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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:
21-11-2014 to 15-12-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
other: Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test, China Environmental Press, China (2013)
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 [M]. Second Edition. Beijing: China Environmental Press. 2013: 30-36. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Range-Finding Test: Nominal loading rates: 10 mg/L, 18 mg/L, 32 mg/L, 58 mg/L and 105.0 mg/L as WAFs (geometric series factor 1.8)
Definitive Test: Nominal loading rates: 7.80 mg/L, 11.0 mg/L, 15.4 mg/L, 21.4 mg/L and 30.0 mg/L as WAFs (geometric series factor 1.4)
- Sampling method: 50 mL test solution (after filtration with 0.22 µm filter membrane) was added to separation funnel, and 20 mL, 15 mL, 10 mL ethyl acetate were added respectively to extract three times (5 min for each time). Each extraction was collected and transferred respectively into three 50 mL volumetric flasks, and then diluted with ethyl acetate to 50 mL.
- Sample storage conditions before analysis: The actual concentrations of test item in ethyl acetate extractions were immediately measured. Otherwise the samples (ethyl acetate extractions) are stored at 4 °C until analysis within one week.
Vehicle:
no
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 FDr20141023-1
- Source: Recognised supplier (recorded in full study report) located in China
- 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 2.497 ±0.142 cm (meeting the requirement of 2.0±1.0 cm according to guideline) and 0.134±0.014 g (loading was 0.336 g/L ; i.e. < 1.0 g fish/litre).
- Weight at study initiation (mean and range, SD): See above.
- Method of breeding: Not reported.
- Maintenance of the brood fish: Not reported.

ACCLIMATION
- Acclimation period: 40 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 > 80% ASV.
- Type and amount of food: Not reported.
- Feeding frequency: At least daily until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

FEEDING DURING TEST
- Food type: Feeding was conducted during acclimation (twice daily) until 24-hours prior to test initiation.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
In accordance with guideline.
Hardness:
118 mg/L expressed as CaCO3; Test water (dechlorinated water): Conductivity: 500±100μs/cm) as specified in China Water quality standard for fisheries in GB-11607-89.
Test temperature:
Temperature 22.0 - 23.8 °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 7.0±0.5 as specified in China Water quality standard for fisheries in GB-11607-89.
The pH was in the range of 7.57 - 7.88 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 day 1 to 7: 73 - 102% ASV during the definitive test.
Nominal and measured concentrations:
- Preliminary range-finding test performed on semi-static test system with renewal every 24 hour intervals:
Nominal loading rates: 10 mg/L, 18 mg/L, 32 mg/L, 58 mg/L and 105.0 mg/L as WAFs (geometric series factor 1.8)
Semi-static exposure method was performed in the range-finding test with renewal of test solution every 24 hours. The range-finding test showed that there was 10%mortality observed at the nominal loading rate concentration of 10 mg/L WAF and 100% mortality was observed at or above the nominal loading rate concentration of 32 mg/L WAF within the test period of 96 hours.
- Definitive test performed on semi-static test system with renewal every 24 hour intervals:
Nominal loading rates: 7.80 mg/L, 11.0 mg/L, 15.4 mg/L, 21.4 mg/L and 30.0 mg/L as WAFs (geometric series factor 1.4)
During the test, the dissolved test material concentration analytically were determined in the freshly prepared solution/medium (t=0h, 72 h) and in the aged medium/solution before renewal (t=24h*, 96h*).
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L wide-mouth vessel with stopper
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass. 4 Litre fill volume.
- Aeration: Not reported.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Renewal every 24 hours.
- No. of organisms per vessel: Ten (10) 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 (ca. 0.336 g fish/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used in the test were purified declorination. (Certified annually).
- Conductivity: 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 after renewal

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours light / 10 hours dark photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortalities and abnormalities; measured at 3h, 6h, 24h, 48h, 72h and 96h as applicable.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study
- Test concentrations: 10 mg/L, 18 mg/L, 32 mg/L, 58 mg/L and 105.0 mg/L as WAFs (geometric series factor 1.8) (10% mortality at 10 mg/L WAF was reported)
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nominal laoding prepared as WAF
Basis for effect:
mortality
Remarks on result:
other: based on nominal loading rate prepared as WAF
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
12.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nominal laoding prepared as WAF
Basis for effect:
mortality
Remarks on result:
other: C.I.11.2 - 13.2 mg/L; based on nominal loading rate prepared as WAF
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
15.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
nominal laoding prepared as WAF
Basis for effect:
mortality
Remarks on result:
other: based on nominal loading rate prepared as WAF
Details on results:
- Behavioural abnormalities: There were no abnormal responses of test fish in the blank control. Observed abnormal responses are reported in table 3.
- Observations on body length and weight: Not reported.
- Mortality of control: None.
- Other adverse effects control: Yes. None reported.
- Abnormal responses: Observed abnormal responses are reported in table 3.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported. The measured concentrations of the main components of test item were followed as a tracer in the nominal loading rates of 7.80 mg/L. 11.0 mg/L, 15.4 mg/L, 21.4 mg/L and 30.0 mg/L WAFs at the start of the exposure and after the renewal ranged from 5.54 mg/L to 11.0 mg/L. Before the renewal and at the end of the exposure, the measured concentrations of the main components were followed as a tracer ranged from 3.02mg/L to 7.62mg/L (ranged from 50% to 80% of the initial concentration). During the renewal period of 24h, the measured concentrations of the main components of the test item in the old solutions could not be maintained at ±20% of initial concentrations.
- 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: 252 mg/L

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

Observation times

LC50 (mg/L) #1

95% confidence limits (mg/L)

24 h

12.2

11.2 - 13.2

48 h

12.2

11.2 - 13.2

72 h

12.2

11.2 - 13.2

96 h

12.2

11.2 - 13.2

#1: LC50 based on nominal loading concentrations prepared as WAF

 

Table 2. Cumulative mortality

Cumulative mortality

Group #1

Exposure duration (hours)

3

6

24

48

72

96

Control

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

7.8 mg/L

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

11.0 mg/L

0.0%

0.0%

20.0%

20.0%

20.0%

20.0%

15.4 mg/L

0.0%

0.0%

100.0%

100.0%

100.0%

100.0%

21.4 mg/L

0.0%

0.0%

100.0%

100.0%

100.0%

100.0%

30.0 mg/L

0.0%

0.0%

100.0%

100.0%

100.0%

100.0%

#1: Group is control or nominal loading concentration prepared as WAF group.

 

Table 3. Observed abnormal responses

Visible abnormalities

Group #1

Exposure duration (hours)

3

24

48

72

96

Control

N (10/10)

N (10/10)

N (10/10)

N (10/10)

N (10/10)

7.8 mg/L

N (10/10)

IMM (4)

RA (4)

IMM (3)

RA (7)

RA (6)

RA (4)

11.0 mg/L

RA (4)

IMM (6)

RA (2)

IMM (6)

LB (2)

IMM (7)

LB (1)

RA (8)

15.4 mg/L

RA (6)

-

-

-

-

21.4 mg/L

LB (2)

RA (8)

-

-

-

-

30.0 mg/L

LB (3)

RA (7)

-

-

-

-

Note: N: means normal; RA: means reduced activity; IMM: immobilized at bottom ; LB: means lying on bottom.

#1: Group is control or nominal loading concentration prepared as WAF group. 

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 12.2 (C.I. 11.2 - 13.2) mg/L based on nominal loading rate concentrations prepared as WAF.
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 Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test (2013) under GLP. Based on the results of range finding tests the nominal concentrations of 0 (control), 4.0 , 4.8, 5.8, 7.0 and 8.5 mg/L (geometric series with a factor of 1.4) were selected for exposure to five fish groups. One control containing no test item was also performed. The test item was renewed at 24 hour intervals within a 96 hour semi-static test system. Observations were made at 3, 24, 48, 72 and 96 hours after the start of the exposure. Analytical measurements of test item concentration were made every 24 hours, pre- and post- medium renewal and the end of the exposure by GC-FID analysis. The measured concentrations were determined to be outside the ±20% of initial concentration limits during the test period. Effect concentrations were based on nominal loading rates prepared as WAFs. Probit analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 24h, 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 23.0 – 23.8 °C, pH was 7.57 - 7.88 and air saturation was 73 - 102% indicating that environmental conditions were adequately maintained. Behavioural abnormalities and biological effects were reported during the course of the study. No abnormalities were observed in the control group. The 96h highest nominal loading rate concentration causing no mortality was 7.8 mg/L WAF. The 96h median LC50 was 12.2 (C.I. 11.2 – 13.2) mg/L based on the nominal loading rate concentrations prepared as WAF.

Description of key information

LC50 (fish) =  7.13 (C.I. 11.2 - 13.2) mg/L based on nominal loading rate concentrations prepared as WAF, 96-hour, freshwater, OECD TG 203, 2014

LC0 (fish) = 7.8 mg/L based on nominal loading rate concentrations prepared as WAF, 96-hour, freshwater, OECD TG 203, 2014

Key value for chemical safety assessment

LC50 for freshwater fish:
12.2 mg/L

Additional information

Key study : OECD TG 203, 2014 : 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 Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test (2013) under GLP. Based on the results of range finding tests the nominal concentrations of 0 (control), 4.0 , 4.8, 5.8, 7.0 and 8.5 mg/L (geometric series with a factor of 1.4) were selected for exposure to five fish groups. One control containing no test item was also performed. The test item was renewed at 24 hour intervals within a 96 hour semi-static test system. Observations were made at 3, 24, 48, 72 and 96 hours after the start of the exposure. Analytical measurements of test item concentration were made every 24 hours, pre- and post- medium renewal and the end of the exposure by GC-FID analysis. The measured concentrations were determined to be outside the ±20% of initial concentration limits during the test period. Effect concentrations were based on nominal loading rates prepared as WAFs. Probit analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 24h, 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 23.0 – 23.8 °C, pH was 7.57 - 7.88 and air saturation was 73 - 102% indicating that environmental conditions were adequately maintained. Behavioural abnormalities and biological effects were reported during the course of the study. No abnormalities were observed in the control group. The 96h highest nominal loading rate concentration causing no mortality was 7.8 mg/L WAF. The 96h median LC50 was 12.2 (C.I. 11.2 – 13.2) mg/L based on the nominal loading rate concentrations prepared as WAF.