Assessment of the toxicity (48h LL50) of 

to the marine copepod Acartia 

tonsa

 

 
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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) 16

 

 
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Confidentiality statement 

The information contained in this document is confidential and proprietary and is the property of 

 The contents must not be disclosed to any third party without the express 

and written approval of 

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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 LL50 (mg.l-1) 55 

Definitive test period 31 January – 3 February 2012 

24h LL50 (mg.l-1) 45.37 

48h LL50 (mg.l-1) 21.68 

48h LL90 (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 O2 range ≥ 80% 89 – 98 

Informal reference toxicant 48h LC50 range 0.4 - 1.5 mg.l-1 1.09 

Control mortality ≤ 10% 10 
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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. Appendix B indicates the nature of test material 

preparation methods and Appendix C contains 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 therefore 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 0.252 
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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 LL50 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 0.02490 
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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 distribution (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/L 

Response
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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 distribution (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/L 

Response
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Table 1.5 Calculated LL50 values with 95% confidence limits, and 48h LL90 and NOELR 

values 

 95% Confidence limits

(mg.l-1) 

Test 

material 

LL50

(mg.l-1) Lower Upper 

48h LL90

(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 LL50 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 was 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.l1

 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. 
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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. 
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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 days 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 conducted at 1000, 100, 10 and 1 mg.l-1, plus four control vessels. Definitive 

test concentrations depend on the magnitude of response evident in the Rangefinding test and employ 

five concentrations, plus four control vessels. 

In the Rangefinding tests, five animals were exposed 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 and 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 LL50, 48h LL90 and 48h NOELR values are 

calculated using an appropriate statistical method from the ToxCalc Version 5 software.
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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 material was observed to be soluble a dilution series 

was prepared, where an appropriate weight of test material 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 hour and its behaviour assessed (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 Water 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 ‰. 
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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 89 
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ToxCalc analysis of Acartia mortality after 48 hours exposure to 3,5 DCP and control media 

Table C4 Calculated 3,5 DCP LC50 (mg.l-1) values with 95% confidence limits

 95% confidence limits 

Test material 48h LC50 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/L 

Response
Study no Page no 16 

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Appendix D Certificate of Analysis (client supplied)