Appendix 6 Sediment Toxicity

Appendix 6

Sediment Toxicity

Appendices

Chapter 6 Sediment Toxicity

2018-2019 Biennial Report

Appendix 6.1 Standard Operating Procedure (SOP) for Assessing the Toxicity of Sediment-Associated Contaminants with Eohaustorius estuarius

Appendix 6.1 Standard Operating Procedure (SOP) for Assessing the Toxicity of Sediment-Associated Contaminants with Eohaustorius estuarius

This SOP has been reviewed by the Supervisors and Quality Assurance Group. Printed and/or digital copies of this document are considered uncontrolled unless a copy number for controlled document is issued by the QA group. No update will be provided to externally distributed uncontrolled copy.

App 6.1 - 1

San Jose Creek WQL Sediment Testing- 1 Version 15.1.1 Approved 11/02/15

B26. METHODS FOR ASSESSING THE TOXICITY OF SEDIMENT-ASSOCIATED CONTAMINANTS WITH Eohaustorius estuarius

Introduction

Many contaminants in aquatic systems adhere to particles and may subsequently accumulate in sediments. Sediment contamination is a widespread environmental problem that can potentially pose a threat to a variety of aquatic ecosystems. Sediment functions as a reservoir for common chemicals such as pesticides, herbicides, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and metals such as lead, mercury, and arsenic. Contaminated sediment can result in poorly developed benthic communities, while disposal of contaminated dredged material can potentially exert adverse effects on both pelagic and benthic systems.

In 2008, the first phase of the California Sediment Quality Objectives (SQO) program was implemented in many California monitoring programs. Soon after in 2009, the United States Environmental Protection Agency (USEPA) approved the State Water Control Board Water Quality Control Plan for enclosed Bays and Estuaries. Historically, assessment of sediment quality has been limited to chemical characterizations. The implementation of this plan however, will yield sediment quality data consisting of the measurement and integration of three lines of evidence: sediment toxicity, benthic community conditions, and sediment chemistry.

Eohaustorius estuarius is a free-burrowing estuarine amphipod within the family Haustoriidae. They are found on beaches exclusively on the Pacific coast from British Columbia to the central coast of California. E. estuarius is a favorable sediment toxicity testing organism for reasons including their ability to tolerate drastic salinity changes, they can test in a variety of grain size, they are easily visible, and are thought to be deposit-feeders which makes maintenance of a mass culture fairly simple. Previous studies have successfully used E. estuarius to evaluate the toxicity of estuarine or marine sediments. Endpoints typically monitored in a sediment toxicity test with E. estuarius include survival and reburial (optional).

Uncontrolled Document

App 6.1 - 2

San Jose Creek WQL

Sediment Testing- 2 Version 15.1.1 Approved 11/02/15

B26. METHODS FOR ASSESSING THE TOXICITY OF SEDIMENT-ASSOCIATED CONTAMINANTS WITH Eohaustorius estuarius

1. Scope and Application

1.1. This method evaluates the toxicity of contaminants associated with whole sediments using E. estuarius. Procedures described in this method are for conducting a 10-day, non-renewal sediment toxicity test measuring the survival of E. estuarius and also for conducting the concurrently run 96-hour non-renewal reference toxicant test. Procedures are primarily described for testing estuarine or marine sediments. The effects include the synergistic, antagonistic, and additive effects of the chemical, physical and biological components of which adversely affect the physiological and biochemical functions of the test organisms.

2. Summary of Procedure

2.1. For the sediment test, sediment should be thoroughly homogenized and added to each test chamber. Overlying water is added to the chambers in a manner that minimizes suspension of sediment. A test begins when the organism E. estuarius are added to all test chambers. Survival is measured after the test terminates on day 10.

2.2. For the reference toxicant test, no sediment is added to the test chambers and tests are not exposed to light. To prevent light exposure, the test should be covered with a box at all time, except during daily counts and water quality readings. Reference toxicant concentrations are added to each test chamber. Survival is measured after 96 hours when the test terminates.

3. Interferences

3.1. Testing-specific interferences

3.1.1. Toxic substances may be introduced by contaminants in dilution water, glassware, sample hardware, and testing equipment.

3.1.2. Adverse effects of low dissolved oxygen (DO) concentrations, high concentrations of suspended and/or dissolved solids, and extremes of pH, alkalinity, or hardness, may mask the presence of toxic substances.

3.1.3. Improper sample handling may adversely affect test results. 3.1.4. Pathogenic and/or predatory organisms in the dilution water may affect test

organism survival and confound test results. 3.1.5. Changes in chemical bioavailability as a function of sediment manipulation or

storage.

3.1.5.1. Mixing, homogenization, and sieving may temporarily disrupt the equilibrium of organic compounds in sediment.

3.1.5.2. Conducting sediment tests at temperatures different from the field might affect contaminant solubility, partitioning coefficients, or other physical and chemical characteristics.


App 6.1 - 3

San Jose Creek WQL


3.1.5.3. The addition of food, water or solvents to the test chamber might obscure the bioavailability of contaminants in sediment or might provide a substrate for bacterial or fungal growth.

3.2. Sediment-specific interferences

3.2.1. Non-contaminant Factors 3.2.1.1. Ultraviolet light may breakdown chemical bonds altering potential

toxicants and/or altering the chemical make-up of the sediment. However, lighting typically used to conduct laboratory tests does not include the appropriate spectrum of ultraviolet radiation to photo-activated compounds and thus laboratory tests may not account for toxicity expressed by this mode of action.

3.2.1.2. Natural geomorphological and physico-chemical characteristics (e.g. sediment texture and/or grain size) may influence the response of test organisms if the characteristics are not within the tolerance limits of the test organism.

3.2.2. The presence of indigenous organisms

3.2.2.1. Native organisms may be present in field-collected sediments and may make interpretations of treatment effects difficult.

4. Apparatus and Equipment

4.1. Environmental chamber or equivalent facility with temperature controlled at 15±1º C and constant light at 50-100 Fc

4.2. Water purification system - Millipore Milli-Q, deionized water or equivalent 4.3. Light meter – with a range of 500 – 1000 lux (50-100 FC) 4.4. Fixed angle rotor centrifuge – for extracting pore water 4.5. Aeration device- to continuously aerate sediment tests (not the reference toxicant test) 4.6. Test chambers – 1000 mL wide-mouth glass jars with lids, borosilicate glass or

equivalent 4.7. Volumetric flasks, Class A, borosilicate glass, 2000 mL – for making reference

toxicant test solutions 4.8. 4 L beakers- for holding test solutions 4.9. Graduated Cylinder, Class A, borosilicate glass, 2000 mL – for making reference

toxicant test solutions 4.10. Volumetric pipettes, Class A, 1-100 mL 4.11. Pipette bulbs and fillers 4.12. Carboys, 20 L or 50 L – for holding dilution water 4.13. Squirt bottles - for aiding in making up sample concentrations 4.14. Thermometers, glass or electronic, laboratory grade, NIST traceable - for measuring

water temperatures 4.15. Temperature Chart Recorder - for continuously recording temperature


App 6.1 - 4

San Jose Creek WQL


4.16. Meters, pH, DO - for routine physical and chemical measurements 4.17. Ruler- for determining organisms 3-5mm and for marking 2 cm on sediment test

chambers 4.18. Refractometer or conductivity meter - for determining salinity 4.19. Equipment for measuring overlying water and/or pore water ammonia -Hach® water

quality test strips for ammonia (optional) 4.20. Dissection trays – for aiding in test termination 4.21. Sieve, 1 mm or equivalent – for press sieving sediment prior to test 4.22. Sieve, 500 µm or equivalent – aiding in test termination 4.23. 10 L fish tanks – for holding mass culture 4.24. Polycarbonate aeration racks – used to aerate and cover sediment test chambers

5. Reagents and Consumable Materials

5.1. Data sheets (one set per test) - for recording data 5.2. Disposable polyethylene pipettes - for counting, moving, and/or removing dead E.

estuarius 5.3. Markers, waterproof - for marking containers, etc. 5.4. Disposable 1 mL pipette – for using as a stir-rod 5.5. Glass capillary tubes – for aeration rack tubing 5.6. 50 mL disposable cups 5.7. Reference toxicant solutions – 10,000 ppm ammonia stock 5.8. Reagent water - deionized water 5.9. Sediment sample ~ 1L for a 5-replicate test 5.10. Dilution water- Scripps seawater (adjusted to appropriate salinity) 5.11. Nitex screen (30 μm mesh or similar) – for filtering dilution water 5.12. Large box- to cover reference toxicant test 5.13. Tetramarin Saltwater Flakes 5.14. Test organisms, Eohaustorius estuarius

5.14.1. Organisms should be 3-5mm in length. 5.14.2. E. estuarius and control sediment currently obtained through:

Northwestern Aquatic Sciences 3814 Yaquina Bay Road Newport, OR 97365 Tel. No. 1-541-265-7225

6. Procedure

6.1. Bioassay Preparation

6.1.1. Test Chambers

6.1.1.1. Reference Toxicant Tests


App 6.1 - 5

San Jose Creek WQL


6.1.1.1.1. 12 clean glass 1000 mL wide-mouth jars are used as test chambers (five concentrations plus a control, each consisting of two replicates).

6.1.1.1.2. 12 lids are needed to cover the mouth of each beaker to prevent evaporative cooling of the sample, as well as contamination (e.g. debris and splashing) from the outer environment of the test chamber.

6.1.1.1.3. Label test chambers and the associated lids 1 through 12 with an identifier indicating origin of contents (e.g., RT1 for Reference Toxicant 1).

6.1.1.2. Sediment Tests

6.1.1.2.1. 10 clean glass 1000 mL wide-mouth jars are used as test chambers for a single sediment sample test (sediment control and sediment from sampling site, each consisting of five replicates). In addition, 2 clean glass 1000 mL wide-mouth jars are used as surrogate test chambers for each site to record overlying water quality data throughout the test and pore water quality data at T0 and Tf.

6.1.2. Sediment collection and homogenization

6.1.2.1. Sediment should be collected using a van-veen sampler. Sediment sampling is conducted directly out of the van veen.

6.1.2.1.1. Using a polypropylene scoop, remove only the top 5 cm of sediment from the van-veen sampler and composite into sample container(s) until they are filled.

6.1.2.1.2. Store sample(s) on ice or in a cooler. 6.1.2.1.3. Homogenization can occur in the field or laboratory.

6.1.2.1.3.1. Push the entire sample (all sample containers) through a 1mm

stainless steel sieve into a 4L plastic beaker or stainless steel bucket. Use only the water within the sample.

6.1.2.1.3.2. Large indigenous organisms and large debris can be removed using forceps.

6.1.2.1.3.3. Once all sediment has been sieved, mix the entire sample until it is homogenized.

6.1.2.1.3.4. Distribute the homogenized sediment back into its container(s). 6.1.2.1.3.5. Label containers with the jar # and date it was sieved.

6.1.3. Sample preparation

6.1.3.1. Sediment Test preparation should be conducted the day before the test is

initiated (Day -1).


App 6.1 - 6

San Jose Creek WQL


6.1.3.2. Sediment tests should be initiated as soon as possible following sediment sample collection to limit the storage time of the sample.

6.1.3.2.1. The maximum holding time for core samples is 4 weeks; however,

sediment samples should be used before this time. 6.1.3.2.2. Hold the sediments after collection in the dark at 4ºC.

6.1.3.3. Record the sample date used on the water quality bench sheets. 6.1.3.4. All replicates should be randomized. 6.1.3.5. Using a stainless steel tool, add approximately 2 cm (around 175 ml) of

sediment sample or control sand into each beaker. 6.1.3.6. The sediment added to the test chamber should be settled either by tapping

the side of the test chamber against the side of the hand or by smoothing the sediment surface using a spatula.

6.1.3.7. On day -1, gently add overlying water to allow the particulate matter to settle before test initiation.

6.1.3.7.1. Add 800 mL of dilution water to the test chambers in a manner that minimizes disturbance of the sediment. A buffering apparatus or squirt bottle should be used to avoid disturbing the sediment.

6.1.3.8. An aeration apparatus providing gently aeration should be placed in the

sediment test chambers on day -1 and allowed to aerate overnight before the addition of the organisms. Use a board aeration rack attached to a splitter for optimal control. Deliver gentle aeration toe each chamber (~1 bubble/second).

6.1.4. Collection and Preparation of Scripps Dilution Seawater

6.1.4.1. Dilution water is prepared by filtering full strength Scripps seawater with

a 30 µm filter and diluting the seawater to the appropriate salinity with DI water.

6.1.4.1.1. Reference toxicant seawater salinity should be at 20 ppt. 6.1.4.1.2. Sediment test seawater

6.1.4.1.2.1. If salinity in the field is 20-24 ppt, then testing range is 22 ± 2 ppt

6.1.4.1.2.2. If salinity in the field is 25-29 ppt, then testing range is 27 ± 2 ppt

6.1.4.1.2.3. If salinity in the field is ≥ 30 ppt, then testing range should be 32 ± 2ppt

6.1.4.2. 60-µm filtered Scripps seawater is collected from Scripps Pier in La Jolla.

Refer to the “Standard Operating Procedure (SOP) for Sampling


App 6.1 - 7

San Jose Creek WQL


Collection Methods for Acute and Chronic Bioassay Testing” for details on the collection and handling procedures.

6.1.4.3. Fill out a chain of custody (COC) form when collecting dilution seawater in order to document the collection date and temperature.

6.1.4.4. Seawater should be collected 96 hours before first use and used within 14 days of collection.

6.1.4.5. The day prior to use, place an appropriate volume of the seawater in the testing area in order to acclimate the seawater to the correct temperature.

6.1.4.6. Prior to use, read the salinity using a calibrated refractometer and adjust the salinity to the desired level by dilution with D.I. water using the following formula:

V1 C1 = V2 C2 V1 = [(V2 C2)/( C1)] - V2

Where: V1 = DI water volume to add to carboy

V2 = Seawater volume desired C1 = Actual seawater salinity (approx. 33-35ppt)

C2 = Seawater salinity desired

6.1.4.7. Mix the seawater and retest the salinity. Adjust as necessary until the target salinity is obtained.

6.1.5. Electronic Test Setup

6.1.5.1. Initiate test(s) and generate bench sheets for the appropriate tests

according to the Biology Laboratory Standard Operating Procedure (SOP) for “Bioassay Test Initiation and Data Entry Using CETIS and LIMS”.

6.1.5.2. Place the bench sheets for each test on a clipboard and place in the testing area.

6.1.6. Test Organisms

6.1.6.1. E. estuarius are shipped from Northwestern Aquatic Sciences and

delivered by overnight mail so they arrive to the laboratory approximately one week prior to test initiation.

6.1.6.2. Arrangements must be made with Northwestern Aquatic Sciences ahead of time (at least one week prior to receipt) to ensure proper delivery and supply of organisms.

6.1.6.3. Once the organisms arrive in the lab, they must be immediately pooled and transferred into 10 L tank(s) along with the sediment they were shipped in and acclimated to testing temperature and salinity.

6.1.6.3.1. It is recommended that each 10 L tank not exceed a storage

capacity of approximately 300 organisms (or 1 amphipod/cm2).


App 6.1 - 8

San Jose Creek WQL


6.1.6.3.2. Pour the collection site sediment into the 2-10 L tanks so that it contains a 2 to 4 cm layer.

6.1.6.4. The initial temperature and salinity of the holding sediment and water

should be recorded on the organism history data sheet.

6.1.6.4.1. Temperature acclimation should not exceed more than 3 degrees per day during acclimation.

6.1.6.4.2. Salinity acclimation should not exceed 5 ppt per 24 hours.

6.1.6.5. Once organisms are acclimated to the proper test temperature and salinity, amphipods must be maintained at that temperature for a minimum of 2 days.

6.1.6.6. Feeding organisms

6.1.6.6.1. Once the E. estuarius are placed into the appropriate holding chamber and water quality readings have been taken, the organisms should be fed.

6.1.6.6.2. Make a solution of 0.25 g of Tetramarin slurry in 100 mL of seawater, for approximately 1000 amphipods.

6.1.6.6.2.1. Weigh out the prescribed amount of Tetramarin Saltwater Flakes, and add to approximately 100 mL of the appropriate salinity seawater.

6.1.6.6.2.2. Mix on a magnetic stir plate for about 10 minutes, or until the flakes have softened and started to dissolve.

6.1.6.6.3. Add the entire slurry to the holding container. 6.1.6.6.4. If the animals are split among multiple containers, the slurry may

be split accordingly. 6.1.6.6.5. The amphipods will not be fed during testing.

6.1.6.7. Renew overlying water in the holding tank when needed. 6.1.6.8. Record daily mortality of the amphipods in the holding tank on the

organism history data sheet. 6.1.6.9. Lighting must be constant at 50-100 Fc throughout the holding and

acclimation period. 6.1.6.10. Amphipods should not be held longer than 10 days. 6.1.6.11. If the mortality is >10 %, the entire batch of E. estuarius cannot be used.

Notify a senior staff member if mortality is >10%. 6.1.6.12. Upon receiving the organisms, fill out the “Eohaustorius estuarius receipt

form”. Note the organism lot number on the holding tank, and place the receipt form in the “Organism Source History” binder.

6.2. Test Initiation


App 6.1 - 9

San Jose Creek WQL


6.2.1. Reference Toxicant Solution Preparation

6.2.1.1. Ammonium Chloride as Reference Toxicant

6.2.1.1.1. The reference toxicant used for this test is 10,000 ppm ammonia stock

6.2.1.1.2. To make 10,000 ppm ammonia stock, add 31.34g ammonium chloride (NH4Cl) to 1 L DI water and mix for 30 minutes.

6.2.1.2. Preparing reference toxicant concentrations

6.2.1.2.1. Prepare the highest reference toxicant concentration (500 ppm NH3) by adding 200 mL of the 10,000 ppm ammonia stock to a 2-liter volumetric flask using class “A” volumetric pipettes and bring it up to volume with 20 ppt 30 μm filtered seawater.

6.2.1.2.2. Invert and shake the volumetric flask at least three times to ensure well mixing.

6.2.1.2.3. Pour the 2-liter volumetric flask into a 4-liter beaker. 6.2.1.2.4. Add an additional 2 liters of 20 ppt 30 μm filtered seawater to

make a total of 4 liters of the 500 ppm NH3 concentration. 6.2.1.2.5. Stir the concentration completely with a stirring rod. 6.2.1.2.6. Measure out 2 liters of the well-mixed 500 ppm concentration

using a 2 L graduated cylinder. Transfer the 2 liters into a 4-liter beaker labeled “RT 250 ppm”. Add an additional 2 liters of 20 ppt 30 μm filtered seawater.

6.2.1.2.7. Repeat steps 6.2.1.2.4 through 6.2.1.2.6 until the following reference toxicant concentrations and volumes have been prepared.

6.2.1.2.7.1. 500 ppm NH3 concentration = 2 L 6.2.1.2.7.2. 250 ppm NH3 concentration = 2 L 6.2.1.2.7.3. 125 ppm NH3 concentration = 2L 6.2.1.2.7.4. 62.5 ppm NH3 concentration = 2L 6.2.1.2.7.5. 31.25 ppm NH3 concentration = 2L + 2L not used 6.2.1.2.7.6. Control = 2L (dilution water only)

6.2.1.2.8. Using the randomization bench sheet, organize the test chambers

by concentration and add 800 mL of appropriate reference toxicant concentrations into each beaker.

6.2.1.2.9. Place prepared test chambers back into numerical order. 6.2.1.2.10. Write the LIMS ID number of the reference toxicant on the CETIS

water quality data sheets.

6.2.2. Chemical/Physical Observations for day 0


App 6.1 - 10

San Jose Creek WQL


6.2.2.1. Reference Toxicant

6.2.2.1.1. Measure the pH, temperature, DO, and salinity (see respective Biology Laboratory Instrumentation SOP for maintenance and calibration) of each test concentration and record them, along with the time, meter number and analyst’s initials on the water quality bench sheets under the appropriate day.

6.2.2.1.1.1. A single duplicate pH, DO, salinity and temperature is taken on

each series of measurements and recorded on the water quality bench sheets.

6.2.2.2. Sediment Test

6.2.2.2.1. Overlying water

6.2.2.2.1.1. Initial pH, DO, salinity and temperature for the sediment test

overlying water should be taken directly from the surrogate test chamber before the introduction of organisms.

6.2.2.2.1.2. Perform a duplicate water quality measurement on one test concentration.

6.2.2.2.1.3. An aeration apparatus should already be set into place to maintain the oxygen levels within the sediment tests.

6.2.2.2.1.4. Before the addition of the organisms, check that the ammonia levels are not above the ammonia tolerance of for E. estuarius (≥ 60 mg/L total ammonia)

6.2.2.2.1.4.1. Use the Hach® water quality test strips for ammonia to

get an immediate quantification. Follow the directions on the bottle.

6.2.2.2.1.4.2. A subsample can also be submitted to the chemistry group for analysis.

6.2.2.2.1.4.3. If the ammonia result for the overlying water is less than 60 mg/L, no further steps need to be taken. If the ammonia result for the overlying water is equal to or greater than 60 mg/L, the overlying water in all exposure chambers in the test batch should be replaced until none exceed this level.

6.2.2.2.2. Pore water.

6.2.2.2.2.1. Pore water can be sampled from the initial surrogate test

chamber. 6.2.2.2.2.1.1. Siphon/decant the overlying water without disturbing

the surface of the sediment.


App 6.1 - 11

San Jose Creek WQL


6.2.2.2.2.1.2. Put the remaining sediment in 250 mL centrifuge tubes and place them in the fixed angle rotor of the centrifuge located in the Microbiology Laboratory.

6.2.2.2.2.1.3. Centrifuge samples at 10,000 x g at 15° C for 30 minutes to extract pore water.

6.2.2.2.2.2. Measure the initial pH, and salinity readings for the sediment

test pore water. 6.2.2.2.2.3. Perform a duplicate water quality measurement on one test

concentration. 6.2.2.2.2.4. Record the meter numbers used to take water quality readings

on the water quality bench sheets. 6.2.2.2.2.5. Before the addition of the organisms, check that the ammonia

levels are not above the ammonia tolerance of for E. estuarius (≥ 60 mg/L total ammonia)

6.2.2.2.2.5.1. Use the Hach® water quality test strips for ammonia to

get an immediate quantification. Follow the directions on the bottle.

6.2.2.2.2.5.2. A subsample can also be submitted to the Chemistry Group for analysis.

6.2.3. Test Organism Placement

6.2.3.1. Ten random E. estuarius are placed into each reference toxicant test

chamber and twenty random E. estuarius are placed into each sediment test chamber. Do not place organisms into the surrogate chambers.

6.2.3.1.1. Test organisms should be handled as little as possible. 6.2.3.1.2. Place a 1mm sieve onto a dissection tray and add enough dilution

water so the sieve is submerged. Scoop a small amount of sediment from the holding tank and place it onto the sieve. Gently agitate the sieve so the sediment passes and the organisms are revealed.

6.2.3.1.3. Gently, using a generously cut pipette, randomly choose organisms 3-5mm in length and place them into 50 mL disposable cups. Avoid choosing colorless or white E. estuarius or amphipods that look unhealthy or impaired. Minimize the amount of excess water and sediment in each cup.

6.2.3.1.4. Once all cups are filled with the correct amount of organisms, transfer one disposable cup at a time into each of the replicates for the reference toxicant and the sediment tests proceeding in random order

6.2.3.1.4.1. Gently submerge the disposable cup containing the amphipods

into each test chamber in sequential order until every chamber contains organisms. Remove the empty cup.


App 6.1 - 12

San Jose Creek WQL


6.2.3.1.4.2. Do not place organisms into the surrogate chambers.

6.2.3.1.5. Cover the reference toxicant test chambers with lids and cover the entire test with a box to shield it from light.

6.2.3.1.6. Aeration in the sediment test should be ceased during organism addition and for a period of one hour after to allow time for organisms to burrow.

6.2.3.1.6.1. Organisms remaining in the water column after one hour or exhibiting abnormal behavior will be replaced. Make a note on the bench sheet of how many were replaced in each replicate.

6.2.3.1.7. After you have checked for proper burrowing in the sediment test, ensure that each chamber is being gently aerated.

6.2.3.1.8. Record the initiation time on the bench sheet after the last E. estuarius is placed into the test.

6.2.4. Initial Light Level

6.2.4.1. The ambient light level for each test is taken at the time of test initiation.

6.2.4.1.1. Place the light meter in the central region of the testing area. 6.2.4.1.2. The light level should be between 500 to 1000 lux (50-100 Fc). If

it is not within this range, the test beakers should be moved to another location or the light levels need to be altered (addition or removal of bulbs) until the light levels are within specified range.

6.2.4.1.3. Note and initial the light level on the bench sheet. 6.2.4.1.4. The light levels for the sediment tests should be continuous, set for

24 hours of light and 0 hours of darkness each day. 6.2.4.1.5. The reference toxicant tests should be covered with a large box so

that the tests are kept in the dark at all times.

6.3. Daily Bioassay Maintenance

6.3.1. Biological Observations 6.3.1.1. Record daily survival counts on the raw data sheet.

6.3.1.1.1. Organism counting should be done in numerical order. 6.3.1.1.2. Discard any dead individuals daily. Ensure bodies removed are the

organisms themselves and not molts.

6.3.1.1.2.1. A light table or dissection microscope can be used to aid in determine whether an organism is are dead or alive.


App 6.1 - 13

San Jose Creek WQL


6.3.1.1.3. Use a disposable pipette to remove any dead organisms and rinse the pipette immediately after removing any dead organisms to minimize any cross contamination.

6.3.1.1.4. Note any other significant observations on the raw data sheet (i.e. a significant number of buried Eohaustorius or neonates present).

6.3.2. Chemical/Physical Observations

6.3.2.1. Temperature readings should be taken daily. 6.3.2.2. Daily observations should be made to double-check that aeration is

working properly

6.3.3. Daily data entry

6.3.3.1. Daily entry of biological and water quality data should be done by the person who makes the observations.

6.3.3.2. See the Biology Laboratory SOP for “Bioassay Test Initiation and Data Entry Using CETIS and LIMS” for details regarding daily data entry.

6.4. Test Termination

6.4.1. Test Duration

6.4.1.1. E. estuarius are exposed to test solutions for 96 hours ± 2 hours for the reference toxicant test and for 10 days ± 4 hours for the sediment test.

6.4.1.2. Water quality measurements can be taken prior to breakdown.

6.4.2. Chemical Observations

6.4.2.1. Dissolved oxygen (DO), pH, salinity and temperature are determined at each concentration of the reference toxicant test and sediment test using calibrated meters.

6.4.2.1.1. Take readings out of the surrogate test chambers for the sediment

test. 6.4.2.1.2. Take readings out of randomly selected test chambers for the

reference toxicant test. 6.4.2.1.3. Record the meter number used on the water quality bench sheets. 6.4.2.1.4. A single duplicate pH, DO, salinity and temperature is taken on

each series of measurements and recorded on the water quality bench sheets.

6.4.2.2. Measure Final Ammonia Levels (sediment test only)

6.4.2.2.1. A sample of the final overlying water from each sediment site will

be tested for ammonia and recorded on the raw data sheets.


App 6.1 - 14

San Jose Creek WQL


6.4.2.2.1.1. Use the Hach® water quality test strips for ammonia to get an

immediate quantification. Follow the directions on the bottle. 6.4.2.2.1.2. A subsample can also be submitted to the chemistry group for

analysis.

6.4.2.3. Check final water quality from the pore water.

6.4.2.3.1. Pore water can be sampled from the final surrogate test chambers.

6.4.2.3.1.1. Siphon or decant the overlying water without disturbing the surface of the sediment.

6.4.2.3.1.2. Put the remaining sediment in 250 mL centrifuge tubes and place them in the fixed angle rotor of the centrifuge located in the Microbiology Laboratory.

6.4.2.3.1.3. Centrifuge samples at 10,000 x g at 15° C for 30 minutes to extract pore water.

6.4.2.3.1.4. Measure the pH, and salinity (see respective Biology Laboratory Instrumentation SOP for maintenance and calibration) of each sampling site(s) pore water and record them, along with the time, meter number and analyst’s initials on the water quality bench sheets under the appropriate day.

6.4.2.3.1.5. Measure final ammonia levels

6.4.2.3.1.5.1. Use the Hach® water quality test strips for ammonia to get an immediate quantification for the overlying water and the pore water. Follow the directions on the bottle.

6.4.2.3.1.5.2. A subsample can also be submitted to the chemistry group for analysis.

6.4.3. Physical Observations for the reference toxicant test

6.4.3.1. Count the number of organisms alive.

6.4.3.1.1. Organism counting should be done in numerical order. 6.4.3.1.2. A light table or dissection microscope can be used to aid in

determine whether an organism is are dead or alive. 6.4.3.1.3. Note any other significant observations on the raw data sheet (i.e.

neonates present).

6.4.4. Physical Observations For Sediment test

6.4.4.1. Count the number of organisms alive.

6.4.4.1.1. Observe any dead or live organisms in the water column or on the surface of the sediment.


App 6.1 - 15

San Jose Creek WQL


6.4.4.1.2. Use a 500 µm sieve to separate the sediment from the organisms.

6.4.4.1.2.1. Slowly, pour the contents of the beaker into the 500 µm sieve. 6.4.4.1.2.2. Use a squirt bottle or a D.I. hose to slowly pass the sediment

through the sieve. 6.4.4.1.2.3. Once organisms are exposed, record the number of live

organisms on the bench sheet under the appropriate column.

6.4.4.1.3. If there are still organisms that have not been accounted for, consider these organisms dead. Dead organisms decompose fairly quickly and if they are not found within the sediment after conducting the above-mentioned routine, consider them dead.

6.4.4.1.4. Make sure to count the organisms and not the molts; a dissecting scope may be helpful in deciphering the two.

6.4.4.1.5. It is possible for E. estuarius to reproduce during the test. If neonates are observed, it should be noted somewhere on the raw data sheet.

6.4.5. Final Data Entry

6.4.5.1. Daily entry of biological and water quality data should be done by the

person who makes the observations. 6.4.5.2. See the Biology Laboratory SOP for “Bioassay Test Initiation and Data

Entry Using CETIS and LIMS” for details regarding final data entry.

7. Calculations

7.1. Data Analysis

7.1.1. Bioassay data are analyzed according to the procedures outlined in the SOP for “Bioassay Data Analysis, Report Generation, and Result Verification”.

8. Quality Assurance Guidelines

8.1. Quality Control

8.1.1. Bioassay data quality control procedures are outlined in the “Biology Section

Quality Assurance Plan”. 9. Method Performance

9.1. The following minimum test acceptability criteria must be met for the bioassay to be considered valid under the recommended test conditions for conducting sediment and reference-toxicant tests.


App 6.1 - 16

San Jose Creek WQL


9.1.1. Mean control survival must be ≥ 90 % for both the sediment and the reference toxicity test.

10. References

10.1. Water Quality Control Plan For Enclosed Bays and Estuaries-Part I Sediment Quality. August 2009. California Environmental Protection Agency, State Water Resources Control Board. P.O. Box 100, Sacramento, CA 95812-0100.

10.2. Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods. June 1994. U.S. Environmental Protection Agency, Office of Research and Development. Narragansett, Rhode Island, 02882. EPA 600/R-94/025


App 6.1 - 17

Documents

Appendix 6 Sediment Toxicity