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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:
2016
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: Nominal: 4.0 mg/L, 4.8 mg/L, 5.8 mg/L, 7.0 mg/L and 8.5 mg/L (geometric series factor 1.2); nominal concentrations were analytically confirmed during the study.
- Sampling method: 20 ml extract from test solution.
- Sample storage conditions before analysis: Actual concentrations of test substance in were immediately measured.
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 FZA000-20160507-03
- 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 2.798 - 2.911 cm (meeting the requirement of 2.0±1.0 cm according to guideline) and 0.235 - 2.55g (loading was 0.434 g/L in the blank control). In the treated groups the minimum and maximum loading was 0.405 - 0.434 g/L respectively (i.e. < 1.0g fish/litre).
- Method of breeding: Not reported.
- 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.

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. pH was marginally outside the exposure duration range: acclimatisation: pH 7.31-7.58 and exposure 7.11-7.54; which is considered to have no impact on the study.
- Type and amount of food: Not reported.
- Feeding frequency: At twice daily until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
75 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 23.0 - 23.9 °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 7.11 to 7.54 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: 96.2 - 98.7% ASV during acclimatisation and 72.8 to 102.3% during the definitive test.
Nominal and measured concentrations:
- Preliminary range-finding test performed on semi-static test system with renewal every 24 hour intervals:
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 no mortality observed at the nominal concentration of 5 mg/L and 100% mortality was observed at the nominal concentration of 10 mg/L within the test period of 96 hours.
- Definitive test performed on semi-static test system with renewal every 24 hour intervals:
Nominal concentrations: 0 (blank control), 4.0, 4.8, 5.8, 7.0 and 8.5 mg/L (geometric series with a factor of 1.2)
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 packers 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: 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 (0.405 - 0.434 g/L minimum and maximum, respectively)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used in the test were purified by the reverse osmosis equipment. (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 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: 1.2
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study
- Test concentrations: Not reported (no mortality at < 5 mg/L 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.:
4.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: nominal - analytically confirmed concentration
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.128 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: C.I. 6.346 - 8.295 mg/L; nominal - analytically confirmed concentration
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 8.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: nominal - analytically confirmed concentration
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: Body weight and length were recorded at the end of the observation period for all groups. 2.392-2.979 cm, meeting the requirement of 2.0±1.0 cm within the guidelines.
- 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: 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: 189.258 mg/L

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

Recommended observation times

LC50 (mg/L)

95% confidence limits (mg/L)

24 h

> 8.5

-

48 h

8.536

7.433 - 30.477

72 h

7.128

6.346 - 8.295

96 h

7.128

6.346 - 8.295

#1: LC50 based on nominal concentrations; analytically confirmed concentrations

 

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%

4.0 mg/L

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

4.8 mg/L

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

5.8 mg/L

0.0%

0.0%

0.0%

0.0%

14.3%

14.3%

7.0 mg/L

0.0%

0.0%

0.0%

28.6%

42.9%

42.9%

8.5 mg/L

0.0%

0.0%

0.0%

42.9%

85.7%

85.7%

#1: Group is control or nominal concentration group.

 

Table 3. Observed abnormal responses

Visible abnormalities

Group

Exposure duration (hours)

3

6

24

48

72

96

Control

A

A

A

A

A

A

Treated 1

A

A

A

A

A

A

Treated 2

A

A

F

F

F

F

Treated 3

A

A

F I

F I

F I

F I

Treated 4

F

F

D F I

D F

D F

D F

Treated 5

F

D F

D F E

D F I

D

D

Note: A: means no abnormality; D: means rollover; E: means loss balance; F: means diminished swimming capacity; I: means abnormal static.

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 7.13 mg/L with a 95% confidence interval from 6.35 - 8.30 mg/L based on nominal concentrations that were analytically confirmed.
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 1.2) were selected for exposure to 7 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 in response to ensuring adequate stability of exposure to test item. Observations were made at 3, 6, 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 HPLC analysis. The measured concentrations were determined to remain within the (±20%) limit range of 83.33 – 107.18% during the test period which were within the guideline limit range. Therefore effect concentrations were based on nominal concentrations that are analytically confirmed. Probit analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 23.0 – 23.9 °C, pH was 7.11 – 7.54 and air saturation was 72.8 – 102.3% 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 concentration causing no mortality was 4.8 mg/L. The 96h median LC50 was 7.13 (C.I. 6.35 – 8.30) mg/L based on the nominal concentrations that were analytically confirmed.

Description of key information

LC50 (fish) =  7.13 (C.I. 6.35 - 8.30) mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2016

LC0 (fish) = 4.8 mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2016

Key value for chemical safety assessment

LC50 for freshwater fish:
7.13 mg/L

Additional information

Key study: OECD TG 203, 2016 : 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 1.2) were selected for exposure to 7 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 in response to ensuring adequate stability of exposure to test item. Observations were made at 3, 6, 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 HPLC analysis. The measured concentrations were determined to remain within the (±20%) limit range of 83.33 – 107.18% during the test period which were within the guideline limit range. Therefore effect concentrations were based on nominal concentrations that are analytically confirmed. Probit analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 23.0 – 23.9 °C, pH was 7.11 – 7.54 and air saturation was 72.8 – 102.3% 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 concentration causing no mortality was 4.8 mg/L. The 96h median LC50 was 7.13 (C.I. 6.35 – 8.30) mg/L based on the nominal concentrations that were analytically confirmed.