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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:
2014-10-27 to 2014-11-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 203 with GLP certificate. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Storage condition of test material: Keep in cool temperature (4 °C) and under the protection of nitrogen
- Stability: Stable in water
- Vapour Pressure: 41.4Pa (calculated)
- Solubility in Water: 709mg/L (Predicted Value)
- Solubility in Solvent: 709mg/L (Predicted Value)
- Partition Coefficient LogPow=3.48-3.53
Analytical monitoring:
no
Details on sampling:
Measurement of actual concentrations of test substance in test solution: The actual concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups at 0, 48 and 96 h (before and after the renewal) after the start of the test and blank control at 0 h (new solution) 48 h (old solution) were measured by Gas chromatograph (GC).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Preparation of test stock solution: 0.535 g test substance was obtained in a small amount of test water with ultrasonication treatment for 10 min and transferred into 5 L-jar, test water was added to 5350 mL and the bottle cap was covered (there was no headspace on the test suspension). Then the nominal concentration of 100 mg/L suspension was obtained. And then the suspension was stirred with a magnetic stirrer for about 48 h in dark condition, so the nominal concentration of 100 mg/L stock solution was obtained. Four replicates of stock solution were prepared by the same method above and fully mixed before the start of the test.
Note: The replicates of test stock solutions were adjusted according to the mortalities of fish during the test exposure.
Preparation of test solution: 1391, 2048, 3010, 4420 and 6500 mL volumes of test stock solutions were taken and allocated into the five 10 L tanks respectively, then 5109, 4452, 3490, 2080 and 0 mL volumes of test water were added. After stirred well, the nominal concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test solutions were obtained. Then the test solutions of each concentration were divided into the 5L brown jars, after the test fish were put into the solutions, the test solutions were also added into the test containers. When the test solutions were overflowed the test containers, the bottle caps were covered to start the test. The total volumes of each test container were about 6100mL.
- Controls: A blank control (test water without test substance) was selected in parallel to the test concentration.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra Fish
- Source: Guangzhou Yangshi Breeding Farm
- Zebra rish (Danio rerio) were obtained on August 27, 2014. The Batch number was FDr20140827-1.

ACCLIMATION
- Acclimation period: 63 days
- Before the start of the test, Zebra fish were acclimated for 63 days by flow-through under the same conditions (water quality: dechlorinated tap water, temperature: 23 ± 2 °C, phoioperiod: dark condition). The dissolved oxygen concentraiion during acclimation was kept at not less than 80 % of the air saturation value.
- Feeding: The test fish were fed at least three times per week until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): The mortality was 0% during 7 days before the start of the test, which met the requirement.
- Allocation to the test groups: Zebra fish (Danio rerio) were randomly picked out and allocated at random to each test group within 30 minutes.

FEEDING DURING TEST
- Feeding: No feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
The hardness of dechlorinated tap water was 123 mg/L (expressed as CaCO3).
Test temperature:
22.3-23.9 °C
pH:
The test was performed without adjustment of pH. The pH was in the range of 7.62 to 8.08.
Dissolved oxygen:
The tesl solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test was kept at not less than 60 % of the air saturation value.
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 21.4, 31.5, 46.3, 68.0 and 100 mg/L
Details on test conditions:
The result of stability test showed that the measured concentration of 10 mg/L test solution could be maintained within ±20% of the initial concentration during 120 h, but the result also indicated that there was a slowly decline of the concentration of test substance. So the definitive test was conducted with semi-static test at 48 h intervals.

TEST SYSTEM
- Test vessel: Vessel (5L brown jar with a ground-in stopper: 16.6 cm x 25.0 cm, 10 L tank), 5 L flask with a ground-in stopper, beaker, measuring cylinder, volumetric flask and straw and so on.
- Test volume: About 6.1 L/test vessel
- Aeration: No aeration
- Renewal rate of test solution: 24 h
- No. of fish: 10 fish/test group
- Replicates: No replicates/test group
- No. of vessels per control (replicates): 1
- Loading rate: 10 fish/test vessel, 0.203 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated water

OTHER TEST CONDITIONS
- Photoperiod: Dark condition

EFFECT PARAMETERS MEASURED
- Observation of test organisms: Mortalities and visible abnormalities of the test fish were recorded at 3, 24, 48, 72 and 96 h after the start of the test during the test period. The dead fish were removed as soon as they were found. A fish is considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction.
- Body weight and body length of test fish: The body weight and length of 10 test fish in blank control were measured at the end of the test.
- Observation of test solutions appearance: The changes of color and appearance of the test solutions were recorded daily.
- Measurement of temperature, pH and dissolved oxygen of test solution: The temperature, pH and dissolved oxygen of each test group were measured at 0, 24, 48, 72 and 96 h (before and after the renewal) after the start of the test. Measurements of pH were determined with a portable pH meter. Measurements of temperature and dissolved oxygen were determined with an oxygen meter. And the temperature of test water was continuously monitored during the exposure by dual thermometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.47
- Range finding study: Static method - 3.05, 9.76, 31.2 and 100 mg/L; 5 fish/test group
- Results used to determine the conditions for the definitive study: The range-finding test showed that, the cumulative mortality on Zebra fish in the nominal concentrations of 3.05, 9.76, 31.2 and 100 mg/L test groups was 0, 0, 0 and 100% respectively after 96 h exposure.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: based on analytically confirmed nominal concentration of the test substance
Remarks:
(95% CL: 47.1-57.3 mg/L)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
31.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: based on analytically confirmed nominal concentration of the test substance
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
68 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: based on analytically confirmed nominal concentration of the test substance
Details on results:
Mortality: The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups were 0, 0, 20, 100 and 100%, respectively at the end of the test.

Observed abnormal response: There were no abnormal responses of test fish in the blank control. Sub-lethal effects responses during the test included reduction of activity, spasmodic swimming, overturn and loss of equilibrium, immobilized at the bottom of the vessel and so on. After the start of the test on 3 h, some of the test fish performed reduction of activity in the test group of 21.4, 31.5 and 46.3 mg/L. Sub-lethal effects of reduction of activity, swimming and immobilized at the bottom and spasmodic swimming were observed in the test groups of 68 and 100 mg/L. During the test period of 24 h, all test fish died in the test groups of 68 and 100 mg/L. Some test fish performed reduction of activity, immobilized at the bottom of the vessel in the group of 21.4, 31.5 and 46.3 mg/L. From 48 to 72 h, no sub-lethal effects responses were observed in the test group of 21.4 mg/L. Sub-lethal effects responses including reduction of activity, overturn and loss of equilibrium lying on the bottom of the vessel were observed in the test groups of 31.5 and 46.3 mg/L. At the end of the test, the test fish performed reduction of activity, overturn and loss of equilibrium lying on the bottom of the vessel were observed in the test groups of 21 .4, 31.5 and 46.3 mg/L.

Body weight and total length of test fish: The body weight and total length of test fish in blank control were measured when the test was finished. The average and standard deviation of test fish's body weight and total length were 26.14 mm ± 1.83 mm and 0.124 g ± 0.011 g, respectively.
Results with reference substance (positive control):
Sensitivity Test of potassium dichromate with Zebra fish (Danio rerio) (Study No.: 2014GST0015) showed that the 24 h-LC50 of the reference substance (potassium dichromate) was 275 mg/L and the 95% confidence limits were 239-322 mg/L. The results were kept at the value ranging from 200 to 400 mg/L, which met the criterion for the validity of the test.
Reported statistics and error estimates:
Cumulative mortalities of each group at 3, 24, 48, 72 and 96 h after the start of the test were calculated and the concentration - response curve was rendered at the same time. The LC50 values and the 95% confidence limits at 24, 48, 72 and 96 h after the start of the test were calculated by Trimmed Spearman-Karber software (US EPA: Trimmed Spearman-Karber Program Version 1.5).

Table 6.1.1/1: Cumulative mortality

 

Nominal concentration (mg/L)

No. of test fish

Cumulative mortality (%)

3 h

24 h

48 h

72 h

96 h

Blank control

10

0

0

0

0

0

21.4

10

0

0

0

0

0

31.5

10

0

0

0

0

0

46.3

10

0

20

20

20

20

68.0

10

0

100

100

100

100

100

10

0

100

100

100

100

  

Measurement of temperature, pH and dissolved oxygen of test solution: During the test, the measured values of pH were in the range of 7.62 to 8.08 and the dissolved oxygen were in the range of 76 to 96%. The temperatures of test water environment were in the range of 22.3-23.9 °C. The dissolved oxygen concentrations were kept at not less than 60% of the air saturation value, which met the criterion for the validity of the test.

 

Observation of test solutions: During the test period, the appearances of blank control and test substance groups were observed to be clear and colorless.

 

Results of analytical measurements: The measured concentrations of test substance in blank control were all lower than the limit of detection (LOD=0.185 mg/L) at 0 h (new solution) and 48 h (old solution) after the start of the test. The measured concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups at 0 and 48 h (new solutions) ranged from 21.9 to 105 mg/L (88 to 108% of analytically confirmed nominal concentration of the test substance). At 48 h (old solutions) and 96 h, the measured concentrations of old solutions ranged from 21.0 to 102 mg/L (89 to 115% of analytically confirmed nominal concentration of the test substance). The test results showed that the measured concentrations of test substance could be maintained within 20% of the nominal concentrations, and the test results would be described based on analytically confirmed nominal concentrations of the test substance.

 

Validity of the test:

The mortality of the control was less than 10% at the end of the test, which met the criterion for the validity of the test.

The dissolved oxygen concentration in the test solutions was kept at not less than 60% of the air saturation value throughout the test, which met the criterion for the validity of the test.

All validity criteria were met according to the guidelines.
Validity criteria fulfilled:
yes
Conclusions:
Based on analytically confirmed nominal concentration of the test substance, the 96 h-LC50 to Zebra fish was 52.0 mg/L (95% Cl: 47.1 to 57.3 mg/L). LC0 and LC100 at 96 h were 31.5 and 68.0 mg/L, respectively.
Executive summary:
The acute toxicity of the test item to zebra fish (Danio rerio) was conducted according to OECD guideline 203 with GLP compliance.

 

Test item was tested at five nominal concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L under semi-static conditions for 96 h. A blank control (test water without test substance) was selected in parallel to the test concentration. Ten fish per group were used at the test concentrations and in the control.

 

The actual concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups at 0, 48 and 96 h (before and after the renewal) after the start of the test and blank control at 0 h (new solution) 48 h (old solution) were measured by Gas chromatograph.

 

The measured concentrations of test substance in blank control were all lower than the limit of detection (LOD=0.185 mg/L) at 0 h (new solution) and 48 h (old solution) after the start of the test. The measured concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups at 0 and 48 h (new solutions) ranged from 21.9 to 105 mg/L (88 to 108% of analytically confirmed nominal concentration of the test substance). At 48 h (old solutions) and 96 h, the measured concentrations of old solutions ranged from 21.0 to 102 mg/L (89 to 115% of analytically confirmed nominal concentration of the test substance). The test results showed that the measured concentrations of test substance could be maintained within ± 20% of the analytically confirmed nominal concentrations, and the test results would be described based on analytically confirmed nominal concentrations of the test substance.

 

The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups were 0, 0, 20, 100 and 100%, respectively at the end of the test.

 

Based on analytically confirmed nominal concentration of the test substance, the 96 h-LC50 was 52.0 mg/L (95% Cl: 47.1 to 57.3 mg/L). The highest concentration causing 0% mortality at 96 h (LC0) was 31.5 mg/L and the lowest concentration causing 100% mortality at 96h (LC100) was 68.0 mg/L.

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LC50 (Danio rerio) = 52.0 mg/L (95% CI: 47.1 -57.3 mg/L) based on analytically confirmed nominal concentrations

Key value for chemical safety assessment

LC50 for freshwater fish:
52 mg/L

Additional information

One key study is available (Guangdong, 2015) to assess the 96h acute toxicity of the registered substance to zebra fish (Danio rerio) under semi-static conditions according to OECD guideline 203 with GLP compliance. Although not requested under REACH Annex VII, the acute fish study was performed to comply with worldwide regulations. The nominal test substance concentrations tested were 21.4, 31.5, 46.3, 68.0 and 100 mg/L. A blank control (test water without test substance) was selected in parallel to the test concentration. The analytical monitoring was performed by Gas chromatograph at 0, 48 and 96h (before and after the renewal) after the start of the test and blank control at 0h (new solution) and 48h (old solution). Ten fish per group were used at the test concentrations and in the control.

The measured concentrations of test substance in blank control were all lower than the limit of detection (LOD=0.185 mg/L) at 0 h (new solution) and 48 h (old solution) after the start of the test. The measured concentrations of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups at 0 and 48 h (new solutions) ranged from 21.9 to 105 mg/L (88 to 108% of analytically confirmed nominal concentration of the test substance). At 48 h (old solutions) and 96 h, the measured concentrations of old solutions ranged from 21.0 to 102 mg/L (89 to 115% of analytically confirmed nominal concentration of the test substance). The test results showed that the measured concentrations of test substance could be maintained within ± 20% of the analytically confirmed nominal concentrations, and the test results would be described based on analytically confirmed nominal concentrations of the test substance.

The cumulative mortality of blank control was 0% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 21.4, 31.5, 46.3, 68.0 and 100 mg/L test groups were 0, 0, 20, 100 and 100%, respectively at the end of the test.

Based on analytically confirmed nominal concentration of the test substance, the 96 h-LC50 was 52.0 mg/L (95% Cl: 47.1 to 57.3 mg/L). The highest concentration causing 0% mortality at 96 h (LC0) was 31.5 mg/L and the lowest concentration causing 100% mortality at 96h (LC100) was 68.0 mg/L.