. The Stormwater Specialist (or

designee) will place the "In house" samples and laboratory samples into separate coolers and deliver the

laboratory samples to the laboratory. Once the samples arrive at the laboratory, the Stormwater Specialist will

sign the COC at the bottom of the data sheet. The laboratory will make copies of all the field data sheets and COC

forms for their records with all the original data returned to the Stormwater Specialist. The Stormwater Specialist

will bring the remaining "In-house" samples to the BBAC office for testing. The results of the "In house" testing

will be placed on the original field data sheets. The original field data sheets will then be filed at the Buzzards

Bay Action Committee office with copies sent to appropriate municipal DPWs. The monitoring data will be

entered on a spreadsheet, organized by municipal name, stormdrain system, and then specific location (discharge

points) within each stormdrain system.

18

Table 3. Project Schedule

2015 2016 2017

Oct

No

v

De

c

Jan

Feb

Mar

Ap

r

May

Jun

July

Au

g

Sep

Oct

No

v

De

c

Jan

Feb

Mar

Ap

r

May

Jun

Jul

Au

g

Sep

Oct

No

v

1 Sampling Program Design

X X X X X X X

2 QAPP X X X

3 Hiring of Staff X1 X

2 X

3 X

3

4 Implementation: Field Mapping and Monitoring

X X X X X X X X X X X X X X X X

5 Reports and Analysis

X4 X

5 X

6

Notes: 1BBAC Project Manager,

2BBAC Stormwater Specialist,

3summer interns,

4annual progress meeting and interim report,

5Draft prioritization,

6Final

prioritization and data report

Table 4. Laboratory Tests - Bacteria, New Bedford Health Lab and Barnstable Co. Health and Environment

Parameter

Sample Volume/

Container

Maximum

Holding Time

Field Processing/

Preservation

Laboratory

Precision Method Units

Lower Detection

Units

MDLs

Fecal Coliform 100 ml sterilize

polyethylene

6 hours Collect, label, store on

blue ice

Logarithm range of

duplicates, PT tests for

bacterial analysis

SM9222D, 21th Edition

2005

cfu/100ml <1

Enterococci 100 ml sterilize

polyethylene

6 hours Collect, label, store on

blue ice

Logarithm range of

duplicates, PT tests for

bacterial analysis

EPA Office of Water,

Method 1600,

Membrane Filter Test

EPA 821-R-97-004.

cfu/100ml <1

19

Table 6. "In house" Field Tests

(Samples to be taken to the BBAC Office for analysis)

Analyte(s) Measured Equipment Operating Range Resolution Accuracy Holding time

Surfactants (detergents as

MBAS) CHEMetrics K-9400

1 0-3 ppm + 1 color

standard

increment

+ 30% error at 0.25 ppm, 0.63

ppm, and 1.88 ppl. 48 hours

Nitrates LaMotte Nitrate-Nitrogen

test kit (3615-01) 0.00 to 1.00 ppm 0.1ppm 0.1ppm 24 hours

1Organic sulfonates, sulfates, carboxylates, phosphates, and phenols as well as inorganic cyanates and thiocyanates may interfere. Sulfides may interfere negatively. Nitrate interferes positively; 10

ppm NO3-N may read as approximately 0.2 ppm. Isopropanol at up to 0.1% does not interfere. Chloride at up to 100 ppm does not interfere significantly. However, because higher chloride levels will

interfere positively, this test kit is not recommended for the analysis of brine or seawater samples unless additional sample manipulation is performed.

Table 5. "In the field" Field Tests

(Sample analysis to be completed onsite)

Analyte(s) Measured Equipment Operating Range Resolution Accuracy

Ammonia Hach Test Strips 0-6ppm 0.25ppm +/- one half of a color block

Conductivity YSI Model 30 0 to 200 mS/cm (auto-range) 0.0001 to 0.1 mS/cm

(range dependent)

1 m, 4 m cables:

±1.0% of the reading or 1.0 μS/cm, whichever is

greater

Salinity

Calculated from

Conductivity and Temp

YSI Model 30

0 to 70 ppt 0.01 ppt ±1% of reading or ±0.1 ppt, whichever is greater

Temperature

(°C, °F, K)

YSI Model 30 -5 to 70°C (23 to 158°F) 0.1°C ±0.2°C;

±0.3°C cables over 45-meters

Chlorine1 Hach Pocket Colorimeter II

#5870024

Range: 0.02 to 2.00 mg/L CI2

Range 2: 0.1 to 8.0 mg/L CI₂ 0.02mg/l 0.2 mg/l

1 Chlorine monitoring will be conducted as decided by the Project Manager in consultation with the Technical Advisor and Stormwater Specialist. Decision will be made based upon previous

monitoring results and available funding.

20

Table 7. Other Laboratory Tests (future optional tests), Barnstable County Health and Environment

Parameter1 Max. Holding Time Process &Storage Method (Ref) Units Lower Detection Units (MDLS)

Nitrates as Nitrogen 48 Hours Filtered and stored in the

dark at 4°C

EPA 300.0, Rev11 mg/L 0.1

Orthophosphate 28 Days 120 ml glass bottle, preserve

with H2SO4 to a pH cool to

4° C 50 ml Glass Bottle

EPA 365.1 mg/L 0.05

Total Kjeldahl Nitrogen 28 Days Sulfuric Acid to pH <2, cool

to 4° C, plastic or glass

bottle

351.2 Rev001 mg/L 0.05

Oil and Grease 28 Days Sample acidified and stored

at 4°C

EPA 1664A, Ref: US EPA-

821-R-98-002, Feb 1999 mg/L <0.5

1 The Project Manager in consultation with the QA officer and the TPA will decide on the necessity of monitoring these parameters based upon previous monitoring results and available funding.

Table 8. Monitoring Events by Municipality

Town Discharge

Pipes

Monitoring

Events

Acushnet 9 63

Dartmouth 44 308

Fairhaven 77 539

Mattapoisett 41 287

Wareham 167 1169

Total 2366

21

2.0 Data Generation and Acquisition

2.1 Sampling Process Design

The Buzzards Bays Stormwater Collaborative water quality monitoring program is designed to meet the project

quality objectives discussed in Section 1.7 ‐ Quality Objectives and Criteria. This monitoring program involves

wet and dry weather sampling of outfall pipes or roadcuts and associated stormdrain systems that discharge within

100 feet of either a shellfish closed embayment and/or bacteria impaired waters (Massachusetts 2012 Integrated

Waters List, Category 4 and 5). If future funding is available, the BBAC plans to expand this sampling program to

include other municipalities along with a more extensive “within the pipe network” monitoring program.

Approximately 338 outfall pipes/roadcuts will be sampled at least twice - one dry weather and one wet weather.

Upon the receipt of the laboratory analysis of the first wet and dry weather sampling events, the Project Manager

in consultation with the QA Officer and TPA may decide the discharge pipe and/or stormdrain system warrants

further investigation to determine potential sources of pollution. The Project Manager and/or Stormwater

Specialist will discuss the test results with the municipal collaborative representative and DPW to determine the

next sampling location. It is estimated that each discharge pipe/roadcut or associated stormdrain system will be

sampled seven times (3 wet weather and 4 dry weather).

Sampling Description:

Stormwater water quality sampling is expected to be undertaken beginning in April 2016 and will continue for

one or two years depending upon additional supplemental funding. In general, water samples will be collected

during periods of elevated bacteria survival and will be either a wet weather or dry weather event. Some samples

may be collected during cold weather for parameters less likely to be affected by temperature such as inorganic

nutrients or optical brighteners.

Wet weather defined:

Antecedent conditions where no more than 0.1inches of rainfall have occurred 72‐hours prior to the start of

the rainfall event;

During a rain event with a minimum rainfall of 0.25‐inches and a minimum duration of 1 hour before the

sample is collected

Dry weather defined:

Antecedent conditions where no more than 0.1inches of rainfall have occurred at least 48 hours prior to the

sampling event;

Currently not raining and no rainfall is predicted for that day.

All data collection will be taken as close to the discharge point as possible and as close to low tide as feasible.

Many of the pipe discharges in Buzzards Bay are partially or totally submerged even during low tides. Tides will

be monitored using data provided by the National Oceanic and Atmospheric Administration (NOAA)

https://NOAA Tide Predictions.

Antecedent rain conditions and predicted rainfall will be checked prior to an event by the Stormwater Specialist.

Precipitation forecasts will be based on meteorological forecast models provided by the National Weather Service

at the New Bedford Station. A final decision on whether or not a sample will be collected will be decided by the

Project Manager or Stormwater Specialist.

Prior to the first monitoring event, all the pre-selected discharge pipes/roadcuts will be assigned a unique site

name (i.e. Buzzards Bay Stormwater Atlas point identifier). The Stormwater Specialist (or designee) will then

conduct visual inspections of the sites, collecting GPS coordinates, photographs, and information regarding

outfall pipe /roadcut(material, size, condition, etc.). During each monitoring event, the Field Teams will confirm

https://tidesandcurrents.noaa.gov/tide_predictions.html?gid=1403

22

the pipe/roadcut location using 11x 17 paper copies of GIS map (includes aerial map of discharge point, catch

basins, flow direction, and street names) of each site and take additional photographs. Data collection will be in

accordance with Appendix B - Water Quality Grab Sample Collection Standard Operating Procedure and

Appendix C – SOP: Field Water Quality Measurements. Environmental conditions (weather, antecedent rain, tide

conditions, etc.) as indicated on the monitoring data sheet will be filled out and flow characteristics noted along

with any observations (odor, color, waste products).

To minimize potential sediment contamination, data collection will begin with the bacterial samples. Once the

bacteria samples are collected they will immediately be placed in a cooler with blue ice. Separate bottles will then

be used to collect water samples for all "in the field" tests (conductivity, temperature, salinity, and ammonia) and

the "in house" (analysis to be done at BBAC Office) tests (surfactants and nitrates). All "in house" samples will be

placed on blue ice. Additional samples will be collected for any other laboratory testing, followed by appropriate

storage protocol. Stormwater will be analyzed for chlorine on an as needed basis at the discretion of the Project

Manager following the examination of previous testing results. During each sampling event, up to two duplicate

samples and one field blank will be collected and analyzed by the appropriate laboratory identically to grab

samples.

If the discharge pipe is submerged due to tidal conditions, a notation will be made on the data sheet as to the depth

of submersion. The Field Team will then proceed up to the next available upstream manhole (or catchbasin if the

stormdrain system is catchbasin to catchbasin without any manholes). Under typical situations, this first manhole

or catchbasin will be the last of the stormwater entering the stormdrain system and will still be representative of

stormwater flow to the discharge pipe. At this first manhole, the Field Teams will take samples from every inlet

pipe discharging under free-flow conditions into this first manhole (junction manhole). If the inlet pipe or pipes in

the first manhole remains submerged, the Field Team will note the condition of the inlet pipe (submerged) and the

outlet pipe (submerged or not) and collect the samples from the manhole sump. If the discharge pipe in this first

manhole is submerged and the salinity is over 3.0 ppt (indicating tidal influence), the Field Team will proceed up

to the next upstream manhole to collect additional samples/data. Any manhole with elevated salinity test results

and does not appear to be tidally influenced (outlet pipe is free flowing) will be investigated further for potential

illicit discharges.

In addition to non-tidal salinity, all other parameters will be evaluated by the Project Manager and the Buzzards

Bay NEP Quality Assurance Officer to determine the necessity of further investigation of individual stormdrain

systems for illicit discharges and/or pollution sources. As a minimum, efforts will be made to evaluate stormdrain

systems whose previous samples were in a manhole sump (as opposed to free-flowing conditions). The water

quality within the stagnant water of the sump may not be the best bacterial representation of the water quality

within the stormdrain system. In these circumstances, the Field Teams will recheck the manhole during the next

sampling event, if the inlet(s) is still submerged, the Field Teams will proceed to the next upstream manhole to

collect samples/data.

Subsequently if the water quality parameters indicate the potential presence of an illicit discharge and/or pollution

source under free-flowing conditions, this will trigger an immediate and more detailed search within the

stormdrain system to determine the source of the problem.

2.2 Sampling Methods

Water quality sampling performed during this monitoring program will include the collection of grab samples,

field measurements (both in the field and at the BBAC office), and quality control (QC) samples. Samples will be

collected and handled according to the Standard Operating Procedures (SOPs) listed in Table 9 and included in

the associated Appendices of this Plan.

23

Table 9. Project SOPs

Appendix Project SOP

A Laboratory SOPs and QAPs

B Water Quality Grab Sample Collection

C Field Water Quality Measurements

D Wet and Dry Weather Field Data Sampling Sheet

Table 10. Water Quality Parameters and Indicators

Parameter Indication of:

Surfactants Washing machine or sewer connection

Ammonia as Nitrogen Sewer connection

Temperature Impact to aquatic life

Conductivity Saline conditions, impacts on surfactant tests

Nitrates as Nitrogen Sewer connection

Enterococci Presence of pathogens in fresh water

Fecal Coliform Pathogen contamination in shellfish areas

Orthophosphate (optional) Washing machine or sewer connection

Total Kjeldahl Nitrogen (optional) Sewer connection

Oil and Grease(optional) Connection to high vehicle areas

Chlorine (optional) Connection to swimming pools

Note: all water quality parameters, with the exception of the optional parameters, will be tested for both the dry and wet-weather

monitoring events. Optional parameters will be monitored at the discretion of the Project Manager in consultation with the Technical

Advisor and Stormwater Specialist. Decision will be made based upon previous monitoring results and available funding.

Pre-Sampling Activities

Preparation for sampling activities will be the responsibility of the Project Manager and the Stormwater

Specialist. Responsibilities include assignment of site location identification, confirmation of discharge point

location and safe access, collecting GPS coordinates, documenting point data (size, material, and condition),

procurement, and field testing of field equipment, training of Field Team members, weather tracking, review

of SOPs, and laboratory coordination. Once in the field, the Stormwater Specialist (or designee) will establish

the sample staging areas, equipment distribution, confirmation of bottle labeling, and distribution of bottles

and coolers provided by the subcontracted laboratories.

Sampling Procedure

All samples sent to the laboratory will be grab samples collected in accordance with the laboratories SOP

found in Appendix A. Sample bottles will be pre-preserved and supplied by the assigned analytical

laboratory. All data will be recorded by the Field Team on the appropriate monitoring field data sheets.

Sample worksheets are provided in Appendix D-Wet and Dry Weather Field Data Sampling Sheet. Pre-

labeled bottles will be provided to each Field Team for each station and sampling round. Upon collection,

sample bottles will be handled in accordance with this QAPP and the sample handling procedures provided by

the assigned analytical laboratory. COC at the bottom of the field data sheets will be signed and dated by the

either the DPW Director (or designee) or the Stormwater Specialist (or designee) before samples are

transported to the appropriate laboratory and then again once the samples arrive at the laboratory. An example

label is shown in Figure 8, which shows the date and time the sample was taken, identification label,

parameter being tested for, any pre-charged preservatives and the initials of the person who obtained the

sample.

24

Field bottles sample identification labels (to be analyzed by the laboratories) will consist of the Project ID

(BBAC), Date and Time, Sample ID, Sample Type (wet or dry), Laboratory Analysis, Preservative (if any), and

the collectors initials. The Sample ID will follow the point identification protocol as established in the Buzzards

Bay NEP Stormdrain Atlas.

Figure 8. Sample Identification Label

Figure 9. Laboratory Sample Identification - Field Code Designation

The laboratory identification protocol is as follows:

Project ID: BBAC Quality Sampling Program (BBAC-WQSP)

Sample ID:

Event Type:W (Wet Weather) or D (Dry Weather)

Sampling Type QG (Water Quality Grab Sample), FD (Field Duplicate Grab Sample) or FB (Field Blank)

"In the field" field measurements will include color, odor, and waste products observations in addition to,

ammonia as nitrogen, temperature, conductivity, and occasionally chlorine. "In house" (analysis in the BBAC

office) will include surfactants (detergents as MBAS) and nitrates. All data will be collected in accordance with

the procedures in Appendix C – SOP: Field Water Quality Measurements and Appendix E Field Equipment

Instructions. Field measurements for conductivity and temperature (YSI 30 meter) will be taken with a calibrated

hand-held probe following the bacteria sampling and other laboratory samples. The hand-held probe will be

submerged directly into the center of the water column or, when necessary, in a secondary container..

Corrective Actions

Corrective action in the field may be required when conditions require a modification to this QAPP. Any member

of the Field Team may identify a problem requiring corrective action. The Field Team, in consultation with the

Stormwater Specialist, TPA, or Project Manager, will implement the minor corrective actions and document the

action taken on the field data worksheets (under comments). Major corrective actions including revision of field

procedures, resampling, or retesting will require approval by the Project Manager prior to implementation. All

major corrective actions will be relayed to the individual Field Teams prior to the next sampling event to ensure

consistency in the monitoring procedures. Corrective actions by the analytical laboratory will be identified

according to the laboratory Quality Assurance Plan. Any deviations from standard procedures, including

BBAC

Water Quality Sampling Program

Sample Date/Time: ______________________________

Sample ID: _____________________________________

Sample Type: _____________________________ _____

Laboratory Analysis: _____________________________

Preservative _______ ________

Collected By: ___________________________________

25

corrective actions, which may jeopardize the integrity of the sample analysis, will be communicated to the Project

Manager or Stormwater Specialist and the TPA immediately and will be reported in the final laboratory QA/QC

report.

2.3 Sample Handling and Custody

Pre-Sampling Activities

All sample containers will be clean, pre-preserved bottles supplied by the contracted laboratory in accordance

with the applicable SOPs. Prior to the collection of samples for laboratory analysis, the sample containers will

be labeled using pre-printed, water-resistant labels. The labels will include the sample ID, analysis requested

and preservative added and will provide locations for collection date and time, and initials of the person who

obtained the sample (see Figure 8).

Chain of Custody (COC)

COC is located at the bottom of each data sheet will be signed and dated by the DPW director (or designee) at

the end of each sampling event. When transferring sample custody, the individuals relinquishing and

receiving the samples will sign and date the COC. This COC documents the transfer of sample custody from

the sampler to another person, to the laboratory, or to/from a secure storage area. The BBAC Project Manager

will retain all the original data sheets with the COC. Copies of the data sheets will be given to the appropriate

laboratory.

Sample Handling

Laboratory samples will be placed in coolers with sufficient blue ice to meet preservation and holding

requirements. The COC form for the samples will be placed in a waterproof plastic bag and will accompany

the samples in the coolers. The coolers will be delivered by the Stormwater Specialist or designee.

2.4 Analytical Methods and Quality Control

Monitoring parameters, analytical methodology, sample volumes, containers, sample processing, and storage

for this project are listed in Table 4 through Table 7

Laboratory SOPs used for each analysis and associated QAs manuals are listed in Appendix A. Barnstable

County Health and Environment lab and the New Bedford Health Department lab will conduct the bacteria

analysis, all other parameters will be done through the Barnstable Co. lab.

Laboratory Analytical Methods/Quality Control

The analytical laboratory will use the procedures outlined in their QA Plan to ensure the reliability and validity of

analytical results (Appendix A). Compliance with the laboratory QA plan will be the responsibility of the

laboratory QA Officer. Laboratory reports will be sent to the BBAC Project Manager and the Technical Advisor

within 28 days of the monitoring event and will include:

Laboratory duplicates and/or blanks

Matrix spikes and matrix spike duplicates (MS/MSDs)

Laboratory Control Standard and Laboratory Control Standard Duplicates (LCS/LCSDs)

Specific information regarding the laboratory analysis of the water quality analysis can be found in the laboratory

SOPs in Appendix A. Specific criteria for the evaluation of laboratory precision and accuracy are provided in

Table 5 and Section D1 – Data Review, Verification, and Validation. Any samples that do not meet the laboratory

QC criteria will be reanalyzed in the respective laboratory if sufficient sample volume is available.

2.5 "In the field" and "In House" Field Measurements/Quality Control

Field measurements will be performed at either monitoring site locations ("In the field") or at the BBAC office

("In house") using portable field units, test kits, and test strips (Table 4). The instruction manuals for the usage of

the portable meter and test strips is included in Appendix C. Specific procedures for quality control of the portable

meter are discussed in Sections B6 – Instrument Testing, Inspection, and Maintenance and B7 – Instrument

Calibration and Frequency.

26

Sample quality of the field parameters will be assessed through the use of field duplicates. The location of the

field duplicate will be determined before the sample round by the Stormwater Specialist. At least one field

duplicate will be taken in each municipality during a sampling event. If more than 10 samples are taken per event

(per municipality), then field duplicates will be taken for every 10 samples. This duplicate sample will be

collected, handled, and analyzed/recorded for the same parameters as the original sample.

2.6 Instrument Testing, Inspection, and Maintenance

The laboratory will perform routine preventative maintenance in accordance with their respective QA Plans and

with manufacturer’s specifications. The laboratory will maintain factory-trained repair staff or will maintain

service contracts with applicable vendors.

The inspection, testing, and maintenance of all field equipment, instruments, and test kits will be performed in

accordance with the respective manufacturers' procedures. The Stormwater Specialist (or designee) will be

responsible for ensuring calibration documentation is provided by the equipment supplier and will be responsible

for testing, inspection, and maintenance of all equipment prior to the first sampling event

.

At the first sampling event, the Field Team will visually inspect the field probes and perform a calibration check,

as described in Section B7 – Instrument Calibration and Frequency. Results of that calibration check will be noted

on the field calibration sheets and Equipment Inspection, Testing, and Maintenance Sheets provided in Appendix

E and any calibration errors will be corrected at that time. Each DPW Director (or designee) will be responsible

for the proper maintenance and storage of the portable meters.

2.7 Instrument Calibration and Frequency

Laboratory Instruments

Calibration procedures and frequencies of all laboratory equipment will be performed in accordance with the

respective laboratory’s QAPs, manufacturer’s specifications, analytical SOPs, and written procedures approved

by laboratory management. Records of calibration method and frequency will be filed and maintained by the

designated laboratory Quality Assurance Officers.

Field Instruments

Instruments and equipment used to conduct the field measurements will be calibrated before each sampling event

in accordance with manufacturer specifications. Any deviations in calibration which may affect final results will

be noted on the equipment worksheets and corrective actions will be taken, as determined by the Stormwater

Specialist and/or Project Manager.

2.8 Inspection/Acceptance for Supplies and Consumables

All supplies to be used during the field sampling program will be inspected (Table 11) by the Stormwater

Specialist or Field Team prior to the monitoring event to ensure that supplies are functioning properly, in

satisfactory condition, and free of defects or contamination in accordance with the methods specified in Table 5.

The Stormwater Specialist and or Field Team will keep extra supplies, stored and maintained in accordance with

the respective manufacturers or laboratory protocol.

27

Table 11. Supply Inspection Requirements and Acceptance Criteria

Supplies Inspection Requirements

Sample Bottles Visual inspection for cracks, breakage, cleanliness, and

preservation solution (if needed)

Chemicals and Reagents (test strips) Visual inspection for proper labeling, expiration dates, and

appropriate grade

Water Quality Monitors (portable meters) Calibration and operational check

Sampling equipment Functional check and visual inspection for cracks, breakage,

damage, cleanliness/contamination

2.9 Non-Direct Measurements

Monitoring data will be collected for both dry and wet weather events for each discharge point location. Rainfall

will be tracked using the NOAA weather station (www.erh.noaa.gov/box/dailystns.shtml) in New Bedford,

Massachusetts . Whenever possible, the sampling collection will coincide with low tide using tidal data from the

NOAA website (http://NOAA Tide Predictions), with supplemental data from the local harbormaster's office.

2.10 Data Management

Laboratory analysis results and QA documentation will be provided by the analytical laboratory to the BBAC

Project Manager and the Technical Advisor. Field measurements collected by the Field Team during sampling

events will be documented on the field data sheets (under Field Tests). Copies of the data sheets will be provided

to the appropriate laboratory and the originals delivered to the Project Manager upon completion of the sampling

event.

The field and laboratory data will be linked to the Buzzards Bay NEP Stormdrain Atlas and associated GIS

database. The GIS database will include information regarding watershed size, impervious surface contribution,

and land use characteristics associated with each discharge point. The GIS database will also include discharge

pipe/roadcut characteristics (pipe material, size, and condition) and other stormdrain data collected in the field.

Utilizing the GIS and monitoring data, the Project Manager and the Technical Advisor will be continually

reassessing the monitoring program and making changes as needed. EPA will be kept informed of any changes

either verbally (phone call) or in writing (email). All water quality monitoring data and GIS data will be available

on the Buzzards Bay NEP website at stormwater.buzzardsbay.org

3.0 Assessment and Oversight

3.1 Assessments and Response Actions

The Project Manager will be responsible for ensuring both laboratory and field activities are performed in

accordance with the procedures established in this QAPP. Throughout the monitoring program, the Stormwater

Specialist will work with the Field Teams directly, coordinating the day to day activities, ensuring on‐site review

of field activities including:

Checking field data sheets, and field equipment operation and maintenance

Checking/calibrating field equipment

Checking status of sample collection, handling, and packaging procedures

Ensuring Quality Assurance (QA) procedures are followed

Ensuring Chain‐of‐Custody procedures are followed

Corrective actions in the field will be implemented on a case-by-case basis. Minor response actions taken in the

field to immediately correct a problem will be conveyed to the Stormwater Specialist (if unavailable then TP or

Project Manager) and documented on the data sheets. Major corrective actions will require approval by the

http://www.erh.noaa.gov/box/dailystns.shtml

http://tidesandcurrents.noaa.gov/tide_predictions.html?gid=1403#listing

http://stormwater.buzzardsbay.org/

28

Project Manager prior to implementation. Such actions may include revising procedures in the field, resampling,

or retesting. All major corrective actions will be relayed to the individual Field Teams prior to the next sampling

event to ensure consistency in the monitoring procedures. Any corrective action undertaken by the laboratory will

be completed in accordance the procedures outlined in their QA Plan. Any deviations from standard procedures,

including corrective actions which may jeopardize the integrity of the sample analysis, will be communicated

(verbally and in writing) to the Project Manager and the Technical Advisor immediately and will be reported in

the final laboratory QA/QC report.

3.2 Reports

All field measurements collected by the Field Team during sampling events will be recorded on the data sheets

and provided to the Project Manager upon completion of the field sampling event. The laboratory analysis data

will be sent to the Project Manager within 28 days of the sampling event. The Project Manager will organize and

compile all the data based on site location onto a spreadsheet.

The field and laboratory data (BBAC spreadsheet) will be linked to the Buzzards Bay NEP Stormdrain Atlas and

associated GIS database. The GIS database will include information regarding watershed size, impervious surface

contribution and land use characteristics associated with each discharge point. The GIS database will also include

discharge point characteristics (pipe material, size, and condition) and other stormdrain data collected in the field.

Once the final monitoring event is completed, the Project Manager and the Technical Advisor will assess the

monitoring and GIS data and provide a final report (including links to the Buzzards Bay NEP Stormdrain Atlas)

to EPA and other interested parties. The report will make recommendations with regards to future monitoring

needs including potential expansion of the program into other municipalities.

4.0 Data Validation and Usability

4.1 Data Review, Verification, and Validation

As discussed in Section 2, the analytical laboratories will use the procedures outlined in their QA Plan to ensure

the reliability and validity of analytical results (Appendix A). Laboratory reports will be sent to the BBAC Project

Manager and the BBNEP QA Officer within 28 days of the monitoring event and will include:

Laboratory duplicates and/or blanks

Matrix spikes and matrix spike duplicates (MS/MSDs)

Laboratory Control Standard and Laboratory Control Standard Duplicates (LCS/LCSDs).

Upon receipt of the laboratory reports both the Project Manager and the Technical Advisor will review the

laboratory and field test results to determine the general validity of sample results.

The accuracy of YSI 30 meter (parameters: conductivity and temperature) and other field parameters will be

assessed using QA procedures outlined in Section 2 and the accuracy/precision data as depicted in Table 5. Prior

to each monitoring event, the Stormwater Specialist will select a site (or sites) for collection of filed duplications

Each field teams will conduct at least one field duplication for each monitoring event in each municipality. If

more than 10 samples are taken per event (per municipality), then field duplicates will be taken for every 10

samples. This duplicate sample will be collected, handled, and analyzed/recorded for the same parameters as the

original sample.

4.2 Verification and Validation Methods

General data evaluation will include review of holding times, laboratory duplicates, matrix spikes/matrix spike

duplicates (MS/MSDs), laboratory control samples/laboratory control sample duplicates (LCS/LCSDs), sampling

procedures, field blank results, equipment blank results as applicable, field duplicate results, and review of the

analytical laboratory QA report. If the Field Team identifies the need for a corrective action, the Stormwater

Specialist will be responsible for approving the implementation of the response action. Problems that are

attributed to laboratory quality assurance issues will be brought to the attention of the Project Manager.

Appendix A

1. Barnstable County Health and Environment

Quality Assurance Plan

SOP's - Fecal Coliform (SM9222D), Enterococci (EPA1600), Nitrate (EPA300), TKN

(EPA351.2 Rev001), Oil/Grease (EPA EPA 1664A contracted to Envirotech Laboratories Inc.,

and Orthophosphate (EPA 365.1 contracted to Mircobac

2. New Bedford Health Department Laboratory

Quality Assurance Manual

SOP's Fecal Coliform and Enterococci (EPA1600)

Barnstable County Laboratory, Massachusetts

April 10, 2015

- 1 -


(Revision 017)

Revised on April 10, 2015

County of Barnstable

Barnstable County Department of Health and the Environment

Water Quality Laboratory

Superior Court House

Route 6A

P.O. Box 427

Barnstable, MA 02630

Appendix A

1. Barnstable County Health and Environment


SOP's - Fecal Coliform (SM9222D), Enterococci (EPA1600), Nitrate (EPA300), TKN

(EPA351.2 Rev001), Oil/Grease (EPA EPA 1664A contracted to Envirotech Laboratories Inc.,

and Orthophosphate (EPA 365.1 contracted to Mircobac

2. New Bedford Health Department Laboratory

Quality Assurance Manual

SOP's Fecal Coliform and Enterococci (EPA1600)


April 10, 2015

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Table of Contents

Page

I. Introduction 3

II. Quality Assurance Policy and Quality Assurance Objectives 3

III. Responsibilities and Authorities 5

IV. Quality Assurance for Precision and Accuracy 8

V. Control Charts 11

VI. Laboratory Corrective Actions 13

VII. Analytical Methods 15

VIII. Sample Management 17

IX. Laboratory Equipments and their Calibration procedures 19

X. Preventive Maintenance 23

XI. Standard Operating procedures 26

XII. Data Reports, Validation and Review 26

XIII. Record Keeping, Logbook Review and Standard Traceability 28

XIV. Safety 30

XV. References 30


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1. Introduction

This Quality Assurance (QA) manual has been developed to describe the overall quality

assurance program employed by the Barnstable County Department of Health and the

Environment Water Quality Laboratory. The County Laboratory performs environmental

analyses of volatile organic compounds, metals, and wet chemistry parameters using Methods

and Guidance for the Analysis of Water (Version 2, June 1999)1 approved by the United

States Environmental Protection Agency, and Standard Methods for the Examination of

Water and Wastewater (22th

Edition, 2012)2 approved by American Public Health

Association, American Water Works Association and Water Environment Federation. The

County Lab also performs analyses of total coliform, fecal coliform, e. coli, and heterotrophic

plate count in potable and/or non potable water using the Standard Method2. The County

laboratory has also carried out its testing and calibration activities in such a way as to meet

the requirements of the International Organization for Standardization (ISO) and the

International Electrotechnical Commission (IEC) 17025:2005 3. The Laboratory specializes

in analysis of drinking water and groundwater whose continuous testing and quality

maintenance are of great interest to the residents of the County.

This plan describes our quality assurance organization and responsibilities, our quality

assurance objectives for precision and accuracy, the format for generating our standard

operating procedures, and the procedure for maintaining our records. The objective of the

program is to assess and maintain the quality, accuracy and precision of the generated data

and to provide a permanent record of instrument performance and overall data quality and

reliability by implementing well defined QA / Quality Control (QC) procedures.

2. Quality Assurance Policy and Quality Assurance Objectives

2.1 Statement of Policy

It is the policy of the Barnstable County Health Laboratory to provide the possible

highest quality analysis of drinking water. We are committed to maintaining a strict

QA program and adhering to all policies required by regulatory and accrediting

agencies and other organizations3. To achieve this high standard of quality, we have

implemented the following quality assurance plan for our analytical services.

Our laboratory employs modern analytical instrumentation and is fully automated to

provide results of high quality in a timely fashion. The analytical professionals and

technicians are well trained with many years of work experience in the respective

area of analysis. Therefore this program assures that the results provided by the

County Laboratory are as accurate as possible and highly reliable.


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2.2 Quality Assurance Objectives

In order to ensure the production of high quality data and clients’ full satisfaction,

Barnstable County Laboratory has established the following Quality Assurance

Objectives:

Full compliance with certification requirements;

Full compliance with regulatory agencies;

Full compliance with contract requirements;

Full compliance with published methodologies;

All personnel concerned with analysis and calibration activities in the

Laboratory are required to familiarize themselves with the quality

documentation and implement the policy as stated above.

2.3 Subcontracted Laboratories

Barnstable County Laboratory also subcontracts some analyses to other

laboratories. Barnstable County Laboratory makes sure these subcontracted

laboratories also meet Quality Assurance Policy and Quality Assurance Objectives

as stated above. Especially Barnstable County Laboratory makes sure that the

subcontracted laboratories must comply with ISO/IEC 17025:20053 when the

samples for National Sanitation Foundation internal projects are analyzed.


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3. Responsibilities and Authorities

Barnstable County does have its Personnel Policies and Procedures5 for all County

employees to follow. Two of major purposes of the Personnel Policies and Procedures are:

To ensure that its management and personnel are free from any undue internal and

external commercial, financial and other pressures and influences that may adversely

affect the quality of their work;

To avoid involvement in any activities that would diminish confidence in its

competence, impartiality, judgment or operational integrity.

Barnstable County laboratory employees must follow not only the County’s Personnel

Policies and Procedures, but also the requirements of the Laboratory’s quality assurance and

quality control program. The quality assurance and quality control program requires an

effective chain of command within the laboratory. The maintenance of this requirement is

the responsibility of the laboratory analysts, office staff and the Laboratory Director.

3.1 Laboratory Director

The Laboratory Director supervises all analysts, technicians, and laboratory

administrative personnel. Additional responsibilities include supporting the

implementation of the quality assurance plan within the laboratory, maintaining and

enforcing standard operating procedures, and maintaining good laboratory practices.

The Director may substitute for any analyst when questions arise and performs any

analysis as required.

The Laboratory Director also acts as the Quality Assurance Officer of the laboratory.

All data are finally reviewed by the Laboratory Director prior to release. Data that

fall outside of quality control limits can be accepted if in the judgement of the

Director there are suitable technical reasons for these to be accepted. However, these

cases are well documented and the reasons for acceptance are fully explained.

If the Laboratory Director is not around for a few days, the organic chemist will

conduct final review and signing of reports. All other issues will go to Department

Director. If there is still anything else that must need the Laboratory Director, the

Laboratory Director will be contacted immediately, and the Laboratory Director will

respond as soon as possible to make sure that the laboratory is operated smoothly.

3.1.1 Internal Audits:

One of the major responsibilities of the Laboratory Director is to conduct the

laboratory audit once a year. The main purpose of the internal audit is to


April 10, 2015

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verify that the laboratory operations continue to comply with the

requirements of the management system, quality assurance policy and quality

assurance objectives. The internal audit covers sampling, sample log-in,

sample analysis and data reporting, etc. The checklist of the Laboratory

internal audits is attached (Attachment 1).

3.1.2 Management Reviews

Laboratory Director also work with Department Director together to conduct

a review of the laboratory’s management system and testing and/or

calibration activities to ensure their continuing suitability and effectiveness,

and to introduce necessary changes or improvements. The review will take

account of the following areas:

The suitability of policies and procedures;

Reports from managerial and supervisory personnel;

The outcome of recent internal audits;

Corrective and preventive actions;

Assessments by external bodies;

The results of interlaboratory comparisons or proficiency tests;

Changes in the volume and type of the work;

Customer feedback;

Customer complaints;

Recommendations for improvements;

Other relevant factors, such as quality control activities, resources and

staff training.

A period for conducting a management review is once a year.

3.2 Laboratory Analysts

All analysts conduct sample analysis and maintain quality assurance by following the

laboratories quality assurance plan. This is achieved by a thorough knowledge of the

appropriate standard operating procedure of each method employed by the analyst.

Especially analysts must closely track the holding times of all analyses, and it is

analysts’ primary responsibility to make sure that all analyses are done within their

holding times. Additional responsibilities include complete and accurate work

records, immediate notification of quality control problems, and the authority to

accept or reject data based on defined quality control acceptance criteria.


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3.2.1 The bacteriologist is the primary microbiological resource for the Laboratory.

The primary duties of the bacteriologist include performing or supervising all

bacteriological analyses, all media and buffer preparation, and all QA/QC

record keeping for the microbiology laboratory. The bacteriologist also

performs and oversees sample container preparation, preservation,

sterilization, and distribution. The bacteriologist also generates the final

reports. If there is any positive identification of total coliform in drinking

water, the bacteriologist will inform customers right away.

3.2.2 The primary duties of the Inorganic Chemist and Inorganic Analyst are

performing all metal analyses by flame or graphite furnace AA and

Inductively Coupled Plasma Mass Spectrometer (ICP-MS), all inorganic

anion analyses by ion chromatography, and all wet chemistry analyses by

closely following Standard Operating Procedures (SOP). The Inorganic

Chemist and Inorganic Analyst also generate final data reports and conduct

data reviews. Secondary duties include supervision of sample container

preparation, preservation, and distribution.

3.2.3 The Organic Chemist is the primary resource for questions regarding organic

methodology and analyses. The primary duties of the organic chemist are to

analyze volatile organic compounds using Gas Chromagraph – Mass

Spectrometer (GC/MS) and Total Organic Carbon (TOC) Analyzer. The

organic chemist is responsible for maintaining proper documentation for any

modified methods and SOP’s. The Organic Chemist also generates data

reports and conducts data reviews. Secondary duties include performing

sample container preparation, preservation, and distribution.

3.2.4 Analytical training is required for all analysts, and training includes new

analyst training and cross training. When a new analyst is hired, a systematic

training will be conducted by an experienced analyst and/or laboratory

Director. All analysts are required and encouraged to be cross trained for each

other to ensure the analysis is done properly when its primary analyst is

absent for a period of time. All training processes will be recorded in Lab

Staff Training Log Book (Attachment 2).

3.2.5 All analysts need to enter their own sample data to the Laboratory

Information Management System (LIMS), and also cross review data entry to

ensure data information are correct in sample received date and time,

customer ID, lab ID, analytical date and time, units, and analytical method.


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3.3 Information Specialist/Billing Clerk

Information specialist/billing clerk has the following three kinds of duties:

Maintains customer accounts receivable records and processes payments as

received.

Prepare invoices as scheduled and as needed basis.

Develop and maintain database applications as needed and directed.

3.4 Administrative Staff

The administrative staff is primarily responsible for sample log-in, inquiries of

sample status, final report generation, taking and filling bottle order requests, keeping

track of inventory and ordering supplies, and typing and calling in purchase order.

Some of the staff's secondary responsibilities include sample container preparation

and sample pick-up.

3.5 Laboratory Personnel

Laboratory Director/Chemist Gongmin Lei

Microbiologist Ryan Grady

Microbiologist Bethany Sadlowski (part-time)

Inorganic Chemist Lacey Prior

Inorganic Analyst Diane Brown

Organic Chemist Yuankun Ni

Inorganic Chemist Kelby Karnes (part-time)

Information Specialist/Billing Clerk Howard Weigel

Administrative Staff Elna Hughes

Laboratory Assistant Jennifer Doherty

Laboratory Assistant Veronica Tavares (part-time)

4. Quality Assurance for Precision and Accuracy

Quality assurance is used to establish and maintain confidence in the precision and accuracy

of the data generated by the Laboratory. The routine procedures utilized in assessing

precision and accuracy are based on established SOPs.

4.1 All data are recorded in the pertinent logbooks, charts, and Laboratory Information

Management System (LIMS). These records are periodically boxed and stored for

easy retrieval. LIMS data are archived yearly. All of the data are maintained for a

period of ten years.


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4.2 Forms used in reporting results are generated by the LIMS and are designed to

convey all pertinent information to the client. Report forms include date of

collection, date of receipt by the laboratory, date of analysis, client name, client I.D.

name or number, and laboratory sample I.D. number. All analyses required by the

Massachusetts Department of Environmental Protection (DEP), Division of Water

Supply (DWS) are reported on forms supplied by DWS. Included in any report, if

requested by the client, are the recoveries of all QC samples and matrix spikes.

4.3 If any analyte exceeds its MCL, the local Health Agent is informed by the report and

the client is telephoned as soon as possible and advised of the results. All

recommended limits exceeded are explained on the report form sent to the client

along with the recommended limit. If any MCL is exceeded for an analysis required

by a State or Federal Agency, the client is contacted immediately (24 hours) and

advised of the result. The client has the primary responsibility to contact the

regulatory agency involved; however, the Laboratory will make the contact within 48

hours if based on follow up conversations with the client, there is no indication of the

results being reported.

4.4 A number of different methods for internal quality control checks are used. The

internal controls include daily instrument blanks, daily method standards, matrix

spikes, duplicates, and annual Proficiency Testing (PT) samples analyses, and PT

samples are ordered from an accredited PT vendor meeting the criteria of the current

policies made by Massachusetts Department of Environmental protection, Laboratory

Certification office. Please check the following website for the detailed information:

http://www.mass.gov/dep/bspt/wes /wespubs.htm.

4.5 Calibration curves must consist of at least three points and are used to calculate

analyte concentration. A separate calibration curve is generated for each analyte

included in the analytical method. Instrument calibration is performed each day

samples are analyzed. The instrument may be completely calibrated with at least

three calibration standards or a single calibration standard may be used to check an

existing calibration curve. Again all analytes of interest are included in the

calibration.

4.6 Method blanks are analyzed daily and represent all sample preparation procedures

excluding any target analytes. The method blank contains all appropriate surrogate

and internal standards, diluents, and modifiers.

4.7 Laboratory fortified blanks (LFB) or Laboratory Control samples (LCS) are analyzed

to monitor accuracy of the method. The LFB is a spiked method blank sample

containing all of the analytes of interest. The recoveries of the analytes are charted


April 10, 2015

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and used as a diagnostic tool to monitor system performance. When these recoveries

approach either the upper or lower limits established by the method, corrective action

is taken before the system fails to meet calibration criteria.

4.8 Quality Control (QC) check samples or Continuing calibration Verification (CCV)

are analyzed to monitor the accuracy of the method. QC check sample is from an

alternative source containing all of the analytes of interest. The recoveries of the

analytes are charted and used as a diagnostic tool to monitor system performance.

When these recoveries approach either the upper or lower limits established by the

method, corrective action is taken before the system fails to meet calibration criteria.

4.9 Duplicate analyses are performed on each batch of samples analyzed. The frequency

is generally 10 % of all samples in the batch, but at least one sample if less than 10

samples are analyzed. The duplicate samples are prepared and analyzed using

exactly the same procedures as the original sample. The recovery of the duplicate

analysis is used to monitor the reproducibility of the entire procedure.

4.10 Matrix spikes are performed to account for any matrix effects in an environmental

sample. The frequency of matrix spike analysis is 10 % of a sample batch. Duplicate

matrix spike analyses are used to monitor reproducibility.

4.11 Bottle sterility check for microbiology laboratory: The laboratory checks at least

one bottle per lot of commercially prepared sample containers for sterility by adding

approximately 25 ml of sterile non-selective broth to each bottle. The bottle is capped

and rotated so that the broth comes in contact with all surfaces and is incubated at

35±0.5ºC and checked for growth at 24 and 48 hours and the results are recorded.

4.12 Precision Criterion of Duplicate Analysis:

In order to determine the acceptability of duplicate analysis of Fecal Coliform, E.coli,

Enterococci, and Hetertrophic Plate Count (HPC), their Precision Criterion of

Duplicates analysis are obtained by calculating the range of logs of most recent 15

samples and their corresponding duplicates as follows:

4.12.1 Collect the most recent 15 sets of the results of the original

samples and their duplicates.

4.12.2 Calculate the Logarithms of each set of the results, and record

them as L1 and L2. If any result is <1, add 1 to both values

before calculating the Logarithms.

4.12.3 Range of Logarithms (Rlog) is calculated using the following

equaltion:


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21log LLR

4.12.4 The mean ( R ) of Rlog is calculated as follows:

n

RR

log

logR = the sum of the range of Logs;

n = the number of sets of duplicates.

4.12.5 Precision Criterion is calculated as follows:

RterionecisionCri 27.3Pr

4.13.6 If any Range of Logarithms is greater than the precision Criterion,

there is a greater than 99% probability that the analysis has exceeded

variability limits. For any samples that fall outside the acceptable

limits, the acceptability of the imprecision will be determined. If the

data are not acceptable, all results since the last precision check must

be rejected. The analytical problems will be determined and

corrective actions will be taken to resolve problem.

Corrective action based on internal quality control samples or

external samples such as performance evaluation sample is used to

maintain precision and accuracy of the analytical results. The process

for corrective action includes a review of the history of the problem

by checking standard preparation logs, instrument maintenance logs,

and QC charts. Based on the historical information the cause of the

problem is narrowed. The next steps are to change a defective part,

clean a dirty part, or if necessary call a service engineer for advice or

a visit.

5. Control Charts2

Two types of control charts are used in the County Laboratory: (1) accuracy (or means) chart;

and (2) precision (or range) chart.

Accuracy Chart:


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The accuracy chart for QC samples is constructed from the average and standard

deviation of a specified number of measurements of the analyte of interest. The

accuracy chart includes upper and lower warning levels (WL) and upper and lower

control levels (CL). ±2s and ±3s are used for the WL and CL, respectively, where s

represents standard deviation. These values are derived from stated or measured

values for reference materials. The number of measurements, n or n-1, used to

determine the standard deviation, s, is specified relative to statistical confidence

limits of 95% for WLs and 99% for CLs. The County Laboratory is using the

Accuracy Chart for Laboratory Control Sample (LCS)/Laboratory Fortified Blank

(LFB), Matrix Spike (MS), and sample surrogate recovery. A chart is constructed for

each analytical method. The results are entered on the chart each time the QC sample

is analyzed. Everything is done in EXCEL Spreadsheet.

Precision Chart:

The precision chart also is constructed from the average and standard deviation of a

specified number of measurements of analyte of interest. Precision chart is used for

percent differences of LCS/LCSD, MS/MSD, and sample and sample duplicate.

Perfect agreement between replicates or duplicates results in a difference of zero

when the values are subtracted, so the baseline on the chart is zero. Therefore for

precision charts, only upper warning limits and upper control limits are meaningful.

A chart is constructed for each analytical method. The results are entered on the chart

each time the QC sample is analyzed. Everything is done in EXCEL Spreadsheet.

Updating of Control Charts:

If measurements never or rarely exceed the WL, recalculate the WL and CL using the

20 to 30 most recent data points. Trends in precision can be detected sooner if

running averages of 10 to 20 are kept. Trends indicate systematic error; random error

is revealed when measurements randomly exceed warning or control limits.

Application:

Control Limit – If one measurement exceeds a CL, repeat the analysis

immediately. If the repeat measurement is within the CL, continue analyses;

if it exceeds the CL, discontinue analyses and correct the problem.

Warning Limit – If two out of three successive points exceed a WL, analyze

another sample. If the next point is within the WL, continue analyses; if the

next point exceeds the WL, evaluate potential bias and correct the problem.

Trending – If seven successive samples are on the same side of the central


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line, discontinue analyses and correct the problem.

Barnstable County Laboratory has calculated our own in-house control limits

and warning limits for drinking water and wastewater. Each analyst closely

reviews his or her in-house control limits for each analysis based on the

procedures stated as above. The laboratory director must review control

charts monthly.

6. Lab Corrective Actions

Quality control data outside the acceptance limits or exhibiting a trend are evidence of

unacceptable error in the analytical process. The County laboratory takes corrective action

promptly to determine and eliminate the source of the error. The laboratory do not report data

until the cause of the problem is identified and either corrected or qualified.

The County Laboratory records any problems and issues that occur and affect data integrity,

lab safety and lab operation. If there are any unusual things happened in the Lab, the Lab

Director is informed right away, and the Originator must describe the detailed information on

these problems and issues in the Laboratory Corrective Action Log Book (See Attachment

3). The Originator needs to address the following three things:

(1) Describe what happened;

(2) Describe how and why they happened;

(3) What actions should be taken to prevent or eliminate them happening again?

The following three categories of problems and issues can be written down in the lab

Corrective Action Log Book:

(1) Improper Lab Practices:

Definition: A scientifically unsound or technically unjustified omission,

manipulation, or alteration of procedures or data that bypasses the

required QC parameters, making the results appear acceptable.

Any alteration of data such that the data are unauthentic or untrue

representations of the experiment or test performed.

Peak integrations are not done properly;

Quality Control Samples do not meet criteria;

Initial Calibrations do not meet criteria;

Sample holding times are out;

Method Blank is contaminated;

Loss of sample;


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Equipment malfunction;

Etc.

(2) Standard Operating Procedures (SOP) Modifications:

Incorporate new equipment into the SOP;

Correct the wrong or inappropriate procedures in the SOP;

Etc.

(3) Any other actions affecting data quality and lab operations:

6.1 Corrective Actions:

Corrective actions begin with the analyst, who is responsible for knowing when the

analytical process is out of control. The analyst must initiate corrective action when a

QC check exceeds the acceptance limits or exhibits trending and must report an out

of control event to the supervisor. The corrective actions to be used when QC data

are unacceptable are as follows:

Check data for calculation or transcription error. Correct results if error

occurred.

Check to see if sample(s) was prepared and analyzed according to the

approved method and SOP. If it was not, prepare and/or analyze again.

Check calibration standards against an independent standard or reference

material. If calibration standards fail, reprepare calibration standards and/or

recalibrate instrument and reanalyze affected sample(s).

If a LFB fails, reanalyze another laboratory-fortified blank.

If a second LFB fails, check an independent reference material. If the second

source is acceptable, reprepare and reanalyze affected sample(s).

If a LFM fails, check LFB. If the LFB is acceptable, qualify the data for the

LFM sample (Table 1 lists the data qualifiers) or use another method or the

method of standard addition.

If a LFM and the associated LFB fail, reprepare and reanalyze affected

samples.

If reagent blank fails, analyze another reagent blank.

If second reagent blank fails, reprepare and reanalyze affected sample(s).

If the surrogate or internal standard known addition fails and there are no

calculation or reporting errors, reprepare and reanalyze affected sample(s).


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6.2 Customer Complaints:

Barnstable County laboratory is totally customer-focused organization, and it has

been understood that customer complaints represent valuable information about

recurrent problems. Laboratory secretary and assistant are always front-line staff to

handle customer complaints, and they must give customers their full and undivided

attention. Laboratory secretary and assistant must resolve complaints promptly if

they are able to. Laboratory secretary and assistant must report any customer

complaints to laboratory director. If the customer complaints can not be resolved

right away, laboratory secretary and/or lab assistant must report to laboratory director

right away. The laboratory director must talk to the customers to find out the root

causes for complaints if necessary. Laboratory director must take actions to have any

mistakes corrected properly and promptly, and call back to customers to explain the

final resolutions for the complaints. All these processes must be recorded in the

Laboratory Customer Complaint Logbook (Attachment 4).

If there are any customers of Barnstable County Test Center to complaint to the Test

Center Staff about laboratory testing, the Test Center Staff will pass the complaints to

the laboratory. Barnstable County Test Center has its own Customer Complaint

Logbook. All the resolutions of the Customer Complaints for the Test Center must be

reviewed by Department Director.

7. Analytical Methods

The analytical methods performed by the Barnstable County Laboratory have come from two

sources1, 2

. The first one is Methods and Guidance for the Analysis of Water (Version 2) by

EPA, and the second one is Standard Methods for the Examination of Water and

Wastewater, 22th

Edition, 2012 by American Public Health Association, American Water

Works Association and Water Environment Federation. Barnstable County Laboratory has its

own modified standard operation procedures (SOP) based on the methods stated above.

These SOP including any modifications are approved and certified by the Laboratory

Certification Office (LCO) of Massachusetts Department of Environmental Protection. All

non-approved methods used are for informational purposes only and are documented as such.

The Massachusetts Laboratory Certification Identification Number of Barnstable County

Laboratory is M-MA009.


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7.1 A list of methodologies used for specific analysis is noted below:

Parameters Methodologies 1, 2

Bacteria Potable Non-Potable

Total Coliform MF-SM9222B

Total Coliform ENZ.SUB.SM9223

Total Coliform EPA 1604

Fecal Coliform MF-SM9222D MF-SM9222D

E. Coli NA-MUG-SM9222G EPA 1604

E. Coli EPA 1604

E. Coli ENZ.SUB.SM9223

Heterotrophic Plate Count SM9215B

Enterococci EPA 1600 EPA 1600

Metals Potable Non-Potable

Aluminum 200.8

Antimony 200.8 200.8

Arsenic 200.8 200.8

Barium 200.8

Beryllium 200.8 200.8

Cadmium 200.8 200.8

Chromium 200.8 200.8

Calcium SM3111B SM3111B

Cobalt 200.8

Copper 200.8, SM 3111B 200.8, SM 3111B

Iron SM 3111B

Lead 200.8 200.8, SM 3111B

Magnesium SM 3111B

Manganese 200.8, SM 3111B

Mercury 200.8

Nickel 200.8 200.8

Potassium SM 3111B

Selenium 200.8 200.8

Sodium SM 3111B SM 3111B

Thallium 200.8 200.8

Vanadium 200.8

Zinc 200.8, SM 3111B

Inorganics


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Total Alkalinity SM 2320B SM 2320B

Biochemical Oxygen Demand SM 5210B

Specific Conductivity 120.1, SM2510B

Chemical Oxygen Demand HACH 8000

Total Organic Carbon SM 5310B

Chloride 300.0

Fluoride 300.0 300.0

Nitrate-N 300.0 300.0

Nitrite-N 300.0

Sulfate 300.0 300.0

pH SM 4500-H-B SM 4500-H-B

Non-Filterable Residue (TSS) SM 2540D

Total Dissolved Solids SM 2540C

Turbidity 180.1

Total Hardness (CaCO3) SM 2340B

Perchlorate 314.0

Organics

VOC 524.2 624

Trihalomethanes 524.2

7.2 In order to clearly distinguish in the analytical reports between those analyses for

which it holds Massachusetts Department of Environment Protection Certification

and those for which it does not hold Massachusetts Department of Environmental

Protection Certification, Barnstable County Laboratory has started attaching a

summary of the laboratory certifications as stated in Section 7.1 to each of analytical

reports to all customers.

8. Sample Management

8.1 The generation of quality data begins with the collection of the sample. The integrity

of the sample collection is therefore of importance to the laboratory. Samples must

be collected in such a way so as not to disrupt the integrity of the sample by the

introduction of foreign material or the release of any material of interest. The

laboratory maintains sample integrity by supplying the appropriate sample containers,

ensuring that the sample containers are properly cleaned and contain the appropriate

preservative, enforcing sample holding times to allow adequate time for analysis, and

ensuring that adequate volumes of the sample are collected.


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8.2 Upon receipt of a batch of samples, the Sample Reception Office examines the

samples for breakage or damage while checking the accompanying documents for

conformance with sampling procedure. The Sample Receiving Person also insures

that the type of preservative is noted. Then the samples are logged into the LIMS by

the Sample Receiving Person, and an unique laboratory ID is assigned to each

sample. The Sample Receiving Person must ensure all information is entered into the

LIMS correctly. All information and documentation are relayed to the analyst for

his\her review and if any further sample manipulation is required, it is performed in a

timely fashion.

8.3 All chain-of-custody samples received by the laboratory are examined for breakage or

damage and sample integrity. Once the chain-of-custody form has been reviewed for

clarity and accuracy, it is signed and the samples are received into the laboratory.

After receipt, the samples are logged into the sample log book by the Sample

Receiving Person, given a laboratory identification number, and stored in a secured

area. The internal report form follows the sample through the laboratory until all

analyses are complete. At the end of each day when the sample was being analyzed,

it is returned to the secured area until all analyzes have been completed.

8.4 If the samples are not properly collected, preserved and handled, they will not be

accepted by the laboratory. The following are the Laboratory’s policies for the sample

rejection:

The samples are stored in wrong containers such as non-sterile bottles for

total coliform;

The samples are out of the holding times;

There is no clear identification of the sample matrix;

There are Bubbles in VOC vials;

There are no enough sample volumes such as less than 100 ml for Total

Coliform analysis;

The samples are preserved with wrong preservatives;

Perchlorate samples are not filtered with sterile filters and syringes;

The samples are not kept in coolers when they are received.

NOTE: When the samples are rejected, the samples receiving officers will

make notes on the chain of custody for any rejection reasons.

8.5 There are the following circumstances under which the received samples need to be

subcontracted to another certified laboratory:

There are too many samples, and the number of the samples received exceeds


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the capacity of the laboratory.

The instruments break down, and they cannot be fixed right away. Then the

samples may need to be subcontracted out.

The samples received contain the uncertified parameters, and they will be

subcontracted out.

The samples requiring total metals will be subcontracted out for the time

being.

8.6 If there are any contaminants detected which exceed their Maximum Contaminant

Levels, Maximum Residual Disinfectant Level or reportable concentration, the

subcontracted laboratories are required to notify Barnstable County Laboratory within

24 hours of obtaining valid data. When it prepares the chain of custody to the

subcontracted laboratory, Barnstable County Laboratory will stamp on it with

“NOTIFY FOR ANY MCL EXCEEDANCES”.

8.7 If the final analytical reports need to be submitted to Massachusetts Department of

Environmental Protection, Barnstable County Laboratory will stamp on the chain of

custody to the subcontracted laboratory with “STATE FORM”. The subcontracted

laboratory will have to report the data with MA DEP required format.

9. Laboratory Equipments and their Calibration Procedures

Calibration of the laboratory's equipment is performed on a regular basis. The calibration is

performed in accordance with the manufacturer's instruction or in accordance with the

calibration procedures outlined in the appropriate analytical methodology and, as needed.

Each piece of equipment requires preventive maintenance to ensure optimal performance of

the instrument. The preventive maintenance schedule is supported by vendor service

maintenance contracts and an inventory of spare parts. All major instruments have separate

service contracts that include a yearly preventive maintenance visit. The minor instruments

and equipment are covered by a general laboratory preventive maintenance contract where a

service representative will annually check and calibrate all ovens, incubators, thermometers,

refrigerators, autoclaves, UV-VIS spectrophotometers, and fume hoods.

9.1 Flame Atomic Absorption - This instrument is calibrated daily if samples are

analyzed with five calibration standards prepared fresh. The calibration is checked

with a QC check sample of an alternative source and a LFB sample. Both of these

analyses occur before any samples are analyzed and at the end of the analysis. Also,

interferences are analyzed for with a method blank.

9.2 Total Organic Carbon Analyzer (TOC-V CPH/CPN) - This instrument is calibrated daily


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with a six point calibration curve and checked for interferences with a method blank.

The calibration is checked with QC and LFB samples at a frequency of 10% of the

sample load.

9.3 Inductively Coupled Plasma-Mass Spectrometer (ICP-MS):

There are the following few tuning steps must be done before the ICP-MS is

calibrated for analysis:

Tuning for sensitivity daily by using a 2 wt% nitric acid solution containing

10 ng/ml of each of the elements: Li, Co, Y, Ce, and Tl. Sensitivity after

tuning must meet criteria set by ICP-MS manufacturer;

Tuning for detector daily by setting P/A factor of each target element;

Checking of mass calibration, mass resolution and instrument stability.

Initial calibration is composed of six different levels of standards, and initial

calibration verification standards are run following the initial calibration to verify the

accuracy of the initial calibration. Please refer to the SOP for EPA Method 200.8.

9.4 Ion Chromatograph - Each day a five-point calibration curve of the analytes of

interest is generated from freshly prepared standards for nitrite-N, o-phosphate,

bromide, nitrate-N, sulfate, chloride, and fluoride standards. Each day a one point

calibration standard (continuing calibration) is analyzed to check an existing

calibration curve or as the daily single point standard. Interferences are checked with

reagent water blank. Checks include QC check and LFB samples at a frequency of

20% and matrix spikes to identify any environmental sample matrix effects.

9.5 Gas Chromatographs - Each day a one point calibration standard (continuing

calibration) is analyzed to check an existing calibration curve or as the daily single

point standard. For the daily single point standard method, if an analyte is detected,

another single point calibration standard closer to the suspected concentration is

analyzed. Daily method blanks including all steps in sample and standard

preparation are run to insure the absence of interferences. QC check and LFB

samples are analyzed to check the calibration and system and method performance.

9.6 GC\MS - Each day the mass spectrometer tune settings are checked with 25 ng of

p-bromofluorobenzene (method 524.2) or, 50 ng of p-bromofluorobenzene (for

method 624). The background subtracted spectra with the highest abundance is

compared to and must meet established criteria for analysis to continue. When a

successful BFB analysis is achieved, a single point calibration standard (Continuing

Calibration) is analyzed to check the validity of the existing initial calibration curve.


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Whenever the MS is tuned, a new five point calibration curve may need to be

generated. A method blank is analyzed daily to check for interferences and matrix

spikes are analyzed for matrix effects.

9.7 PC-Titrate – The PC-Titrate is used to analyze pH, conductance, and alkalinity in

aqueous samples:

PH is calibrated daily using standard solutions of pH = 4.0, pH = 7.0, and pH

= 10. A beginning and an end QCs (pH = 7.0) are run every ten samples.

Alkalinity measurement is based on the pH calibration. A beginning and an

end QCs (Alk = 25 mg/L) are run every ten samples.

Conductance probe is calibrated using seven levels of standards. A beginning

and an end QCs (Cond = 100 µmohs/cm) are run every ten samples.

9.8 Refrigerators - Each day the refrigerator is used, the temperature is recorded and must

be within + 2oC of the required 4

oC. The thermometers used to measure the

temperatures are calibrated yearly with a NIST certified thermometer.

9.9 Balances - Each day the balance is used, the calibration of the balance is checked

with two class S weights that bracket the expected weight to be measured. The

deflection test is performed for the top-loader balances. The analysts make sure the

balances are capable of detecting 100 mg at 150 g. The results of the deflection test

are recorded daily. The range of weights available is 0.10 mg to 100 g, and they are

all NIST traceable. The accuracy of the reference weights are verified annually.

9.10 Incubators (bacteriology) - The temperature for each incubator is recorded twice each

day with readings separated by at least four hours. The temperatures must be within

+ 0.2oC for the 44.5

oC water bath and + 0.5

oC for the 35

oC incubator and + 0.5

oC for

the 41.5oC incubator. The thermometers used to measure the temperatures are

calibrated yearly with a NIST certified thermometer.

9.11 Incubators (BOD, CBOD) - Each day the incubator is used, the temperature is

recorded twice per day. The temperatures must be within + 0.5oC for incubators to

be maintained at 20oC. The thermometers used to measure the temperatures are

calibrated yearly with a NIST certified thermometer.

9.12 Ovens - During use the temperature is monitored to insure that the required

temperature is reached and maintained for the appropriate length of time.

9.13 Thermometers - All thermometers are made of glass material and are calibrated


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yearly with a NIST certified thermometer. Any correction factor to be used in

reading the temperature of the thermometer is indicated on the record sheet taped to

the refrigerator or incubator where the thermometer is located. The reference

thermometers are calibrated annually.

9.14 Reagent Grade Water – The distilled water is produced by using EMD Millipore

water purification system. There are three water purification systems from EMD

Millipore:

Two Milli-Qs (Model);

One Direct-Q (Model).

The quality of the purified reagent water used in microbiology laboratory as stated in

the following paragraph must be met1:

The specific conductivity of the reagent water is monitored and recorded

daily, and the values of specific conductivity must be less than 2 mhos/cm at

25C.

The resistivity of the reagent water must be ≥ 16.5 megohm-cm (temperature

compensated to 25ºC), and the old cartridges will be replaced if the reading is

below 16.5 megohm-cm. The daily resistivity is recorded.

Residual chlorine level is tested monthly. The amount of the chlorine

detected must be less than 0.01 mg/L.

Heterotrophic plate count (HPC) is performed monthly to check if HPC is

less than 500 colony forming units (CFU)/ml. If HPC ≥ 500 colony forming

units (CFU)/ml, the distilled water system will be checked and maintained.

Heavy metal content is tested annually, and no single metal (Cd, Cr, Cu, Ni,

Pb and Zn) may be present at 0.05 mg/L. Total heavy metals must be less

than 0.1mg/L.

Biosuitability is tested annually, and it must have a ratio of 0.8-3.0.

9.15 Autoclaves: There are three autoclaves in the laboratory:

Market Forge: Model: STM-E;

Tuttnauer: Model: 3870M;

Tuttnauer: Model: 3850E-B/L.

The following parameters are recorded in the lab Autoclave Logbook when any

autoclaves are used for sterilization:

Start time;

Pressure;

Temperature;

Items which are placed in the inside of the autoclaves for sterilization;


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Cycle length;

Stop time;

Maximum Temperature

Initial and date

10. Preventive Maintenance

Each analytical system or piece of equipment is required to be maintained according to the

manufacturer’s recommendations. Regular maintenance checks ensure that the systems are

able to operate properly and efficiently on a consistent basis.

Each major piece of equipment is covered by a service contract offered by the manufacturer

(or a similar company). These contracts include an annual preventive maintenance visit. In

addition, more frequent maintenance is performed as recommended by the manufacturer on a

regular basis by the laboratory staff as needed.

Maintenance logbooks are utilized to document major preventive as well as emergency

maintenance procedures as these are performed. These logbooks are also used to document

any routine maintenance / repair procedures.

The following outlines the major and minor preventive maintenance routines for each

analytical system in operation within the laboratory:

10.1 Gas Chromatograph

Cut the chromatographic column on a regular basis.

Change injection port septum, o-ring and the glass wool in the liner, when necessary.

Clean by baking, and/or solvent rinsing the Electron Capture Detector, when signal is

high.

Service the Electron Capture Detector, when signal high after cleaning the detector.

Cycle/Bake out the entire system once a week, when not in use.

10.2 Mass Spectrometer

Check rough pump oil leads, routinely.

Clean the source when the instrument fails to tune.

Change the electron multiplier when the applied voltage is too high.

Routinely verify that adequate calibration fluids are available for automatic

instrument tuning.

Change the o-rings and transfer lines when inspection indicates these are degrading or

weakening.

10.3 Inductively Coupled Plasma-Mass Spectrometer (ICP-MS) -


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See attachment 5 for preventive maintenance of the ICP-MS.

10.4 Purge and Trap Apparatus -

Purge each line prior to analysis.

Bake the trap once all lines are purged.

Check for leaks in the purge and trap system using the pressurization technique when

air leaks are detected.

Change the ferrules when leak check supports a leak; this procedure may be done

frequently at most susceptible sites.

Replace the trap when data indicate its degradation.

10.5 Ion Chromatograph

Regularly check the eluent reservoir to see if it needs to be filled.

Daily check the component-mounting panel for leaks or spills. Wipe up spills. Isolate

and repair leaks. Rinse off any dried eluent with deionized water.

Weekly check fluid lines for crimping or discoloration. Relocate any pinched lines.

Replace damaged lines.

Check the pump and replace the pump piston rinse seals and piston seals if necessary.

Replace the AS40 Automated sampler tip and tubing once a year.

10.6 Flame Atomic Absorption

10.6.1 Burner Head:

Clean and polish the top surface using mild abrasive soap cleaner;

Clean the burner slot with the Burner Cleaning tool supplied or a piece of

stiff card. Do not use an abrasive material inside the slot;

Wash the Burner with detergent solution and rinse with deionized water. Dry

the Burner carefully before using it again;

To clean the interior surface of the Burner head, dismantle and use an

ultrasonic bath with deionized water, dilute detergent solution or 5% (v/v)

solution of nitric acid.

10.6.2 Spray Chamber:

Aspirate a solution of 1% (v/v) hydrochloric acid, or a solution of laboratory

detergent to clean the Spray Chamber. Aspirate the solution for 5 minutes,

then aspirate deionized water.

For more thorough cleaning, dismantle the Spray Chamber, clean the

individual components with lab detergent, rinse thoroughly with deionized

water, dry and reassemble.

10.6.3 Nebulizer:


April 10, 2015

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Use the cleaning probe and push through the nebulizer to dislodge to remove

any blockage in the nebulizer capillary.

10.7 pH Meter

Clean the electrode prior to and after analysis.

Change the electrode, when necessary.

Store the electrode in pH 7 buffer solution between analyses.

Clean and maintain the magnetic stirrer and magnetic stirring bars, whenever used.

Examine the KCl level in the electrode prior to use, add more solution, when

necessary.

10.8 PC-Titrate

Clean the electrodes/probes prior to after analysis.

Change the electrodes/probes when necessary.

Replace all tubings once a year.

10.9 Water Purification Unit

Check the conductivity of the finished water daily.

Change the cartridges when resistivity starts to decrease, approximately every 2-4

months.

10.10 Quebec Colony Counter and Microscope

Microscopes and colony counters are maintained in a clean condition and checked for

defects on each use. All glass surfaces are periodically cleaned as needed. Moving

parts of all microscopes are lubricated on an-needed basis.

10.11 Total Organic Carbon Analyzer (TOC-V CPH/CPN)

10.11.1 Daily Inspection:

Check the level of dilution water;

Drain vessel water and humidifier water;

10.11.2 Periodic Inspections:

Catalyst Regeneration;

Washing or replacing catalyst;

Replacing the carrier gas purification tube and catalyst;

Washing/replacing the combustion and carrier gas purification tubes;

Replacing the CO2 absorber;

Replacing the Halogen Scrubber;

Syringe replacement.


April 10, 2015

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10.11.3 Autosampler (ASI-V) Maintenance

Please see the USER’s MANUAL 5 for the details.

Generally if any instruments have problems, the analysts or technical director will try fix

them first. If the problem still persists, service maintenances will be called.

11. Standard Operating Procedures

Each method utilized by the laboratory has a standard operating procedure (SOP) developed

by the laboratory (Attachment 6). The analyst follows this procedure at all times to ensure

proper operation accuracy and precision. The SOP's include the analytes to be measured, the

detection limits of the method, and the applicable matrices. Each SOP also includes a

detailed description of the method procedure.

The safety issues involved in the analysis are discussed to insure the safety of the operator

and the laboratory environment. All reagents and standards are described as well as the

recipes for their preparation. When applicable, sample holding times, appropriate containers,

preservatives, and methods of collection are discussed. A step-by-step procedure is detailed

with all sample preparation, instrument calibration, and sample analysis steps described.

After completion of the analysis, the SOP describes the calculation procedure and the

reporting procedure for the analysis to ensure accurate reporting of the results. Following

this section is a description of the QA/QC requirements of the method. Described within this

section are the QC samples to be analyzed, the acceptance criteria for each QC sample, and

potential corrective action to be taken if the criteria are not met.

All corrections or updates to existing SOP's are approved by the Laboratory Director before

incorporated into the daily routine. All analysts involved in the analysis by the revised SOP

must read and acknowledge the revised SOP.

12. Data Report, Validation and Review

12.1 Data reporting is performed through the Laboratory Information Management System

and a custom created system to report data to Massachusetts Department of

Environmental Protection (MA DEP), Drinking Water Program. There are three

sections on any lab reports produced by the Lab: (1) Customer Information, (2)

Analytical Information, and (3) Signature and Date.

12.1.1 Regarding the Customer Information, the following items must show on any

report:


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Reporting mailing address;

Public Water Supply (PWS) ID# (for reporting to MA DEP);

PWS Name (for reporting to MA DEP);

City/Town (for reporting to MA DEP);

Class: COM, NTNC or TNC (for reporting to MA DEP);

Multiple or Single (for reporting to MA DEP);

Raw or Finished (for reporting to MA DEP);

Date collected;

Collected by (or Sampler);

Routine Sample or Special Sample (for reporting to MA DEP);

Original, Resubmitted or Confirmation (for reporting to MA DEP);

Reason for resubmission (for reporting to MA DEP);

Collection Date of Original Sample (for reporting to MA DEP);

Sample Notes.

12.1.2 Regarding the Analytical Information, the following items must show on any

report:

Primary Laboratory Name;

Primary Laboratory Massachusetts Certification Number (Barnstable

County Health Laboratory: M-MA 009);

Subcontracted Laboratory Name if any samples are sent out;

Subcontracted Laboratory Massachusetts Certification Number;

Sample Matrix;

Specific Analytes and their Respective Results, Maximum

Contaminant Levels (MCL), Maximum Detection Limits (MDL),

Analytical Methods, Analytical Dates and time, Analysis Lab MA

Certification Numbers, Analysis Lab Name, and Lab Sample ID#;

Information on the sample composited by the Lab and lab notes;

12.2 Data validation is the process by which data are accepted or rejected based on a set of

specific criteria. This process is performed to insure possible accuracy of the data

and the calculations in data reduction process.

12.2.1 The initial review of all data is performed by the analyst. The analyst checks

the raw sample results and compares them to the daily standard(s), daily

method blank, and all QC results. If all of the QC results meet the criteria

established in the method SOP, the completed report sheet for that sample is

submitted for validation. All relevant daily QC samples (standards, blanks,


April 10, 2015

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check samples, and LFBs) are included with the report sheet. This

information is reviewed by the Laboratory Director. Any analysis performed

by the Director will be submitted to the Chemist in charge of that area.

12.2.2 The secondary review of all data is preformed by an Analyst or the laboratory

director who did not conduct the associated analyses. The review covers the

following areas:

All raw data supporting the report are included;

All data in LIMS are correctly entered;

All criteria of QA/QC are met;

The subcontract lab name is indicated on the report if any parameters

were subcontracted out;

Any comments on the report are properly addressed.

12.2.3 The final review is conducted by the Laboratory Director, and the content of

review is the same as ones described in Section 12.2.2. The reports are then

signed and checked off in the LIMS as being completed.

12.2.4 Data reduction includes adjusting reporting limits for sample amount and any

dilutions required, rounding of the results occurs after all calculations have

been made, all results are reported in two or three significant figures, and no

results are reported below the method reporting limit unless requested by

customer. Volatile organic results are reported in ug/L or ug/Kg, metal

results are reported in mg/L, and bacteriological results are reported in

CFU/100 mL (CFU = colony forming units). Inorganic results are generally

reported in mg/L except for pH (0.1 pH units), specific conductivity

(umhos/cm), alkalinity and total hardness (mg/L CaCO3), and turbidity (NTU

or nephelometric turbidity units).

12.2.5 For customers’ reference, Barnstable County Laboratory attaches its certified

parameter list approved by Massachusetts Department of Environmental

Protection to any analytical report.

13. Record Keeping, Logbook Review and Standard Traceability

13.1 As part of the QA/QC plan, the procedure for maintaining the records for all aspects

of the laboratory operation are well established. Each instrument has a log book in

which each analysis is recorded. Also, the printouts of each analysis are filed

according to the intra-laboratory identification number. Included in these files are

copies of the appropriate chain-of-custody forms, quality control reports, and all


April 10, 2015

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calculations of the data. All of this material is available upon request by the client.

13.2 The results of quality control checks such as temperature records for ovens and

refrigerators, quality control results for laboratory glassware, distilled water, and

microbiological media are stored as permanent records to maintain quality

control in the laboratory. Instruments such as pH meters, analytical balances, and

thermometers are calibrated daily or prior to use, and the records of these

calibrations are also maintained.

13.3 The laboratory maintains copies of all analytical reports, logs, charts, and any

other documentation for a minimum of 10 years.

13.4 Standard Traceability and Logbook Review:

13.4.1 All standards and reagents received will be called as primary

standards that will be recorded in the laboratory primary logbook.

Any standards made from the primary standards will be called as

either intermediate or working standards which will be recorded in

the laboratory working standard logbooks. All primary standards,

intermediate and working standards will be assigned unique

identification numbers (ID) which will be also recorded. These

unique IDs will be recorded on their Certificate of Analyte will be

kept in a three hole binder. The intermediate, working standards and

primary standards will be labelled clearly on their containers with the

unique IDs.

13.4.2 There are also the following logbooks:

Instrument running logbook

Instrument maintenance logbook

Media preparation logbook

Refrigerator and freezer temperature logbook

Conductance logbook for lab water purification logbook

Buffer preparation logbook

pH meter calibration logbook

Incubator and water bath temperature logbook

Lab corrective action logbook

Customer complaint logbook

Lab staff training logbook

13.4.3 Logbook Review


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Laboratory Director or an analyst assigned by the Laboratory Director

will review the logbooks quarterly to make sure all recordings are

done correctly and properly.

14. Safety

A formal safety program for the Laboratory is issued to each new employee. All

personnel are introduced to the safety equipment available in the laboratory and

instructed as to its use. All analysts are made aware of the safety consideration of the

chemicals they use by Right-to-Know training and their review of the individual

material safety data sheets. If an analyst requests additional safety measures, the

request is acted on immediately.

14.1 All gas tanks such as Helium, Argon, Oxygen, Hydrogen, Acetylene, and Air

must be chained and secured.

15 References

1. Environmental Protection Agency, Methods and Guidance for the Analysis of

Water, Version 2, June 1999.

2. American Public Health Association, American Water Works Association

and Water Environment Federation, Standard Methods for the Examination

of Water and Wastewater, 20th

Edition, 1998, and 22th

Edition, 2012.

3. International Organization for Standardization (ISO) and International

Electrotechnical Commission (IEC), General Requirements for the

Competence of Testing and Calibration Laboratories, ISO/IEC

17025:2005(E), Second Edition, 2005-05-15.

4. Barnstable County, Personnel Policies and Procedures, Effective July, 2005.

6. SHIMADZU CORPORATION, User’s manual for Total Organic Carbon

Analyzer (TOC-V CPH/CPN) (For TOC-Control V Ver.2), Part# 638-94536,


April 10, 2015

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Table 1: QC Data Qualifiers

Symbol Explanation

B Analyte found in reagent blank, Indicate possible reagent or

background contamination.

E Reported value exceeded calibration range.

J Reported value is an estimate because concentration is less than

reporting limit and greater than method detection limit or because

certain QC criteria were not met.

N Organic constituents tentatively identified. Confirmation is needed.

PND Precision not determined.

R Sample results rejected because of gross deficiencies in QC or

method performance. Re-sampling and/or reanalysis is necessary.

RND Recovery not determined.

U Compound was analyzed for, but not detected.


April 10, 2015

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ATTACHMENT 1: The Checklist of Laboratory Internal Audits

1. Sample Management

Sampling

Shipping

Receiving

Holding times

Preservatives

Temperature

Log-in

Storage

2. Analysis

Certified methods

Standard operating procedures (SOPs)

Holding times

3. Quality Assurance and Quality Control (QA/QC)

Runlog/Sequence

Method blank, Lab Control Sample and Lab Control sample Duplicate

(LCS/LCSD), and Matrix Spike and Matrix Spike Duplicate (MS/MSD)

Method Detection Limit (MDL) study

Accuracy and Precision

Control Charts

Second Sources

Continuing Calibration Verification (CCV)

Maintenance logbook

Corrective Action Logbook

4. Data Entry, Data Reporting and Data Backup

Data entry

Report generation

Data backup

5. Data Review

Analyst level

Cross review

Final review

6. Report Mailing, Filing and Storage


April 10, 2015

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ATTACHMENT 2: Lab Staff Training Log Book

DATE: TRAINEE: TRAINER:

TRAINING START DATE:

TRAINING ENDING DATE:

PURPOSES:

GOALS:

SPECIFIC TRAINING ACTIONS:

DOES THE TRAINING MEET THE INITIAL GOALS? EXPLAIN IF NECESSARY.

COMMENT:


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ATTACHMENT 3: Lab Corrective Action Log Book

Corrective Action Log Book

Date: Lab: Originator:

Problem/Issue Description:

Reasons for the Problem/Issue:

Actions Taken to Eliminate The Problem/Issue in the Future:


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ATTACHMENT 4: Customer Complaint Log Book

Customer Complaint Log Book

Date: Name of Customer: Originator:

Content of the Complaint:

Reasons for the Complaint:

Actions Taken to Satisfy the Customer/Make Things RIGHT:

Comment by Lab Director or Department Director:


April 10, 2015

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ATTACHMENT 5: ICP-MS Routine Maintenance Schedule

Time* Component Task Subsystem

D Argon gas Check for argon gas

pressure and volume

D Peristaltic pump

tube

Check for damage

D Sampling cone,

Skimmer cone

Check orifice

WN Sampling cone,

Skimmer cone

Clean/replace Interface

WN Nebulizer Clean/replace Sample

introduction

WN Pelistaltic pump

tube

Replace Sample

introduction

WN Torch Clean/replace Torch box

WN Water filter Check/replace Cooling

support

1 W Torch, spray

chamber, End cap

Clean

1 W Nebulizer Clean

1 W Cooling water Check water volume

and for contaminants

1 M Rotary pump Check oil level and

color

Vacuum

1 M Cooling water

filter

Check

1 M Extraction Lens (If

necessary)

Check

1 M Sample tubing Replace

6 M Rotary pump Change oil vacuum

1 Y Oil mist filter of

Rotary pump

Check/replace mist

filter

vacuum

2 Y Argon gas filter Replace (2 years

after installation)

* D = day; WN = when needed; W = week; M = month; Y = year.

Attachment 6: A List of Standard Operating Procedures


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Lab Method Code Description

Microbiology EPA 1604 Total Coliforms and Escherichia coli in Water by Membrane Filtration

Using a Simultaneous Detection Technique (MI Medium)

Microbiology SM 9222B,

SM9222G

Determination of Total Coliform in Potable and Non Potable Water Using

SM 9222B and SM9222G

Microbiology SM 9223 Determination of Total Coliform Bacteria and Escherichia coli (E.coli) in

Potable Water

Microbiology SM 9222D Determination of Fecal Coliform in Potable and Non Potable Water Using

SM 9222D

Microbiology SM 9215B Heterotrophic Plate Count for Potable Water Using SM 9215B

Microbiology EPA 1600 Determination of Enterococci in Water using membrane-Enterococcus

Indoxyl-β-D-Glucoside Agar (mEI)

Microbiology EPA 1603

Determination of Escherichia coli (E.coli) in Water by Membrane

Filtration Using Modified membrane-Thermotolerant Escherichia coli

Agar (Modified mTEC)

Organics EPA 524 Determination of Volatile Organic Compounds in Aqueous Samples

Using Gas Chromatography/Mass Spectrometry

Organics EPA 624 Determination of Volatile Organic Compounds in Non-potable Water

Using Gas Chromatography/Mass Spectrometry

Organics SM 5310 B Determination of Total Organic Carbon (TOC) in Aqueous Samples Using

High-Temperature Combustion Method

Organics SM 5210B

Determination of Biochemical Oxygen Demand (BOD5)

and Carbonaceous Biochemical Oxygen Demand (CBOD5) in Aqueous

Samples Using SM 5210 B

Organics HACH 8000 Determination of Chemical Oxygen Demand (COD) in Aqueous Samples

Inorganics EPA 200.8 Determination of Trace Elements in Aqueous Samples by Inductively

Coupled Plasma – Mass Spectrometry

Inorganics SM 3111B

Determination of Sodium, Copper, Iron, Manganese,

Zinc, Nickel, Potassium, Calcium, Magnesium

in Aqueous Samples Using SM 3111B

Inorganics EPA 314.0 Determination of Inorganic Anions in Aqueous Samples Using Ion

Chromatography

Inorganics EPA 300.0 Determination of Inorganic Anions in Aqueous Samples Using Ion

Chromatography

Inorganics SM 2320B Determination of Alkalinity in Aqueous Samples

Inorganics SM 2510 B

EPA 120.1 Determination of Conductance in Aqueous Samples

Inorganics SM 4500-H-B Determination of pH in Aqueous Samples

Inorganics SM 2540 C Determination of Total Dissolved Solids in Aqueous

Samples

Inorganics SM 2540 D Determination of Total Suspended Solids in Aqueous

Samples

Inorganics EPA 180.1 Determination of Turbidity in Aqueous Samples

Inorganics SM 2340B Determination of Hardness in Aqueous Samples

Barnstable County Department of Health and the Environment Laboratory

STANDARD OPERATING PROCEDURE

For

Determination of Fecal Coliform in Potable and Non Potable Water Using

SM 9222D

Revision (003)

November 4, 2015

Signature Date

Bacteriologist: Ryan Lucier ____________________________

Laboratory Director: Gongmin Lei ____________________________

Barnstable County Health Laboratory

November 5, 2015

2

Table of Contents

1.0 Scope and Application 3

2.0 Summary of Method 3

3.0 Interference and Contamination 3

4.0 Safety 3

5.0 Equipment and Supplies 4

6.0 Reagents and Standards 4-9

7.0 Preservation and Storage 9-10

8.0 Quality Control 10-16

9.0 Procedure 16-19

10.0 Data Analysis and Calculations 19- 20

11.0 References 21

12.0 Waste Disposal 21

13.0 Appendix A 21-22

14.0 Appendix B 23


November 5, 2015

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For

Determination of Fecal Coliform in Potable and Non Potable Using

SM 9222D

1.0 Scope and Application

1.1 This method describes a membrane filter (MF) procedure and verification

procedure for the detection and enumeration of Fecal Coliform in potable

and non potable waters.

1.2 Since a wide range of sample volumes or dilutions can be analyzed by the

MF technique, a wide range of levels in water can be detected and

enumerated.

2.0 Summary of Method

2.1 The MF method provides a direct count of fecal coliform bacteria in water

based on the development of colonies on the surface of the membrane

filter. A water sample is filtered through the membrane which retains the

bacteria. After filtration, the membrane containing the fecal coliform is

placed on mFC agar and then placed in a water bath at 44.5±0.2°C for 24

±2.0 hours.

3.0 Interferences and Contamination

3.1 Water samples containing colloidal or suspended particulate material can

clog the membrane filter and prevent filtration, or cause spreading of

bacterial colonies which could interfere with identification of target

colonies.

4.0 Safety

4.1 The analyst/technician must know and observe the normal safety

procedures required in a microbiology laboratory while preparing, using,

and disposing of cultures, reagents, and materials and while operating

sterilization equipment.

5.0 Equipment and Supplies

5.1 Stereoscopic Microscope (vendor: Fisher Scientific, Stereomaster)

5.2 Hand tally or electronic counting device

5.3 Plastic or glass 1 and 10 mL graduated serological pipets


November 5, 2015

4

5.4 Graduated cylinders, 50 mL, 100mL, 250 mL, 500mL, and 1000mL

5.5 Ultraviolet light box for sanitization of stems and funnels

5.6 Electronic vacuum pump (Model # SKH33DN16GX, Vendor: VWR)

5.7 Flask, filter, with appropriate tubing

5.8 A five or six manifold filtration assembly (glass stems and funnels

Vendor: Fisher)

5.9 Flat tipped forceps

5.10 Ethanol or isopropanol

5.11 Burner , Bunsen or Fisher type, or propane fuel lighter

5.12 National Institute of Standards and technology (NIST) certified

thermometer (Thermometer, Cat. # 1005-3FC, range (-1+101°C, 1, 1°C)

5.13 Thermometers for incubators

5.14 Incubators (Vendor: VWR, Model: 1550 and 1545), Waterbaths (Vendor:

Blue M, Model MW-1130A-1, and Vendor: VWR , Model 1265PC and

1203) with appropriate temperature range (35.5°, 41.0°, and 44.5°C)

5.15 Millipore membrane filters, sterile, white grid marked 0.45µm pore size

47mm diameter

5.16 Sterile Millipore petri dishes 9x50mm designed for 47mm filters

5.17 Flasks 100mL, 500mL, 1000mL, 2000mL

5.18 Plastic Inoculation loops at least 3mm diameter

5.19 Pyrex Borosilicate glass Test Tubes with autoclavable caps, size 20x

150mm

5.20 Whirl-Pak® bags

5.21 Hot plate (Vendor: Corning PC-620)

5.22 Stir bars

5.23 Safety glasses

5.24 Gloves

5.25 Lab coat

5.26 Balances (Vendor: Scout, Item # SC6010)

5.27 Autoclaves (Vendor: Tuttnauer Brinkmann, Model 3870M, Vendor:

Market Forge, Model:STM-E)

6.0 Reagents and Standards

6.1 Purity of Reagents: Reagent-grade chemicals shall be used in all tests.

Whenever possible, use commercial culture media as a means of quality

control. Use Reagent grade water from either the Milli-Q or Solution

2000 system to assure quality of water.

6.2 Phosphate Buffer Solution

6.2.1 Purpose and Use:

The phosphate buffer solution is used as a diluting and rinsing

solution for the bacteria samples. The Magnesium Chloride is

added to the buffer to increase the recovery of organisms with


November 5, 2015

5

metabolic injury due to high quality waters or to waters containing

significant concentrations of metals.

6.3 Preparation of Stock Magnesium Chloride Solution (MgCl2):

Add 32.44g of Magnesium Chloride Solution (MgCl2 • 6H2O) to 400mL

of reagent-Grade distilled or deionized water. Transfer to 500mL bottle

with a loose cap. Put autoclave tape on bottle and autoclave at 121°C for

15 minutes. Label bottle as “MgCl2 Stock” with date and initials. Store

this in the refrigerator for future use.

6.4 Preparation of Stock Potassium Dihydrogen Phosphate Solution

(KH2PO4 ): Add 13.6g of Potassium Dihydrogen Phosphate to 200mL of

reagent-grade distilled or deionized water. Adjust pH to 7.2±0.5 with 1N

NaOH. Add remaining distilled water to bring to 400mL. Transfer to

500mL bottle with a loose cap. Put autoclave tape on bottle and autoclave

at 121°C for 15 minutes. Label as “KH2PO4 Stock” with date, pH and

initials.

6.5 Phosphate Buffer Solution Preparation

Composition:

Reagent-grade Distilled or Deionized water 2L

MgCl2 Stock Solution 10mL

KH2PO4 Stock Solution 2.5mL

6.5.2 Preparation: Always take the initial pH of the KH2PO4 before

you prepare the buffer. The initial pH should be 7.2±0.5. Always

take the initial pH of the reagent grade distilled or deionized water

before making the buffer, the pH should be 5.5-7.5. Dissolve all

ingredients in 2L of Reagent-Grade distilled or deionized water by

stirring in a 2L flask. The final pH should be 7.2 ±0.2. If the pH is

not within range, add 1-2 drops of 2N NaOH. Stir and check pH.

Keep adjusting pH until it is within the range. Transfer buffer

solution into 1L bottles filling them only ¾ of the way full. Affix

autoclave tape to caps and cap bottles loosely. Autoclave the

buffer for 30 minutes at 121°C. Allow bottles to cool down before

tightening caps. Date and initial bottles and store at room

temperature for future use. Perform sterility checks on each batch

of buffer before use (refer to section 8.18 in Quality Control

section).

6.6 Tryptic Soy Broth(TSB) (Becton Dickenson:211825)

Composition:


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Pancreatic Digest of Casein 17.0g

Enzymatic Digest of Soybean Meal 3.0g

Dextrose 2.5g

Sodium Chloride 5.0g

Dipotassium Phosphate 2.5g

6.6.1 Preparation: To prepare 1L measure out 30g of TSB using top

loading balance. Suspend in 1L of reagent-grade deionized water

in an Erlenmeyer flask. Warm slightly with stirring to dissolve

completely. Add 25 mL of broth to 20, 30mL screw cap tubes.

Before sterilization add 100mL of TSB to 5 clear 250mL bottles

(for buffer sterility checks). Autoclave for 15 minutes at 121°C;

do not exceed 45 minutes in autoclave. The final pH is 7.3±0.2 at

25ºC. The TSB can be stored in the refrigerator for three months

and then discarded.

6.7 R2A agar (Reference number: Becton Dickinson 218263)

Composition:

Yeast Extract 0.5 g

Proteose Peptone No.3 0.5 g

Casamino Acids 0.5 g

Dextrose 0.5 g

Soluble Starch 0.5 g

Sodium Pyruvate 0.3 g

Dipotassium Phosphate 0.3 g

Magnesium Sulfate 0.05 g

Agar 15.0 g

6.7.1 Preparation: To prepare one liter of media suspend 18.2 g R2A

agar in one liter of reagent grade deionized water in a 2 liter

Erlenmeyer flask. Heat to boiling with stirring until completely

dissolves. Be careful that the media does not burn on the

bottom of the Erlenmeyer flask. Dispense in 12 ml

amounts after tempering into 16 x 150 mm screw cap tubes. The

media can last up to 3 months being stored at 4°. Autoclave agar

for 15 minutes at 121oC. Do not exceed 45 minutes in

autoclave. Final pH should be 7.2±0.2 at 25.0ºC.

6.8 mFC agar (Reference #: Becton Dickinson 267720)

Composition:


Proteose Peptone No.3 9.0g


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Yeast Extract 3.0g

Lactose 12.5g

Bile Salts No. 3 1.5g


Agar 15.0g

Aniline Blue 0.1g

6.8.1 Preparation: This media cannot be autoclaved. All utensils and

containers must be sterilized prior to use. To make 1L of media,

prepare 10mL of 1% Rosolic acid solution. Add 0.1g Rosolic acid

(cat. # 83910) and dissolve in 9.9mL 0.2 NaOH. Store at 4° C in a

screw cap tube for up to two weeks; discard if solution becomes

brown or muddy. Measure 1 L of reagent-grade deionized water in

a graduated cylinder and pour into a 2 liter Erlenmeyer flask

containing a stir bar. Add 52g of mFC agar to the flask. Boil to

dissolve completely. Add 10mL of a 1% solution of Rosolic acid in

0.2N NaOH and continue heating for 1 minute. The final pH

should be 7.4±0.2 at 25 °C, adjust with 1N HCL if necessary.

Dispense 4-5 mL of tempered media (45° C) into sterile petri

dishes. Allow to solidify and invert and store in labeled box at

4°C. Media can last up to two weeks.

6.9 EC Medium ( Reference #: Becton Dickinson 231420)

Composition:

Tryptose 20.0g

Lactose 5.0g

Bile Salts No. 3 1.5g


Monopotassium Phosphate 1.5g

Sodium Chloride 5.0 g

6.9.1 Preparation of EC Medium: To prepare 400 mL of broth (makes

approximately 40 tubes). Add 14.8g of EC Medium in 400mL of

deionized water in a sterile 600mL beaker. Warm the broth

slightly with stirring to dissolve agar completely. Use Durham

tubes for EC media. Place test tubes into test tube racks that can

withstand the autoclave. Place an inverted Durham fermentation

tube into each test tube. Dispense 10mL amounts into each test

tube. Cap loosely, affix autoclave tape onto tubes. Sterilize in

the autoclave for 15 minutes at 15psi and 121°C. Limit the time in

the autoclave to 45 minutes. Before opening autoclave, allow the

temperature to drop below 75° C to avoid entrapping air bubbles in

the inverted tubes. After the tubes have cooled, tighten the caps of

the tubes and check to see that no air bubbles are present in the


November 5, 2015

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Durham tubes. If air bubbles are present in the Durham tubes, the

media must be discarded. The final pH when cooled to 25°C

should be 6.9±0.2. Label the rack with date, type of media, and

initials. Media should be stored in a dark cabinet at room

temperature to be used within 3 months.

6.10 Laurly Tryptose Broth (LTB) (Becton, Dickinson and Company;

Reference # 224140)

Composition:


Proteose Peptone No. 3 8.0g

Lactose 5.0g


Monopotassium Phospate 2.75g


Sodium Lauryl Sulfate 0.1g

6.10.1 Preparation of LTB: to prepare 400mL of LTB (makes

approximately 40 tubes) add 14.2g of LTB in 400mL of reagent-

grade deionized water in a sterile 600mL beaker. Dissolve broth

completely by heating and stirring but do not allow it to boil. Use

Durham tubes for LTB. Place test tubes into test tube racks that

can withstand the autoclave. Place an inverted Durham

fermentation tube into each test tube. Dispense 10mL amounts into

each test tube. Cap loosely, affix autoclave tape onto tubes.

Sterilize in the autoclave at 121°C and 15psi for 15 minutes. Limit

the time in the autoclave to 45 minutes. Before opening autoclave,

allow the temperature to drop below 75° C to avoid entrapping air

bubbles in the inverted tubes. After the tubes have cooled, tighten

the caps of the tubes and check to see that no air bubbles are

present in the Durham tubes. If air bubbles are present in the

Durham tubes, the media must be discarded. The final pH when

cooled to 25°C should be 6.8±0.2. Label the rack with date, type

of media, and initials. Media should be stored in a dark cabinet at

room temperature and needs to be used within 3 months.

6.11 Holding Times for Media:


November 5, 2015

9

Media Holding time

MF agar in plates with tight-fitting lids in

refrigerator

Agar or broth in tightly closed screw-capped

tubes in refrigerator

Poured agar plates with loose fitting covers in

sealed plastic bags in refrigerator.

Agar or broth in loose-cap tubes at 4o C

Dehydrated Media opened in dessicator

Dehydrated Media opened not in dessicator

2 weeks

3 months

2 weeks

1 week

1 year from date

opened

6 months from

date opened

7.0 Preservation and Storage

7.1 Start microbiological analysis of water samples as soon as possible after

collection to avoid unpredictable changes in the microbial population. For

most accurate results, ice samples during transport to the laboratory and do

not submerge samples in water or ice during transport to the laboratory.

This could cause cross contamination in the samples.

7.2 Drinking water for compliance purposes: Preferably hold samples <10°C

during transit to the laboratory. Analyze samples on day of receipt

whenever possible and refrigerate overnight if arrival is too late for

processing on same day. Do not exceed 30 hour holding time from

collection on analysis for coliform bacteria.

7.3 Nonpotable water for compliance purposes: Hold source water, stream

pollution, recreational water, and wastewater samples below 10°C during

a maximum transport time of 6 hours. Refrigerate these samples upon

receipt in the laboratory and process within 2 hours. When transport

conditions necessitate delays in delivery of samples longer than 6 hours,

consider using either field laboratory facilities located at the site of

collection or delayed incubation procedures.

7.4 Other water types for noncompliance purposes: Hold samples below 10°C

during transport and until time of analysis. Do not exceed 24h holding

time.


November 5, 2015

10

8.0 Quality Control

8.1 Thermometers and temperature recording equipment- The accuracy of

each thermometer is checked annually against a certified NIST

thermometer at the temperature used. In general the graduations on the

thermometer should be in increments of 0.5 degrees or less. The

maximum thermometers and NIST thermometers are checked annually by

Alert Scientific. The result of each thermometer check is recorded in the

thermometer logbook.

8.2 Balances- Balances are checked daily with the provided class S weights.

The top loading balance is for weighing 3g or more. For this balance the

100.0g, 50.0g, 1.0g, and 100mg weights are used. Each weight is weighed

independently, and in addition, a deflection test is performed prior to

usage. The results of the check are recorded in the assigned balance book.

The S class weights and balance are checked annually by Alert Scientific.

8.3 pH Meter- The pH meter is to be calibrated daily with at least three

standards: 4.00, 7.00, and 10.00. The standards need to be changed daily.

The temperature, slope, and pH are recorded in the temperature logbook.

The pH meter is checked annually by Alert Scientific.

8.4 Media dispensing apparatus- Check accuracy of volume by pipetting the

amount into a weighing dish on the top loader balance. Adjust the setting

accordingly. When sterilizing, put the glass barrel in the apparatus and

tighten finger tight and then loosen quarter of a turn, this lets airflow

through the setup so the pressure, from autoclaving, won’t build up and

break the glass. Also, fill an Erlenmeyer flask with reagent grade water

and sterilize. This water is used to pump through the syringe at the end of

each use. If there is any visible staining at the end of use, boil in reagent

grade water.

8.5 Autoclave (Tuttnauer Brinkmann Model 3870M and Market Forge Model

# STM-E)- Record items being sterilized, temperature (maximum

thermometer), pressure and time for each run. Use BTSure ampoules

(catalog # AY759X3) weekly by placing the ampoule in the autoclave for

15 or 20 minutes at or above 121°C. After autoclaving is complete, let

ampoule cool and then gently squeeze and break the glass ampoule. The

ampoule will now be activated. It must immediately be placed in a screw

cap tube and placed in the small water bath at 55°C for 48 hours. Also

activate and incubate one ampoule that has not been autoclaved as a

control. The control will turn yellow after incubation and if there is no

color change (ampoule remains purple) for the autoclaved ampoules then

it is concluded that the sterilization process of the autoclave is working

properly. Make sure there is enough water in the water bath so BT Sure


November 5, 2015

11

ampoules are completely submerged throughout the incubation period.

Autoclave tape is used on every sample bottle before autoclaving. A

maximum temperature thermometer is used and temperature is recorded

after each run. The Autoclaves are checked annually by Alert Scientific.

8.6 Refrigerators- Check and record all temperatures daily. Use a refrigerator

maintaining a temperature of 1 to 4.4°C to store samples, media, reagents,

etc.

8.7 Water baths- Temperature is checked and recorded twice daily with a

four hour separation of time between readings and adjusted when needed.

The time of the temperature check will also be recorded.

8.8 Incubators- Temperature is checked and recorded twice daily with a

four hour separation of time between readings and adjusted when needed.

The time of the temperature check is also recorded.

8.9 Membrane filter equipment- Wrap filter units in sterile wrap and

autoclave daily for 20 minutes at 121°C. Before each set of samples

expose stems and funnels to 2 minutes of UV light. Wash filter units

weekly with detergent and rinse with deionized water before the daily

autoclaving. Record the lot number and date of receipt of new Millipore

membrane filters in the microbiology primary standard logbook when they

are received.

8.10 Ultraviolet sterilization lamps- Clean lamps with ethanol monthly and

record in quality control logbook. Do a sterility check to assure proper

sterilization. Either stock organisms or a known positive water source can

be used.

8.10.1 Procedure for UV sterility check- Label R2A plates as follows:

1. Control (swabbed)

2. UV left

3. UV center

4. UV right

Pouring Plates: Melt solid R2A agar (refer to section 6.7 for media

preparation) in boiling water and put one tube of water or an extra

R2A tube in the boiling water and then transfer to 46°C water bath.

Place a thermometer in the water tube or extra R2A tube to

indicate medium temperature. Melted agar must be used within

three hours and can not be re-melted. Agar can be used when temp

cools to between 44-46°C. Allow no more than 20 minutes to

elapse between plating sample and pouring medium. Before

pouring agar wipe the tube dry with a paper towel and flame the

neck of the tube. Carefully pour the contents of one tube (12mL)


November 5, 2015

12

into the 100x15mm petri dish, and mix contents by rotating

clockwise and then counter clockwise to cover the bottom surface

of the petri dish. Allow agar to solidify. Pipet 0.1mL or a five-

fold dilution of the broth culture or 0.1mL of a two-fold dilution of

the raw sewage sample onto each of the five plates, using a sterile

glass spreader, spread the inoculums evenly over the entire surface

of the agar of each plate. Plate #1 is to be left out of the UV box as

the control. Remove the lids of plates 2-4; place the plate into the

light box. Set the timer to two minutes and turn on the UV lights.

At the end of two minutes turn off the UV lights and remove the

plates. Incubate the UV sterilized plates and the controls for 48

hours at 35±0.5° C. After the incubation period check plates for

growth, control plates should show heavy growth, (>200 colonies)

and sterilized plates should show little to no growth (0-2CFU).

The kill rate from the lamps should be 98%. If more than 2

colonies form on a plate then lamps need replacing.

8.11 Solution 2000 and Milli-Q deionized water - The sterility of reagent water

should be checked monthly using pour plate method with R2A.

8.11.1 Sterility Procedure: 1mL of the Solution 2000 water is pipetted

into a large sterile petri dish. Sample is run in duplicate on another

plate. Heat tempered R2A (refer to section 6.7 for media

preparation) is poured over sample and swirled clockwise and

counterclockwise. Repeat process for Milli-Q water. After the

plates solidify they are inverted and incubated for 48 hours at 35.0

± 0.5°C. After the incubation period check plates for

growth; this water should be sterile.

8.12 The Total Chlorine residual of the reagent water is checked monthly.

8.12.1 Total Chlorine Procedure: Using chlorimeter fill one test cell

with reagent water to 10mL line. Add contents of one DPD Total

Chlorine pouch to the sample cell; cap and shake for 20 seconds.

Set timer for 3 minutes; during this time fill the other cell with

10mL of the reagent water and cap (this is the blank). Place blank

in chlorimeter with cover on and press ZERO. Remove blank cell

and within 3 minutes after the 3 minute period place prepared

sample cell in chlorimeter, cover, and press READ. Record result

in QC Log (result must be less than 0.1mg/l).

8.13 The pH of the reagent water is checked before each use. Fill clean beaker

with reagent water and stir. Measure the pH with a calibrated meter.

Record result in media preparation log with the media (pH of water must

be 5.5-7.5).


November 5, 2015

13

8.14 Air Quality- The air quality in the laboratory is monitored monthly.

Plates of R2A media are left open in different parts of the laboratory for

15 minutes. A sterile plate is also poured and left closed for the 15

minutes. Incubate plates @ 35±0.5°C for 48 hours and observe for growth

and record in appropriate book.

8.15 Glassware- Glassware must have the pH checked because some

cleaning solutions are difficult to remove completely, spot check batches

of clean glassware for pH reaction, especially if soaked in alkali or acid.

This is done by adding a few drops of 0.04% bromthymol blue (BTB) to a

bottle and observing for a blue-green color indicating neutral pH. One of

the bottles from each batch of washed and dried bottles needs to be tested

by adding 3 drops of 0.04% BTB to the bottle. Observe for any color

reaction. If the color is blue-green then the bottle from the batch can be

used. If the color is yellow (acid) or blue (basic) then that batch would

need to be rewashed. The results are to be recorded in the QA/QC log. If

0.04% of bromthymol blue needs to be prepared: add 16 mL 0.01N

NaOH to 0.1g BTB and dilute to 250mL with distilled water.

8.16 All Microbiological bottles need to be autoclaved. Non-disposable sample

bottles are washed in the dishwashers (using a distilled water rinse

cycle).Bottles and caps are then removed from the dishwasher. All caps

need heat activated autoclave tape affix on them and then they are placed

in a clean beaker and covered with aluminum foil. Bottles and all caps are

autoclaved for 20 minutes at 121-134°C. Make sure the tape has turned to

a black color. When handling caps after being autoclaved use aseptic

techniques when taking them out of the beaker and tightening caps on

bottles when removed from the autoclave and cooled.

8.17 Autoclaved and sterile bottles need a sterility check. For each new batch

of bottles received and at least one batch of autoclaved bottles per day

perform a sterility check by adding approximately 25mL of sterile non

selective broth (Tryptic Soy Broth) to at least one bottle. Cap bottle and

rotate so that broth comes into contact with all surfaces. Check for any

growth after incubation at 35°C at 24 hours and at 48hours. Record

results in Quality Control Log. If TSB does not remain clear rewash and

autoclave all bottles from the batch/batches in question.

8.18 Buffer Sterility Checks- The sterility of the buffer needs to be checked

after it has been autoclaved and about to be put away for use. After the

Tryptic Soy Broth has been made according to TSB preparation in section

6.6, add 20mL of prepared buffer water to a TSB bottle using a top loader

balance. Once combined shake the bottle and mix the buffer with the

TSB. Label the bottle with the date and time it enters the incubator with

the tech’s initials. Incubate at 35oC checking for any growth and/or

cloudiness at 24 hours and at 48 hours. If solution appears cloudy it was


November 5, 2015

14

not sterilized properly and the batch must then be re-sterilized. If the TSB

is clear then buffer can be put away for future use in cabinet. This

information is then documented in the QC logbook.

8.19 Utensils and containers for media preparation- Only use utensils and

containers of borosilicate glass, stainless steel, aluminum, or other

corrosion-resistant material. Do not use copper utensils. Use glassware

that is clean and free of residues, dried agar, or other foreign materials that

may contaminate media.

8.20 Culture Media- Culture methods depend on properly prepared media; use

the best available materials and techniques in media preparation, storage,

and application. Order media in quantities that last no longer than a year.

When media is received record kind, amount, lot number, expiration date

and date received. Store media in a cool and dry place away from

sunlight. Store opened containers of media in a desiccator immediately

after use. Discard media that cake, discolor, or show other signs of

deterioration. If expiration date is given by manufacturer, discard unused

media after that date. Discard any opened media after one year. Never

combine different lots of media.

8.21 New Lot testing- Pure culture comparison testing and positive and

negative controls need to be performed whenever a new lot of culture

media is opened.

Batch testing- Each batch of laboratory-prepared media must be tested

prior to use with at least one pure culture control of a positive and one

pure culture of a negative (non-target reaction, as appropriate to method)

reaction.

Target Bacteria Positive Control Negative Control

Fecal coliforms Escherichia coli E. aerogenes

E. faecalis

8.22 Comparative Testing Procedure: Use the old batch as your control group.

Make parallel tests between the control group and the test group. As a

minimum make a single analysis using five pure positive control stock

organism samples (Example: E.coli). After incubation, compare bacterial

colonies from the two lots for size and appearance. If colonies on the test

lot plates are atypical or noticeably smaller than colonies on the reference

lot plates, record the evidence of inhibition or other problem, regardless of

count differences. Re-test media and if the differences still occur discard

media.

8.22.1 Positive and negative controls: Using known organisms test each

new lot of media to ensure proper growth. Swab new media plates

or tubes with appropriate control cultures and incubate at


November 5, 2015

15

appropriate time and temperatures. Record results in Quality

Control logbook.

8.23 For routine performance evaluation, repeat counts on one or more positive

samples at least monthly and compare the counts with those of other

analysts testing the same samples. Replicate counts for the same analyst

should agree within 5% and those between analysts should agree within

10%.

8.24 Analysts must perform analysis of samples for various parameters in a

uniform fashion. Example: If a sample needs fecal coliform, total

coliform and E.coli run, then the analyst will perform all three tests with

appropriate dilutions first before moving on to next sample.

8.25 Precision of Duplicate Analysis: Duplicates are run on 10% of samples for

all analyses requiring enumeration. For each most recent set of 15 samples

and its corresponding duplicate (for each type of sample analysis i.e. fecal,

e.coli, enterococci), the range of logs is calculated in order to determine

the acceptance criteria of precision. Once established, the criteria of

precision will be used to determine if the following duplicate analyses are

within acceptable limits. At the end of each week the criteria are updated

to include the most recent set of 15 samples run in duplicate. The updated

precision is used to determine precision acceptability for the following

week.

8.25.1 Obtain the recorded numbers of the most recent 15 samples run in

duplicate (for the same type of analysis).

8.25.2 Calculate the logarithm of each result (and if any result is <1 then

add 1 to both values before calculating the logarithm) and record in a

spreadsheet as L1 and L2.

8.25.3 Range of Logarithms (Rlog) is calculated using the following

equation:

Rlog= │ L1-L2 │ (1)


(2) ∑R log = The sum of the range of logs. n = The number of sets of transformed duplicates. 8.25.5 Precision Criterion is calculated as follows: __

n

RR

log


November 5, 2015

16

Precision Criterion = 3.27 x R (3)

8.25.6 Any samples run in duplicate (10% of all samples) have their

Range of Logarithms (Rlog). If the range is greater than the Precision

Criterion, there is a greater than 99% probability that the analysis has

exceeded variability limits. For any samples that fall outside the

acceptable limits, determine if the imprecision is acceptable. If data is not

acceptable, all results since the last precision check should be rejected.

The analytical problem(s) should be determined and corrective action

should be taken to resolve problem.

8.25.6 See Appendix B for an example of the precision criterion.

9.0 Procedure

9.1 Procedure for membrane filtration:

9.1.1 Analyze samples within 8 hours of collection unless customer

specifies otherwise. Samples must be preserved in a refrigerator or

cooler until analysis.

9.1.2 Use mFC agar for fecal coliform identification.

9.1.3 Sterilize work area by washing down with a solution of 50%

Bleach, 2%Lysol, or Conflikt.

9.1.4 CAUTION: UV light is carcinogenic and damaging to eyes.

Sterilize filter funnel and bases in UV light box for 2 minutes.

Turn light off before lifting wooden cover. Daily wrap the filter

funnels in steriwrap and sterilize in autoclave for 20 minutes at

121°C.

9.1.5 Label plates on the bottom (media side) with bottle # and dilution

if other than 100mL of sample is used. Record time on each plate.

9.1.6 Place the filter funnel base in manifold. Be sure valve is open.

9.1.7 Dip the forceps in alcohol (95% ETOH) and pass through the

flame.

9.1.8 Using aseptic technique, remove sterile white gridded 0.45µm

(pore size) 47mm (diameter) Millipore membrane filter with flat

forceps from package touching only 1/8” of the filter’s outside

perimeter.


November 5, 2015

17

9.1.9 Place filter on base.

9.1.10 Place the funnel on the base.

9.1.11 Shake sample 25 to 30 times vigorously immediately before

pouring.

9.1.12 Place the appropriate amount of sample into funnel:

9.1.12.1 When the sample volume used for dilution ranges

from 1.0 mL to 10 mL, add 10-30mL of sterile

buffered water to the funnel, and using disposable

graduated pipet to transfer certain amount of the

samples (1.0 mL-10.0 mL) to the funnel.

9.1.12.2 When the sample volume used for dilution is less

than 1.0 mL, dilutions will be conducted in sterile

bottles and then transfer diluted samples from the

bottles to the funnels containing 10-30 mL sterile

buffer solution.

9.1.12.3 When diluting a sample follow the diagram in

Appendix A. Step #1 explains how to dilute the

sample, and Steps 2-4 explain how to filter the

diluted sample to arrive at the appropriate dilution.

9.1.13 For drinking water samples, shake sample and pour 100mL of

sample into funnel. Sample volume is measured by the line on the

funnels that has been calibrated using a 100mL volumetric flask.

For non potable water sources use appropriate volume.

9.1.14 Turn vacuum pump on (Millipore Vacuum/Pressure Pump), after

sample has filtered through, rinse funnels 3 times with

approximately 20-30 mL of sterile buffered water.

9.1.15 Run duplicate bacteria analysis on 10% of all samples known to

give positive results.

9.1.16 Using sterile flat forceps remove filter from the filter base again

being careful to touch only the outside edge of filter. Place the

filter on an mFC plate and make sure there are no air spaces

between filter and agar surface (the filter can be picked up and

reseated if necessary). Place plates in water bath bag (stacked up

to three plates high maximum) and close bag so that it is watertight

and place in water for 24 hours ± 2 hours at 44.5±0.2°C so the

media side of the plates is on top. The media side of the plates has


November 5, 2015

18

to be on top, so if moisture builds up in the petri dish it will not

collect on the filter side and interfere with bacterial growth. Plates

must be placed into water bath within 30 minutes after membrane

filtration procedure. Make sure samples are submerged under

water at all times by placing samples under the water bath rack and

then place bricks or weights on top of the water bath rack.

9.1.17 Sterile controls are run by filtering 100mL of buffered water at the

beginning and end of each series, when a different type of media is

being used and when switching between water sources (sewage to

surface waters). Label the plates as “Sterile” and the time.

9.1.18 Examine plates after the required incubation using the stereo

microscope with light source at 10-20 X magnification. Count all

blue colonies.

9.2 Verification of Fecal coliform:

9.2.1 Verify colonies monthly (year round) from a known positive

source.

9.2.2 Pick at least 10 blue colonies from membranes on mFC medium

and transfer to separate Lauryl Tryptose Broth (LTB) tubes and

incubate at 35° C checking for gas production at 24 and 48 hours.

9.2.3 Transfer growth from positive LTB tubes to EC broth and incubate

in water bath at 44.5°C for 24 hours.

9.2.4 Growth and gas in EC broth tubes verifies colony as fecal

coliform.

9.2.5 To determine false negatives pick atypical colonies and verify as

above.

10.0 Data Analysis and Calculations

10.1 General information

10.1.1 Record all results in appropriate logbook and the chain of custody

on the same day as sample analysis.

10.1.2 Record date, time of analysis and incubation, media, dilution,

result, and analysts initials for all samples and steriles.

10.1.3 If a plate is run in duplicate, only use the plate that is not the

duplicate. Use the duplicate results for QC data.


November 5, 2015

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10.1.4 If there are any mistakes when writing in any log book cross out

error with a single line and initial and date the change.

10.1.5 Enter results into the LIMS for report processing.

10.2 Calculation of Colony Density

10.2.1 Compute the count, using membrane filters with colony counts

within the ideal range (fecal coliform:20-60 colonies per 100 mL),

by the following equation:

Colonies/100mL = colonies counted × 100

mL sample filtered

10.2.2 If no filter has a plate falling in the ideal range, compute the count

using the following equation:

Colonies/ 100 mL = [(Sum of colonies counted on each plate) × 100]

(Sum of each volume analyzed in mL)

Example: If 50, 25, and 10-mL portions were examined and the

counts were 15, 6, and <1 colony, respectively, follow the equation:

mLcolonies 100/25)102550(

]100)0615[(

10.2.3 Follow the table below to calculate dilutions using 100mL:

Anything under 1mL will need a dilution prepared.

10.2.4 Use the excel spreadsheet when calculating the colony densities.

When more than one dilution gives a zero result, use only the first

zero result (this will

correspond to the

highest volume of

sample with no

growth.). Even when

a duplicate is run on a

sample and both

results are zero, still

use only one zero in

the final calculation.

Dilution Factor Volume of Sample (mL)

10 10.0

102

1.0

103

0.1

104 0.01

105 0.001

106 0.0001

107 0.00001

108 0.000001


November 5, 2015

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10.2.5 If plates have distinguished recognizable colonies, but seem to

have greater than the ideal counting range, try and count the plate.

Use the number in the above equation. If confluent growth occurs,

covering either the filtration area of the membrane or a portion

thereof, and colonies are not discrete, report results as “too

numerous to count” (TNTC). These results will not be included in

the equation above.

10.3 Data Reporting Limits (RL): All the final data are entered manually into

the laboratory Information Management System (LIMS) and printed out

through the LIMS. The reporting limits on the reports are calculated

based on the sample dilution. Factors as follows:

10.3.1 If the sample is needed for dilution and more than one dilution are

needed for calculating the final concentration, the smallest dilution

factor will be used for the RL. The dilution factor can be

calculated using the following equation:

)(

)(100

mLDilutionforTakenAmountSample

mLFactorDilution

For example:

100mL and 10mL were taken and the final result was obtained based on

calculation of the two individual results. Then RL= 0 CFU/100mL

1mL, 0.1 mL and 0.01 mL were used and the final result was obtained

based on calculation of the three individual results.

Then RL= 100 CFU / 100mL

11.0 Disposal of Wastes

11.1 Biological wastes, such as petri dishes with bacterial growth, cotton-tipped

applicators swabbed with bacteria, and confirmation and/or verification

test tubes, shall be autoclaved at 121°C for thirty minutes and disposed of

with standard trash. Little chemical waste is generated in association with

the microbiological analyses. Those chemical wastes generated shall be

stored in a designated area and segregated from non-compatible wastes

and removed annually by a licensed Hazardous Materials Disposal

Company.


November 5, 2015

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12.0 References

12.1 American Public Health Association, American Water Works

Association, Water Environment Federation; Standard Methods,

“Examination of Water and Wastewater’, Revision 20th

Edition, 1998,

Methods Quality Assurance /Quality Control 9020; Laboratory

Apparatus9030; Preparation of Culture Media 9050; Samples 9060;

Swimming Pools 9213B; Whirlpools 9213C; Natural Bathing Beaches

9213 D; Membrane Filter Technique for Pseudomonas aeruginosa 9213

E; Fecal Coliform Procedure 9221E; Escherichia coli Procedure

(Proposed) 9221 F; Membrane filter technique for members of the

coliform group 9222; Membrane Filter Techniques 9230C.

Original

Sample

Dilution

Bottle #1

10ˉ3, 10ˉ

4

Dilution

Bottle #2

10ˉ5, 10ˉ

6

Dilution

Bottle #3

10ˉ7, 10ˉ

8

Take 1 mL

of sample

1mL from Bottle #1 1 mL from Bottle #2

99 mL of Buffer

99 mL of Buffer

STEP #1: Diluting the Original

sample. 13.0 Appendix A


November 5, 2015

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Dilution

Bottle #2

Funnel

#3

10ˉ5

Funnel

#4

10ˉ6

Take 10 mL

from Bottle #2

Take 1ml from

Bottle #2

STEP #3: Filtering the

diluted sample.

Dilution

Bottle #1

Funnel

#1

10ˉ3

Funnel

#2

10ˉ4

Take 10 mL

from Bottle #1

Take 1mL from

Bottle #1


diluted sample.

Dilution

Bottle #3

Funnel

#5

10ˉ7

Funnel

#6

10ˉ8

Take 10 mL from

Bottle #3

Take 1ml from

Bottle #3


diluted sample.


November 5, 2015

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13.0

Appendix

B

mTEC- Escherichia coli Duplicate Analyses

Logarithms of

Counts

Range of

Logarithms

(Rlog)

Sample

No. Analytical Date D1 D2 L1 L2 |L1-L2|

1 50031-11/14/08 4 1 0.60206 0 0.60206

2 50017- 11/13/08 12 10 1.079181 1 0.079181

3 49928- 11/4/08 9 5 0.954243 0.69897 0.255273

4 49886- 10/30/08* 1 1 0 0 0

5 49863-10/29/08** 1 1 0 0 0

6 49814- 10/23/08 20 23 1.30103 1.361728 0.060698

7 49764- 10/21/08 5 5 0.69897 0.69897 0

8 49728- 10/16/08 6 7 0.778151 0.845098 0.066947

9 49666- 10/14/08 1 2 0 0.30103 0.30103

10 49591- 10/7/08 7 6 0.845098 0.778151 0.066947

11 49557- 10/2/08 11 10 1.041393 1 0.041393

12 49512- 9/30/08 10 7 1 0.845098 0.154902

13 49512- 9/30/08 2 2 0.30103 0.30103 0

14 49456- 9/25/08 13 17 1.113943 1.230449 0.116506

15 49402- 9/23/08*** 1 1 0 0 0

1) Σ Rlog = 1.744935

2)

0.116329

3)

Precision

criterion

= 0.380396

*= Value of D2 is 0

**=Value of D1 and D2

is 0

***=Value of D1 is 0

n

RR

log

Barnstable County Department of Health and the Environment Laboratory


For

Determination of Enterococci in Water using membrane-Enterococcus Indoxyl-β-D-

Glucoside Agar (mEI)

EPA 1600

Revision (003)

November 4, 2015

Signature Date

Bacteriologist: Ryan Lucier ____________________________

Laboratory Director: Gongmin Lei ____________________________

Barnstable County Laboratory

November 5, 2015

1

Table of Contents

1.0 Scope and Application 2

2.0 Summary of Method 2-3

3.0 Definitions 3

4.0 Interferences and Contamination 3

5.0 Safety 3

6.0 Equipment and Supplies 3-4

7.0 Reagents and Standards 4-7

8.0 Preservation and Storage 8

9.0 Quality Control 8-14

10.0 Procedure 14-17

11.0 Data Analysis and Calculations 17- 19

12.0 Disposal of Waste 19

13.0 References 20

14.0 Appendix A 21

15.0 Appendix B

22


November 5, 2015

2


For

Determination of Enterococci in Water using membrane-Enterococcus Indoxyl-β-D-

Glucoside Agar (mEI)

EPA 1600


1.1 This method describes a membrane filter (MF) procedure for the detection and

enumeration of the enterococci bacteria in water. Enterococci are commonly

found in the feces of humans and other warm-blooded animals. Although some

strains are ubiquitous and not related to fecal pollution, the presence of

enteroccoci in water is an indication of fecal pollution and the possible presence

of enteric pathogens.

1.2 The enterococci test measures the bacteriological quality of recreation waters.

Epidemiological studies have led to the development of criteria which can be used

to promulgate recreational water standards based on the established relationship

between health effects and water quality. The significance of finding enterococci

in recreational water samples is the direct relationship between the density of

enteroccoci in the water and swimming-associated gastroenteritis studies of

marine and fresh water bathing beaches (Reference 18.2).

1.3 The test for enterococci can be applied to potable, fresh, estuarine, marine, and

shellfish growing waters.

1.4 Since a wide range of sample volumes or dilutions can be analyzed by the MF

technique, a wide range of enterococci levels in water can be detected and

enumerated.

2.0 Summary of Method

2.1 The MF method provides a direct count of bacteria in water based on the

development of colonies on the surface of the membrane filter (Reference 18.2).

A water sample is filtered through the membrane which retains the bacteria.

Following filtration, the membrane containing the bacterial cells is placed on a

selective medium, mEI agar, and incubated for 24 hours at 41 ±0.5C. All colonies

(regardless of color) with a blue halo are recorded as enterococci colonies.

Magnification and a small fluorescent lamp are used for counting to give

maximum visibility of colonies.


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3.0 Definitions

3.1 In this method, enterococci are those bacteria which produce colonies with a blue

halo after incubation on mEI agar. Enterococci include Streptococcus faecalis,

Streptococcus faceium, Streptococcus avium, and their variants.

4.0 Interferences and Contamination

4.1 Water samples containing colloidal or suspended particulate material can clog the

membrane filter and prevent filtration, or cause spreading of bacterial colonies

which could interfere with identification of target colonies.

5.0 Safety

5.1 The analyst/technician must know and observe the normal safety procedures

required in a microbiology laboratory while preparing, using, and disposing of

cultures, reagents, and materials and while operating sterilization equipment.

6.0 Equipment and Supplies

6.1 Stereoscopic Microscope (vendor: Fisher Scientific, Stereomaster)

6.2 Hand tally or electronic counting device

6.3 Plastic or glass 1 and 10 mL graduated serological pipets

6.4 Graduated cylinders, 50 mL, 100mL, 250 mL, 500mL, and 1000mL

6.5 Ultraviolet light box for sanitization of stems and funnels

6.6 Electronic vacuum pump (Model # SKH33DN16GX, Vendor: VWR)

6.7 Flask, filter, with appropriate tubing

6.8 A six manifold filtration assembly (glass stems and funnels Vendor: Fisher)

6.9 Flat tipped forceps

6.10 Ethanol or isopropanol

6.11 Burner , Bunsen or Fisher type, or propane fuel lighter

6.12 National Institute of Standards and technology (NIST) certified thermometer

(Thermometer, Cat. # 1005-3FC, range (-1+101°C, 1, 1°C)

6.13 Thermometers for incubators

6.14 Incubators ( Vendor: VWR, Model: 1550, Vendor: Blue M, Model MW-1130A-

1) with appropriate temperature range (35.5°, 41.0°, and 44.5°C)

6.15 Millipore membrane filters, sterile, white grid marked 0.45µm pore size 47mm

diameter

6.16 Sterile Millipore petri dishes 9x50mm designed for 47mm filters

6.17 Flasks 100mL, 500mL, 1000mL, 2000mL

6.18 Plastic Inoculation loops at least 3mm diameter

6.19 Pyrex Borosilicate glass Test Tubes with autoclavable caps, size 20x 150mm

6.20 Whirl-Pak® bags

6.21 Hot plate (Vendor: Corning PC-620)


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4

6.22 Stir bars

6.23 Safety glasses

6.24 Gloves

6.25 Lab coat

6.26 Balances (Vendor: Scout, Item # SC6010)

6.27 Autoclaves (Vendor: Tuttnauer Brinkmann, Model 3870M, Vendor: Market

Forge, Model:STM-E)

7.0 Reagents and Standards

7.1 Purity of Reagents: Reagent-grade chemicals shall be used in all tests. Whenever

possible, use commercial culture media as a means of quality control. Use

Reagent grade water from either the Solution 2000 or Milli-Q system to assure

quality of water.

7.2 Phosphate Buffer Solution

7.2.1 Purpose and Use:

The phosphate buffer solution is used as a diluting and rinsing

solution for the bacteria samples. The Magnesium Chloride is

added to the buffer to increase the recovery of organisms with

metabolic injury due to high quality waters or to waters containing

significant concentrations of metals.

7.3 Preparation of Stock Magnesium Chloride Solution (MgCl2): Add 32.44g of

Magnesium Chloride Solution (MgCl2 • 6H2O) to 400mL of reagent-Grade

distilled or deionized water. Transfer to 500mL bottle with a loose cap. Put

autoclave tape on bottle and autoclave at 121°C for 15 minutes and do not exceed

45 minutes in autoclave. Label bottle as “MgCl2 Stock” with date and initials.

Store this in the refrigerator for future use.

7.4 Preparation of Stock Potassium Dihydrogen Phosphate Solution (KH2PO4 ):

Add 13.6g of Potassium Dihydrogen Phosphate to 200mL of reagent-grade

distilled or deionized water. Adjust pH to 7.2±0.5 with 1N NaOH. Add

remaining distilled water to bring to 400mL. Transfer to 500mL bottle with a

loose cap. Affix autoclave tape and autoclave at 121°C for 15 minutes and do not

exceed 45 minutes in autoclave. Label bottle as “KH2PO4 Stock” with date, pH

and initials. Store this in the refrigerator for future use.

7.5 Phosphate Buffer Solution Preparation

7.5.1 Composition:

Reagent-grade Distilled or Deionized water 2L

MgCl2 Stock Solution 10mL

KH2PO4 Stock Solution 2.5mL


November 5, 2015

5

7.5.2 Preparation: Always take the initial pH of the KH2PO4 before you

prepare the buffer. The initial pH should be 7.2±0.5. Always take the

initial pH of the reagent grade distilled or deionized water before making

the buffer, the pH should be 5.5-7.5. Dissolve all ingredients in 2L of

Reagent-Grade distilled or deionized water by stirring in a 2L flask. The

final pH should be 7.2 ±0.2. If the pH is not within range, add 1-2 drops

of 2N NaOH. Stir and check pH. Keep adjusting pH until it is within the

range. Transfer buffer solution into 1L bottles filling them only ¾ of the

way full. Affix autoclave tape to caps and cap bottles loosely. Autoclave

the buffer for 30 minutes at 121°C. Allow bottles to cool down before

tightening caps. Date and initial bottles and store at room temperature for

future use. Perform sterility check on each batch of buffer before use, by

pouring 20ml into a 100ml bottle of Tryptic Soy Broth and incubating at

35°C and checking for growth and/or cloudiness at 24 hours and at 48

hours. If solution appears cloudy it was not sterilized properly and the

batch must then be re-sterilized. If the TSB is clear then buffer can be put

away for future use in cabinet. Record results in Quality Control Log

7.6 Tryptic Soy Broth(TSB) (Becton Dickenson:211825)

7.6.1 Composition:


Enzymatic Digest of Soybean Meal 3.0g

Dextrose 2.5g



7.6.2 Preparation: To prepare 1L measure out 30g of TSB using top loading

balance. Suspend in 1L of reagent-grade deionized water in an

Erlenmeyer flask. Warm slightly with stirring to dissolve completely. Add

25 mL of broth to 20, 30mL screw cap tubes. Before sterilization add

100mL of TSB to 5 clear 250mL bottles (for buffer sterility checks).

Autoclave for 15 minutes at 121°C; do not exceed 45 minutes in

autoclave. The final pH is 7.3±0.2 at 25.0 º C.

7.7 mEI agar (Reference number: BD 214881-mEI Agar)

7.7.1 Composition(Approximate Formula* Per Liter:

Peptone 10.0 g

Sodium Chloride 15.0 g

Esculin 1.0 g

Cycloheximide 0.05 g


November 5, 2015

6

Sodium Azide 0.15 g

Yeast Extract 30.0 g

Indoxyl-β-D-glucoside 0.75g

Agar 15.0 g

* Adjusted and/or supplemented as required to meet performance criteria.

7.7.2 Preparation of mEI agar: To prepare one liter of media suspend 72.0g of

mEI media and in one liter of reagent grade water. Boil to dissolve.

Sterilize media and all utensils to be used after at 121oC for 15 min.

Duration of “autoclave running time” from beginning to finish should take

no longer than 45 minutes. Cool in a water bath to 45 +/-1oC. Add 0.24g

of nalidixic acid to 5ml reagent grade water with a few drops 0.1N NaOH

to dissolve in a screw cap tube. Add solution to the media and swirl.

Separately add 0.02g triphenyltetrazolium chloride (TTC) and swirl to

mix. Final pH should be 7.1±0.2 at 25 oC. Dispense in 4-5ml amounts into

sterile 9 x 50 mm petri dishes. Invert and store at 4oC. Media expires after

two weeks.

7.8 Brain Heart Infusion Broth (BHIB)(Reference number: Becton Dickinson

237400)

7.8.1 Composition:

Calf Brains, Infusion from 200 g 7.7 g

Beef Heart, Infusion from 250 g 9.8 g

Proteose Peptone 10.0 g

Dextrose 2.0 g

Sodium Choloride 5.0 g

Disodium Phosphate 2.5 g

7.8.2 Preparation of BHIB: to prepare tubes dissolve 37g of hydrated brain heart

infusion in 1L of reagent grade water. Dispense in 10 mL amounts into

screw-cap tubes and sterilize @ 121o C for 15 min. Duration of “autoclave

running time” from beginning to finish should take no longer than 45

minutes. Note: If the media is not used the same day it is prepared and

sterilized, heat in boiling water for several minutes to remove absorbed

oxygen. Cool in water bath just prior to inoculation. The final pH should

be 7.4 +/-0.2.

7.9 Brain Heart Infusion broth (BHIB) w/6.5% NaCl

7.9.1 Preparation: Follow same procedure as above for BHI broth then add

60.0g NaCl per 1L of media. Because most media already contain

sodium chloride this amount is taken into consideration in the

percentage calculated above.

7.10 Brain Heart Infusion Agar (BHIA)


November 5, 2015

7

7.10.1 Preparation: contains the same components as BHI broth (above) with the

addition of 15.0g of agar per 1L of BHI broth.

7.11 BEA (Reference number: Becton Dickinson (299068)

Composition:

Beef Extract 3.0 g

Pancreatic Digest of Gelatin 5.0 g

Esculin 1.0 g

Oxgall 20.0 g

Ferric Citrate 0.5 g

Agar 14.0 g

7.11.1 Preparation: Add 43.5g of dehydrated Bile Esculine Agar to 1L of

reagent-grade deionized water. Mix and boil the contents until dissolved.

Dispense 10 mL amounts into tubes for slants. Autoclave @ 121oC for 15 min.

Autoclave cycle must be completed within 45 minutes. The tubes must be placed in

slant racks for cooling. The final pH should be 6.8 ±0.2. Store @ 4oC.

7.12 Holding Times for Media:

Media Holding time

MF agar in plates with tight-fitting lids in

refrigerator

Agar or broth in tightly closed screw-capped

tubes in refrigerator

Poured agar plates with loose fitting covers in

sealed plastic bags in refrigerator.

Agar or broth in loose-cap tubes at 4o C

Dehydrated Media opened but in dessicator

Dehydrated Media opened but not in dessicator

2 weeks

3 months

2 weeks

1 week

1 year from date

opened

6 months from

date opened

8. Preservation and Storage

Start microbiological analysis of water samples as soon as possible after

collection to avoid unpredictable changes in the microbial population. For most

accurate results, ice samples during transport to the laboratory and do not

submerge samples in water. This could cause cross contamination in the samples.


November 5, 2015

8

8.1 Drinking water for compliance purposes: Preferably hold samples <10°C

during transit to the laboratory. Analyze samples on day of receipt

whenever possible and refrigerate overnight if arrival is too late for

processing on same day. Do not exceed 30 hour holding time from

collection on analysis for coliform bacteria.

8.2 Nonpotable water for compliance purposes: Hold source water, stream

pollution, recreational water, and wastewater samples below 10°C during

a maximum transport time of 6 hours. Refrigerate these samples upon

receipt in the laboratory and process within 2 hours. When transport

conditions necessitate delays in delivery of samples longer than 6 hours,

consider using either field laboratory facilities located at the site of

collection or delayed incubation procedures.

8.3 Other water types for noncompliance purposes: Hold samples below 10°C

during transport and until time of analysis. Do not exceed 24h holding

time.

9 Quality Control

9.1 Thermometers and temperature recording equipment: The accuracy of

thermometers are checked annually against a certified NIST thermometer

at the temperature used. In general the graduations on the thermometer

should be in increments of 0.5° or less. The maximum thermometers and

NIST thermometers are checked annually by Alert Scientific. The results

of each thermometer check are recorded in the thermometer logbook.

9.2 Balances are checked daily with the provided S class weights. The top

loading balance is for anything weighing 3g or more. For this balance the

100.0g, 50.0g, 1.0g, and 100mg weights are used. Each weight is weighed

independently, and in addition, a deflection test is performed prior to

usage. The results of the check are recorded in the assigned balance book.

The S class weights and balance are checked annually by Alert Scientific.

9.3 The pH meter is to be calibrated daily with at least three standards: 4.00,

7.00, and 10.00. The standards must be changed daily. The temperature,

slope, and pH are recorded in the temperature logbook. The pH meter is

checked annually by Alert Scientific.

9.4 Media Dispensing Apparatus: Check accuracy of volume by pipetting the

amount into a weighing dish on the top loader balance, adjust the setting

accordingly. When sterilizing, put the glass barrel in the apparatus and

tighten finger tight and then loosen quarter of a turn, this lets airflow

through the setup so the pressure from autoclaving won’t build up and

break the glass. Fill a beaker with reagent grade water and sterilize with


November 5, 2015

9

syringe. This water is used to pump through syringe at the end of each

use. If there is any visible staining at the end of use boil in reagent grade

water.

9.5 Autoclave (Tuttnauer Brinkmann Model 3870M and Market Forge Model

# STM-E): Record items being sterilized, temperature (maximum

thermometer), pressure and time for each run. Use BTSure ampoules

(catalog # AY759X3) weekly by placing the ampoule in the autoclave for

15 or 20 minutes at or above 121°C. After autoclaving is complete, let

ampoule cool and then gently squeeze and break the glass ampoule. The

ampoule will now be activated. It must immediately be placed in a screw

cap tube and placed in the small water bath at 55°C for 48 hours. Also

activate and incubate one ampoule that has not been autoclaved as a

control. The control will turn yellow after incubation and if there is no

color change for the autoclaved ampoules then it is concluded that the

sterilization process of the autoclave is working properly. Make sure there

is enough water in the waterbath so BTSure ampoules are completely

submerged throughout the incubation period. Autoclave tape is used on

every sample bottle before autoclaving. A maximum temperature

thermometer is used and temperature is recorded after each run. The

Autoclaves are checked annually by Alert Scientific.

9.6 Refrigerators: Check and record all temperatures daily. Use a refrigerator

maintaining a temperature of 1.0 to 4.4°C to store samples, media,

reagents, etc.

9.7 Water baths: The time and temperature is checked and recorded twice

daily with a four hour separation of time between readings and adjusted

when needed.

9.8 Incubators: The time and temperature is checked and recorded twice

daily with a four hour separation of time between readings and adjusted

when needed.

9.9 Membrane filter equipment: Wrap filter units in sterile wrap and autoclave

daily for 20 minutes at 121°C. Before each set of samples expose stems

and funnels to 2 minutes of UV light. Wash filter units weekly with

detergent and rinse with deionized water. When new Millipore membrane

filters are received record the lot number and date of receipt in the

microbiology primary standard logbook.

9.10 Ultraviolet sterilization lamps: Clean lamps with ethanol monthly

and record in quality control logbook. Do a sterility check to assure

proper sterilization. Use a known positive water source or stock organism.

9.10.1 Procedure for UV sterility check:


November 5, 2015

10

Label R2A plates as follows:

1. Control (swabbed)

2. UV Left

3. UV Center

4. UV Right

Pouring plates: Melt solid R2A agar in boiling water and put

one tube of water or extra R2A tube in the boiling water and then transfer

to 46°C water bath. Place a thermometer in the water tube or extra R2A

tube to indicate medium temperature. Agar can be used when temp cools

to 44-46°C. Allow no more than 20 minutes to elapse between plating

sample and pouring medium. Before pouring agar wipe the tube dry with

a paper towel and flame the neck of the tube. Carefully pour the contents

of one tube (12mL) into the 100 x 15 mm petri dish, and mix contents by

rotating clockwise and counter clockwise to cover the bottom surface of

the petri dish. Allow agar to solidify. Pipet 0.1 mL of a five fold dilution

of culture broth or 0.1mL of a two fold dilution of the raw sewage sample

onto each of the five plates, using a sterile glass spreader, spread the

inoculums evenly over the entire surface of the agar of each plate. Plate

#1 is to be left out of the UV box as the control. Remove the lids of plates

2-4 and place in a light box. Set the timer to two minutes and turn on the

UV lights. At the end of two minutes turn off the UV lights and remove

the plates. Incubate the UV sterilized plates and the controls for 48 hours

at 35±0.5°C. After the incubation period check plates for growth, control

plates should show heavy growth (>200 colonies) and sterilized plates

should show little to no growth (0-2 CFU). The kill rate from the lamps

should be 98%. If more than 2 colonies form on a plate then lamps need

replacing.

9.11 Solution 2000 and Milli-Q deionized water: The sterility of reagent water

should be checked monthly using pour plate method with R2A.

9.11.1 Procedure: 1mL of the Solution 2000 water is pipetted into a large

sterile petri dish. Sample is run in duplicate on another plate. Heat

tempered R2A is poured over sample and swirled clockwise and

counterclockwise. Repeat process for Milli-Q water. After the plates

solidify they are inverted and incubated for 48 hours at 35°C. After

incubation period check for growth on plate. There should not be any

growth.

9.12 The Total Chlorine residual of the reagent water is checked monthly:

9.12.1 Total Chlorine Procedure: Using a colorimeter fill one test cell

with reagent water to 10mL line. Add contents of one DPD Total

Chlorine pouch to the sample cell; cap and shake for 20 seconds. Set

timer for 3 minutes; during this time fill the other cell with 10mL of the


November 5, 2015

11

reagent water and cap (this is the blank). Place blank in colorimeter with

cover on and press ZERO. Remove blank cell and within 3 minutes after

the 3 minute period place prepared sample cell in colorimeter, cover, and

press READ. Record result in QC log (result must be less than 0.1 mg/L).

9.13 The pH of the reagent water is checked before each use. Fill clean beaker

with reagent water and stir. Measure the pH with a calibrated meter.

Record result in media preparation log with media (pH of water must be

5.5-7.5).

9.14 Air Quality: The air quality in the laboratory is monitored monthly.

Plates of R2A media are left open in different parts of the laboratory for

15 minutes. A sterile plate is also poured and left closed for the 15

minutes. Incubate plates for 48 hours at 35.0±0.5°C and observe for

growth and record in quality control logbook.

9.15 Glassware: Glassware must have the pH checked because some

cleaning solutions are difficult to remove completely, spot check batches

of clean glassware for pH reaction, especially if soaked in alkali or acid

solutions. This is done by adding a few drops of 0.04% bromthymol blue

(BTB) to a bottle and observing for a blue green color indicating neutral

pH. One of the bottles from each batch of washed and dried bottles needs

to be tested by adding 3 drops of 0.04% BTB to the bottle. Observe for

any color reaction. If the color is yellow (acidic) or blue (basic) then that

batch needs to be rewashed. The results are to be recorded in the QA/QC

log. If 0.04% of bromthymol blue needs to be prepared: Add 16mL of

0.01N NaOH to 0.1g BTB and dilute to 250mL with distilled water.

9.16 All microbiological bottles need to be autoclaved: Non-disposable sample

bottles are washed in the dishwashers (using a distilled water rinse cycle).

Bottles and caps are then removed from the dishwasher. All caps need

heat activated autoclave tape affix on them and then they are placed in a

clean beaker and covered with aluminum foil. Bottles and all caps are

autoclaved for 20 minutes at 121°-134°C. Make sure the tape has turned

to a black color. When handling caps after being autoclaved use aseptic

techniques when taking them out of the beaker and tightening caps on

bottles when removed from autoclave and cooled.

9.17 Autoclaved and sterile bottles need a sterility check: For each new batch

of bottles received and at least one batch of autoclaved bottles per day

perform a sterility check by adding approximately 25ml of sterile non

selective broth (Tryptic Soy Broth) to at least one bottle. Cap bottle and

rotate so that broth comes into contact with all surfaces. Check for any

growth after incubation at 35°C at 24 hours and 48 hours. Record results

in Quality Control Log. If TSB does not remain clear rewash and

autoclave all bottles from the batch/batches in question.


November 5, 2015

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9.18 Utensils and containers for media preparation: Only use utensils and

containers of borosilicate glass, stainless steel, aluminum, or other

corrosion-resistant material. Do not use copper utensils. Use glassware

that is clean and free of residues, dried agar or other foreign materials that

may contaminate media.

9.19 Culture Media: Culture methods depend on properly prepared

media. Use the best available materials and techniques in media

preparation, storage, and application. Order media in quantities that last

no longer than a year. When media is received record type, amount, lot

number, expiration date and date received. Store dehydrated media in a

cool and dry place away from direct sunlight. Store opened containers of

media in a desiccator immediately after use. Discard media that cake, are

discolored, or show other signs of deterioration. If expiration date is given

by manufacturer, discard unused media after that date. Discard any

opened media after one year. Never combine different lots of media.

9.20 New Lot Testing: Pure culture comparison testing and positive and

negative controls need to be performed whenever a new lot of culture

media is opened.

9.21 Comparative testing procedure: Use the old batch as your control group.

Make parallel tests between the control group and the test group. As a

minimum make single analysis on five pure positive control stock

organism samples listed below:

After incubation, compare bacterial colonies from the two lots for size and

appearance. If colonies on the test lot plates are atypical or noticeably

smaller than colonies on the reference lot plates, record the evidence of

inhibition or other problem, regardless of count differences. Re-test media

and if the differences still occur discard media.

9.21.1Positive and negative controls: using known organisms (shown

above in the table) test each new lot and batch of media prior to use to

ensure proper growth. Swab new media plates or tubes with appropriate

control cultures and incubate at appropriate time and temperatures.

Record results in Quality Control logbook.

Group Positive Control Negative Control

Enterococci E. faecalis S. mitis/ salvivarius, E.Coli


November 5, 2015

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9.22 Positive and negative Controls: Using known organisms test each

new lot of media to ensure proper growth. Swab new media plates or

tubes with appropriate controls cultures and incubate at appropriate time

and temperatures. Record results in quality control logbook.

9.23 For routine performance evaluation, repeat counts on one or more positive

samples at least monthly and compare the counts with those of other

analysts testing the same samples. Replicate counts for the same analyst

should agree within 5% and those between analysts should agree within

10%.

9.24 Analyst must perform testing samples for various parameters in a uniform

fashion. Example: If a sample needs fecal coliform, total coliform and

E.coli run on it, then the analyst will perform all three tests with

appropriate dilutions first before moving on to the next sample.

9.25 Precision of Duplicate Analysis: Duplicates are run on 10% of samples for

all analyses requiring enumeration. For each most recent set of 15 samples

and its corresponding duplicate (for each type of sample analysis i.e. fecal,

e.coli, enterococci), the range of logs is calculated in order to determine

the acceptance criteria of precision. Once established, the criteria of

precision will be used to determine if the following duplicate analyses are

within acceptable limits. At the end of each week the criteria are updated

to include the most recent set of 15 samples run in duplicate. The updated

precision is used to determine precision acceptability for the following

week.

9.25.1 Obtain the recorded numbers of the most recent 15 samples run in

duplicate (for the same type of analysis).

9.25.2 Calculate the logarithm of each result (and if any result is <1 then

add 1 to both values before calculating the logarithm) and record in a

spreadsheet as L1 and L2.

9.25.3Range of Logarithms (Rlog) is calculated using the following

equation:

Rlog= │ L1-L2 │ (1)


(2) ∑R log = The sum of the range of logs.

n

RR

log


November 5, 2015

14

n = The number of sets of transformed duplicates. 9.25.5 Precision Criterion is calculated as follows: __ Precision Criterion = 3.27 x R (3)

9.25.6 Any samples run in duplicate (10% of all samples) have their

Range of Logarithms (Rlog). If the range is greater than the Precision

Criterion, there is a greater than 99% probability that the analysis has

exceeded variability limits. For any samples that fall outside the

acceptable limits, determine if the imprecision is acceptable. If data is not

acceptable, all results since the last precision check should be rejected.

The analytical problem(s) should be determined and corrective action

should be taken to resolve problem.

9.25.6 See Appendix B for an example of the precision criterion.

10.0 Procedure

10.1 Procedure for membrane filtration:

10.1.1 Analyze samples within 8 hours of collection. Samples must be

preserved in a refrigerator or cooler until analysis.

10.1.2 Use mEI agar for the identification of enterococcus.

10.1.3 Sterilize work area by washing down with a solution of 50%

bleach, 2% Lysol, or Conflikt.

10.1.4 CAUTION: UV light is carcinogenic and damaging to eyes.

Sterilize filter funnel and bases in UV light box for 2 minutes.

Turn light off before lifting wooden cover. Daily wrap the filter

funnels in steriwrap and sterilize in autoclave for 15 minutes at

121°C.

10.1.5 Label mEI plates on the bottom (media side) with bottle # and

dilution if other than 100mL of sample is used. Record time on

each plate.

10.1.6 Place the filter funnel base in manifold. Be sure valve is open.

10.1.7 Dip forceps in alcohol (95% ETOH) and pass through the flame.

10.1.8 Using aseptic technique, remove sterile white gridded 0.45µm

(pore size) 47 mm (diameter) Millipore membrane filter with flat


November 5, 2015

15

forceps from package touching only 1/8” of the filter’s outside

perimeter.

10.1.9 Place filter on base.

10.1.10 Place the funnel on the base.

10.1.11 Shake sample 25-30 times vigorously immediately before

pouring.

10.1.12 Place the appropriate amount of sample into funnel:

10.1.12.1 Measure 50mL of sample for bathing beach samples

using sterile graduated cylinder. Use lower volume

if high sediments or algae present. Pour into funnel

and filter. For all other samples use appropriate

volume.

10.1.12.2 When the sample volume used for dilution ranges

from 1.0- 10mL, add 10-30mL of sterile buffered

water to the funnel and use a disposable graduated

pipet to transfer appropriate amount of sample (1.0-

10.0mL) to the funnel.

10.1.12.3 When the sample volume used is less than1.0mL,

dilutions will be conducted in sterile dilution

bottles. The diluted samples will then be

transferred into funnels containing 10-30mL of

sterile buffer water.

10.1.12.4 When diluting a sample follow the diagram in

Appendix A. Step #1 explains hot to dilute the

sample, and steps 2-4 explain how to filter the

diluted sample to arrive at the appropriate dilution.

10.1.13 Drinking water samples: Shake sample and pour

100mL of sample into funnel. Sample volume is measured by the

line on the funnels that has been calibrated using a 100mL

volumetric flask. For non potable water sources use appropriate

volume listed above.

10.1.14 Turn vacuum pump on (Millipore Vacuum/Pressure Pump), after

the sample has been filtered, rinse funnels 3 times with

approximately 20-30mL of sterile buffer water.


November 5, 2015

16

10.1.15 Run duplicate bacterial analysis on 10% of all samples known to

give positive results.

10.1.16 Using sterile flat forceps remove filter from the base again being

careful to touch only the outside edge of the filter. Place the filter

on a mEI plate and make sure there are no air spaces between filter

and agar surface (the filter can be picked up and reseated if

necessary). Place plates in incubator (stacked three high

maximum) for 24±2 hours at 41.0 ±0.5°C so the media side of the

plates is on top. The media side of the plates has to be on top so if

moisture builds up in the petri dish it will not interfere with

bacterial growth.

10.1.17 Sterile controls are run by filtering 100mL of buffer water at the

beginning and end of each series, when a different type of media is

being used and when switching between water sources (sewage to

surface waters). Label the plates with a time and the word

“Sterile”.

10.1.18 Examine plates after 24±2 hours using the stereo microscope

with light source at 10-20 X magnification. Enterococcus colonies

are any colonies with a blue halo, regardless of colony color.

10.1.19 Enter the raw results into the sample run logbook with the

appropriate dilution that was used. To report multiply according to

dilution, if at 50 mL then multiply by two to equal

CFU/100mL.

10.2 Verification of Enterococcus:

10.2.1 Verify colonies monthly (when Enterococcus is being regularly

analyzed) from a known positive source.

10.2.2 Transfer cells from the centers of at least 10 well isolatter typical

colonies into a BHIB tube and onto a BHIA slant.

10.2.3 Incubate for broth for 24hrs and slants for 48hrs @ 35 ±0.5oC.

10.2.4 After 24 hr incubation, transfer a loopful of material from each

BHIB tube to each of the following media:٭BEA and incubate @

35±0.5oC for 48hrs.٭BHIB and incubate @ 45±0.5

oC for 48 hrs.

@ detabucni lCaN %5.6 htiw BIHB0.5±35٭oC for 48hrs.

10.2.5 Observe for growth on all media.


November 5, 2015

17

10.2.6 After 48hr incubation, apply a Gram stain to the growth from each

BHIA slant.

10.2.7 Gram positive cocci that grow and hydrolyze esculin on BEA

and grow in BHIB @ 45±0.5oC and BHIB with 6.5% NaCl @

35±0.5oC are verified as enterococci.

11. Data Analysis and Calculations

11.1 General Information

11.1.1 Record all results in appropriate logbook and the chain of custody

on the same day as sample analysis.

11.1.2 Record date, time of analysis, media, dilution, result, time out of

the incubator and the analyst initials for all samples and steriles.

11.1.3 If a plate is run in duplicate only use the original plate and not the

duplicate. The duplicate will only be used for quality control

purposes.

11.1.4 If there are any mistakes when writing in any log book, cross out

error with a single line and initial change with date and initials.

11.1.5 If there are any mistakes when writing in a logbook cross out error

with a single line and initial the change with the date.

11.1.6 Enter results into LIMS for report processing.

11.1.7 Call Water Supplier immediately for any samples that exhibit

colonies in exceedance. If a sample is found to be TNTC or

exhibits confluent growth call water department immediately for

re-sampling.

11.2 Calculation of Colony Density

11.2.1 Compute the count using membrane filters with colony counts

within the ideal range (total coliform 20-60 CFU/100mL) using the

following equation:

Colonies/100mL = colonies counted × 100

mL sample filtered

11.2.2 If no filter has a plate falling in the ideal range, compute the count

using the following equation:


November 5, 2015

18

Colonies/ 100 mL = [(Sum of colonies counted on each plate) × 100]

(Sum of each volume analyzed in mL)

Example: If 50, 25, and 10 mL portions were examined and the

counts were 15, 6, and <1 colony, respectively follow the

equation:

mLcolonies 100/25)102550(

]100)0615[(

11.2.3 Follow the table below to calculate dilutions using 100mL.

Anything under 1mL will need a dilution prepared.

11.2.4 Use the excel spreadsheet when calculating the colony densities.

When more than one dilution gives a zero result, use only the first

zero result (this will correspond to the highest volume of sample

with no growth). If a duplicate is run and you have two zeros use

only one zero in the calculation.

11.2.5 If plates have distinguished recognizable colonies but seem to have

greater that the ideal counting range, try and count the plate. Use

that number in the equation. If confluent growth occurs covering

either the filtration area of the membrane or a portion thereof with

non discrete colonies, report result as “too numerous to count”

(TNTC). These results will not be included in the equation above.

11.3 Data Reporting Limits (RL): All the final data are entered manually into

the laboratory Information Management System (LIMS) and printed out

through the LIMS. The reporting limits on the reports are calculated based

on the sample dilution. Factors as follows:

Dilution Volume of Sample (mL)

10 10.0

1.0 1.0

10ˉ3

0.1

10ˉ4 0.01

10ˉ5 0.001

10ˉ6 0.0001

10ˉ7 0.00001

10ˉ8 0.000001


November 5, 2015

19

11.3.1 If the sample is needed for dilution and more than one dilution are

needed for calculating the final concentration, the smallest dilution factor

will be used for the RL. The dilution factor can be calculated using the

following equation:

)(

)(100

mLDilutionforTakenAmountSample

mLFactorDilution

For example:

If 100mL and 10mL were taken and the final result was obtained

based on the calculation of the two individual results, then the

RL=1.

If 1.0mL, 0.1mL, and 0.01mL were used and the final result was

obtained based on the calculation of the three individual results,

then the RL = 100.

12.0 Disposal of Wastes

12.1 Biological wastes, such as petri dishes with bacterial growth, cotton-tipped

applicators swabbed with bacteria, and confirmation and/or verification test tubes,

shall be autoclaved at 121°C for thirty minutes and disposed of with standard

trash. Little chemical waste is generated in association with the microbiological

analyses. Those chemical wastes generated shall be stored in a designated area

and segregated from non-compatible wastes and removed annually by a licensed

Hazardous Materials Disposal Company.

13.0 References

13.1 American Public Health Association, American Water Works

Association, Water Environment Federation; Standard Methods,

“Examination of Water and Wastewater’, Revision 20th

Edition, 1998,

Methods Quality Assurance /Quality Control 9020; Laboratory

Apparatus9030; Preparation of Culture Media 9050; Samples 9060;

Swimming Pools 9213B; Whirlpools 9213C; Natural Bathing Beaches

9213 D; Membrane Filter Technique for Pseudomonas aeruginosa 9213

E; Fecal Coliform Procedure 9221E; Escherichia coli Procedure

(Proposed) 9221 F; Membrane filter technique for members of the

coliform group 9222; Membrane Filter Techniques 9230C.

13.2 U.S. Environmental Protection Agency, “Enterococci in Water by

Membrane Filtration Using membrane-Enterococcus Indoxyl-ß-D-

Glucoside Agar (mEI )


November 5, 2015

20

.

mTEC- Escherichia coli Duplicate Analyses

Logarithms of

Counts

Range of

Logarithms

(Rlog)

Sample

No. Analytical Date D1 D2 L1 L2 |L1-L2|

1 50031-11/14/08 4 1 0.60206 0 0.602059991

2 50017- 11/13/08 12 10 1.079181 1 0.079181246

3 49928- 11/4/08 9 5 0.954243 0.69897 0.255272505

4 49886- 10/30/08* 1 1 0 0 0

5 49863-10/29/08** 1 1 0 0 0

6 49814- 10/23/08 20 23 1.30103 1.361728 0.06069784

7 49764- 10/21/08 5 5 0.69897 0.69897 0

8 49728- 10/16/08 6 7 0.778151 0.845098 0.06694679

9 49666- 10/14/08 1 2 0 0.30103 0.301029996

10 49591- 10/7/08 7 6 0.845098 0.778151 0.06694679

11 49557- 10/2/08 11 10 1.041393 1 0.041392685

12 49512- 9/30/08 10 7 1 0.845098 0.15490196

13 49512- 9/30/08 2 2 0.30103 0.30103 0

14 49456- 9/25/08 13 17 1.113943 1.230449 0.116505569

15 49402- 9/23/08*** 1 1 0 0 0

1)

Σ Rlog =

1.744935

2) 0.116329

3)

Precision

criterion

= 0.380396

*= Value of D2 is 0

**=Value of D1 and D2

is 0

***=Value of D1 is 0

n

RR

log


November 5, 2015

21

Continue in this pattern for more dilutions.

Original

Sample

Dilution

Bottle #1

10ˉ3, 10ˉ

4

Dilution

Bottle #2

10ˉ5, 10ˉ

6

Dilution

Bottle #3

10ˉ7, 10ˉ

8

Take 1 mL

of sample

1mL from Bottle #1 1 mL from Bottle #2

STEP #1: Diluting the Original

sample.

14.0 Appendix A

99 mL of Buffer

99 mL of Buffer

15.0 Appendix B

Dilution

Bottle #1

Funnel

#1

10ˉ3

Funnel

#2

10ˉ4

Take 10 mL

from Bottle #1

Take 1mL from

Bottle #1


diluted sample.

Dilution

Bottle #2

Funnel

#3

10ˉ5

Funnel

#4

10ˉ6

Take 10 mL

from Bottle #2

Take 1ml from

Bottle #2


diluted sample.


September 29, 2015

Page 1 of 16

Barnstable County Department of Health and the

Environment Laboratory

EPA Method 351.2


For

Determination of Total Kjeldahl Nitrogen in Aqueous Samples

by Semi-Automated Colorimetry

(Revision 001)

September 29, 2015

Signature Date

Analyst: Kelby Karnes

Laboratory Director: Gongmin Lei


September 29, 2015

Page 2 of 16

STANDARD OPERATING PROCEDURE (SOP)

For

Determination of Total Kjeldahl Nitrogen in Aqueous Samples

by Semi-Automated Colorimetry

1.0 SCOPE AND APPLICATION

1.1 This SOP provides procedure for determination of total Kjeldahl nitrogen

in drinking, ground, and surface waters, domestic and industrial wastes.

The procedure converts nitrogen components of biological origin such as

amino acids, proteins and peptides to ammonia, but may not convert the

nitrogenous compounds, hydrazones, oximes, semicarbazones and some

refractory tertiary amines.

1.2 The applicable range is 0.1 – 20 mg/L TKN. The range may be extended

with sample dilution.

2.0 SUMMARY OF METHOD

2.1 The sample is heated in the presence of sulfuric acid, H2SO4 for three

hours. The residue is cooled, diluted to 25 mL and analyzed for ammonia.

The digested sample may also be used for phosphorus determination.

2.2 Total Kjeldahl nitrogen is the sum of free-ammonia and organic nitrogen

compounds which are converted to ammonium sulfate (NH4)2SO4, under

the conditions of digestion described.

2.3 Organic Kjeldahl nitrogen is the difference obtained by subtracting the

free-ammonia value from the total Kjeldahl nitrogen value.

3.0 DEFINITIONS

3.1 Calibration Blank (CB) – A volume of reagent water fortified with the

same matrix as the calibration standards, but without the analyte .

3.2 Calibration Standard (CAL) – A solution prepared from the primary

dilution standard or stock standard solutions.

3.3 Instrument Performance Check Solution (IPC) – A Solution of one or

more method analytes or other test substances used to evaluate the

performance of the instrument system with respect to a defined set of

criteria.


September 29, 2015

Page 3 of 16

3.4 Laboratory Fortified Blank (LFB) – An aliquot of reagent water or other

blank matrices to which known quantities of the method analytes are

added in the laboratory. The LFB is analyzed exactly like a sample, and its

purpose is to determine whether the methodology is in control, and

whether the laboratory is capable of making accurate and precise

measurements.

3.5 Laboratory Fortified Sample Matrix (LFM) – An aliquot of an

environmental sample to which known quantities of the method analytes

are added in the laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix contributes bias

to the analytical results. The background concentrations of the analytes in

the sample matrix must be determined in a separate aliquot and the

measured values in the LFM corrected for background concentrations.

3.6 Laboratory Reagent Blank (LRB) – An aliquot of reagent water or other

blank matrices that are treated exactly as a sample including exposure to

all glassware, equipment, solvents, and reagents that are used with other

samples. The LRB is used to determine if method analytes or other

interferences are present in the laboratory environment, the reagents, or

the apparatus.

3.7 Linear Calibration Range (LCR) – The concentration range over which the

instrument response is linear.

3.8 Safety Data Sheets (SDS) [Used to be called as Material Safety Data Sheet

(MSDS)] – Written information provided by vendors concerning a

chemical’s toxicity, health hazards, physical properties, fire, and reactivity

data including storage, spill, and handling precautions.

3.9 Method Detection Limit (MDL) – The minimum concentration of an

analyte that can be identified measured and reported with 99% confidence

that the analyte concentration is greater than zero.

3.10 Quality Control Sample (QCS) – A solution of method analytes of known

concentrations that is obtained from a source external to the laboratory and

different from the source of calibration standards. It is used to check

laboratory performance with externally prepared test materials.

3.11 Stock Standard Solution (SSS) – A concentrated solution containing one

or more method analytes prepared in the laboratory using assayed

reference materials or purchased from a reputable commercial source.

4.0 INTERFERENCES


September 29, 2015

Page 4 of 16

4.1 High nitrate concentrations (10x or more than the TKN level) result in low

TKN values. If interference is suspected, samples should be diluted and

reanalyzed.

4.2 Method interferences may be caused by contaminants in the reagent water,

reagents, glassware, and other sample processing apparatus that bias

analyte response.

5.0 SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not

been fully established. Each chemical must be regarded as a potential

health hazard and exposure must be as low as reasonably achievable.

Cautions are included for known extremely hazardous materials or

procedures.

5.2 Barnstable County Health Laboratory maintains a current awareness file

of OSHA regulations regarding the safe handling of the chemicals

specified in this method. Reference files of Safety Data Sheets (SDS) are

available to all personnel involved in the chemical analysis. The

preparation of a formal safety plan is also advisable.

5.3 The following chemicals have the potential to be highly toxic or hazardous,

consult SDS.

5.3.1 Sulfuric acid.

5.3.2 Sodium nitroprusside.

6.0 EQUIPMENT AND SUPPLIES

6.1 Balance – Analytical, capable of accurately weighing to the nearest

0.0001g. Fisher Scientific, Model ACCU-124D Dual Range.

6.2 Glassware – Class A volumetric flasks and pipets as required.

6.3 Block Digestor with Tubes – TKN 50 well AIM600 Block/Controller with

rack and 100 mL glass digestion tubes (Environmental Express, Item#:

SC900).

6.4 Automated Continuous Flow Analysis Equipment – QuickChem 8500

Series 2 Flow Injection Analysis System (LACHAT Instruments, A Hach

Company Brand)

6.4.1 LACHAT XYZ Autosampler.


September 29, 2015

Page 5 of 16

6.5 BD Kjeldahl Digestion Granules from Environmental Express, Item#.

8032178

6.6 Seal Analytical Teardrop Stoppers, Item No. SC9703

7.0 REAGENTS, CHEMICALS AND STANDARDS

7.1 Potassium Sulfate (K2SO4). Fisher, Cat No. P305-500

7.2 Copper (II) Sulfate (CuSO4). Fisher, Cat No. AC422871000

7.3 Sulfuric Acid (H2SO4). Fisher, Cat No. A300-212

7.4 Sodium Hypochlorite (NaClO). Cat No. 19-546-929

7.5 Sodium Salicylate C6H4(OH)(COO)Na. Fisher, Cat No. 50-700-6201

7.6 Sodium Nitroprusside [sodium nitroferricyanide dehydrate,

Na2Fe(CN)5NO∙2H2O]. Fisher, Cat No. AC21164-1000

7.7 Sodium Phosphate dibasic heptahydrate (Na2HPO4∙7H2O). Fisher, Cat

No. AC20651-5000

7.8 disodium EDTA (ethylenediaminetetracetic acid salt). Fisher, Cat No.

BP120500

7.9 Sodium Hydroxide (NaOH). Fisher, Cat No. S613-3

7.10 Ammonium Chloride (NH4Cl). Fisher, Cat No.s AC199975000 and

A661-3

7.11 Reagent Water: Ammonia free deionized water produced from

Millipore Milli-Q Water Purification System.

7.12 Degassing with Helium:

7.2.1 To prevent bubble formation, degas the carrier and buffer with

helium. Use He at 140 kPa (20 lb/in2) through a helium degassing

tube. Bubble helium through one liter of solution for one minute.

7.2.2 All reagents used in heated chemistry must be degassed.

7.13 Reagent 1: Digestion Solution

In a 1.0 liter volumetric flask, add 134 g potassium sulfate (K2SO4) and

7.3 g copper sulfate (CuSO4) in 800 mL water. Then add 134 mL conc.


September 29, 2015

Page 6 of 16

Sulfuric acid (H2SO4) and dilute to the mark with reagent water. Stir to

mix.

7.14 Reagent 2: Hypochlorite Solution

In a 250 mL volumetric flask, dilute 15 mL 5.25% sodium hypochlorite

(NaOCl) to the mark with reagent water. Invert to mix.

7.15 Reagent 3: Salicylate Nitroprusside

In a 1.0 liter volumetric flask, dissolve 150 g sodium salicylate [salicylic

acid sodium salt, C6H4(OH)(COO)Na] and 1.0 g sodium nitroprusside

[sodium nitroferricyanide dehydrate, Na2Fe(CN)5NO∙2H2O] in about 800

mL reagent water. Dilute to the mark with reagent water and invert to mix.

Store in a dark bottle and prepare fresh monthly.

7.16 Reagent 4: Buffer

In a 1 liter volumetric flask containing 900 mL reagent water, completely

dissolve 35 g sodium phosphate dibasic heptahydrate (Na2HPO4∙7H2O).

Next, add 20 g disodium EDTA (ethylenediaminetetracetic acid salt). The

EDTA will not dissolve but will form a turbid solution. Finally, add 50 g

sodium hydroxide (NaOH), dilute to the mark with reagent water and

invert to mix. Degas weekly and prepare fresh monthly.

7.17 Reagent 5: Sodium Hydroxide (0.8M)

In a 1.0 liter volumetric flask, dissolve 32 g sodium hydroxide (NaOH) in

about 800 mL reagent water. Dilute to the mark with reagent water and stir

to mix.

7.18 Reagent 6: Digestion Diluent (for Carrier and Simulated Standards)

In a 1.0 liter volumetric flask, dissolve 400 mL digestion solution

(Reagent 1) in about 600 mL reagent water. Dilute to the mark with

reagent water and shake to mix.

7.19 Calibration Standards

7.19.1 Stock Standard: 1000 mg/L

In a 1.0 liter volumetric flask, dissolve 3.819 ammonium chloride

(NH4Cl) that has been dried for two hours at 110˚C in about 800

mL reagent water. Dilute to the mark with reagent water and invert

to mix.


September 29, 2015

Page 7 of 16

7.19.2 Working Stock Standard: 20 mg N/L

In a 250 mL volumetric flask, dilute 5.0 mL of the stock standard

(1000 mg/L) to the mark with reagent water or digestion diluent:

Depending on which process utilized: Digested (use reagent water)

for Samples, LFB, Blanks, and Simulated Digestion (use

digestion diluent) for Calibration Standards and QCS, and invert to

mix.

7.19.3 Calibration Standards: For Simulated Digestion

There are six levels calibration standards and their respective

concentrations and preparation procedures are listed as follows:

Level Concentration

(mg N/L)

Volume (mL) of Working

Stock Standard (20 mg N/L) to

50 mL with Digestion Diluent

6 20 50

5 10 25

4 5.0 12.5

3 1.0 2.5

2 0.50 1.25

1 0 0

8.0 SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1 Samples are collected in plastic or glass bottles. All bottles must be

thoroughly cleaned and rinsed with reagent water. Volume collected must

be sufficient to insure a representative sample, allow for replicate analysis,

and minimize waste disposal.

8.2 Samples must be preserved with H2SO4 to a pH<2 and cooled to 4˚C at the

time of collection.

8.3 Samples should be analyzed as soon as possible after collection. If storage

is required, preserved samples are maintained at 4˚C and may be held for

up to 28 days.

9.0 QUALITY CONTROL

9.1 Barnstable County Health Laboratory operates a formal quality control

(QC) program. The QC program for this method consists of an initial

demonstration of laboratory capability, and the periodic analysis of

laboratory reagent blanks and other laboratory solutions as a continuing


September 29, 2015

Page 8 of 16

check on performance. The laboratory maintains performance records that

define the quality of the data that are generated.

9.2 INITIAL DEMONSTRATION OF PERFORMANCE

9.2.1 The initial demonstration of performance is used to characterize

instrument performance (determination of linear calibration ranges

and analysis of QCS) and laboratory performance (determination

of MDL) prior to performing analyses by this method.

9.2.2 Linear Calibration Range (LCR) – The LCR is determined initially

and verified every 6 months or whenever a significant change in

instrument response is observed or expected. The initial

demonstration of linearity uses a blank and five calibration

standards. If any verification data exceeds the initial values by

±10%, linearity will be reestablished.

9.2.3 Quality Control Sample (QCS) – The QCS is analyzed right after

initial calibration (Section 9.2.2) to verify the calibration standards

and acceptable instrument performance with preparation and

analysis of a QCS. If the determined concentrations are not within

±10% of the stated values, performance of the determinative step

of the method is unacceptable. The source of the problem must be

identified and corrected before either proceeding with the initial

determination of MDLs or continuing with on-going analyses.

9.2.4 Method Detection Limit (MDL) – MDL must be established using

reagent water (blank) fortified at a concentration of two to three

times the estimated instrument detection limit. To determine MDL

values, seven replicate aliquots of the fortified reagent water are

taken and processed through the entire analytical method. The

following equation is used to calculate the MDL:

)()( StMDL

Where

t = Student’s value for a 99% confidence level and a

standard deviation estimate with n-1 degrees of

freedom [t = 3.14 for seven replicates].

S = Standard deviation of the replicate analyses.

9.2.4.1 The Standard deviation (S) can be calculated using the

following equation:


September 29, 2015

Page 9 of 16

1

)(2

2

n

nS

xx

Where, n = number of samples;

x = concentration in each sample.

9.2.4.2 MDLs must be determined every six months, when a new

operator begins work, or whenever there is a significant

change in the background or instrument response.

9.2.4.3 One set of MDLs is listed as follows:

9.3 ASSESSING LABORATORY PERFORMANCE

9.3.1 Laboratory Reagent Blank (LRB) – The laboratory analyzes at

least one LRB with each batch of samples. Data produced are used

to assess contamination from the laboratory environment. Values

that exceed the MDL indicate laboratory or reagent contamination

must be suspected and corrective actions must be taken before

continuing the analysis.

9.3.2 Laboratory Fortified Blank (LFB) – The laboratory analyzes at

least one LFB with each batch of samples. Calculate accuracy as

percent recovery as follows:

100

S

RCCs

Where, R = percent recovery;

Cs = recovered fortified blank concentration;

C = blank background concentration;

S = concentration equivalent of analyte added to

blank.

9.3.2.1 If the recovery of any analyte falls outside the required

control limits of 90-110%, the result is judged out of

control, and the source of the problem must be identified

and resolved before continuing analysis.

Unit: mg/L

Spiking

Level 8/11/15 8/11/15 8/11/15 8/31/15 8/31/15 8/31/15 9/1/15 MEAN STDEV MDL

0.5 0.391 0.419 0.346 0.268 0.344 0.362 0.399 0.361 0.050 0.16


September 29, 2015

Page 10 of 16

9.3.3 The laboratory also uses LFB analyses data to assess laboratory

performance against the required control limits of 90-110%. When

sufficient internal performance data become available (usually a

minimum of 25 analyses), optional control limits and control charts

can be developed from the percent mean recovery (x) and the

standard deviation (S) of the mean recovery. These data can be

used to establish the upper and lower control limits as follows:

UPPER CONTROL LIMIT = x + 3S

LOWER CONTROL LIMIT = x – 3S

The optional control limits must be equal to or better than the

required control limits of 90-110%. After each five to ten new

recovery measurements, new control limits can be calculated using

only the most recent 20-30 data points. Also the standard deviation

(S) data must be used to establish an on-going precision statement

for the level of concentration included in the LFB. These data are

kept on file and be available for review.

9.3.4 Instrument Performance Check Solution (IPC) – For all

determinations the laboratory must analyze the IPC (a mid-range

check standard) and a calibration blank immediately following

daily calibration, after every 10th

sample (or more frequently, if

required), and at the end of the sample run. Analysis of the IPC

solution and calibration blank immediately following calibration

must verify that the instrument is within ±10% of calibration.

Subsequent analyses of the IPC solution must verify the calibration

is still within ±10%. If the calibration cannot be verified within the

specified limits, the IPC solution is reanalyzed. If the second

analysis of the IPC solution confirms calibration to be outside the

limits, the sample analysis must be discontinued, the cause

determined and/or in the case of drift the instrument recalibrated.

All samples following the last acceptable IPC solution must be

reanalyzed. The analysis data of the calibration blank and IPC

solution are kept on file with the sample analysis data.

9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY

9.4.1 Laboratory Fortified Sample Matrix (LFM) – The laboratory adds

a known amount of analyte to a minimum of 10% of the routine

samples. In each case the LFM aliquot must be a duplicate of the

aliquot used for sample analysis. The analyte concentration must

be high enough to be detected above the original sample and

should not be less than four times the MDL. The added analyte


September 29, 2015

Page 11 of 16

concentration should be the same as that used in the laboratory

fortified blank.

9.4.2 The percent recovery for TKN is calculated and corrected for

concentration measured in the unfortified sample using the

following equation:

100

S

RCCs

Where, R = percent recovery;

Cs = fortified sample concentration;

C = sample background concentration;

S = concentration equivalent of analyte added to

sample.

Acceptable range of R is 90-110%.

9.4.3 If the recovery falls outside the designated LFM recovery range

(90-110%) and the laboratory performance is shown to be in

control (Section 9.3), the recovery problem encountered with the

LFM is judged to be matrix related, not system related.

10 CALIBRATION AND STANDARDIZATION

10.1 Prepare reagents and standards as described in Section 7.

10.2 Set up TKN manifold as is shown in Section 17.3 of the Lachat

Instruments Methods Manual (reference 14.5.)

10.3 Input data system parameters as shown in Section 17 of the Lachat

Instruments Methods Manual (reference 14.5.)

10.4 Pump reagent water through all reagent lines and check for leaks and

smooth flow. In order to avoid precipitate forming in the manifold tubing:

Add the Buffer Line First and allow to pump through manifold for at

least 5 minutes. Then add reagent lines one by one, ending with the

salicylate nitroprusside added last. For removal after analysis, reverse this

order with the salicylate nitroprusside line disconnected first, and the

buffer line last. When finished, place all respective reagent lines into

water and allow to pump through manifold for ten minutes.

10.5 Place standards in the sampler and sequence the required information in

the data system.


September 29, 2015

Page 12 of 16

10.6 Calibrate the instrument by injecting the standards. The system will then

associate the concentrations with the peak area for each standard to

determine the calibration curve.

10.7 The initial calibration is deemed acceptable if the following criteria are

met:

10.7.1 R ≥ 0.995

10.7.2 Quality Control Sample (QCS) standard is run right after the initial

calibration. The concentration of the QCS is 10 mg/L. This

standard (Ammonium chloride) is ordered from Fisher Scientific,

Acros Organics, ACS reagent grade. The procedure for making

the QCS is the same as the one for Level 5 of the calibration

standards described in (Section 7.9.1, 7.9.2, and 7.9.3). The QCS

concentration must fall within ± 10% of the stated value.

10.7.3 Instrument Performance Check (IPC) refer to (Section 9.3.4).

10.8 Figure 1. Lists a set of initial calibration peaks and a linear calibration

curve

11 PROCEDURE

11.1 Barnstable County Health Laboratory prepares the standards using the

simulated digestion process with the digestion diluent. At a minimum,

two blanks and one standard (LFB) should be prepared in reagent water

and carried through the digestion procedure.

11.2 DIGESTION PROCEDURE

11.2.1 To a 25.0 mL sample add 10 mL digestion solution (Reagent 1)

and mix.

11.2.2 Add 2 to 4 BD Kjeldahl Digestion Granules to each tube.

11.2.3 Place tubes in the preheated block digester for one hour at 200oC.

Water from the sample must be boiled off before increasing the

temperature.

11.2.4 Place the cold finger, teardrop stopper on the top of the sample

tube.


September 29, 2015

Page 13 of 16

11.2.5 Continue to digest for 2 hours at 380oC. This includes the ramp

time (approximately 50 minutes) for the block temperature to come

up to 380oC.

11.2.6 Remove the sample tubes from the block and allow about 3

minutes to cool.

11.2.7 Dilute to 25.0 mL with reagent water (add 23.5 mL) to each tube

and vortex to mix.

11.2.8 If the samples are not run immediately they should be covered

tightly and refrigerated at 4oC.

11.3 SYSTEM START-UP PROCEDURE

11.3.1 Prepare reagent and standards as described in section 7.

11.3.2 Set up manifold as shown in Section 17.3 of the Lachat

Instruments Methods Manual.

11.3.3 Input peak timing and integration window parameters as specified

in section 17.2 of the Lachat Instruments Methods Manual.

11.3.4 Pump reagent water through all the reagent lines and check for

leaks and smooth flow. Switch to reagent lines - add buffer first

and pump through the system for 5 minutes, followed by the other

reagents, adding salicylate nitroprusside last – and allow the

system to equilibrate until a stable baseline is achieved.

11.3.5 Place the standards in the autosampler, and fill the sample tray.

Input the information required by the data system, such as

concentration, replicates and QC scheme.

11.3.6 Calibrate the instrument by injecting the standards. The data

system will then associate the concentrations with responses for

each standard.

11.3.7 After a stable baseline has been obtained, start the sampler and

perform the analysis.

11.4 TROUBLESHOOTING AND SYSTEM NOTES

11.4.1 Allow at least 15 minutes for the heating unit to warm up to 60oC.

11.4.2 If sample concentrations are greater than the high standard the

digested sample should be diluted with Reagent 6 (diluent.) Do


September 29, 2015

Page 14 of 16

not dilute digested samples or standards with reagent water, as this

will cause a problem with matix-matching, pH differences.

11.4.3 If the salicylate reagent is merged with a sample containing

sulfuric acid in the absence of the buffer solution, the salicylate

reagent will precipitate. If this occurs NaOH can be run through

the system to attempt to clear clogs, flush system with NaOH for

20 minutes. If clogged tubing cannot be cleared, the tubing should

be replaced. To prevent this, prime the system by first placing the

buffer transmission line in the buffer solution.

11.4.4 In normal operation nitroprusside gives a yellow background color

which combines with the blue indosalicylate to give an emerald

green color. This is the normal color of the solution in the waste

container.

11.4.5 If the block digestor tubes are not completely dry and have water

droplets on them, there exists the possibility of ammonia

contamination in the water droplets.

11.4.6 If phosphorus is also determined with the Lachat System, a second

helium degassing tube should be used and segregated for the

individual chemistries.

11.4.7 If baseline drifts, peaks are too wide, or other problems with

precision arise, clean the manifold by the following procedure:

11.4.7.1 Place transmission lines in water and pump to clear

reagents first.

11.4.7.2 Place reagent lines in 1M HCl and pump for several

minutes

11.4.7.3 Place all lines back into water and pump out HCl.

11.4.8 If digested samples contain turbidity allow to settle prior to

analysis, decant sample slowly into test tube.

11.4.9 Alternatively, if turbid conditions persist, filter the digested sample

with 0.45uM filter.

12 POLLUTION PREVENTION

12.1 Pollution prevention encompasses any technique that reduces or eliminates

the quantity or toxicity of waste at the point of generation. Numerous

opportunities for pollution prevention exist in the laboratory operation.


September 29, 2015

Page 15 of 16

The EPA has established a preferred hierarchy of environmental

management techniques that places pollution prevention as the

management option of first choice.

12.2 Quantity of the chemicals purchased should be based on the expected

usage during its shelf life and disposal cost of unused material. Actual

reagent preparation volumes should reflect anticipated usage and reagent

stability.

13 WASTE MANAGEMENT

13.1 The laboratory waste management practices are conducted consistent with

all applicable rules and regulations as stated in the laboratory’s Sample

and Waste Disposal (Revision 001) on February 25, 2004. Excess

reagents, samples and method process wastes are characterized and

disposed of in an acceptable manner in this SOP.

14 REFERENCES

14.1 U.S. Environmental Protection Agency, Methods for Chemical Analysis

of Water and Wastes, EPA-600/4-79-020, Revised March 1993, Method

351.2

14.2 ASTM, Water(I), Volume 11.01, Method D3590-89, Test Methods for

Kjeldahl Nitrogen in Water, p. 447

14.3 Code of Federal Regulation 40, Chapter 1, Part 136, Appendix B

14.4 Guidelines and Format for EMSL-Cincinnati Methods. EPA-600/8-83-

020, August 1983.

14.5 Lachat Instruments Methods Manual, QuikChem Method 10-107-06-2-L,

Revision Date, 14 May 2008


September 29, 2015

Page 16 of 16

Figure 1.

Conc = 0.631 * Area + 0.0687

Area = 1.59 * Conc - 0.109

Correlation Coefficient (r) = 0.99998

Weighting : None

1

Envirotech Laboratories, Inc

8 Jan Sebastian Dr unit 12

Sandwich, MA 02563

SPE-DEX AUTOMATED SOLID PHASE EXTRACTOR SYSTEM

METHOD 1664A, HEM; OIL AND GREASE

Ref: US EPA-821-R-98-002, Feb 1999

2

Scope and Application

This method is for determination of n-hexane extractable material (HEM; oil and grease)

and n-hexane extractable material that is not adsorbed by silica gel (SGT-HEM; non-

polar material) in surface and saline waters and industrial and domestic aqueous wastes.

Extractable materials that may be determined are relatively non-volatile hydrocarbons,

vegetable oils, animal fats, waxes, soaps, greases and related materials. The method is

based on prior Environmental Protection Agency (EPA) methods for determination of

―oil and grease‖ and ―total petroleum hydrocarbons‖.

This method is not applicable to measurement of materials that volatilize at temperatures

below approximately 85 C. Petroleum fuels from gasoline through #2 fuel oil may be

partially lost in the solvent removal operation.

Some crude oils and heavy fuel oils contain a significant percentage of materials that are

not soluble in n-hexane. Accordingly, recoveries of these materials may be low.

This method is capable of measuring HEM and SGT-HEM in the range of 5 to 1000

mg/L, and may be extended to higher levels by analysis of a smaller sample volume

collected separately.

For HEM and SGT-HEM in this method, the method detection limit (MDL) is 1.4 mg/L

and the minimum level of quantitation (ML) is 2.0 mg/L.

Summary of Method

Theory of Operation

The SPE-DEX 4750 Extractor System automates the extraction of analytes from aqueous

samples by using a 47mm SPE disk. The SPE disk offers significant advantages over

other forms of solid phase extraction.

To successfully use the SPE disk, there are three steps that must be performed:

Prewet the SPE disk with solvents

Introduce the water sample to the SPE disk

Solvent rinses the sample container and extracts the SPE disk

Prewet the SPE Disk with Solvents

The Prewet step is very important as it:

Removes manufacturing and handling impurities from the SPE disk

Activates and prepares the disk in preparation of the water sample

The necessary solvent soaking and air drying times for each prewet solvent are entered by

the user and can be changed to optimize the extraction efficiency of the disk. In no case,

3

however, during the final Prewet step, i.e., the Methanol and Reagent Water prewets,

should the SPE disk be allowed to go dry before the introduction of water sample. This is

accomplished by entering a ―0‖ time for the air dry times for the Methanol and Reagent

Water steps.

Introduce the Water Sample to the Disk

This step automatically introduces the water sample to the SPE disk. The disk extracts

the organics or Oil & Grease out of solution and onto the active chemical surface of the

disk.

Solvent Rinses the Sample Container and Extracts the SPE Disk

The solvent rinse step uses organic solvents to rinse the sample container, wash the

Extractor’s internal surfaces, and extract the analytes of interest off the SPE disk and

back into solution. The rinse solvents and analytes pass through the SPE disk and into

the collection tube.

Definitions

HEM and SGT-HEM are method-defined analytes; i.e., the definitions of both HEM and

SGT-HEM are dependent on the procedures used. The nature of the oils and/or greases,

and the presence of extractable non-oily matter in the sample will influence the material

measured and interpretation of results.

Interferences

Solvents, reagents, glassware, and other sample processing hardware may yield artifacts

that affect results. Specific selection of reagents and purification of solvents may be

required.

All materials used in the analysis shall be demonstrated to be free from interferences by

running laboratory blanks.

Safety

The toxicity or carcinogenicity of each reagent in this method has not been precisely

determined; however, each chemical should be treated as a potential health hazard.

Exposure to these chemicals should be reduced to the lowest possible level. It is

suggested that the laboratory perform personal hygiene monitoring of each analyst that

uses this method. This monitoring should be performed using Occupational Safety and

Health Administration (OSHA) or National Institute of Occupational Safety and Health

(NIOSH) approved personal hygiene monitoring methods. Results of this monitoring

should be made available to the analyst.

n-Hexane has been shown to have increased neurotoxic effects over other hexanes and

some other solvents. OSHA has proposed a time-weighted average (TWA) of 50 parts-

per-million (ppm); NIOSH concurs that an 8-hour TWA/permissible exposure limit

(PEL) of 50 ppm is appropriate for n-hexane; and the American Conference of

4

Governmental Industrial Hygienists (ACGIH) has published a threshold limit value

(TLV) of 50 ppm for n-hexane. Inhalation of n-hexane should be minimized by

performing all operations with n-hexane in an explosion-proof hood or well-ventilated

area.

N-hexane has a flash point of –23 C (-9 F), has explosive limits in air in the range of 1-7

percent, and poses a serious fire risk when heated or exposed to flame. N-hexane can

react vigorously with oxidizing materials. The laboratory should include procedures in

its operations that address the safe handling of n-hexane.

Sampling Equipment

Sample collection bottles glass, approximately 1-L, with PTFE-lined screw cap.

Equipment for calibration

Analytical Balance—Capable of weighing 0.1 mg

Volumetric flasks—Glass, 100-mL

Vials—Assorted sizes, with PTEFE-lined screw caps

Volumetric pipette—Glass, 5-mL

SPE-DEX 4700 Automated Solid Phase Extractor System (Horizon)

Speed-Vap ll 9000 Sovent Evaporation System (Horizon).

Reagents and Standards

Reagent water—Water in which HEM is not detected at or above the minimum level

(ML) of this method. Bottled distilled water or water prepared by passage of tap water

through activated carbon have been shown to be acceptable sources of reagent water.

Hydrochloric acid or sulfuric acid—ACS. Mix equal volumes of concentrated HCI and

reagent water to produce an approximately 6N solution.

n-Hexane-ACS (>85% N Hexane)

Acetone—ACS, residue less than 1 mg/L

Methanol-ACS

SPE disks (SSI #1047TL)

Fastflo Prefilters (CPI #4350-010089)

Sample Collection, Preservation and Storage

Collect approximately 250 ml, 500 ml or one liter of representative sample in a glass

bottle acidified with sulfuric acid to pH < 2 following conventional sample practices,

except that the bottle must not be pre-rinsed with sample before collection. To allow for

potential QC failures, it is recommended that additional sample aliquots be collected.

5

All samples must be refrigerated at 0-4C from the time of collection until extraction (40

CFR 136, Table II). Preservation using Sulfuric Acid is used. Before samples are

analyzed pH is performed to ensure pH is < 2. This is recorded on raw data worksheet.

All samples must be analyzed within 28 days of the date and time of collection (40 CFR

136, Table II).

Quality Control

The laboratory operates a formal quality assurance program. The program consists of an

initial demonstration of laboratory capability, ongoing analyses of standards and blanks

as a test of continued performance, and analysis of a matrix spike (MS) to assess

recovery. Laboratory performance is compared to established performance criteria to

determine if the results of analyses meet the performance characteristics of the method.

For each analytical batch the laboratory analyzes a blank, a check standard (2 mg/ml std

CPI cat# 4401-1664-2). Range 1.8-2.2 mg/L

Sample duplicates, and matrix spike duplicate (add 1.0 ml of 4 mg/ml std (NSI cat# QC-

003LSIIP to sample = 4.0 mg/ml) is performed every 10 samples (±10%).

Initial Demonstration of Laboratory Capability

Method Detection Limit (MDL)—To establish the ability to detect HEM and Sgt-HEM,

the laboratory shall determine the MDL per the procedure in 40 CFR 136, Appendix B

using the apparatus, reagents, and standards that will be used in the practice of this

method. An MDL less than or equal to the MDL in Section 1.6 or less than 1/3 the

regulatory compliance limit must be achieved prior to the practice of this method.

Matrix spikes—The laboratory must spike a minimum of 5 percent of all samples from a

given sampling site or, if for compliance monitoring, from a given discharge/waste

stream (matrix spike). The sample aliquot shall be spiked with the hexadecane/stearic

acid spiking solution. The concentration of the spike in the sample shall be determined as

follows: The concentration of the spike of HEM or SGT-HEM in the sample, shall be at

the concentration of the precision and recovery standard or at 1 to 5 times higher than the

background concentration, whichever concentration is higher.

Note: Samples containing high concentrations (> 100 mg/L) of HEM will require

a large volume of spiking solution) for the MS (and MSD). If the concentration

of HEM is expected to exceed 1000mg/L, smaller sample volumes should be

6

collected for the background measurement and MS (and MSD) so that the amount

of HEM plus the amount spiked does not exceed 1000 mg/L.

Spike the additional sample aliquot(s) with the spiking solution and analyze the

aliquot(s) to determine the concentration after spiking (A).

Calculate the percent recovery (P) of HEM or SGT-HEM in each aliquot using the

following equation:

Equation 2

P= 100(A-B)

T

Where:

A=Measured concentration of analyte after spiking

B=Measured background concentration of HEM or SGT-HEM

T=True concentration of the spike

When determining SGT-HEM, the true concentration (T) must be divided by 2 to reflect

the concentration of hexadecane that remains after removal of stearic acid.

Compare the percent recovery of the HEM or SGT-HEM with the corresponding QC

acceptance criteria in Table 1.

If the results of the spike fail the acceptance criteria, and the recovery of the QC

standard in the ongoing precision and recovery test for the analytical batch is

within the acceptance criteria in Table 1, an interference is present. In this case,

the result may not be reported or used for purposes regulatory compliance

purposes and the laboratory must assess the potential cause for the interference. If

the interference is attributable to sampling, the site or discharge/waste stream

should be resampled. If the interference is attributable to a matrix problem, the

laboratory must modify the method, repeat the tests required, and repeat the

analysis of the sample of the MS (and MSD, if performed). Most matrix

interference problems are attributable to the formation of emulsions in the

extraction.

If the results of both the spike and the ongoing precision and recovery test fail the

acceptance criteria, the analytical system is judged to be out of control, and the

problem shall be identified and corrected, and the sample batch reanalyzed. All

samples must be associated with a valid MS (and MSD, if performed).

Laboratory blanks—Laboratory reagent water blanks are analyzed to demonstrate

freedom from contamination.

Extract and concentrate a laboratory reagent water blank initially and each

analytical batch. The blank must be subjected to the same procedural steps as a

sample.

If material is detected in the blank at a concentration greater than the minimum

level, analysis of samples is halted until the source of contamination is eliminated

7

and a blank shows no evidence of contamination. All samples must be associated

with an uncontaminated method blank before the results may be reported for

regulatory compliance purposes.

Calibration verification—Verify calibration of the balance before and after each

analytical batch. If calibration is not verified after measurement of the analytical batch,

recalibrate the balance and reweigh the batch.

Ongoing precision and recovery—To demonstrate that the analysis system is in control,

and acceptable precision and accuracy is being maintained with each analytical batch, the

laboratory shall perform the following operations:

Extract and concentrate a precision and recovery standard with each analytical

batch.

Compare the recovery with the limits for ongoing precision and recovery. If the

recovery is in the range specified, the extraction, weighing processes are in

control and analysis of blanks and samples may proceed. If, however, the

recovery is not in the specified range, the analytical process is not in control. In

this event, correct the problem, re-extract the analytical batch, and repeat the

ongoing precision and recovery test.

The specifications contained in this method can be met if the apparatus used is

scrupulously cleaned and dedicated for the determination of HEM and SGT-HEM. The

standards used for initial precision and recovery, matrix spike, and ongoing precision and

recovery should be identical, so that the most precise results will be obtained.

Calibration and Standardization

Calibrate the analytical balance at 2 mg and 1000 mg using class ―S‖ weights.

Calibration shall be within 10% at 2 mg and 0.5% at 1000 mg. If values are not

within these limits, recalibrate the balance.

Procedure using SPE-DEX 4750 Extraction

1. Bring the analytical batch of samples, including the sample aliquots for the MS, to

room temperature. Use 500 ml of sample: Lower volumes may be used if high

levels are expected.

2. Place approximately 500 ml of reagent water in a clean sample bottle to serve as

the laboratory blank.

3. Prepare standard and spiked sample using identical procedure as samples

4. Verify that the pH of the sample is less than 2

5. Check the levels of all the solvent bottles and fill them if necessary

6. Empty all waste bottles (water and solvent)

7. Turn on the vacuum source (25 hg min. at pump and 15 at solvent/waste

recovery bottle)

8

8. Turn the gas supply on (check the regulator bracket and make sure there is 35 psi

on the extractor pressure side, and 8-10 psi on the solvent bottle pressure side)

9. Turn on controller (the display will show active extractors-make sure it matches

the number of extractors plugged in)

10. Free up all 3 check valves with a tie wrap (tech tip t24 in users manual)

11. Run purge method:

Place the purge gasket into the disk area, and load an empty collection

vessel and sample bottle

Select the extractor to be purged by pushing SELECT on the controller

and then entering the extractor #

Select the purge method # when prompted by controller and press ENTER

Press the PURGE key on the extractor

Repeat a second time if necessary

Select the next extractor to be purged and repeat

Remove the collection vessel, purge gasket, and sample bottle

12. Run samples:

Place the support screen and the disk in the disk area, or the O-ring and JT

Baker speedisk or CPI SPE disc.

Close the extractor mouth and snug the assembly by hand to ensure a tight

seal

Load empty collection vessel (hold by top of the adapter not the vial and

gently give a ¼ turn to ensure a good seal)

Select an extractor by pushing SELECT on the controller and then

entering the extractor # (pushing the . key will activate all extractors)

Select 1664 when prompted by the controller and press ENTER

Put foil over the top of the sample bottle and screw the adapter in place. If

larger bottles are being used, put the large adaptor on the bottle, then put

the foil over the smaller opening and screw the small cap adapter over the

foil.

Load the sample bottle onto the extractor

Press start

Repeat procedure for each sample, standard, blank, and matrix spike.

13. Sampling Handling Technique using Speed-VAP II 9000 Solvent Evaporation

System

Step 1:

Remove a clean, empty aluminum pan from the cardboard container. As

cardboard ―dust‖ can collect on the inside of the pan, use a kimwipe to

gently wipe the inside of the pan. If desired, turn the pan over, and using

a hard point pen, write the sample identification on the bottom of the pan.

This will eliminate any confusion once multiple pans are in use.

Note: From this point on only use the Pan Transfer Tongs or forceps to move or

transfer the pan. Do not touch the pan with your fingers, as finger oils

will contribute to the pan weight.

9

Step 2: Once the pan is clean and labeled, use the tongs or forceps to transfer the

pan to the analytical balance. Record the weight 3 times on the

worksheet.

Step 3: Transfer the pan to the evaporator and place the pan into any one of the

open positions.

Note: One drop of concentrated food color added to the collection vessel will

visually identify the water layer. If any water is pulled into the pipette

it can be dispensed back into the collection vessel.

Step 4: Using a polypropylene transfer pipette, carefully transfer the top, hexane

layer from the extract collection vessel into the pan. If any colored

liquid is pulled into the pipette tip, dispense the colored liquid back into

the collection vessel. Be careful not to spill any of the solvent during the

transfer to the pan.

Step 5: Using fresh n-hexane and a new transfer pipette or squeeze bottle rinse

down the walls of the collection vessel. Using the pipette originally

used to transfer the hexane into the pan, transfer the rinse hexane into

the pan. Repeat this operation a second time to ensure all of the extracted

Oil and Grease is transferred into the pan.

Step 6: Close the cover of the evaporator and turn the Vac On / Off switch to on.

Set temperature control to 40ºC.

If necessary, adjust the Control Knob on the Vacuum Generator to

achieve the desired swirling rate in the pans.

Step 7: When the solvent has visually appeared to have evaporated, press the

Cover Release button and use the tongs or forceps to transfer the pan back

to the balance. Wait 5 min. before taking weight readings. If the weight is

stable, record the new weight 3 times on worksheet. If the weight is

slowly

dropping, this may indicate solvent is still evaporating off the pan. The

pan can either be left on the balance until a stable weight is achieved, or

the pan can be returned to the evaporator for a short period of time

(approximately 1 minute).

Step 8: Repeat the above steps for all samples to be run.

Step 9: When all samples are run, open the cover by pressing and holding the

Cover Release button until the vacuum level inside the unit is vented.

If the unit is to be shut down, turn the main power off, then turn the Vac

On / Off switch to off, turn the Control Knob on the Vacuum Generator

counter-clockwise to 0 or shut off the vacuum pump/source.

Note: If the vacuum source will be turned off for more than 10 minutes, it is

10

recommended to turn off the evaporator. This will minimize any safety

issue due to solvent vapors.

14. Calculate results using equation:

HEM mg/L = weight of extractable material (mg)

SampleVolume (L)

Reporting—Report results to three significant figures for HEM and SGT-HEM found at

or above 10 mg/L, and report results to two significant figures for HEM and SGT-HEM

found below 10 mg/L.

Samples—Report results for HEM and SGT-HEM found below the MDL as <2.0

mg/L.

Blanks—Report results for HEM and SGT-HEM found below the MDL as <1.4

mg/L. Do not report results below the MDL unless required by the permitting

authority or permit.

Results from tests performed with an analytical system that is not in control must

not be reported or otherwise used for permitting or regulatory compliance

purposes but do not relieve a discharger or permittee of timely reporting.

Method Performance

The method detection limit (MDL) and minimum level of quantitation (ML) are based on

five studies conducted by EPA and described at proposal of Method 1664A and as

verified by data submitted in comments on the proposal of Method 1664A. The MDL is

performed yearly.

Waste Management

It is the laboratory’s responsibility to comply with all Federal, State, and local regulations

governing waste management, particularly the hazardous waste identification rules and

land disposal restrictions, and to protect the air, water, and land by minimizing and

controlling all releases from fume hoods and bench operations. Compliance with all

sewage discharge permits and regulations is also required.

Samples preserved with HCI or H2SO4 to pH<2 are hazardous and must be neutralized

before being disposed, or must be handled as hazardous waste.

City of New Bedford Health Department Laboratory Quality Assurance Manual

..

Reviewed for Revision April 2014 1

QUALITY ASSURANCE MANUAL

Page

Section 1 - Laboratory Facilities, Personnel and Analytical Test Procedures 2

Section 2 - Laboratory Equipment and Instrumentation 4 Section 3 - Laboratory Supplies 8 Section 4 - Media and Reagents 11 Section 5 - Sampling Procedures 13 Section 6 - Laboratory QA/QC Testing 14 Section 7 - Standard Operating Procedures 17 Section 8 - Documentation and Recordkeeping 18


..


Section 1 - Laboratory Facilities and Personnel 1.1 Ventilation Central air conditioning is used to promote a well-ventilated environment which is free of dust, drafts and extreme temperature changes. Temperatures are maintained between 16 and 27oC. Air conditioning is used to reduce contamination, permit more stable operation of incubators and decrease possible moisture problems with medias or analytical balances. 1.2 Organization of Laboratory Space Entrance to the laboratory area is posted to minimize through traffic and visitors. Apart from the main laboratory area, a separate room, the chemical prep and incubation room, is used for the weighing and preparation of medias and reagents as well as various incubators and waterbaths for incubation purposes. The main laboratory area is divided into working sections which include a glassware preparation and storage area, a water purification and dishwashing area with safety shower and eyewash, and a laboratory work-bench area with analytical meters and filtration apparatus where samples are processed for analysis. The main laboratory area also has two fume hoods for working with hazardous or volatile materials. One fume hood is specific for working with acids and the other fume hood is reserved for use with organics. Sterilization is performed in the autoclave located in the Wastewater Treatment Laboratory. 1.3 Laboratory Bench Areas A minimum of 2m linear bench space is provided per analyst in addition to areas specific for preparation and support activities. The laboratory bench tops provide a smooth, impervious surface which is chemically inert, corrosion resistant and has a minimum number of seams. The bench working surfaces are illuminated with even, glare-free lighting. 1.4 Walls and Floors Walls are covered with a smooth finish that is readily cleaned and disinfected. Floors are coated with an impervious, washable surface which is textured to prevent slipping accidents. 1.5 Air Monitoring Laboratory air quality is monitored on a monthly basis using Plate Count Agar in sterile dishes,


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set on lab bench surfaces (exposed) for a 15 minute period. Following 48 hours incubation at 35oC, the number of colonies on the air density plates must not exceed 15 colonies/plate/15 minutes. Records of air quality monitoring are maintained in the lab Quality Assurance Manual. 1.6 Laboratory Cleanliness and Maintenance Laboratory rooms are cleaned regularly (weekly). Routine cleaning includes washing of benches, shelves, floors and other areas prone to dust accumulation. Floors are wet mopped and treated with a disinfectant solution. Sweeping of floors and/or accumulation of clutter is not allowed. Benchtops are treated with a disinfectant solution before and after each use. Laboratory equipment is maintained with regular inspection and servicing as necessary. Procedures specific for maintenance of equipment are outlined in Standard Operating Procedures (SOPs) and records of repair and service are kept. 1.7 Laboratory Personnel All bacteriological testing is performed by trained personnel, currently consisting of a lab director/supervisor/analyst (Leslie Aubut) and a part-time analyst (Jane Wurm). The Lab Director monitors and shares in defined bench-top work assignments to ensure that test procedures are precisely followed and that quality control measures are incorporated into the analyses. The Lab Director is responsible for ensuring the laboratory quality assurance program is followed. Job descriptions and training records for laboratory personnel are maintained on file, as a resume of pertinent experience, and are updated at least annually. Any additional training received between updates is documented in the personnel files. Laboratory personnel are subject to annual performance evaluation reviews. Copies of performance evaluations are maintained on file with the training records. Lab personnel are encouraged to take additional training for the advancement of skills and knowledge. The Lab Director is responsible for record keeping for laboratory services and archival of lab data. The Lab Director reviews all reports generated by support personnel for accuracy, and monitors record keeping procedures to ensure compliance with SOPs and appropriate regulatory guidelines. 1.8 Analytical Test Procedures For the analysis of potable waters, the analytes and methods employed are: Total Coliform by MF-SM9222B and E.coli by NA-mug -SM9222G, and for ambient and wastewaters the Enterococcus method used is EPA Method 1600.


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Section 2 - Laboratory Equipment and Instrumentation 2.1 Autoclaves The autoclave is of sufficient size to prevent crowding of the interior. It is constructed to provide uniform sterilizing temperature of 121oC within the chamber. The autoclave is equipped with digital readouts of pressure, temperature and time. Autoclave cycles must be completed within 45 minutes when a 15 minute sterilization cycle time is used, such as with sugar broths. Records are maintained in the Autoclave Logbook for each sterilization cycle and include items sterilized, date, start-up time, time temperature reached, time temperature off, time removed from autoclave, total time in autoclave, maximum registering thermometer temperature, heat sensitive tape usage and user initials. Sterilization effectiveness is monitored using spore strips weekly (Standard Methods, 20th Ed., 9020B section 3h) with results maintained in the Autoclave Logbook. The timing operation of the autoclave is checked against a stopwatch quarterly, with results recorded and maintained in the Autoclave Logbook. Periodic inspection is performed by qualified service representatives. Servicing is conducted whenever a deviation of sterilization time or effectiveness is observed and records of corrective action are maintained in the Autoclave Logbook. 2.2 Balances Operation and routine maintenance is performed in accordance with manufacturers instructions. Balances and weights are serviced and checked annually by the Sealer of Weights and Measures. Balances are calibrated monthly with a set of S weights traceable to NIST and records of calibrations are maintained in the Balance Logbook. If balance calibrations vary from true values, adjustments are made to re-center the bubble and re-calibration is performed. Continued variance from true values will result in servicing, with records maintained in the Balance Logbook. In weighing less than a gram, an analytical balance with a sensitivity less than 1 mg at a 10 g load is used. For larger quantities, a top-loading balance with sensitivity of 0.1 g at a 150 g load is used. The balance is wiped before and after each use with a soft brush. Spills are immediately cleaned-up with a damp towel. Weights are inspected with each use and are discarded if corrosion is observed. Weights are protected from manual contact, corrosion and laboratory atmosphere. 2.3 Colony Counters and Tallies A standard colony counter, Quebec model, providing magnification and good visibility is used for heterotrophic plate counts. Grid plates are inspected for scratches. Internal mirror placement


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and cleanliness is maintained to ensure optimal illumination. Hand-held colony tallies are used and checked with each use to ensure accuracy. For 50mm plates, a Spencer dissection microscope with 10X magnification and fluorescent light source is used in counting colonies. maintenance records are maintained in the Microscope Logbook. 2.4 pH Meter An electronic pH meter (Orion) graduated in 0.01 pH units with temperature compensation and reference electrode is used. Commercial buffer solutions are used to standardize the meter daily when in use. Three buffers which bracket the pH of interest are standard practice. Also included in the calibration is a record of the slope and mV readings as a check of electrode accuracy. If the slope falls outside the range of 95% to 105%, the cause is investigated, corrective action is taken and documented in the pH meter Logbook. Records of pH meter calibrations are maintained in the pH Meter Logbook. Buffer solutions are dated when opened and are used prior to the manufacturers expiration date. The lot numbers for each set of buffers in use is recorded at the end of the pH Meter Logbook. Buffer solutions are discarded after each use. Glass electrodes are stored in pH buffer solution when not in use. All samples and buffers are brought to room temperature prior to measurements. In between each sample or buffer, the electrodes are thoroughly rinsed with distilled water and wiped with a soft tissue. The maintenance of electrodes is performed as described in Standard Methods, 20th Ed., 4500-H+ section 5b. Precision and accuracy checks are conducted at least daily when the pH meter is in use and results are recorded in the Laboratory Precision and Accuracy Logbook. When unacceptable results are obtained from precision or accuracy measurements, the cause is investigated, corrective action taken and recorded in the Precision and Accuracy Logbook. 2.5 Laboratory Refrigerators The temperature of the laboratory refrigerators is checked and recorded twice daily. Each laboratory refrigerator is assigned a unique number and has its own temperature recording logbook. Refrigerators are cleaned monthly. All materials stored are identified and dated. The lab refrigerators are used to cool and maintain samples between 1 to 4oC until tested and to store prepared media, reagents and control cultures. Refrigerator temperatures are measured using two thermometers with bulbs immersed in glycerin solution, in glass vials, on upper and lower shelves. If refrigerators show unacceptable temperature ranges, a service call is made to a repair technician and servicing is recorded in the Refrigerator Logbook. 2.6 Water Distillation and Deionization Systems The laboratory water still (Barnstead MP-1) is fed from New Bedford city tap water. The still is


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drained and cleaned after each 3-5 days usage using a 10% HCl acid solution according to SOP #3.31 (1) and manufacturers instructions. Cleaning dates are recorded, with technician initials, in the system logbook. The distilled water produced is used to feed the laboratory water deionization system (Barnstead Nanopure II). The Nanopure system combines pre-filtration, mixed-bed resins, activated carbon and aseptic final filtration with a 0.22 um pore membrane to produce a reagent grade microbiologically suitable water. The water deionization system is monitored daily when in use, by observing the resistivity reading displayed on the electronic meter and recording results in the system logbook. The reagent grade water produced is analyzed annually for trace metals (Cd, Cr, Cu, Pb, Zn and Ni). The Nanopure system cartridges are replaced at intervals recommended by the manufacturer or as indicated by analytical results. The reagent grade water is tested monthly for conductivity, and each time a new lot of media is prepared (must be < 2 uS/cm at 25oC), residual chlorine (must be non-detectable), pH and heterotrophic plate count (must contain < 500 cfu/mL). All reagent grade water quality control tests are performed in accordance with 310 CMR 42.08(5)(c)12 and Standard Methods for the Examination of Water and Wastewater, 21st Ed., 2005 and pertinent laboratory SOPs. Records are maintained in the laboratory Water Systems Logbook. Any deviations from prescribed test parameter limits will result in investigation accordingly and corrective action with records maintained. 2.7 Waterbaths Each laboratory waterbath is equipped with a gable cover and is identified with a unique number. Waterbaths have an adequate capacity for the workload. The level of water in the baths is kept above the level of liquid in incubating tubes. Waterbath temperatures are maintained at 44.5oC +/- 0.2oC. Temperatures are recorded twice daily with records maintained in the Waterbaths Logbook. Deviation from prescribed temperature maintenance is investigated and rectified by repair from an authorized service representative with records maintained. The thermometers used to monitor the temperatures of the waterbaths are graduated in 0.1oC increments. Waterbath working thermometers are calibrated semi-annually against the laboratory standards thermometer (traceable to NIST) at 44.5oC and calibration records are maintained in the Thermometers Logbook. 2.8 Incubators All laboratory incubators are identified with a unique number. Incubator temperatures are checked twice daily (morning and afternoon) on the shelf areas in use (top and bottom). Records are maintained in the logbooks specific for each incubator. Thermometers are immersed in water to the stem marking. Laboratory incubators are kept in areas where the temperature is maintained at 16 to 27oC. The incubator working thermometers are calibrated annually against the laboratory


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standards thermometer (traceable to NIST) at the appropriate operational temperature, and calibration records are maintained in the Thermometers Logbook. Thermometers used in air incubators are graduated in 0.1oC increments. Incubators are loaded so that there will be at least 2.54 cm of space between stacks of plates and from incubator walls. Humidity levels of incubators are maintained such that SPC agar plates do not lose more than 15% of agar weight after 48 hours of incubation at 35oC. Agar weight loss is determined quarterly and records are maintained in the laboratory Quality Assurance Manual. Deviation from prescribed temperatures or agar weight loss is investigated and remedied by servicing if necessary, with records maintained. 2.9 Laboratory Fume Hoods Hoods are located away from high traffic areas as a fire safety and hood velocity integrity measure. Fume hood face velocity should be at least 30.5 linear meters/minute and are checked annually with an airflow measurement device. No airflow restrictive devices shall be placed in the hood. The laboratory fume hoods are made of corrosion resistant materials. 2.10 Membrane Filtration Equipment Before use, filtration units are assembled and checked for leaks. Units are discarded if inside surfaces are scratched or cracked. Filtration assemblies are washed and rinsed thoroughly after use, wrapped in non-toxic paper or foil, and sterilized by autoclaving. Membrane filter funnels are numbered and marked at appropriate graduations. Volume accuracy is determined by weight annually and documented in the Funnel Logbook. Tolerance must not exceed 2.5%. 2.11 Laboratory Thermometers Accuracy of thermometers is checked annually against a NIST traceable thermometer. The NIST traceable thermometer is checked annually for accuracy at ice-point (distilled water ice). For incubators and waterbaths, thermometers with 0.1oC increments are used. All thermometer incubation records are maintained in the laboratory Thermometers Logbook. Calibration corrections for each thermometer are marked on the thermometer identification tag, including calibration date, ID number and calibration temperature. Working thermometers are appropriately immersed in stoppered glass tubes containing water, or in waterbaths above the stem marking. Metal field thermometers are calibrated against a NIST traceable working thermometer quarterly. The calibration check is documented in the Field Thermometer Logbook. A field thermometer which has greater than a 5o correction factor is discarded. 2.12 Pipettors


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Eppendorf fixed and adjustable volume pipettors are used throughout the laboratory for dispensing of aqueous aliquots in the range of 10 uL to 1000 uL. The pipettors are assigned a unique ID number and are calibrated quarterly, using ten weighings each, on an analytical balance. Calibration results are recorded and maintained in the laboratory Pipettor Logbook. Pipet tips (Eppendorf) are placed into autoclavable trays and are sterilized prior to use. 2.13 Conductivity Meter A YSI Model 30 Handheld SCT meter, SN 04F10421, is dedicated to the use of monitoring low level conductivity of the laboratory reagent grade water. The meter is calibrated monthly, using single-use, low level calibration standard. The conductivity probe is maintained with extraordinary cleanliness by thorough cleaning with distilled/deionized water and cleaning with a mild acid solution (per manufacturer instruction manual) as dictated by any deviation observed during calibration checks. If erroneous measurements are observed despite these measures, the meter is returned to the manufacturer for servicing. Records of calibration, maintenance and servicing are maintained in the laboratory Conductivity Meter Logbook. 2.14 Dissection Microscope A Spencer Cycloptic Dissection Microscope with 10X-15X objective and Cyclospot Illuminator is used to count sheen colonies on m-Endo agar medium or enterococcus colonies on mEI medium. The microscope and cool light illuminator are maintained according to the Manufacturer Reference Manual (American Optical Co.) and serviced when necessary (ie; inability to focus) by an authorized factory representative. The microscope lenses are inspected for smears and dust particles with use and cleaned with lens cleaner and a lint free cloth. Maintenance and service records are maintained in the laboratory Microscope Logbook. Section 3 - Laboratory Supplies 3.1 Glassware and Plasticware Prior to each use, glassware is examined and items which are scratched, cracked or have chipped edges are discarded. Dilution bottles are made of borosilicate glass and are indelibly etched at the graduation level of 99 +/- 1 mL. Leakproof stopper closures are used with dilution bottles. Petri dishes are 12 mm deep and have bottoms of at least 80 mm inside diameter. Petri dishes for membrane filtration work have a 47 mm inside diameter. Petri dishes, whether sterilized glass or sterile plastic, are inspected for bubbles, scratches or cracks. Pipets are of glass or sterile plastic and are operated manually with a pipet aid. Pipets are marked to sharply contrast with pipetted


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solutions. As a general rule, for delivery of 1 mL volumes, pipets of no greater than 10 mL are used and for 0.1 mL volumes, pipets no greater than 1 mL are used. Manual use pipets have straight walls and tips are inspected for cracks to ensure accurate volume delivery. Disposable pipets and Eppendorf pipet tips are used one time only, placed in a disinfectant solution and are disposed of. Reusable glassware and plasticware is decontaminated if necessary and washed. In washing reusable sample containers, glassware and plasticware, a minimum of four fresh water rinses and a final rinse in distilled water deionized water is used to thoroughly rinse off all residual detergent. This is accomplished by running two complete wash and rinse cycles (the second cycle does not utilize detergent) in the lab dishwasher, with a final purified water rinse. To ensure glassware cleanliness, the following quality control checks for clean glassware are made: a) pH check - batches of clean glassware are spot checked for pH reaction by adding a few

drops of 0.04% bromthymol blue (BTB) and observing the color reaction. BTB should be blue-green, in the neutral range. BTB indicator solution is purchased commercially or is prepared by adding 16 mL 0.01N NaOH to 0.1 g BTB and diluting to 250 mL with distilled deionized water. Records of pH checks are maintained in the laboratory Glassware Washing Logbook. If deviation from the prescribed pH range is observed, the cause is investigated by replacing reagents and reviewing the rinse procedure.

b) Check for Inhibitory Residues - the test is performed before using a new supply of

detergent, to ensure the effectiveness of the rinsing procedure. If pre-washed, pre-sterilized plasticware is used it is also tested for inhibitory residues. The inhibitory residue test is performed according to Standard Methods for the Examination of Water and Wastewater, 20th Ed., page 9-6, and laboratory SOP #4.11(1). Records of the Inhibitory Residue Test are maintained in the Glassware Washing Logbook. If inhibitory residue results, the cause is investigated by reviewing the detergent in use and the rinse procedure, with records of corrective action maintained.

After washing, glassware is inspected for excessive water beads and rewashed if necessary. All glassware and plasticware (polypropylene), including reusable sample containers, which have been exposed to bacteriological agents are autoclaved for 30 minutes prior to washing and are re-autoclaved for 30 minutes prior to storage. The sterility of reusable sample containers is determined with each autoclaved batch by addition of Tryptic Soy Broth to a sample container followed by incubation at 35oC for 24 and 48 hours. Results of sterility checks are recorded and maintained in the laboratory Autoclave Logbook. The sterility of irradiated disposable plasticware is determined with each new manufacturer lot. Laboratory disposables are also subjected to lot comparison/productivity tests with records maintained in the Laboratory Disposables Logbook. Lack of sterility will prompt immediate investigation into the sterilization procedures and autoclave maintenance, with servicing by an authorized service technician.


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Servicing and maintenance records are maintained. 3.2 Membrane Filter Equipment Membrane filtration units are assembled and checked for leaks prior to use. The filtration units contain clearly marked graduations for measuring sample aliquots. Following use, filtration units are decontaminated by autoclaving. The filtration units are washed and re-autoclaved prior to storage and reuse. Filtration assemblies are autoclaved in non-toxic paper or aluminum foil for 30 minutes. Membrane filters are purchased through the Millipore Corp. and come provided with certification for each new lot, which includes retention, pore size, flow rate, sterility, pH, percent recovery and limits for specific inorganic chemical extractables. The lot number of the filters and usage dates is recorded in the Membrane Filters Logbook. The manufacturer’s certification report for each lot is maintained on record in the laboratory Membrane Filter Logbook.. Use of the membrane filters and apparatus is performed in accordance with laboratory SOP’s. When a new lot of membrane filters is used, comparison tests of the lot in use is made against the new lot as described in Standard Methods for the Examination of Water and Wastewater, 20th Ed., page 9-7. 3.3 Reagent Grade Water Quality The laboratory reagent grade water supply is monitored for acceptability criteria as outlined in Standard Methods, 20th Ed., Table 9020:II, page 9-6. The resistivity reading on the electronic display of the Nanopure deionization system is checked with each use to ensure a value of >16.7 megaohms/cm (per manufacturer instructions). Conductivity is monitored to ensure a value of <2 umhos/cm at 25oC. Records are maintained in the Conductivity/Salinity Logbook. The pH is checked with each pH meter calibration to be between 5.5 and 7.5 and recorded in the pH Meter Logbook. A heavy metals analysis (Cd, Cr, Cu, Ni, Pb and Zn) is performed annually and records are maintained in the Reagent Grade Lab Water Logbook. The metals analyzed must be < 0.05 mg/L. The total residual chlorine is determined monthly and records are maintained in the Reagent Grade Lab Water Logbook. The value obtained for residual chlorine must be below detectable limits. A heterotrophic plate count (HPC) is conducted monthly and records are maintained in the Reagent Grade Lab Water Logbook. HPC results must be <1000 CFU/mL. An annual Biosuitability Test (also called the Water Quality Test) is conducted annually (if conductivity tests exceed acceptability criteria) as described in Standard Methods, 19th Ed., 9020 B 3c1, page 9-5 through 9-7. The Water Quality Test is performed by NH Dept. Of Environmental Services, State of New Hampshire, 1-603-271-3503 (lab 1-603-271-3445) and results must be within the acceptable range of a 0.8 - 3.0 ratio. If water quality test results do not meet acceptability criteria, the problem is investigated and corrective measures are documented.


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3.4 Laboratory Control Cultures Laboratory control cultures are purchased commercially as inoculated swabs, or equivalent, bearing an ATCC reference number. The control cultures are revived and incubated on appropriate agar media slant tubes for 24 hours at 35oC. Control cultures are stored refrigerated and are aseptically transferred monthly to fresh agar slants. Each new set of control culture slants is assigned a lot number based on the preparation date. Records for control cultures are maintained in the Control Cultures Logbook. For each new lot of prepared culture medium, the analytical procedures are tested with known positive and negative control cultures for the organism(s) under test. Control cultures used for each type of test are as follows:

Test Positive Control Negative Control Total Coliforms E. coli or Enterobacter Staphylococcus Fecal Coliforms E. coli Enterobacter E. coli E. coli Enterobacter Fecal Streptococci Enterococcus faecalis Staphylococcus or E. coli Enterococci Streptococcus faecalis Staphylococcus or E. coli Section 4 Media and Reagents 4.1 Reagents Only reagents and chemicals of ACS grade are used for analyses of test samples. All reagents are prepared in distilled/deionized water and clean glassware, following pertinent laboratory SOP’s. All chemicals and reagents are dated when received and when first opened for use. Reagents are made to volume using Class A volumetric glassware. Preparation details are recorded in the laboratory Media and Reagents Preparation Logbook. For storage, good-quality inert plastic or borosilicate glass bottles with tight fitting lids are used. Prepared reagents are labeled with name, concentration, date prepared, lot number, preparer initials, expiration date, storage conditions, and chemical hazard rating (if applicable) according to NFPA recommendations. 4.2 Culture Media Commercially prepared media is used wherever possible for control of quality. Media is ordered in quantities to last no longer than 1 year. Upon receipt, media bottles are labeled with the date


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of receipt and the date when opened. The new media is logged into the Media Receipt Logbook with Manufacturer, lot number, amount received, date received, date opened and date discarded. Media that appears caked or exhibits signs of deterioration are discarded. The manufacturer’s lot numbers are recorded on media preparation forms (maintained as raw data in the Media Preparation Logbook). Opened bottles of media are used within 1 year of opening. Culture medias are stored in a Fisher Scientific Desiccator Cabinet containing desiccant to protect dehydrated medias from humidity. Storage of unopened medias is no longer than 2 years. Newly purchased lots of media are compared against proven lots using recovery of pure culture isolates and natural samples. a) Media Preparation - Medias are prepared in containers that are at least twice the volume

of the media being prepared. Media are stirred while heating, either on a hot plate or in a waterbath. Prepared media is identified, dated, and assigned a lot number in the Media Preparation Logbook. Media lot numbers are recorded on raw data worksheets. All medias are prepared in distilled, deionized water which has been measured with pipets or graduated cylinders conforming to NIST and APHA standards. The pH of a portion of media from each lot is checked after sterilization and recorded in the Media Preparation Logbook. Minor adjustments in pH are made with 1M NaOH or HCl solution. The media is remade if large deviations in pH are observed (> 0.5 pH units). Prepared medias are examined for unusual color, darkening or precipitation and discarded if problems are noted.

b) Media Sterilization - Media is sterilized at 121oC for the minimum time specified. A

double walled autoclave maintaining full pressure and temperature in the jacket between loads is used to reduce chances of heat damage. Manufacturer’s directions are followed for sterilization of specific medias. Autoclave times other than specified by the manufacturer are in accordance with Standard Methods, 20th Ed., pg 9-8, Table 9020:III. Sterilized media is removed from the autoclave as soon as chamber temperature reaches zero. Media is never re-autoclaved. An Autoclave Logbook is kept detailing specifics of each batch, including actual sterilization time and total autoclave time. Effectiveness of sterilization is monitored using indicator tape, an autoclave thermometer and spore strip suspensions. Weekly spore strip testing for autoclave effectiveness is documented in the Autoclave Logbook. If growth of autoclave spores occurs, then sterilization procedures are deemed inadequate and an authorized service representative is called for corrective action. As a further check of sterility, media controls are run with each series of test samples and media controls are run for each new lot of media prepared. Results of controls are recorded on raw data worksheets accompanying test samples. If growth occurs on media controls, the source of contamination is investigated, preparation of the media and analytical procedures are reviewed and new samples are requested.


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c) Media Use - Melted agars are tempered in a waterbath until use but are not held longer

than 3 hours. A control bottle of media containing a thermometer is exposed to the same heating and cooling conditions as the media to be used in pouring plates. Sterile fermentation tubes are carefully handled and examined before use to ensure that gas bubbles are absent. Fermentation tubes stored refrigerated are incubated overnight and checked for the presence of gas prior to use. Tubes showing gas are rejected and a new lot of fermentation media is prepared.

d) Media Storage - Sterile media is prepared in amounts that will be used within holding

time limits. If fermentation tube media is refrigerated, the tubes are incubated overnight before use and checked for false positives. Media that can be stored for more than one week is prepared in tightly capped screw top tubes or bottles. Pre-poured media plates are sealed in plastic bags, inverted and refrigerated to retain moisture. Holding time for pre-poured media plates is two weeks, except for NA-mug which can be held for one week. Media containing dyes are protected from light and discarded if color changes are observed. Holding times for prepared media follow those specified in Standard Methods, 20th Ed., pg 9-9, Table 9020:IV.

e) Media Quality Control - Media preparation records are maintained in a bound logbook

with the name and date of preparer, name and lot number of the medium, amount weighed, volume and solvent used, pH measurements, balance used and any other adjustments. Sterility and positive and negative control culture checks are included for each lot of media used in sample analysis. Deviations from expected results are investigated by reviewing preparation and analytical procedure, and documenting corrective action.

4.3 Dilution Water Stock phosphate buffer solutions are stored refrigerated in tightly capped 100 mL screw top bottles for up to 1 year. Working phosphate buffer solutions are prepared from the stock, in reagent grade water, and are sterilized by autoclaving 15 minutes. Each batch of dilution/rinse water is checked for sterility by adding 50 mL of sterile, double-strength non-selective broth, which is then incubated at 35 +/- 0.5oC and checked for turbidity at 24 and 48 hours and the results recorded. Dilution water is stored at room temperature in stoppered bottles for up to 6 months. Any bottles observed to have turbidity are discarded. Dilution water controls are checked with each use and the lot is discarded if any growth is observed. Dilution water is prepared in accordance with laboratory SOP # 4.13.(1).


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Section 5 - Sampling Procedures 5.1 Sampling Containers Samples are collected in clean, distilled water rinsed, sterilized bottles of borosilicate glass or non-toxic polypropylene plastic, with screw cap closures. Sodium thiosulfate (0.1 N) is added, prior to sterilization, to containers intended for the collection of water samples containing residual chlorine or other halogen requiring neutralization. Sample containers are sterilized by autoclaving for 30 minutes. The sterility of sample containers is verified, per each batch, by adding 25 mL sterile, non-selective nutritive broth to a container, rotating the media within the container and incubating for 24 and 48 hours. Results are recorded at each interval and maintained in the QA Logbook. If growth is observed, the sterilization process is reviewed, with corrective action documented in the QA Logbook. 5.2 Sample Collection Samples are collected leaving ample air space in the bottle (2.5 cm) to facilitate mixing by shaking prior to examination. Sample ports are flushed or disinfected in order to collect samples representative of the water being tested. Sample bottles are kept closed until ready to be filled. Contamination of the inner surface of the cap or neck of the bottle is avoided. Sample containers are filled and immediately capped. The volume of samples collected is sufficient to carry out all tests required. Sample containers are labeled with an identifying number or location, time and date of collection. Other pertinent sampling information is included on a field data collection form. In collecting samples from recreational areas, samples are taken at a uniform depth of approximately 1 m. Waders and shoulder length disposable gloves are available for sampling in contaminated areas. When sampling from other non-potable sources (rivers, streams, lakes or ponds) samples are taken by holding the bottle near its base in the hand and plunging it, neck downward, below the surface. The bottle is turned until the neck points slightly upward and directed towards the current, or pushing bottle forward in a direction away from the hand. A chain of custody form must accompany all samples including those shipped by mail or courier. Upon receipt of samples in the laboratory, the chain of custody is completed and signed. Samples are then logged in with a unique identification number, which follows the samples throughout the testing and reporting procedures. For this purpose, a Sample Receipt & Identification Logbook is maintained in the laboratory. The sample ID numbering system uses the month and year of sample receipt with the chronological number of samples received for the month attached.


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5.3 Sample Preservation and Storage Microbiological examination of water samples is initiated as soon as possible after collection. A iced cooler and a temperature control blank is used for transportation to the laboratory if analysis cannot be initiated within one hour. Holding times for specific water types are as follows: a) Drinking water for compliance purposes - Samples are iced in a cooler during transit to

the laboratory and refrigerated upon receipt. Samples are analyzed on the day of receipt. Maximum holding time will not exceed 24 hours from the time of collection to the time of analysis for coliform bacteria. For heterotrophic plate counts the maximum holding time is 8 hours.

b) Nonpotable water for compliance purposes - Source water, stream pollution, recreational

water and wastewater samples are kept iced in a cooler during a maximum transport time of 4 hours. Upon arrival at the laboratory, a temperature control blank is checked for adherence to the storage temperature requirement of 1 – 4oC. Samples are refrigerated upon receipt at the laboratory and processed within 2 hours.

Samples which fail to meet the preservation and storage requirements are rejected. The sampling entity is notified and new samples are requested. Section 6 - Laboratory QA/QC Testing 6.1 Microbiological Quality Control Procedures a) Plate Count Comparison - Counts on one or more positive samples is conducted

monthly and compared with those of other analysts testing the same samples. Replicate counts for the same analyst should agree within 5% and those between analysts should agree within 10%. Statistical calculation of data precision is performed in accordance with Standard Methods Section 9020B.5b. If replicate counts do not fall within prescribed limits, colony counting equipment is inspected, the plate counting procedure is reviewed and corrective action is documented.

b) Control Cultures/Positive and Negative Controls - Each new lot of medium is checked

by testing with known positive and negative control cultures for the organism(s) under test. Specific control cultures are referenced in Section 3.4 of this manual. If a new lot of medium does not perform appropriately, control cultures are replaced with fresh cultures


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and the preparation of the media employed is reviewed. Any corrective action taken is documented in the Control Culture Logbook.

c) Duplicate Analysis - Duplicate analyses are performed on 10% of samples and on at

least one sample per test run - defined as an uninterrupted series of analyses. Calculation of data precision is as described in part e) below. If duplicates do not fall within data precision criteria, the analytical procedure is reviewed and corrective action is documented in the Precision and Accuracy Logbook.

d) Sterility Checks - For each series of analyses, the sterility of media, pipets, rinse water

and glassware, as appropriate, is checked. If any contamination is indicated, the cause is determined and the analytical data is rejected.

e) Precision of Quantitative Microbial Methods - Duplicate analyses is performed on 15

positive samples undergoing quantitative microbial analyses by membrane filtration procedures. The logarithm of each duplicate result is calculated and used to determine the range (R) for each pair of transformed duplicates. The mean range of the transformed duplicates is used in the equation 3.27 (R) to establish precision criterion. If the defined precision criterion is exceeded then analytical results are discarded and the problem is identified and resolved prior to making further analyses. Precision criterion is updated for each new set of 15 duplicate results.

f) Method Verification - For membrane filtration procedures with new lots of commercial

media, positive samples may be verified by picking at least 10 representative colonies for transfer to selective mediums. Results are scored and counts are adjusted based on percentage of verification.

g) Laboratory Precision and Accuracy for Physical/Chemical Parameters - The

laboratory establishes acceptance limits for precision and accuracy and maintains quality control charts for the various physical/chemical parameters commonly analyzed in conjunction with microbial analyses (ie; pH, salinity, turbidity, chlorine, etc.) to assess the validity of the analytical process. For precision, duplicate analyses are employed as described in section c) above, for calculation of the closeness of agreement between repeated measurements. Standard deviations are applied to mean percent deviation in establishing upper and lower control limits for acceptable precision criterion. Laboratory standards are utilized to establish accuracy criterion, as the closeness of a measurement to the true value, in the assessment of possible systematic error. Standard deviations are applied to mean percent recoveries in defining upper and lower control limits. Measurements are acceptably accurate when both systematic and random errors are low.


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QC results outside the acceptability limits warrant investigative and corrective action prior to further analyses. Equipment and the analytical procedures used are examined and corrective actions are documented in the laboratory Precision and Accuracy Logbook.

h) Annual Proficiency Tests - The laboratory participates in annual Proficiency Tests with

a DEP approved Proficiency Test Provider, to demonstrate microbiological proficiency in the analysis of total coliform and fecal coliform in potable water samples and enterococcus in ambient water samples. The laboratory reports the specific analytical method and media used for each PT round to the Provider, and reports results of each sample analysis as coliform present or absent, and if coliform present, either present or absent for fecal coliform. The acceptability of results is determined by the PT Provider. Results must be sent directly from the PT Provider to the DEP Lab Certification Office (LCO). Acceptable performance for total and fecal coliform is defined as the correct analysis of at least 90% of the samples in each testing round with no false negatives. If a PT round is determined by the PT Provider to be not acceptable, the laboratory, within 30 days of receiving notification of the failure, will determine the cause of error by checking media preparation, reviewing the analytical procedures used and checking for possible contamination. Upon taking corrective action, the DEP LCO will be notified in writing describing the corrective actions taken. In this instance, the laboratory will participate in a new PT round and report results to the Provider and the DEP LCO. The follow up PT round is completed within the same calendar year, from 30-90 days after the initial failed PT notice.

Section 7 - Standard Operating Procedures 7.1 Written Standard Operating Procedures (SOPs) are maintained in a Laboratory SOP Manual, and are provided to each analyst. The SOPs describe in a detailed step-wise fashion all laboratory operations involving instrumentation, reagents, analytical methods, QA/QC, calculations and reporting requirements to assure uniform operations between analysts and analyses. The SOPs specific for recreational water testing are: SOP # 4.42 - Monitoring New Bedford Bathing Beaches: Collection, Analysis, Quality

Assurance, Regulations and Reporting. SOP # 4.48 - Bacteriological Examination of Environmental and Recreational Waters for 24

Hour Enterococci Using EPA Method 1600 and the Membrane Filtration


..


Technique. SOP # 4.50 - Data Reduction, Handling and Reporting Specific To Contractual Compliance

With The MDPH Beach Program Quality Assurance Project Plan. The current complete list of Laboratory SOPs are as follows: 1.1 How to Write an SOP 1.2 Data Recording, Handling and Storage 1.3 Ensuring Compliance with GLPs 1.4 Sample Intake Procedures for Well Waters 3.15 Orion Star Meter 3 Series pH Meter 3.16 Denver Instrument Company Model XL-1800 Balance 3.17 SOP for Incubators 3.18 SOP for Waterbaths 3.21 SOP for Thermometers Used in Testing 3.31 Barnstead Mega-Pure Water Still and Barnstead Nanopure II D3077 Series 3.32 Hach DR 2010 Spectrophotometer Test for Free and Total Chlorine 3.34 Use, Calibration and Maintenance of the YSI Model 57 Dissolved Oxygen Meter 3.39 Calibration of Eppendorf Pipettors 3.45 Use of the LaMotte 2020 Turbidometer 3.48 Use of the YSI Model 30 Handheld Salinity, Conductivity & Temperature Meter 4.9 Bacteriological Examination of Shellfish Samples 4.10 Bacteriological Examination of Seawater by the Medium A-1 Method for Fecal Coliform 4.11 Washing Glassware Used in Shellfish and Waters Testing 4.12 SOP for Heterotrophic Plate Counts 4.13 Preparation of Phosphate Buffered Dilution Water 4.26 Bacteriological Examination of Potable Water for Members of the Total Coliform Group

on mEndo Agar LES, SM 9222B,Using the Membrane Filtration Technique and Rapid Confirmation of E.coli using SM 9222G on NA-Mug medium.

4.27 Bacteriological Examination of Environmental Waters for Members of the Fecal Coliform Group Using the Membrane Filtration Technique

4.28 Bacteriological Examination of Environmental, Recreational, Drinking and Ground Waters for Confirmatory Testing of Total and Fecal Coliforms for E. coli

4.29 Bacteriological Examination of Seawater and Pollution Sources for E. coli Utilizing the mTec Direct Testing Method

4.41 LYFO-DISK Microorganisms for Bacteriological Control Cultures 4.42 Monitoring New Bedford Bathing Beaches, Collection, Analysis, Quality Assurance,

Regulations and Reporting 4.43 Bacteriological Examination of Environmental Waters for the 48 hour Fecal


..


Streptococci/Enterococci Using the Membrane Filtration Technique 4.44 Bacteriological Examination of Seawater Holding Tanks by the Double Strength Media

MPN Total Coliform Test 4.48 Bacteriological Examination of Environmental, Wastewater and Recreational Waters for

24 hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique 4.49 Total Suspended Solids EPA Method 160.2 4.50 Data Reduction, Handling and Reporting Specific to Contractual Compliance with the MDPH Beach program Quality Assurance Project Plan Section 8 - Documentation and Recordkeeping 8.1 The documentation and recordkeeping system provides needed information on sample collection and preservation, analytical methods, raw data, calculations through reported results, and a record of persons responsible for sampling and analyses. All records are kept in bound notebooks, with entries in ink, and corrections made by drawing a single line through any change with the correction entered next to it. Laboratory raw data worksheets are bound with copies of computer generated reports. Reports generated from laboratory raw data are checked for accuracy, and are signed and dated. All bound laboratory notebooks are archived by fiscal year and maintained on laboratory premises for a period of ten years. Electronic databases are kept secure in a locked office.

New Bedford Health Department Laboratory SOP # 4.48(13) Page 1 DATE EFFECTIVE:______February 12, 2008_____________ APPROVALS: __Leslie Aubut____________March 12, 2013__ Laboratory Director Date of Review/Update

Standard Operating Procedure for

Bacteriological Examination of Environmental and Recreational Waters for 24 Hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique

OBJECTIVE: This SOP describes the procedures used in the analysis of enterococci by membrane filtration, using EPA Method 1600. The procedure is intended for the analysis of fresh, marine and wastewater. The SOP also describes laboratory methods, reporting requirements and quality control criteria in accordance with 310 CMR 42.00 and the MDPH Beach Project QAPP. EQUIPMENT AND SUPPLIES:

1. Dissection Microscope with 10X magnification and cool, white fluorescent light for counting plates.

2. Hand Tally counting device.

New Bedford Health Department Laboratory SOP # 4.48(13) Page 2

3. Plastic pipets, bacteriological, sterile, calibrated To Deliver. 4. Graduated cylinders, 100 – 1000 mL, sterile. 5. Membrane Filtration Units, sterile, plastic. 6. Electric vacuum pump with 1L filter flask and safety trap. 7. Forceps with smooth tips. 8. Alcohol and Bunsen Burner for flame sterilization. 9. Working thermometer checked against a NIST Standards thermometer. 10. Field use thermometer. 11. Petri dishes, sterile, plastic, 9x50 mm, with tight lids. 12. Dilution bottles, sterile, glass, marked at 99 mL (1:100) and 90 mL (1:10). 13. Membrane Filters, sterile, grid marked, 47 mm with 0.45 um pore size. 14. Incubator maintained at 41 +/- 0.5oC. 15. Waterbath maintained at 45 +/- 0.5oC for tempering agar. 16. Sample Bottles, 250 mL, autoclavable polypropylene.

REAGENTS AND MEDIA: Stock phosphate buffer solution: 3.4 g phosphate dihydrogen phosphate 50 mL reagent grade water Adjust the pH of the solution to 7.2 with 1N NaOH and bring the volume to 100 mL with reagent grade water. Autoclave at 121oC for 15 minutes. Store refrigerated and discard if turbidity or evidence of contamination is observed. Stock magnesium chloride solution: 8.1 g MgCl2

.6H2O 100 mL reagent grade water Sterilize the solution by autoclaving at 121oC for 15 minutes. Store refrigerated. Discard if turbidity or contamination is observed. Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5 mL of the magnesium chloride stock per liter of reagent grade water. Dispense in appropriate amounts for dilutions or in containers for rinse water. Autoclave at 121oC for 15 minutes. Final pH should be 7.0+/-0.2. Store at room temperature and discard if turbidity or contamination is observed. Perform a sterility check with each new batch using 50 mL of double strength TSB and 50 mL of the dilution/rinse water incubated at 35+/-0.5oC for 24 and 48 hour checks for growth (G) or no growth (NG). Record the

New Bedford Health Department Laboratory SOP # 4.48(13) Page 3 results in the lab Quality Assurance Logbook. A sterility control is also run each time the dilution/rinse water is used in an analysis and results are recorded on the lab raw data worksheet. MEI Agar: Per 100 mL of media - Add 7.12 g of dehydrated basal medium (mE agar, Difco 0333) plus 0.075 g of indoxyl B-D glucoside (Sigma # I3750) to 100 mL of reagent grade water. Heat to boiling to dissolve ingredients. Autoclave at 121 C for 15 minutes and cool to 45 oC in a waterbath. After sterilization and cooling, add 0.520 mL of Nalidixic Acid solution* to the mEI medium. Add 0.002 g triphenyl tetrazolium chloride (TTC) separately to the 100 mL of prepared mEI medium and mix. Pour 4.0 ml of the mEI agar into 50 mm Petri dishes and allow to solidify. The final pH should be 7.1 +/- 0.2. *Nalidixic Acid solution - Add 0.48 g of nalidixic acid and 0.400 mL of 10N NaOH to 10 mL of sterile reagent grade water and mix. Use 0.520 mL per 100 mL of mEI medium. QUALITY CONTROL CHECK: Prior to the first use of the media, the laboratory shall test each batch with a pure culture of a known positive reaction and a known negative reaction. The results of the quality control check are recorded in the Media Preparation Logbook. STORAGE: Prepared plates are labeled with the lot# (date of preparation) and the name of the medium. Prepared mEI plates are stored inverted, in sealed bags @ 2 - 4 degrees C for up to two weeks. PROCEDURE:

1. Label Petri dishes and report forms with sample identification and volume used. Select sample amount to give preferably 20-60 colonies on the membrane surface.

2. Place a 0.45um sterile membrane filter on the filter base, with the grid side up, and attach the funnel to the base.

3. Shake the sample bottle vigorously 25 times to distribute the bacteria evenly and measure the desired volume into the filter funnel.

4. Filter the sample and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse water. Turn off the vacuum.

5. Transfer the filter to the mEI Agar Petri dish using sterile forceps. Roll the filter onto the agar surface to avoid the formation of bubbles between the membrane and the agar surface.

6. Cover the dish, invert and incubate at 41o +/- 0.5oC for 24 hours +/- 2 hours. 7. Count all colonies with a blue halo as enterococci regardless of colony color. Count

colonies using a fluorescent lamp and magnification. The positive control used for this method is Enterococcus faecalis and the negative control used is Staphylococcus


epidermidis. CALCULATION AND REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution: Enterococci/100 mL = 100 (number of enterococci colonies) (volume of sample filtered, in mL) Report the results as Enterococci per 100 mL. QUALITY CONTROL: Laboratory Duplicates: Analyze at least one sample in duplicate for each analytical batch of 10 samples. Calculate the logarithm of each duplicate result. If either duplicate value is <1, add 1 to both results before calculating the logarithms. Calculate the range of logarithms for each duplicate set. When 15 sets of duplicate results are obtained, calculate the precision criterion with equals 3.27 times the mean range of logarithms for the 15 duplicate sets. Thereafter, if the logarithm range of a duplicate set is greater than the precision criterion, the analytical results for this batch of samples must be reported to the sampling entity as “unacceptable precision”, and fresh samples requested for analysis. Prior to the analysis of new samples, the laboratory must derive a new precision criterion using the most recent sets of 15 duplicate results. Negative Controls (sterile blanks): At the beginning and end of the filtration series and after every 10th sample, filter sterile buffered dilution water and process as a sample to check the sterility of all components in the analytical system and to check for cross contamination. If enterococcus colonies are found in the negative control plates, the analytical results for the batch of samples must be reported to the sampling entity as “unacceptable background contamination”, and fresh samples requested for analysis. The laboratory must investigate and resolve the contamination problem prior to analysis of any new samples. Positive Controls: With each analytical batch of 10 or fewer samples, filter sterile, buffered dilution water spiked with Streptococcus faecalis and process as a sample to assess the performance of the media and check for inhibitors. If no enterococcus colonies grow on the positive control plates, the

New Bedford Health Department Laboratory SOP # 4.48(13) Page 5 analytical batch is reported to the sampling entity as “unacceptable positive control failure” and fresh samples are requested for analysis. The laboratory must investigate and resolve the failure prior to the analysis of new samples. External Proficiency (PT) Tests: To evaluate the ability of the laboratory to produce accurate and precise results within specified acceptance criteria, the laboratory will conduct enterococcus profiency testing annually on an unknown sample obtained from Environmental Resource Associates. The results of PTs are kept on file in the laboratory and sent to MADEP Lab Certification Office. WASTE MANAGEMENT: Bacterial plates, positive and negative controls and dilutions thereof, will be autoclaved prior to disposal. The bacterial plates are placed into biohazard bags prior to autoclaving at 121oC for 30 minutes. REFERENCES: Standard Methods for the Examination of Water and Wastewater, 20th Edition 1998. EPA Office of Water, Method 1600, Membrane Filter Test for Enterococci in Water , EPA 821-R-97-004. MDPH Beach project QAPP, Revision # 5, 5/15/2007.

NEW BEDFORD HEALTH DEPARTMENT LABORATORY SOP # 4.27 (12) Page 1 of 2

DATE EFFECTIVE: __December 6, 2012 APPROVALS: _Leslie Aubut 12/6/12

Authorized By Date


Bacteriological Examination of Environmental and Recreational Waters for Members of the Fecal Coliform Group Using the Membrane Filtration Technique

Objective: This SOP describes the procedures used to examine environmental and recreational waters for members of the fecal coliform bacteria group by membrane filtration using mFC medium and describes the quality control procedures for this methodology.

SAMPLE HOLDING TIME AND TEMPERATURE: Samples are held below 10oC during a maximum transport time of 6 hours. Upon receipt of samples, chain of custody and logging-in at the laboratory, samples are refrigerated and processed within 2 hours.

MEDIA PREPARATION: Prepare commercial, dehydrated mFC agar with 1% rosolic acid solution addition according to manufacturer’s instructions. Record preparation details and assigned lot number in the laboratory Media and Reagent Preparation Logbook. Pipet 5 mL of media into each sterile plastic, 60 x 15 mm Petri plate. Prepared mFC agar is stored in the refrigerator for up to two weeks, inverted, in sealed containers under refrigeration.

NEW BEDFORD HEALTH DEPARTMENT LABORATORY

SOP # 4.27 (12) Page 2 of 2

PROCEDURE: Select sample amount to give preferably 20-60 colonies on the membrane filter surface. Filter sample through a 0.45um, gridded, sterile membrane. For sample dilutions use sterile, phosphate-buffered dilution water. Rinse the sides of the filtration apparatus between successive filtrations with the dilution water. Transfer filter to the mFC agar Petri dish, avoiding air bubbles beneath the membrane. Prepared plates are sealed with laboratory parafilm, placed inverted inside a glass beaker of appropriate size and placed in a waterbath for incubation at 44.5oC for a 24+/-2 hour period. Count all various shades of blue colonies as fecal coliform. Do not count non-fecal coliform colonies which are grey to cream colored. Count colonies using a 10X magnification and calculate fecal coliform density based on dilution factors applied.

REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution, and report as fecal coliform per 100 mL of sample.

QUALITY CONTROL

Positive and Negative Controls: For each new lot of m FC broth used. Samples are analyzed concurrently with known positive and negative pure cultures. The positive control culture used is E. coli and the negative control culture is Enterobacter aerogenes. The pure cultures are purchased commercially and maintained in-house with records kept in the laboratory Control Cultures Logbook.

Sterility Checks: Check sterility of media, filters and rinse water at the start and at the end of each series of samples. Use sterile reagent water as the sample to check equipment sterility as applicable (filters, flasks, pipets, etc.). Record results of sterility checks on the laboratory raw data sheets.

Duplicate Analyses: Perform duplicate analyses on 10% of samples and on at least one sample per test run. Record results in the laboratory Precision and Accuracy Logbook. Calculate precision of duplicate analyses for the quantitative test method according to Standard Methods for the Examination of Water and Wastewater, 21th Ed., Section 9020B, pg 9-10.

REFERENCES: Standard Methods for the Examination of Water and Wastewater, Section 9222D, 21th Edition 2005.

New Bedford Health Department Laboratory SOP # 4.48(13) Page 1 DATE EFFECTIVE:______February 12, 2008_____________ APPROVALS: __Leslie Aubut____________March 12, 2013__ Laboratory Director Date of Review/Update


Bacteriological Examination of Environmental and Recreational Waters for 24 Hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique

OBJECTIVE: This SOP describes the procedures used in the analysis of enterococci by membrane filtration, using EPA Method 1600. The procedure is intended for the analysis of fresh, marine and wastewater. The SOP also describes laboratory methods, reporting requirements and quality control criteria in accordance with 310 CMR 42.00 and the MDPH Beach Project QAPP. EQUIPMENT AND SUPPLIES:

1. Dissection Microscope with 10X magnification and cool, white fluorescent light for counting plates.

2. Hand Tally counting device.


3. Plastic pipets, bacteriological, sterile, calibrated To Deliver. 4. Graduated cylinders, 100 – 1000 mL, sterile. 5. Membrane Filtration Units, sterile, plastic. 6. Electric vacuum pump with 1L filter flask and safety trap. 7. Forceps with smooth tips. 8. Alcohol and Bunsen Burner for flame sterilization. 9. Working thermometer checked against a NIST Standards thermometer. 10. Field use thermometer. 11. Petri dishes, sterile, plastic, 9x50 mm, with tight lids. 12. Dilution bottles, sterile, glass, marked at 99 mL (1:100) and 90 mL (1:10). 13. Membrane Filters, sterile, grid marked, 47 mm with 0.45 um pore size. 14. Incubator maintained at 41 +/- 0.5oC. 15. Waterbath maintained at 45 +/- 0.5oC for tempering agar. 16. Sample Bottles, 250 mL, autoclavable polypropylene.

REAGENTS AND MEDIA: Stock phosphate buffer solution: 3.4 g phosphate dihydrogen phosphate 50 mL reagent grade water Adjust the pH of the solution to 7.2 with 1N NaOH and bring the volume to 100 mL with reagent grade water. Autoclave at 121oC for 15 minutes. Store refrigerated and discard if turbidity or evidence of contamination is observed. Stock magnesium chloride solution: 8.1 g MgCl2

.6H2O 100 mL reagent grade water Sterilize the solution by autoclaving at 121oC for 15 minutes. Store refrigerated. Discard if turbidity or contamination is observed. Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5 mL of the magnesium chloride stock per liter of reagent grade water. Dispense in appropriate amounts for dilutions or in containers for rinse water. Autoclave at 121oC for 15 minutes. Final pH should be 7.0+/-0.2. Store at room temperature and discard if turbidity or contamination is observed. Perform a sterility check with each new batch using 50 mL of double strength TSB and 50 mL of the dilution/rinse water incubated at 35+/-0.5oC for 24 and 48 hour checks for growth (G) or no growth (NG). Record the

New Bedford Health Department Laboratory SOP # 4.48(13) Page 3 results in the lab Quality Assurance Logbook. A sterility control is also run each time the dilution/rinse water is used in an analysis and results are recorded on the lab raw data worksheet. MEI Agar: Per 100 mL of media - Add 7.12 g of dehydrated basal medium (mE agar, Difco 0333) plus 0.075 g of indoxyl B-D glucoside (Sigma # I3750) to 100 mL of reagent grade water. Heat to boiling to dissolve ingredients. Autoclave at 121 C for 15 minutes and cool to 45 oC in a waterbath. After sterilization and cooling, add 0.520 mL of Nalidixic Acid solution* to the mEI medium. Add 0.002 g triphenyl tetrazolium chloride (TTC) separately to the 100 mL of prepared mEI medium and mix. Pour 4.0 ml of the mEI agar into 50 mm Petri dishes and allow to solidify. The final pH should be 7.1 +/- 0.2. *Nalidixic Acid solution - Add 0.48 g of nalidixic acid and 0.400 mL of 10N NaOH to 10 mL of sterile reagent grade water and mix. Use 0.520 mL per 100 mL of mEI medium. QUALITY CONTROL CHECK: Prior to the first use of the media, the laboratory shall test each batch with a pure culture of a known positive reaction and a known negative reaction. The results of the quality control check are recorded in the Media Preparation Logbook. STORAGE: Prepared plates are labeled with the lot# (date of preparation) and the name of the medium. Prepared mEI plates are stored inverted, in sealed bags @ 2 - 4 degrees C for up to two weeks. PROCEDURE:

1. Label Petri dishes and report forms with sample identification and volume used. Select sample amount to give preferably 20-60 colonies on the membrane surface.

2. Place a 0.45um sterile membrane filter on the filter base, with the grid side up, and attach the funnel to the base.

3. Shake the sample bottle vigorously 25 times to distribute the bacteria evenly and measure the desired volume into the filter funnel.

4. Filter the sample and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse water. Turn off the vacuum.

5. Transfer the filter to the mEI Agar Petri dish using sterile forceps. Roll the filter onto the agar surface to avoid the formation of bubbles between the membrane and the agar surface.

6. Cover the dish, invert and incubate at 41o +/- 0.5oC for 24 hours +/- 2 hours. 7. Count all colonies with a blue halo as enterococci regardless of colony color. Count

colonies using a fluorescent lamp and magnification. The positive control used for this method is Enterococcus faecalis and the negative control used is Staphylococcus


epidermidis. CALCULATION AND REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution: Enterococci/100 mL = 100 (number of enterococci colonies) (volume of sample filtered, in mL) Report the results as Enterococci per 100 mL. QUALITY CONTROL: Laboratory Duplicates: Analyze at least one sample in duplicate for each analytical batch of 10 samples. Calculate the logarithm of each duplicate result. If either duplicate value is <1, add 1 to both results before calculating the logarithms. Calculate the range of logarithms for each duplicate set. When 15 sets of duplicate results are obtained, calculate the precision criterion with equals 3.27 times the mean range of logarithms for the 15 duplicate sets. Thereafter, if the logarithm range of a duplicate set is greater than the precision criterion, the analytical results for this batch of samples must be reported to the sampling entity as “unacceptable precision”, and fresh samples requested for analysis. Prior to the analysis of new samples, the laboratory must derive a new precision criterion using the most recent sets of 15 duplicate results. Negative Controls (sterile blanks): At the beginning and end of the filtration series and after every 10th sample, filter sterile buffered dilution water and process as a sample to check the sterility of all components in the analytical system and to check for cross contamination. If enterococcus colonies are found in the negative control plates, the analytical results for the batch of samples must be reported to the sampling entity as “unacceptable background contamination”, and fresh samples requested for analysis. The laboratory must investigate and resolve the contamination problem prior to analysis of any new samples. Positive Controls: With each analytical batch of 10 or fewer samples, filter sterile, buffered dilution water spiked with Streptococcus faecalis and process as a sample to assess the performance of the media and check for inhibitors. If no enterococcus colonies grow on the positive control plates, the

New Bedford Health Department Laboratory SOP # 4.48(13) Page 5 analytical batch is reported to the sampling entity as “unacceptable positive control failure” and fresh samples are requested for analysis. The laboratory must investigate and resolve the failure prior to the analysis of new samples. External Proficiency (PT) Tests: To evaluate the ability of the laboratory to produce accurate and precise results within specified acceptance criteria, the laboratory will conduct enterococcus profiency testing annually on an unknown sample obtained from Environmental Resource Associates. The results of PTs are kept on file in the laboratory and sent to MADEP Lab Certification Office. WASTE MANAGEMENT: Bacterial plates, positive and negative controls and dilutions thereof, will be autoclaved prior to disposal. The bacterial plates are placed into biohazard bags prior to autoclaving at 121oC for 30 minutes. REFERENCES: Standard Methods for the Examination of Water and Wastewater, 20th Edition 1998. EPA Office of Water, Method 1600, Membrane Filter Test for Enterococci in Water , EPA 821-R-97-004. MDPH Beach project QAPP, Revision # 5, 5/15/2007.

Appendix B - Water Quality Grab Sample Collection Standard Operating Procedure

Water Quality Grab Sample Collection Standard Operating Procedure

1.0 Scope and Application This standard operating procedure establishes the protocol to be followed for collecting water quality grab

samples for both the wet and dry weather monitoring events. Samples will be collected for bacteria (Enterococci

and Fecal Coliform) and possibly other optional parameters (Nitrates as Nitrogen, Orthophosphate, Total Kjeldahl

Nitrogen, and Oil and Grease).

2.0 Method Summary

Sample Location

When feasible, surface water samples will be collected directly from the outfall of the discharge pipe into the

laboratory analysis bottles. If the discharge pipe is inaccessible or if tidal water is back flowing into the

discharge pipe, the Field Teams will move the sampling site to the next available upstream manhole (or

catchbasin if no manholes exist in the drainage system). The sample in the manhole will be taken at the inlet

of the main stormdrain pipe. If the sample cannot be taken under free-flowing conditions, the Field Team will

take the sample as close to the inlet pipe as possible and make a notation on the comment section of field data

sheet (preferably with a photograph) as to the problems encountered at the site (tidal back flowing, inlet/outlet

elevations prevent free-flowing conditions, etc)

Sample Collection

To minimize potential sediment contamination, sample collection will begin with the bacterial samples. Once

the bacteria samples are collected, they will immediately be placed in a cooler on blue ice. Separate bottles

will then be used to collect water samples for all field tests (surfactants, ammonia, nitrates, and total chlorine)

and then any other laboratory testing (followed by appropriate storage protocol). If the sample location is not

accessible, samples will be collected using either a laboratory analysis bottle or a clean (rinsed in the field

with de-ionized water or purified water from the laboratories) sample collection container attached to a

sampling rod. If available, the Field Teams may also use a hand-held vacuum pump to collect samples. Once

collected, the container and/or tubing from the vacuum pump will be used to distribute the sample directly to

the appropriate lab bottles. The collection container and or tubing will be decontaminated (rinsing with de-

ionized water or purified laboratory water) prior to use at the next grab sample site to prevent cross

contamination.

3.0 Safety, Restrictions, and Limitations When accessing all monitoring sites, Field Teams must be aware of accessibility and safety issues, especially

during adverse weather conditions. At outfall pipes, Field Teams should not attempt collecting samples if the

gradient is too steep or slippery, water velocity too high, or water depth is over three feet. Field Teams must

follow all safety precautions as outlined by the local Department of Public Works when lifting

manholes/catchbasins grates or when entering/exiting manholes or catchbasins.

While collecting grab samples, Field Teams should wear clean latex gloves to minimize their contact with

contaminated water and also to prevent sample contamination. The Field Teams should never touch the inside of

any bottle or cap, especially the sterilized containers for the bacteria samples. If the sample is collected using a

sampling pole or vacuum pump, the sampling pole bottle or tubing must be decontaminated in the field prior to

use.

The laboratories should supply the proper size sample bottles to meet the required volume for testing. If the

containers are not adequate, multiple samples will be needed to supply the necessary volume. If possible, each

laboratory analysis bottle should be filled from a single grab sample. In addition, the QA/QC duplicate samples

should be filled from the same grab as the original sample bottles.

4.0 Sample Collection, Handling, and Preservation Samples to be analyzed for bacteria will be collected first using a sterile sampling container that has been

provided by the laboratory. Following the bacterial collection, a second sample will be taken for the analysis of

the on-site "in the field" (temperature/conductivity/salinity and ammonia) parameters. Once the "in the field"

analysis has been completed, this second bottle will be placed on ice and used for the "in house" analysis

(surfactants and nitrates). Additional samples will then be collected for any remaining laboratory parameters.

Once collected, all samples will be handled and preserved in accordance with Tables 3 through 7 in the QAPP.

The preferred method of sample collection is to fill the sample containers directly from the discharge under free-

flowing conditions. If a free-flowing sample is not feasible, the Field Team will collect the sample as close to the

discharge as possible. If the discharge is inaccessible, a sampling pole container or vacuum pump will be used to

collect the sample. Once the sample is retrieved, it will be completely mixed and then poured into the analysis

containers. If the water quality sample cannot be collected under free-flowing conditions and/or must be collected

with a sampling pole, the Field Team will document the testing conditions/methods on the field data sheets and

the chain of custody forms so that the laboratory results can be flagged for review.

5.0 Equipment and Materials .

6.0

Procedures Analysis laboratory should be contacted prior to sampling date.

Laboratory analysis bottles and equipment should be organized prior to each grab at the site.

New, clean, latex gloves should be worn at all times when handling the sample collection bottles and

obtaining samples in the field.

The caps from the lab analysis bottles should be removed just prior to collecting or receiving a sample.

They should be re-capped just after a sample is collected or received. The amount of time an opened

bottle is exposed to the environment should be minimized.

The bacteria samples should be collected first, followed by the field measurements and then any other

laboratory samples.

If the discharge pipe is free-flowing, obtain the laboratory sample directly from the pipe while minimizing

sediment disturbance. If the discharge pipe is not free-flowing, take the sample as close to the pipe as

possible. Note on the monitoring data sheet the conditions under which the sample was taken (not free-

flowing, tidal backflow, manhole, etc.) There should be a minimum of two people collecting samples at

all times, one to take all notes, fill out labels and forms, etc. while the other collects the samples.

coolers with blue ice hip-waders

pens and permanent markers manhole hook

field log books latex gloves

data collection sheet paper towels

chain of custody forms sealable bags

sampling pole or hand-held vacuum pump measuring tape

pre-labeled laboratory bottles camera

sampling pole with clean collection containers safety glasses

de-ionized water or laboratory purified water

Appendix C - Field Water Quality Measurements Standard Operating Procedure

Field Water Quality Measurements Standard Operating Procedure


This standard operating procedure addresses the procedures to collect field water quality measurements using YSI

Model 30 meters (temperature, conductivity, salinity), Hach test strips (ammonia), Chemetrics K-9400

(surfactants), LaMotte Nitrate-Nitrogen test kit (nitrate) and Hach Pocket Colorimeter (chlorine).

2.0 Equipment Inspection, Maintenance, and Calibration

Prior to each sampling event, all test kits and equipment will be inspected to ensure the availability of testing

materials (Hach strips, test kits, and Chemetrics) and operability of equipment (YSI meter and Colorimeter).

Calibration of the YSI meter and Colorimeter will be in accordance with manufacturer's instructions. If any parts

of the testing equipment need to be repaired or replaced, this will be noted in the standard Equipment Inspection,

Testing and Maintenance Log Sheet.

Calibration checks on the YSI meters and colorimeter will be performed by the Field Team prior to each sampling

event with the equipment being re-adjusted as needed in accordance to manufacturer's instructions. Calibration

checks will be recorded on the standard Equipment Calibration Form and in the data log book.

3.0 Field Measurement Procedure

Field measurements will be collected at each sampling location after the filling of analysis bottles. Whenever

possible, the field measurements sample will be taken at the center of the discharge flow, at half of the depth and

upstream of the sample collector. For the YSI meter, care will be taken not to allow the probe to contact any

accumulated sediment.

As with the bacterial samples, if free-flowing conditions do not exist, the sample will be taken as close to the

discharge as possible or moved upstream at the discretion of the Stormwater Specialist (or designee). If the

discharge pipe is inaccessible, water quality samples will be collected using a sampling pole or hand held vacuum

pump. The sample collection point, collection conditions, and accessibility will be noted on the field data sheet.

All field data collections will be in accordance with manufacturer's instructions.

The Stormwater Specialist or Field Team Leader will be responsible for equipment maintenance and cleaning

following each sampling event. Any issues regarding the equipment or other testing materials will be reported

immediately to the Stormwater Specialist.

4.0 Quality Assurance

For quality assurance purposes, each Field Team will duplicate at least one sample or ten percent of all samples

collected (whichever is more) as part of each sampling event. A duplicate is a second reading of the same water

sample.

5.0 Field Water Quality Measurements Standard Operating Procedure

Prior to each sample round all of the YSI meters will be calibrated by the Field Team Leader in accordance with

the manufacturer's instructions.. The time and date of the calibration will be recorded on the standard Equipment

Quality Assurance Form.

References

YSI Pro 30 Users Manual (2011) https://www.ysi.com/File%20Library/Documents/Manuals/606082A-YSI-

Pro30-Manual-English.pdf

LaMotte Nitrate Nitrogen Test Kit (2013) http://www.lamotte.com/en/industrial/individual-test-kits/3615-01.html

Pocket Colorimeter II User Manual, Edition 1 (2014)

http://www.hach.com/pocket-colorimeter-ii-chlorine-free-and-total/product-downloads?id=7640442953

Chemtrics Instructions http://www.chemetrics.com/Detergents+(anionic+surfactants,+MBAS)/Visual+Kits/K-

9400/R-9400

Hach test strips http://www.hach.com/teststrips

https://www.ysi.com/File%20Library/Documents/Manuals/606082A-YSI-Pro30-Manual-English.pdf

https://www.ysi.com/File%20Library/Documents/Manuals/606082A-YSI-Pro30-Manual-English.pdf

http://www.lamotte.com/en/industrial/individual-test-kits/3615-01.html

http://www.hach.com/pocket-colorimeter-ii-chlorine-free-and-total/product-downloads?id=7640442953

http://www.chemetrics.com/Detergents+(anionic+surfactants,+MBAS)/Visual+Kits/K-9400/R-9400

http://www.chemetrics.com/Detergents+(anionic+surfactants,+MBAS)/Visual+Kits/K-9400/R-9400

http://www.hach.com/teststrips

Appendix D - Wet and Dry Weather Field Data Sampling Sheet


Water Quality Sampling Sheet

Low tide time Low tide height Wind Dir Wave action

Manhole ID:

Station ID Time Bottle ID Pipe

Flow1 F

C

En

t

N Op

TK

N

O/G

Temp

(0C)

Cond

(mS/cm)

Sal

(PPT)

NH4

(PPM)

Sensory2 Cl

(Mg/L)

NO3

(PPM)

Surf

(PPM)

1 Stormwater in pipe : <1/4, 1/4, 1/2, 3/4, F-Full

2 Visual C-Color, O-Odor, W-Waste products

2 Sensorary C-Color, O-Odor, W-Waste products, G-Garbage

In the Field Tests In House Tests

Sample Type: WW or DW

Lab Tests (check):

Date: Logger: Collectors:

Todays Weather Prev 72 hr ppt:

Pipe Conditions (submerged) Check one

F- Free Flowing, P - Partially Submerged

T -Totally Submerged

Stat F P T

1

2

3

4

Outlet

Field Team Comments

Location Sketch (use back if needed)

Location Schematic (use back if needed)

Custody1: Custody2: Custody3:


Water Quality Sampling Sheet