US ARMY CORPS OF ENGINEERS - ACUTE TOXICITY ...Department of the Army US Army Engineer Research and Development Center Environmental Laboratory 3909 Halls Ferry Rd, EP-R Vicksburg,

Acute Toxicity Evaluation of Duck and Otter Creek Sediments with Chironomus dilutus (Confluence Site Investigation) Prepared for: U.S. EPA Great Lakes National Program Office Atn: Brenda Jones 77 West Jackson Blvd. Chicago, IL 60604 Prepared by: J. Daniel Farrar Burton Suedel Department of the Army US Army Engineer Research and Development Center Environmental Laboratory 3909 Halls Ferry Rd, EP-R Vicksburg, MS 39180

25 February 2011

ACUTE TOXICITY EVALUATION OF DUCK AND OTTER CREEK SEDIMENTS WITH CHIRONOMUS DILUTUS

2

TABLE OF CONTENTS EXECUTIVE SUMMARY ............................................................................................................ 3

INTRODUCTION .......................................................................................................................... 4

TEST SEDIMENTS........................................................................................................................ 4

METHODS ..................................................................................................................................... 5

Chironomus dilutus whole sediment toxicity bioassay .............................................................. 5

Water quality parameters ............................................................................................................ 6

Statistical analysis ....................................................................................................................... 6

RESULTS ....................................................................................................................................... 6

REFERENCES ............................................................................................................................... 8

Appendix A. Photos from Chironomus dilutus bioassays ............................................................. 9

Appendix B. Water quality parameters for Chironomus dilutus bioassays ................................. 11

Appendix C. Survival and Growth Endpoint data for Chironomus dilutus bioassays ................. 14

Appendix D. Statistical analyses for Chironomus dilutus sediment bioassays ........................... 19

Appendix E. Chain of Custody sheets ......................................................................................... 34

Appendix F. Data Sheets for Chironomus dilutus sediment toxicity bioassays .......................... 39


3

EXECUTIVE SUMMARY

The Great Lakes National Program Office (GLNPO) requested the Army Engineer Research and Development Center (ERDC) to conduct acute toxicity testing on sediments from Duck and Otter Creeks as part of its Great Lakes Legacy Act initiative. Eleven (11) sediments were evaluated using the midge Chironomus dilutus. This report summarizes the biological testing of sediment collected from Duck and Otter Creeks confluence sites conducted in basic accordance with guidance provided in “Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates” (USEPA 2000). Three separate experiments were conducted. Significant reduction in survival was detected in OC-26 and OC-27 sediment samples. Significant reductions in ash-free biomass per initial organism and ash-free dry weight per surviving organism were detected for sediment samples OC-26, OC-29 and OC-30. A significant reduction in ash-free dry weight per surviving organism was observed for sediment sample OC-28 (Tables 2-4). No significant effects on survival or biomass were detected for the remaining sediment samples evaluated.


4

INTRODUCTION At the request of the USEPA Great Lakes National Program Office (GLNPO), the US Army Engineer Research and Development Center (ERDC) conducted sediment toxicity tests of bottom sediment collected from sites at the confluence of Duck and Otter Creeks. Duck and Otter Creeks are located in an industrialized area of Northwest Ohio. The creeks are part of the Maumee River watershed which empties into Lake Erie. The evaluations were conducted under the Great Lakes Legacy Act with support from the GLNPO. The evaluation consisted of whole sediment acute toxicity tests with the midge Chironomus dilutus. Studies were conducted in basic accordance with the guidance provided in “Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates” (USEPA 2000). The evaluation was conducted blind, in that the ERDC was unaware of the amounts or types of contaminants in the sediment samples and where they were collected.

TEST SEDIMENTS Eleven (11) sediments were received by the ERDC in two (2) separate shipments. Shipments were received the 5th and 8th of November 2010. Sediments were immediately inspected, logged and placed in cold storage at 4°C where they remained until test initiation. Chain of custody sheets are provided in Appendix D. A list of sediment samples evaluated is provided in Table 1.

Table 1. Summary of the test sediments and sediment identification

Sample Name Customer ID

DC-13 DC-13

DC-14 DC-14

DC-16 DC-16

DC-17 DC-17

DC-19 DC-19

OC-26 OC-26

OC-27 OC-27

OC-28 OC-28

OC-29 OC-29

OC-30 OC-30

OC-31 OC-31


5

METHODS

Chironomus dilutus whole sediment toxicity bioassay Chironomus dilutus 10-day sediment exposures were conducted in basic accordance with guidance provided in “Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates” (USEPA 2000). Due to logistics related to the number of samples tested, the study was divided into 3 separate experiments. Testing was initiated on the 19th of November 2010 and was completed on 23rd December 2010. Egg masses were obtained from Environmental Consulting and Testing (Superior, WI) and maintained in culture until the correct age (~ 10 days old) organisms were available to initiate testing. On the day prior to test initiation, approximately 75 mL of each sediment was added to eight (8) replicate 300 mL beakers. Sediment from the University of Mississippi field station, (Oxford, MS) was included as a negative control. Beakers were placed in a temperature controlled water bath or environmental chamber at 23 °C and allowed to equilibrate over night. Pore water ammonia measurements were taken on sediments prior to addition to beakers. Pore water was collected by centrifuging 50 mL of each sediment at 3000 RPM for 15 minutes. Pore water analysis was performed on the supernatant of the centrifuged sediment. All pore water total ammonia concentrations were below the 20 mg/L guidance for freshwater organisms provided in the Inland Testing Manual (USEPA /USACE 1998) (Appendix B; Table B1). Overlying water quality (temperature, pH, dissolved oxygen, hardness, alkalinity, conductivity and ammonia) was recorded for each replicate beaker at test initiation. Ten organisms were added to each replicate and 1 mL of a Tetrafin® food mixture was provided. Initial weights of all organisms added were within the 0.08 to 0.23 mg/individual dry weight range indicative of the correct developmental stage (USEPA 2000). Temperature and dissolved oxygen were measured daily on a single replicate of each sediment throughout the exposure period. Animals were fed 1 mL of a Tetrafin® food mixture and two full water renewals were conducted daily. Water renewals were conducted using a modified Zumwalt water renewal system (USEPA 2000) (Appendix A; Photo A1 and A2). To maintain dissolved oxygen levels, aeration was provided to each replicate. On day 10, overlying water quality was measured and each replicate beaker was terminated by passing the sediment through a 425 µm screen. Surviving organisms were recovered and enumerated. Following enumeration organisms from each replicate were placed on pre-weighed pans and placed in a drying oven for 24 hours at 60 °C. Following the drying period, pans were removed from the oven and dry mass for each replicate was recorded. Pans were then placed in a muffle furnace at 550 °C for 2 hours. After 2 hours, pans were removed from the furnace allowed to cool and ash-free mass was recorded for each replicate.


6

Water quality parameters The water quality during bioassay testing was measured using a Thermo Scientific Orion 4Star meter (Thermo Scientific, Beverly, MA) for electrical conductivity, a model 315i meter (WTW; Weilheim, Germany) for pH, and a model Oxi 330 meter (WTW; Weilheim, Germany) for dissolved oxygen (D.O.). Total overlying water ammonia, hardness and alkalinity were measured using LeMotte titration kits (Chestertown, MD, USA). Total pore water ammonia was measured using a 720A ion-selective electrode (ISE) meter (Thermo Orion Electron Corp., Beverly, MA) equipped with a 95-12 ammonia-sensitive electrode (Thermo Orion Electron Corp., Beverly, MA).

Statistical analysis Data normality (Kolmogorov–Smirnov test), homogeneity (Levene’s Test), and sediment differences compared to the control were conducted using SigmaStat software (Version 3.5, SPSS, Chicago, IL). Statistical significance was determined at α = 0.05. Survival data were arcsine-square-root transformed prior to analysis. Where data were normal and homogenous, a One Way ANOVA was conducted followed by means comparison to the control using the Dunnett’s mean comparison method. When normality was not achieved, the Kruskal–Wallis one-way ANOVA on ranks was applied followed by means comparison to the control using the Dunn’s mean comparison procedure. Data endpoints analyzed were survival, weight per initial organism, ash-free weight per initial organism, dry weight per surviving organism and ash-free weight per surviving organism.

RESULTS

Water quality parameters for all bioassays were within the acceptability ranges specified by testing guidance (US EPA 2000) (Appendix B; Tables B2-B4). Survival in the negative control exceeded 78% in all tests and met the 70% minimum performance requirement indicated in testing guidance (US EPA 2000) (Tables 2-4). Survival ranged from 0% to 87.5% in the sediment samples tested. Statistically significant reductions in survival relative to the control were detected in OC-26 and OC-27 sediments (Tables 3 and 4). Significant effects on ash-free biomass per initial organism were detected for sediments OC-26, OC-29 and OC-30. A significant reduction in ash-free dry weight per surviving organism was observed for sediment sample OC-28 (Tables 3 and 4). No significant effects on survival or the biomass endpoints examined were detected for sediments DC-13, DC-14, DC-16, DC-17, DC-19 and OC-31 (Tables 2-4). Significant reductions in ash-free biomass per initial organism and ash-free dry weight per surviving organism were detected for sediment samples OC-26, OC-29 and OC-30. A significant reduction in ash-free dry weight per surviving organism was observed for sediment


7

sample OC-28 (Tables 2-4). No significant effects on survival or biomass were detected for the remaining sediment samples evaluated. Table 2. Test 1 Chironomus dilutus mean survival and mass (± one standard deviation). Asterisks denote a statistically significant reduction in survival or mass relative to the control sediment.

Sample Name (% Survival)

Ash-free Biomass per

Initial Organism

(mg)

Ash-free Dry weight per Surviving

organism (mg)

Control 87.5 ± 14.9 2.84 ± 1.40 3.10 ± 1.12 DC-13 87.5 ± 16.7 2.59 ± 1.02 2.92 ± 0.89

Table 3. Test 2 Chironomus dilutus mean survival and mass (± one standard deviation). Asterisks denote a statistically significant reduction in survival or mass relative to the control sediment.



Initial Organism

(mg)


organism (mg)

Control 81.3 ± 6.4 1.76 ± 0.64 2.39 ± 0.81 DC-14 81.3 ± 19.6 1.68 ± 0.79 2.04 ± 0.68 DC-16 83.8 ± 16.0 1.93 ± 0.42 2.35 ± 0.58 DC-17 83.8 ± 14.1 1.86 ± 0.57 2.24 ± 0.68 DC-19 73.8 ± 16.0 1.78 ± 0.58 2.41 ± 0.45 OC-26 *17.5 ±20.5 *0.35 ± 0.05 *0.46 ± 0.09


8

Table 4. Test 3 Chironomus dilutus mean survival and mass (± one standard deviation). Asterisks denote a statistically significant reduction in survival or mass relative to the control sediment.



Initial Organism

(mg)


organism (mg)

Control 78.8 ± 9.9 1.17 ± 0.51 1.45 ± 0.48 OC-27 *0 ± 0 N/A N/A OC-28 83.8 ± 14.1 0.46 ± 0.18 *0.54 ± 0.16 OC-29 66.3 ± 13.0 *0.09 ± 0.01 *0.15 ± 0.02 OC-30 60.0 ± 21.4 *0.11 ± 0.05 *0.19 ± 0.06 OC-31 70.0 ± 28.3 0.68 ± 0.37 0.90 ± 0.25

REFERENCES US Environmental Protection Agency / US Army Corps of Engineers (US EPA / USACE). 1998. Evaluation of Material Proposed for Discharge to Waters of the U.S. - Testing Manual (Inland Testing Manual). EPA-823-B-98-004, Office of Water, Washington DC. US Environmental Protection Agency. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, Second Edition. EPA 600/R-99/064, Office of Water, Washington, D.C.


9

Appendix A. Photos from Chironomus dilutus bioassays

Photo A1. Zumwalt Water Delivery System used during Bioassay Testing.


10

Photo A2. Close-up of Zumwalt Water Delivery System and Exposure Chambers used during Bioassay Testing.


11

Appendix B. Water quality parameters for Chironomus dilutus bioassays Table B1. Total pore water ammonia concentrations at test initiation.

Treatment Total Ammonia

(mg/L)

Unionized Ammonia

(mg/L)

pH (SU)

Temperature (°C)

DC-13 5.38 0.175 7.83 23.1

DC-14 11.0 0.133 7.40 22.8

DC-16 7.41 0.154 7.63 23.1

DC-17 7.29 0.098 7.44 23.0

DC-19 9.77 0.134 7.45 23.0

OC-26 5.38 0.072 7.44 23.0

OC-27 2.80 0.057 7.62 23.1

OC-28 9.92 0.208 7.64 22.9

OC-29 4.12 0.095 7.68 23.0

OC-30 4.18 0.071 7.54 23.1

OC-31 3.86 0.081 7.64 22.9


12

Table B2. Test 1 overlying water quality parameters. Means and one standard deviation from the mean are indicated, with the minimum and maximum range of the data provided in parentheses.

Treatment Temperature

( C) pH

(SU)

Dissolved oxygen (mg/L)

Conductivity (µS/cm)

*MaximumAmmonia

(mg/L)

*Alkalinity(mg/L)

*Hardness(mg/L)

Control 23.3 ± 0.2

(23.1 - 23.6) 7.4 ± 0.2 (7.1 - 7.7)

7.7 ± 0.4 (6.9 - 8.6)

230 ± 7 (221 - 245)

<1 35 ± 7

( 30 - 40) 78 ± 4

(75 - 80)

DC-13 23.0 ± 0.2

(22.7 - 23.2) 8.0 ± 0.1 (7.9 - 8.1)

7.3 ± 0.4 (6.7 - 8.0)

386 ± 20 (360 - 420 )

<1 105 ± 21.2 (90 -120)

125 ± 7.1 (120 - 130)

*Measurement taken on composite sample; concentrations <1 are below method detection limit Table B3. Test 2 overlying water quality parameters. Means and one standard deviation from the mean are indicated, with the minimum and maximum range of the data provided in parentheses.


( C) pH

(SU)



*MaximumAmmonia

(mg/L)

*Alkalinity (mg/L)

*Hardness(mg/L)

Control 23.6 ± 0.2

(23.3 - 24.0) 7.7 ± 0.6

(6.3 – 8.4) 8.5 ± 0.2

(8.2 – 8.9)238 ± 9

(225 - 256) 3

42 ± 2.8 (40 - 44)

60 ± 0 (60-60)

DC-14 23.4 ± 0.2

(23.2 - 23.7) 8.3 ± 0.4

(6.8 – 8.5) 8.6 ± 0.2

(8.3 – 8.8)208 ± 9.4

(195 - 231) 1

74 ± 20 (60 - 88)

107 ± 52 (70 - 144)

DC-16 23.4 ± 0.2

(23.1 - 23.7) 8.3 ± 0.3 (7.3 - 8.5)

8.2 ± 0.8 (6.3 - 8.9)

349 ± 11 (325 – 369)

1 92 ± 12

(83 -100) 105 ± 35 (80 - 130)

DC-17 23.4 ± 0.2

(23.1 - 23.7) 8.3 ± 0.1 (8.2 - 8.4)

8.2 ± 0.5 (7.4 – 8.7)

347 ± 14 (327 - 368)

1 80 ± 0

(80 -80) 102 ± 32 (80 - 125)

DC-19 23.3 ± 0.2

(23.1 – 23.7) 8.2 ± 0.4 (7.1 - 8.5)

8.4 ± 0.4 (7.7 – 8.9)

365 ± 12 (349 - 380)

1 90 ± 0.7 (89 - 90)

98 ± 39 (70 - 125)

OC-26 23.4 ± 0.3

(23.1 - 23.8) 8.4 ± 0.1 (8.2 - 8.6)

8.6 ± 0.2 (8.1 – 8.8)

325 ± 17 (298 - 343)

1 96 ± 7.8

(90 - 101) 111 ± 44 (80 - 142)

*Measurement taken on composite sample; concentrations <1 are below method detection limit


13

Table B4. Test 3 overlying water quality parameters. Means and one standard deviation from the mean are indicated, with the minimum and maximum range of the data provided in parentheses.


( C) pH

(SU)



*MaximumAmmonia

(mg/L)

*Alkalinity (mg/L)

*Hardness (mg/L)

Control 23.3 ± 0.4

(22.9 - 23.8) 7.1 ± 0.3 (6.7 - 7.7)

6.9 ± 0.5 (6.6 - 8.1)

206 ± 28 (166 - 270)

2 60 ± 0

( 60 - 60) 68 ± 11 (60 - 75)

OC-27 23.3 ± 0.4

(22.9 – 24.1) 7.9 ± 0.1 (7.7 - 8.0)

6.3 ± 1.2 (5.0 - 7.8)

418 ± 42 (343 - 463)

<1 114 ± 20

(100 -128) 120 ± 0

(120 - 120)

OC-28 23.1 ± 0.3

(22.8 – 23.7) 8.1 ± 0.1

(8.0 – 8.3) 6.4 ± 1.0 (5.2 - 7.5)

406 ± 45.5 (322 - 456)

<1 129 ± 13

(120 - 138) 139 ± 1.4

(138 - 140)

OC-29 23.2 ± 0.4

(22.9 - 23.8) 8.1 ± 0.1 (8.0 - 8.2)

6.7 ± 0.8 (5.8 – 7.7)

461 ± 53 (333 - 550)

1 135 ± 21

(120 -140) 130 ± 14

(120 - 140)

OC-30 23.1 ± 0.4

(22.3 – 23.7) 7.8 ± 0.3 (7.5 - 8.1)

6.3 ± 0.7 (5.5 – 7.7)

359 ± 41 (303 - 420)

1 110 ± 14

(100 - 120) 115 ± 7.1

(110 – 120)

OC-31 23.2 ± 0.4

(22.4 – 23.8) 7.7 ± 0.5 (7.1 - 8.2)

6.9 ± 0.6 (6.2 – 7.7)

360 ± 29 (316 - 412)

<1 100 ± 0

(100 - 100) 108 ± 3.5

(105 - 110) *Measurement taken on composite sample; concentrations <1 are below method detection limit


14

Appendix C. Survival and Growth Endpoint data for Chironomus dilutus bioassays


15


16


17


18


19

Appendix D. Statistical analyses for Chironomus dilutus sediment bioassays TEST 1 Test 1 Survival One Way Analysis of Variance Sunday, January 09, 2011, 2:43:02 AM Data source: Data 5 in Notebook 1 Normality Test: Failed (P = <0.001) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, January 09, 2011, 2:43:02 AM Data source: Data 5 in Notebook 1 Group N Missing Median 25% 75% Control 8 0 1.410 0.991 1.571 DC-6/7 8 0 1.178 1.049 1.571 DC-11/12 8 0 1.249 0.997 1.571 DC-13 8 0 1.249 1.178 1.571 OC-22 8 0 1.410 1.107 1.571 OC-24/25 8 0 1.178 1.049 1.571 H = 1.042 with 5 degrees of freedom. (P = 0.959) The differences in the median values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.959)


20

Test 1 Dry Biomass per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:01:15 PM Data source: Test 3 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: DBM/IO Normality Test: Passed (P = 0.877) Equal Variance Test: Failed (P < 0.050) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, February 13, 2011, 6:01:15 PM Data source: Test 3 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Group N Missing Median 25% 75% Control 8 0 3.837 2.021 5.483 DC-6/7 8 0 2.636 2.073 2.818 DC-11/12 8 0 1.979 1.770 2.451 DC-13 8 0 3.867 2.298 4.280 OC-22 8 0 3.071 2.780 3.976 OC-24/25 8 0 3.237 2.507 3.881 H = 10.028 with 5 degrees of freedom. (P = 0.074) The differences in the median values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.074)


21

Test 1 Ash-free Biomass per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:02:38 PM Data source: Test 3 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: ABM/IO Normality Test: Passed (P = 0.597) Equal Variance Test: Failed (P < 0.050) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, February 13, 2011, 6:02:38 PM Data source: Test 3 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Group N Missing Median 25% 75% Control 8 0 2.831 1.486 4.177 DC-6/7 8 0 1.956 1.555 2.071 DC-11/12 8 0 1.431 1.332 1.899 DC-13 8 0 2.922 1.578 3.179 OC-22 8 0 2.374 2.095 2.936 OC-24/25 8 0 2.290 1.895 2.944 H = 8.749 with 5 degrees of freedom. (P = 0.120) The differences in the median values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.120)


22

Test 1 Individual Dry Mass One Way Analysis of Variance Sunday, January 30, 2011, 8:48:13 PM Data source: Test 3 Mass Data in Stats Dependent Variable: D-Mass Normality Test: Passed (P = 0.153) Equal Variance Test: Passed (P = 0.202) Group Name N Missing Mean Std Dev SEM Control 8 0 4.164 1.400 0.495 DC-6/7 8 0 3.263 1.010 0.357 DC-11/12 8 0 2.597 0.915 0.323 DC-13 8 0 3.893 1.049 0.371 OC-22 8 0 3.558 0.743 0.263 OC-24/25 8 0 3.755 0.855 0.302 Source of Variation DF SS MS F P Between Groups 5 12.211 2.442 2.364 0.056 Residual 42 43.396 1.033 Total 47 55.607 The differences in the mean values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.056).


23

Test 1 Individual Ash-Free Mass One Way Analysis of Variance Sunday, January 09, 2011, 2:44:01 AM Data source: Data 6 in Notebook 1 Normality Test: Passed (P > 0.050) Equal Variance Test: Passed (P = 0.143) Group Name N Missing Mean Std Dev SEM Control 8 0 3.098 1.118 0.395 DC-6/7 8 0 2.424 0.732 0.259 DC-11/12 8 0 1.921 0.718 0.254 DC-13 8 0 2.915 0.895 0.316 OC-22 8 0 2.642 0.548 0.194 OC-24/25 8 0 2.733 0.582 0.206 Source of Variation DF SS MS F P Between Groups 5 6.845 1.369 2.196 0.073 Residual 42 26.188 0.624 Total 47 33.033 The differences in the mean values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.073).


24

TEST 2 Test 2 Survival One Way Analysis of Variance Sunday, January 09, 2011, 2:48:46 AM Data source: Data 7 in Stats Normality Test: Passed (P > 0.050) Equal Variance Test: Failed (P = 0.006) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, January 09, 2011, 2:48:46 AM Data source: Data 7 in Stats Group N Missing Median 25% 75% Control 8 0 1.107 1.107 1.178 DC-14 8 0 1.178 1.049 1.410 DC-16 8 0 1.178 0.991 1.571 DC-17 8 0 1.178 1.049 1.410 DC-19 8 0 1.049 0.991 1.178 OC-26 8 0 0.232 0.000 0.632 H = 21.174 with 5 degrees of freedom. (P = <0.001) The differences in the median values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001) To isolate the group or groups that differ from the others use a multiple comparison procedure. Multiple Comparisons versus Control Group (Dunnett's Method) : Comparison Diff of Ranks q' P<0.05 OC-26 vs Control 184.000 3.286 Yes DC-19 vs Control 38.500 0.688 No DC-17 vs Control 22.000 0.393 Do Not Test DC-16 vs Control 20.000 0.357 Do Not Test DC-14 vs Control 18.500 0.330 Do Not Test Note: The multiple comparisons on ranks do not include an adjustment for ties.


25

Test 2 Dry Biomass per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:06:44 PM Data source: Test 4 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: DBM/IO Normality Test: Passed (P = 0.524) Equal Variance Test: Passed (P = 0.126) Group Name N Missing Mean Std Dev SEM Control 8 0 2.553 0.841 0.297 DC-14 8 0 2.498 1.306 0.462 DC-16 8 0 2.864 0.482 0.170 DC-17 8 0 2.736 0.822 0.291 DC-19 8 0 2.637 0.914 0.323 OC-26 4 0 0.234 0.0831 0.0415 Source of Variation DF SS MS F P Between Groups 5 22.041 4.408 5.754 <0.001 Residual 38 29.114 0.766 Total 43 51.155 The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Multiple Comparisons versus Control Group (Dunnett's Method) : Comparisons for factor: Sediment Comparison Diff of Means q' P P<0.050 Control vs. OC-26 2.319 4.326 -- Yes Control vs. DC-16 0.311 0.710 -- No Control vs. DC-17 0.183 0.418 -- Do Not Test Control vs. DC-19 0.0841 0.192 -- Do Not Test Control vs. DC-14 0.0551 0.126 -- Do Not Test Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01. A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist.


26

Test 2 Ash-free Biomass Per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:08:24 PM Data source: Test 4 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: ABM/IO Normality Test: Passed (P = 0.743) Equal Variance Test: Passed (P = 0.225) Group Name N Missing Mean Std Dev SEM Control 8 0 1.759 0.639 0.226 DC-14 8 0 1.679 0.791 0.280 DC-16 8 0 1.925 0.420 0.148 DC-17 8 0 1.858 0.571 0.202 DC-19 8 0 1.785 0.581 0.205 OC-26 4 0 0.155 0.0514 0.0257 Source of Variation DF SS MS F P Between Groups 5 10.139 2.028 5.867 <0.001 Residual 38 13.133 0.346 Total 43 23.272 The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Multiple Comparisons versus Control Group (Dunnett's Method) : Comparisons for factor: Sediment Comparison Diff of Means q' P P<0.050 Control vs. OC-26 1.604 4.457 -- Yes Control vs. DC-16 0.166 0.564 -- No Control vs. DC-17 0.0982 0.334 -- Do Not Test Control vs. DC-14 0.0806 0.274 -- Do Not Test Control vs. DC-19 0.0255 0.0868 -- Do Not Test Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01. A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist.


27

Test 2 Individual Dry Mass One Way Analysis of Variance Sunday, January 30, 2011, 8:46:23 PM Data source: Test 4 Mass Data in Stats Dependent Variable: D-Mass Normality Test: Passed (P = 0.514) Equal Variance Test: Passed (P = 0.329) Group Name N Missing Mean Std Dev SEM Control 8 0 3.469 1.050 0.371 DC-14 8 0 3.038 1.172 0.414 DC-16 8 0 3.514 0.828 0.293 DC-17 8 0 3.295 0.956 0.338 DC-19 8 0 3.545 0.684 0.242 OC-26 4 0 0.700 0.187 0.0933 Source of Variation DF SS MS F P Between Groups 5 27.383 5.477 6.523 <0.001 Residual 38 31.906 0.840 Total 43 59.290 The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Multiple Comparisons versus Control Group (Dunnett's Method) : Comparisons for factor: Sediment Comparison Diff of Means q' P P<0.050 Control vs. OC-26 2.769 4.935 -- Yes Control vs. DC-14 0.432 0.942 -- No Control vs. DC-17 0.174 0.380 -- Do Not Test Control vs. DC-19 0.0754 0.165 -- Do Not Test Control vs. DC-16 0.0448 0.0979 -- Do Not Test Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01. A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist.


28

Test 2 Individual Ash-free Mass One Way Analysis of Variance Sunday, January 09, 2011, 2:50:42 AM Data source: Data 8 in Stats Normality Test: Passed (P > 0.050) Equal Variance Test: Passed (P = 0.486) Group Name N Missing Mean Std Dev SEM Control 8 0 2.388 0.811 0.287 DC-14 8 0 2.045 0.679 0.240 DC-16 8 0 2.346 0.583 0.206 DC-17 8 0 2.241 0.681 0.241 DC-19 8 0 2.408 0.455 0.161 OC-26 4 0 0.458 0.0911 0.0455 Source of Variation DF SS MS F P Between Groups 5 12.861 2.572 6.549 <0.001 Residual 38 14.925 0.393 Total 43 27.786 The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001). Multiple Comparisons versus Control Group (Dunnett's Method) : Comparisons for factor: Sediment Comparison Diff of Means q' P P<0.050 Control vs. OC-26 1.930 5.030 -- Yes Control vs. DC-14 0.343 1.096 -- No Control vs. DC-17 0.147 0.468 -- Do Not Test Control vs. DC-16 0.0418 0.133 -- Do Not Test Control vs. DC-19 0.0196 0.0626 -- Do Not Test Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01. A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist.


29

TEST 3 Test 3 Survival One Way Analysis of Variance Sunday, January 09, 2011, 2:55:01 AM Data source: Data 9 in Stats Normality Test: Passed (P > 0.050) Equal Variance Test: Passed (P = 0.339) Group Name N Missing Mean Std Dev SEM Control 8 0 1.102 0.128 0.0451 OC-28 8 0 1.216 0.250 0.0883 OC-29 8 0 0.960 0.150 0.0532 OC-30 8 0 0.922 0.299 0.106 OC-31 8 0 1.033 0.366 0.129 Source of Variation DF SS MS F P Between Groups 4 0.442 0.111 1.701 0.172 Residual 35 2.274 0.0650 Total 39 2.716 The differences in the mean values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.172).


30

Test 3 Dry Biomass per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:11:05 PM Data source: Test 5 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: DBM/IO Normality Test: Failed (P < 0.050) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, February 13, 2011, 6:11:05 PM Data source: Test 5 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Group N Missing Median 25% 75% Control 8 0 1.376 1.092 1.745 OC-28 8 0 0.641 0.448 0.715 OC-29 8 0 0.130 0.111 0.137 OC-30 8 0 0.128 0.0946 0.200 OC-31 8 0 1.007 0.553 1.278 H = 27.697 with 4 degrees of freedom. (P = <0.001) The differences in the median values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001) To isolate the group or groups that differ from the others use a multiple comparison procedure. Multiple Comparisons versus Control Group (Dunn's Method) : Comparison Diff of Ranks Q P<0.05 OC-29 vs Control 25.125 4.298 Yes OC-30 vs Control 24.875 4.256 Yes OC-28 vs Control 11.250 1.925 No OC-31 vs Control 8.750 1.497 Do Not Test Note: The multiple comparisons on ranks do not include an adjustment for ties.


31

Test 3 Ash-free Biomass per Initial Organism One Way Analysis of Variance Sunday, February 13, 2011, 6:12:52 PM Data source: Test 5 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Dependent Variable: ABM/IO Normality Test: Failed (P < 0.050) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, February 13, 2011, 6:12:52 PM Data source: Test 5 Dry and Ash-free Biomass per Initial Organism Data in Stats(2-13-11) Group N Missing Median 25% 75% Control 8 0 1.010 0.836 1.308 OC-28 8 0 0.480 0.332 0.539 OC-29 8 0 0.0964 0.0833 0.104 OC-30 8 0 0.0970 0.0699 0.148 OC-31 8 0 0.760 0.420 0.944 H = 27.799 with 4 degrees of freedom. (P = <0.001) The differences in the median values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001) To isolate the group or groups that differ from the others use a multiple comparison procedure. Multiple Comparisons versus Control Group (Dunn's Method) : Comparison Diff of Ranks Q P<0.05 OC-29 vs Control 25.375 4.341 Yes OC-30 vs Control 25.000 4.277 Yes OC-28 vs Control 11.750 2.010 No OC-31 vs Control 9.125 1.561 Do Not Test Note: The multiple comparisons on ranks do not include an adjustment for ties.


32

Test 3 Individual Dry Mass One Way Analysis of Variance Sunday, January 30, 2011, 8:36:52 PM Data source: Test 5 Mass Data in Stats Dependent Variable: D-Mass Normality Test: Failed (P < 0.050) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, January 30, 2011, 8:36:52 PM Data source: Test 5 Mass Data in Stats Group N Missing Median 25% 75% Control 8 0 1.743 1.551 2.054 OC-28 8 0 0.789 0.593 0.807 OC-29 8 0 0.186 0.171 0.221 OC-30 8 0 0.276 0.192 0.310 OC-31 8 0 1.218 0.922 1.537 H = 34.458 with 4 degrees of freedom. (P = <0.001) The differences in the median values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001) To isolate the group or groups that differ from the others use a multiple comparison procedure. Multiple Comparisons versus Control Group (Dunn's Method) : Comparison Diff of Ranks Q P<0.05 OC-29 vs Control 29.125 4.983 Yes OC-30 vs Control 25.375 4.341 Yes OC-28 vs Control 14.250 2.438 No OC-31 vs Control 7.500 1.283 Do Not Test Note: The multiple comparisons on ranks do not include an adjustment for ties.


33

Test 3 Individual Ash-Free Mass One Way Analysis of Variance Sunday, January 09, 2011, 3:06:42 AM Data source: Data 10 in Stats Normality Test: Failed (P = <0.001) Test execution ended by user request, ANOVA on Ranks begun Kruskal-Wallis One Way Analysis of Variance on Ranks Sunday, January 09, 2011, 3:06:42 AM Data source: Data 10 in Stats Group N Missing Median 25% 75% Control 8 0 1.305 1.163 1.541 OC-28 8 0 0.588 0.441 0.607 OC-29 8 0 0.139 0.128 0.168 OC-30 8 0 0.207 0.146 0.234 OC-31 8 0 0.915 0.701 1.151 H = 34.570 with 4 degrees of freedom. (P = <0.001) The differences in the median values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001) To isolate the group or groups that differ from the others use a multiple comparison procedure. Multiple Comparisons versus Control Group (Dunnett's Method) : Comparison Diff of Ranks q' P<0.05 OC-29 vs Control 234.000 5.004 Yes OC-30 vs Control 204.000 4.363 Yes OC-28 vs Control 115.000 2.459 Yes OC-31 vs Control 62.000 1.326 No Note: The multiple comparisons on ranks do not include an adjustment for ties.


34

Appendix E. Chain of Custody sheets


35


36


37


38


39

Appendix F. Data Sheets for Chironomus dilutus sediment toxicity bioassays Testing for Duck and Otter Creeks data gap and confluence site investigations were conducted simultaneously. The test number in the data sheets corresponds to when the sediments were evaluated. For the confluence site investigation, tests 1, 2, and 3 described in the report correspond with test 3, 4, and 5 in the data sheets. TEST 1


40


41


42


43


44


45


46


47


48


49


50


51


52


53


54

TEST 2


55


56


57


58


59


60


61


62


63


64


65


66


67


68


69

TEST 3


70


71


72


73


74


75


76


77


78


79


80


81


82


83

Documents

US ARMY CORPS OF ENGINEERS - ACUTE TOXICITY ...Department of the Army US Army Engineer Research and Development Center Environmental Laboratory 3909 Halls Ferry Rd, EP-R Vicksburg,