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Long-term toxicity to fish

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Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-18 to 2018-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: 20170214
Purity: >99%
Analytical monitoring:
yes
Details on sampling:
Samples were taken for analysis from the freshly prepared solutions and the old solution at least twice a week.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: Propagated in the test facility. Their parental generation were introduced from Hangzhou Hunter Biotechnology Inc.
- Weight at study initiation (mean and range, SD): 0.050 ~ 0.100 g
For the whole batch of fish used in the test, the range in individual weights at the start of the test should ideally be kept to within ±10% of the arithmetic mean weight and, in any case, should not exceed 25%. In this test, the individual weights were -10.2% ~ 12.9% of the arithmetic mean weight.

FEEDING DURING TEST
- Food type: Brine shrimp
- Amount: A rate of 2% ~ 4% of their body weight per day. The concentration of the brine shrimp dried weight was 0.0716g/ml, and 0.480 ml condensed brine shrimp was feed per day according to the initial fish weight.
- Frequency: The daily ration may be once a day or divided into two equal portions and given to the fish in two feeds per day, separated by at least 5 hours
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Hardness:
170 mg/L ~ 200 mg/L (as CaCO3)
Test temperature:
21.8 ~ 22.9 °C
pH:
7.74 ~ 8.19
Dissolved oxygen:
74.2 ~ 94.0 %ASV
Nominal and measured concentrations:
Norminal: 2.5, 5.0 and 10 mg/L
Measured: 0.00291, 0.00678 and 0.0144 mg/L (The overall arithmetic mean determined concentration of each batch geometrical mean determined concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L cylinder glass bottle
- Test solution volume: 3L
- Aeration: Before usage, the water was aerated for more than 24 hours to attain dissolved oxygen saturation.
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One replicate for test concentration
- No. of vessels per control (replicates): One
- Biomass loading rate: 0.2 ~ 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: treated with Aquapro Pure Water System
- Total organic carbon: < 2 mg/L
- Intervals of water quality measurement: The indicators of the test water should be inspected externally by a qualified company once a year, there were no indicators affecting the test quality when last checked.

OTHER TEST CONDITIONS
- Photoperiod: 12 hr light: 12 hr dark

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 10 mg/L
- Results used to determine the conditions for the definitive study: Based on the preliminary test, the definitive test were conducted with the nominal concentration of 10, 5, 2.5 mg/L (supersaturated solution with 10 mg/L), concurrently with a blank control group. Because the weight of the test group fish and pseudo specific growth rate were no obverse differences with the control, three concentrations in a geometric series with a factor 2 were used.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
During the 28 days exposure period, no death and visible toxic signs were observed in the control group and test groups.
At the start and end of the test all fish (surviving) were weighed as wet weights (blotted dry) individually. The P value of average r3 (pseudo specific growth rate) for each test concentration was compared with the average r3 for the control, followed by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The p-value in each group was higher than 5%, so there was no significance difference between test group and control group.
The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 2.5, 5.0 and 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0048, 0.010 and 0.020 mg/L. The concentrations below the LOQ (0.001mg/L) were calculated as 0.5 LOQ. The arithmetic mean determined concentration in old media was 0.0020, 0.0046 and 0.010 mg/L. The concentrations in old solution were 40.9%, 45.3% and 50.2% of the fresh concentrations.
The overall arithmetic mean determined concentration of each batch geometrical mean determined concentrations were 0.00291, 0.00678 and 0.0144 mg/L.
Results with reference substance (positive control):
24-hour LC50: 296.35 mg/L
95% confidence limits: 236.01~372.11 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The 28 days NOEC of the test substance to Zebra fish on the growth of Juvenile phase was 0.0144 mg/L.
Executive summary:

A Fish, Juvenile Growth Test of the test substance was conducted with Zebra fish (Danio rerio) over 28 days exposure period according to OECD Guideline 215.

Based on the results of the preliminary test, the definitive test was carried out at concentration of 2.5, 5.0, 10 mg/L. The actual concentrations of test solutions were measured by HPLC-MS/MS method. A concurrent blank control was performed in parallel. 10 fish were used for the test groups and the control group.

 

During the 28 days exposure period, no death and visible toxic signs were observed in the control group and test groups.

At the start and end of the test all fish (surviving) were weighed as wet weights (blotted dry) individually. The average r3 (pseudo specific growth rate) for each test concentration was compared with the average r3 for the controls, followed by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The P value in each group was higher than 5%, so there was no significance difference between test group and control group.

The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 2.5, 5.0 and 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0048, 0.010 and 0.020 mg/L. The concentrations below the LOQ (0.001mg/L) were calculated as 0.5 LOQ. The arithmetic mean determined concentration in old media was 0.0020, 0.0046 and 0.010 mg/L. The concentrations in old solution were 40.9%, 45.3% and 50.2% of the fresh concentrations.

The overall arithmetic mean determined concentration of each batch geometrical mean determined concentrations were 0.00291, 0.00678 and 0.0144 mg/L.

Based on the results, the 28 days NOEC of the test substance to Zebra fish on the growth of Juvenile phase was 0.0144 mg/L.

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-06 to 2019-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch No.: 1902
Purity: 98.8%
Analytical monitoring:
yes
Details on sampling:
An analysis of the test solution was performed in the controls and each exposure concentration on Day 0 (at the start), 10, 13, 20, 27, 33, 40 and 44 (at the end) during the study.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All of the test solutions were prepared by dissolving of the test item in DMF, respectively. Each weighed the test item was dissolved in 200 mL DMF to prepare stock solutions of 60, 100, 180, 330 and 600 mg/L. Prepared stock solutions of each concentrations were supplied to the mixing chamber at the rate of 10 μL/min by means of a syringe pump. The dilution water was supplied simultaneously at the rate of 100 mL/min to the mixing chamber via a peristaltic pump. In the 1st mixing chamber, the dosed volume and the dilution water were mixed under continuous stirring, and then mixed solution was transferred into 2nd mixing chamber. Test solution was re-stirred in the 2nd chamber before it entered the vessel. Thus, each test solution of different concentration prepared was 0.006, 0.010, 0.018, 0.033 and 0.06 mg/L (nominal concentration).
- Controls: For solvent control, the dilution water was supplied and 10 μL of DMF was added to the same quantity of the 100 mL of dilution water (solvent concentration: 0.1 mL/L) and only the dilution water was used for dilution water control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Ricefish
- Source: Fish culture room, Ecotoxicology Research Group, Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology (KIT)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Culturing method: Broodstock fish were cultured in fish culture room and then parent fish were produced through natural spawning to get eggs for toxicity test. Parent fish were cultured under 16 h light: 8 h dark with 30-minute dawn and dusk transition periods at 21-25 °C (water temperature) in the fish culture room. Fish were fed daily with brine shrimp nauplii in the morning and Tetramin flake in the afternoon.
- Subsequent handling of eggs: Initially, fertilized eggs, embryos and larvae were exposed within the main vessel in a smaller glass, fitted with mesh side to permit flow of the test solution in the vessel. Non-turbulent flowthrough in these small vessels was induced by suspending them from an arm arranged to move the vessel up and down but always keeping the organisms submerged. Where the egg containers, meshes were used to hold the eggs within the main test vessel, these egg containers were removed from the test vessels, when all of the eggs have hatched.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
44 d
Hardness:
Between 17 and 26 mg/L as CaCO3
Test temperature:
24.2-25.8 °C
pH:
7.31-7.82
Dissolved oxygen:
7.01-7.99 mg/L (85-97% of Air Saturation Value)
Nominal and measured concentrations:
Norminal: 0.006, 0.010, 0.018, 0.033 and 0.06 mg/L
Measured: 0.0071, 0.0118, 0.0193, 0.0412 and 0.0657 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Teflon aquaria, with approximate dimensions of 16.0 × 7.0 × 9.0 cm (length, width, height) fitted with a surface overflow (7.0 cm above the base), to hold an approximate volume of 800 mL of exposure medium.
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): Flow rates of dilution water to the mixing chambers, that supplied each solvent control and test vessel, ranged between 95 and 104 mL/min during the definitive test; equivalent to 95 to 104% of the nominal flow rate (100 mL/min) during the study.
Dosing accuracy of the solvent stock solutions was estimated from the weight of solvent stock solution that supplied to each mixing chamber. The weights of stock solutions ranged between 10 and 11 μL/min during the definitive test; equivalent to between 100 and 110% of the nominal flow rate (10 μL/min) during the study.
- No. of fertilized eggs/embryos per vessel: Twenty
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four
- No. of vessels per vehicle control (replicates): Four
- Biomass loading rate: The loading rate (biomass per volume of test solution) did not exceed 0.5 g/L wet weight per 24 hours and 5 g/L of solution at any time.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water was passed through a membrane filter (5, 1, 0.2 μm) to remove particulate matter and then a high-grade activated carbon filter to remove any organic contaminants.
- Chlorine: The residual chlorine of dilution water were <0.05 mg/L (not detected)
- Intervals of water quality measurement: 3 times per year

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of dark, including 30-minute dawn and dusk transition periods during the study
- Light intensity: From 853 to 950 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Daily observations were made on development of the embryos prior to hatching, together with the number of dead larvae and any abnormalities in appearance or behavior after hatching (30 days ‘post-hatch’ phase). Size of fish (total length and dry weight) was assessed at the end of the exposure period.

POST-HATCH DETAILS
- Begin of post-hatch period: 30 days ‘post-hatch’ phase was counted from 14 days after study initiation
Key result
Duration:
44 d
Dose descriptor:
EC50
Effect conc.:
> 0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
44 d
Dose descriptor:
LOEC
Effect conc.:
> 0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
44 d
Dose descriptor:
NOEC
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
44 d
Dose descriptor:
EC50
Effect conc.:
> 0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other:
Remarks:
Dry weight
Key result
Duration:
44 d
Dose descriptor:
LOEC
Effect conc.:
> 0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other:
Remarks:
Dry weight
Key result
Duration:
44 d
Dose descriptor:
NOEC
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other:
Remarks:
Dry weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: Post-hatch survival (%) was 93.4-96.2% and the overall survival was 88.8-93.8% in all test
concentrations and there was no adverse effect to survival in all test concentrations.
- Days to hatch or time to release of young: Hatching commenced 10 days after the eggs were introduced into the test system from all test concentrations and hatching was completed on day 21.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Hatching success (rate) was 90~100 % in all test concentrations.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: There was no significant effect on total length and dry weight of fry exposed all test concentrations
- Number of healthy fish at end of test: There were no sub-lethal effects to the all survival fry exposed to the test item. All survival fish were normal and healthy in all test concentrations during study period.
- Hatching success: A few eggs in solvent control, 0.010, 0.06 mg/L were hatched late (21 day) other than eggs. 2-4 eggs were dead at the all test concentrations until hatching completed date (21 day). Hatching success (rate) was 97.5% in control, solvent control, 0.006, and 0.010 mg/L (measured concentration: 0.0071, 0.0118 mg/L), 96.3% in 0.018 mg/L (measured concentration: 0.0193 mg/L) and 95.0% in 0.033 and 0.06 mg/L (measured concentration: 0.0412, 0.0657 mg/L). As the result of statistical analysis, embryo development was not affected by exposure to the test item at any concentrations employed in this test. EC50 (95% confidence limits) of hatching rate: >0.0657 mg/L (95% confidence limits were not available.); LOEC for hatching rate: >0.0657 mg/L; NOEC for hatching rate: 0.0657 mg/L
- Post-hatch survival: 3 fry (3.8%) in control, solvent control and 0.006 mg/L (measured concentration: 0, 0, 0.0071 mg/L) of test solution were dead, respectively, 4 fry (5.1-5.3%) were dead in 0.010, 0.018 and 0.033 mg/L (measured concentration: 0.0118, 0.0193, 0.0412 mg/L) and 5 fry (6.6%) in 0.06 mg/L (measured concentration: 0.0657 mg/L) were dead during post-hatching study period. From those result, there was no adverse effect to post-hatch survival in all test concentrations in this study. LC50 (95% confidence limits) of post-hatch survival: >0.0657 mg/L (95% confidence limits were not available.); LOEC for post-hatching survival: >0.0657 mg/L; NOEC for post-hatching survival: 0.0657 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The LC (or EC)50, LOEC (Lowest observed effect concentration) and NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all >0.0657 mg/L (the geometric mean measured concentration). The NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all 0.0657 mg/L (the geometric mean measured concentration).
Executive summary:

The study was conducted to assess toxicity of the test item to the early life stage of ricefish, Oryzias latipes under a continuous flow-through condition according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test).

Groups of 80 fertilized eggs were exposed to the test item dispersed in dilution water at nominal concentrations of 0.006, 0.010, 0.018, 0.033 and 0.06 mg/L. All of the test solutions were prepared by dissolving the test item in dimethylformamide (DMF), respectively. Control fish were exposed to the dilution water alone and to solvent control (concentration of DMF: 0.1 mL/L).

 

In this study, hatching was started on day 10 and all eggs were hatched on day 21. 90% of eggs in control, solvent control were hatched on day 14. Therefore, 30 days "post-hatch" phase was counted from 14 days after study initiation (total exposure period was 44 days including prehatched phase)

Hatching commenced 10 days after the eggs were introduced into the test system from all test concentrations and hatching was completed on day 21. Hatching success (rate) was 90~100 % in all test concentrations.

Post-hatch survival (%) was 93.4-96.2% and the overall survival was 88.8-93.8% in all test concentrations and there was no adverse effect to survival in all test concentrations. All survival fish were all normal and healthy during study period. No significant effects on the total length and dry weight of survival fish were observed at the end of the study.

 

The LC (or EC)50, LOEC (Lowest observed effect concentration) and NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all >0.0657 mg/L (the geometric mean measured concentration). The NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all 0.0657 mg/L (the geometric mean measured concentration).

Description of key information

Fish, juvenile growth test:

The 28 days NOEC of the test substance to Zebra fish on the growth of Juvenile phase was 0.0144 mg/L.

Fish early-life stage toxicity:

The LC (or EC)50, LOEC (Lowest observed effect concentration) and NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all >0.0657 mg/L (the geometric mean measured concentration). The NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all 0.0657 mg/L (the geometric mean measured concentration).

Key value for chemical safety assessment

Additional information

Fish, juvenile growth test:

A Fish, Juvenile Growth Test of the test substance was conducted with Zebra fish (Danio rerio) over 28 days exposure period according to OECD Guideline 215.

Based on the results of the preliminary test, the definitive test was carried out at concentration of 2.5, 5.0, 10 mg/L. The actual concentrations of test solutions were measured by HPLC-MS/MS method. A concurrent blank control was performed in parallel. 10 fish were used for the test groups and the control group.

During the 28 days exposure period, no death and visible toxic signs were observed in the control group and test groups.

At the start and end of the test all fish (surviving) were weighed as wet weights (blotted dry) individually. The average r3 (pseudo specific growth rate) for each test concentration was compared with the average r3 for the controls, followed by a two-sided Student T-test using a 5% significance level to obtain the no observed effect concentration (NOEC). The P value in each group was higher than 5%, so there was no significance difference between test group and control group.

The concentrations of test item were determined in samples of test media by HPLC-MS/MS method. The results showed that for the test solutions of 2.5, 5.0 and 10 mg/L, the arithmetic mean determined concentration in fresh media was 0.0048, 0.010 and 0.020 mg/L. The concentrations below the LOQ (0.001mg/L) were calculated as 0.5 LOQ. The arithmetic mean determined concentration in old media was 0.0020, 0.0046 and 0.010 mg/L. The concentrations in old solution were 40.9%, 45.3% and 50.2% of the fresh concentrations.

The overall arithmetic mean determined concentration of each batch geometrical mean determined concentrations were 0.00291, 0.00678 and 0.0144 mg/L.

Based on the results, the 28 days NOEC of the test substance to Zebra fish on the growth of Juvenile phase was 0.0144 mg/L.

Fish early-life stage toxicity:

The study was conducted to assess toxicity of the test item to the early life stage of ricefish, Oryzias latipes under a continuous flow-through condition according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test).

Groups of 80 fertilized eggs were exposed to the test item dispersed in dilution water at nominal concentrations of 0.006, 0.010, 0.018, 0.033 and 0.06 mg/L. All of the test solutions were prepared by dissolving the test item in dimethylformamide (DMF), respectively. Control fish were exposed to the dilution water alone and to solvent control (concentration of DMF: 0.1 mL/L).

In this study, hatching was started on day 10 and all eggs were hatched on day 21. 90% of eggs in control, solvent control were hatched on day 14. Therefore, 30 days "post-hatch" phase was counted from 14 days after study initiation (total exposure period was 44 days including prehatched phase)

Hatching commenced 10 days after the eggs were introduced into the test system from all test concentrations and hatching was completed on day 21. Hatching success (rate) was 90~100 % in all test concentrations.

Post-hatch survival (%) was 93.4-96.2% and the overall survival was 88.8-93.8% in all test concentrations and there was no adverse effect to survival in all test concentrations. All survival fish were all normal and healthy during study period. No significant effects on the total length and dry weight of survival fish were observed at the end of the study.

The LC (or EC)50, LOEC (Lowest observed effect concentration) and NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all >0.0657 mg/L (the geometric mean measured concentration). The NOEC (No observed effect concentration) values of the test item in Ricefish, Oryzias latipes for hatching success rate, post-hatching survival, overall survival, total length, dry weight were all 0.0657 mg/L (the geometric mean measured concentration).