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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to guideline
Guideline:
other: refer below principle
Principles of method if other than guideline:
Short term combine multigeneration reproductive and genotoxicity study of methyl methanesulfonate in zebrafish was conducted.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Methyl methanesulfonate
- Molecular formula (if other than submission substance): C2H6O3S
- Molecular weight (if other than submission substance): 110.1324 g/mol
- Smiles notation (if other than submission substance): COS(=O)(=O)C
- InChI: 1S/C2H6O3S/c1-5-6(2,3)4/h1-2H3
- Substance type: Organic
- Physical state: Liquid
Analytical monitoring:
yes
Details on sampling:
Details on sampling
- Concentrations: 0, 2, 4 and 8 mg/L
- Sampling method: 200 mL tap water containing 0, 2, 4 and 8 mg/L MMS in glass beakers at 26 ± 1 ˚C
- Sample storage conditions before analysis: 26 ± 1 ˚C and a photoperiod of 12 h:12 h
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mL tap water containing 0, 2, 4 and 8 mg/L MMS in glass beakers at 26 ± 1˚C and a photoperiod of 12 h: 12 h, which was maintained throughout the experiment.
- Differential loading: Yes, flow of stock solutions (52, 104, 208 mg/L MMS) was adjusted by means of peristaltic pumps
- Controls: Tap water
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio, West Aquarium wild
- Source: Laboratory
- Age at study initiation (mean and range, SD): 110 fertilized eggs
- Length at study initiation (length definition, mean, range and SD): No data available
- Weight at study initiation (mean and range, SD): No data available
- Method of breeding: Fertilization
- Feeding during test: Yes
- Food type: Artemia nauplii, powder food and flakes.
- Frequency: Artemia nauplii fed once, powder food, three times and flakes two times a day


ACCLIMATION
- Acclimation period: 1–2 h
- Acclimation conditions (same as test or not): Same
- Type and amount of food: Artemia nauplii fed once, powder food, three times and flakes two times a day
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No data available
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
169 days post fertilization
Hardness:
12–18˚dH
Test temperature:
25 ± 2 ˚C
pH:
7.0–8.3
Dissolved oxygen:
No data available
Salinity:
No data available
Nominal and measured concentrations:
Measured concentration: 0, 2, 4 and 8 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Tank
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 10 L
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Renewal rate of test solution (frequency/flow rate): Renewed every 48 hours
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): Triplicate
- No. of vessels per control (replicates): Triplicate
- No. of vessels per vehicle control (replicates): Triplicate
- Biomass loading rate: No data available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data available
- Total organic carbon: No data available
- Particulate matter: No data available
- Metals: No data available
- Pesticides: No data available
- Chlorine: No data available
- Alkalinity: No data available
- Ca/mg ratio: No data available
- Conductivity: 522–828 lS/cm
- Culture medium different from test medium: No data available
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: No data available
- Photoperiod: 12 h:12 h
- Light intensity: No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Growth, Body weight, length, Fertility and fertilization, Sex distribution of F1 generation, Teratogenicity in embryos and early larval stages, Genotoxicity and histopathology were measured.

TEST CONCENTRATIONS
No data available
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Viability and malformations
Remarks on result:
other: Effect on survival , growth, length of fish, Fertility and fertilization, Sex distribution of F1 generation, Teratogenicity in embryos and early larval stages, Genotoxicity and Histopathology
Details on results:
Details on result
- Behavioural abnormalities: No data available
- Observations on body length and weight: Body weight: No significant effects were observed on body weight of treated fish as compared to control.

Length: Significantly decreased total length of fish were observed in 2 mg/L dose as compared to control

- Other biological observations:
Fertilization: In 2 mg/L dose group, the time point of the first spawning was delayed by 51 days and the number of eggs was also decreased relative to the controls.

Sex distribution:
Female fish were more prevalent in 2, 4 and 8 mg/L dose group as compared to control.

Teratogenicity in embryos and early larval stages: Significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control.

Genotoxicity:
Percentage of DNA significantly elevated in tail and liver cells from sexes as well as female gills and male gonads of 2 mg/L treated fish as compared to control.

Histopathology:
Micronucleus frequencies were significantly increased in liver, gills and gonads of both male and female fish in 2 mg/L treated fish as compared to control.

- Mortality of control: No data available
- Other adverse effects control: No data available
- Abnormal responses: No data available
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No data available
- Effect concentrations exceeding solubility of substance in test medium: No data available
Results with reference substance (positive control):
No data available

Reported statistics and error estimates:
One-Way-ANOVA
Validity criteria fulfilled:
yes
Conclusions:
In a short term combined multigeneration reproductive and genotoxicity study, Danio rerio, was treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hrs.

Executive summary:

In a short term combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. Overall mortality, embryo and larval stages mortality were observed in 2, 4 and 8 mg/L dose. Similarly, Significantly decreased total length of fish, Premature gonads, delayed time point of the first spawning by 51 days and decreased number of eggs were observed in 2 mg/L dose. More prevalent female fish and Significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours.

Description of key information

In a short term combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. Overall mortality, embryo and larval stages mortality were observed in 2, 4 and 8 mg/L dose. Similarly, Significantly decreased total length of fish, Premature gonads, delayed time point of the first spawning by 51 days and decreased number of eggs were observed in 2 mg/L dose. More prevalent female fish and Significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
2 mg/L

Additional information

Based on the experimental data for the target chemical from various journals and publications, study have been reviewed to determine the toxic nature of target chemical methyl methanesulfonate (MMS) (66-27-3) on the growth, mortality and embryo development of fishes. The studies are as mentioned below:  

First experimental study (Ecotoxicology (2013) 22:825–837) suggest in a short term combine multigeneration reproductive and genotoxicity study, Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) in the concentration of 0, 2, 4 and 8 mg/L for 96 hours. Overall mortality, embryo and larval stages mortality were observed in 2, 4 and 8 mg/L dose. Similarly, significantly decreased total length of fish, premature gonads, delayed time point of the first spawning by 51 days and decreased number of eggs were observed in 2 mg/L dose. More prevalent female fish and significantly more lethal and sublethal teratogenic effects in embryos and larvae were observed in 2 mg/L treated fish as compared to control. In addition, significantly elevated percentage of DNA in tail and liver cells from sexes as well as female gills and male gonads and significantly increased micronucleus frequencies in liver, gills and gonads of both male and female fish in 2 mg/L treated fish were observed as compared to control. Therefore, EC 50 was considered to be 2 mg/L when Danio rerio, West Aquarium wild Zebrafish treated with Methyl methanesulfonate (MMS) for 96 hours.

 

Whereas another authorative database (ECOTOX database by US EPA; 2017) indicate in Acute fish toxicity data the lowest observable effect concentration determine of test substance methyl methanesulfonate for exposure period of 24 hrs on the basis of Genetics effects. Test organism was used as Australoheros facetus (Chameleon Cichlid) in static freshwater with test temp. 18 deg.C, pH 8.2,Hardness 270.2 mg/L CaCO3 and alkalinity 160 mg/L CaCO3.In acute fish toxicity study the Lowest observable effect concentration (LOEC) on the basis of Genetics effect was observed to be 10 mg/l for exposure period of 24 hrs with significance level <0.05.

 

Similarly study from same data source suggest the lowest observable effect concentration of test substance methyl methanesulfonate for exposure period of 24 hrs. on the basis of Genetics damage effects. Test organism was used as Oreochromis niloticus (Nile Tilapia) in static freshwater with test temp. 25 deg.C and pH 7.0.In acute fish toxicity study the Lowest observable effect concentration (LOEC) on the basis of Genetics damage effect was observed to be 7.5 mg/l for exposure period of 24 hrs with significance level <0.0001.

 

In the fourth experimental study from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) study was carried out and the effect was determine on the viability of fish for total exposure period of 2 hrs. The objective of this study was to determine the in vitro developmental toxicity of methyl methanesulfonate (MMS) on the (medaka) fish embryo culture systems as models for screening these developmental toxin. MMS were dissolved in 11 mM sodium citrate buffer (pH 6.0) immediately before use. The test solution was added directly to embryo rearing medium at final concentrations ranging from 0.75 mM to 120 mM (82 .59 mg/l to 13215 mg/l). Medaka (Oryzias latipes) was obtained from Carolina Biological, Burlington, North Carolina and were maintained in freshwater aquaria in our laboratory at a temperature range of 25 to 28°C. Fish fed Tetra-Min flakes and newly hatched brine shrimp larvae twice daily. In 60 mm x 15 mm Petri dishes Embryos were collected from female fish from two aquaria each containing 60 fish in a ratio of two males to three females. Eggs were pooled and then randomly distributed among experimental groups. All embryo experiments were conducted in isotonic rearing media at 20°C. Control embryos were exposed to buffer vehicle only. Treatment was initiated when embryos reached stage 20 (period of early organogenesis, approximately 66 h post fertilization at 20°C). After 2 h of exposure in the dark, embryos were extensively rinsed and transferred to Petri dishes containing fresh embryo rearing medium. All dishes were coded for blind observation. In addition, each experiment contained an uncoded control dish for comparative evaluation and photography. These values and their 95% confidence limits were calculated according to the modified probit analysis method. Based on the significant concentration -dependent decreases in viability, as expressed by the percentage of embryos that survived until hatching, the LC50 was determine to be 1740 mg/l after the exposure of chemical methyl methanesulfonate (MMS) with the medaka fish for 2 hrs. Based on the LC50 chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

Similarly fifth study was taken from the same from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) in which effect of chemical on the embryonic development of Medaka (Oryzias latipes) was studied for the target chemical. The objective of this study was to determine the in vitro developmental toxicity of methyl methanesulfonate (MMS) on the (medaka) fish embryo culture systems and malformations. MMS were dissolved in 11 mM sodium citrate buffer (pH 6.0) immediately before use. The test solution was added directly to embryo rearing medium at final concentrations ranging from 0.75 mM to 120 mM (82 .59 mg/l to 13215 mg/l). Medaka (Oryzias latipes) was obtained from Carolina Biological, Burlington, North Carolina and were maintained in freshwater aquaria in our laboratory at a temperature range of 25 to 28°C. Fish fed Tetra-Min flakes and newly hatched brine shrimp larvae twice daily. In 60 mm x 15 mm Petri dishes Embryos were collected from female fish from two aquaria each containing 60 fish in a ratio of two males to three females. Eggs were pooled and then randomly distributed among experimental groups. All embryo experiments were conducted in isotonic rearing media at 20°C. Control embryos were exposed to buffer vehicle only. Treatment was initiated when embryos reached stage 20 (period of early organogenesis, approximately 66 h post fertilization at 20°C). After 2 h of exposure in the dark, embryos were extensively rinsed and transferred to Petri dishes containing fresh embryo rearing medium. All dishes were coded for blind observation. In addition, each experiment contained an uncoded control dish for comparative evaluation and photography. These values and their 95% confidence limits were calculated according to the modified probit analysis method. Based on the significant concentration -dependent increases in frequency of malformations the MC50 was 1376 mg/l after the exposure of chemical methyl methanesulfonate (MMS) with the medaka fish for 2 hrs. Based on the MC50 chemical was consider as nontoxic and not classified as per the CLP classification criteria.

 

Thus by considering all the studies. As the above three studies from Ecotoxicology (2013) 22:825–837) and secondary source ECTOX database suggest that the chemical was toxic as the effect concentration EC50 ranges from 2 mg/l to 10 mg/l which indicates that the chemical was consider as toxic and classified in aquatic chronic category 2. But the remaining 2 studies from peer reviewed journal (Environmental Toxicology and Chemistry, 1987) for the target chemical indicates and supports the nontoxic and not classified nature of target chemical methyl methanesulfonate (MMS). In the fourth and fifth studies, total exposure period of chemical with the test organism was very less (2 hrs.) and the study was not performed according to the standard OECD guidelines. Thus based on the overall criteria chemical was consider as toxic and can be consider to be classified in aquatic chronic category 2 as per the CLP classification criteria.

 

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