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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:
22 September to 14 October, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 203 with GLP statement. Confidence limits were not reliable
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Storage condition of test material: Kept in the dark, at 4 °C and under nitrogen
- Stability: Stable in container after opening/in light
- Melting point: -20 ± 0.5 °C
- Vapour Pressure: 0.33 Pa (20 °C)
- Solubility in Water: 2.16 g/L (20 °C, pH 4, flask method, 2014)
- Partition Coefficient: 2.69 (HPLC, 2013
- Densit: 1.101 kg/m^3 , 20 ± 0.5 °C
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of 9.53, 17.1, 30.9, 55.6 and 100 mg/L test groups at 0 h, 72 h (new solution) and 24 h, 96 h (old solution) after the start of the test and the blank control was measured at 0 h (new solution) and 24 h (old solution) were measured by Gas chromatograph (GC).
25 mL test solution was added in the separatory funnel, and 20 mL, 15 mL, 10 mL ethyl acetate were added respectively to extract for three times (5 min for each time). After that, each extraction was collected and transferred respectively into three 50 mL volumetric flasks, and then diluted with ethyl acetate to 50 mL (meaning that test solutions were diluted for 2·folds). The actual concentrations of test substance in ethyl acetate extractions were immediately measured. Otherwise the samples (the ethyl acetate extractions) would be stored at 4 °C until being analyzed in four days.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Stock solution: 0.400 g test substance was obtained in a small amount of test water with ultrasonication treatment for 10min and transferred into a 5 L-jar and diluted with test water to 4000 mL to obtain the nominal concentration of 100 mg/L suspension. Then the suspension was stirred with a magnetic stirrer for about 24 h, obtaining the nominal concentration of 100 mg/L stock solution (based on the test substance's purity of 98.9%, the nominal concentration of main constituent was 98.6 mg/L). Two more jars of stock solution were prepared by the same method above and fully mixed before the start of the test.
Test solution: 476 mL, 855 mL, 1545 mL, 2780 mL and 5000 mL test stock solutions were taken and allocated into five 5 L-jars respectively, then all solutions were diluted with test water to 5000 mL. After a certain extent of stirring, the nominal concentrations of 9.53, 17.1, 30.9, 55.6 and 100 mg/L test solutions were obtained (based on the test substance's purity of 98.9%. the nominal concentrations of main constituent in test solutions were 9.43, 16.9, 30.6, 55.0 and 98.6 mg/L, respectively). During the test period, all test vessels were lidded.
- 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

ACCLIMATION
- Acclimation period: 39 days
- Before the start of the test, Zebra fish were acclimated for 39 days by flow-through under the same conditions (water quality: dechlorinated tap water, temperature: 23 ± 2 °C, photoperiod: 14 h light / 10 h dark). The dissolved oxygen concentration 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) and the pH was 8.02.
Test temperature:
22.4-24.0 °C
pH:
The test was perfonned without adjustment of pH. The pH was in the range of 7.64 to 8.01.
Dissolved oxygen:
The test 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 (the dissolved oxygens were in the range of 80 to 96%).
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 9.53, 17.1, 30.9, 55.6 and 100 mg/L (based on the test substance's purity of 98.9%. the nominal concentrations of main constituent in test solutions were 9.43, 16.9, 30.6, 55.0 and 98.6 mg/L, respectively)
Details on test conditions:
TEST SYSTEM
- Test vessel: Vessel (5L jar: 16.6 cm x 25.0 cm)
- Volume of test solution: 5 L/group
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of fish: 10 fish/test group
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Loading rate: 10 fish/test vessel, 0.258 g fish/L

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

OTHER TEST CONDITIONS
- Photoperiod: 14 h light / 10 h darkness
- Light intensity: Artificial light of white fluorescent lamp was used. At the start of the test, the intensities of illumination of test environment were 102, 90 and 100 lux (97 lux on average). At the end of the test, the intensities of illumination of test environment were 96, 88 and 100 lux (95 lux on average).

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: 1.80
- 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 were 0, 0, 40 and 100% respectively after 96 h exposure.
The results of the range-finding test indicated that, the measured concentration of the nominal concentration of 10 mg/L test solution declined to 82% of the nominal concentrations of main constituent after 48 h. Meanwhile, the measured concentration of test substance in the test group containing test fish was significantly lower than other groups without any fish. So to maintain the actual concentration of test substance in test solution within ± 20% of the nominal concentrations of main constituent, the test would be conducted with semi-static test at 24 h intervals.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
17.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: based on analytically confirmed nominal concentration of the test substance
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: based on analytically confirmed nominal concentration of the test substance
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
30.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
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 9.53, 17.1, 30.9, 55.6 and 100 mg/L test groups were respectively 0, 0, 100, 100 and 100% 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, immobilized and swimming at the bottom of the vessel. At 3 h after the start of the test, some of the test fish performed reduction of activity in the test group of 9.53 mg/L. All the test fish in the test groups of 17.1 mg/L and 30.9 mg/L responded reduction of activity and immobilized at the bottom. All test fish died in the test groups of 55.6 mg/L and 100 mg/L. At 24 h after the start of the test, all test fish died in the test group of 30.9 mg/L. From 48 h to 96 h, some test fish performed reduction of activity in the group of 9.53 mg/L, some of the test fish performed reduction of activity and immobilized and also responded swimming at the bottom in test group of 17.1 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 respectively 24.83 mm ± 1.33 mm and 0.129 g ± 0.014 g.
Results with reference substance (positive control):
Sensitivity Test of potassium dichromate with Zebra fish (Danio rerio) (Study No.: 2014GST0013) showed that the 24 h-LC50 of the reference substance (potassium dichromate) was 261 mg/L to Zebra fish (Danio rerio) and the 95% confidence limits were 235-289 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 (L95) 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).
Sublethal observations / clinical signs:

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

9.53

10

0

0

0

0

0

17.1

10

0

0

0

0

0

30.9

10

0

100

100

100

100

55.6

10

100

100

100

100

100

100

10

100

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 7.64 to 8.01 and the dissolved oxygens were in the range of 80 to 96%. The temperatures of test water environment were in the range of 22.4-24.0 °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=0.186 mg/L) at 0 h (new solution) and 24 h (old solution) after the start of the test.

The measured concentrations of 9.12 mg/L, 12.4 mg/L, 16.9 mg/L, 22.9 mg/L and 31.2 mg/L test groups at 0 h and 72 h (new solution) ranged from 9.24 mg/L to 96.0 mg/L (90 to 100 % of the nominal concentrations of main constituent) and at 24 h and 96 h, the measured concentrations of old solutions ranged from 8.22 mg/L to 96.2 mg/L (83% to 98% of the nominal concentrations of main constituent). The test results showed that the measured concentrations of test substance could be maintained within ±20% of the nominal concentrations of the main constituent, and the test results would be described based on analytically confirmed nominal concentrations of the test substance.

 

Validity of the test

(1) 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.

(2) The dissolved oxygen concentration in the test solutions was kept at not less than 60 percent 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 was 23.0 mg/L. The highest concentration causing 0% mortality at 96 h (LC0) was 17.1 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 30.9 mg/L.
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 9.53, 17.1, 30.9, 55.6 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 9.53, 17.1, 30.9, 55.6 and 100 mg/L test groups at 0 h, 72 h (new solution) and 24 h, 96 h (old solution) after the start of the test and blank control at 0 h (new solution) and 24 h (old solution) were measured by GC.

 

The measured concentrations of test substance in blank control were all lower than the limit of detection (LOD=0.186 mg/L) at 0 h (new solution) and 24 h (old solution) after the start of the test. The measured concentrations of 9.12 mg/L, 12.4 mg/L, 16.9 mg/L, 22.9 mg/L and 31.2 mg/L test groups at 0 h and 72 h (new solution) ranged from 9.24 mg/L to 96.0 mg/L (90 to 100 % of the nominal concentrations of main constituent) and at 24 h and 96 h, the measured concentrations of old solutions ranged from 8.22 mg/L to 96.2 mg/L (83% to 98% of the nominal concentrations of main constituent). The test results showed that the measured concentrations of test substance could be maintained within ±20% of the nominal concentrations of the main constituent, 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 9.53, 17.1, 30.9, 55.6 and 100 mg/L test groups were respectively 0, 0, 100, 100 and 100% at the end of the test.

 

Based on analytically confirmed nominal concentration of the test substance, the 96 h-LC50 was 23.0 mg/L. The highest concentration causing 0% mortality at 96 h (LC0) was 17.1 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 30.9 mg/L.

Description of key information

OECD Guideline 203, GLP, key study, validity 2:

96 h-LC50 (Danio rerio) = 23.0 mg/L based on analytically confirmed nominal concentration of the test substance.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
23 mg/L

Additional information

One key study is available (Guangdong, 2014) 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 9.53, 17.1, 30.9, 55.6 and 100 mg/L and organisms were exposed under semi-static conditions for 96 h. 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 h, 72 h (new solution) and 24 h, 96 h (old solution) after the start of the test and blank control at 0 h (new solution) and 24 h (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=0.186 mg/L) at 0 h (new solution) and 24 h (old solution) after the start of the test.The measured concentrations of 9.12 mg/L, 12.4 mg/L, 16.9 mg/L, 22.9 mg/L and 31.2 mg/L test groups at 0 h and 72 h (new solution) ranged from 9.24 mg/L to 96.0 mg/L (90 to 100 % of the nominal concentrations of main constituent) and at 24 h and 96 h, the measured concentrations of old solutions ranged from 8.22 mg/L to 96.2 mg/L (83% to 98% of the nominal concentrations of main constituent). The test results showed that the measured concentrations of test substance could be maintained within ±20% of the nominal concentrations of the main constituent, 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 9.53, 17.1, 30.9, 55.6 and 100 mg/L test groups were respectively 0, 0, 100, 100 and 100% at the end of the test.

 Based on analytically confirmed nominal concentration of the test substance, the 96 h-LC50 was 23.0 mg/L. The highest concentration causing 0% mortality at 96 h (LC0) was 17.1 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 30.9 mg/L.