Results of Sediment Toxicity Testing for

Bight 18 - Santa Margarita River Estuary

Sample IDs: B18-10167 and B18-10166

Monitoring Period: August 2018

Prepared for: Weston Solutions, Inc. 5817 Dryden Place, Suite 101 Carlsbad, CA 92008

Prepared by: Nautilus Environmental 4340 Vandever Avenue

San Diego, CA 92120 (858) 587-7333

Date Submitted: December 11, 2018

Data Quality Assurance:

o Nautilus Environmental is accredited in accordance with NELAP by the State of Oregon Environmental Laboratory Accreditation Program (Certificate No. 4053). It is also certified by the State of California Department of Health Services Environmental Laboratory Accreditation Program (Certificate No. 1802) and the State of Washington Department of Ecology (Lab ID C552).

o All data have been reviewed and verified.

All test results have met minimum test acceptability criteria under their respective o EPA protocols, unless otherwise noted in this report.

o All test results have met internal Quality Assurance Program requirements.

Verified by:

California 4340 Vandever Avenue San Diego, California 92120 858.587.7333 Kasey Skrivseth, Project Manager fax: 858.587.3961

Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

TABLE OF CONTENTS

1.0 INTRODUCTION ...... 1

2.0 MATERIALS AND METHODS ...... 1 2.1 Sample Collection, Receipt, and Preparation ...... 1 2.2 Toxicity Test Methodology ...... 1 2.3 Statistical Analyses ...... 4 2.4 Testing Schedule ...... 4

3.0 RESULTS ...... 4 3.1 Solid-Phase Toxicity Tests ...... 5 3.2 Sediment-Water Interface Toxicity Tests ...... 5

4.0 QUALITY ASSURANCE ...... 6 4.1 Sample Receipt, Handling and Holding Time ...... 6 4.2 Solid-Phase Toxicity Tests ...... 6 4.3 Sediment-Water Interface Tests ...... 6 4.4 Reference Toxicant Tests ...... 6

5.0 REFERENCES ...... 7

Nautilus Environmental, San Diego, CA i Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

LIST OF TABLES

Table 1. Toxicity Test Methodology and QA/QC Requirements for the 10-day Solid Phase Amphipod Toxicity Test ...... 2

Table 2. Toxicity Test Methodology and QA/QC Requirements for Sediment Water Interface Bivalve Larvae Development Toxicity Tests ...... 3

Table 3. Toxicity Testing Schedule ...... 4

Table 4. Summary of Amphipod Solid-Phase Toxicity Test Results ...... 5

Table 5. Summary of Mussel Sediment-Water Interface Toxicity Test Results ...... 5

Table 7. Total and Un-ionized Ammonia Concentrations in Bulk Sediment - Sediment Porewater ...... Appendix D

Table 8. Total and Un-ionized Ammonia Concentrations in Solid-Phase Amphipod Toxicity Tests – Overlying Water ...... Appendix D

Table 9. Total and Un-ionized Ammonia Concentrations in Sediment-Water Interface Toxicity Tests – Overlying Water ...... Appendix D

LIS T OF APPENDICES Appendix A: Chain-of-Custody Forms and Sample Check-in Information

Appendix B: Statistical Analyses and Raw Data Packages for Eohaustorius

Appendix C: Statistical Analyses and Raw Data Packages for Mytilus

Appendix D: Ammonia tables and Raw Data Packages

Appendix E: Reference Toxicant Raw Data and Analyses

Appendix F: List of Laboratory Qualifier Codes

Nautilus Environmental, San Diego, CA ii Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

1.0 INTRODUCTION Weston Solutions, Inc. (Weston) has partnered with Nautilus Environmental (Nautilus) to perform sediment toxicity testing as part of the Santa Margarita River Estuary monitoring portion of the Southern California Bight 2018 Regional Marine Monitoring Survey (Bight’18) within the counties of San Diego and Riverside, which is coordinated by the Southern California Coastal Water Research Project (SCCWRP). Specific testing guidelines provided by SCCWRP for Bight’18 as well as the Sediment Quality Objectives (SQO) program were followed for this testing event (SCCWRP 2018 and 2009). This report summarizes results of the August 2018 sampling event. Two sediment samples were collected and evaluated using two test methods. These included: • 10-day amphipod Eohaustorius estuarius (Eohaustorius) solid-phase test (SCCWRP 2018 and 2009; USEPA 1994) • 48-hour mussel Mytilus galloprovincialis (Mytilus) larvae development sediment-water interface (SWI) test (SCCWRP 2018 and 2009; Anderson et al., 1996)

2.0 MATERIALS AND METHODS The methods employed to evaluate sediment toxicity are described below. 2.1 Sample Collection, Receipt, and Preparation Test site sediments for the Bight'18 program were collected on August 29, 2018 by Weston personnel. Approximately six to eight liters of sediment were provided from each site, in one double layered high density polyethylene plastic bag. Samples were delivered in coolers with wet ice to Nautilus by Weston personnel on August 30, 2018. Upon receipt, contents of coolers were verified against chain of custody forms. Temperatures were measured and recorded on a sample check-in form. All samples were stored at 4 ± 2°C in the dark until used for testing. Copies of chain of custody forms and sample check-in sheets are provided in Appendix A. For all testing, an aliquot of each sample was sieved through a 1.0-mm Nitex® mesh screen to remove potential native organisms and ensure reliable test organism recovery. Following homogenization, interstitial pore water samples were collected and analyzed for total ammonia content. Prior to testing, each sample was thoroughly homogenized. 2.2 Toxicity Test Methodology Test methods and acceptability criteria for the 10-day amphipod and the mussel SWI are described in Tables 1 and 2, respectively.

Nautilus Environmental, San Diego, CA 1 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

Table 1. Toxicity Test Methodology and QA/QC Requirements for the 10-day Solid Phase Amphipod Toxicity Test Test organism Marine Amphipod – Eohaustorius estuarius

Test organism source Northwest Aquatic Sciences; Newport, OR

Test organism size at initiation 3-5 mm

Test duration; endpoint 10 days; survival

Test solution renewal None

Feeding Prior to test initiation only

Test chamber 1-L glass jar

Sediment depth 2 cm

Overlying water volume 800 mL

Test temperature 15 ± 1°C test-wide mean, 15 ± 3°C instantaneous

Overlying water quality monitoring pH, temperature, salinity, and dissolved oxygen daily, total ammonia on days 0 and 10

Overlying water Natural seawater collected offshore SIO Pier in La Jolla, CA. 20-µm filtered. Seawater was diluted to 17 or 32 ppt with deionized water prior to testing.

Number of organisms per chamber 20 5, plus 1 surrogate test chamber for water quality Number of replicates readings Sieved sand collected near organism home site Negative controls (Lab Control #1 32 ppt)

Photoperiod Continuous light (24 hour)

Aeration Continuous (3-4 bubbles per second) SCCWRP 2018, 2009, and USEPA 600/R-94/025 Test Protocol (EPA 1994) Test acceptability criteria ≥ 90 percent mean survival in controls Reference toxicant Ammonium chloride

cm – centimeter °C – degrees Celsius L - Liter mL – milliliter mm - millimeter ppt – parts per thousand SIO – Scripps Institution of Oceanography µm - micrometer USEPA – United States Environmental Protection Agency

Nautilus Environmental, San Diego, CA 2 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

Table 2. Toxicity Test Methodology and QA/QC Requirements for Sediment Water Interface Bivalve Larvae Development Toxicity Tests Test organism Mediterranean mussel - Mytilus galloprovincialis

Test organism source Field collected locally; San Diego, CA and Taylor Shellfish; Shelton, WA Test duration, endpoint 48 hours, Normal-alive embryos Test solution renewal None Feeding None 250-mL polycarbonate screen tube inside 1-L Test chamber glass jar Screen Size 25-micron Test temperature 15 ± 1°C test-wide mean, 15 ± 3°C instantaneous Sediment depth 4 cm Overlying water volume 300 mL Overlying water quality monitoring pH, temperature, salinity, and dissolved oxygen daily, total ammonia on days 0 and 2 Overlying water 20-µm filtered natural seawater collected offshore Scripps Pier in La Jolla, CA. Seawater was diluted to 32 ppt with deionized water prior to testing.

Number of organisms per chamber ~300 larvae Number of replicates 5, with 1 surrogate chamber for daily water quality Negative controls Water only with screen tube Photoperiod 16 hours light:8 hours dark Aeration Continuous (1-2 bubbles per second) Test Protocol SCCWRP 2018, 2009, and Anderson et al. 1996 Test acceptability criteria Mean percent normal-alive ≥70% in controls Reference toxicant Ammonium chloride

cm – centimeter °C – degrees Celsius L - Liter mL – milliliter ppt – parts per thousand SIO – Scripps Institution of Oceanography µm - micrometer USEPA – United States Environmental Protection Agency

Nautilus Environmental, San Diego, CA 3 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

2.3 Statistical Analyses For the amphipod tests, mean survival in each sample was compared to the mean survival in the associated lab control. Amphipod survival data, expressed as a proportion, were arcsine square-root transformed prior to analysis to normalize the distribution of the data and satisfy statistical assumptions for analysis. Statistical assumptions were evaluated prior to analysis using Bartlett’s Test or F-test for differences in variance and D’Agostino Pearson Omnibus test for normality. A One-Way Analysis of Variance (ANOVA) was performed to determine if significant differences existed within groups of means of test sediments and associated lab controls. In addition, unpaired t-test comparisons with Welch's correction, as per SCCWRP guidance, were performed to identify significant differences between means of an individual sample compared to the associated lab control unless sample means matched or outperformed control mean. For the mussel tests mean percent normal-alive (number normal embryos/ initial number of embryos) in each sample was compared to the mean percent normal-alive of the lab control. Following guidance from SCCWRP, unpaired t-test comparisons with a Welch’s correction were performed for each sample in comparison to control unless sample means matched or outperformed the control means. Statistical analyses of test data were performed using GraphPad Prism, Version 6.05. Statistical analyses of the reference toxicant data were performed using Comprehensive Environmental Toxicity Information System Software (CETIS™), version 1.8.7.20 (Tidepool Scientific Software 2013). 2.4 Testing Schedule A summary of the testing schedule is provided in Table 3.

Table 3. Toxicity Testing Schedule

Toxicity Test Initiation Date

Solid-Phase Tests

Eohaustorius 10-Day September 4, 2018

Sediment-Water Interface Tests

Mytilus Larvae 48-Hour September 5, 2018

3.0 RESULTS Summaries of toxicity test results are discussed below and detailed results are provided in Appendix B and C.

Nautilus Environmental, San Diego, CA 4 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

3.1 Solid-Phase Toxicity Tests Mean survival of Eohaustorius in the sediment laboratory controls was 95 for Lab Control (32 ppt), meeting the Bight’18 and USEPA minimum control survival criterion of 90 percent. Mean amphipod survival in the two test sites associated the control ranged from 95 to 100 percent (Table 4, Appendix B). For the control and associated samples, a One-way ANOVA failed to detect a statistically significant difference among sites (p=0.0813). Un-paired t-tests with Welch's correction failed to find any samples to be significantly reduced from the lab control. All statistical comparisons and raw data sheets for Eohaustorius can be found in Appendix B.

Table 4. Summary of Amphipod Solid-Phase Toxicity Test Results Mean Percent Mean Percent Standard Standard Site ID Survival Survival Deviation Deviation (Control Adjusted) Lab Control (32 ppt) 95 5.0 -- -- B18-10167 97 4.5 100 4.6 B18-10166 100 0.0 103 0.0

3.2 Sediment-Water Interface Toxicity Tests Mean normal-alive of Mytilus in the water only laboratory control was 99 percent, meeting Bight’18 minimum control normal-alive criterion of 70 percent. Mean mussel normal-alive in the two test sites were 90 and 93 percent, respectively (Table 5, Appendix C). An un-paired t-test with Welch's correction detected a significant difference compared to the lab control for sample B18-10167 (p=0.0346). All statistical comparisons and raw data sheets for Mytilus can be found in Appendix C.

Table 5. Summary of Mussel Sediment-Water Interface Toxicity Test Results Mean Percent Mean Percent Testing Standard Standard Site ID Normal-Alive Normal-Alive Embryos Initiation Date Deviation Deviation Embryos (Control Adjusted) Lab Control 99 7.5 -- -- 9/5/18 B18-10167 90* 5.1 91 5.1 B18-10166 93 7.5 94 7.6

* Values in bold with asterisk are statistically reduced compared to their respective control (α=0.05)

Nautilus Environmental, San Diego, CA 5 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

4.0 QUALITY ASSURANCE All of the data presented have been thoroughly reviewed and are deemed acceptable for reporting in accordance with relevant protocols and Nautilus’ internal QA/QC program. Any deviations with respect to test conditions and acceptability criteria are summarized below. All were determined to be minor with no meaningful impact on the final data or its interpretation. 4.1 Sample Receipt, Handling and Holding Time Samples were received and processed according to protocol described in previous sections. All tests were initiated within the recommended holding time of two weeks as suggested by SCCWRP guidance (SCCWRP 2018 and 2009). All samples were transported on ice. Receipt temperatures ranged from 6.0 to 8.0°C. Upon receipt, all samples were moved to cold storage and held at 4 ± 2°C in the dark. All overlying ammonia values were below any reported thresholds for both test species (SCCWRP 2018; Tang 1997; USEPA 1994). Total ammonia values ranged from 1.0 to 1.3 mg/L in porewater from bulk composited test sediments prior to testing. Total ammonia values ranged from <0.5 to 0.7 mg/L in overlying water from days 0 and 10 of the Eohaustorius test. Total ammonia values ranged from ˂0.5 to 0.9 mg/L in overlying water for days 0 and 2 for the Mytilus SWI test. Total and unionized ammonia tables (Appendix D, Tables 6-8) and all calculation and raw ammonia data sheets can be found in Appendix E. 4.2 Solid-Phase Toxicity Tests All test acceptability criteria were met and all measured water quality values were within acceptable ranges as defined by the test protocol. 4.3 Sediment-Water Interface Tests All test acceptability criteria were met and all measured water quality values were within acceptable ranges as defined by the test protocol. 4.4 Reference Toxicant Tests Reference toxicant test results are provided in Appendix E. Ammonium chloride median effect concentration (EC50) values for the Eohaustorius and Mytilus reference toxicant tests (respectively) were within ± 2 standard deviations of internal control chart means for both species. A list of laboratory qualifier codes can be found in Appendix F.

Nautilus Environmental, San Diego, CA 6 Toxicity Testing Report for Bight 18 - Santa Margarita River Estuary Weston Solutions, Inc Test IDs: 1809-S018 to S021 Monitoring Period: August 2018

5.0 REFERENCES Anderson B.S., J.W. Hunt, M. Hester, B.M. Phillips. 1996. Assessment of sediment toxicity at the sediment-water interface. In: G.K. Ostrander (ed.) Techniques in Aquatic Toxicology. Lewis Publishers, Ann Arbor, MI. GraphPad Software Inc. 1992-2014. GraphPad Prism, version 6.05. Kohn N.P., J.Q. Word, D.K. Niyogi. 1994. Acute toxicity of ammonia to four species of marine amphipod. Marine Environmental Research. 38; 1-15. SCCWRP, 2018. BIGHT’18 Toxicology Laboratory Manual. June, 2018 SCCWRP, 2009. Sediment Quality Assessment Draft Technical Support Manual. Tang A., J.G. Kalocai, S. Santos, B. Jamil, J. Stewart. (1997). Sensitivity of blue mussel and purple sea urchin larvae to ammonia. Poster presentation at Society of Environmental Toxicology and Chemistry, 18th Annual Meeting, San Francisco, CA. Tidepool Scientific Software. 2000-2013. CETIS™ Comprehensive Environmental Toxicity Information System Software, Version 1.8.7.20 USEPA 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine Amphipods. June 1994. Environmental Protection Agency, Office of Research and Development. EPA 600/R-94/025. USEPA 1995. Short-term methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to West Coast Marine and Estuarine Organisms. August 1995. Environmental Protection Agency, Office of Water. EPA 600/R-95/136.

Nautilus Environmental, San Diego, CA 7

Appendix A

Chain of Custody Forms and Sample Receipt Information

Appendix B

Statistical Analyses and Raw Data Packages for Solid Phase Testing (Eohaustorius)

Weston Solutions - Bight 18 10-Day Eohaustorius Survival Bioassay Summary of Survival Results Sampling Period: 8/21-29/18 Test Initiation Date: 9/4/18

Percent Mean Percent Random Number Percent Mean Percent Standard Survival Survival Standard Site ID Rep No. Alive Survival Survival Deviation (Control (Control Deviation Adjusted) Adjusted) A 28 18 90 -- B 37 18 90 -- Lab Control C 14 19 95 95 5.0 ------(32ppt) D 9 20 100 -- E 49 20 100 -- A 3 20 100 -- B 53 20 100 -- Lab Control C 10 20 100 100 0.0 ------(17ppt) a D 7 22 100 -- E 45a 21 100 -- A 21 20 100 105 B 8 17 85 89 B18-10176 C 50 20 100 97 6.7 105 102 7.1 D 39 20 100 105 E 2 20 100 105 A 13 20 100 105 B 34 20 100 105 B18-10196 C 52 20 100 99 2.2 105 104 2.4 D 44 20 100 105 E 48 19 95 100 A 26 20 100 105 B 23 20 100 105 B18-10197 C 1 18 90 98 4.5 95 103 4.7 D 24 20 100 105 E 5 20 100 105 A 35 19 95 100 B 54 17 85 89 B18-10198 C 33 19 95 91 4.2 100 96 4.4 D 4 18 90 95 E 19 18 90 95 A 17 20 100 105 B 51 20 100 105 B18-10177 C 16 20 100 97 4.5 105 102 4.7 D 20 19 95 100 E 32 18 90 95 A 6 19 95 98 B 42 19 95 98 B18-10193 C 18 20 100 98 2.7 103 101 2.8 D 55 20 100 103 E 41 20 100 103 A 12 20 100 103 B 27 20 100 103 B18-10176- C 15 20 100 97 6.7 103 100 6.9 Dup D 46 20 100 103 E 38 17 85 88 A 30 20 100 103 B 36 18 90 93 B18-10167 C 22 20 100 97 4.5 103 100 4.6 D 31 19 95 98 E 40 20 100 103 A 47 20 100 103 B 43 20 100 103 B18-10166 C 11 20 100 100 0.0 103 103 0.0 D 25 20 100 103 E 29 20 100 103 a Greater than 20 organisms recovered. Additional organisms of a smaller size and were likely inadvertently added at initiation. Control adjusted values compared to the 32ppt Lab Control except for sample B18-10193 which is compared to the 17ppt Lab Control.

Nautilus Environmental San Diego, CA

Appendix C

Statistical Analyses and Raw Data Packages for Sediment-Water Interface Testing (Mytilus)

Weston Solutions - Bight 18 48-Hour Bivalve Larval Sediment Water Interface Bioassay Summary of Normal-Alive Results Sample Collection: 8/29/18 Test Initiation Date: 9/5/18

Percent Percent Normal Mean Percent Rand Initial Total Number Mean Percent Standard Standard Site ID Rep Normal Alive (Control Normal Alive No. Density Counted Normal Normal Alive Deviation Deviation Alive Adjusted) (Control Adjusted) A 2 394 352 368 93.4 -- B 11 394 343 349 88.6 -- Lab Control C 4 394 416 418 106 99 7.5 ------(glass jar) D 7 394 401 403 102 -- E 10 394 404 409 104 -- A 6 394 328 332 84.3 85 B 15 394 335 335 85.0 86 B18-10167 C 5 394 367 370 93.9 90 5.1 95 91 5.1 D 3 394 366 373 94.7 96 E 12 394 361 366 92.9 94 A 8 394 332 342 86.8 88 B 9 394 324 343 87.1 88 B18-10166 C 1 394 382 400 102 93 7.5 103 94 7.6 D 13 394 345 352 89.3 90 E 14 394 384 399 101 102 Values in bold indicate a significant statistical reduction compared to the Lab Control (unequal variance t-test p<0.05, untransformed data per SCCWRP 2018 Bight instructions)

Nautilus Environmental San Diego, CA

Appendix D

Ammonia Tables and Raw Data

Table 6. Total and Un-ionized Ammonia Concentrations in Bulk Sediment - Sediment Porewater

Subsample Sample ID Date Total Ammonia (mg/L) Un-ionized Ammonia (mg/L) Pre-test Pre-test B18-10167 1.0 0.051 8/30/18 B18-10166 1.3 0.014 Threshold Effect Levels Ammonia NOEC (mg/L) Species Total Un-ionized a Eohaustorius 60 0.8 a No Observed Effect Concentration (NOEC) values from EPA 1994 & Kohn et al 1994

Table 7. Total and Un-ionized Ammonia Concentrations in Solid-Phase Amphipod Toxicity Tests – Overlying Water

Test Initiation Total Ammonia (mg/L) Un-ionized Ammonia (mg/L) Sample Date Day 0 Day 10 Day 0 Day 10 Lab Control (32ppt) <0.5 <0.5 <0.013 <0.011 Lab Control (17ppt) <0.5 <0.5 <0.011 <0.012 9/4/18 B18-10167 <0.5 <0.5 <0.012 <0.013 B18-10166 <0.5 0.7 <0.013 0.019 Threshold Effect Levels Ammonia NOEC (mg/L) Species Total Un-ionized a Eohaustorius 60 0.8 a NOEC values from EPA 1994 & Kohn et al 1994

Table 8. Total and Un-ionized Ammonia Concentrations in Sediment-Water Interface Toxicity Tests – Overlying Water

Test Initiation Total Ammonia (mg/L) Un-ionized Ammonia (mg/L) Sample Date Day 0 Day 2 Day 0 Day 2 Lab Control <0.5 1.0 <0.010 0.023 9/5/18 B18-10167 0.6 0.9 0.013 0.024 B18-10166 0.7 0.9 0.013 0.021 Threshold Effect Levels Ammonia NOEC (mg/L) Species Total Un-ionized Mytilus 4.0a 0.26 and 0.14b a Tang et al, 1997 b NOEC values from the concurrent ammonia reference toxicant tests, respectively.

Appendix E

Reference Toxicant Information

Appendix F

Laboratory Qualifier Codes

Glossary of Qualifier Codes:

Q1 - Temperatures out of recommended range; corrective action taken and recorded in Test Temperature Correction Log Q2 - Temperatures out of recommended range; no action taken, test terminated same day Q3 - Sample aerated prior to initiation or renewal due to dissolved oxygen (D.O.) levels below 6.0 mg/L Q4 - Test aerated; D.O. levels dropped below 4.0 mg/L Q5 - Test initiated with aeration due to an anticipated drop in D.O. Q6 - Airline obstructed or fell out of replicate and replaced; drop in D.O. occurred Q7 - Salinity out of recommended range Q8 - Spilled test chamber/ Unable to recover test organism(s) Q9 - Inadequate sample volume remaining, 50% renewal performed Q10 - Inadequate sample volume remaining, no renewal performed Q11 - Sample out of holding time; refer to QA section of report Q12 - Replicate(s) not initiated; excluded from data analysis Q13 - Survival counts not recorded due to poor visibility or heavy debris Q14 - D.O. percent saturation was checked and was ≤ 110% Q15 - Did not meet minimum test acceptability criteria. Refer to QA section of report. Q16 - Percent minimum significant difference (PMSD) was below the lower bound limit for acceptability. This indicates that statistics may be over-sensitive in detecting a difference from the control due to low variability in the data set. Q17 - Percent minimum significant difference (PMSD) was above the upper bound limit for acceptability. This indicates that statistics may be under-sensitive in detecting a difference from the control due to high variability in the data set. Q18 - Incorrect Entry Q19 - Illegible Entry Q20 - Miscalculation Q21 - Other (provide reason in comments section) Q22 - Greater than 10% mortality observed upon receipt and/or in holding prior to test initiation. Organisms acclimated to test conditions at Nautilus and ultimately deemed fit to use for testing. Q23 - Test organisms received at a temperature greater than 3°C outside the recommended test temperature range. However, due to age-specific protocol requirements and/or sample holding time constraints, the organisms were used to initiate tests upon the day of arrival. Organisms were acclimated to the appropriate test conditions upon receipt and prior to test initiation. Q24 - Test organisms received at salinity greater than 3 ppt outside of the recommended test salinity range. However, due to age-specific protocol requirements and/or sample holding time constraints, the organisms were used to initiate tests upon the day of arrival. Organisms were acclimated to the appropriate test conditions upon receipt and prior to test initiation.

Updated: 6/30/15