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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency (1985). Hazard Evaluation Division. Standard Evaluation Procedure EPA-540/9-85-006. Acute Toxicity Test for Freshwater Fish.
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 92/69/EEC (1992). Methods for the determination of ecotoxicity. C2, Acute toxicity for Fish. L383A.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling: samples collected from the center of test solutions at 0, 48, and 96 hours of the definitive test
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock concentrate of 10 g/L was prepared by dissolving 0.10 g of the test substance in 10 ml of an organic solvent, dimethylformamide (DMF). The test exposure solutions were prepared from this concentrate by addition of appropriate volumes to 10 litres of dilution water in each exposure vessel and stirring thoroughly. Equalizing additions of DMF were made such that all test concentrations and the solvent control contained 0.1 mL of DMF per litre. The control consisted of dilution water only. The test solutions were observed to be clear and colourless and were not changed during the 96 hour exposure period.
- Controls: The vehicle control consisted of DMF and dilution water only. The control consisted of dilution water only.
- Chemical name of vehicle: Dimethylformamide and dilution water
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas
- Source: Aquatic Research Organisms Inc., 1 Lafayette Road, PO Box 1271, Hampton, New Hampshire, U.S.A.
- Length at end of exposure period: 14 - 22 mm (mean: 18 mm)
- Weight at end of exposure period: 0.038 - 0.18 g (mean: 0.094 g)

ACCLIMATION
- Acclimation period: 68 days
- Type and amount of food during acclimation: Fish were fed with Tropical flake daily until approximately 48 hours prior to their use in the test
- Health during acclimation: No mortality was observed in the fish in the 10 weeks prior to the study. The fish showed no evidence of disease during holding.

FEEDING DURING TEST
- Frequency: Unfed
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Hardness:
43 mg CaCO3/L
Test temperature:
21.3 to 22.0°C
pH:
7.88 to 8.08
Dissolved oxygen:
8.6 to 9.4 mg/L
Conductivity:
245 μS/cm
Nominal and measured concentrations:
Nominal: 18, 32, 56, 100 and 180 μg/L
Mean measured: 18, 32, 58, 100 and 180 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Borosilicate glass vessels (external dimensions; 360 mm x 230 mm x 230 mm; length x width x height) with a maximum capacity of 16.5 L and a working volume of 10 L were used to hold the test fish.
- Aeration: gently areated
- No. of organisms per concentration: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. The water was then passed through an ultra violet sterilizer to a second set of filters of 25 and 10 µm and then to a third storage tank with a capacity of 13.5 m³. The treated water was then delivered via a ring circuit to a temperature controlled header tank in the test laboratory set to a nominal temperature of 22°C and finally filtered to 5 µm before use.
- Alkalinity: 30.2 mg CaCO3/L
- Chlorine: <4 µg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hr photoperiod and 8 hr darkness with 20 min transition period

EFFECT PARAMETERS MEASURED: Observations for mortalities and symptoms of toxicity were made at 24, 48, 72, and 96 hours.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
65 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 50-87 μg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
32 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: No symptoms of toxicity, other than mortalities, were observed during the course of this study.
- Mortality of control: No mortality were observed in the dilution water control or vehicle control.
Reported statistics and error estimates:
The LC50 results calculated using the moving average angle method were selected from the computer program.
Validity criteria fulfilled:
yes
Conclusions:
96-hour LC50: 65 μg/L (mortality) (95% CI: 50-87 μg/L)
Executive summary:

The acute toxicity of the test substance to the Fathead minnow (Pimephales promelas), was determined in a 96-hour static test according to OECD guideline 203.

The study was conducted with five nominal concentrations of the test substance (18, 32, 56, 100 and 180 μg/L), a dilution water control, and a solvent control at a temperature range of 21.3 to 22.0°C.

Mean measured concentrations of the test substance during the exposure were <LOD (control), <LOD (vehicle), 18, 32, 58, 100 and 180 μg/L.

After 96-hours, mortality was 0, 0, 0, 0, 20, 100, and 100% in the 0 (control), 0 (solvent control), 18, 32, 58, 100 and 180 μg/L treatments, respectively. No mortality were observed in the dilution water control or solvent control.

The lowest nominal concentration at which there was 100% mortality was 100 μg/L. The 96 hour nominal no observed effect concentration (NOEC) of the test substance, based on mortalities, was 32 μg/L.The 96-hour LC50 was 65 μg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling: samples collected from the center of test solutions at 0, 48, and 96 hours of the definitive test
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- A stock concentrate was prepared by dissolving 0.05 g of the test substance in 10 ml of dimethylformamide (DMF), resulting in a clear and colourless solution. The test exposure solutions were prepared from this concentrate by addition of the required volumes to 24 L of freshwater in each exposure vessel and stirring thoroughly.
A solvent control was similarly prepared by the addition of DMF to freshwater such that all test exposure concentrations and the solvent control contained 0.036 mL of solvent per litre of test solution. The test solutions were observed to be clear and colourless. The test solutions were not changed during the 96 hour exposure period.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Bibury Trout Farm, Bibury, Gloucestershire, UK
- Length at end of exposure period: 40 - 50 mm (mean: 49 mm)
- Weight at end of exposure period: 0.85 - 2.28 g (mean: 1.61 g)

ACCLIMATION
- Acclimation period: 14 days
- Type and amount of food during acclimation: Fish were fed with Promin daily until approximately 48 hours prior to their use in the test
- Health during acclimation: The batch of fish were observed to be in good condition prior to use in the study. Less than 1% mortality was observed in the fish in the week prior to the test. The fish showed no evidence of disease during holding; therefore no medication was necessary.

FEEDING DURING TEST
- Frequency: Unfed
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Hardness:
15.7 mg CaCO3/L
Test temperature:
14.7 - 15.1°C
pH:
7.1 to 7.5
Dissolved oxygen:
9.2 to 10.2 mg/L
Conductivity:
97 μS/cm
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56, 100 and 180 μg/L
Mean measured: 7.8, 14, 27, 49, 100 and 190 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Borosilicate glass vessels (external dimensions; 400 mm x 280 mm x 280 mm; length x width x height) with a maximum capacity of 27.5 L and a working volume of 24 L were used to hold the test fish.
- Aeration: gently areated
- No. of organisms per concentration: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. The water was then passed through an ultra violet sterilizer to a second set of filters of 25 and 10 µm and then to a third storage tank with a capacity of 13.5 m³. The treated water was then delivered via a ring circuit to a temperature controlled header tank in the test laboratory set to a nominal temperature of 15°C and finally filtered to 5 µm before use.
- Alkalinity: 9.4 mg CaCO3/L
- Chlorine: <4 µg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hr photoperiod and 8 hr darkness with 20 min transition period

EFFECT PARAMETERS MEASURED: Observations for mortalities and symptoms of toxicity were made at 24, 48, 72, and 96 hours.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
75 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 60 - 95 μg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
18 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Symptoms of toxicity
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: The only general symptom of toxicity noted in this study was dark discolouration. No mortalities, were observed during the course of this study in the nominal concentrations of 18, 32 and 56 μg/L.
- Mortality of control: No mortality were observed in the dilution water control or vehicle control.
Validity criteria fulfilled:
yes
Conclusions:
96-hour LC50: 75 μg/L (mortality) (95% CI: 60 - 95 μg/L)
Executive summary:

The acute toxicity of the test substance to the Rainbow trout (Oncorhynchus mykiss), was determined in a 96-hour static test according to OECD guideline 203.

The study was conducted with six nominal concentrations of the test substance (10, 18, 32, 56, 100 and 180 μg/L), a dilution water control, and a solvent control at a temperature range of 14.7 - 15.1°C.

Mean measured concentrations of the test substance during the exposure were <LOD (control), <LOD (vehicle), 7.8, 14, 27, 49, 100 and 190 μg/L.

No mortality were observed in the dilution water control or solvent control and in the nominal concentrations of 18, 32 and 56 μg/L.

The lowest nominal concentration at which there was 100% mortality was 100 μg/L. The 96-hour nominal no observed effect concentration (NOEC) of the test substance, based on symptoms of toxicity, was 18 μg/L. The 96-hour LC50 was 75 μg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.5, 13, 25, 50, and 100 μg a.s./L
- Sampling: samples collected at 0, 48, and 96 hours of the definitive test
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 1.0 mg a.s./mL primary standard solution was prepared at test initiation by adding 0.0101 g of the test substance to a 10-mL glass volumetric flask and bringing the flask to volume with dimethylformamide (DMF). The working standard was serially diluted with 5.0 mL volumes of DMF to produce working standard solutions at nominal concentrations of 0.065, 0.13, 0.25, and 0.50 mg a.s./mL. Aliquots of the working standard solutions were used to prepare the 6.5, 13, 25, 50, and 100 μg a.s./L test solutions by diluting a 1.8 mL aliquot with 18.0 L of dilution water. A 1.8 mL aliquot of the primary standard solution was diluted with 18.0 L of dilution water to prepare the 100 μg a.s./L test solution. There was a single replicate test chamber per treatment.
- Controls: The vehicle control consisted of DMF and dilution water only. The control consisted of dilution water only.
- Chemical name of vehicle: Dimethylformamide and dilution water
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio
- Source: In-house culture
- Age at dosing: Juvenile
- Length at study initiation: 17 to 24 mm in standard length (mean and standard deviation = 20 ± 2.1 mm)
- Weight at study initiation: 0.0520 to 0.125 g in blotted wet weight (mean and standard deviation = 0.0698 ± 0.0239 g)

ACCLIMATION
- Acclimation period: 14 days
- Type and amount of food during acclimation: Fish were fed brine shrimp daily until approximately 50 hours prior to their use in the definitive test
- Health during acclimation: No diseases were observed or treated during the 14-day period prior to use in the test and no mortalities were observed.

FEEDING DURING TEST
- Frequency: Unfed
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
146 mg CaCO3/L
Test temperature:
23.4 to 24.3°C
pH:
8.3 to 8.4
Dissolved oxygen:
6.3 to 7.8 mg/L (77 to 96% saturation)
Conductivity:
353 μS
Nominal and measured concentrations:
Nominal: 6.5, 13, 25, 50, and 100 μg a.s./L
Mean measured: 5.79, 12.2, 24.7, 48.3, and 102 μg a.s./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 21-L glass jar containing 18 L of test solution (32-cm test solution depth), covered with a glass lid.
- No. of organisms per vessel: 7 fish per test chamber
- No. of vessels per concentration (replicates): One
- Biomass loading rate: 0.0271 g of fish tissue per liter of test solution

TEST MEDIUM / WATER PARAMETERS
- preparation of dilution water: laboratory freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis
- Total organic carbon: < 2.0 mg/L
- Alkalinity: 158 mg CaCO3/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hr photoperiod (708 lux) and 8 hr darkness which included 30 min transitional light preceding and following the 16-hr light interval

EFFECT PARAMETERS MEASURED: Observations for mortality and sublethal responses were made at 6 hours, and then once every 24 hours (±1 hour) for the duration of the test.

TEST CONCENTRATIONS
- Range finding study
A range-finding test was conducted under static test conditions. The nominal concentrations were 0 (control), 1.0, 10, 100, and 1000 μg a.s./L. The control and all test substance treatment solutions were clear and colorless with no visible precipitate, surface film, or undissolved test substance at initiation and throughout the test. After 96 hours, mortality was 0, 0, 0, 100, and 100% in the 0 (control), 1.0, 10, 100, and 1000 μg a.s./L treatments, respectively. No sublethal responses were observed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
92.9 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 60.5 to 143 μg a.s./L
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
48.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Observations: After 96 hours of exposure, all surviving fish in the test substance treatment groups appeared normal with no behavioral or morphological abnormalities.
- Mortality of control: No mortality or sublethal effects were observed in the dilution water control or vehicle control.
Reported statistics and error estimates:
Estimates of LC50 values and their 95% confidence limits were calculated using the Probit method and Trimmed Spearman-Karber method
Validity criteria fulfilled:
yes
Conclusions:
96-hour LC50: 92.9 μg a.s./L (mortality) (95% CI: 60.5 to 143 μg a.s./L)
Executive summary:

The acute toxicity of the test substance to the zebrafish, Danio rerio, was determined in a 96-hour static test. The test was conducted in accordance with the U.S. EPA Guideline 850.1075 and the Organization of Economic Cooperation and Development (OECD) Guideline 203.

The study was conducted with five nominal concentrations of the test substance (6.5, 13, 25, 50, and 100 μg a.s./L), a dilution water control, and a vehicle control at a temperature range of 23.4 to 24.3°C. One replicate with seven fish was used per test substance concentration, vehicle control, and dilution water control.

Mean measured concentrations of the test substance during the exposure were <LOD (control), <LOD (vehicle), 5.79, 12.2, 24.7, 48.3, and 102 μg a.s./L or 89 to 102% of the nominal concentrations. The biological response results are based on mean measured concentrations.

After 96-hours, mortality was 0, 0, 0, 0, 0, 0, and 57% in the 0 (control), 0 (vehicle control), 5.79, 12.2, 24.7, 48.3, and 102 μg a.s./L treatments, respectively. No mortality or sublethal effects were observed in the control or vehicle control treatments. The highest mean measured concentration causing no mortality at test end was 48.3 μg a.s./L. The 96-hour LC50 was 92.9 μg a.s./L based on mean measured concentrations of the test substance.

Description of key information

96 -hour LC50 (Pimephales promelas): 66 µg./L; OECD 203; Reliability = 1

96 -hour LC50 (Poecilia reticulata): 59.7 µg//L; OECD 203; Reliability = 1

96 -hour LC50 (Danio rerio): 92.9 µg//L; OECD 203; Reliability = 1

96 -hour LC50 (Oncorhynchus mykiss): 75 µg/L; OECD 203; Reliability = 1

96 -hour LC50 (Cyprinodon variegatus): 330 µg/L; EPA OPPTS 850.1075; Reliability = 1

Key value for chemical safety assessment

LC50 for freshwater fish:
0.066 mg/L
LC50 for marine water fish:
0.33 mg/L

Additional information

Acute fish toxicity tests were conducted in eight freshwater species and one marine species. Acute 96 -hour LC50 values in freshwater species ranged from 0.060 mg a.s./L (mean measured) to 0.228 mg a.s./L (mean measured) in Poecilia reticulata and Oryzias latipes, respectively. The marine fish species tested, Cyprinodon variegatus showed similar toxicity to freshwater species, the mean measured acute LC50 = 0.330 mg a.s./L. The most sensitive acute 96 h LC50 for fish is less than 1 mg a.s./L.