Registration Dossier

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Short-term toxicity to fish:

Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra fish (Danio rerio) was consider to be > 100 mg/L as no effects were observed on the test concentration of 100 mg/l. Thus, on the basis of LC50 value, it can be concluded  that the chemical was non toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

An acute immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The stock solution 100 mg/l was prepared in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. 5.0 , 10.0 , 20.0 , 40.0 , 80.0 mg/l, respectively nominal concentrations were used in this study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 16.5 mg/l. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified as chronic 3 category as per the CLP classification criteria. The test substance is rapidly degradable in aquatic environment , hence it can be considered to have no adverseeffect on aquatic environment and can not be classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

An algal growth inhibition study was conducted for 72 hrs on green algae. The test was performed in accordance with the‘’OECD Guideline 201 (Alga, Growth Inhibition Test)’’. Chlorella vulgaris was used as a test organism. The test solution was prepared in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l  respectively nominal test concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 2. Test conducted under the static system. 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. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test was considered to be valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Toxicity to microorganisms:

As in the second weight of evidence study after the exposure of Vibrio fischeri with a test chemical for 15 min, bioluminescence inhibition of microorganism was observed and the EC50 was determined to be at 284 mg/l with 95% confidence limit of 241–336 mg/l, whereas in the third study minimum inhibition concentration (MIC) was determined to be  > 4.2 umol/cm2. Thus based on the above all studies, it is observed that the test chemical was non toxic.

Additional information

Short-term toxicity to fish:

Data available for the test chemicals and structually and functionally similar read across chemicals have been reviewed to determine the short term toxicity of the test chemical on fish.The studies are as mentioned below:

An acute study was carried out to assessing the effect of test chemical on freshwsater fishes. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used a test organism. The stock solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous one hour stirring for achieving test concentrations of 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L, respectively. Potable water (passed through reverse osmosis system) was used. Test conducted under the static system for 96 hours. Fishes were exposed to these concentrations for 96 hours. The Stability of Test substance was determined as per the Spectrophotometric method and found to be stable for 4 days at 25°C. Aquaria used in the study having the water capacity of 5 liters filled with 2 liters of water and were loaded with 8 fishes. Test consider to be valid as it fulfills all necessary criteria. The mortality in the control was found to be 0 percent at the end of the test. Constant conditions were maintained as far as possible throughout the test and, static procedures used. The dissolved oxygen concentration was found to be 79.76 per cent of the air saturation value throughout the test. After 96 hours of exposure to test chemical with fishes, no effects were observed upto the higher test concentration of 100 mg/l, thus LC50 value consider to be > 100 mg/l. Thus based on the LC50 value, it was concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.

Above study further supported by the second study from experimental source. In an experimental study, an acute test was conducted for 96 hours for assessing the effect of test chemical on the fishes.This study was conducted by following the OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra Fish Danio rerio was used a test organism. The test solution was prepared by dissolving 400 mg of the test substance in 4 liters of potable water (passed through reverse osmosis system) with 1 hr continuous stirring for achieving test concentrations of 100 mg/L respectively. Study conducted under the static system. Mortality were used a test parameter on which the effects were calculated after the exposure period of 96 hours. After 96 hours of exposure periods, effects were calculated. No effects (NOEC) was observed at the limit test concentration of 100 mg/l, thus on the basis of NOEC value, LC50 was consider to be > 100 mg/l and chemical consider th be nontoxic and not classified as per the CLP classification criteria.

Based on the LC50 value, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

Data available for the test chemicals and structually and functionally similar read across chemicals have been reviewed to determine the short term toxicity of the test chemical on aquatic invertebrates.The studies are as mentioned below:

An acute immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The stock solution 100 mg/l was prepared in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. 5.0 , 10.0 , 20.0 , 40.0 , 80.0 mg/l, respectively nominal concentrations were used in this study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 16.5 mg/l. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified as chronic 3 category as per the CLP classification criteria. The test substance is rapidly degradable in aquatic environment , hence it can be considered to have no adverseeffect on aquatic environment and can not be classified as per CLP criteria.

Above study further supported by the second study from experimental source. An acute immobilisation test was conducted for 48 hrs to determine the short term toxicity of test chemical on aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The stock solution 100 mg/l was prepared in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 1, 2, 4, 8 and 16 mg/l, respectively nominal concentrations were used in this study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 9.9 mg/l. Based on the EC50 value, substance consider to be toxic but as the classification also depends on the biodegradation rate and the test chemical consider to be readily biodegradable in water, thus on the basis of readily biodegradable criteria, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Similar acute immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The stock solution 100 mg/l was prepared in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 6.2, 12.5, 25, 50 and 100 mg/l, respectively nominal concentrations were used in this study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 18.7 mg/l. Based on the EC50 value, substance consider to be hazardous to aquatic invertebrate and can be classified as chronic 3 category as per the CLP classification criteria. But as the test chemical was rapidly degradable in aquatic environment , hence it is considered that test chemical have no adverse effect on aquatic environment and cannot be classified as per CLP classification criteria.

Hence by considering all data from various sources, test chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the test chemicals and structually and functionally similar read across chemicals have been reviewed to determine the toxicity of the test chemical on aquatic algae.The studies are as mentioned below:

An algal growth inhibition study was conducted for 72 hrs on green algae. The test was performed in accordance with the‘’OECD Guideline 201 (Alga, Growth Inhibition Test)’’. Chlorella vulgaris was used as a test organism. The test solution was prepared in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l  respectively nominal test concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 2. Test conducted under the static system. 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. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test was considered to be valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Above study further supported by the second study from experimental source. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/l was prepared in OECD growth medium. Test conducted under the static system for 72 hours on limit test concentration of 100 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of test chemical, effect concentration the differences in the mean of control and sample were estimated by the t-test for independent groups at a 95% confidence level, all individual replicates were used (STATISTICA CZ - data analysis software system, version 9.0, StatSoft, Inc) Statistically significant differences are for p < 0.05. Effects on the growth rate of algae was determined after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical for 72 hours, only 2.1% inhibition was observed at limit test concentration of 100 mg/l. Thus on the basis of % inhibition, EC50 was consider to be > 100 mg/l. Based on the % inhibition value, test chemical was consider likely to be nonhazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

Similar study was conducted to assess the effect of test chemical on green algae. Test was conducted according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 160 mg/l was prepared in OECD growth medium. Test solution of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted under the static system and tested at the various nominal concentrations 0, 10, 20, 40, 80 and 160 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration ErC50 was calculated using nonlinear regression by the software Prism 4.0. Growth rate inhibition of Desmodesmus subspicatus were used a key parameter to determine the effect of chemical on algae by providing exposure period of 72 hours. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical for 72 hours, the ErC50 value was determined to be 259.5 mg/l with the 95% CI of 226.7 mg/l to 297.5 mg/l. Thus on the basis of ErC50 value, test chemical consider to be nontoxic and not classified as per the CLP classification criteria.

On the basis of above all studies and effects observations from various sources, it was concluded that the test chemical was non toxic and not classified as per the CLP classification criteria.

Toxicity to microorganisms:

Data available for the test chemicals has been reviewed to determine the toxicityof the test chemical on microorganisms. The studies are as mentioned below:

Toxicity to V. fischeri was measured as inhibition of bioluminescence using Microtoxs M500 Rapid Toxicity Testing System equipment and consumables.The assay was carried out as described in the Microtox user’s manual. Nine concentrations were tested in a 1 : 2 dilution series. Test conducted in triplicates. Light emission of the bacteria was measured after 0, 5 and 15 min of exposure using a Microtoxs Model 500 Toxicity Analyzer. The differences tested by ANOVA are highly significant (P < 0.001). After the exposure of Vibrio fischeri with a test chemical for 15 min, bioluminescence inhibition of microorganism was observed and the EC50 was determined to be at 284 mg/l with 95% confidence limit of 241–336 mg/l.

Above study further supported by the study from peer reviewed journal. Principle of this study was to determine the effect of test chemical on the Perenniporia tephropora. Test chemical analytically monitorized by NMRspectroscopy, ES-MS and X-ray crystallography. The toxicity test, cellulose papers i.e. Whatman No. 41, diameter 5.5 cm, thickness 510μm were treated with solutions of the compounds in acetonitrile or water (0.8–8.4μmol cm−2) and allowed to air-dry. Solvent controls were also prepared in the same manner but without addition of the test compound. The cellulose papers were sterilized by gamma irradiation (25 kGy). Under the sterile conditions, the cellulose papers loaded with test chemical were placed in center on 90 mm malt agar plates and inoculated with a small plug (7mm × 5 mm) of the test fungi Perenniporia tephropora. The fungi were sub-cultured on malt agar plates from existing stocks and used within about 7–9 days. After inoculation process, the plates were incubated at 25°C and at 29% relative humidity for three weeks. Fungal growth was scored periodically by measuring its growth diameters. Test was performed in quadruplicate. After the exposure of test chemical with the fungi, chemical did not inhibit fungi (Perenniporia tephropora) growth under bioassay conditions and minimum inhibition concentration (MIC) was determined to be  > 4.2 umol/cm2.

Thus based on the above all studies, it is observed that the test chemical was non toxic.

On the basis of above all studies from various sources, it was observed that the test chemical was readily biodegradable in water and consider to be nontoxic and not classified as per the CLP classification criteria.