### Assessment of the toxicity (10 day LC 50 

### ) of Berol R648 NG to the Marine crustacean Corophium volutator

Opus Project No: 1648-2 Opus Study No: P10187Study no 1648-2 Page no 2

Project no P10187 Issue no 1.0 

### Contents Page nos

Confidentiality statement 3

Study director and quality assurance statements 4

Summary 5

Section 1: Berol R648 NG 6

Retention & archiving of test documentation 9

References 9

Appendix A Test methods 10

Appendix B Test material preparation 12

Appendix C Control data 13

Certificate of Analysis 14Study no 1648-2 Page no 3

Project no P10187 Issue no 1.0 

### Confidentiality statement

The information contained in this document is conf idential and proprietary and is the property of Akzo Nobel Surface Chemistry AB. The contents must not be disclosed to any third party without the express and written approval of Akzo Nobel Surface Chemistry AB.Study no 1648-2 Page no 5

Project no P10187 Issue no 1.0 

### Summary

Sponsor

name Akzo Nobel Surface Chemistry AB. Test personnel Mrs Brenda Hudson, Study Director Mr Nicolas Pirie, Technician Mr William Scott, Technician Miss Rosalind Murphy, Technician

Sponsor
address Stenunge Allè 3
SE-444 85 Stenungsund
Sweden 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 Bengt Fjällborg Test guidelines OSPARCOM guidelines (2006) Part A. A sediment bioassay using an amphipod Corophium sp

.

Study number 1648-2 was commissioned by Akzo Nobel Surface Chemistry AB to determine the sediment phase toxicity of Berol R648 NG to the intertidal amphipod Corophium volutator . A summary of all testing conducted is given below:

Test material Berol R648 NG

Behaviour in seawater Soluble Preparation method Via treated seawater Definitive test period 16 – 27 February 2012 10d LC 50 ( mg.kg - 1 ) 1028
10d LC 90 (mg.kg -1 ) 8558 10d NOEC (mg.kg -1 ) 446

Tests were assessed for compliance by the following guideline criteria:

Test material Berol R648 NG

Dissolved O 2 range ( ≥ 85%) 90 – 99 pH range (7.5 – 8.5) 7.88 – 8.10 Temperature range (15 ± 2 °C) 14.0 – 15.5 Salinity range (maintained at ± 4 ppt from the initial 0hr figure during the test) 36 – 44*
0hr: 37– 39

Control mortality ( ≤ 15%) 7 * Some observed salinities above ±4ppt limit. See Interpretation.Study no 1648-2 Page no 6

Project no P10187 Issue no 1.0 

### Section 1

Test organisms and sediment were collected on 9 F ebruary 2012 from a site in the Bay of Suckquoy, Toab in Orkney and received on site on 10 February 2012, and acclimated to laboratory conditions for 17 days prior to testing. Details of maintenance, 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.

Table 1.1 Description and characterisation (SOP 402)

Property MSDS supplied Observed

Form Liquid Liquid

Colour Brown Brown/Amber Density 1065 Kg/m 3 @ 25°C 1.0591g/cm 3 @ 20°C

Odour No data available Fatty/Grease Viscosity Not stated Slight to moderate pH 8 (1% in water) Treated Seawater = 7.83 (1000 mg.l -1 stock)

Aqueous solubility Soluble Soluble at 1000 mg.l -1 in seawater after 1 hour stirring Preparation

method Via sea water

Flash point 100-199°C Melting point No data available Boiling point >100°C Name, CAS No, Percentage composition

Chemical Quaternary C12-14 alkyl methyl amine ethoxylate methyl chloride CAS No not stated, 60-70% Description(s) Water, CAS No 7732-18-5, 30-50%

Berol R648 NG was characterised as soluble and t herefore was added to the test system in a small
volume of seawater.

Table 1.2 Test material preparation (according to SOP 403)

Actual wet weight
of sediment (g) Target wet weight
nominal conc. (mg.kg -1 ) Weight of test
material

added

(g) Volume of TSW (ml) Nominal dry weight
Concentration (mg.kg -1 )

750.05 10 0.00751 200 13.96

750.16 100 0.07499 200 139.42

750.12 320 0.240 200 446.23

750.00 1000 0.750 200 1394.68

750.08 10000 7.50 200 13945.34Study no 1648-2 Page no 7

Project no P10187 Issue no 1.0 

### Results

The wet to dry ratio of sediment used in this st udy was 1.39 and this was used to correct nominal wet
sediment concentrations to nomi nal dry sediment concentrations.

Corophium Survival Test-10 day survival Start Date: 2/15/2012 Test ID: 1648-2017 Sample ID: R12/017 End Date: 2/27/2012 Lab ID: FLOTTA-Opus Plus Limited Sample Type: OC-Offshore Chemical Sample Date: 3/8/2012 Protocol: OSPAR 2006 Test Species: CO-Corophium volutator
Comments: Conc-mg/Kg 1 2 3 4 5
D-Control 1.0000 0.9000 0.9000 0.9000 0.9500
13.96 0.9000 0.9000 0.9000
139.42 0.7500 0.8500 0.9500
446.23 0.9000 0.9000 0.8000
1394.68 0.2000 0.2500 0.2000
13945.34 0.0000 0.0000 0.0000

Transform: Untransformed 1-Tailed Isotonic Conc-mg/Kg Mean N-Mean Mean Min Max CV% N t-Stat Critical MSD Mean N-Mean
D-Control 0.9300 1.0000 0.9300 0.9000 1.0000 4.809 5 0.9300 1.0000
13.96 0.9000 0.9677 0.9000 0.9000 0.9000 0.000 3 0.806 2.624 0.0976 0.9000 0.9677
139.42 0.8500 0.9140 0.8500 0.7500 0.9500 11.765 3 2.150 2.624 0.0976 0.8583 0.9229
446.23 0.8667 0.9319 0.8667 0.8000 0.9000 6.662 3 1.702 2.624 0.0976 0.8583 0.9229
*1394.68 0.2167 0.2330 0.2167 0.2000 0.2500 13.323 3 19.174 2.624 0.0976 0.2167 0.2330
*13945.34 0.0000 0.0000 0.0000 0.0000 0.0000 0.000 3 24.997 2.624 0.0976 0.0000 0.0000

Auxiliary Tests Statistic Critical Skew Kurt Shapiro-Wilk's Test indicates normal di stribution (p > 0.01) 0.94474 0.868 0.05284 1.40364 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 446.23 1394.68 788.89 0.09764 0.10499 0.53401 0.0026 1.4E-12 5, 14

Linear Interpolation (200 Resamples) Point mg/Kg SD 95% CL(Exp) Skew
IC50 1027.62 22.4306 928.54 1109.94 -0.2486
IC90 8558.21 339.05 7351.9 9711.55 0.2121

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0 5000 10000 15000 Dose mg/KgResponseStudy no 1648-2 Page no 8

Project no P10187 Issue no 1.0 Table 1.3 Calculated LC 50 values with 95% confidence limits

95% confidence limits (mg.kg -1 )

Test material 10 day LC 50 (mg.kg -1 ) Lower Upper 10d LC 90 (mg.kg -1 ) 10d NOEC (mg.kg -1 )
Berol R648 NG 1027 929 1110 8558 446

Interpretation

The test was conducted in accordance with the study plan except for the following deviation(s) but met all other relevant validity criteria.

On day 5, salinities 5ppt above the day 0 leve l were recorded in vessels 22,23,24 (all 320mg.kg -1 ), 25 (1000mg.kg -1 ), 28 & 30 (10000mg.kg -1 ). As no unusual effects in the animals were observed at the time or at Day 10, this was deemed to have had no effect on the test result.

Berol R648 NG exhibited a 10 day LC 50 value of 1027mg.kg -1 (via treated seawater) to the marine amphipod Corophium volutator in the sediment phase.

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

There were no interferences in this test.Study no 1648-2 Page no 9

Project no P10187 Issue no 1.0 

### Retention and archiving of test documentation

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



### References

OSPARCOM Guidelines (2006) Part A. A sediment Bioassay using an amphipod Corophium sp.

ToxCalc Version 5 Tidepool Scientific Software.Study no 1648-2 Page no 10

Project no P10187 Issue no 1.0 

### Appendix A

Test organisms

The test population of Corophium volutator was collected from an uncontaminated site in Orkney. The site location at Bay of Suckquoy, Toab, Orkney was chosen because of its high natural densities of Corophium and, in accordance with the guidance document ‘Biological Effects of Contaminants; Corophium sp. sediment bioassay and toxicity test’, BD Roddie & JE Thain, published by ICES in 2001. Initial parameters of the holding water were recorded upon arrival at the Opus laboratory. Specimens were gently sieved from their nat ive sediment and held in plastic containers (approximately 5l capacity) containing some ambient water, with a small amount of detritus, until transfer to the laboratory. On return to the laboratory, the Corophium were transferred in ambient water to polythene tanks of approximately 200 litre capacity, gentle aeration wa s supplied. The tanks were held at approximately 15±2 ° C.

The stock animals were gradually acclimated from t he ambient salinity of less than 5‰ to that of undiluted seawater (approximately 35‰) in in crements of approximately 10‰ per day. Once acclimation is complete, the stock tanks were maintained under semi-static conditions until the initiation of the test. The holding period in the l aboratory is between 3 and 14 days. Sufficient detrital material is retained in each tank to provide f ood and some bottom cover, but not of a density that prevents daily observation of mortality and morbidit y. Dead or impaired animals are removed when
observed.

Specimens of approximately 5 mm in body length (excl uding rostrum) were used in the toxicity tests.

Test sediment

Sediment for use in toxicity testing was co llected from a point adjacent to the site of Corophium collection. On return to the laboratory, the s ediment was wet-sieved through 0.6 mm mesh to remove larger infaunal organisms and Corophium within the test size range, and allowed to settle in tall polyethylene containers. After settling, the s upernatant water was decanted and the sediment stored at approximately 4 ± 2 °C, until required for testing.

Sediment

Particle size analysis characterised the sediment as well-sorted, fine sand with a silt/clay content of 31.6% by weight. Median particle diameter was 100 μ m. The organic material content was estimated from weight loss on ignition to be 2%.

Immediately before the initiation of the te st, the sediment was thoroughly homogenised and a representative sample taken for wet and dry weight determination. The ratio of wet weight to dry weight was subsequently used to convert nominal ex posure concentrations on a wet weight basis to nominal exposure concentrations on a dry weight basis.

Test method and guidelines

Test methods were conducted in accordance with SOP 102, which conforms to the Paris Commission guidelines for conducting sediment toxicity tests with the amphipod Corophium volutator (OSPARCOM 2006). The method estimate s the effect on survival of Corophium over a period of ten days exposure in sediment amended with the test materials.Study no 1648-2 Page no 11

Project no P10187 Issue no 1.0 Test procedure

Tests were conducted in 1 litre capacity glass beakers each containing 2 cm depth (approximately 150 ml) of amended sediment and 850 ml of overlying seawater (1 μm filtered ultra violet treated seawater). Test beakers were maintained in a c ontrolled temperature room for a test period of 10 days.

Three replicates were prepared for each test concentra tion; controls are replicated five times. The beakers were assigned positions within the test area, arranged in rows of three to five and spaced to maintain effective separation of different treatment s. Each row was covered with a rectangular sheet of perspex perforated with a small hole above the centre of each beaker. Aeration was provided and
a stream of air bubbles was released at a depth of approximately 6 cm.

Twenty animals were added to receiving vessels in two groups of ten and then dispensed into a test vessel. Sixty organisms were exposed in total per concentration and one hundred in five vessels for
controls. The final level of water in each te st vessel was marked with a permanent marker.

Water quality measurements are made in accordance with the following regime.

Day of

commencement Measurement day Dissolved oxygen (%) pH Temperature (°C) Salinity (‰) Friday Day 0 9 9 9 9 Day 3 9 9 9 9 Day 5 9 Day 7 9 9 9 9 Day 10 9 9 9 9

Salinity is maintained at ±4 ppt from the initial 0h figure during the test. A 5 mm drop in water level is equivalent to ~2 ppt increase in salinity.

Any visible signs of animals on the sediment surface whether alive or dead were recorded.

At the end of the test period, the contents of each beaker were gently sieved through a 0.6 mm mesh, and all surviving animals separated and counted. Missing animals were counted as dead; early mortalities will either have been consumed by surv ivors or have decomposed during the test period.

Statistical methods

The number of animals responding after 10 days in each replicate are expressed as a proportion of the nominal number exposed (20). The 10 day LC 50 , LC 90 and NOEC values are calculated using an appropriate statistical method from the ToxCalc Version 5 software.Study no 1648-2 Page no 12

Project no P10187 Issue no 1.0 

### 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 com parison of the reported and observed physical characteristics (eg form, colour, odour, pH and dens ity) of the test material was carried out.

In order to determine an appropriate test prepar ation method, an assessm ent was made of its 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 then its behaviour was assessed. If it was poorly soluble after one hour t hen it was stirred for approximately a further 19 hrs
and then left to settle for one hour and its behaviour assessed in accordance with SOP 402.

Test material preparation was conducted in accordance with SOP 403. Materials which were assessed to be poorly solubl e were prepared by mixing initially with a small quantity of dry sediment. The dried sediment and mix ed test material was then incorporated with the wet sediment within the mixing container. If a material was classified as soluble or dispersi ble it was dissolved in a small quantity of sea water and added directly to the mixing container, containing the wet sediment.

The mixing vessels were then placed on a platform s haker at approximately150 rpm for 3 hours. After this period, the contents of eac h container were equally distribut ed between the replicate vessels,
three for test concentrations and five for control vessels.

The nominal test concentrations empl oyed were 10, 100, 320, 1000 and 10000 mg.kg -1

.

Seawater for testing was supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 1 μm before being used for testing.Study no 1648-2 Page no 13

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### Appendix C

Control data

Table C1 Observed mortality after 10 days in the controls

Replicate Number
dead Total
added Proportional response (10d)

1 0 20 0

2 2 20 0.1

3 2 20 0.1

4 2 20 0.1

5 1 20 0.05

Table C2 Maximum and minimum values of measured control water quality variables over 10 days

DO 2 (% saturation) pH Temperature (°C) Salinity (‰)

Control minimum 92 7.88 13.8 36
maximum 99 8.06 15.2 41Study no 1648-2 Page no 14

Project no P10187 Issue no 1.0 Certificate of Analysis (Client supplied)