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Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Short term toxicity to fish:

A 96 h study was conducted for test chemical on Danio rerio, at the test concentrations of 5mg/L, & 7.5 mg/L. In5mg/L, & 7.5 mg/L, the fishes were found to be freely swimming in the bowl aquaria. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 50 mg & 75 mg of the test substance in 10 liters deionized water with continuous stirring for achieving test concentrations of 5 mg/L and 7.5 mg/L, respectively. No mortalities were found in the control aquaria. Hence the LC50 value was considered to be > 7.5 mg/L and the LC0 value was determine to be 7.5 mg/l. Thus considering CLP Criteria for aquatic classification of the substance , it is concluded that test chemical exhibits short term toxicity to fish (Danio rerio) and classified in the chronic 2 classification category. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Long term toxicity to fish:

1. After the exposure of test chemical with fish for 28 days, NOEC was observed at 0.134 mg/l.

2. After the exposure of test chemical with fish for 28 days, NOEC was observed at 0.173 mg/l.

Thus on the basis of above available data, it can be concluded that the test chemical was toxic to fish and classified as aquatic chronic 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

1. After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2.

2. After the exposure of 48 hrs, effect on the daphnia magna were observed and measured on the basis of immobility. The EC50 was determine to be 5.7 mg/l.

3. The EC50 value of test chemical was observed to be 3.7 mg/L when daphnia magna was exposed to for 48 hrs. on the basis of value it can be concluded that the test chemical is toxic to the aquatic environment.

Long term toxicity to aquatic invertebrates:

1. On the basis of no effects observed in a static freshwater system, the NOEC value for the test substance is estimated to be 0.136 mg/l for daphnia for 21 days of exposure duration.

2. On the basis of no effects observed on the static freshwater daphnia, the NOEC value for the test substance is estimated to be 0.170 mg/l for daphnia for 21 days of exposure duration.

Thus based on the above data it was concluded that the chemical was toxic and as it is readily biodegradable in water tus classified as aquatic chronic 3 category as per the CLP classification criteria.

Toxicity to algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Toxicity to microorganisms:

1. The Minimum Inhibitory Concentration (MIC) value of test chemical on the fungi  Colletotrichum musae DAR 24962 was determine to be 894.195 mg/L.

2. Based on the antimicrobial effect of test chemical on the growth of Bacillus subtilis after the exposure of chemical for 2-5 days, the MIC was observed at 100 mg/l.

Thus based on the above studies, MIC ranges from 100 mg/l to 894.195 mg/l after the exposure of microorganisms with the test chemical for 2-10 days.

Additional information

Summarized result for the toxicity of test chemical and structually and functionally similar read across chemicals on the growth and mortality of aquatic life’s including fish, invertebrates, algae and microorganism were studied and are as follows:

Short term toxicity to fish:

Based on the various experimental data for the test chemical study have been reviewed to determine the toxic nature of test chemical on the mortality of fish. The studies are as mentioned below:  

A 96 h study was conducted for test chemical on Danio rerio, at the test concentrations of 5mg/L, & 7.5 mg/L. In5mg/L, & 7.5 mg/L, the fishes were found to be freely swimming in the bowl aquaria. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 50 mg & 75 mg of the test substance in 10 liters deionized water with continuous stirring for achieving test concentrations of 5 mg/L and 7.5 mg/L, respectively. No mortalities were found in the control aquaria. Hence the LC50 value was considered to be > 7.5 mg/L and the LC0 value was determine to be 7.5 mg/l. Thus considering CLP Criteria for aquatic classification of the substance, it is concluded that test chemical exhibits short term toxicity to fish (Danio rerio) and classified in the chronic 2 classification category. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

First study was supported by the second experimental study for test chemical. Short term toxicity of test chemical was studied in fathead minnows (Pimephales promelas) fish. Before testing chemical was analytically monitorized by gas liquid chromatography. Stock solution of test chemical (42.3 mg/l) were prepared by blending. 29 days old fishes were used for the study under the flow-through system. The 96 hrs Lethal concentration (LC50) and effective concentration (EC50) value of test chemical on fish Pimephales promelas was observed at dose concentration of 5.09 mg/l and 4.71 mg/l respectively. Thus considering CLP Criteria for aquatic classification of the substance, it is concluded that tes test chemical exhibits short term toxicity to fish (Pimephales promelas) and classified in the chronic 2 classification category.

 

Based on the above studies, it was conclude that the chemical was toxic and can be consider to be classified in aquatic chronic 2 category as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Long term toxicity to fish:

Various long term studies available for the test chemical and structually and functionally similar read across chemicals were reviewed to determine the toxic nature of test chemical on the mortality and other long term effect of fish. The studies are as mentioned below:

Based on the prediction, the long term toxicity on fish was predicted for test substance. On the basis of no effects were observed in a freshwater system, the NOEC value for the test substance is estimated to be 0.134 mg/l for fish for 28 days of exposure duration. Based on this value, it can be concluded that the test chemical can be considered as toxic to fish at environmentally relevant concentrations and can be considered to be classified as aquatic chronic 3 category as per the CLP classification criteria. 

Similarly in the another study based on the prediction, the long term toxicity on fish was predicted for test substance. On the basis of no effects observed in a freshwater system, the NOEC value for the test substance is estimated to be 0.173 mg/l for fish for 28 days of exposure duration. Based on this value, it can be concluded that the test chemical can be considered as toxic to fish at environmentally relevant concentrations and chemical was readily biodegradable in water thus on that basis it can be considered to be classified as aquatic chronic 3 category as per the CLP classification criteria. 

Thus on the basis of above available data, it can be concluded that the test chemical was toxic to fish and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Short term toxicity to aquatic invertebrates:

Data available for the test chemical and structually and functionally similar read across chemicals was reviewed to determine its toxic nature upon the growth and mobility of aquatic invertebrate. The studies are as mentioned below:

 

The first study was to determine the effect of test chemical on the immobilisation of test organism daphnia magna. Test conducted in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample conducted by using HPLC/UV detector. Juvenile daphnia (<24 hours old) produced from an in-house culture of adults were maintained at the contract laboratory under test conditions for 45 days. During the 48 hours prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions for 48 hours. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7days prior to the beginning of the test. The test substance was provided via an intermittent flow proportional diluter. 5 nominal concentrations 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L were used for the toxicity testing. 10 daphnia per vessel added and test conducted in duplicates. After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Similarly in the second study a Daphnia immobilization static study was conducted following Guideline “Acute Toxicity for Daphnia” (C.2), Directive 92/69/EEC by providing the exposure period of 48 hrs. Test conducted under the static system. After the exposure of 48 hrs, effect on the daphnia magna were observed and measured on the basis of immobility. The EC50 was determine to be 5.7 mg/l.

 

Above studies was supported by another third study. To study the effects of test chemical on aquatic invertebrates test was carried out according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The 48-h LC50 value is based on measured data. A synthetic fresh water was used for culture and test purposes. Properties of this medium: Total hardness: 2,20 - 3,20 mmol/l, Alkalinity up to pH 4.3: 0,80 - 1,00 mmol/l, Molar ratio Ca:Mg: about 4 : 1, pH value: 7,5 - 8,5, Conductivity: 550 - 650 μS/cm. After preparation the M4 medium was aerated for approximately 24 h until saturation. The test substance was tested in the range of concentrations between 16 and 1 mg/l. The dilution factor was 2. The number of mobile daphnid after 24 and 48 hours of exposure in the different test concentrations was measured. The EC50 (48 h) calculated using the Probit analyses The EC 50 value of test chemical was observed to be 3.7 mg/L when daphnia magna was exposed to for 48 hrs. On the basis of this value it can be concluded that the test chemical is toxic to the aquatic environment and can be considered as Aquatic acute 2 as per the classification criteria for aquatic environment.

 

Thus based on the overall studies, chemical consider to be toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrates:

Data for the long term toxicity of test chemical and structually and functionally similar read across chemicals on the mobility and mortality of test organism daphnia were summarized and studies are as mention below:

 

Based on the prediction, the long term toxicity on aquatic invertebrate daphnia species was predicted for test substance. On the basis of no effects observed in a freshwater daphnia, the NOEC value for the test substance is estimated to be 0.136 mg/l for daphnia for 21 days of exposure duration. Based on this value, it can be concluded that the test chemical was toxic to daphnia at environmentally relevant concentrations and chemical was readily biodegradable in water thus can be considered to be classified as aquatic chronic 3 category as per the CLP classification criteria. 

 

Above toxic nature of test chemical was supported by the second study. Based on the prediction, the long term toxicity on aquatic invertebrate daphnia magna was predicted for test substance. On the basis of no effects observed in a freshwater daphnia, the NOEC value for the test substance is estimated to be 0.170 mg/l for daphnia for 21 days of exposure duration. Based on this value, it can be concluded that the test chemical can be considered as toxic to daphnia at environmentally relevant concentrations and chemical was readily biodegradable in water thus can be considered to be classified as aquatic chronic 3 category as per the CLP classification criteria. 

 

Thus based on the above data it was concluded that the chemical was toxic and as it is readily biodegradable in water thus chemical classified as aquatic chronic 3 category as per the CLP classification criteria.

 

 

Toxicity to algae and cyanobacteria:

Based on the various experimental data for the test chemical study have been reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:  

 

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Above study was supported by the second study from peer reviewed journal. Toxicity of test chemical was accessed on the growth of green algae Microcystis aeruginosa. Experiments were carried out in 250mL conical flasks containing 94.9mL CT liquid medium, and 100L of test solutions in dimethyl sulfoxide (DMSO). Prior to these experiments, 0.2% (v/v) DMSO had no obvious effect on the growth and photosynthetic processes of M. aeruginosa NIES-843. Five milliliters of exponential-phase cultures of M. aeruginosa NIES-843 (in CT medium) were added to the conical flasks in order to reach a final volume of 100 mL. The final test concentrations were set as 0, 6.67, 10, 15, 22.5, 33.75 mg/L, respectively, and the initial cellular concentrations of M. aeruginosa NIES-843 were 6.5×100000 cells/mL. Each treatment was replicated three times. All treatments were cultured under the same conditions as mentioned above. The increase in M. aeruginosa cell number was calculated after 48 h using a hemocytometer. Chl a, and carotenoid content were determined. Cultures were sampled 48 h after inoculation to measure content of photosynthetic pigments. The EC50 of test chemical on the growth of M. aeruginosa NIES-843 was shown to be 21.23±1.87 mg/L. Results based on transcript expression of genes, polyphasic Chl a fluorescence transients and ultrastructural examinations through TEM indicated that the reaction centre of PS II and electron transport at the acceptor side of PS II are the targets responsible for the toxicity of test chemical on the PS II of M. aeruginosa NIES-843. Based on the EC50 value, chemical consider to be toxic and consider to be classified as per the CLP classification criteria.

 

Similarly in the third study short term toxicity study of test chemical was performed on the growth of aquatic algae Scenedesmum subspicatus. Test conducted under the static system for total exposure period of 96 hrs, effect after 72 hrs were also observed. After the exposure of test chemical for 96 hrs effect were measured on the basis of growth rate inhibition and biomass of Scenedesmum subspicatus. On the basis of biomass and growth rate for 72 hrs, the EC50 was determine as 20.9 and 22.9 mg/l, respectively. After the exposure period of 96 hrs the effect were observed at 12.2 and 21.9 mg/l.

 

Thus based on the above experimental data it was concluded that the chemical was toxic and consider to be classified as aquatic chronic category 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to microorganisms:

Based on the various experimental data for the test chemical and structually and functionally similar read across chemicals study have been reviewed to determine the toxic nature of test chemical on the growth of microorganisms. The studies are as mentioned below:  

The effects of test chemical on the growth of Colletotrichum musae on agar medium were evaluated. Test was performed on the agar medium. Agar plugs (5.5-mm diam.) were picked up from the 3-day-old cultures of decay fungi using the bottom end of a sterilized Pasteur pipet and then transferred onto the centers of new PDA media, in 9-cm plastic Petri dishes. The Petri dishes were then inverted and 7-cm Whatman No. 1 filter papers were attached onto the inner surface of their lids. Ethanol, the first tested volatile in this experiment, was impregnated into the filter paper with varying volumes from 0.1 to 1.0 mL/dish in the 4°C room. Immediately after the impregnation, the Petri dishes were sealed by wrapping them with plastic film and incubated for 10 days at 25 °C. Experiments were repeated two times with four replications for each experiment. The minimum concentration of ethanol (expressed as mmol/dish) required to give complete control or the minimum inhibitory concentration (MIC) for each microorganism was determined. The MIC of ethanol for target decay microorganism was used as the initial level to identify the MIC of other tested volatiles. If the MIC level of ethanol used for other volatiles failed to stop the growth of pathogen, the level was increased until the MIC was found. However, if the volume of 1.5 mL/dish still failed to stop the growth of pathogen, the compound was considered ineffective as a vapor to stop the growth of pathogens. When the tested compounds had the same effect as the MIC of ethanol, the concentration was decreased until the MIC of the compound for each microorganism was determined. All the unit concentrations of MIC were then expressed as mmol/dish. After the incubation of 10 days, the Minimum Inhibitory Concentration (MIC) value of test chemical on the fungi, Colletotrichum musae DAR 24962 was determine to be 894.195 mg/L.

 

First study was supported by the second experimental study. Aim of this study was to evaluate the effect of test chemical on the growth of Bacillus subtilis and other fungi. The antimicrobial activity of test compounds against various bacteria and fungi was examined by the broth dilution method. Solution of the test compound was added to 2-day-old cultures of the microorganisms. After 2-5 days of incubation, growth of the microorganisms was checked. Minimal inhibitory concentrations (MICs) were measured by two fold serial broth dilution. Based on the antimicrobial effect of test chemical on the growth of Bacillus subtilis after the exposure of chemical for 2-5 days, the MIC was observed at 100 mg/l.

 

Thus based on the above studies, MIC ranges from 100 mg/l to 894.195 mg/l after the exposure of microorganisms with the test chemical for 2-10 days.

Based on the effect observed on the growth, mobility and other abnormal behavior of test organisms by the chemical exposure for long term duration, chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria.