Anaheim_Bay_SAR_Appendices_Part_3_Final.pdf

Appendix J Bioassay Report

Amy Howk November 9, 2017 Kinnetic Laboratories, Inc.

307 Washington St.

Santa Cruz, CA 95060

Dear Ms. Howk:

Please find attached an electronic copy of the report “Biological Testing of the Sediment Samples Collected from Anaheim Bay” in PDF format. Hard copies of this report can be provided upon request.

If you have any questions, please give my colleague Jeff Cotsifas or myself a call at (707) 207- 7760.

Sincerely, Mike McElroy Project Manager

Pacific EcoRisk is accredited in accordance with NELAP (ORELAP ID 4043). Pacific EcoRisk certifies that the test results reported herein conform to the most current NELAP requirements for parameters for which accreditation is required and available. Any exceptions to NELAP requirements are noted, where applicable, in the body of the report. This report shall not be reproduced, except in full, without the written consent of Pacific EcoRisk. This testing was performed under Lab Order 27827.

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DATA REPORT

Biological Testing of the Sediment Samples

Collected from Anaheim Bay

Prepared for

Kinnetic Laboratories, Inc.

307 Washington St.

Santa Cruz, CA 95060

Prepared by

Pacific EcoRisk

2250 Cordelia Road Fairfield, CA 94534

November 2017

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Pacific EcoRisk Environmental Consulting and Testing i

Table of Contents Page

1. INTRODUCTION

2. METHODS

2.1 Biological Testing Procedures

2.2 Receipt and Handling of Sediment Samples

2.3 Source of Natural Seawater

2.4 Sediment Porewater Characterization

2.5 Solid-Phase Sediment Toxicity Testing with Ampelisca abdita

2.5.1 Reference Toxicant Testing of the Ampelisca abdita

2.6 Solid-Phase Sediment Toxicity Testing with Neanthes arenaceodentata

2.6.1 Reference Toxicant Testing of the Neanthes arenaceodentata

2.7 Water Column (Sediment Elutriate) Toxicity Test Procedures

2.7.1 Preparation of Sediment Elutriate

2.7.2 Sediment Elutriate Toxicity Testing with Mytilus galloprovincialis

2.7.2.1 Preparation of Bivalve Embryos

2.7.2.2 Embryo Development Toxicity Test Procedures

2.7.2.3 Reference Toxicant Testing of the Mytilus galloprovincialis Embryos

2.7.3 Sediment Elutriate Toxicity Testing with Americamysis bahia

2.7.3.1 Reference Toxicant Testing of the Americamysis bahia

2.7.4 Sediment Elutriate Toxicity Testing with Menidia beryllina

2.7.4.1 Reference Toxicant Testing of the Menidia beryllina

2.8 Sediment Bioaccumulation Testing with the Bivalve Macoma nasuta and the Polychaete Nereis virens

3. BIOLOGICAL TESTING RESULTS

3.1 Effects of Anaheim Bay Sediments on Ampelisca abdita

3.1.1 Reference Toxicant Toxicity to Ampelisca abdita

3.2 Effects of Anaheim Bay Sediments on Neanthes arenaceodentata

3.2.1 Reference Toxicant Toxicity to Neanthes arenaceodentata

3.3 Toxicity of Anaheim Bay Sediment Elutriates to Mytilus galloprovincialis

3.3.1 Reference Toxicant Toxicity to Mytilus galloprovincialis Embryos

3.4 Toxicity of Anaheim Bay Sediment Elutriates to Americamysis bahia

3.4.1 Reference Toxicant Toxicity to Americamysis bahia

3.5 Toxicity of Anaheim Bay Sediment Elutriates to Menidia beryllina

3.5.1 Reference Toxicant Toxicity to Menidia beryllina

3.6 Sediment Bioaccumulation Test Data for Macoma nasuta

3.7 Sediment Bioaccumulation Test Data for Nereis virens

3.8 Biological Testing QA/QC Summary

4. REFERENCES

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Appendices

Appendix A Chain-of-Custody Records for the Collection and Delivery of Anaheim Bay

Sediments

Appendix B Water Quality Characteristics for the Whole Sediment Test Porewater and

Overlying Water

Appendix C Test Data and Summary of Statistics for the Toxicity Evaluation of Anaheim Bay

Sediments with the Amphipod, Ampelisca abdita

Appendix D Test Data and Summary of Statistics for the Reference Toxicant Evaluation of the

Amphipod, Ampelisca abdita

Appendix E Test Data and Summary of Statistics for the Toxicity Evaluation of Anaheim Bay

Sediments with the Polychaete, Neanthes arenaceodentata

Appendix F Test Data and Summary of Statistics for the Reference Toxicant Evaluation of the

Polychaete, Neanthes arenaceodentata

Appendix G Test Data and Summary of Statistics for the Toxicity Evaluation of Anaheim Bay

Sediment Elutriates with Bivalve (Mytilus galloprovincialis) Embryos

Appendix H Test Data and Summary of Statistics for the Reference Toxicant Evaluation of the

Mytilus galloprovincialis Embryos

Appendix I Test Data and Summary of Statistics for the Toxicity Evaluation of Anaheim Bay

Sediment Elutriates with Mysids (Americamysis bahia)

Appendix J Test Data and Summary of Statistics for the Reference Toxicant Evaluation of the

Mysid, Americamysis bahia

Appendix K Test Data and Summary of Statistics for the Toxicity Evaluation of Anaheim Bay

Sediment Elutriates with the Inland Silverside (Menidia beryllina)

Appendix L Test Data and Summary of Statistics for the Reference Toxicant Evaluation of the

Inland Silverside, Menidia beryllina

Appendix M Test Data for Anaheim Bay Sediment Bioaccumulation Tests with the Bivalve, Macoma nasuta

Appendix N Test Data for Anaheim Bay Sediment Bioaccumulation Tests with the Polychaete, Nereis virens

Appendix O Bioassay Standard Test Conditions

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List of Tables Page

Table 2-1. Sediment Porewater Initial Water Quality Characteristics Table 3-1. Ampelisca abdita Survival in Anaheim Bay Sediments Table 3-2. Reference Toxicant Testing: Effects of KCl on Ampelisca abdita Table 3-3. Neanthes arenaceodentata Survival in Anaheim Bay Sediments Table 3-4. Reference Toxicant Testing: Effects of KCl on Neanthes arenaceodentata Table 3-5. Effects of ABHVC-17-A Sediment Elutriate on Mytilus galloprovincialis Table 3-6. Effects of ABHVC-17-B Sediment Elutriate on Mytilus galloprovincialis Table 3-7. Effects of ABHVC-17-C Sediment Elutriate on Mytilus galloprovincialis Table 3-8. Effects of ABHVC-17-D Sediment Elutriate on Mytilus galloprovincialis Table 3-9. Reference Toxicant Testing: Effects of KCl on Mytilus galloprovincialis Table 3-10. Effects of ABHVC-17-A Sediment Elutriate on Americamysis bahia Table 3-11. Effects of ABHVC-17-B Sediment Elutriate on Americamysis bahia Table 3-12. Effects of ABHVC-17-C Sediment Elutriate on Americamysis bahia Table 3-13. Effects of ABHVC-17-D Sediment Elutriate on Americamysis bahia Table 3-14. Reference Toxicant Testing: Effects of KCl on Americamysis bahia Table 3-15. Effects of ABHVC-17-A Sediment Elutriate on Menidia beryllina Table 3-16. Effects of ABHVC-17-B Sediment Elutriate on Menidia beryllina Table 3-17. Effects of ABHVC-17-C Sediment Elutriate on Menidia beryllina Table 3-18. Effects of ABHVC-17-D Sediment Elutriate on Menidia beryllina Table 3-19. Reference Toxicant Testing: Effects of KCl on Menidia beryllina Table 3-20 Sediment Bioaccumulation Test with Macoma nasuta Table 3-21. Sediment Bioaccumulation Test with Nereis virens

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1. INTRODUCTION

Kinnetic Laboratories, Inc., has contracted Pacific EcoRisk (PER) to perform whole sediment and water column (sediment elutriate) bioassay testing of sediments in support of Anaheim Bay maintenance dredging sampling and testing program. The performance and results of this testing are presented in this report.

2. METHODS

2.1 Biological Testing Procedures

There were up to seven different biological tests performed for each site composite sample:

1. a 10-day sediment amphipod survival test with Ampelisca abdita;

2. a 10-day sediment juvenile polychaete survival test with Neanthes arenaceodentata;

3. a 48-hr standard elutriate bivalve embryo survival and development test with Mytilus galloprovincialis;

4. a 96-hr standard elutriate mysid survival test with Americamysis bahia;

5. a 96-hr standard elutriate larval fish survival test with Menidia beryllina;

6. a 28-day bioaccumulation test with the clam Macoma nasuta; and

7. a 28-day bioaccumulation test with the polychaete Nereis virens.

The methods used in conducting these tests followed established guidelines:

• Method E1367-03. Standard Test Method for Measuring the Toxicity of Sediment-

Associated Contaminants with Estuarine and Marine Invertebrates. (ASTM 2015);

• Method E724-98. Standard Guide for Conducting Static Acute Toxicity Tests Starting with

Embryos of Four Species of Saltwater Bivalve Molluscs. (ASTM 2015);

• Method E1611-00. Standard Guide for Conducting Sediment Toxicity Tests with

Polychaetous Annelids. (ASTM 2015);

• Method E1688-10. Standard Guide for Determination of the Bioaccumulation of Sediment-

Associated Contaminants by Benthic Invertebrates. (ASTM 2015);

• Testing Manual for the Evaluation of Dredged Material Proposed for Discharge in Waters of the U.S. (Inland Testing Manual, US EPA/ACOE, 1998);

• Testing Manual for the Evaluation of Dredged Material Proposed for Ocean Disposal.

(Ocean Testing Manual, US EPA/ACOE, 1991);

• Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to

Freshwater and Marine Organisms, Fifth Edition. (US EPA, 2002); and

• Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods. (US EPA 1994).

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2.2 Receipt and Handling of Sediment Samples

During the period of August 18-23, sediment samples were collected from Anaheim Bay; a ‘site water’ sample was also collected for use in preparing the sediment elutriates. These samples were delivered to the PER testing lab in Fairfield, CA, on ice and under chain-of-custody, on August 29, 2017. Upon receipt at the PER testing laboratory, all samples were logged in and stored at 4°C in the dark until needed. The chain-of-custody records for the collection and delivery of these samples are provided in Appendix A.

In addition, sediment from Paradise Cove, located in San Francisco Bay, was collected by PER for use as Lab Control sediment in the A. abdita and N. arenacoedentata toxicity tests and the M.

nasuta and N. virens bioaccumulation tests.

2.3 Source of Natural Seawater

The natural seawater used in these tests was obtained from the UC Davis Granite Canyon Marine Laboratory, and is characterized as “pristine”; this water was stored at the PER laboratory in a 3000-gallon insulated HDPE tank maintained at 4˚C. This seawater was 1-µm filtered and then adjusted to the desired test salinity (e.g., 30 ppt) via addition of Type 1 lab water (reverse-osmosis, de-ionized water) prior to use in these tests (these diluted natural seawaters are referred to using the adjusted salinity level [e.g., ‘30 ppt seawater’]).

2.4 Sediment Porewater Characterization

Upon receipt, each Anaheim Bay sediment sample was homogenized in a large stainless steel bowl. Aliquots of the homogenized site sediments were centrifuged at 2,500 rpm for 15 minutes;

the resulting supernatant porewaters were carefully collected and analyzed for routine water quality characteristics (Table 2-1).

Table 2-1. Sediment Porewater Initial Water Quality Characteristics.

Sample ID pH Salinity (ppt) Total Ammonia (mg/L N)

Total Sulfide (mg/L)

ABH-17-LA2 7.66 31.1 5.92 0.050

ABHVC-17-A NM NM 9.20 0.550

ABHVC-17-B NM NM 13.4 0.328

ABHVC-17-C 7.91 32.4 6.89 0.056

ABHVC-17-D NM NM 14.1 0.438

*Note – Chemistry was not measured due to a lack of porewater volume.

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2.5 Solid-Phase Sediment Toxicity Testing with Ampelisca abdita

The A. abdita used in these tests were obtained from a commercial supplier (Aquatic Research Organisms [ARO], Hampton, NH). The A. abdita were maintained at a salinity of 28 ppt at 20˚C prior to use in the testing.

These tests were initiated on September 11, 2017. On the day preceding test initiation, the test replicates for the Lab Control sediment were set-up. Five replicates were established for each of the sediment treatments, each replicate consisting of a 1-L glass beaker to which homogenized sediment was added to a depth of approximately 2-cm; additional “porewater” test replicates were similarly prepared for the determination of sediment porewater water quality characteristics at test initiation and at test termination. The overlying water for this testing consisted of 28 ppt seawater; approximately 800 mL of the 28 ppt seawater was carefully poured into each test replicate so as to minimize disturbance of the sediment. These test replicates were maintained in a temperature-controlled room at 20˚C under continuous illumination from fluorescent lighting.

Each test replicate was gently aerated.

The following day, and immediately prior to test initiation, routine water quality characteristics (temperature, pH, dissolved oxygen [D.O.], and salinity) were determined for the overlying water in each test replicate; in addition, a small sample of the overlying water was collected from each replicate and composited for each treatment for determination of the total ammonia in the overlying water at that treatment. At this time, one of the “porewater” test replicates was sacrificed for the determination of “initial” porewater water quality characteristics (Appendix B).

The tests were then initiated with the allocation of 20 randomly selected A. abdita into each replicate container (aeration was shut off until the amphipods re-buried themselves, approximately 1 hr after their introduction). Each day, for the next nine days, the temperature, pH, D.O., and salinity of the overlying water were measured in one test replicate for each treatment.

After 10 days exposure, routine water quality characteristics (temperature, pH, D.O., and salinity) were again determined for each test replicate; in addition, a small sample of the overlying water was collected from each replicate and composited for each treatment for determination of the total ammonia in the overlying water at that treatment. At this time, the remaining “porewater” test replicate was sacrificed for the determination of “final” porewater water quality characteristics (Appendix B). The contents of each replicate beaker were then sieved and examined, and the surviving amphipods were collected and counted. The resulting survival data were statistically analyzed using the CETIS® statistical software (Tidepool Scientific, McKinleyville, CA). The results of these tests are summarized in Section 3.1.

2.5.1 Reference Toxicant Testing of the Ampelisca abdita

In order to assess the sensitivity of the organisms used in these tests to chemical stress, concurrent reference toxicant testing was performed. The reference toxicant test was performed

8/181 as a 96-hr static waterborne exposure using test solutions consisting of 20 ppt seawater spiked with potassium chloride (KCl) at test concentrations of 0.25, 0.5, 1, 2, and 4 g/L.

There were two replicates at each treatment, each replicate consisting of 400 mL of test solution in a 600-mL HDPE beaker. The test was initiated by randomly allocating 10 amphipods into each replicate beaker. The beakers were placed in a temperature-controlled room at 20˚C under continual darkness. Routine water quality characteristics (D.O., pH, and temperature) of the treatment waters were measured and recorded for one randomly selected replicate per treatment each day.

After ~96 hrs, the number of live amphipods in each replicate beaker was determined. The resulting test response data were statistically analyzed to determine key concentration-response point estimates (e.g., EC50); all statistical analyses were made using the CETIS® software. These response endpoints were then compared to the typical response range established by the mean ± 2 SD of the point estimates generated by the 20 most recent previous reference toxicant tests performed by this lab. The results of these tests are summarized in Section 3.1.1.

2.6 Solid-Phase Sediment Toxicity Testing with Neanthes arenaceodentata

The N. arenaceodentata used in these tests were obtained from a commercial supplier (Aquatic Toxicology Support [ATS], Bremerton, WA), and were maintained at a salinity of 30 ppt prior to shipment to the testing lab; upon receipt, the test organisms were held in 30 ppt seawater at 20˚C.

These sediment tests were initiated on September 12, 2017. On the day preceding test initiation, the test replicates for the Lab Control sediment were set-up. Five replicates were established for each of the sediment treatments, each replicate consisting of a 1-L glass beaker to which approximately 200 mL (approximately 2.5 cm depth) of homogenized sediment was added;

additional test replicates were set up for the determination of sediment porewater water quality characteristics at test initiation and at test termination. The overlying water consisted of 30 ppt seawater; approximately 800 mL of this water was carefully poured into each test replicate so as to minimize disturbance of the sediment. The test replicates were then placed in a temperature-controlled room at 20˚C, under cool white fluorescent lighting on a 12L:12D photoperiod. Each test replicate was gently aerated.

The following day, and immediately prior to test initiation, routine water quality characteristics (temperature, pH, D.O., and salinity) were determined for the overlying water in each test replicate; in addition, a small sample of the overlying water was collected from each replicate and composited for each treatment for determination of the total ammonia in the overlying water at that treatment. At this time, one of the “porewater” test replicates were sacrificed for the determination of “initial” porewater water quality characteristics (Appendix B). The tests were then initiated with the allocation of 10 randomly selected polychaetes into each replicate container (aeration was shut off until the polychaetes re-buried themselves, approximately 1 hr

9/181 after their introduction). Each day, for the next 10 days, the temperature, pH, D.O., and salinity of the overlying water were measured in one test replicate for each treatment.

After 10 days exposure, routine water quality characteristics (temperature, pH, D.O., and salinity) were again determined for each test replicate; in addition, a small sample of the overlying water was collected from each replicate and composited for each treatment for determination of the total ammonia in the overlying water at that treatment. At this time, the remaining “porewater” test replicate was sacrificed for the determination of “final” porewater water quality characteristics (Appendix B). The contents of each replicate beaker were then sieved and examined, and the surviving polychaetes were collected and counted. The resulting survival data were statistically analyzed using the CETIS® statistical software. The results of these tests are summarized in Section 3.2.

2.6.1 Reference Toxicant Testing of the Neanthes arenaceodentata In order to assess the sensitivity of the organisms used in these tests to chemical stress, concurrent reference toxicant testing was performed. The reference toxicant test consists of a static acute 96-hr survival toxicity test of waterborne KCl, at test treatment concentrations of 0.25, 0.5, 1, 2, and 4, g/L.

There were two replicates at each treatment, each replicate consisting of 400 mL of test media in a 600-mL HDPE beaker. The test was initiated by randomly allocating five polychaetes into each replicate beaker. The beakers were placed in a temperature-controlled room at 20˚C under continual darkness. Each replicate container was examined daily, and the number of live polychaetes in each was recorded at this time. Routine water quality characteristics (temperature, pH, D.O., and salinity) of the treatment waters were measured and recorded for one randomly selected replicate per treatment each day.

After ~96 hrs, the number of live organisms in each replicate beaker was determined. The resulting test response data were statistically analyzed to determine key concentration-response point estimates (e.g., EC50); all statistical analyses were made using the CETIS® software. These response endpoints were then compared to the typical response range established by the mean ± 2 SD of the point estimates generated by the 20 most recent previous reference toxicant tests performed by this lab. The results of this test are summarized in Section 3.2.1.

2.7 Water Column (Sediment Elutriate) Toxicity Test Procedures

2.7.1 Preparation of Sediment Elutriate

Sediment elutriates were prepared for each of the site composite sediments by mixing each sediment with site water at a 1:4 ratio for 30 minutes. After mixing, the sediment-site water slurries were allowed to settle for 1 hour. The resulting supernatant comprised the 100% elutriate test solution for each sample.

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2.7.2 Sediment Elutriate Toxicity Testing with Mytilus galloprovincialis The sediment elutriate toxicity test with M. galloprovincialis consists of exposing the mussel embryos to the sediment elutriates for ~ 48 hrs, after which the effects on embryo survival and development are determined. The specific procedures used in these tests are described below.

The sediment elutriate tests with M. galloprovincialis embryos were initiated on September 14, 2017.

2.7.2.1 Preparation of Bivalve Embryos - The adult bivalves used to provide the embryos for these tests were obtained from commercial suppliers (Dave Gutoff, San Diego, CA and Taylor Shellfish, Shelton, WA); upon receipt in the laboratory, the adult bivalves were placed in small tanks of 30 ppt seawater at 12˚C where they were held until used to produce embryos later that same day.

The adult bivalves were rinsed thoroughly with 30 ppt seawater and then placed into holding tanks containing 30 ppt seawater at 20˚C in order to induce spawning; spawning individuals were subsequently placed into separate beakers containing 30 ppt seawater in order to isolate and collect gametes.

Samples of the gametes from spawning males and females were collected and evaluated for quality by visual inspection. For males exhibiting good sperm viability, overlying waters (containing the sperm) from the beakers containing spawning males were decanted off and pooled. The overlying waters (containing eggs) from the females exhibiting the best egg quality were also pooled, and then concentrated (or diluted) to provide an egg suspension of ~50 eggs/mL.

Fertilization of the eggs was accomplished by addition of sperm at a density of 105 to 107 sperm/mL. The resulting embryos were then placed in a temperature-controlled room at 15°C until being used for inoculation of test solutions (inoculation was initiated within 4 hrs of fertilization).

2.7.2.2 Embryo Development Toxicity Test Procedures - The Lab Water Control medium for these bioassays consisted of 30 ppt seawater. The Lab Water Control medium and the 100% sediment elutriate solutions were used to prepare fresh test solutions at additional interim test treatment concentrations of 1%, 10%, and 50% elutriate for each of the site sediments; as the sediment porewater concentrations of ammonia exceeded 25 mg/L (Table 2-1) for each of the site sediments, an additional test treatment of 25% elutriate was also prepared and tested for each sediment. The site water that was used to prepare the elutriates was also tested (at the 100% concentration only). As an additional QA measure, and in order to assess any potential inhibitory effects resulting from the addition of the artificial sea salt to the elutriates, a “Salt Control” consisting of Type 1 lab water adjusted to the test salinity of 30 ppt using the artificial sea salt was also prepared and tested. Routine water quality characteristics (pH, D.O., salinity, and total ammonia) were measured for each treatment test solution prior to use in these tests.

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There were five replicates at each treatment level, each replicate consisting of 10 mL of test solution in a 20-mL glass scintillation vial. Additional vials were established at each treatment for determination of final water quality characteristics; additional Lab Control treatment “observation” vials were established for confirmation of appropriate embryo development prior to test termination. The tests were initiated by randomly inoculating approximately 150-300 fertilized bivalve embryos into each vial. The vials were then randomly positioned in a temperature-controlled room at 16˚C under a 16L:8D photoperiod.

After 48 (±2) hrs, the Lab Control treatment “observation” vials were examined to confirm that the test organisms had achieved an acceptable degree of embryo development, after which the test was terminated. Routine water quality characteristics were determined for the ‘water quality’ replicates. The embryos in the test vials were immediately preserved with the addition of 1 mL of a 5% glutaraldehyde solution in filtered seawater.

The contents of each preserved vial were subsequently examined microscopically and the number of embryos that survived and developed normally or abnormally were determined. The resulting survival and development data were then statistically analyzed and key concentration-response EC point estimates determined for each site sediment elutriate using the CETIS® statistical software. The results of these tests are summarized in Section 3.3.

2.7.2.3 Reference Toxicant Testing of the Mytilus galloprovincialis Embryos - In order to assess the sensitivity of the mussel embryos to toxic stress, a reference toxicant test was performed. This reference toxicant test was performed similarly to the water column toxicity test, except that test solutions consisted of Lab Water Control medium (30 ppt seawater) spiked with KCl at concentrations of 0.5, 1, 2, 3, and 4 g/L. The resulting test response data were analyzed to determine key concentration-response point estimates (e.g., EC50); all statistical analyses were made using the CETIS® software. These response endpoints were then compared to the typical response range established by the mean ± 2 SD of the point estimates generated by the 20 most recent previous reference toxicant tests performed by this lab. The results of this test are summarized in Section 3.3.1.

2.7.3 Sediment Elutriate Toxicity Testing with Americamysis bahia The sediment elutriate toxicity test with A. bahia consists of exposing the mysids to the sediment elutriates for ~ 96-hrs, after which the effects on survival determined. The specific procedures used in this test are described below. The sediment elutriate tests with A. bahia were initiated on September 14, 2017.

The A. bahia used in these tests were obtained from commercial suppliers (Aquatic Indicators [AI], St. Augustine, FL); upon receipt in the laboratory, the mysids were maintained in small tanks of 25 ppt seawater at 20°C, and were fed brine shrimp nauplii ad libitum.

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The Lab Water Control medium for these tests consisted of 25 ppt seawater. The Lab Water Control medium and the 100% sediment elutriates were used to prepare test solutions at additional interim test treatment concentrations of 1%, 10%, and 50% elutriate for each of the site sediments; as the sediment porewater concentrations of ammonia exceeded 25 mg/L (Table 2-1) for each of the site sediments, additional test treatments of 25% elutriate were also prepared and tested. The site water that was used to prepare the elutriates was also tested (at the 100% concentration only). Initial routine water quality characteristics (temperature, pH, D.O., and salinity) were measured for each treatment test solution prior to use in these tests.

There were five test replicates at each treatment, each replicate consisting of a 400-mL glass beaker containing 200 mL of appropriate test solution. The tests were initiated with the allocation of 10 randomly selected 5-day old mysids into each test replicate. The test replicates were then placed into a temperature-controlled room at 20°C under a 16L:8D photoperiod.

Each day, water quality conditions were determined for one randomly selected replicate per treatment, and the test replicates were examined to determine the number of surviving organisms, with any dead organisms being removed via pipette. Each replicate was fed brine shrimp nauplii daily.

After 96 (±2) hrs exposure, the tests were terminated. At test termination, the final water quality conditions were determined for one randomly selected replicate per treatment, after which each of the test replicates was examined to determine the number of surviving mysids. The resulting survival data were then statistically analyzed and key concentration-response EC point estimates determined for each site sediment elutriate using the CETIS® statistical software. The results of these tests are summarized in Section 3.4.

2.7.3.1 Reference Toxicant Testing of the Americamysis bahia - In order to assess the sensitivity of these test organisms to chemical stress, a reference toxicant test was performed concurrently with the elutriate tests. The reference toxicant test was performed similarly to the sediment elutriate tests, but used test solutions consisting of Lab Water Control medium spiked with waterborne KCl at test concentrations of 0.125, 0.25, 0.5, 1, and 2 g/L, instead of elutriate dilutions. The resulting test response data were statistically analyzed to determine key concentration-response point estimates (e.g., EC50); all statistical analyses were made using the CETIS® software. These response endpoints were then compared to the typical response range established by the mean ± 2 SD of the point estimates generated by the 20 most-recent previous reference toxicant tests performed by this lab. The results of this test are summarized in Section 3.4.1.

2.7.4 Sediment Elutriate Toxicity Testing with Menidia beryllina The sediment elutriate toxicity test with M. beryllina consists of exposing the larval fish to the sediment elutriates for ~ 96-hrs, after the effects on survival are determined. The specific

13/181 procedures used in this test are described below. The sediment elutriate toxicity tests with M.

beryllina were initiated on September 13, 2017.

The M. beryllina used in the sediment elutriate tests were obtained from commercial suppliers (AI); upon receipt in the laboratory, the larval fish were maintained in small tanks of 25 ppt seawater at 20°C, and were fed brine shrimp nauplii ad libitum.

The Lab Water Control medium for these tests consisted of 25 ppt seawater. The Lab Water Control medium and the 100% sediment elutriates were used to prepare test solutions at additional interim test treatment concentrations of 1%, 10%, and 50% elutriate for each of the site sediments; as the sediment porewater concentrations of ammonia exceeded 25 mg/L (Table 2-1) for each of the site sediments, an additional test treatment of 25% elutriate was also prepared and tested for each sediment. The site water that was used to prepare the elutriates was also tested (at the 100% concentration only). Initial routine water quality characteristics (temperature, pH, D.O., and salinity) were measured for each treatment test solution prior to use in these tests.

There were five test replicates at each treatment, each replicate consisting of a 400-mL glass beaker containing 200 mL of appropriate test solution. These tests were initiated with the allocation of 10 randomly selected 12-day old fish into each test replicate. The test replicates were then placed into a temperature-controlled room at 20°C under a 16L:8D photoperiod.

Each day, the water quality conditions were determined for one randomly selected replicate per treatment, and the test replicates were examined to determine the number of surviving organisms, with any dead organisms being removed via pipette. Each replicate was fed brine shrimp nauplii daily.

After 96 (±2) hrs exposure, the tests were terminated. At test termination, the final water quality conditions were determined for one randomly selected replicate per treatment, after which each of the test replicates was examined to determine the number of surviving fish. The resulting survival data were then statistically analyzed and key concentration-response EC point estimates determined for each site sediment elutriate using the CETIS® statistical software. The results of these tests are summarized in Section 3.5.

2.7.4.1 Reference Toxicant Testing of the Menidia beryllina - In order to assess the sensitivity of these test organisms to chemical stress, a reference toxicant test was performed concurrently with the elutriate tests. The reference toxicant test was performed similarly to the sediment elutriate tests, but used test solutions consisting of Lab Water Control medium spiked with waterborne KCl at test concentrations of 0.125, 0.25, 0.5, 1, and 2 g/L, instead of elutriate dilutions. The resulting test response data were analyzed to determine key concentration-response point estimates (e.g., EC50); all statistical analyses were made using the CETIS® software. These response endpoints were then compared to the typical response range established

14/181 by the mean ± 2 SD of the point estimates generated by the 20 most-recent previous reference toxicant tests performed by this lab. The results of this test are summarized in Section 3.5.1.

2.8 Sediment Bioaccumulation Testing with the Bivalve Macoma nasuta and the Polychaete

Nereis virens

Sediment bioaccumulation testing was conducted with the bivalve M. nasuta and the polychaete N. virens. Negative Lab Control treatments were tested concurrently with the bioassays and consisted of sediment collected during the collection of the test organisms.

The adult M. nasuta used in the sediment bioaccumulation tests were provided by a commercial supplier (J&G Gunstone Clams Inc., Port Townsend, WA), and were obtained from Sequim Bay, WA; upon receipt in the laboratory, the clams were maintained in small tanks of seawater at 15°C.

The N. virens used in the sediment bioaccumulation tests were provided by a commercial supplier (ARO, Hampton, NH); upon receipt in the laboratory, the polychaetes were maintained in small tanks of seawater at 15°C.

The M. nasuta and N. virens tests were initiated on September 6 and September 8, respectively.

There were five replicates for the Control treatment and each of the site sediments. Each replicate consisted of a 16-L HDPE tank containing approximately 4 L of homogenized sediment. Approximately 6 L of full-strength seawater was carefully poured into each tank so as to minimize disturbance of the sediment. The replicate tanks were then placed into a temperature-controlled room at 15°C. Each of the test replicates was gently aerated.

After ~24 hrs equilibration, routine water quality characteristics (temperature, pH, D.O., and salinity) were determined for each test replicate at each treatment. Then, 20 randomly-selected adult clams were loaded into each of the M. nasuta test replicates and 10 randomly-selected adult polychaetes were placed into each N. virens test replicate container. Additional bivalves and polychaetes were depurated for 24 hrs and collected for determination of T0 tissue concentrations (five replicates of 20 clams and 10 polychaetes each, placed inside HDPE bags which were sealed and labeled for identification, and then frozen for later analyses). Each day, for the next 28 days, the water quality characteristics of the overlying water were measured in one test replicate for each treatment; when observed, dead organisms or empty shells were removed from the test replicates. Approximately 80% of the overlying water in each replicate was carefully replaced three times per week.

After 28 days exposure, the sediment in each replicate was sieved and the surviving bivalves and polychaetes were collected and enumerated. The organisms were rinsed with clean seawater and then placed back into their original plastic tubs (now emptied of sediment and rinsed out) containing only 0.45 µm-filtered seawater to allow for depuration of the gastro-intestinal tracts.

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Approximately 24 hrs later, these organisms were collected and placed inside pre-cleaned sample containers that were sealed and labeled for identification. These organisms were then frozen and stored in a sample freezer. The frozen test organisms were subsequently shipped, on dry ice and under chain-of-custody, to Eurofins for remaining tissue processing and chemical analyses.

Summary tables of the total ammonia concentrations measured in the test overlying waters during the performance of the testing are presented in Appendix B.

The test organism survival results of the bioaccumulation test exposures are presented in Sections 3.6 and 3.7 for M. nasuta and N. virens, respectively.

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3. BIOLOGICAL TESTING RESULTS

There were up to seven different biological tests performed for each site composite sample:

1. a 10-day sediment amphipod survival test with Ampelisca abdita;

2. a 10-day sediment juvenile polychaete survival test with Neanthes arenaceodentata;

3. a 48-hr standard elutriate bivalve embryo survival and development test with Mytilus galloprovincialis;

4. a 96-hr standard elutriate mysid survival test with Americamysis bahia;

5. a 96-hr standard elutriate larval fish survival test with Menidia beryllina;

6. a 28-day bioaccumulation test with the clam Macoma nasuta; and

7. a 28-day bioaccumulation test with the polychaete Nereis virens.

A summary table of the whole-sediment tests’ water quality characteristics and sediment porewater water quality characteristics at test initiation and test termination are presented in Appendix B. Summaries of test conditions and test acceptability criteria are provided in Appendix O.

3.1 Effects of Anaheim Bay Sediments on Ampelisca abdita.

The results of these tests are summarized in Table 3-1. There was 91% survival in the Lab Control sediment, indicating an acceptable survival response by the test organisms. There were no significant reductions in survival in any of the site sediments. The test data and summary of statistical analyses for these tests are attached as Appendix C.

Table 3-1. Ampelisca abdita Survival in Anaheim Bay Sediments.

Sediment Site % Survival in Test Replicates Mean % Survival Rep A Rep B Rep C Rep D Rep E

Lab Control 90 85 90 95 95 91

ABH-17-LA2 95 80 90 100 95 92

ABHVC-17-A 80 70 75 80 85 78

ABHVC-17-B 70 80 85 90 85 82

ABHVC-17-C 85 40 80 90 85 76

ABHVC-17-D 90 85 90 85 95 89

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3.1.1 Reference Toxicant Toxicity to Ampelisca abdita

The results of this test are presented in Table 3-2. The LC50 for this test was consistent with the “typical response” range established by the reference toxicant test database for this species, indicating that these test organisms were responding to toxic stress in a typical fashion. The test data and summary of statistical analyses for this test are attached as Appendix D.

Table 3-2. Reference Toxicant Testing: Effects of KCl on Ampelisca abdita.

KCl Treatment (g/L) Mean % Survival

Lab Control 90

0.25 95

0.5 90 1 60* 2 0* 4 0*

LC50 = 1.09 g/L KCl Typical Response Range (mean ± 2 SD) = 0.53 – 1.98 g/L KCl

* The survival response at this treatment was significantly less than the Lab Control response at p < 0.05.

3.2 Effects of Anaheim Bay Sediments on Neanthes arenaceodentata

The results of these tests are summarized in Table 3-3. There was 100% survival in the Lab Control sediment, indicating an acceptable survival response by the test organisms. There were no significant reductions in survival in any of the site sediments. The test data and summary of statistical analyses for these tests are attached as Appendix E.

Table 3-3. Neanthes arenaceodentata Survival in Anaheim Bay Sediments.

Sediment Site % Survival in Test Replicates Mean

% Survival Rep A Rep B Rep C Rep D Rep E Lab Control 100 100 100 100 100 100

ABH-17-LA2 100 100 90 100 100 98

ABHVC-17-A 90 100 100 100 100 98

ABHVC-17-B 100 100 100 100 100 100

ABHVC-17-C 100 100 90 100 100 98

ABHVC-17-D 100 100 90 100 100 98

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3.2.1 Reference Toxicant Toxicity to Neanthes arenaceodentata The results of this test are presented in Table 3-4. The LC50 for this test was consistent with the “typical response” range established by the reference toxicant test database for this species, data and summary of statistical analyses for this test are presented in Appendix F.

Table 3-4. Reference Toxicant Testing: Effects of KCl on Neanthes arenaceodentata.

Lab Control 100

0.5 100 1 100 2 100 3 0* 4 0*

LC50 = 2.45 g/L KCl Typical Response Range (mean ± 2 SD) = 1.15 – 2.49 g/L KCl

* The survival response at this treatment was significantly less than the Lab Control response at p < 0.05.

3.3 Toxicity of Anaheim Bay Sediment Elutriates to Mytilus galloprovincialis

The results of these tests are summarized below in Tables 3-5 through 3-8. There was ≥94% embryo survival and ≥92% normal development in the Lab Control treatment, indicating acceptable responses by the test organisms. The test data and the summary of statistical analyses for these tests are presented in Appendix G.

Table 3-5. Effects of ABHVC-17-A Sediment Elutriate on Mytilus galloprovincialis.

Elutriate Treatment Mean % Survival Mean % Normal Development

Lab Control 96.6 92.0 1% 99.3 91.9 10% 94.4 92.5 50% 99.9 96.0 100% 94.5 93.6

Salinity Control 96.5 91.5 Site Water 94.5 90.3

Survival LC50 or Development EC50 = >100% Elutriatea >100% Elutriatea a - Due to the absence of significant impairment, both the LC50 and the EC50, could not be calculated but can be determined by inspection to be >100% elutriate.

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Table 3-6. Effects of ABHVC-17-B Sediment Elutriate on Mytilus galloprovincialis.

Elutriate Treatment Mean % Survival Mean % Normal Development

Lab Control 94.9 92.3 1% 95.7 93.1 10% 100 95.3 50% 99.3 93.7 100% 96.9 92.3

Salinity Control 96.5 91.5 Site Water 94.5 90.3

Survival LC50 or Development EC50 = >100% Elutriatea >100% Elutriatea a - Due to the absence of significant impairment, both the LC50 and the EC50, could not be calculated but can be determined by inspection to be

Table 3-7. Effects of ABHVC-17-C Sediment Elutriate on Mytilus galloprovincialis.

Development

Lab Control 98.7 93.3 1% 92.4 91.6 10% 97.7 91.5 50% 99.6 91.9 100% 93.7 91.0

Salinity Control 96.5 91.5 Site Water 94.5 90.3

Survival LC50 or Development EC50 = >100% Elutriatea >100% Elutriatea a - Due to the absence of significant impairment, both the LC50 and the EC50, could not be calculated but can be determined by inspection to be

Table 3-8. Effects of ABHVC-17-D Sediment Elutriate on Mytilus galloprovincialis.

Development

Lab Control 94.0 92.7 1% 99.0 92.3 10% 97.1 93.0 50% 95.5 94.5 100% 91.0 91.6

Salinity Control 96.5 91.5 Site Water 94.5 90.3

Survival LC50 or Development EC50 = >100% Elutriatea >100% Elutriatea a - Due to the absence of significant impairment, both the LC50 and the EC50, could not be calculated but can be determined by inspection to be

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3.3.1 Reference Toxicant Toxicity to Mytilus galloprovincialis Embryos The results of this test are presented in Table 3-9. The EC50 for this test was consistent with the “typical response” range established by the reference toxicant test database for this species, data and summary of statistical analyses for this test are attached as Appendix H.

Table 3-9. Reference Toxicant Testing: Effects of KCl on Mytilus galloprovincialis.

KCl Treatment (g/L) Mean % Normal Embryo Development

Lab Control 91.7

0.5 91.9 1 92.8 2 89.5 3 0* 4 0*

EC50 = 2.41 g/L KCl Typical Response Range (mean ± 2 SD) 1.42 – 2.95 g/L KCl

* The normal development response at this treatment was significantly less than the Lab Control response at p < 0.05.

3.4 Toxicity of Anaheim Bay Sediment Elutriates to Americamysis bahia

The results of these tests are summarized below in Tables 3-10 through 3-13. There was ≥96% survival in the Lab Control treatments, indicating acceptable survival responses by the test organisms; there was 96% survival in the Site Water Control treatment. The LC50 was >100% elutriate for each of the sediment elutriates. The test data and summary of statistical analyses for these tests are attached as Appendix I.

Table 3-10. Effects of ABHVC-17-A Sediment Elutriate on Americamysis bahia.

Elutriate Treatment Mean % Survival

Lab Control 96 1% 98 10% 98 50% 96 100% 98

Site Water 96 Survival LC50 = >100 % Elutriatea a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

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Table 3-11. Effects of ABHVC-17-B Sediment Elutriate on Americamysis bahia.

Elutriate Treatment Mean % Survival

Lab Control 96 1% 100 10% 98 50% 98 100% 100

Site Water 96 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

Table 3-12. Effects of ABHVC-17-C Sediment Elutriate on Americamysis bahia.

Lab Control 98 1% 98 10% 94 50% 98 100% 100

Site Water 96 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

Table 3-13. Effects of ABHVC-17-D Sediment Elutriate on Americamysis bahia.

Lab Control 96 1% 98 10% 98 50% 98 100% 90

Site Water 96 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

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3.4.1 Reference Toxicant Toxicity to Americamysis bahia

The results of this test are summarized in Table 3-14. The LC50 for this test was consistent with the “typical response” range established by the reference toxicant test database for this species, indicating that these test organisms were responding to toxic stress in a typical fashion. The test data and summary of statistical analyses for this test are attached as Appendix J.

Table 3-14. Reference Toxicant Testing: Effects of KCl on Americamysis bahia.

KCl Treatment (g/L) Mean % Survival Lab Control 100

0.125 95

0.25 97.5

0.5 67.5* 1 0* 2 0*

LC50 = 0.56 g/L KCl Typical Response Range (mean ± 2SD) = 0.28 – 0.62 g/L KCl

* The response at this test treatment was significantly less than the Control treatment response at p < 0.05.

3.5 Toxicity of Anaheim Bay Sediment Elutriates to Menidia beryllina

The results of these tests are summarized below in Tables 3-15 through 3-18. There was ≥98% survival in the Lab Control treatments, indicating acceptable survival responses by the test organisms; there was 100% survival in the Site Water Control treatment. The LC50 was >100% elutriate for each of the sediment elutriates. The test data and summary of statistical analyses for these tests are attached as Appendix K.

Table 3-15. Effects of ABHVC-17-A Sediment Elutriate on Menidia beryllina.

Lab Control 100 1% 100 10% 100 50% 100 100% 96

Site Water 100 Survival LC50 or Development EC50 = >100 % Elutriatea a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

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Table 3-16. Effects of ABHVC-17-B Sediment Elutriate on Menidia beryllina.

Elutriate Treatment Mean % Survival

Lab Control 100 1% 100 10% 100 50% 98 100% 98

Site Water 100 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

Table 3-17. Effects of ABHVC-17-C Sediment Elutriate on Menidia beryllina.

Lab Control 98

1% 100 10% 96 50% 96 100% 94

Site Water 100 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

Table 3-18. Effects of ABHVC-17-D Sediment Elutriate on Menidia beryllina.

Lab Control 100

1% 98 10% 96 50% 96 100% 96

Site Water 100 a - Due to the absence of significant impairment, the LC50 could not be calculated but can be determined by inspection to be >100% elutriate.

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3.5.1 Reference Toxicant Toxicity to Menidia beryllina

The results of this test are presented in Tables 3-19. The LC50 for this test was consistent with the “typical response” range established by the reference toxicant test database for this species, data and summary of statistical analyses for this test are attached as Appendix L.

Table 3-19. Reference Toxicant Testing: Effects of KCl on Menidia beryllina.

Lab Control 100

0.125 100

0.25 100

0.5 100 1 90 2 0*

LC50 = 1.32 g/L KCl Typical Response Range (mean ± 2SD) = 0.93 – 1.46 g/L KCl

* The response at this test treatment was significantly less than the Control treatment response at p < 0.05.

3.6 Sediment Bioaccumulation Test Data for Macoma nasuta

The survival responses for the bivalves in each of the sediment test treatments are summarized below in Table 3-20. The test data for these tests are attached as Appendix M.

Table 3-20. Sediment Bioaccumulation Test with Macoma nasuta.

Sample I.D.

Percent of Bivalves that Survived

Mean Rep A Rep B Rep C Rep D Rep E

Lab Control 100 100 100 90 95 97

ABH-17-LA2 97 100 95 95 100 97

ABHVC-17-A 100 95 90 100 100 97

ABHVC-17-B 90 95 100 100 100 97

ABHVC-17-C 100 90 100 95 95 96

ABHVC-17-D 100 95 100 95 90 96

Note - Replicate A of treatment ABH-17-LA2 was initiated with 30 clams. All other replicates were initiated with 20 clams per replicate.

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3.7 Sediment Bioaccumulation Test Data for Nereis virens

The survival responses for the polychaetes in each of the sediment test treatments are summarized below in Table 3-21. The test data for these tests are attached as Appendix N.

Table 3-21. Sediment Bioaccumulation Test with Nereis virens.

Sample I.D.

Percent of Polychaetes that Survived

Mean Rep A Rep B Rep C Rep D Rep E Lab Control 90 90 100 90 100 94

ABH-17-LA2 100 100 100 100 90 98

ABHVC-17-A 100 100 100 100 100 100

ABHVC-17-B 100 90 100 90 90 94

ABHVC-17-C 100 100 90 100 70 92

ABHVC-17-D 100 100 100 90 100 98

Note - All treatments were initiated with 10 polychaetes per replicate.

3.8 Biological Testing QA/QC Summary

The biological testing of Anaheim Bay sediments incorporated standard QA/QC procedures to ensure that the test results were valid, including the use of negative Lab Controls, positive Lab Controls, test replicates, and measurements of water quality during testing.

Quality assurance procedures that were used for sediment testing are consistent with methods described in the US EPA/ACOE (1991) and US EPA/ACOE (1998). Sediments for the bioassay testing were stored appropriately at ≤4°C and were used within the 8-week holding time period.

Sediment interstitial water characteristics were within test acceptability limits at the start of the tests. Sediment elutriates were prepared using site water. The toxicity test overlying waters consisted of high-quality natural seawater.

All measurements of routine water quality…