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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:
5-23 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 203 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The actual concentrations of 4.9% LS, 7.4% LS, 11% LS and 17% LS test groups were measured at 0, 96 h and 24, 48 and 72 h (before and after the renewal) after the start of the test. And the blank control and 25% LS test group were measured at 0 h and 24 h after the start of the test by HPLC.
“LS” : Limit solution => Nominal 50 mg/L but measured as approx. 45 mg/L at T0 [close to water saturation value of 51 mg/L]
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Preparation of test stock solution: 0204 g test substance was dissolved in appropriate test water with ultrasonic treatment for 10 min, and then transferred into 5 L-jar, diluted with the test water to 4000 mL and was stirred with a magnetic stirrer for about 2 h. After filtered with 0.45 µm filter membrane and abandoned the insoluble substance, the saturation concentration of filtered test solution of nominal concentration of 51.0 mg/L (50.0 mg/L calculated based on the 98.1 % purity of the substance main constituent) test substance solution (Limit solution, abbreviated to LS) was obtained. According to the above methods two replicates of the test stock solution would be prepared.
Note: As all test fish in the 25 % LS test group had died at 24 h after the start of the test, the test stock solution was prepared with one replicate at 48 h after the start of the test.
Preparation of test substance solution: 147, 222, 330, 510 and 750 mL of test stock solution was taken into the test container, and then 2853, 2778, 2670, 2490 and 2250 mL of test water was added. Stir well to obtain the nominal concentration of 4.9%, 7.4%, 11%, 17% and 25% LS test substance solution. Three replicates were prepared in each test group.
- Controls: A blank control (test water without test substance) was set 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: 30 days
- Before the start of the test, Zebra fish were acclimated for 30 days by flow-through under the same conditions (water quality: dechlorinated tap water, temperature: 23 ± 2 °C, light and dark period (14 h light/10 h dark). The dissolved oxygen concentraiion during acclimation was kept at least 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 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 100 mg/L (expressed as CaCO3)
Test temperature:
23.0-23.8 °C
pH:
The test was performed without adjustment of pH. The pH was in the range of 7.29 to 8.23.
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 least 60 % of the air saturation value (62-96%).
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 4.9%, 7.4%, 11%, 17% and 25% LS (measured geometric mean concentrations of 1.51, 2.39, 3.60, 6.23 and 10.2 mg/L, respectively)
Details on test conditions:
The test results of stability test of test substance in test water showed that, there were no significant differences of solubility of test substance in three different waters. And the nominal concentration of 50 mg/L test solution prepared by dechlorinated tap water was stable after standing for 72 h, and the measured concentration was 90.9% of the nominal value.

TEST SYSTEM
- Test vessel: Vessel (5 L jar with plug: 16.6 cm x 25.0 cm)
- Test volume: 9 L/test group (3 L/test vessel)
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of fish: 21 fish/test group
- No. of fish per test vessel: 7
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Loading rate: 7 fish/test vessel, 0.502 g fish/L

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

OTHER TEST CONDITIONS
- Photoperiod: Artificial light of white fluorescent lamp, 14 h light/10 h darkness

EFFECT PARAMETERS MEASURED
- Observation of test organisms: Mortality and visible abnormality 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 was 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.
- Measurement of temperature, pH and dissolved oxygen of test solution: The temperature, pH and dissolved oxygen of each test group were measured at 0, 96 h and 24, 48, 72 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study: Static method - 0.1, 1.0, 10 and 50 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 was 0, 0, 100 and 100% at 0.1, 1.0, 10 and 50 mg/L test groups, respectively based the nominal concentrations.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
2.39 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 7.4% LS test group
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.07 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% Cl: 3.63-4.55 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
10.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 25% LS test group
Details on results:
Mortality: The cumulative mortality of blank control was 4.76% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 4.9%, 7.4%, 11%, 17% and 25% LS test groups were 0, 0, 38.1, 95.2 and 100% at the end of the test.
Observed abnormal response: There was one test fish died in the blank control at the test period of 48 h and no abnormal responses of the other test fish in blank control were observed. Observed abnormal responses during the test were included activity reduced, immobilized at the bottom, lay on the bottom, swimming at the bottom, spasmodic swimming and overturn and Ioss of equilibrium and so on. After the start of the test at 3 h, there were abnormal responses observed of test fish in the five test groups. All the test fish died in the 25% LS test group at the start of 24 h. And the abnormal responses of test fish including activity reduced, lay on the bottom, swimming at the bottom, immobilized at the bottom and spasmodic swimming were observed in the 11% LS and 17% LS test groups, but the test fish in the 4.9% LS and 7.4% LS got back to normal. At the test period of 48 h, there is one test fish died in the blank control. At the end of the test at 96 h, the test fish in 11% LS test group was still observed the abnormal response of activity reduced, and there was only one test fish alive in the 17% LS test group and observed Iaying on the bottom.
Body weight and total length of test fish: The body weight and total length of 10 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 0.215 g ± 0.036 g and 23.64 mm ± 1.60 mm, respectively.
Results with reference substance (positive control):
Sensitivity test of potassium dichromate with Zebra fish (Brachydanio rerio) (Study No.: 2013GST0010) showed that the 24 h-LC50 of the reference substance (potassium dichromate) was 222 mg/L to Zebra fish (Brachydanio rerio) and the 95% confidence limits was 193-253 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 mortality of each group at 3, 24, 48, 72 and 96 h after the start of the test were calculated. 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).

Table 6.1.1/1: Cumulative Mortality

 

Setting Concentration (mg/L)

Geometric mean of measured

Concentration (mg/L)

No. of test fish

Cumulative Mortality %

3 h

24 h

48 h

72 h

96 h

Blank control

-

21

0

0

4.76

4.76

4.76

4.9% LS (Limit solution)

1.51

21

0

0

0

0

0

7.4% LS

2.39

21

0

0

0

0

0

11% LS

3.60

21

0

23.8

38.1

38.1

38.1

17% LS

6.23

21

0

61.9

85.7

95.2

95.2

25% LS

10.2

21

4.76

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 7.29 to 8.23 and the dissolved oxygen were in the range of 62 to 96%. The temperatures of test water environment were in the range of 23.0-23.8 °C. The dissolved oxygen concentrations were kept at least 60% of the air saturation value, which met the criterion for the validity of the test.

 

Concentration of test substance in test solution:

The sample processing and preservation: The test solutions were filtered through 0.45 µm filter first and were diluted with acetonitrile for 2-fold before testing. The samples were analyzed immediately. Otherwise the samples were stored at -20°Cuntil analysis and must be analyzed during two days. 

Results of analytical measurements: The measured concentrations of test substance in blank control were all lower than the LOD (limited of detection) 0.0155 mg/L at 0 h, 24 h after the start of the test.

The measured concentrations of test substance in 4.9% LS, 7.4% LS, 11% LS, 17% LS and 25%LS test groups at the start of the exposure and after the renewal ranged from 2.252 mg/L to 11.52 mg/L. Those before the renewal and at the end of the exposure ranged from 0.8214 mg/L to 9.072 mg/L. The measured concentrations of the test substance in old solutions could not be maintained within 20% of the initial concentrations (in the new solutions) during the renewal period of 24 h. So, the test results were described based on the geometric means of measured concentrations.

  

Validity of the test

(1) The mortality of the control was 4.76% which was less than 10% at the end of the test.

(2) The dissolved oxygen concentration in the test solutions was kept at least 60 percent of the air saturation value throughout the test.

All validity criteria were met according to the guidelines.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions and based on geometric mean measured concentration, the 96 h-LC50 of the test substance is 4.07 mg/L (95% Cl: 3.63-4.55 mg/L). The highest concentration causing 0% mortality at 96 h (LC0) was 2.39 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 10.2 mg/L.
Executive summary:

The acute toxicity of the test item to zebra fish (Brachydanio rerio) was conducted according to OECD guideline 203 with GLP compliance.

 

Test item was tested at five concentrations of 4.9%, 7.4%, 11%, 17% and 25% LS (measured geometric mean concentrations of 1.51, 2.39, 3.60, 6.23 and 10.2 mg/L, respectively) under semi-static conditions for 96 h. A blank control (test water without test substance) was selected in parallel to the test concentration. 21 fish per group were used at the test concentrations and in the control.

The actual concentrations of 4.9% LS, 7.4% LS, 11% LS and 17% LS test groups were measured at 0, 96 h and 24, 48 and 72 h (before and after the renewal) after the start of the test. And the blank control and 25% LS test group were measured at 0 h and 24 h after the start of the test by HPLC.

 

The measured concentrations of test substance in blank control were all lower than the LOD (limited of detection) 0.0155 mg/L at 0 h, 24 h after the start of the test. The measured concentrations of test substance in 4.9% LS, 7.4% LS, 11% LS, 17% LS and 25%LS test groups at the start of the exposure and after the renewal ranged from 2.252 mg/L to 11.52 mg/L. That before the renewal and at the end of the exposure ranged from 0.8214 mg/L to 9.072 mg/L. The measured concentrations of the test substance in old solutions cannot maintain +20% of the initial concentrations (in the new solutions) during the renewal period of 24 h. So, the test results would be described based on the measured concentrations in geometric mean.

 

The cumulative mortality of blank control was 4.76% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 4.9%, 7.4%, 11%, 17% and 25% LS mg/L test groups were 0, 0, 38.1, 95.2 and 100% at the end of the test.

 

Under the test conditions and based on geometric mean measured concentration, the 96 h-LC50 of the test substance is 4.07 mg/L (95% Cl: 3.63-4.55 mg/L). The highest concentration causing 0% mortality at 96 h (LC0) was 2.39 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 10.2 mg/L.

Description of key information

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

96h-LC50 (Danio rerio) = 4.07 mg/L (95% CI: 3.63 -4.55 mg/L) based on the geometric mean of measured concentrations

Key value for chemical safety assessment

LC50 for freshwater fish:
4.07 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.

Test item was tested at five concentrations of 4.9%, 7.4%, 11%, 17% and 25% Limit Solution (measured geometric mean concentrations of 1.51, 2.39, 3.60, 6.23 and 10.2 mg/L, respectively) under semi-static conditions for 96 h. A blank control (test water without test substance) was selected in parallel to the test concentration. 21 fish per group were used at the test concentrations and in the control.

The actual concentrations of 4.9% LS (Limit Solution), 7.4% LS, 11% LS and 17% LS test groups were measured at 0, 96 h and 24, 48 and 72 h (before and after the renewal) after the start of the test. And the blank control and 25% LS test group were measured at 0 h and 24 h after the start of the test by HPLC.

 The measured concentrations of test substance in blank control were all lower than the LOD (Limited Of Detection) 0.0155 mg/L at 0 h, 24 h after the start of the test. The measured concentrations of test substance in 4.9% LS, 7.4% LS, 11% LS, 17% LS and 25%LS test groups at the start of the exposure and after the renewal ranged from 2.252 mg/L to 11.52 mg/L. That before the renewal and at the end of the exposure ranged from 0.8214 mg/L to 9.072 mg/L. The measured concentrations of the test substance in old solutions cannot maintain +20% of the initial concentrations (in the new solutions) during the renewal period of 24 h. So, the test results would be described based on the measured concentrations in geometric mean.

 The cumulative mortality of blank control was 4.76% at the end of the test, which met the criterion for the validity of the test. The cumulative mortalities of 4.9%, 7.4%, 11%, 17% and 25% LS mg/L test groups were 0, 0, 38.1, 95.2 and 100% at the end of the test.

 Under the test conditions and based on geometric mean measured concentration, the 96 h-LC50 of the test substance is 4.07 mg/L (95% Cl: 3.63-4.55 mg/L). The highest concentration causing 0% mortality at 96 h (LC0) was 2.39 mg/L and the lowest concentration causing 100% mortality at 96 h (LC100) was 10.2 mg/L.