effluent toxicity testing results · purple sea urchins, strongylocentrotus purpuratus, used in the...

Effluent Toxicity Testing Results

for

July 2010

Prepared for Alyeska Pipeline Service Company

Prepared by RoyK. Kropp

Battelle Pacific Northwest Division 1529 W. Sequim Bay Road

Sequim, W A 98382

August 4, 2010

PNWD-4194

Introduction

This report describes the toxicity testing of an effluent sample collected from the Alyeska Ballast Water Treatment Plant (BWTP) on July 6, 2010 (AF86912). The testing complied with the August I, 2004 NPDES permit requirements for effluent testing at Alyeska's BWTP. Toxicity testing conducted under this permit includes: 1) one test per year using the mysid, Americamysis bahia; 2) two quarterly sperm fertilization tests per year with an echinoderm (sea urchinStrongylocentrotus purpura/us or sand dollar-Dendraster excentricus); and 3) two quarterly tests per year with a chronic bivalve survival and development test (Mytilus spp. or Crassostrea gigas). The January 2010 testing included the once-yearly mysid test. Because the Alyeska Pipeline Service Company would like to compare the toxicity findings for echinoderms and bivalves, the company has chosen to conduct, when possible, chronic tests with both in each quarter of 2010. The January 2010 testing also included echinoderm and bivalve tests (Kropp 2010a), whereas the April2010 test included only the bivalve test (Kropp 2010b).

Methods

The methods used in this testing program follow the most recent guidelines provided by the U.S. Environmental Protection Agency (1995, 2002). The results of all tests were expressed as the percentage of effluent producing a specific effect, including the no-observed-effect concentration (NOEC), the lowest-observed-effect concentration (LOEC), the concentration resulting in a 25% or 50% inhibition of a response (IC2s, ICso), a 25% or 50% reduction in survival (LC2s, LCso), a 25% or 50% reduction in the maximum effect (EC2s, ECso), and the toxic unit (TU) evaluation that relates the observed response to a predetermiiied baseline.

Tests with echinoderms (Strongylocentrotus purpuratus) and bivalve larvae (Mytilus galloprovincialis) were initiated on July 7, 2010. The effluent sample (AF86912) arrived at a temperature of 4.0°C and a salinity of24.8 parts per thousand (ppt). This salinity was lower than the range specified for the echinoderm (34 ± 2 ppt) and bivalve (30 ± 2 ppt) assays. Thus, it was necessary to use concentrated seawater brine to raise the effluent salinity to the acceptable range for each assay. Because of the addition of brine, the highest concentration of effluent that could be tested for the each test was 85%. A brine control was included in the experimental design to evaluate the effects of brine addition.

Purple sea urchins, Strongylocentrotus purpuratus, used in the Second Quarter effluent testing were obtained from Mr. Dave Gutoff, San Diego, California. Sea urchin sperm were exposed to dilutions of the effluent for 20 minutes, then eggs were added and fertilization was allowed to occur for an additional 20 minutes. At the end of this exposure period, the percentage of fertilized eggs was determined. The test temperature for this assay was 13.0°C and the test salinities ranged from 31.5 to 33.0 ppt. Dissolved oxygen concentrations ranged from 7.2 to 8.0 mg/L and pH ranged from 7.7 to 8.2. The reported test conditions met the QA/QC guidelines.

2

Effluent concentrations for this test were 85%, 50%, 25%, 12.5%, and 6.25%, with control (0%) and brine-control series.

Mussels, Mytilus gal/oprovincialis, used in the testing were obtained from Taylor Shellfish Farms of Shelton, Washington. Gametes were collected from male and female mussels to conduct the assay with bivalve larvae. After fertilization and the first division of the embryo, larvae were exposed to the dilutions of effluent for 48 hours. At the end of the exposure period, larvae were examined by using a compound microscope to determine the overall percent survival and percent normal development. The bivalve larval testing was conducted at salinities ranging from 29.5 to 30.7 ppt and temperatures from 14.9°C to 16.3°C. Dissolved oxygen concentrations ranged from 6.8 to 8.0 mg!L and pH ranged from 7.6 to 8.3. The reported test conditions met the QA/QC guidelines for water quality. Effluent concentrations for this test were 85%, 50%, 25%, 12.5% and 6.25%, with control (0%) and brine-control series.

Results

Details of the test results are contained in the attached Weston Solutions Report in the Appendix.

The results of the July 2010 echinoderm fertilization assay ofthe BWTP effluent showed NOEC and LOEC values for fertilization of 50% and 85% effluent concentration, respectively (Table 1). Echinoderm fertilization was at least 86% in all concentrations of BWTP effluent tested in July 2010 (Table 2). Echinoderm fertilization success in the highest effluent concentration was 86%, which was significantly different from the 90% success in the brine control treatment although the absolute difference was only 4%. Copper reference toxicant test results were compared to the long-tenn control charts for responses to copper. Echinoderm fertilization success in the reference toxicant test resulted in an ICso of 29.3 ppb copper. This result was within 2 standard deviations of the laboratory's long-term mean ICso of 41.5 ppb copper, indicating the echinoderm gametes were appropriately sensitive.

The results of the July 2010 bivalve larval toxicity assays of the BWTP effluent showed NOEC/LOEC values of 85% and >85% effluent concentration for survival and of <6.25% and 6.25% for percentage normal development (Table 1 ). The bivalve larvae response to a known reference toxicant (copper) for survival (LCso = 23.1 ppb) was slightly greater than, but was within the range of two standard deviations of the long-term mean (LCso = 22.8 ppb ), indicating the larvae sensitivity to copper was similar to that observed during previous tests at Weston (Table 1). The bivalve larvae reference toxicant response for percentage normal development (ECso = 6.86 ppb) was greater than, but was within the range of two standard deviations of, the long-term mean (EC5o = 6.20 ppb ), indicating that the developing bivalve larvae sensitivity to copper was similar to that observed during previous tests at Weston (Table 1).

Bivalve larvae mean survival and mean normal development were at least 63% and 95%, respectively, in all concentrations ofBWTP effluent tested in July 2010 (Table 2). The percentage normal development in all effluent concentrations were statistically different from

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that in the brine control treatment. However, the percentage normal development in the three lowest effluent concentrations were 93% to 96%. The statistical significance between these treatments and the brine control probably resulted from the very low variability within the four treatments (Coefficients of Variation< 1.7% for all four treatments) and was not likely biologically important. Therefore, the more appropriate NOEC and LOEC values for this test are 25% and 50% effluent, respectively. Bivalve larval survival was at least 95% in all treatments, with no significant differences detected among treatments.

Table 1. Test Summary for Echinoderm and Bivalve Larval Toxicity Assays for the BWTP Effluent Sample Collected July 6, 2010 (AF86912).

I NOEC I LOEC I ECzs orLCzs I ECsoorLC!!G I TU

Echinoderm Fertilization Eftluent (%) I 50 I 85 I >85 I >85 I 1.2 Copper ( ppb) I <5 I 5 I 16.6 I 29.3 I -

Bivalve Larvae Percentage Normal Development Effluent(%) I <6.25 I 6.25 I 65 I >85 I 1.5 Copper (ppb) I 2.5 I 5 I 5.95 I 6.86 I -

Bivalve Larvae Survival Eftluent (%) I 85 I >85 I >85 I >85 I 1.2 Cop~r(ppb) I 10 I 20 I 19.9 I 23.1 1 -

Table 2. Test Results for Echinoderm and Bivalve Larval Toxicity Assays for the BWTP Effluent Sample Collected July 6, 2010 (AF86912).

Brine Emuent Concentration_{% Parameter Control Control 6.25 12.5 25 so 85

Mean Echinoderm Fertilization(%) 95 90 92 89 90 87 86* Mean Bivalve Larvae Normal 97 98 96* 95* 93* 81* 63* Development (%) Mean Bivalve Larvae Survival (%) 97 99 100 99 99 95 98 .. * Statlsttcally stgntficant reduction m normal development relative to bnne control (Dunnett's 1-taJied, a = 0.05).

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Conclusions

The effluent sample (AF96912) from the Alyeska Pipeline Service Company ballast water treatment plant tested in July 2010 showed little to no effect on echinodenn fertilization or toxicity to bivalve larvae. Echinodenn fertilization in the highest effluent concentration (86%) was significantly different from that in the brine control treatment (90%) although the absolute difference was only 4%. Although bivalve larval normal development in all effluent concentration significantly differed from that in the brine control treatment (98% ), the differences for the three lowest concentrations (93%-96% normal development) were not likely biologically important. Bivalve larval survival ranged from 95% to I 00% in the effluent treatments, none of which differed statistically from that in the brine control treatment (99%). The LC50 or EC5o values of echinoderm gametes bivalve larvae exposed to copper were within ± two standard deviations of the long-term mean, indicating the sensitivity of the organisms was near normal. The overall results of the July 2010 effluent testing generally were similar to those observed during testing of the January 2010 effluent (Kropp 2010a), except that the July testing identified an effect on bivalve larval development at the two highest effluent concentrations tested. These results demonstrate that the BWTP effluent continues to exhibit low toxicity for sensitive marine organisms.

References

Kropp, R.K. 2010a. tffluent Toxicity Testing Results for January2010. Prepared for Alyeska Pipeline Service Company. Battelle Pacific Northwest Division, Sequim, Washington. PNWD-4155.

Kropp, R.K. 2010b. Effluent Toxicity Testing Results for January 2010. Prepared for Alyeska Pipeline Service Company. Battelle Pacific Northwest Division, Sequim, Washington. PNWD-4180.

U.S. Environmental Protection Agency. 1995. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to West Coast Marine and Estuarine Organisms. First Edition. Office of Research and Development. Cincinnati, Ohio. EP A/600/R-95-136

U.S. Environmental Protection Agency. 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. Office of Water (4303T). Washington, DC. EPA-821-R-02-012

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July 26, 2010

Roy Kropp BatteJle Pacific Northwest National Laboratory Marine Sciences LaboratOJy 1529 W. Sequim Bay Road Sequim, W A 98382

Re: Alyeska 3111 Quarter 2010 Testing Results

Dear Mr. Kropp:

WESTON SOLUTIONS, INC. 2433 Impala Drive Carlsbad, CA 92010 (760) 795-6900 I (760) 931-1580 FAX www.westonsolutlons.com

Enclosed please find the final reports for the bioassay tests performed on test substance AF86912, received on the 71

h of July, 2010 and processed between July 71h and 91

h. The results of these tests are summarized in the tables below.

Test I.D. Number Fertilization in

1~/ICso NOEC LOEC Control Water

Echinoderm C100707.0145 94.5% >85%/>85% 50% 85% Fertilization

Development Survival NOEC LOEC Test I.D. Number Development/ Development/

EC25 / ECso LC2s/LCso Survival Survival

Bivalve Larvae Cl00707.0142 65.5%/>85% >85%/>85% <6.25%/85% 6.25o/ol>85% Chronic

All testing was perfom1ed consistent with our laboratory's quality assurance program. All results are intended to be considered in their entirety, and Weston Solutions is not responsible for use of less than the complete report. Results apply only to the samples tested.

If you have any questions regarding these results, or require additional testing, please call me at (760) 795-6959. Thank you for using the aquatic testing services ofWeston Solutions, Inc.

Sincerely,

Client Project: Client Sample ID: Weston Test ID:

Weston Solutions, Inc. Analytical Report

Alyeska Pipeline Service Co. 3rd Quarter WET

Date Received: Date Test Started:

AF 86912 Date Test Ended: C100707.0145 Matrix:

Echinodenn Sperm Fertilization Bioassay Weston Testing Protocol No.: 810045

EPA/600/R-95/136

07 Jul10 07 Jul10 07 Jul10 Liquid

Note: The sample was received at a salinity of 24.8 ppt, which is below the test protocol limit of 34 ± 2 ppl A hypersaline brine was used to increase the salinity of the sample to within protocol limits. As a result, the highest concentration of sample which could be achieved was 85 percent.

Test Organism: Strongylocentrotus purpuratus

Treatments: Control, Brine Control, 6.25. 12.5, 25, 50, and 85 percent test substance.

Results

Chronic Toxicity Statement Fertilization data met the assumptions of normality (Shapiro-Wilk's Test, p > 0.01). Variances were equal (Bartlett's Test, p = 0.59). Dunnett's Multiple-Comparison Test provided a NOEC (No Observed Effect Concentration) of 50 percent test substance and a LOEC (Lowest Observed Effect Concentration) of 85 percent test substance.

The chronic toxicity IC25 and IC50 were both greater than 85 percent test substance.

Protocol Deviations: None.

?Jw}t.~ QA Officer

112MO Date

Page 1 of3

Client Project: Client Sample ID: Weston Test 10:




AF 86912 Date Test Ended: C100707.0145 Matrix:

Analyte:

EPA Method:

Echinoderm Sperm Fertilization Bioassay Weston Testing Protocol No.: 810045

EPNSOO/R-95/136

Test Organism: Strongylocentrotus purpuratus

Test Solution Physical and Chemical Data

Dissolved Oxygen

360.1

pH

150.1

Method Reporting Limit: 0.1 mg/L 0.1

07 Jul10 07 Jul10 07 Jul10 Liquid

* Water quality measurements were taken from surrogate containers.

Page 2 of3


Client Project:



07 Ju110 07 Jul10 07 Jul10 liquid

Client Sample 10: AF 86912 Date Test Ended:

Weston Test 10: C100707.0145 Matrix:

TEST:

DILUTION WATER:

TEST ORGANISM:

TEST CHAMBER:

EXPERIMENTAL DESIGN:

REFERENCE TOXICITY: (Control Chart Attached)

EFFECT CRITERIA:

ACCEPTABILITY CRITERIA:

STUDY DIRECTOR: INVESTIGATORS:

APPENDIX Pertinent Test Data

Echinoderm Sperm Cell Fertilization Bioassay, Weston Testing Protocol 810045, EPA/600/R-95/136

Treated seawater from Scripps Institution of Oceanography, La Jolla, CA Salinity 33.0 ppt pH 8.2 Dissolved Oxygen 7.5 mg/L Temperature 13.0 oc

Purple sea urchins, Strongylocentrotus purpuratus from Dave Gutoff, San Diego, CA.

20ml borosilicate glass scintillation vials, four replicates for each of five concentrations of6.25, 12.5, 25, 50, and 85 percent test substance, plus a seawater control, and a brine control. Test chambers contained 5ml test volume each.

1. Alyeska Pipeline Service Co. personnel collected a sample at 0440 hours on July6, 20'10. Weston Solutions personnel received one 4-L plastic cubitainer of sample at 1030 hours on thefollowingc::lay. Sample temperature upon receipt was 4;00C. 2. The temperature ofthe effluent was adjusted to 12 ± 1°C. 3. Dissolved oxygen in the sample was within protocol limits. 4. Approximately 1120 eggs were inoculated into each chamber. The sperm to egg ratio used was 800:1. 3 females and 3 males were used. 5. Test chambers were held at 12 ± 1 °C during testing, lighting was continuous.

1. CuS0,11 Lot No. 1907619, received: 3/16/10, opened: 3/25/10, expires: 7/31/11. 2. EC25: 16.56 ppb EC50: 29.33 ppb 3. Laboratory Mean LC50: 41.45 ppb Within 95% Confidence Limits

4. Test Date: 07/07/10

lack of fertilization membrane.

~ 70% fertilization in control; MSD < 25%; fertilization at NOEC within 80% of control.

J. Hansen J. Hansen, D. Sowersby, E. Batliner, K. Curry

Page 3 of 3 Page 3

Sperm Cell Fertilization Test-Proportion Fertilized Start Date: 7n/2010 14:45 • Test ID: C100707.0145 • Sample ID: AF 86912 • End Date: 7n/2010 15:25· Lab ID: CCA-Weston, Carlsbad· Sample Type: DMR-Discharge Monitoring Report· Sample Date: 7/6/2010 04:40. Protocol: EPAW 95-EPA West Coast • Test Species: SP-Strongylocentrotus purpuratus • Comments: compared to Brine Control.

Cone-% 1 2 3 Control 0.9400 0.9600 0.9500

Brine Control 0.8800 0.9200 0.9200 6.25 0.9100 0.9200 0.9300 12.5 0.9400 0.8900 0.8700

25 0.8800 0.8900 0.8900 50 0.8400 0.8600 0.8800 85 0.8500 0.8700 0.8800

4 0.9300 0.8900 0.9300 0.8700 0.9200 0.8900 0.8300

Transform: Arcsin Sguare Root Cone-% Mean N-Mean Mean Min Max CVO/o N

Control 0.9450 1.0471 1.3353 1.3030 1.3694 2.140 4 Brine Control 0.9025 1.0000 1.2545 1.2171 1.2840 2.770 4

6.25 0.9225 1.0222 1.2891 1.2661 1.3030 1.375 4 12.5 0.8925 0.9889 1.2400 1.2019 1.3233 4.632 4

25 0.8950 0.9917 1.2416 1.2171 1.2840 2.35,3 4 50 0.8675 0.9612 1.1991 1.1593 1.2327 2.714 4

*85 0.8575 0.9501 1.1845 1.1458 1.2171 2.666 4

Auxilia!l Tests Statistic Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.92784 Bartlett's Test indicates equal variances (p = 0.59) 3.71637 The control means are significant!~ different {E = 0.01} 3.59256 H~eothesis Test {1-tall, 0.05) NOEC LOEC ChV TU MSDu

1-Talled Number Total t-Stat Critical MSD Resp Number

39 400 -~.363 2.410 0.0612 31 400 0.571 2.410 0.0612 43 400 0.505 2.410 0.0612 42 400 2.182 2.410 0.0612 53 400 2.758 2.410 0.0612 57 400

Critical Skew Kurt 0.884 0.69713 0.31546

15.0863 2.44691

MSDe MSB MSE F-Prob df Dunnett's Test 50· 85· 65.192 2 0.03909 0.04328• 0.00577 0.00129 0.00794 5, 18

Maximum Likellhood..Probit Parameter Value SE 95% Fiducial Limits Control Chi-Sg Critical P-value Mu Siama Iter Slope 0.98647 0.7094 -0.404 2:3769 0.0975 1:87804· 7.81473. 0.6 3.48653 1.01371 18 Intercept 1.56063 1.26991 -0.9284 4.04965 TSCR 0.09009 0;0125 0.0656 0.11459 1.0 Point Problts % 95% Fiducial Limits 0.9 EC01 2.674 13.4364 EC05 3.355 65.9358 0.8

EC10 3.718 153.959 0.7 EC15 3.964 272.826 :o.s EC20 4.158 429.901 c EC25 4.326 635.027 &. 0.5

EC40 4.747 1697.11 II)

&! 0.4 EC50 5.000 3065.71 0.3 ECSO 5.253 5538 EC75 5.674 14800.3 0.2

EC80 5.842 21862.2 0.1 EC85 6.036 34449.1 0.0 EC90 6.282 61046 100 10000 1000000 EC95 6.645 142541 EC99 7.326 699490

Dose%

Page 1 ToxCalc v5.0.23 Reviewed by:..t!f!tt:_

Test: SC-Sperm Cell Fertilization Test" Test ID: C100707.01'15. Species: SP-Strongylocentrotus purpuratus . Protocol: EPAW 95-EPA West Coast· Sample ID: AF 86912 • • Sample Type: DMR-[)i~charge Monitoring Report • Start Date: 7/7/2010 14:45 • End Date· 1m2010 15:2!Lab ID: CCA-Weston, Carlsbad.

Total Number Pos ID Rep Group Counted Fertilized Notes

1 1 Control 100 94 2 2 Control 100 96 3 3 Control 100 95 4 4 Control 100 93 5 1 Brine Control 100 88 6 2 Brine Control 100 92 7 3 Brine Control 100 92 8 4 Brine Control 100 89 9 1 6.250 100 91 10 2 6.250 100 92 11 3 6.250 100 93 12 4 6.250 100 93 13 1 12.500 100 94 14 2 12.500 100 89 15 3 12.500 100 87 16 4 12.500 100 87 17 1 25.000 100 88 18 2 25.000 100 89 19 3 25.000 100 89 20 4 25.000 100 92 21 1 50.000 100 84 22 2 50.000 100 86 23 3 50.000 100 88 24 4 50.000 100 89 25 1 85.000 100 85 26 2 85.000 100 87 27 3 85.000 100 88 28 4 85;000 100 83

Comments:

Page1 ToxCalc5.0 Reviewed by:...J;;J_

CHRONIC TOXICITY DATA SHEET FOR ECHINODERM FERTILIZATION

CLIENT: ll/111~~ Pti?dtlu.. fl.vw/, tA. PROJECT: ~Y4 Quu!k.v \jJ U CLIENT SAMPLE ID: IJP ~~'!!?-WESTON SAMPLE ID: C/0() 7 () 7. b ( lffi STUDY DIRECTOR: J._/kn_!Ul

Concentration

Day 0 (0 Hours) Control Brine Control

Date: 7/7/fb /U5_ 12.f\

Time: llf~ ~s

~ Technician: JH ff5

Sperm to Egg Ratio Fertilized

/IJIJ : I (l)o

JQ~: J 61 lf-110 ~ I d8 tJ40: I 1l./

/:>«;:I qq tl!. CIIJ: I '15 ~oo: I

Number of Females:

DATE RECEIVED: 1[1 'Jb DATE TEST STARTED: '"1/1/JO DATE TEST ENDED: '1i'11Jo WESTON SOP NO.: (J/() d'l5 SPECIES: ,~).,..,, ~JAJO•~Iz.u

NO. ORGANISMS~ER CHAMBER DO !} Temp Salinity

(mg/L) JH (•C) (ppt)

1.6 L3·D -6~.0 '7.'1/ I~.IJ "f>~.b '1.v l~.o ~2..tlt "1.'< /g.o !,~.~ '}.( /~.{) ~>'1-.u .., .Jt l~-D ... ~.-~ #6.o /B.n ~1.6

TRIAL FERTILIZATION Unfertilized

4-0 /?J I~ lR fl 5' 'f

SPAWNING RECORD Number of Males:

Percent Fertilized

(,{) 61 ee qlf ql}. &16

810045

_,, vA,. /AJ/u. ~ 11/~

pH

$.1. ~.1_

r/.P., ~.~.-

e ·' 'l.~

.-.."7

Concentration Selected for Test

~

FERTILIZATION COUNT DATA ~ l'i2 IJ;o E!H li~_d/DlJ Rep1 Rep2 Rep3 Rep4 RepS

Cone. NC?rmal Abnormal N.onnal Abnonnal Normal Abnormal Normal Abnormal Normal Abnormal Initials

Control qq w 'J_(g_ lf 15 5 q?J _7 Jl-f t?nnc ¢ ~JI.lr gg 11! q;,l._ ~ q~ f ~q _L} JJ.I

b.26 q] ~ q'),_ { (iJP. 'ii '13 _-z ~~ 2 JJ/ /j.S '14- I# _$~' II 87 /3 R1 r; JH ~s tlL~ 1?-- _9_1: II gq II q;;, g Jlf 5l) fJ4 t(g $Jg~ t4_ At !?" B'f II Jlf 65 617 1'5 6_7 ,~ AS {";>- e~ 1"1_ JH

tb lOt./ID ~==~.-......:.:.;.;;.....,-._~-=~=~~=--~==~-~~---'--"-'-=~_,...~~-.__,

START TIME: ENDTIME: /6~5 ~

HOBO TEMP. NO.:

TEST LOCATION:

DILUTION WATER BATCH: 510 06t>lf-(

H: f,,_. DO: 7. ~ TEMP: 1-:;. REFTOX: (I)()Og 1~·~0 LOT NO.: I~ 01lll~ LC50:J. .~~ } TESTDATE: 7 7 j(J TEST ACCEPTABIUTY: - 70% FERTILIZATION IN CONTROL ~ Fertilization at the NOEC > BOO~ of control fertilization Ill MSD <25%

Figure 1: Chronic Toxicity Data Sheet for Echinoderm Fertilization. (t) f.unp~r.t ~ ~ A. f~p VJ~ I ?f:;.J ;o J/1-

Strongylocentrotus purpuratus Reference Toxicant Control Chart:

CV% = 50.9 Proportion Fertilized

90

80 +2 SD

-.:- 70 8. +1 SD Q. 60 0

~ 50 .D Q.

40 Mean Q. .. ., 30 0 w 20 -1 SD

10

0 -2 SD

Dates Values Mean -1 so ·2SD +1 SD +2SD 04/08/09 7.9545 41.4536 20.3466 0.0000 62.5605 83.6675 05/20/09 12.8290 41.4536 20.3466 0.0000 62.5605 83.6675 07/14/09 51.3015 41.4536 20.3466 0.0000 62.5605 83.6675 08118/09 14.0651 41.4536 20.3466 0.0000 62.5605 83.6675 09/02/09 32.7640 41.4536 20.3466 0.0000 62.5605 83:6675 09/19/09 67.9646 41.4536 20.3466 0.0000 62.5605 83.6675 10/27/09 15.3600 41.4536 20.3466 0.0000 62.5605 83.6675 11113109 28.5970 41.4536 20.3466 0.0000 62.5605 83.6675 11/17/09 38.4620 41.4536 20.3466 0.0000 62.5605 83.6675 12109/09 62.8865 41.4536 20.3466 0.0000 62.5605 83.6675 12/14/09 62.3350 41.4536 20.3466 0.0000 62.5605 83.6675 01/06/10 47.8518 41.4536 20;3466 . 0.0000 62.5605 83.6675 01/19/10 56.9500 41.4536 20.3466 0.0000 62.5605 83.6675 02/09/10 61.8527 41.4536 20.3466 0.0000 62.5605 83.6675 02122110 28.6400 41.4536 20.3466 0.0000 62.5605 83.6675 02/27/10 54.0030 41.4536 20.3466 0.0000 62.5605 83.6675 03109/10 60.7870 41.4536 20.3466 0.0000 62.5605 83;6675 04/07/10 75.2507 41.4536 20.3466 0.0000 62.5605 83.6675 04/16110 19.8840 41.4536 20.3466 0.0000 62.5605 83.6675 07/07/10 29.3330 41.4536 20.3466 0.0000 62.5605 83.6675

Updated 7/12/10 JH

Sperm Cell Fertilization Test-Proportion Fertilized Start Date: 7/7/2010 14:45 • Test ID: C100316.38 Sample ID: REF-RefToxicant / End Date: 7f7/2010 15:25 • Lab ID: CCA-Weston, Carlsbad • Sample Type: CUSO-Copper sulfate · Sample Date: Protocol: EPAW 95-EPA West Coast • Test Species: SP-Strongylocentrotus purpuratus Comments:

Conc-ppb Control

5 10 20 40 80

1 0.8800 0.8400 0.6700 0.6700 0.2200 0.0100

2 0.9700 0.7600 0.7800 0.6800 0.1900 0.0400

3 4 0.9800 0.9700 0.6700 0.8600 0.8000 0.8100 0.7900 0.6000 0.0900 0.4400 0.0500 0.0500

Transform: Arcsin Square Root ConC·J.!(!b Mean N-Mean Mean Min Max

Control 0.9500 1.0000 1.3598 1.2171 1.4289 *5 0.7825 0.8237 1.0911 0.9589 1.1873

"10 0.7650 0.8053 1.0671 0.9589 1.1198 "20 0.6850 0.7211 0.9773 0.8861 1.0948 *40 0.2350 0.2474 0.4923 0.3047 0.7253 ·so 0.0375 0.0395 0.1881 0.1002 0.2255

Auxiliary Tests Shapiro-Wilk's Test indicates normal distribution {p > 0.01) Bartlett's Test indicates equal variances (p = 0.56) Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV Dunnett's Test <5 5

CV% 7.089 9.530 6.915 8.865

35.414 31.755

TU

N 4 4 4 4 4 4

Statistic 0.96778 3.9105 MSDu

0.10144

t-5tat

3.597 3.917 5.119

11.609 15.679

MSDp 0.10609 ~

Linear Interpolation (200 Resamples} Point ppb SO 95% CL(Exp) Skew IC05* 1.418 0.387 0.835 2.959 1.2423 IC10" 2.836 0.759 1;671 5.919 1.0439 IC15* 4.254 2.248 2.506 15.918 1.7321 IC20 10.625 4.201 0.370 25.811 0.5530 IC25 16.563 3.786 3.177 25.622 -0.4635 IC40 25.111 1.584 20.861 30.409 0.4284 IC50 29.333 ., 1.791 24.800 35.855 0.6623 • indicates IC estimate less than the lowest concentration

:l 0.6 r:: &. 0.5 crJ ~ 0.4

0.3

0.2

0.1

0.0 0 20

Page 1 ToxCalc v5.0.23

1-Tailed Isotonic Critical MSD Mean N-Mean

0.9500 1.0000 2.410 0.1801 0.7825 0.8237 2.410 0.1801 0.7650 0.8053 2.410 0.1801 0.6850 0.7211 2.410 0.1801 0.2350 0.2474 2.410 0.1801 0.0375 0.0395

Critical Skew Kurt 0.884 0.09162 0.63948

15.0863 MSB MSE F-Prob df

0.75716 0.01117 4.9E-11 5, 18

40 60

Dose ppb 80 100

Reviewed by:~

Test: SC-Sperm Cell Fertilization Test • Test ID: C100316.38 . Species: SP-Strongylocentrotus purpuratus Protocol: EPAW 95-EPA West Coast -· Sample ID: REF-RefToxicant . . Sample Type: CUSO-Copper sulfate Start Date: 7/7/2010 14:45 ~ End Date: 7/7/2010 15:2!Lab ID: CCA-Weston Carlsbad

Total Number Pos ID Rep Group Counted Fertilized Notes

1 1 Control 100 88 2 2 Control 100 97 3 3 Control 100 98 4 4 Control 100 97 • 5 1 5.000 100 84 6 2 5.000 100 76 7 3 5.000 100 67 8 4 5.000. 100 86 .. 9 1 10.000 100 67 10 2 10.000 100 78 11 3 10.000 100 80 12 4 10.000 • 100 81 . 13 1 20.000 100 67 14 2 20.000 100 68 15 3 20.000 100 79 16 4 20.000 • 100 60 . 17 1 40.000 100 22 18 2 40.000 100 19 19 3 40.000 100 9 20 4 40.000' 100 44 -21 1 80.000 100 1 22 2 80.000 100 4 23 3 80.000 100 5 24 4 80.000 100 5

Comments:

Page 1 ToxCalc5.0 Reviewed by:E._

Test ID:

t:tDOj'({g .3f Dilution Water

40 Minute Echinoderm Fertilization Reference Toxicant Test

Replicates: 4 Study Director: Location:

J.I/A41SAA f2oo1Yl ') eaft1 I Organism: Organism Batch: Associated Test(s):

No. Males:

~ No. Females:

Batch: C! b IJI.t ol/ I 0 J. furualz< J D~ IJ70itiD fJ(g.tJ JL lr' '3 Toxicant: Toxicant Lot No.: Da;f~h~: Initials: Copper Sulfate 1'10'1bt'1 ~ (0.509gCuiLCuSO.)_

Target Quantity ofStock: Quantity ·of Diluent: Concentrations: Target: .. Target:

80ppb 0.0.18mL 500mL

80ppb Actual: o. o.-,7'( tAL Actual: 5o<), 00 . . . - . . ' .·

Serial Dilute·by ~to obtain co.qeentrations of 40, 20, 10, and 5 ppb. . .

Date7{ { WQTime: Start Time: /illfJJJfEnd Time: /5:1-6 Initials: Jlf I 110 I {<l<J ~

STOCK

Control 5 10 20 40 80

D.O.(%) 7.? 7.5 7/5 7.5 7 .. 5 7.5 Tempw;e /~.0 I~. o /3.0 /8.0 /~. 0 1~. 0

Salinity ~~.0 ?Z.Cf 31. .<=r -; t .Gf )2 .~ 3>.0 pH <0.2, <6.2. 1> ."1- 4,. 2. ~~z. <:r6 ' '2-

0Pass 0 Fail Notes:

~. 40 Minute Echinoderm Fertilization Reference Toxicant Test

Cone. Rep Number Number

Date Fertilized Unfertilized

1 88 /;); 1/f /1{) 2 !11 0_

I I

Control 3 '1» ;), 4 0;7 3 .. 1 ~f /(p '1 h'L }JD 2 7~ ~tf

I

5 ppb l.t7 3 ?~

4 0(( 14 1 l.t7 ~? 2 1Z g-;r

10ppb 80 3 ~

4 91 l&f 1 lo1 '?? 2 lib '??-"

20ppb 1'1 ?--l 3

4 {;,() 4-D 1 ~~ 16 2 1'1 91 40ppb

~ t:t/ 3

4 4-_4 ~ 1 I qq 2 4 '/(p

80ppb ? 3 P/5

4 6 qt) "I.>

Initials

Jll-

-~

JH

11

-

-

12.

OA Wt.Wi

2-S/77 ?jJL/

-r-

~. Echinoderm RT Random Number Sheet

7/7/10

1 14

~ 2 19 3 18 4 15 5 24 6 8 0 7 2 8 9 9 6

10 11 )0 11 22 12 20 13 1 14 13

~D 15 17 16 10

18 12 19 3 20 4 21 21 22 16 bD 23 23 24 7

BIOASSAY SAMPLE RECEIPT

Client: -A-lt :kA- 'Rr· I s-; ---&>--------- - - ~L~c - -t <;- _,h_r_ -e-rvu:-£ • _fl-roject:-------~ra:--Q \..UI rk we.r Weston Sampll ID: 9~0~ ':0_!: '? ~ .11J. I..._U/,

Client Sample ID: AF~fR1t:z. Renewal Sample (YIN): N Date/Time Received: -r /-:,f,lJ lo7b Airbfll #: "7qatzor-r~Dd't.J-Sample Tracking Information Kept for y Records: (YIN)

Collection Date/Time: !1 ft.lw D~ Condition of Shipping Container: IJ.{)DJ.

Type and Capacity of Sample Container: OJ

'1- l- ~l&c:-kc. ~~ Total Sample Volume (L): lfL Condition of Sampling Container: IJ.(}Dd_

Sample Container Appropriate: (YIN) ""'y Custody Seals Intact: (YIN) y Ice or Frozen Blue Ice Present During y ShlpmentiTransl)ort: _(YIN)

Samplers Name Present on COC Form: (YJN) ~"~ ..-

TAKE THE FOLLOWING MEASUREMENTS UPON ARRIVAL

Temp. (•C) Dissolved Conductivity Hardness Alkafinity Total Total WESTONID Oxygen pH (mSicm) or Chlorine Ammonia Tech (o-&•cJ•

Cm91i.J. Salfr!lti{ppt) (mgCaCOJL) (mgC.COJL)

{mg/L) (mgNHafl.)

ClbD'":I-07-.0l 'f;o 'f.~ 7.2.. .2lf~nnt D.oD <D.f5 JM!f'.A II #

*Notify project manager or study director of temperatures- above 6°C. Client must be notified ASAP.

lfthere are sample receipt problems, complete the following:

Reason for unacceptability:

Name of Client Contact: Contacted by:

Client Response and/or Action to be Taken: Date Action Taken:

(j;)~. ":!-{1/tD ~ -:rJ?fdt(Jw 0:\Biousay WQ&Repons\BLANK FOR.MS\Fonns will\ Weston Logo\SAMPLE REC£1PT Form- Weston 2006.doe Last printed J/9/lOOIIO:S!ii:OO AM

Client: Project: Sample Matrix: Sample Name/10:

Weston Solutions, Inc.

Alyeska Pipeline Service Co. 2010 3rc~ Quarter Chronic WET Liquid

Date Received: Date Test Started: Date Test Ended:

AF86912 Test 10 No.:

Bivalve Larvae Chronic 48-Hour Bioassay Weston Testing Protocol No. BI0042

EPN600/R-95/136 Test Organism: Mytilus galloprovincialis

7July10 7 July 10 9 July 10 C100707.0142

Note: Due to the relatively low salinity (24.8 ppt) of the sample upon receipt, hypersaline brine was added to increase the salinity to 30 ppt. A Brine Control was run concurrently with this test and included in the analysis. Brine was prepared by freezing natural seawater, and then collecting the hypersaline liquid as the frozen seawater was thawed. Since it was necessary to add brine to the sample, the highest concentration of the sample achieved was 85 percent.

Control

Brine Control

6.25%

12.5%

25%

50%

85%

'4~1?.~~ QAUnit

w/;a oate

96.8

97.8

96.0

95.4

93.0

80.9

62.7

Results

97.2

98.9

100

99.2

98.8

95.0

97.7

112~110 Date

Page 1 of4

Client: Project: Sample Matrix: Sample Name/ID:


Alyeska Pipeline Service Co. 2010 3rt1 Quarter Chronic WET liquid


AF86912 Test ID No.:

Bivalve Larvae Chronic 48-Hour Bioassay Weston Testing Protocol No. 810042

EPA/600/R-95/136 Test Organism: Mytilus ga/loprovincialis

Chronic Toxicity - Development

7July10 7 July 10 9 July 10 C100707.0142

Development data met the assumptions of normality (Shapiro-Wilk's Test, p > 0.01). Variances were equal (Bartlett's Test, p = 0.50). Dunnetfs Multiple-Comparison Test provided a NOEC (No Observed Effect Concentration) of less than 6.25 percent and a LOEC (Lowest Observed Effect Concentration} of 6.25 percent test substance.

The Maximum Likelihood-Probit Method was used to calculate a point estimate for the concentrations causing 5, 10, 15, 20, 25,40 and 50 pereent reductions in normal development. The EC25 was estimated to be 65.5 percent test substance while the EC50 was estimated to be greater than 85 percent test substance.

Toxicity, expressed as Toxic Units Chronic (TUc), was greater than 16.

Chronic Toxicity- Survival

Survival data did not meet the assumptions of normality (Shapiro-Wilk's Test, p ~ 0.01 ). Equality of variances could not be confirmed. Steel's Many-One Rank Test provided a NOEC of 85 percent test substance and a LOEC of greater than 85 percent test substance.

The Linear Interpolation Method was used to calculate a point estimate for the concentrations causing 5, 10, 15, 20, 25, 40 and 50 percent reductions in survival. The IC25 and IC50 were both estimated to be greater than 85 percent test substance.

Toxicity, expressed as Toxic Units Chronic(TUc), was 1.18.

Myti/us galloprovincialis 48 hrs

Protocol Deviations: None.

<6.25%

Summary of Results·

>85%

Page2 of4

85% >85% AF86912


Client: Date Received: Project: Date Test Started: Sample Matrix:

Alyeska Pipeline Service Co. 2010 3ro Quarter Chronic WET liquid Date Test Ended:

Sample Name/ID: AF86912

Control

Brine Control

6.25

12.5

25

50

Test ID No.:

Bivalve Larvae Chronic 48-Hour Bioassay Weston Testing Protocol No. BI0042

EPA/600/R-95/136 Test Organism: Mytilus ga/loprovincialis.

Test Solution Physical and Chemical Data

7.3 15.7 30.0

6.8 15.8 29.6

7.4 15.3 30.1

~4 14~ 3Q1

7.4 15.2 29.9

7.7 15.1 29.8

85 7.9 14.9 29.5

Brine Control 7.4 16.1 29.8

6.25 7.6 15.8 30.3

12.5 7.5 15.5 30.7

25 7.5 16.3 30.2

50 7.8 16.1 29.9

85 7.2 16.1 30.1

"'Water quality measured on surrogate chambers.

Page 3 of4

7July10 7 July 10 9 July 10 C100707.0142

. ' . -\ . · ... ·. ~ ... . .

8.2

8.3

8.2

8.2

8.1

7.9

7.6

7.9

7.8

7.8

7.8

7.8

7.8


Client: Project: Sample Matrix:

Alyeska Pipeline Service Co. 2010 3rt1 Quarter Chronic WET Liquid


7 July 10 7 July 10 9 July 10 C100707.0142 Sample Name/ID: AFB6912

TEST:

DILUTION WATER:

TEST ORGANISM:

TEST CHAMBER:

APPENDIX Pertinent Test Data

Test ID No.:

Chronic 48-Hour Survival/Growth Bioassay with Bivalve Larvae (Mytilus galloprovincialis.), EPA/600/R-951136, Weston Testing Protocol No. 810042

Control water (zero time). Treated Seawater, Scripps Institution of Oceanography, La Jolla, CA.

Salinity pH Dissolved Oxygen Temperature

30.0 ppt 8.2 7.3 mg/L 15.7°C

Mussel larvae, Myti/us ga//oprovincialis. Source: Taylor Shellfish Farms, Shelton, WA.

Four replicates, concentrations of 6.25, 12.5, 25, 50, and 85 percent, plus a seawater control and a brine control. Test substance volume per replicate= 10ml.

EXPERIMENTAL DESIGN: 1. Alyeska Pipeline Service Co. personnel collected a sample on July 6, 2010, at 0440 hours. The sample was received in one 4-liter plastic cubitainer at the Weston Solutions, Inc. laboratory on the following day at 1030 hours. The temperature of the sample upon receipt was 4.0°C.

MORTALITY CRITERIA:

2. The temperature of the test substance was adjusted to 15 ± 1 oc. 3. Approximately 270 test organisms were placed into each chamber. 4. Test chambers were held at 15 ± 1°C for 48 hours with a photoperiod of 16

hours light, 8 hours darkness. 5. Test substance was not renewed.

Absence of larvae, or completely developed shells without meat.

ACCEPTABILITY CRITERIA: ~70% survival of controls; ~90% normal shell development in surviving controls; minimum significant difference < 25%.

REFERENCE TOXICITY:

(control chart included)

STUDY DIRECTOR:

INVESTIGATORS:

Toxicant:

Species: 48 hr EC2s: 48 hrECso:

CuS04, Lot No. 1907619, received 03/16/10, opened 03/25/10, expires 07/31/11. Myti/us gal/oprovincialis, larvae

Laboratory Mean (EC50):

19.9 ppb survival, 5.95 ppb proportion normal 23.1 ppb survival, 6.86 ppb proportion normal 22.8 ppb survival, 6.20 ppb proportion normal 7/7/10 (within 95% confidence limits) Test Date:

D. Sowersby

D. Sowersby, K. Skrivseth, E. Batliner

Page 4 of4

Bivalve Larval Survival and Development Test-Proportion Nonnal Start Date: 7n12010 16:25 • Test JD: C100707.0142 • Sample ID: AF 86912 • End Date: 7/9/2010 16:05• Lab 10: CCA-Weston, Carlsbad· Sample Type: DMR-Discharge Monitoring Report· Sample Date: 7/6/2010 04:40 • Protocol: EPAW 95-EPA West Coast. Test Species: MG-Mytilus galloprovincialis • Comments: compared to brine control •

Cone-% 1 2 3 Control 0.9662 0.9715 0.9633

Brine Control 0.9713 0.9834 0.9811 6.25 0.9590 0.9673 0.9624 12.5 0.9549 0.9407 0.9618

25 0.9219 0.9502 0.9333 50 0.7561 0.8388 0.8204 85 0.6552 0.5985 0.5904

4 0.9705 0.9771 0.9508 0.9583 0.9146 0.8222 0.6655

Transfonn: Arcsin Square Root Cone-% Mean N-Mean Mean Min Max CV% N


*6.25 0.9598 0.9812 1.3696 1.3470 1.3889 1.283 4 "12.5 0.9539 0.9752 1.3552 1.3248 1.3741 1.584 4

"25 0.9300 0.9507 1.3043 1.2742 1.3457 2.395 4 ·so 0.8094 0.8274 1.1202 1.0543 1.1577 4.044 4 "85 0.6274 0.6413 0.9145 0.8763 0.9540 4.349 4

Auxiliarx Tests Statistic Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.95874 Bartlett's Test indicates equal variances (p = 0.50) 4.3296 The control means are significant!~ different {e = 0.022 3.12792 H~pothesls Test ~1-tall, 0.05) NOEC LOEC ChV TU MSDu Dunnett's Test <6.25. 6.25. ~1, . 0.01786

. Maximum Likelihood-Problt Parameter Value SE 95% Fiducial Limits Control Chi..Sq

1-Tailed t..Stat Critical MSD

2.472 2.410 0.0525 3.130 2.410 0.0525 5.468 2.410 0.0525

13.918 2.410 0.0525 23.356 2.410 0.0525

Critical 0.884

15.0863 2.44691

MSDp MSB MSE 0.01825. 0.15025 0.00095

Critical P-value Mu Slope 2.50933 0.18657 2.14366 2.875 0~02199 3.01399· 7.81473. 0.39. 2.08488 Intercept -0.2316 0.33446 -0.8872 0.4239 TSCR 0.03274 0.00359 0.0257 0.03977 1.0 Point Pro bits % 95% Fiducial Limits

0.9 EC01 2.674 14.3813 11.008 17.5889 EC05 3.355 26.8771 22.7704 30.5163 0.8 EC10 3.718 37.5113 33.42 41.0904 0.7 EC15 3.964 46.9727 43.101 50.4502 EC20 4.158 56.1669 52.4469 59.7407 :g 0.6

EC25 4.326 65.4763· 61.6125 69.5695 c B. 0.5

EC40 4.747 96.3641 89.4079 105.598 XI EC50 5.000 121.584· 110.401 137.519 0:: 0.4

EC60 5.253 153.405 135.841 179.727 0.3 EC75 5.674 225.772 191.111 281.384 0.2 EC80 5.842 263.193 218.71 336.369 EC85 6.036 314.709 255.886 414.262 0.1

EC90 6.282 394.087 311.687 538.528 0.0 EC95 6.645 550.012 417.389 794.747 10 100 1000 EC99 7.326 1027.91 721.351 1650.35

Dose%

Number Res~

23 43 48 74

183 380

Skew -0.5043

F-Prob 3.2E·14

Sigma 0.39851

10000

Total Number

1046 1073 1049 1051 958

1020

Kurt -0.1015

df 5, 18

Iter 7

Page 1 ToxCalc v5.0.23 Reviewed by:M_

Bivalve Larval Survival and Development Test-Proportion Alive Start Date: 1n12010 16:25• Test ID: C100707.0142 · Sample ID: AF 86912 • End Date: 7/9/2010 16:05· Lab ID: CCA-Weston, Carlsbad· Sample Type: DMR-Discharge Monitoring Report· Sample Date: 7/6/2010 04:40 • Protocol: EPAW 95-I:PA West Coast· Test Species: MG-Mytilus galloprovincialis • Comments: compared to brine control •

Cone-% 1 2 3 Control 1.0000 0.9762 0.9722

Brine Control 1.0000 0.9563 1.0000 6.25 1.0000 1.0000 1.0000 12.5 0.9683 1.0000 1.0000

25 1.0000 1.0000 0.9524 50 0.9762 0.9603 0.9722 85 0.9206 1.0000 0.9881

4 0.9405 1.0000 1.0000 1.0000 1.0000 0.8929 1.0000

Transfonn: Arcsin Sguare Root Cone-% Mean N-Mean Mean Min Max CV% N


6.25 1.0000 1.0110 1.5393 1.5393 1.5393 0.000 4 12.5 0.9921 1.0030 1.5024 1.3917 1.5393 4.913 4

25 0.9881 0.9990 1.4922 1.3508 1.5393 6.316 4 50 0.9504 0.9609 1.3567 1.2373 1.4159 6.035 4 / 85 0.9772 0.9880 1.4563 1.2852 1.5393 8.228 4

Auxilia!l Tests Statistic Shapiro-Wilk's Test indicates non-normal distribution (p <= 0.01) 0.79134 Equality of variance cannot be confirmed The control means are not significant!~ different ~E = 0.29~ • 1.17114 Hypothesis Test {1-tall, 0.051 NOEC LOEC ChV TU Steel's Many-One Rank Test 85. >85· 1.17647

Rank Sum

20.00 18.50 17.50 13.00 16.00

linear Interpolation (200 Resamples) Point· IC05 IC10 IC15 IC20 IC25 IC40 IC50

Page 1

% so >85 >85 >85 >85 >85 >85 >85·

95% Cl(Exp) Skew

ToxCalc v5.0.23

1.0

0.9

0.8

0.7

Gl 0.6 ctJ 6 0.5

~ 0.4 Gl a: 0.3

0.2

0.1

0.0 ~

-o.1 0 20

1-Tailed Isotonic Critical Mean N-Mean

0.9945 1.0000 10.00 0.9945 1.0000 10.00 0.9921 0.9975 10.00 0.9881 0.9935 10.00 0.9638 0.9691 10.00 0.9638 0.9691

Critical Skew Kurt 0.884 -1.2388 0.22771

2.44691

--~

40 60 80 100

Dose%

Reviewed by:l:fd_

Test: BV-Bivalve Larval Survival and Development Test· Test ID: C100707.01¥2. • Species: MG-Mytilus galloprovincialis • Protocol: EPAW 95-EPA West Coast· Sample ID: AF 86912 • Sample Type: DMR-Discharge Monitoring Report • Start Date: 7n/2010 16:25 • End Date: 7/9/2010 16:0'! Lab ID: CCA-Weston, Carlsbad·

Initial Final Total Number Pos ID Rep Group Density Density Counted Normal Notes

1 1 Control 252 266 266 257 2 2 Control 252 246 246 239 3 3 Control 252 245 245 236 4 4 Control 252 237 237 230 5 1 Brine Control 252 279 279 271 6 2 Brine Control 252 241 241 237 7 3 Brine Control 252 264 264 259 8 4 Brine Control 252 262 262 256 9 1 6.250 252 268 268 257 10 2 6.250 252 275 275 266 11 3 6.250 252 266 266 256 12 4 6.250 252 264 264 251 13 1 12.500 252 244 244 233 14 2 12.500 252 253 253 238 15 3 12.500 252 288 288 277 16 4 12.500 252 264 ' 264 253 17 1 25.000 252 269 269 248 18 2 25.000 252 261 261 248 19 3 25.000 252 240 240 224 20 4 25.000 252 281 281 257 21 1 50.000 252 246 246 186 22 2 50.000 252 242 242 203 23 3 50.000 252 245 245 201 24 4 50.000 252 225 225 185 25 1 85.000 252. 232 232 152 26 2 85.000 252 264 "2SJI 158 27 3 85.000 252 249 249 147 28 4 85.000 252 275 275 183

Comments: .

Page 1 ToxCalc5.0 Reviewed by:../JJ__

BIVALVE 48-HOUR CHRONIC TOXICITY TEST 810042

DATE RECEIVED:

PROJECT: 3 ...-d. Qv.D.r..J..L.r WE-T

CLIENT SAMPLE ID: A F g (;. <1tl. DATE TEST ENDED:

WESTON SOP NO.:

SPECIES: f'l. STUDY DIRECTOR:

. bO* .. : T(tmp* Salinity• Concentration (mgll) (oC) . •(ppt) .. J)I:'i*

Dar- 0 (0 Hours) Control j,"J fif.l ?a.{) ~.2-Date: '7 ; (1 0

Brine Control '· .sr ,c;j -;,,,IJ ?,3 Sample ID: C.lDO(O(.O( ID.L c:; -,_'{- 15.7 jl). ( ~.2..

12·5 "7.~ I'E-.q J"D I f-.. 2.. Dilutions (Tech)::U,

2C: '"1.Lf- ~.2- zq.lf t-A WQ Time: {52.5 50 '·' IS·. I :z,.r ...,.,_ Technician: 1J.4, 8'5 1.'1 14-'t 2'tt; -z .. fo

24 Hours Control ... .. . ·., IS.~ ··· .. . . . . ''{,_:.:;;;:. .•

Brine Control , 5./: ,.

!:-_?:·-~{-. ·. .,.- .,=

t-fcr,/JD L ~t; ·.' .. ~ . . ·'· .. L5.4 .... ··: ·.'·: ··:_ ::_._;. Date:

_, . . . . : :

·~.s- I S.o · .. .'.· .. _::.· . .· .. :· .. ·;:· ...

.. ··· . '.:~:

:·_.' :':

ry S'L .2S cs.'f ·-.·.·

WQTime: ... '':: ·.·:. · .. ·~ ..

\;! . . ..

Technician: ¥? Q) : .. ' 19.4 (fie; ·:

15.~ . '~. :· : .

l'r . .:,-~·-"'·'•';· ..... ·.: ..... ;":'_: · .. , .. ·' ·. ., . ...... .··.·

48Hours Control a.o //,.1- ,g n.1.. 7.~

Date: 1/'1/llJ Brine Control 7.-fw /1,./ ~~~. « 7.&:f

(,.~~ -,:<, 1~.! 3A.~ 7.A WQTime: JISO /.2.6 7.6 /5.5 3()./ ·7.5

~ "25' Ji.~ 'Zfi. '1- 7. 'I Technician: (j/J 50 7-fJ lk. I ~'I., 7.9

B5 /.1.. '"·' 5o. I 7.8

*Water quality measurements taken in surrogate water quality chambers.

START TIME:

END TIME:

HOBO TEMP. NO.: l. "l..'j b TEST LOCATION: ~

DILUTION WATER BATCH:

TEST ACCEPT ABILITY:

)( <!:70% SURVIVAL IN CONTROL (oysters) or 50% SURVIVAL FOR MUSSELS

1i( <!: 90"k NORMAL SHELL DEVELOPMENT IN SURVIVING CONTROLS

'Q MSD < 25'Yo

Initial Spawning Time:

1.

Stocking Volume Calculation:

Rep1 Cone.

Normal Abnormal

Control 25""t _'j Brine 211 I> &. z.t;_ 25""1 J l I 2 .. t; 23'3 I t 25 2~P Z(

5D 1~/n ~D

lrS Jc;"2_ ~6 -

.. . .

Concentration I Replicate

Total#

#Normal

Date /Initials

BIVALVE 48-HOUR CHRONIC TOXICITY TEST 810042

Client: Client Sample 10:

f~ 6lfl2-

SPAWNING DATA Fertilization Time: No. of Males:

:t 2 Average:

., .··.t.ARVAk."COUNT DATA.':=:;

Re~{:2. =

·-· ... . . .. .. ;.:.··.:

·. '· -='Rep .3 , ~· ·- fl~p4 .~ep5 · Date Initials

Normal Abnormal Normal Abnormal Normal Abnormal Normal Abnormal

21'1 +- 2.3lD q 2'30 1- jl/1'~.(10 ltM> 211 .Lt 25'f 5 25ih _fD

2/e{, - q 2.5~ lO 2.5_1 13 2'1P. IC::: 21-i- ll 253

{ ' "" IL

2tf-~ L3 22't 1k 251 .2.'f 'r71titb li..i . 203 3CJ 2Dl lflf 11'5 l.fO I c; 8' r C>_(q tlfl tn~ If'? qz .. v l.f

. : 'QA. COtJ,~T:'CttJ:C~S : . ' ·. : ··· .... .. . . . · ..

.. ..... ... - ... '· .. 'qAcheck#1

' ·9.~.check 12 ·.

I, : ·QA Check#~ QACheck#4

~- I + 2.5 I J IJO I I es I 2. t-!8 24Z ~.4-1 2&8 tqo J,f-1 164 //,()

711//;{) I u 7/!1/tl) I~ 7//f/1() I~ 7/lf(/OI -~ QA Check Acceptability: )ia <5% difference in means of QA & orig. counts

~- -·- n -1 "

I

-c Gl Q. Q. 0

~ .c Q. Q.

~ 0 w

Myti/us ga/Joprovincialis Reference Toxicant Control Chart: Proportion Alive

CV% = 21.9

35

30

25

20

15

10

#####~##~##~~~~~~~## ~~~~~·~~~~~~#~~~~-#~~~ a ~ zy ~ ~ ~~ ~ a ~ ~ a ~ ~ ~ ~· ~· ~ ~ a Test Dates

+2SD

+1 so

Mean

-1 so

-2 so

Dates Valt.tes Mean -1 so -2SD +1 so +2SD 07/31/08 15.1480 22.7884 17.7888 12.7893 27.7880 32.7875 10108/08 20.5190 22.7884 17.7888 12.7893 27.7880 32.7875 12/12/08 21.6370 22.7884 17.7888 12.7893 27.7880 32.7875 12/16/08 28.0290 22.7884 17.7888 12.7893 27.7880 32.7875 01/13/09 32.7620 22.7884 17.7888 12.7893 27.7880 32.7875 02/06/09 30.0650 22.7884 17.7888 12.7893 27.7880 32.7875 06/10109 26.2370 22.7884 17.7888 12.7893 27.7880 32.7875 06126109 15.0480 22.7884 17.7888 12.7893 27.7880 32.7875 07/14/09 27.7330 22.7884 17.7888 12.7893 27.7880 32.7875 08/19109 23.0140 22.7884 17.7888 12.7893 27.7880 32.7875 10127109 20.2390 22.7884 17.7888. 12.7893 27.7880 32.7875 01/06110 26.9160 22.7884 17.7888 12.7893 27.7880 32.7875 01121/10 15.1460 22.7884 17.7888 12.7893 27.7880 32.7875 02/28/10 22.9430 22.7884 17.7888 12.7893 27.7880 32.7875 03/12/10 20.4180 22.7884 17.7888 12.7893 27.7880 32.7875 04/02110 23.3960 22.7884 17.7888 12.7893 27.7880 32.7875 04/06/10 20.8750 22.7884 17.7888 12.7893 27.7880 32.7875 04/14/10 16.9500 22.7884 17.7888 12.7893 27.7880 32.7875 06/08/10 25.6340 22.7884 17.7888 12.7893 27.7880 32.7875 07/07/10 23.0590 22.7884 17.7888 12.7893 27.7880 32.7875

Updated 7/14/10 OS

Myti/us gal/oprovlncialis Reference Toxicant Control Chart: Proportion Normal

CV% == 19.5

9 +2 so

-.:- 8 ~ +1 so Q. 0 7 £ ~ Mean Q. 6 Q.

:;: 0 5 ·1 so w

4 ·2 SD

3

Dates Values Mean -1 so -2 so +1 so +2SD 07131/08 5.6218 6.2039 4.9940 3.7840 7.4138 8.6237 10/08/08 5.9754 6.2039 4.9940 3.7840 7.4138 8.6237 12/12/08 4.9351 6.2039 4.9940 3.7840 7.4138 8.6237 12/16/08 4.0833 6.2039 4.9940 3.7840 7.4138 8.6237 01/13/09 4.1374 6.2039 4.9940 3.7840 7.4138 8.6237 02106/09 7.0125 6.2039 4.9940 3.7840 7.4138 8.6237 06/10/09 5.3031 6.2039 4.9940 3.7840. 7.4138 8.6237 06/26/09 7.3577 6.2039 4.9940 3.7840 7.4138 8.6237 07/14/09 7.8205 6.2039 4.9940 3.7840 7.4138 8.6237 08/19/09 7;5955 6;2039 4.9940 3.7840 7.4138 8.6237 10/27/09 6.0112 6.20~9 4.9940 3.7840 7.4138 8.6237 01/06/10 7.7360 6.2039 4.9940 3.7840 7.4138 8.6237. 01/21110 4.8129 6.2039 4.9940 3.7840 7.4138 8.6237 02/28/10 5.9625 6.2039 4.9940 3.7840 7.4138 8.6237 03112/10 6.8784 6.2039 4.9940 3.7840 7.4138 8.6237 04/02/10 7.2994 6.2039 4.9940 3.7840 7.4138 8.6237 04/06/10 6.4404 6.2039 4.9940 3.7840 7.4138 8.6237 04/14/10 4.8596 6.2039 4.9940 3.7840 7.4138 8.6237 06/08/10 7.3768 6.2039 4.9940 3.7840 7.4138 8.6237 07/07/10 6.8581 6.2039 4.9940 3.7840 7.4138 8.6237

Updated 7/14/10 OS

Bivalve Larval Survival and Development Test-Proportion Alive Start Date: 717/2010 16:35 • Test 10: C100316.39 . Sample 10: REF-Ref Toxicant End Date: 7/9/2010 16:15 . Lab 10: CCA-Weston, Carlsbad · Sample Type: CUSO-Copper sulfate Sample Date: Protocol: EPAW 95-EPA West Coast • Test Species: MG-Mytilus galloprovincialis Comments:

Conc-ppb Control

2.5 5

10 20 40

1 1.0000 1.0000 1.0000 1.0000 0.7381 0.0000

2 0.9762 0.9921 1.0000 0.9484 0.7897 0.0079

3 4 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.9603 0.9206 0.7341 0.6825 0.0040 0.0040

Transform: Arcsin Sguare Root Conc:epb Mean N-Mean Mean Min Max CV% N

Control 0.9940 1.0000 1.5084 1.4159 1.5393 4.091 4 2.5 0.9980 1.0040 1.5249 1.4816 1.5393 1.892 4

5 1.0000 1.0060 1.5393 1.5393 1.5393 0.000 4 10 0.9573 0.9631 1.3841 1.2852 1.5393 7.899 4

"20 0.7361 0.7405 1.0323 0.9723 1.0944 4.834 4 *40 0.0040 0.0040 0.0617 0.0315 0.0892 38266 4

Auxilia!l Tests Statistic Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.89521 Eguality of variance cannot be confirmed Hlpothesls Test {1-tail, 0.05! NOEC LOEC ChV TU Steel's Many-One Rank Test 10 20 14.1421

Trimmed Spearman-Karber Trim level EC50 95%CL

O.Oo/o 5.0% 23.715 23.204 24.237

10.0% 24.162 23.569 24.770 1.0 20.0% 24.867 23.911 25,861 0.9

Auto-0.4% 23.059 • 22.575 23.554 0.8

0.7

Cll 0.6 • 5 o.s ~ 0.4 Cll a:: 0.3

0.2

0.1

0.0

-0.1

Rank 1-Tailed Number Sum Critical Rese

6 18.50 10.00 2 20.00 10.00 0 12.50 10.00 43 10.00 10.00 266 10.00 10.00 1004

Critical Skew 0.884 0.70596

10 100

Dose ppb

Total Number

. 1008 1008 1008 1008 1008 1008

Kurt 3.34461

Page 1 ToxCalc v5.0.23 Reriewedby~

Bivalve Larval Survival and Development Test-Proportion Alive Start Date: 1n12010 16:35 • Test ID: C100316.39 • Sample ID: REF-RefToxicant • End Date: 7/9/201016:15 • Lab 10: CCA-Weston, Carlsbad • Sample Type: CUSO-Coppersulfate • Sample Date: Protocol: EPAW 95-EPA West Coast -Test Species: MG-Mytilus galloprovincialis • Comments: for £C25 only ·

Conc-ppb 1 2 Control 1.0000 0.9762

2.5 1.0000 0.9921 5 1.0000 1.0000

10 1.0000 0.9484 20 0.7381 0.7897 40 0.0000 0.0079

Conc-ppb Mean N-Mean Control 0.9940 1.0000

2.5 0.9980 1.0040 5 1.0000 1.0060

10 0.9573 0.9631 *20 0.7361 0.7405 *40 0.0040 0.0040

3 1.0000 1.0000 1.0000 0.9603 0.7341 0.0040

4 1.0000 1.0000 1.0000 0.9206 0.6825 0.0040

Transform: Arcsin Sguare Root Mean Min Max CV% 1.5084 1.4159 1.5393 4.091 1.5249 1.4816 1.5393 1.892 1.5393 1.5393 1.5393 0.000 1.3841 1.2852 1.5393 7.899 1.0323 0.9723 1.0944 4.834 0.0617 0.0315 0.0892 38.266

Rank 1-Tailed Number N Sum Critical Resp 4 6 4 18.50 10.00 2 4 20.00 10.00 0 4 12.50 10.00 43 4 10.00 10.00 266 4 10.00 10.00 1004

Total Number

1008 1008 1008 1008 1008 1008

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test Indicates normal distribution (p > 0.01) 0.89521 0.884 0.70596 3.34461 Equality of variance cannot be confirmed Hypothesis Test (1-tall, 0.05) NOEC LOEC ChV TU Steel's Many-One Rank Test 10 20 14.1421

Maximum Likelihood-Probit Parameter Value SE 95% Fiducial Limits Control Chl-5q Critical P-value Mu Sigma Iter Slope 10.9584 3.21366 0:73115 21.1857 0.00595 94.7059 7.81473 2.1 E-20 1.36116 0.09125 9 Intercept -9.9162 4.25046 -23.443 3.61062 TSCR 0.01261 0.00988 -0.0188 0.04405 Point Problts ppb 95% Fiducial limits EC01 2.674 14.0889 0.05376 17.6508

0.9

EC05 3.355 16.2579 0.45112 19.3735 0.8

EC10 3.718 17.5476 1.38549 20.6033 0.7 EC15 3.964 18.4751 2.91423 21.7689 EC20 4.158 19.247 5.16023 23.1917 EC25 4.326 19.9349' 8.14652 25.3224 eC4o 4.747 21.n94 16.776 48.4917 EC50 5.000 22.9702 19.2115 96.662 EC60 5.253 24.2261 20.6357 205.429

:0.6 c: &_0.5 Cll &! 0.4

0.3

EC75 5.674 26.4676 22.3136 749.184 0.2

EC80 5.842 27.4136 22.8881 1259 0.1 EC85 6.036 28.559 23.5294 2310.29 EC90 6.282 30.0685 24.3126 4968.95 EC95 6.645 32.4537 25.4556 15500.7

0.0 +-....,..,,.,..,..-.t ........ '"""'--r ......... """'"....,..,.,.,.,.mf 0.01 100 1 0000 1000000

EC99 7.326 37.4501 27.6215 131564 Significant heterogeneity detected (p = 2.14E-20)

Dose ppb

Page 1 ToxCalc v5.0.23 Reviewed by:~

Bivalve larval Survival and Development Test-Proportion Normal Start Date: 717/2010 16:35 • Test ID: C100316.39 • Sample ID: REF-RefToxicant • End Date: 71912010 16:15 · Lab 10: CCA-Weston, Carlsbad • Sample Type: CUSO-Copper sulfate Sample Date: Protocol: EPAW 95-EPA West Coast •Test Species: MG-Mytilus galloprovincialis Comments:

Conc-ppb Control

2.5 5

10 20 40

Conc-~b Control

2.5 ·s

"10 20 40

Auxilia~ Tests

1 2 0.9712 0.9715 0.9725 0.9680 0.9061 0.8902 0.0541 0.0377 0.0000 0.0000 0.0000 0.0000

Mean N-Mean 0.9669 1.0000 0.9656 0.9987 0.9010 0.9319 0.0356 0.0368 0.0000 0.0000 0.0000 0.0000

3 4 0.9709 0.9540 0.9611 0.9609 0.9125 0.8953 0.0248 0.0259 0.0000 0.0000 0.0000

Transform: Arcsin Sguare Root Mean Min Max CV% N 1.3889 1.3547 1.4013 1.644 4 1.3848 1.3718 1.4043 1.138 4 1.2511 1.2330 1.2706 1.364 4 0.1874 0.1581 0.2346 19.046 4 0.0368 0.0355 0.0381 2.984 4 0.4695 0.3614 0.5236 19.949 3

Statistic Shapiro-Wilk's Test indicates normal distribution (p > 0.01) 0.94954 Bartlett's Test indicates egual variances~~= 0.51} 2.32379 Hleothesis Test ~1-tail, 0.051 NOEC LOEC ChV TU NiSOu Dunnett's Test 2.5 5 3.53553 0.01533

Maximum Likelihood-Probit Parameter Value SE 95% Fiducial Umlts Contror Chl-5g

t-Stat

0.239 8.070

70.330

MSD(! 0.01585

Critical Slope 10.8876 0.35934 10.1833 11.5919 0.03302 0.01524 7.81473 Intercept -4.1042 0.30717 -4.7063 -3.5022 TSCR 0.03369 0.00396 0.02593 0.04144 1.0 Point Pro bits eeb 95% Fiducial Limits

0.9 EC01 2.674 4.19307 4.01482 4.35977 EC05 3.355 4.84312 4.67462 5.00145 0.8

EC10 3.718 5.2299 5.06736 5.38367 0.7 EC15 3.964 5.50817 5.34946 5.65935 EC20 4.158 5.73985 5.5838 5.88956 :0.6

EC25 4.326 5.94636 5.79211 6.09546 c &. 0.5

EC40 4.747 6.50028 6.34727 6.65187 Ul

EC50 5.000 6.85806 -6.70237 7.01514 ~0.4

EC60 5.253 7.23554 7.07364 7.40211 0.3 EC75 5.674 7.90955 7.72791 8.10259 0.2 EC80 5.842 8.19412 8.00106 8.40173 EC85 6.036 8.53877 8.32971 8. 76643 0.1

EC90 6.282 8.99309 8.75976 9.25075 0.0 EC95 6.645 9.7113 9.4335 10.0234 EC99 7.326 11.2169 10.8278 11.6643

1-Talled Number Critical MSD Rese

35 2.290 0.0391 35 2.290 0.0391 107 2.290 0.0391 937

742 4

Critical Skew 0.844 0.24583

11.3449 MSB MSE F-Prob

1.34864 0.00058 7.8E-17

P-vatue Mu Sigma 1 0.8362 0.09185

r

) 10 100

Dose ppb

Total Number

1060 1018 1081 972 742

4

Kurt -0.0251

df 3,12

Iter 4

Page 1 ToxCalc v5.0.23 Reviewed by:~

Test: BV-Bivalve Larval Survival and Development Test • Test 10: C100316.39 • Species: MG-Mytilus galloprovincialis • Protocol: EPAW 95-EPA West Coast ' Sample ID: REF-Ref Toxicant

. Sample Type: CUSO-Copper sulfate

Start Date: 7n/2010 16:35 • End Date: 7/9/2010 16~1!Lab ID:CCA-VIIeston, Carlsbad·

Initial Final Total Number Pos 10 Rep Group Dens~ty Density Counted Normal Notes

1 1 Control 252 278 278 270 2 2 Control 252 246 246 239 3 3 Control 252 275 275 267 4 4 Control 252 261 261 249 5 1 2.500 252 255 255 248 6 2 2.500 252 250 250 242 7 3 2.500 252 257 257 247 8 4 2.500 252 256 256 246 9 1 5.000 252 277 277 251 10 2 5.000 252 264 264 235 11 3 5.000 252 263 263 240 12 4 5.000 252 277 277 248 13 1 10.000 252 259 259 14 14 2 10.000 252 239 239 9 15 3 10.000 252 242 242 6 16 4 10.000 252 232 232 6 17 1 20.000 252 186 186 0 18 2 20.000 252 199 199 0 19 3 20.000 252 185 185 0 20 4 20.000 252 172 172 0 21 1 40.000 252 0 0 0 22 2 40.000 252 2 2 0 23 3 40.000 252 1 1 0 24 4 40.000 252 1 1 0

Comments:

Page 1 ToxCalc5.0 Reviewed by:$

-~-Bivalve Counts Worksheet

T tID C100316 39 C RT es . opper . Concentration Replicate Number Normal Number Abnormal Total Counted

1 270 8 278

Control 2 239 7 ·-· 246 ·-

3 267 8 275 --4 249 12 261 1 248 7 255 --

2.5 2 242 8 250 3 247 10 -----~7

----246

·---··--···-~ 256 4 10

1 251 26 277 -- -5 2 235 29 264

3 240 23 263 ·-4 248 29 277 1 14 245 259

""

10 2 9 230 239 3 6 236 242 4 6 226 232 1 0 186 186

20 2 0 199 199 3 0 185 185 4 0 172 172 1 0 0 0 ·----

40 1-· 2 0 . - 2 2 3 0 1 -----1

-4 0 1 1

~. 48 Hour Bivalve Development Reference Toxicant Test

TestiDflb03"llf.3 "1 Replicates: 4 StudyDirector:,j'".~~t.l"' Location:~""' 2... Dilution Water Batch: Organism Batch: Associated Jest(s): Organism: 5fb OlnO~IO -r5_F_R7_c;K' Alvf'_sk~ LLo"'r;< '(~'- ~ 1\411. .:. '~ol;'i

Toxicant: Copper ~dl: Date Prepared: ' Initials: ~ ·1

~~;~!~uSO.) \ ~ b 1 \o \~ 1-I 1-lt 0 /%4. Target Quantity ofS.tock: Quantity of Diluent: Concentrations: Target: Target:

40 .ppb 0.039 mL S'OO mL

40ppb Actual: ().07q3 Y~ttl Actual: SPQ.D ,L. Serial Dilute by ~to obtain concentrations of20., 10, 5, and 2.5 ppb.

0 Hours Date: 1{1/10 WQ Time: 15fS Start Time: 1~15 Initialsifft

STOCK Control 2.5 5 10 20 40

/.S _].5 Toempe e · ['). Cj ,,.1 ,c;.? Salinity 10.0 :?0. \ so.z. pH f. I

48 Hours Date: 7/9/10 WQ Time(l.f(} ~ End Time: f{, 15 Initials: 2f4 STOCK

Control 2.5 5 20 40

D.O.~!~ 7_. I 7. _(p _1. 7_ 7.5 TemperatUre / ~. "3 lu. 0 lu.o /~.o I~. I Salinity g 0. t_

pH 7.~

0 Fail

Aryk~!!! _/

;·':!;.-·.·:·····

Chain of Custody

1 :' :· :, '.. • • • :·:.•:. :,_ : • •••••••• :.-·: ••• ,.: ··, .... ··~ •• :.:;: ·.::. ~ • •• ·: ~-, • • • •f•;·· ·.; :,-. •,

. .·.· ... · .·· .· . . .. ·. . . . .. . . .. . GHAIN; QF CUSTODY REd6RD . . !LABORATORY: . .. . I CUSTODY RECORD I REPORT TO: . d • • • • • • ' I d ~AL YS!;~: I WORK ORDER#: I SPECIAL PROJECT#: I

REQUES.TED' . . Weston Solutions, Inc. 2433 Impala Drive

Carlsbad, CA 92008 (760) 931·8081 Attn: Ainy Margolis

Alyeska Pipeline Service Co. Attn: Juliet Cruz P.O. Box 196660 Anchorage, AK 99519-6660 F~: (907)787-8778 Phone: (907) 787-8719

SAMPLER: Complete record, copy, and send original With samples.

u 1: ·c: e ~ I ~

:.·,,.\

On receipt of samples, complete record. On completion of analysis, 1'8tum copy of 1'8cord : 1; .· . · With 1'8port. Notify Analytical Servlcea before disposal of samples. ;

LABORATORY:

Grab, Composite, flow--Jghted, continuous monitoring ~

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~w

_J~~~[~I~:d i(': ·~#:;,~;:·;: .. ; ~ AFBG:t<t (2.. I 'f>A f:J·lP-/0 l)L/t./0 IBWTEflluent

---- -· -. --Grab ifx4L

r..u. 'Cool s; 6 'dWater X

I PROJECT NAME:

.l::'ic. '. 3rd Quarter WET

V10·081

REQUESTED TURN AROUND TIME (DAYS):

Standard

••••• p ·~.~ ,;: REMARKS\ ·

... ,:.:,, ·'·'

JpH=6.93

·J' .... ; .. :.:; '· ·gh:F,':;'~)';;·:,·)!j; .· :::J:~;!{.if;; '~·-:-~~j:j~~·'[ .·.J?·~il: '· ... I;' ·. !Temperature= 8.6 ·c .•71.••.••• :·~- ::·;TJTHL.~. -p~:r.:·:l. · · · ·1: · J,J:>ffcorrected sanntty = 31.2 ppt

. JL.· Ii~~:'c ,::J .: clr:· : .. :.• · .,., ·r':·.·< ·.· 'tl" .. ... ~li .:·•. {/ ::t: ·P::·l·:"F-, . I 1:::, :}:

\ ' :j .· I> · ·I!~'·,: I Run both echlnodenn fertilization test and

·r:r~ ··:'. ?f2·D}~:fl0).·:·.·:·;m;-:w.:-;2-'1l'::: •. ~--~~~J[:I~:·:·, :::::i;Im:k\::'·:;':·.'·:· .. ,1 .. ,, .1:\, .. ,,::J:~ .. ·.·1 '':·:.:.1.·· bivalve embryo-larval development test.

.. .:.QIL:': .. !'.~~IE- .. .PL~? ·' . ,:, ... :,., ..

I' .· .· ~ '- · •• <. •. '~-

IBWT NPDES Pennlt #AKS-002324-8

:::lt;J;?!·:::LjH}:.; :::~:,,:1~)/!:.~;::Y!Y. ;,]·~•• ·>:·:Ii::~:!rlt-.:J.; .. ,; .. <It::\-, .•.. '1:~· ··d.: ...... H\.,,:.

-~LI~!TH.ED BY: I DATE TIME (inm/dd/yy hrhr:mlnmln): I RECEIVED BY: I RELINQUISHED BY: I DATE TIME (mm/dd/yy hrflr:mlnmln):

\$~¥cff- -=\-iP-10 OC900 Cooler RECEIVED BY:

RELINQUISHED BY: I DATE TIME (mmfdd/yy hrhr:mfnmln): I RECEIVED BY:

DATE TIME (mm/dd/yy hrhr:minmfn): LABORATORY ID/BATCH #:

IO:J() ClOOt-0':1.0(

IVED FJR LA~ j3Y:

~ t: -:;r h-fTljD Dn..l\ ~,IAlLr~ ~ ·

FedEx 7988 2017 6867

Am BILL# (attach receipts): REMAR.KS: ()Q?tS9 \

Custody Seal#~

10438 (O.W1)

RELINQUISHED BY:

DISPOSAL DATE:

DATE TIME (mm/dd/yy hrflr:mlnmln): RECEIVED BY:

(·.···. ·.RETURN:, 5AMPL'E'C00lERS: ~

TO:

..... ':. :_:.::~·.::~:: .... ' ;.

DATE:

VMT Lab, MS 704 Alyeska Pipeline Service Co.

300 Dayville Road, Valdez, AK 99686

VALDEZ LABORATORY AL YESKA PIPELINE SERVICE COMPANY

DISCHARGE MONITORING REPORT July 2010

ANALYSIS OF BALLAST WATER TREATMENT PLANT EFFLUENT

TABLE IX- NON-ROUTINE WATER ANALYSIS Day of Sample Sample Test

Month Time Description Performed Results Units Analys1 Notes

6 0437 BWT Effluent-Grab TSS 10.4 mg/L WEQ 1 6 0435 BWT Effluent-Grab Total BETX 0.0030 mg/L ALC 1 6 0441 BWT Effluent-Grab Salinity 24.9 ppt BKA 1 6 0441 BWT Effluent-Meter Reading pH 6.71 su BKA 1 6 0441 BWT Effluent-Meter Reading Temperature 14.09 oc BKA 1 6 0441 BWT Effluent-Meter Reading Flow 800 gpm BKA 1

6 0440 BWT Effluent-Grab WET - Chronic Purple Sea Urc~in TUc 2,3 6 0440 BWT Effluent-Grab WET - Chronic Bivalve Species TUc 2,3

Notes: 1. Sampled concurrently with Whole Effluent Toxicity (WET) sampfing. 2. WET analyses were completed by a contract laboratory.

3. Analyst not provided.

Table IX

August 10, 2010

Michael F. Gear heard Director, Office of Water

P.O Box 300

U.S. Environmental Protection Agency, Region 10 OW-133 1200 Sixth A venue Seattle, W A 98101

VALDEZ. ALASKA 99686

Letter No. 21604 File No. 7.14.3

Subject: Reports Required by NPDES Permit No. AK-002324-8

Attn.: Mike Lidgard

Dear Sir:

Enclosed is a copy pf the following report:

TELEPHONE (907) 834-6480

Effluent Toxicity Testing Results for July 2010, prepared by Roy K Kropp, Pacific Northwest National Laboratory, 1529 W. Sequim Bay Road, Sequim, WA 98382. The toxicity testing was performed by Weston Solutions, Inc. of Carlsbad, California.

This report is required by Part ill.A. of NPDES Permit No. AK-002324-8 for the Alyeska Valdez Marine Terminal at Port Valdez, Alaska.

If you have any questions or require further information, please contact Dave Connor at (907) 787-8806.

Kat nZinn Senior Director Valdez Operations

cc: Shawn Stokes, ADEC/ Anchorage Matt Carr, EP A/JPO Environment and Natural Resources Institute JPO Library RCACNaldez Valdez Fisheries Development Association

P.O Box 300 VALDEZ. ALASKA 99686 TELEPHONE (907) 834-6480

"I certify under penalty of law that this document and all attachments were prepared under my direction or supervision in accordance with a system designed to assure that qualified personnel properly gather and evaluate the information submitted. Based on my inquiry of the person or persons who manage the system, or those persons directly responsible for gathering the information, the information submitted is, to the best of my knowledge and belief, true, accurate and complete. I am aware that there are significant penalties for submitting false information, including the possibility of fines and imprisonment for knowing violations."

Kathleen z· Alyesk peline Service Company Senior Director, Valdez Operations

effluent toxicity testing results · purple sea urchins, strongylocentrotus purpuratus, used in the...

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