### Assessment of the toxicity (48h LL 50 

### ) of

to the marine copepod Acartia tonsaStudy no Page no 2

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### Contents

Page no

Confidentiality statement 3

Study director and quality assurance statements 4

Summary 5

Section 1: 6

Retention and archiving of test documentation 11

References 11

Appendix A Test methods 11

Appendix B Preparation methods 12

Appendix C Control data 13

Appendix D Certificate of Analysis (client supplied) 16Study no Page no 3

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### Confidentiality statement

The information contained in this document is conf idential and proprietary and is the property of The contents must not be disclosed to any third party without the express and written approval ofStudy no Page no 5

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### Summary

Sponsor

name Test personnel Mr William Scott, Study Director Miss Melanie Anderson, Technician Miss Rosalind Murphy, Technician

Sponsor address Test facility Opus Plus Limited Flotta, STROMNESS Orkney, KW16 3NP t +44 1856 702 000 f +44 1856 701 473 admin@opus-results.com www.opus-results.com

Sponsor contact Test guidelines ISO 14669 (1999) Water Quality - Determination of acute lethal toxicity to marine copepods ( Copepoda; Crustacea )

ISO 5667-16 (1998) Water Quality Sampling – Guidance on biotesting of samples

Study number was commissioned by to determine the aquatic phase toxicity of to the marine copepod Acartia tonsa. A summary of all testing conducted is given below:

Test material

Behaviour in seawater Poorly Soluble Preparation method WAFs Range finding test period 10 – 13 January 2012 Provisional 48h LL 50 (mg.l - 1 ) 55 Definitive test period 31 January – 3 February 2012 24h LL 50 (mg.l - 1 ) 45.37 48h LL 50 (mg.l - 1 ) 21.68 48h LL 90 (mg.l - 1 ) 31.09 48h NOELR (mg.l - 1 ) 10

Tests were assessed for compliance by the following guideline criteria:

Parameter Guideline criterion Observed values Salinity at 0h of dilution water 36 ± 4‰ 36 pH at 0h of dilution water 8 ± 0.3 8.01 pH at 0h of the test material stocks 6 - 9 8.05 – 8.16 Temperature range 20 ± 2 °C 20.7 – 21.5 Dissolved O 2 range ≥ 80% 89 – 98 Informal reference toxicant 48h LC 50 range 0.4 - 1.5 mg.l - 1 1.09 Control mortality ≤ 10% 10Study no Page no 6

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### Section 1

Test organisms were obtained from age standardised cohorts and were 19 days old at definitive test commencement. Details of culture methods, in addition to test methods, procedures, guidelines and statistical methods are given in Appendix A. A ppendix B indicates the nature of test material preparation methods and Appendix C cont ains the quality control data.

Characterisation of

Table 1.1 Description and characterisation (SOP 402)

Property MSDS supplied Observed

Form Powder Powder Colour Dark Dark Brown Density 50lbs/ft3 (uncompacted) 55 (compacted) -

Odour Not stated None Viscosity Not stated N/A pH Not stated TSW=7.56, (1000 mg.l -1 stock) Aqueous solubility Insoluble, highly dispersible in oil Floating or settled particles at 1000 mg.l -1 in sea water after 20 hours stirring Preparation method Water Accommodated Fractions (WAFs) Flash point Not stated Melting point 400°F Boiling point Not stated

Chemical Description Name, CAS No, Percentage composition

100%

was characterised as poorly soluble and was ther efore prepared by Water Accommodated Fractions (WAFs).

Rangefinding test preparation

Table 1.2 Test material preparation (Water Accommodated Fractions (WAFs))

Diluent Preparation volumes (ml) Nominal concentrations (mg.l -1 ) Weight (g) added

Treated seawater 1000 1 0.00100 250 10 0.00251 250 100 0.02496 250 1000 0.252Study no Page no 7

Project no Issue no 1.0 Rangefinding test results

Table 1.3 Mortality of Acartia after 48h exposure

Nominal Concentration (mg.l -1 ) Number dead/immobile at test termination Total exposed Proportional response 48h
1 0 5 0.00
10 0 5 0.00
100 5 5 1.00
1000 6 6* 1.00 

### * See interpretation.

The Rangefinding test exhibited a 48h LL 50 of 55mg.l -1 (Water Accommodated Fractions (WAFs)).

Definitive test preparation

Table 1.4 Test material preparation (Water Accommodated Fractions (WAFs))

Diluent Preparation volumes (ml) Nominal concentrations (mg.l -1 ) Weight (g) added

Treated seawater 1000 1.0 0.00100 1000 3.2 0.00321 250 10 0.00249 250 32 0.00800 250 100 0.02490Study no Page no 8

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### Definitive test results

ToxCalc analysis of Acartia mortality after 24 hours

Acartia Survival Test-24h survival

Conc-mg/L 1 2 3 4 5 6 7 8 D-Control 1.0000 0.9000 1.0000 0.9091 1.0000 1.0000 1.0000 1.0000 1 0.9091 1.0000 3.2 1.0000 1.0000 10 1.0000 1.0000 32 0.7000 0.5000 100 0.0000 0.0000

Transform: Arcsin Square Root 1-Tailed Isotonic Conc-mg/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean D-Control 0.9761 1.0000 1.3731 1.2490 1.4195 5.245 8 0.9819 1.0000 1 0.9545 0.9779 1.3383 1.2645 1.4120 7.793 2 0.583 2.681 0.1600 0.9819 1.0000 3.2 1.0000 1.0244 1.4157 1.4120 1.4195 0.372 2 -0.715 2.681 0.1600 0.9819 1.0000 10 1.0000 1.0244 1.4120 1.4120 1.4120 0.000 2 -0.653 2.681 0.1600 0.9819 1.0000 *32 0.6000 0.6147 0.8883 0.7854 0.9912 16.379 2 8.124 2.681 0.1600 0.6111 0.6224 *100 0.0000 0.0000 0.1588 0.1588 0.1588 0.000 2 20.348 2.681 0.1600 0.0000 0.0000

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal dist ribution (p > 0.01) 0.89499 0.858 -0.7319 -0.1244 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 10 32 17.8885 0.08401 0.08738 0.56429 0.0057 2.5E-09 5, 12

Linear Interpolation (200 Resamples) Point mg/L SD 95% CL(Exp) Skew IC50 45.369 6.962 18.105 60.645 -0.2807 IC90 89.074 1.392 83.621 92.129 -0.2807

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0 50 100 150 Dose mg/LResponseStudy no Page no 9

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### Definitive test results

ToxCalc analysis of Acartia mortality after 48 hours

Acartia Survival Test-48h survival

Conc-mg/L 1 2 3 4 5 6 7 8 D-Control 1.0000 0.8000 0.9000 0.8182 0.8889 1.0000 0.9000 0.9000 1 0.9091 0.8000 3.2 1.0000 0.9000 10 0.8000 1.0000 32 0.1000 0.0000 100 0.0000 0.0000

Transform: Arcsin Square Root 1-Tailed Isotonic Conc-mg/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean D-Control 0.9009 1.0000 1.2559 1.1071 1.4195 9.018 8 0.9036 1.0000 1 0.8545 0.9486 1.1858 1.1071 1.2645 9.384 2 0.737 2.681 0.2549 0.9036 1.0000 3.2 0.9500 1.0545 1.3343 1.2490 1.4195 9.032 2 -0.824 2.681 0.2549 0.9036 1.0000 10 0.9000 0.9990 1.2596 1.1071 1.4120 17.115 2 -0.039 2.681 0.2549 0.9000 0.9960 *32 0.0500 0.0555 0.2497 0.1777 0.3218 40.785 2 10.583 2.681 0.2549 0.0556 0.0615 *100 0.0000 0.0000 0.1588 0.1588 0.1588 0.000 2 11.540 2.681 0.2549 0.0000 0.0000

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal dist ribution (p > 0.01) 0.9393 0.858 0.18588 -0.9061 Equality of variance cannot be confirmed Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 10 32 17.8885 0.19509 0.21579 0.69644 0.01446 1.6E-07 5, 12

Linear Interpolation (200 Resamples) Point mg/L SD 95% CL(Exp) Skew IC50 21.677 0.832 16.735 23.148 -0.2417 IC90 31.093 3.365 27.690 48.822 1.1596

-0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0 50 100 150 Dose mg/LResponseStudy no Page no 10

Project no Issue no 1.0 Table 1.5 Calculated LL 50 values with 95% confidence limits, and 48h LL 90 and NOELR values

95% Confidence limits (mg.l -1 )
Test material LL 50 (mg.l -1 ) Lower Upper 48h LL 90 (mg.l -1 ) 48h NOELR (mg.l -1 )

24h 45.37 18.11 60.65

48h 21.68 16.74 23.15 31.09 10



### Interpretation

The test was conducted in accordance with the study plan and met all relevant validity criteria.

exhibited a 48h LL 50 value of 21.68mg.l -1 (Water Accommodated Fractions (WAFs)) to the marine copepod Acartia tonsa in the aqueous phase.

The result is based on nominal concentrations and wa s calculated by Linear Interpolation within the Toxcalc suite of statistical analysis.

The result is taken from a repeat test, which was required due to irregular mortality patterns in the initial definitive test. Initially the lowest concentration (10mg.l -1 ) showed a mort rate of 25% and the next conc. up (18mg.l -1 ) showed 85%. This is both quite a high rate for a bottom concentration and a very large increase over a small concentration range, which is why a second test was started at 1mg.l 1 to 100mg.l -1 in order to get a more representative result.

6 animals were added to the 1000mg/l pot during the rangefinding test due to human error.

There were no interferences in this test.Study no Page no 11

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### Retention and archiving of test documentation

The study plan and all data and records generated during the test are archived at Opus Plus Ltd's offices, and will be retained for a period of three and a half years from the date of the study.



### References

ISO 14669 (1999) Water Quality: Determination of acute lethal toxicity to marine copepods (Copepoda;Crustacea).

ISO 5667-16 (1998) Water Quality Sampling – Guidance on biotesting of samples.

ToxCalc Version 5 Tidepool Scientific Software.Study no Page no 12

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### Appendix A

Test organisms

The Initial culture, CCAP 1077/5, was received from Dunstaffnage Marine Laboratory in 1995.

The test organisms were cultured in accordance with SOP 501 to provide age standardised cohorts. Organisms used for testing were between 17 and 25 day s old at test commencement; stage 5 in their life cycle. The culture system was maintained with flowing 0.45 μm filtered ultra violet treated sea water, supplied by pump from Scapa Flow in Orkney . The cultures were maintained on a mixed algal diet comprising of between 2 and 4 species.

Test method and guidelines

Test methods were consistent with ISO 14669 (1999) Water Quality: Determination of acute lethal toxicity to marine adult copepods ( Copepoda; Crustacea ).

ISO 5667-16 (1998) Water Quality Sampling – Guidance on biotesting samples.

The method estimates the effect of chemicals on the mortality/immobility on adult copepods over a period of 48h at a temperature of 20 ± 2 °C.

Test procedure

Tests were conducted in 100 ml capacity borosilicate glass crystallising dishes, each containing 50 ml of test medium covered with soda glass watch covers.

Rangefinding tests were condu cted at 1000, 100, 10 and 1 mg.l -1 , plus four control vessels. Definitive test concentrations depend on the magnitude of resp onse evident in the Rangefinding test and employ five concentrations, plus four control vessels.

In the Rangefinding tests, five animals were expo sed per concentration and in definitive tests ten animals were exposed per replicate (20 per treatment). Test animals were transferred to test vessels using glass pipettes of three to five millimetres in diameter (internal) and with fire polished rims.

Measurement of water quality (dissolved oxygen, pH and temperature) are carried out in one replicate at each concentration at 0h and 48h. Observation of mortalities are carried out at 24h and 48h. An animal is regarded as immobile if after gentle stimulation no movement is visible after 10 seconds.

A salinity measurement is carried out in the control medium at 0h.

Statistical methods

Mortalities are determined in each vessel at 24 a nd 48h. The number of dead or immobile copepods are expressed as a proportion of the total number exposed.

Where sufficient response is observed, the 24 and 48h LL 50 , 48h LL 90 and 48h NOELR values are calculated using an appropriate statistical method from the ToxCalc Version 5 software.Study no Page no 13

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### Appendix B

Test material preparation

The test material was assessed for risk to health , and appropriate handling and containing procedures were implemented. Following this, a comparison of the reported and observed physical characteristics (eg form, colour, odour, pH and density) of the test material was carried out.

In order to determine an appropriate test preparation method, an assessment was made of the material’s behaviour in seawater. A 1000 mg.l -1 stock was prepared in filtered seawater, and the resulting mixture was stirred for one hour. If the ma terial was observed to be soluble a dilution series was prepared, where an appropriate weight of test ma terial was added to prepare an initial stock. Appropriate volumes were taken from this stock to prepare subsequent test concentrations which were brought to volume with culture medium. If it was poorly soluble then it was stirred again for approximately 19 hrs, then left to settle for one ho ur and its behaviour asse ssed (SOP 402). If, the material produced floating, settled or neutrally buoyant particles or films, it was classified as poorly soluble and exposures were carried out with Wa ter Accommodated Fractions (WAFs). WAFs were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours and a settling period of approximately one hour. After this settling period, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.

A reference test was conducted concurrently using 3,5 Dichlorophenol at 0.10, 0.32, 1.0, 1.8 and 3.2 mg.l -1 which were prepared from a main stock of 100 mg.l -1 . The 100 mg.l -1 stock was stirred for a minimum of one hour, or until completely dissolved.

If, at 0h, the pH of the dilution water is out with the pH range of 8 ± 0.3 then pH will be returned to within these limits by adjustment with either 1M HCl or NaOH as is appropriate.

Natural seawater is supplied by pump from Scapa Flow , Orkney and is UV sterilised and filtered to 0.2 μm. The salinity of the seawater should be 36 ± 4 ‰

.Study no Page no 14

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### Appendix C

Control data

Table C1 Age of test organisms at test commencement

Culture number Date initiated Date sampled Age at 0h test T 2 13 January 2012 1 February 2012 19 Days

Table C2 Test room temperature

0h 24h 48h
Date 1 February 2012 2 February 2012 3 February 2012 Temp (°C) 20.8 19.1 – 20.9 19.7 – 20.2

Table C3 Water quality in 3,5 DCP and control vessels at end of 48h test

Nominal Concentration (mg.l -1 ) pH Temperature (°C) Dissolved Oxygen (%)
Control 8.13 21.7 90

0.10 8.13 21.8 90
0.32 8.13 21.7 91
1.0 8.13 21.6 91
1.8 8.13 21.1 90
3.2 8.11 21.2 89Study no Page no 15

Project no Issue no 1.0 ToxCalc analysis of Acartia mortality after 48 hours exposure to 3,5 DCP and control media

Table C4 Calculated 3,5 DCP LC 50 (mg.l -1 ) values with 95% confidence limits

95% confidence limits Test material 48h LC 50 lower upper 3,5 DCP 1.09 0.54 1.54 Acartia Survival Test-48h survival

Conc-mg/L 1 2 3 4 5 6 7 8 D-Control 1.0000 0.8000 0.9000 0.8182 0.8889 1.0000 0.9000 0.9000 0.1 0.8182 0.7273 0 32 1 0000 0.8889 1 0.3333 0.6667 1.8 0.1000 0.0000 3.2 0.0000 0.0000

Transform: Arcsin Square Root 1-Tailed Isotonic Conc-mg/L Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean D-Control 0.9009 1.0000 1.2559 1.1071 1.4195 9.018 8 0.9012 1.0000 0.1 0.7727 0.8577 1.0758 1.0213 1.1303 7.161 2 1.851 2.681 0.2608 0.8586 0.9527 0 32 0.9444 1.0484 1.3172 1.2310 1.4033 9.255 2 -0.630 2.681 0.2608 0.8586 0.9527

*1 0.5000 0.5550 0.7854 0.6155 0.9553 30.596 2 4.837 2.681 0.2608 0.5000 0.5548 *1.8 0 0500 0.0555 0.2403 0.1588 0.3218 47.963 2 10.442 2.681 0.2608 0.0500 0.0555 *3.2 0.0000 0.0000 0.1551 0.1513 0.1588 3.396 2 11.318 2.681 0.2608 0.0000 0.0000

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal distribu ion (p > 0.01) 0.95258 0.858 0.17359 -0.7252 Bartlett's Test indicates equal variances (p = 0.37) 5.38958 15.0863 Hypothesis Test (1-tail, 0.05) NOEC LOEC ChV TU MSDu MSDp MSB MSE F-Prob df Bonferroni t Test 0 32 1 0.56569 0.20044 0.22172 0.67495 0.01514 2.4E-07 5, 12

Linear Interpolation (200 Resamples) Point mg/L SD 95% CL(Exp) Skew IC50 1 0878 0.1578 0.5390 1.5358 -0.2699 IC90 1.7287 0.1128 1.4162 2.2367 0.6506

-0 2 -0.1 0 0 0.1 0 2 0 3 0.4 0 5 0 6 0.7 0 8 0 9 1 0

01234 Dose mg/LResponseStudy no Page no 16

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### Appendix D Certificate of An alysis (client supplied)