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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:
03-04-2020 to 11-04-2020
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 203 Fish Acute Toxicity Test, China Environmental Press, China (2013)
Version / remarks:
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 and/or 2013 second edition of the aforementioned guideline. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L nominal concentrations (with geometric series factor 1.5)
with equivalent arithmetic mean measured concentrations of : 0 ( control), 3.8, 5.7, 8.4, 12 and 16 mg/L
The mean measured concentrations of test item in the old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations
- Sampling method: ca. 5 mL (n=3) extracts from test solution (three replicate samples, one sample pre-treated and stored refrigerated in case of further analysis being required)
- Sample storage conditions before analysis: The samples were analysed immediately. Following pre-treatment and/or sample preparation. one sample pre-treated and stored refrigerated in case of further analysis being required.
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 (5L) volume test water, to obtain nominal concentrations of 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L. The solution was transferred into a flask and then the suspension was stirred for 2 hours. Semi-static exposure procedure was used, and the test solution was renewed every 24 hours. The containers were kept tight during exposure.
- 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 or Danio rerio)
- Strain: Not reported. Batch generation number C20190715
- 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): The length of fish (randomly selected for measurement at test start from all groups) was in the range of 1.0 - 3.0 cm at test initiation : actual 1.8 to 2.2 cm (mean = 2.0 cm SD = 0.13) (i.e. 2.0 +/- 1.0 cm according to guideline) ; Body weight (wet weight) 209 to 246 mg (mean = 228 mg SD = 12). In the treated/control groups the loading was ca. 0.45 g/L (i.e. < 1.0 g fish/litre) based on the above information.
- 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 (12 hours light; 12 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
Remarks on exposure duration:
In accordance with the OECD TG 203 guideline.
Hardness:
170 mg/L expressed as CaCO3 at test start and maintained at 10 - 250 mgCaCO3/L during the test; Test water (dechlorinated water)
Test temperature:
Temperature 22.0 - 23.4 °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 as specified in China Guidelines for the testing of Chemicals 203 test.
The pH was in the range of 7.42 - 8.08 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: 82.1 to 97.2% 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 static test system:
0 (control), 1.0 and 5.0 mg/L nominal concentrations.
- Definitive (semi-static test system): 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L nominal concentrations (with geometric series factor 1.5)
with equivalent arithmetic mean measured concentrations of : 0 ( control), 3.8, 5.7, 8.4, 12 and 16 mg/L
The mean measured concentrations of test item in the old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: 5 L glass
- Type (delete if not applicable): glass flask (closed)
- Material, size, headspace, fill volume: glass. ca. 5000 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: 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 (Actual: ca. 0.45 to 0.60 g/L based on information in the study)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water 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: 12 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: 1.5
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study: 0 (control), 1.0 and 5.0 mg/L nominal concentrations in static-system utilised. 0% mortality was seen at up to 5.0 mg/L nominal, with sublethal effect – lethargy seen.
- Test concentrations: Definitive Test: 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L nominal concentrations (with geometric series factor 1.5) in semi-static system
with equivalent arithmetic mean measured concentrations of : 0 ( control), 3.8, 5.7, 8.4, 12 and 16 mg/L
The mean measured concentrations of test item in the definitive test old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations
- Results used to determine the conditions for the definitive study: Yes..
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
16.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: C.I. - mg/L
Remarks:
Highest concentration causing no mortality and the lowest concentration producing 100% mortality should be used as an approximation for the LC50 (this being considered the geometric mean of these two concentrations): 20 mg/L and 13 mg/L respectively.
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 up to 4.0 mg/L. See tables for further information on sub-lethal responses in other test groups.
- Observations on body length and weight: Not reported.
- 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 up to 4.0 mg/L. See tables for further information on sub-lethal responses in other 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 (previously conducted sensitivity check - documented in the full study report)
- 24h LC50: 372 (C.I.331 - 419 ) mg/L ; The 24h-LC50 was within the range of 200-400 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).
However, at the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the nominal concentrations of 4.0 mg/L, 6.0 mg/L, 8.9 mg/L, 13 mg/L and 20 mg/L was 0%, 0%, 0%, 0% and 100%, respectively. Therefore as mentioned by OECD 203 (1992) section 22. the data obtained are inadequate for the use of standard methods of calculating the LC50, the highest concentration causing no mortality and the lowest concentration producing 100% mortality should be used as an approximation for the LC50 (this being considered the geometric mean of these two concentrations).

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

Recommended observation times

LC50 (mg/L)

95% confidence limits (mg/L)

24 h

> 16.12

-

48 h

> 16.12

-

72 h

> 16.12

-

96 h

16.12

-

#1: LC50 based on analytically confirmed measured concentrations

#2: At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the nominal concentrations of 4.0 mg/L, 6.0 mg/L, 8.9 mg/L, 13 mg/L and 20 mg/L was 0%, 0%, 0%, 0% and 100%, respectively. As mentioned by in paragraph 22. OECD 203 (1992) the data obtained are inadequate for the use of standard methods of calculating the LC50, the highest concentration causing no mortality and the lowest concentration producing 100% mortality should be used as an approximation for the LC50 (this being considered the geometric mean of these two concentrations).

#3:The mean measured concentrations of test item in the old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations

 

Table 2. Cumulative mortality

Cumulative mortality

Nominal Concentration

/ mg/L

Arithmetic Mean Measured concentration

/ mg/L

Exposure duration (h)

3

24

48

72

96

Control

0.00

Control

0.00

0%

0%

0%

0%

0%

4.0

3.8

0%

0%

0%

0%

0%

6.0

5.7

0%

0%

0%

0%

0%

8.9

8.4

0%

0%

0%

0%

0%

13.0

12

0%

0%

0%

0%

0%

20.0

16

0%

10%

10%

10%

100%

 

 

 

 

 

 

 

Table 3. Observed abnormal responses

Visible abnormalities

Nominal Concentration

/ mg/L

Exposure duration (h)

3

Mortalities

24

Mortalities

48

Mortalities

72

Mortalities

96

Mortalities

Control

0.00

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

4.0

N

0/10

N

0/10

N

0/10

N

0/10

N

0/10

6.0

N

0/10

N

0/10

N

0/10

N

0/10

1A

0/10

8.9

N

0/10

1A

0/10

3A,1L

0/10

4A,2L

0/10

5A,5L

0/10

13.0

N

0/10

2A,3L

0/10

10L

0/10

10L

0/10

10L

0/10

20.0

2A,2L

0/10

-

1/10

-

1/10

-

1/10

-

10/10

 

 

 

 

 

 

 

 

 

 

 

Note:

A: means erratic swimming

L: means lay to one side at the bottom of container

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 16.12 (C.I. -) mg/L based on analytically confirmed nominal 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 (2013) under GLP. Based on the results of preliminary range finding tests, the nominal concentrations of 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L (with geometric series factor 1.5) in semi-static system with equivalent arithmetic mean measured concentrations of : 0 (control), 3.8, 5.7, 8.4, 12 and 16 mg/L were selected for exposure to 10 fish in each single replicate at each concentration. 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 12 hour photoperiod at 22.0 - 23.4°C (23°C ; constant to within 2°C) and pH 7.42 - 8.08. Dissolved oxygen was no less than 60% of ASV (actual: 82.1 to 97.2%). 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. At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the nominal concentrations of 4.0 mg/L, 6.0 mg/L, 8.9 mg/L, 13 mg/L and 20 mg/L was 0%, 0%, 0%, 0% and 100%, respectively. The abnormal behaviours were (n=2) erratic swimming and (n=2) laying to one side at the bottom of container. No abnormal behaviour responses were seen at 4.0 mg/L nominal concentration. The mean measured concentrations of test item in the definitive test old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations. As mentioned by in paragraph 22. OECD 203 (1992) the data obtained are inadequate for the use of standard methods of calculating the LC50, the highest concentration causing no mortality and the lowest concentration producing 100% mortality should be used as an approximation for the LC50 (this being considered the geometric mean of these two concentrations). Under the conditions of this study, the 96h LC50 was 16.12 (C.I. -) mg/L based on analytically confirmed nominal concentrations.

Description of key information

96h-LC50 (fish) = 16.12 (C.I. –) mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2020

Key value for chemical safety assessment

LC50 for freshwater fish:
16.12 mg/L

Additional information

Key study : OECD TG 203, 2020 : 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 (2013) under GLP. Based on the results of preliminary range finding tests, the nominal concentrations of 0 (control), 4.0, 6.0, 8.9, 13 and 20 mg/L (with geometric series factor 1.5) in semi-static system with equivalent arithmetic mean measured concentrations of : 0 (control), 3.8, 5.7, 8.4, 12 and 16 mg/L were selected for exposure to 10 fish in each single replicate at each concentration. 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 12 hour photoperiod at 22.0 - 23.4°C (23°C ; constant to within 2°C) and pH 7.42 - 8.08. Dissolved oxygen was no less than 60% of ASV (actual: 82.1 to 97.2%). 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. At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the nominal concentrations of 4.0 mg/L, 6.0 mg/L, 8.9 mg/L, 13 mg/L and 20 mg/L was 0%, 0%, 0%, 0% and 100%, respectively. The abnormal behaviours were (n=2) erratic swimming and (n=2) laying to one side at the bottom of container. No abnormal behaviour responses were seen at 4.0 mg/L nominal concentration. The mean measured concentrations of test item in the definitive test old solutions were in the range of 93%-109% of those in the fresh solutions at renewal. Therefore, the effect concentration are based on the analytically confirmed nominal concentrations. As mentioned by in paragraph 22. OECD 203 (1992) the data obtained are inadequate for the use of standard methods of calculating the LC50, the highest concentration causing no mortality and the lowest concentration producing 100% mortality should be used as an approximation for the LC50 (this being considered the geometric mean of these two concentrations). Under the conditions of this study, the 96h LC50 was 16.12 (C.I. -) mg/L based on analytically confirmed nominal concentrations.