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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-08-2013 to 11-10-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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: Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 203 Fish Acute Toxicity Test, China Environmental Press, China (2004)

Deviations:

no

Principles of method if other than guideline:

The test was generally conducted in accordance with or equivalent to the following: 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. Beijing: China Environmental Press. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: In refrigerator (4°C) in the dark
- Other: Clear liquid

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 2.0, 2.5, 3.1, 3.9 and 4.9 mg/L nominal concentrations (with geometric series factor 1.25)
with equivalent geometric mean measured concentrations of : 0 ( control), 2.10, 2.42, 3.19, 4.26 and 5.07 mg/L
- Sampling method: 1.5 mL extract from test solution.
- Sample storage conditions before analysis: The samples were analysed immediately.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item was directly weighed into a flask and dissolved with appropriate volume test water, to obtain nominal concentrations of 2, 2.5, 3.1, 3.9 and 4.9 mg/L. The solution was transferred into a 10 L - flask and then the suspension was stirred for 3 hours. Then the solution was directly poured into test container replicates. The solution was prepared freshly every 24 hours.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: A negative/blank control without test item or reference item was also included. A previously conducted sensitivity test with potassium dichromate (positive control/reference item) was performed. Full information provided in the full study report.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): In the definitive test there was no evidence of undissolved test item.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Brachydanio rerio)
- Strain: Not reported. Batch generation number FZA000-20 130226-01
- Source: Recognised supplier (recorded in full study report) located in China.
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): Not reported. The body weight and total length of test fish in blank control were measured when the test was finished. The average and deviation of test fish total length and body weight were respectively 2.099 – 2.9998 cm (i.e. 2.0 +/- 1.0 cm according to guideline) and 0.110 – 0.333 g. In the treated/control groups the loading was 0.540 g/L (i.e. < 1.0g fish/litre).
- Method of breeding: Not reported.
- Feeding during test: No.
- Food type: Feeding was conducted during acclimation (daily) until 24-hours prior to test initiation.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.

ACCLIMATION
- Acclimation period: 7 days (i.e. > 48 hours)
- Acclimation conditions (same as test or not): Yes. Same water quality, temperature 23 ± 2°C and photoperiod as used during the test (14 hours light; 10 hours dark). Oxygen concentration was in the range of the exposure duration (> 80% air saturation).
- Type and amount of food: Not reported.
- Feeding frequency: Daily; until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported (0% during 7 days pre-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

Hardness:

45 mg/L expressed as CaCO3; Test water (dechlorinated water)

Test temperature:

Temperature 21.3 - 23.0 °C during test period, the test was performed in a temperature controlled environment with continuous monitoring and was maintained at 23 ± 2°C.

pH:

Test water (dechlorinated water): pH 6.0 - 8.5 (actual: pH 7.05) as specified in China Guidelines for the testing of Chemicals 203 test.
The pH was in the range of 6.23 - 7.77 during the study.

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. Measured range: 60.3 to 104% ASV during the definitive test.
(Note: minimal ASV occurred prior to renewal of test solutions)

Nominal and measured concentrations:

- Preliminary range-finding test performed on semi-static test system:
0 (control), 2.5 and 5.0 mg/L nominal concentrations were at minimum utilised.
- Definitive: 0 (control), 2.0, 2.5, 3.1, 3.9 and 4.9 mg/L nominal concentrations (with geometric series factor 1.25)
with equivalent geometric mean measured concentrations of : 0 ( control), 2.10, 2.42, 3.19, 4.26 and 5.07 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 L glass
- Type (delete if not applicable): glass flask (open/closed not reported)
- Material, size, headspace, fill volume: glass. ca. 10000 mL fill volume (minimum headspace)
- Aeration: Not continuously aerated although DOC > 60% ASV during exposure.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): Every 24 hours.
- No. of organisms per vessel: Seven (7) per vessel
- No. of vessels per concentration (replicates): None.
- No. of vessels per control (replicates): One (1)
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: < 1.0 g/L (Actual: 0.54 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis water used in the test (indicated in the full study report).
- Conductivity: Typically, would be: Water: 500±100μs/cm) as specified in China Water quality standard for fisheries in GB-11607-89.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Every 24 hour intervals; before and/or after renewal as applicable.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortalities and abnormalities; measured at 24h, 48h, 72h and 96h as applicable.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.25
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study: 0 (control), 2.5 and 5.0 mg/L nominal concentrations were at minimum utilised. 100% mortality was seen at 5.0 mg/L nominal.
- Test concentrations: 0 (control), 2.0, 2.5, 3.1, 3.9 and 4.9 mg/L nominal concentrations (with geometric series factor 1.25)
with equivalent geometric mean measured concentrations of : 0 ( control), 2.10, 2.42, 3.19, 4.26 and 5.07 mg/L
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.254 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: C.I. 4.004 - 4.539 mg/L

Details on results:

- Behavioural abnormalities: There were no abnormal responses of test fish in the blank control. There were no observed abnormalities in test groups.
- Observations on body length and weight: The body weight and total length of test fish in blank control were measured when the test was finished. The average and deviation of test fish total length and body weight were respectively 2.099 – 2.9998 cm (i.e. 2.0 +/- 1.0 cm according to guideline) and 0.110 – 0.333 g. In the treated/control groups the loading was 0.540 g/L (i.e. < 1.0g fish/litre).
- Mortality of control: None.
- Other adverse effects control: Yes. None reported.
- Abnormal responses: There were no abnormal responses of test fish in the blank control. There were no observed abnormalities in test groups.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 24h LC50: 204.922 (C.I. - ) mg/L

Reported statistics and error estimates:

The LC50 values and the 95% confidence limits (LC5) at 72h and 96 h after the start of the test were calculated by Probit Analysis software (US EPA: Probit Analysis Program Version 1.5).

Sublethal observations / clinical signs:

Table 1. Median Lethal Concentration (LC50) and 95% confidence limits

Recommended observation times	LC50 (mg/L) based on geometric mean measured concentrations	95% confidence limits (mg/L)
96 h	4.254	4.004 - 4.539

#1: LC50 based on geometric mean measured concentrations

 

Table 2. Cumulative mortality

Cumulative mortality #2
Nominal Concentration #1	Exposure duration (h)
	24	48	72	96
Control 0.00 mg/L	0.0%	0.0%	0.0%	0.0%
2.0 mg/L	0.0%	0.0%	0.0%	0.0%
2.5 mg/L	0.0%	0.0%	0.0%	0.0%
3.1 mg/L	0.0%	0.0%	0.0%	4.76%
3.9 mg/L	0.0%	0.0%	4.76%	14.3%
4.9 mg/L	14.3%	61.9%	90.5%	90.5%

#1: nominal concentrations with equivalent geometric mean measured concentrations indicated in "nominal and measured concentrations" field.

#2: n = 7 per concentration

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Danio rerio was determined to be 4.254 (C.I. 4.004 - 4.539) mg/L based on geometric mean measured concentrations.

Executive summary:

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 preliminary range finding tests, the nominal loading rates of 0 (control), 2.0, 2.5, 3.1, 3.9 and 4.9 mg/L with a geometric series factor of 1.3 were selected for exposure to 7 fish in each replicate. One control containing no test item was also performed. The test solution was renewed every 24 hours over a 96 hour period by semi-static renewal exposure. The test was performed under 14 hour photoperiod at 23.6 to 24.8°C (constant to within 2°C) and pH 6.23 to 7.77. Dissolved oxygen was no less than 60% of ASV (actual: 60.3 to 103.6%). No feeding was conducted during the exposure. Concentrations of test item were determined at the start (t=0h), 24h, 48h, 72h renewals and at end (t=96h) by GC-FID analysis. Behavioural abnormalities and biological effects were reported during the course of the study. This was poor balance which started at 2.5 mg/L nominal concentration. There was no mortality at 2.5 mg/L nominal concentration. The measured concentrations ranged at the start, after renewals and at the end of the exposure ranged from 2.05 to 5.66 mg/L (or 84.8% to 119% of the nominal concentration). The effect levels were both estimated on nominal concentrations and geometric mean measured concentrations. On the basis the test item was maintained at ±20% of the initial concentrations, the LC50 was based on nominal concentrations that were analytically confirmed. The LC50 was 4.523 mg/L based on geometric mean measured concentrations Under the conditions of this study, the 96h LC50 was 4.254 (C.I. 4.004 – 4.539) mg/L based on analytically confirmed nominal concentrations.

Description of key information

LC50-96h (fish) = 4.254 (C.I. 4.004 – 4.539) mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2013

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.254 mg/L

Additional information

Key study : OECD TG 203, 2013 : 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 preliminary range finding tests, the nominal loading rates of 0 (control), 2.0, 2.5, 3.1, 3.9 and 4.9 mg/L with a geometric series factor of 1.3 were selected for exposure to 7 fish in each replicate. One control containing no test item was also performed. The test solution was renewed every 24 hours over a 96 hour period by semi-static renewal exposure. The test was performed under 14 hour photoperiod at 23.6 to 24.8°C (constant to within 2°C) and pH 6.23 to 7.77. Dissolved oxygen was no less than 60% of ASV (actual: 60.3 to 103.6%). No feeding was conducted during the exposure. Concentrations of test item were determined at the start (t=0h), 24h, 48h, 72h renewals and at end (t=96h) by GC-FID analysis. Behavioural abnormalities and biological effects were reported during the course of the study. This was poor balance which started at 2.5 mg/L nominal concentration. There was no mortality at 2.5 mg/L nominal concentration. The measured concentrations ranged at the start, after renewals and at the end of the exposure ranged from 2.05 to 5.66 mg/L (or 84.8% to 119% of the nominal concentration). The effect levels were both estimated on nominal concentrations and geometric mean measured concentrations. On the basis the test item was maintained at ±20% of the initial concentrations, the LC50 was based on nominal concentrations that were analytically confirmed. The LC50 was 4.523 mg/L based on geometric mean measured concentrations Under the conditions of this study, the 96h LC50 was 4.254 (C.I. 4.004 – 4.539) mg/L based on analytically confirmed nominal concentrations.