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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:
15-03-2019 to 21-03-2019
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
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, 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: 0 (Control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L nominal (geometric series factor 1.778) ; with geometric mean measured equivalent concentrations of: 1.17, 1.99, 3.45, 5.83 and 10.8 mg/L, respectively.
- Sampling method: 50 mL extract from test solution. The actual concentrations were measured at 0 h, 96 h, and 24 h, 48 h, 72 h (after and before the renewal)
- Sample storage conditions before analysis: The test solutions of all test groups were filtered through 0.45 μm aqueous phase PES filter membranes (the first 5 mL - 10 mL filtrations were discarded). Filtrations of blank control were analysed directly and the filtrations of test item groups were diluted with acetonitrile for 2-fold before analysis. Samples were analysed immediately or were stored at 2- 8 °C in the dark within 48 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 5400 mL test water was obtained in about 5 L narrow-mouthed bottle and 6.9 μL, 12.4 μL, 22.0 μL, 38.4 μL and 68.6 μL test item was added (calculated with relative density 0.787, the weight of test item was respectively: 5.4 mg, 9.8 mg, 17.3 mg, 30.2 mg and 54.0 mg). The test item could not be directly weighted due to suspected volatility. The test solutions were covered (glass stopper), with stirring for ca. 3 hours. The test solutions with nominal concentration of 1.0 mg/L, 1.8 mg/L, 3.2 mg/L, 5.6 mg/L and 10.0 mg/L were obtained and used in testing directly.
- 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 FDr20180815-2
- 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 26.54 – 29.21 mm (mean = 27.99 +/- 0.98 mm ; meeting the requirement of 20.0±10.0 mm according to guideline) and 0.100 – 0.115 g (mean = 0.107 +/- 0.006 g). In the treated/control groups the loading was 0.138 6 g/L (i.e. < 1.0 g fish/litre).
- Method of breeding: Not reported.
- Feeding during test: No.
- Food type: Feeding was conducted during acclimation (daily - workdays) until 24-hours prior to test initiation.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.

ACCLIMATION
- Acclimation period: 144 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 (work-day); until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported (0% during 7 days pre-test).
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 guidelines.
Hardness:
135 mg/L expressed as CaCO3; Test water (dechlorinated water)
Test temperature:
Temperature 21.9 - 23.7 °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 8.19) as specified in China Guidelines for the testing of Chemicals (2nd Ed.) 203 test.
The pH was in the range of 7.96 - 8.28 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: 74 to 102% ASV during the definitive test.
Nominal and measured concentrations:
- Preliminary range-finding test performed on semi-static test system: 0 (Control), 1.0, 3.0 and 10 mg/L nominal
During the range-finding test, the actual concentration of 1.0 mg/L, 3.0 mg/L and 10 mg/L test groups were analysed during the test period of 0 h and 24 h by HPLC
The cumulative mortalities of test fish in blank control and test item groups up to 10 mg/L respectively were 0%, 0%, 0%,0% and 100%, respectively
- Definitive test performed on semi-static test system with renewal every 24 hour intervals: 0 (Control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L nominal (geometric series factor 1.778) ; with geometric mean measured equivalent concentrations of: 1.17, 1.99, 3.45, 5.83 and 10.8 mg/L, respectively.
During the test, the dissolved test item concentrations were analytically determined in the freshly prepared solution/medium (t=0h, 24h, 48h, 72 h) and in the aged medium/solution before renewal (t=24h*, 48h*, 72h*, 96h*)
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L narrow-mouth bottle with bottle cap
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass. 5400 Litre fill volume (minimum headspace ca. 0 mL); the report specifies 5 L narrow-mouth bottle but the actual volume is probably ca. 5400 mL
- Aeration: Not continuously aerated although DOC > 60% ASV during exposure.
- 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 (Actual: 0.138 6 g/L).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated Tap Water used in the test were purified by the reverse osmosis equipment. (Certified annually).
- Conductivity: Not reported. 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 after renewal

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours photoperiod daily
- Light intensity: 1086 lx and 1165 lx on average

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: 1.778
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study: Yes. 0 (Control), 1.0, 3.0 and 10 mg/L nominal
During the range-finding test, the actual concentration of 1.0 mg/L, 3.0 mg/L and 10 mg/L test groups were analysed during the test period of 0 h and 24 h by HPLC
The cumulative mortalities of test fish in blank control and test item groups up to 10 mg/L respectively were 0%, 0%, 0%,0% and 100%, respectively
- Test concentrations: See above
- 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.:
0.593 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: C.I. 0.874 - 1.64 mg/L
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
2.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
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: 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 26.54 – 29.21 mm (mean = 27.99 +/- 0.98 mm ; meeting the requirement of 20.0±10.0 mm according to guideline) and 0.100 – 0.115 g (mean = 0.107 +/- 0.006 g).
- 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: Speculated to be volatility driven (although test was conducted in a closed semi-static system).
- 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: 250 (C.I. 214 - 291) mg/L
Reported statistics and error estimates:
The LC50 values and the 95% confidence limits (LC5) at 24h, 72h and 96h after the start of the test were calculated by Probit Analysis software (US EPA: Probit Analysis Program Version 1.5). The LC50 value and the 95% confidence limits (L95) at 48h was calculated by Spearman-Karber software (US EPA: Spearman-Karber Program Version 1.5).
Sublethal observations / clinical signs:

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

Recommended observation times

LC50 (mg/L)

95% confidence limits (mg/L)

24 h

3.58

 -

48 h

1.95

1.66 – 2.28

72 h

1.22

0.882 – 1.64

96 h

1.21

0.874 – 1.64

#1: LC50 based on geometric mean measured concentrations

 

Table 2. Cumulative mortality

Cumulative mortality

Nominal Concentration

/ mg/L

Geometric Mean Measured concentration

Exposure duration (h)

3

24

48

72

96

Control

0.00

Control

0.00 mg/L

0%

0%

0%

0%

0%

1.0

1.17

0%

0%

0%

0%

0%

1.8

1.99

0%

0%

0.0%

43%

43%

3.2

3.45

0%

0%

29%

71%

71%

5.6

5.83

0%

0%

100%

100%

100%

10

10.8

0%

100%

100%

100%

100%

 

 

 

 

 

 

 

Table 3. Observed abnormal responses

Visible abnormalities

Nominal Concentration

Exposure duration (h)

3

Symptom / Survivors

24

Symptom / Survivors

48

Symptom / Survivors

72

Symptom / Survivors

96

Symptom / Survivors

Control

0.00

A

0/7

A

0/7

A

0/7

A

0/7

A

 

1.0

A

0/7

A

0/7

A

0/7

A

0/7

A

 

1.8

A

0/7

A

0/7

A

0/7

RA(2)

2/4

AB(4)

4/4

3.2

A

0/7

AB(7)

7/7

AB(5)

5/5

AB(2)

2/2

AB(2)

2/2

5.6

A

0/7

IMM(7)

7/7

-

7/0

-

-

-

-

10

RA(4)

4/7

-

7/0

-

-

-

-

-

-

 

 

 

 

 

 

 

 

 

 

 

A: means no abnormality

RA : means reduction of activity

AB: means swimming at the bottom,

IMM: means immobilized at the bottom

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 1.21 (C.I. 0.874 - 1.64) 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 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), 1.0, 1.8, 3.2, 5.6 and 10 mg/L nominal concentrations (geometric series with a factor of 1.778) were selected for exposure to fish groups of 7 individuals. 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, 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-VWD analysis. Measured concentrations of old solutions could not be maintained within 80% - 120% of the initial concentrations during the relevant exposure period. On this basis effect levels were indicated based on geometric mean measured concentrations. The corresponding geometric mean measured concentrations were: 0 (control), 1.17, 1.99, 3.45, 5.83 and 10.8 mg/L, respectively. During the definitive test the water temperature was in the 21.3 - 23.7 °C, pH was 8 7.96 - 8.28 and air saturation was 74 to 102% ASV 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. All validity criteria were considered to be met. The 96h median LC50 was 1.21 (C.I. 0.874 - 1.64) based on geometric mean measured concentrations.

Description of key information

LC50 (fish) =  1.21 (C.I. 0.874 - 1.64) mg/L based on geometric mean measured concentrations, 96-hour, freshwater, OECD TG 203, 2019

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
1.21 mg/L

Additional information

Key study : OECD TG 203, 2019 : 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), 1.0, 1.8, 3.2, 5.6 and 10 mg/L nominal concentrations (geometric series with a factor of 1.778) were selected for exposure to fish groups of 7 individuals. 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, 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-VWD analysis. Measured concentrations of old solutions could not be maintained within 80% - 120% of the initial concentrations during the relevant exposure period. On this basis effect levels were indicated based on geometric mean measured concentrations. The corresponding geometric mean measured concentrations were: 0 (control), 1.17, 1.99, 3.45, 5.83 and 10.8 mg/L, respectively. During the definitive test the water temperature was in the 21.3 - 23.7 °C, pH was 8 7.96 - 8.28 and air saturation was 74 to 102% ASV 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. All validity criteria were considered to be met. The 96h median LC50 was 1.21 (C.I. 0.874 - 1.64) based on geometric mean measured concentrations.