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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-07-08 to 2018-07-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 102Z4
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 20.0, 28.3, 40.0, 56.6, 80.0 mg/L
- Sampling method: measured at 0, 48, 96 hours
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Weigh 1.0003 and 1.00001 g test item and add into 10 L test water. The solution was stirred with magnetic stirrer using a stirring rate at 410 and 420 rpm for about 24 hours. The solution was filtrated by 0.45 μm filter and used for diluting to the test solutions. 0.80, 1.13, 1.60, 2.26 and 3.20 L was diluted to 4 L with test water to get the solutions of 20.0, 28.3, 40.0, 56.6 and 80.0 mg/L.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: The fish were reproduced in this facility, and their parents' origin were from Hangzhou Hunter Biotechnology Inc.
- Weight at study initiation: 0.207 - 0.246 g
- Length at study initiation: 2.0 - 2.3 cm

ACCLIMATION
- Acclimation conditions (same as test or not): not same
- Light condition: 12 hours light, 12 hours darkness (the light is cool white)
- Feeding frequency during acclimation: Twice on weekday and one time on weekend until 24 hours before the test was started
- Oxygen concentration: at least 80% of air saturation value
- Health during acclimation (any mortality observed): there were no any sign of disease or mortalities

FEEDING DURING TEST
- Feeding of test fish: not fed during the period of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
160 mg/L (as CaCO3)
Test temperature:
22.8 - 23.3 °C
pH:
7.47 - 8.33
Dissolved oxygen:
78.5 - 94.2 mg/L
Nominal and measured concentrations:
Norminal: 20.0, 28.3, 40.0, 56.6, 80.0 mg/L
Measured: 9.59, 14.1, 19.2, 29.8, 47.9 mg/L (overall mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Rectangle glass tanks (Iength: 30 cm, width: 20 cm, height:30 cm)
- Test solution volume: 4 L
- Aeration: before usage, the water was aerated for more than 24 hours
- Renewal rate of test solution (frequency/flow rate): 48 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Loading: criterion: < 1.0 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the water treated with Aquapro Pure Water System
- Total organic carbon: < 2 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 12 hours light: 12 hours darkness
- Test temperature: 23 ± 2 °C
- Dissolved oxygen concentration (target): not less than 60% of air saturation value (ASV) during the test period without aeration

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 20, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: During the period of 96 hours, no death and any visible toxic signs were observed at group of 20 mg/L. Four fish died at test group of 50 mg/L and all fish died at group of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
24.29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 20.02-29.97 mg/L
Details on results:
- Mortality: dead fish was observed in the vessels of 40.0, 56.6 and 80.0 mg/L
- Mortality of control: 0
- Other adverse effects control: no visible toxic sign observed
- Abnormal responses: The sign of lethargy was observed in the vessels of 40.0, 56.6 and 80.0 mg/L
Results with reference substance (positive control):
- LC50: 318.35 mg/L (95% confidence limits: 252.52 mg/L - 401.33 mg/L)
Validity criteria fulfilled:
yes
Conclusions:
The 96-hour LC50 of test item to zebra fish was 24.49 mg/L, with 95% confidence limit of 20.02-29.97 mg/L.
Executive summary:

The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item in zebra fish according to OECD Guideline 203.

According to the preliminary test results, the definitive test was conducted with 5 test item concentrations of 20.0, 28.3, 40.0, 56.6 and 80.0 mg/L (nominal concentration). A concurrent blank control was performed in parallel. 7 fish were used in each of the test groups and the control group.

 

During the 96-hour exposure period, there was no death and visible toxic sign observed in the vessels of control group and test group of 20.0 and 28.3 mg/L. The sign of lethargy was observed in the vessels of 40.0, 56.6 and 80.0 mg/L, and dead fish was observed in the vessels of 40.0, 56.6 and 80.0 mg/L.

For the test media of 20.0,28.3,40.0,56.6 and 80.0 mg/L,the mean measured concentration of fresh solution was 9.73, 13.6, 18.9, 28.4and 46.2 mg/L, and the mean measured concentration of old solution was 9.44, 14.6, 19.6, 31.1 and 53.2 mg/L. And the mean concentration was 9.59, 14.1, 19.2, 29.8 and 47.9 mg/L, respectively.

 

Basing on the measured concentration and test results, the 96-hour LC50 of test item to zebra fish was 24.49 mg/L, with 95% confidence limit of 20.02-29.97 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2017-11-06 to 2017-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 101Z4
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 66.7, 44.4, 29.6, 19.8% as stock solution content
- Sampling method: the test solution for measurement was taken out from test level (at the start of exposure and every 24 hours)

Test item in test solution:
- Sampling method: the test solution for analysis was taken out from the middle layer of the test vessel (at the start and end of exposure)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test sample and dilution water were mixed to produce the nominal concentration of 100 mg/L, and they were stirred for 48 hours. After stirred for 48 hours, the solution was filtered with a glass fiber filter to prepare the stock solution. The test solutions were prepared by using the stock solution without treatment or by appropriately diluting the stock solution with dilution water.
- Controls: Dilution water without the test item
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Strain: Medaka (Oryzias latipes)
- Source: CERI Kurume (in-laboratory production)
- Age at study initiation (mean and range, SD): five-month-old
- Length at study initiation (length definition, mean, range and SD): total length 2.3 ± 1.2 cm

ACCLIMATION
- Acclimation period: 25 days
- Acclimation conditions:
temperature: 24 ± 1 °C
lighting condition: room light, 16-hour light / 8-hour dark
dissolved oxygen concentration: at least 80% of air saturation value
- Type and amount of food during acclimation: feed of Medaka fry for education (KYORIN), amount corresponding to 3 % of body weight
- Feeding frequency during acclimation: every day
- Feed withdrawal: 24 hours before the start of exposure
- Health during acclimation (any mortality observed): mortality of test groups: 0.4 % (during7 days before the start of exposure)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
32 mg/L (Ca, Mg)
Test temperature:
23.2 - 24.0 °C
pH:
7.7 - 7.9
Dissolved oxygen:
8.3 - 8.7 mg/L
Nominal and measured concentrations:
Nominal: 100, 66.7, 44.4, 29.6, 19.8% as stock solution content
Measured: 74.5, 50.6, 32.1, 21.4, 14.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 3 L Glass tank (diameter: 16 cm, depth: 16cm)
- Aeration: conducted gently
- No. of organisms per vessel: 7 fish / test level
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: < 0.5 mg/L
- Alkalinity: 36 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: room light, 16-hour light / 8-hour dark

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study
- Test concentrations: 10.0, 30.0, 60.0, 100% stock solution content
- Results used to determine the conditions for the definitive study: all test fish died for 100% stock solution
Reference substance (positive control):
yes
Remarks:
Copper (II) sulfate pentahydrate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
70.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 56.0-256 mg/L
Details on results:
- Observations on body length and weight: total length: 2.5 ± 0.1 cm; body weight: 0.15 ± 0.02 g
- Mortality: Minimum concentration causing 100% mortality at 96 hours was estimated as >74.5 mg/L since the mortality 100% was not observed. Minimum concentration causing no mortality at 96 hours was 32.1 mg/L.
- Mortality of control: 0
- Other adverse effects control: no abnormal response was observed
- Abnormal responses: at the surface, complete loss of equilibrium and reduced activity
- Observation and measurement of test solution: The measured concentrations of the test item in the test solutions at start of exposure were 14.0-73.3 mg/L, and those at the end of exposure were 15.0-75.7 mg/L which was 102-108% of the concentration at the start of exposure. The measured concentrations of the test items were kept with ± 20% of the measured concentration at the start of exposure.
Results with reference substance (positive control):
The 96-hour LC50 of the reference substance was 0.33 mg/L. This value was within the stipulated range (mean ± 2S.D.) [mean ± S.D.: 0.52 ± 0.19 mg/L (n=61)] to background data in this laboratory.
Validity criteria fulfilled:
yes
Conclusions:
The 96-hour LC50 of the test item for Medaka was 70.3 mg/L (95% confidence limits: 56.0-256 mg/L).
Executive summary:

The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item in Medaka according to OECD Guideline 203.

Five test concentrations of 100, 66.7, 44.4, 29.6, 19.8% as stock solution content (a geometric series with a factor of 1.5) (74.5, 50.6, 32.1, 21.4, 14.5 mg/L as measured concentrations) and a control were prepared. Seven fish each test level were exposure to the test solutions for 96 hours.

 

Minimum concentration causing 100% mortality at 96 hours was estimated as >74.5 mg/L since the mortality 100% was not observed. Minimum concentration causing no mortality at 96 hours was 32.1 mg/L. Number of dead fish in the control at the end of exposure was 0.

No abnormal response was observed in the control. Observed abnormal responses during exposure were at the surface, complete loss of equilibrium and reduced activity.

The solubility in dilution water was 78.2 mg/L (measured value in preliminary study). The measured concentrations of the test item in the test solutions at start of exposure were 14.0-73.3 mg/L, and those at the end of exposure were 15.0-75.7 mg/L which was 102-108% of the concentration at the start of exposure. The measured concentrations of the test items were kept with ± 20% of the measured concentration at the start of exposure.

The 96-hour LC50 of the test item for Medaka was 70.3 mg/L (95% confidence limits: 56.0-256 mg/L).

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2017-11-13 to 2017-11-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Japanese Industry Standards (JIS) K 0102-2016 Section 71
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 101Z4
Analytical monitoring:
not specified
Vehicle:
yes
Remarks:
N,N-dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test sample (200 mg) and HCO-40 (2.0 g) were dissolved in N,N-dimethylformamide to prepare 10.0 g/L stock solution (20 mL). This stock solution was diluted appropriately with the dilution water to prepare the test solution.
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Source: Kitamura fish farm
- Length at study initiation: 0.20 - 0.23 g
- Weight at study initiation: 2.8 - 3.0 cm
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
16 mg/L (Ca, Mg)
Test temperature:
25.0 °C
pH:
7.5 - 8.2
Dissolved oxygen:
7.8 - 7.9 mg/L
Nominal and measured concentrations:
Norminal: 10.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Round glass vessel
- Volume of test water: 4 L/level
- Aeration: the tank was supplied with air during exposure.
- Renewal rate of test solution (frequency/flow rate): at every 24 hours
- No. of organisms: 10 fish / level

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from the premises of CERI Kurume
- Total organic carbon: 0.7 mg/L
- Alkalinity: 110 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark (artificial light of white fluorescent lamp)
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Adverse effects control: No diseases were observed in control.
Results with reference substance (positive control):
The 48 - hour LC50 value for the fish of the same lot was 10900 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The 96-hour LC50 of test item was more than 10.0 mg/L.
Executive summary:

The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item in Oryzias latipes according to Japanese Industry Standards (JIS) K 0102-2016 Section 71.

The test was conducted with test item concentrations of 10.0 mg/L (nominal concentration). The test sample (200 mg) and HCO-40 (2.0 g) were dissolved in N, N-dimethylformamide to prepare 10.0 g/L stock solution (20 mL). This stock solution was diluted appropriately with the dilution water to prepare the test solution.

No diseases were observed in control.

The 96-hour LC50 of test item was more than 10.0 mg/L.

Description of key information

For this endpoint three studies are available.

In a study to OECD Guideline 203, which was determined in 2017, it was found that the 96-hour LC50 of the test item for Medaka was 70.3 mg/L (95% confidence limits: 56.0-256 mg/L).

In a study to OECD Guideline 203, which was determined in 2018, it was found that the 96-hour LC50 of test item to zebra fish was 24.49 mg/L, with 95% confidence limit of 20.02-29.97 mg/L.

In a study to Japanese Industry Standards (JIS) K 0102-2016 Section 71, which was determined in 2018 and included under the bioaccumulation test, it was found that the 96-hour LC50 of test item to Oryzias latipes was more than 10.0 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
10 mg/L

Additional information

Key study: The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item in zebra fish according to OECD Guideline 203.

According to the preliminary test results, the definitive test was conductedwith 5 test item concentrations of 20.0, 28.3, 40.0, 56.6 and 80.0 mg/L (nominalconcentration). A concurrent blank control was performed in parallel.7 fish were used in each of the test groups and the control group.

During the 96-hour exposure period, there was no death and visible toxic sign observed in the vessels of control group and test group of 20.0 and 28.3 mg/L. The sign of lethargy was observed in the vessels of 40.0, 56.6 and 80.0 mg/L, and dead fish was observed in the vessels of 40.0, 56.6 and 80.0 mg/L.

For the test media of 20.0,28.3,40.0,56.6 and 80.0 mg/L,the mean measured concentration of fresh solution was 9.73, 13.6, 18.9,28.4and 46.2 mg/L,and the mean measured concentration of old solution was 9.44,14.6, 19.6, 31.1 and 53.2 mg/L. And the mean concentration was 9.59, 14.1,19.2,29.8 and 47.9 mg/L,respectively.

Basing on the measured concentration and test results, the 96-hour LC50 of test item to zebra fish was 24.49 mg/L, with 95% confidence limit of 20.02-29.97 mg/L.

Support study (2017): The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item in Medaka according to OECD Guideline 203.

Five test concentrations of 100, 66.7, 44.4, 29.6, 19.8% as stock solution content (a geometric series with a factor of 1.5) (74.5, 50.6, 32.1, 21.4, 14.5 mg/L as measured concentrations) and a control were prepared. Seven fish each test level were exposure to the test solutions for 96 hours.

Minimum concentration causing 100% mortality at 96 hours was estimated as >74.5 mg/L since the mortality 100% was not observed. Minimum concentration causing no mortality at 96 hours was 32.1 mg/L. Number of dead fish in the control at the end of exposure was 0.

No abnormal response was observed in the control. Observed abnormal responses during exposure were at the surface, complete loss of equilibrium and reduced activity.

The solubility in dilution water was 78.2 mg/L (measured value in preliminary study). The measured concentrations of the test item in the test solutions at start of exposure were 14.0-73.3 mg/L, and those at the end of exposure were 15.0-75.7 mg/L which was 102-108% of the concentration at the start of exposure. The measured concentrations of the test items were kept with ± 20% of the measured concentration at the start of exposure.

The 96-hour LC50 of the test item for Medaka was 70.3 mg/L (95% confidence limits: 56.0-256 mg/L).

Support study (2018): The objective of this study is to estimate the 96-hour median lethal concentration (LC50) by conducting an acute toxicity of the test item inOryzias latipesaccording toJapanese Industry Standards (JIS) K 0102-2016 Section 71.

The test was conductedwith test item concentrations of 10.0 mg/L (nominalconcentration).The test sample (200 mg) and HCO-40 (2.0 g) were dissolved in N, N-dimethylformamide to prepare 10.0 g/L stock solution (20 mL). This stock solution was diluted appropriately with the dilution water to prepare the test solution.

No diseases were observed in control.