Assessment of the toxicity (10 day LC50) of 

Berol R648 NG to the Marine crustacean 

Corophium volutator

Opus Project No: 1648-2 

Opus Study No: P10187 

 
Study 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 14 
Study no 1648-2 Page no 3 

Project no P10187 Issue no 1.0 

Confidentiality statement

The information contained in this document is confidential 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 LC50 (mg.kg-1) 1028 

10d LC90 (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 O2 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. 
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Project no P10187 Issue no 1.0 

Section 1

Test organisms and sediment were collected on 9 February 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/m3 @ 25°C 1.0591g/cm3 @ 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 

Description(s) CAS No not stated, 60-70% 

Water, CAS No 7732-18-5, 30-50% 

Berol R648 NG was characterised as soluble and therefore 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 13945.34 
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Project no P10187 Issue no 1.0 

Results

The wet to dry ratio of sediment used in this study was 1.39 and this was used to correct nominal wet 

sediment concentrations to nominal 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 distribution (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/Kg

Response
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Table 1.3 Calculated LC50 values with 95% confidence limits 

 95% confidence limits 

(mg.kg-1) 

 

Test material 

10 day LC50 

(mg.kg-1) Lower Upper 

10d LC90

(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 level 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 LC50 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 was calculated by Linear Interpolation within the 

Toxcalc suite of statistical analysis. 

There were no interferences in this test.
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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.
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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 native 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 was supplied. The tanks 

were held at approximately 15±2 °C.

The stock animals were gradually acclimated from the ambient salinity of less than 5‰ to that of 

undiluted seawater (approximately 35‰) in increments 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 laboratory is between 3 and 14 days. Sufficient detrital 

material is retained in each tank to provide food and some bottom cover, but not of a density that 

prevents daily observation of mortality and morbidity. Dead or impaired animals are removed when 

observed. 

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

Test sediment 

Sediment for use in toxicity testing was collected from a point adjacent to the site of Corophium 

collection. On return to the laboratory, the sediment 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 supernatant 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 μm. The organic material content was estimated 

from weight loss on ignition to be 2%. 

Immediately before the initiation of the test, 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 exposure 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 estimates the effect on survival of Corophium over a period of ten 

days exposure in sediment amended with the test materials.
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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 controlled temperature room for a test period of 10 

days. 

Three replicates were prepared for each test concentration; 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 treatments. 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 test 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 survivors 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 LC50, LC90 and NOEC 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 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 then 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 soluble were prepared by mixing initially with a small 

quantity of dry sediment. The dried sediment and mixed test material was then incorporated with the 

wet sediment within the mixing container. 

If a material was classified as soluble or dispersible 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 shaker at approximately150 rpm for 3 hours. After 

this period, the contents of each container were equally distributed between the replicate vessels, 

three for test concentrations and five for control vessels. 

The nominal test concentrations employed 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. 
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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

 DO2 

(% saturation) pH 

Temperature 

(°C) 

Salinity 

(‰) 

Control minimum 92 7.88 13.8 36 

maximum 99 8.06 15.2 41 
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Certificate of Analysis (Client supplied)