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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-11 - 2010-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Sponsor's identification: Amine C8
Description: dark brown liquid
Batch number: T7-271109
Date received: 21 September 2010
Expiry date: 31 December 2011
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Water samples were taken from the control and the 45 mg/l test group at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis. The 0 and 24-Hour samples were stored at approximately -20°C prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in attached Appendix 3.


Vehicle:
no
Details on test solutions:
Definitive test
The test was conducted according to the threshold approach at a single concentration equivalent to the lowest EC50 value obtained from the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 41003973) and the Algal Inhibition test (Harlan Laboratories Ltd Project Number: 41003974). The results from the Acute Toxicity to Daphnia magna test gave an EC50 of greater than 100 mg/l. The ErC50 for the Algal Inhibition test was 45 mg/l.
Based on these results the test was conducted at a single test concentration of 45 mg/l to determine if the LC50 was higher than this lowest EC50 and hence that fish were not the most sensitive species.

Experimental Preparation
For the purpose of the definitive test the test item was dissolved directly in dechlorinated tap water.
An amount of test item (2000 mg) was dissolved in dechlorinated tap water and the volume adjusted to 500 ml to give a 4000 mg/l stock solution. An aliquot (450 ml) of this solvent stock solution was diluted in a final volume of 40 litres of dechlorinated tap water to give the 45 mg/l test concentration.

The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 (fresh media), 24 and 96 hours (old media) (see attached Appendix 3).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 4 August 2010. Fish were maintained in a glass fibre tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 29 September 2010 to 11 October 2010. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14°C with a dissolved oxygen content of greater than or equal to 9.3 mg O2/l. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was zero mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 6.3 cm (sd = 0.3) and a mean weight of 3.34 g (sd = 0.53) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.58 g bodyweight/litre.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test Water
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in the attached Appendix 2.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Hardness:
Total hardness of approximately 140 mg/l as CaCO3.
Test temperature:
The test vessels were covered to reduce evaporation and maintained at approximately 14ºC
pH:
The pH was measured using a Hach hq30d pH and oxygen meter
pH range of 7.8 - 8.4.
Please see Physico-Chemical Measurements appendix 4 (attached background material).
Dissolved oxygen:
The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Please see Physico-Chemical Measurements appendix 4 (attached background material) for dissolved oxygen results.
Salinity:
Fresh water used.
Nominal and measured concentrations:
Definitive test: 45 mg/l.
Details on test conditions:
Exposure conditions
In the test 40 litre glass exposure vessels were used for each test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14°C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test item remained near nominal and to prevent the build up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence limits not stated
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: CL not stated
Details on results:
-Mortality data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 1.
There were no mortalities in 7 fish exposed to a test concentration of 45 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:
Time (h) LC50 (mg/l)
3 >45
6 >45
24 >45
48 >45
72 >45
96 >45
The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 45 mg/l and the No Observed Effect Concentration (NOEC) to be 45 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

-Sub-lethal effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 45 mg/l for a period of 96 hours.

-Observations on test item solubility
Throughout the duration of the test the control was observed to be a clear, colourless solution whilst the 45 mg/l test concentration was observed to be a very pale straw coloured solution.

-Physico-chemical measurements
The results of the physico-chemical measurements are given in attached Appendix 4. Temperature was maintained at approximately 14ºC throughout the test. While there were no treatment related differences for oxygen concentration, differences in pH were observed between the freshly prepared media for the control and 45 mg/l test concentration throughout the test.

-Verification of test concentrations
Analysis of the test preparations at 0, 24 and 96 hours (see attached Appendix 3) showed measured test concentrations to range from 91% to 94% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
An estimate of the LC50 values was given by inspection of the mortality data.
Sublethal observations / clinical signs:

Table1              Cumulative Mortality Data in the DefinitiveTest

Nominal

Concentration

(mg/l)

Cumulative Mortality
(Initial Population = 7)

%

Mortality

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96 Hours

Control

0

0

0

0

0

0

0

45

0

0

0

0

0

0

0

 

Appendix4      Physico-Chemical Measurements

Nominal

Concentration

(mg/l)

Time (Hours)

0 Hours (Fresh Media)

24 Hours (Old Media)

24 Hours (Fresh Media)

pH

mg O2/l

%ASV*

TºC

pH

mg O2/l

%ASV*

T°C

pH

mg O2/l

%ASV*

T°C

Control

7.8

9.8

95

14

8.3

10.2

99

14

7.8

10.1

98

14

45

8.4

9.8

95

14

8.2

10.1

98

14

8.4

10.1

98

14

 

Nominal

Concentration

(mg/l)

Time (Hours)

48 Hours (Old Media)

48 Hours (Fresh Media)

72 Hours (Old Media)

pH

mg O2/l

%ASV*

TºC

pH

mg O2/l

%ASV*

T°C

pH

mg O2/l

%ASV*

T°C

Control

8.2

10.2

99

14

7.8

10.0

97

14

8.3

10.1

98

14

45

8.3

10.1

98

14

8.4

10.1

98

14

8.3

10.0

97

14

 

Nominal

Concentration

(mg/l)

Time (Hours)

72 Hours (Fresh Media)

96 Hours (Old Media)

pH

mg O2/l

%ASV*

TºC

pH

mg O2/l

%ASV*

T°C

Control

7.8

10.2

99

14

8.3

10.3

100

14

45

8.4

10.2

99

14

8.4

10.2

99

14


*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 45 mg/l. Correspondingly the No Observed Effect Concentration was 45 mg/l. This test was therefore considered valid based on the threshold effect as it showed an LC50 greater than the lowest EC50 from the algae and daphnia studies indicating that fish were not the most sensitive species of three trophic species tested.
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods.

The test was conducted according to the threshold approach at a single concentration equivalent to the lowest EC50value obtained from the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 41003973) and the Algal Inhibition test (Harlan Laboratories Ltd Project Number: 41003974). The results from the Acute Toxicity to Daphnia magna test gave an EC50of greater than 100 mg/l. The ErC50 for the Algal Inhibition test was 45 mg/l. Based on these results, fish were exposed, in a single group of seven, to an aqueous solution of the test item, at a single concentration of 45 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LC50based on nominal test concentrations was greater than 45 mg/l and correspondingly the No Observed Effect Concentration was 45 mg/l.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 91% to 94% of nominal and so the results are based on nominal test concentrations only.

 

CONCLUSION

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 of greater than 45 mg/l. Correspondingly the No Observed Effect Concentration was 45 mg/l.

This test was therefore considered valid based on the threshold effect as it showed an LC50 greater than the lowest EC50 from the algae and daphnia studies indicating that fish were not the most sensitive species of three trophic species tested.

Description of key information

One study (Goodband & Mullee, 2010 - report 41004020), investigating the acute toxicity of the substance to fish according to the OECD guideline 203, was considered as the key study for endpoint coverage. The 96h-LC50 was determined to be > 45 mg/L. The study is given a Klimisch score of 1 and was conducted under GLP.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
45 mg/L

Additional information

The study of Goodband & Mullee (2010) investigates the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss) following the OECD guideline 203. The test was conducted according to the threshold approach at a single concentration equivalent to the lowest EC50 value obtained from the Acute Toxicity to Daphnia magna test (Harlan Laboratories Ltd Project Number: 41003973) and the Algal Inhibition test (Harlan Laboratories Ltd Project Number: 41003974). The results from the Acute Toxicity to Daphnia magna test gave an EC50 of greater than 100 mg/l. The ErC50 for the Algal Inhibition test was 45 mg/l. Based on these results, fish were exposed, in a single group of seven, to an aqueous solution of the test item, at a single concentration of 45 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. No mortality was observed during the 96-hour test period. The 96h-LC50 based on nominal test concentrations was greater than 45 mg/l. Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 91% to 94% of nominal and so the results are based on nominal test concentrations only. This test was therefore considered valid based on the threshold effect as it showed an LC50 greater than the lowest EC50 from the algae and daphnia studies indicating that fish were not the most sensitive species of three trophic species tested.