Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

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:
21-11-2018 to 27-11-2018
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, 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: 10%, 17.8%, 31.6%, 56.2% and 100% LS (limit solution) 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.
- Sample storage conditions before analysis: 50mL test solution were extracted three times with ethyl acetate (15 mL per extraction). Extracts were collected into 50 mL volumetric flasks and made up to volume with ethyl acetate. Then filtered through a 45 µm nylon filter membrane. With discarding of the first 5 to 10 mL filtrate. The samples were analysed immediately, otherwise the sample were pretreated as above and stored at 2°C to 8°C, protected from light within 96 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.538 g test item was weighed into a flask and dissolved with test water, transferred into a 5 L- conical flask and diluted with test water to 5000 mL . After being sealed the test suspension was stirred on a magnetic stirrer for about 24 h (the depth of vortex was maintained at one third of the test solution height). After stirring, the suspension was filtered through 0.45 µm aqueous phase PES filter membrane(s) (pre-saturated with a small amount of test solution before filtration), then the nominal concentration of 108 mg/L (the nominal concentration was 100 mg/L, calculated based on the purity of the test item) saturated solution was obtained and abbreviated as 100%LS. Three replicates of stock solutions were prepared with the above-mentioned method and mixed. Subsequently, 600 mL, 1068 mL, 1896 mL, 3372mL and 6000 mL test stock solution were transferred into 5 L- vessels and mixed with 5400 mL, 4932 mL, 4104 mL, 2628 mL and 0 mL test water respectively, concentrations of 10.0%LS, 17.8%LS, 31.6%LS, 56.2%LS and 100%LS test solutions were obtained
- 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 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 22.00 – 24.10 mm (mean = 23.38 +/- 0.71 mm ; meeting the requirement of 20.0±10.0 3m according to guideline) and 0.064 – 0.095 g (mean = 0.082 +/- 0.01 g). In the treated/control groups the loading was 0.096 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: 22 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).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
145 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 8.10) as specified in China Guidelines for the testing of Chemicals (2nd Ed.) 203 test.
The pH was in the range of 8.00-8.39 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: 80 to 98% ASV during the definitive test.
Nominal and measured concentrations:
- Preliminary range-finding test performed on static test system:
Three concentrations of 3.05% LS (LS=Limit Solution), 9.77%LS, 31.2%LS and 100%LS, respectively and a blank control group
The cumulative mortalities of test fish in blank control and 3.05% LS, 9.77% LS, 31.2% LS and 100% LS test groups were 0%, 0%, 0%,0% and 100%, respectively
- Definitive test performed on semi-static test system with renewal every 24 hour intervals:
10%, 17.8%, 31.6%, 56.2% and 100% LS (limit solution) 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 material concentration analytically were 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 wide-mouth jar with bottle cap
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass. 5 Litre fill volume (minimum headspace ca. 200 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: 0.096 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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: 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: 1.778
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study: Three concentrations of 3.05% LS (LS=Limit Solution), 9.77%LS, 31.2%LS and 100%LS, respectively and a blank control group in a static exposure.
- Test concentrations: The cumulative mortalities of test fish in blank control and 3.05% LS, 9.77% LS, 31.2% LS and 100% LS test groups were 0%, 0%, 0%,0% and 100%, respectively
- Results used to determine the conditions for the definitive study: Yes. The cumulative mortalities of test fish in blank control and 3.05% LS, 9.77% LS, 31.2% LS and 100% LS test groups were 0%, 0%, 0%,0% and 100%, respectively
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
<= 1.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: C.I. - mg/L
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
3.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
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 22.00 – 24.10 mm (mean = 23.38 +/- 0.71 mm ; meeting the requirement of 20.0±10.0 3m according to guideline) and 0.064 – 0.095 g (mean = 0.082 +/- 0.01 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: None reported.
- 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: Trimmed Spearman-Karber Program Version 1.5).

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

Recommended observation times

LC50 (mg/L) based pn geometric mean measured concentrations

95% confidence limits (mg/L)

96 h

2.62

-

#1: LC50 based on geometric mean measured 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%

10%LS

[1.17 mg/L]

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

17.8%LS

[1.99 mg/L]

0.0%

0.0%

0.0%

0.0%

0.0%

0.0%

31.6%LS

[3.45 mg/L]

0.0%

100.0%

100.0%

100.0%

100.0%

100.0%

56.2%LS

[5.83 mg/L]

100.0%

100.0%

100.0%

100.0%

100.0%

100.0%

100%LS

[10.8 mg/L]

100.0%

100.0%

100.0%

100.0%

100.0%

100.0%

#1: geometric mean measured concentrations.

 

Table 3. Observed abnormal responses

 

Observations

Concentration

(geometric mean measured)

3h

 

24h

 

48h

 

72h

 

96h

 

Control

0/7

N

0/7

N

0/7

N

0/7

N

0/7

N

1.17 mg/L

0/7

N

0/7

N

0/7

N

0/7

N

0/7

N

1.99 mg/L

0/7

N

3/7

RA(3)

7/7

RA(7)

7/7

RA(6)

OLE(1)

7/7

RA(4)

AS(3)

3.45 mg/L

7/7

OLE (7)

-

-

-

-

-

-

-

-

5.83 mg/L

-

-

-

-

-

-

-

-

-

-

10.8 mg/L

-

-

-

-

-

-

-

-

-

-

 

 

 

 

 

 

 

 

 

 

 

Note: "N": Normal. "-": All test fish died. "RA": Reduction of activity; "AS": swimming at the surface; "OLE": overturn and loss of equilibrium.

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 2.62 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),10%, 17.8%, 31.6%, 56.2% and 100% LS (limit solution)(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 GC-FID analysis. Measured concentrations of old solutions could be maintained within 80% - 120% of the initial concentrations during the 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 - 23.0 °C, pH was 8.00 to 8.39 and air saturation was 80 to 98% 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 2.62 mg/L (C.I. could not be determined) based on geometric mean measured concentrations.

Description of key information

LC50 (fish) =  2.62 (C.I. - ) mg/L based on geometric mean measured concentrations, 96-hour, freshwater, OECD TG 203, 2019

Key value for chemical safety assessment

LC50 for freshwater fish:
2.36 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),10%, 17.8%, 31.6%, 56.2% and 100% LS (limit solution)(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 GC-FID analysis. Measured concentrations of old solutions could be maintained within 80% - 120% of the initial concentrations during the 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 - 23.0 °C, pH was 8.00 to 8.39 and air saturation was 80 to 98% 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 2.62 mg/L (C.I. could not be determined) based on geometric mean measured concentrations.