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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:
05-09-2012 to 12-04-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, in compliance with GLP, with acceptable restrictions
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test. (2004)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: State Environmental Protection Administration of China: HJ/T 153-2004, The guidelines for the testing of chemicals. China (2004)
Version / remarks:
The test was conducted in accordance with China HJ/T153 - 2004 Chemical Test Guideline stipulations and Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. Second Edition. Beijing: China
Environmental Press. 2013: 30-36. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: November 2010; signature: April 2013
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Analytical recovery samples were taken from all test media.
- Sampling method: Not reported
- Sample storage conditions before analysis: Fresh media were analysed immediately. Samples were stored at 4ºC until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock preparation: direct addition with ultrasonication and stirring. 0.5 g test substance dissolved in 300 mL test water with ultrasonication for 10 mins. Resulting solution transferred to 5-L jar, diluted with test water to 5 L and stirred using magnetic stirrer for 4 h at least to obtain the 100 mg/L stock solution. Test media preparation: Serial dilution of stock used to prepare test media for use in the study.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Not reported
- Source: Recognised supplier (listed in full study report)
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): 26.96 mm +/- 1.37 mm (mean and SD; range not stated)
- Weight at study initiation (mean and range, SD): 0.272 g +/- 0.044 g (mean and SD; range not stated)
- Method of breeding: Not reported

ACCLIMATION
- Acclimation period: 49 days
- Acclimation conditions (same as test or not): same conditions of water quality, temperature, light and dark period and >80% ASV dissolved oxygen.
- Type and amount of food: not reported
- Feeding frequency: at least 3x per week until 24 h before start of test.
- Health during acclimation (any mortality observed): 0% mortality during the 7 days prior to the test.

FEEDING DURING TEST
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none.
Hardness:
112 mg/L (expressed as CaCO3
Test temperature:
23 ± 2ºC
pH:
8.21-8.46 (fresh media); 7.73-7.97 (expired media)
Dissolved oxygen:
95-99% ASV (fresh media); 65-78% (expired media)
Salinity:
not applicable.
Nominal and measured concentrations:
Nominal test concentrations: 4.20, 5.46, 7.10, 9.23 and 12.0 mg/L and blank control.
Measured test concentrations (geometric mean): 3.247, 4.478, 6.061, 7.794, and 10.42 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 5 L glass.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 3L fill volume test media per test vessel
- Aeration: no

- Renewal rate of test solution (frequency): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 0.635 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water; chlorine removed by aeration.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement:Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 h light / 10 h darkness
- Light intensity: white fluorescent lamp, intensity not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at 3 h, 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.3

- Range finding study: yes
- Test concentrations: 5 and 10 mg/L and blank control
- Results used to determine the conditions for the definitive study: at 5 mg/L: 0% cumulative mortality at 96 h time point, visible abnormalities in test fish for the first 72h of the test (not at 96 h time point); at 10 mg/L: 100% cumulative mortality at 96 h time point, visible abnormalities in surviving fish at all observation intervals throughout the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: (95% CL: 3.91 - 4.56 mg/L)
Details on results:
- Behavioural abnormalities: See table 2 and 3
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: 4.8%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: reduced activity, immobilised at the bottom, overturn
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: Not applicable
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: 0% at lowest loading rate (150 mg/L) to 100% at highest loading rate (402 mg/L) in 24 h test period
- LC50: 239 mg/L (95% CL 215-266 mg/L) in 24 h
Sublethal observations / clinical signs:

Table 2: Toxicity to fish: mortality data

Treatment (mg/L) (nominal)

No. of fish at start of study

Observation period

3h

24 h

48 h

72 h

96 h

No. surviving

% mortality

No. surviving

% mortality

No. surviving

% mortality

No. surviving

% mortality

No. surviving

% mortality

Blank control

Replicate #1

7

7

0

7

4.8

7

4.8

7

4.8

7

4.8

Replicate #2

7

7

7

7

7

7

Replicate #3

7

7

6

6

6

6

4.20

Replicate #1

7

7

0

7

0

7

0

7

0

6

9.5

Replicate #2

7

7

7

7

7

7

Replicate #3

7

7

7

7

7

6

5.46

Replicate #1

7

7

0

7

0

6

4.8

5

33

1

71

Replicate #2

7

7

7

7

6

3

Replicate #3

7

7

7

7

3

2

7.10

Replicate #1

7

7

0

7

0

3

38

0

90

0

95

Replicate #2

7

7

7

5

1

0

Replicate #3

7

7

7

5

1

1

9.23

Replicate #1

7

7

0

6

9.5

2

81

0

100

0

100

Replicate #2

7

7

6

1

0

0

Replicate #3

7

7

7

1

0

0

12.0

Replicate #1

7

7

0

2

57

0

95

0

100

0

100

Replicate #2

7

7

4

1

0

0

Replicate #3

7

7

3

0

0

0

Table 3: Toxicity to fish: sublethal effects data

Treatment (mg/L) (nominal)

Observation period

3h

24 h

48 h

72 h

96 h

No. affected

Observations

No. affected

Observations

No. affected

Observations

No. affected

Observations

No. affected

Observations

Blank control

Replicate #1

7

N

7

N

7

N

7

N

7

N

Replicate #2

7

N

7

N

7

N

7

N

7

N

Replicate #3

7

N

6

N

6

N

6

N

6

N

4.20

Replicate #1

7

N

7

N

7

N

7

IMM(2), RA(5)

6

IMM(4), RA(2)

Replicate #2

7

N

7

N

7

N

7

IMM(1), RA(6)

7

IMM(3), RA(4)

Replicate #3

7

N

7

N

7

N

7

RA(7)

6

RA(6)

5.46

Replicate #1

7

N

7

RA(7)

6

IMM(2), RA(4)

5

IMM(1), RA(4)

1

IMM(1)

Replicate #2

7

N

7

RA(7)

7

IMM(2), RA(5)

6

IMM(2), RA(4)

3

IMM(3)

Replicate #3

7

N

7

RA(7)

7

IMM(4), RA(3)

3

IMM(1), RA(2)

2

IMM(1), RA(1)

7.10

Replicate #1

7

N

7

IMM(1), RA(6)

3

IMM(2), RA(1)

-

-

-

-

Replicate #2

7

N

7

RA(7)

5

IMM(4), RA(1)

1

OLE(1)

-

-

Replicate #3

7

N

7

RA(7)

5

RA(5)

1

RA(1)

1

IMM(1)

9.23

Replicate #1

7

RA(7)

6

RA(6)

2

RA(1), OLE(1)

-

-

-

-

Replicate #2

7

RA(7)

6

IMM(1), RA(5)

1

IMM(1)

-

-

-

-

Replicate #3

7

RA(7)

7

IMM(4), RA(3)

1

IMM(1)

-

-

-

-

12.0

Replicate #1

7

RA(7)

2

RA(2)

-

-

-

-

-

-

Replicate #2

7

RA(7)

4

RA(4)

1

RA(1)

-

-

-

-

Replicate #3

7

RA(7)

3

RA(3)

0

 

-

-

-

-

 Notes:

N = normal; - = all fish died; RA = reduced activity; OLE = overturn and loss of equilibrium; IMM = immobilised at the bottom.

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test substance to Danio rerio was determined to be 4.20 mg/L with a 95% confidence interval from 3.91 – 4.56 mg/L based on geometric mean measured concentrations.
Executive summary:

The 96 hour LC50 for the test substance to Danio rerio was determined to be 4.20 mg/L with a 95% confidence interval from 3.91 – 4.56 mg/L based on analytically confirmed geometric mean measured concentrations. The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test according to OECD TG 203 and the China CRC MEP guidelines, effects on Biotic Systems, 203 Fish Acute toxicity test (2004) under GLP. Based on the results of range finding tests, the nominal loading rates of 0 (control), 4.20, 5.46, 7.10, 9.23 and 12.00 mg/L with a geometric series factor of 1.3 were selected for exposure to 21 fish groups (126 total). One control containing no test substance was also performed. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system in response to ensuring adequate stability of exposure to test item. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 4.20, 5.46, 7.10, 9.23 and 12.00 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal) were measured by High Performance Liquid Chromatography (HPLC-DAD). Behavioural abnormalities and biological effects were reported during the course of the study. The measured concentrations ranged before the renewal and at the end of the exposure ranged from 2.386 to 10.24 mg/L (or 57% to 88% of the nominal concentration). On this basis the could not maintained at ±20% of the initial concentrations. Therefore the effect levels were estimate based on geometric mean measured concentrations. Under the conditions of this study, the 96h LC50 was 4.22 (C.I. 3.91 – 4.56) mg/L based on GMM concentrations.

Description of key information

LC50 (fish) = 4.22 (C.I. 3.91 – 4.56) mg/L based geometric mean measured concentrations, 96hour, freshwater, OECD TG 203, 2013

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
4.22 mg/L

Additional information

The 96 hour LC50 for the test substance to Danio rerio was determined to be 4.20 mg/L with a 95% confidence interval from 3.91 – 4.56 mg/L based on analytically confirmed geometric mean measured concentrations. The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test according to OECD TG 203 and the China CRC MEP guidelines, effects on Biotic Systems, 203 Fish Acute toxicity test (2004) under GLP. Based on the results of range finding tests, the nominal loading rates of 0 (control), 4.20, 5.46, 7.10, 9.23 and 12.00 mg/L with a geometric series factor of 1.3 were selected for exposure to 21 fish groups (126 total). One control containing no test substance was also performed. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system in response to ensuring adequate stability of exposure to test item. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 4.20, 5.46, 7.10, 9.23 and 12.00 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal) were measured by High Performance Liquid Chromatography (HPLC-DAD). Behavioural abnormalities and biological effects were reported during the course of the study. The measured concentrations ranged before the renewal and at the end of the exposure ranged from 2.386 to 10.24 mg/L (or 57% to 88% of the nominal concentration). On this basis the could not maintained at ±20% of the initial concentrations. Therefore the effect levels were estimate based on geometric mean measured concentrations. Under the conditions of this study, the 96h LC50 was 4.22 (C.I. 3.91 – 4.56) mg/L based on GMM concentrations.

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