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Ecotoxicological information

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:
2002-01-07 - 2002-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test"
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Method C.l of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 and 0.10 mg/L (nominal)
- Sampling method: Water samples were taken from the solvent control and each replicate test vessel at 0 (fresh test media), 24, 48, 72 (old and fresh test media) and 96 hours (old test media) for quantitative analysis. Two samples of the solvent control and each replicate were taken at each occasion. One sample was analysed uncentrifuged and one sample after centrifugation (40 000 G for approximately 30 minutes).
- Sample storage conditions before analysis: Further samples (in duplicate) were taken and stored frozen for further analysis, if necessary.
Vehicle:
yes
Remarks:
dimethylformamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Solvent control (100 µL/L of dimethylformamide
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test the test material was prepared using a preliminary solution in dimethylformamide. An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 100 mL to give a 100 mg/100 mL solvent stock solution. Aliquots (2.1 mL in duplicate) of this solvent stock solution were each separately dispersed in dechlorinated tap water with the aid of stirring by flat blade mixer and the volume adjusted to 21 litres to give the 0.10 mg/L test concentration replicates Ri and R2. A test volume of 21 litres was prepared initially to allow for the removal of 1 litre for chemical analysis leaving a final test volume of 20 litres.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
- Controls: Control and solvent control groups were maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test preparations were observed to be clear colourless solutions throughout the definitive test. However, it was considered that undissolved test material would not have been visible by eye.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden near Skipton, Yorkshire, UK and maintained in-house since 22 November 2001
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): mean standard length of 4.5 cm (sd = 0.2) at the end of the definitive test
- Weight at study initiation (mean and range, SD): mean weight of 1.17 g (sd = 0.17) at the end of the definitive test
- Maintenance of the brood fish: Fish were maintained in a glass fibre tank with a "single pass" water renewal system. 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 14°C with a dissolved oxygen content of greater than or equal to 9.1 mg O2/L. These parameters were recorded daily.


ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 26 December 2001 to 7 January 2002.
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
- Health during acclimation (any mortality observed): There was zero mortality in the 7 days prior to the start of the test.

FEEDING DURING TEST
The fish received no food during exposure.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
100 mg/L as CaCO3
Test temperature:
14.0 +/- 1.0 °C
pH:
7.3 - 7.6
Dissolved oxygen:
8.4 - 10.2
82 - 99 % ASV (Dissolved oxygen concentration expressed as a percentage of Air Saturation Value)
Nominal and measured concentrations:
nominal: 0 and 0.10 mg/L
mean measured: 0 and 0.064 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: tanks
- Type (delete if not applicable): The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: 20 litre
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.59 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 100 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.
- Culture medium different from test medium: The test water used for both the rangefinding and definitive tests was the same as that used to maintain the stock fish.

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sublethal 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.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a series of nominal test concentrations of 0.010 and 0.10 mg/1. The test material was prepared using a preliminary solution in dimethylformamide
- Results used to determine the conditions for the definitive study:Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 0.10 mg/L to confirm that at the highest attainable test concentration of 0.10 mg/L, no mortalities or sub-lethal effects of exposure were observed. The test concentration of 0.10 mg/1 was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and having due regard to the amount of aujfoiatyjsolvent permitted in the study under the OECD Guidelines. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.064 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.064 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: There were no sub-lethal effects of exposure observed in 20 fish exposed to a test concentration of 0.10 mg/L for a period of 96 hours.
- Mortality of control: no
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: The test preparations were observed to be clear colourless solutions throughout the definitive test. However, it was considered that undissolved test material would not have been visible by eye.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
No statistics and error estimates are reported.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 203 and EU Method C.1 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards fish.
In a semi-static test rainbow trout (Oncorhynchus mykiss) were exposed to the test substance at the limit concentration of nominal 0.10 mg/L, corresponding to an analytocal mean measured concentration of 0.064 mg/L, for 96 hours under defined conditions. No mortality or sub-lethal effects were observed during the test. Accordingly, the 96 h LC50 was determined to be greater than the limit concentration of nominal 0.10 mg/L, corresponding to a mean measured concentration of 0.064 mg/L. Hence, no acute toxicity against fish is recoreded at levels up to the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to fish was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test with rainbow trout (Oncorhynchus mykiss) as test organisms, which is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems (Wetton and Mullee, 2002). The study was conducted under certificated GLP compliance. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on the range-finding study, a "limit test" was conducted at a concentration of nominal 0.10 mg/L. For both purposes the test material was prepared using a preliminary solution in dimethylformamide. The acclimatisation period of the juvenile fish was conducted for 13 days. The stock fish were fed commercial trout pellets which was discontinued 24 h prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.5 cm (sd = 0.2) and a mean weight of 1.17 g (sd = 0.17) at the end of the definitive test. The test vessels were covered to reduce evaporation and maintained at 14.0 +/- 1 °C in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 min dawn and dusk transition periods for a period of 96 h. The test vessels were aerated via narrow bore glass tubes. A semi-static test regime was employed involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products. The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. Any mortalities and sublethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. High Performance Liquid Chromatography (HPLC) was the analytical method of choice.

No mortality or any other effects occurred during the experiment, therefore the 96 h LC50 was determined to be greater than nominal 0.10 mg/L and the 96 h No Observed Effect Concentration (NOEC) was determined to be greater than nominal 0.10 mg/L, corresponding to a mean measured concentration of 0.064 mg/L, respectively. This was the highest obtainable test concentration due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under OECD guidelines.

Description of key information

Short-term toxicity to fish: LC50(96h) > 0.064 mg/L (mean measured concentration) for Oncorhynchus mykiss based on mortality (semi-static, OECD 203, GLP)

Key value for chemical safety assessment

LC50 for freshwater fish:
0.064 mg/L

Additional information

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to fish was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test with rainbow trout (Oncorhynchus mykiss) as test organisms, which is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems (Wetton and Mullee, 2002). The study was conducted under certificated GLP compliance. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on the range-finding study, a "limit test" was conducted at a concentration of nominal 0.10 mg/L. For both purposes the test material was prepared using a preliminary solution in dimethylformamide. The acclimatisation period of the juvenile fish was conducted for 13 days. The stock fish were fed commercial trout pellets which was discontinued 24 h prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.5 cm (sd = 0.2) and a mean weight of 1.17 g (sd = 0.17) at the end of the definitive test. The test vessels were covered to reduce evaporation and maintained at 14.0 +/- 1 °C in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 min dawn and dusk transition periods for a period of 96 h. The test vessels were aerated via narrow bore glass tubes. A semi-static test regime was employed involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products. The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. Any mortalities and sublethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. High Performance Liquid Chromatography (HPLC) was the analytical method of choice.

No mortality or any other effects occurred during the experiment, therefore the 96 h LC50was determined to be greater than nominal 0.10 mg/L and the 96 h No Observed Effect Concentration (NOEC) was determined to be greater than nominal 0.10 mg/L, corresponding to a mean measured concentration of 0.064 mg/L, respectively. This was the highest obtainable test concentration due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under OECD guidelines.

Hence, no acute toxicity against fish is recoreded at levels up to the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).