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Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Short term toxicity to fish:

Fish Acute Toxicity test according to OECD Guideline 203 was conducted for the test substance on Zebra fish.The nominal concentration selected for the experiment were0, 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/Land Zebra Fish Danio rerio were exposed to these concentration for 96 hours. The test substance was sparingly soluble in water. Therefore, the test solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with 24 hours continuous stirring Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. The nominal concentration selected for the experiment was 0, 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/l. and Zebra fish were exposed to this concentration for 96 hours. The lethal concentrations LC50 was found to be >100 mg/l. After 96 hours of exposure to test item to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was non toxic and can be consider to be not classified as per the CLP classification criteria.

Long term toxicity to fish:

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test substance . On the basis of effects observed in a static freshwater system, the NOEC value for the substance is estimated to be1.483mg/l for fish for 28 days of exposure duration.Since, , it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations and can be considered not-classified as per the CLP classification criteria. 

Short term toxicity to aquatic invertebrate:

Determination of the inhibition of the mobility of daphnids was carried out with the test substance according to OECD Guideline 202. The stock solution 200 mg/l was prepared by dissolving colorless  liquid in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. The test substance was tested at the concentrations 0, 0, 10, 20, 40, 80, 160 mg/L. Potassium dichromate (K2Cr2O7) was used as a positive reference substance . Effects on immobilization were observed for 48 hours. EC50 was calculated using non linear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance ethyl butyrate in Daphnia magna was determined to be 116.6 mg/L for immobilization effects. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

Toxicity of aquatic algae and cyanobacteria:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The solution 100 mg/l was prepaired by dissolving colourless liquid in OECD growth medium .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. Effect on the growth of algae was determine after an exposure period of 72 hrs. The inhibition percentage for the test substance in algae was determined to be 1.5% on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as aquatic acute as per the CLP criteria.

Long term toxicity to aquatic invertebrate:

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on aquatic invertebrate was predicted for test substance . On the basis of effects observed in a static freshwater system, the NOEC value for the test substance is estimated to be 28.833mg/l for aquatic invertebrate for 21 days of exposure, it can be concluded that the test chemical can be considered as non-toxic to aquatic invertebrate at environmentally relevant concentrations and can be considered not-classified as per the CLP classification criteria.

Toxicity to microorganism:

In toxicity to microorganism study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope. The toxicity threshold (TT) of test material was determined to be 236 mg/l for Entosiphon sulcatum. Therefore LOEC was considered to be 236mg/l for 72h in Entosiphon sulcatum.

Additional information

Short term toxicity to fish:

The short term toxicity of fish was summarized with detailed experimental data as per OECD guideline along with another experimental data described in peer reviewed journals and handbooks for the test chemical.

Fish Acute Toxicity test according to OECD Guideline 203 was conducted for the test substance on Zebra fish. The nominal concentration selected for the experiment were 0, 6.25mg/L,12.5mg/L, 25mg/L,50mg/L,100mg/L and Zebra Fish Danio rerio were exposed to this concentration for 96 hours.The test substance was sparingly soluble in water. Therefore, the test solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with 24 hours continuous stirring Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. The nominal concentration selected for the experiment was 0, 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/l. and Zebra fish were exposed to this concentration for 96 hours. The lethal concentrations LC50 was found to be >100 mg/l. After 96 hours of exposure to test item to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was non toxic and can be consider to be not classified as per the CLP classification criteria.

 

First study was supported by the second study. Short term toxicity of test material was evaluated on fish golden orfe in static condition for 48 h The median lethal effect LC50 concentration of test material on fish after 48 h was observed to be 17 mg/l. Based on this effect concentration it can be considered that test material is toxic to fish and can be classified in the category aquatic chronic 3 as per CLP criteria .

 

Similarly in short term toxicity to fish study for test substance in Leuciscus idus melanotus was observed for 2 days. No mortality was observed at concentration of 35 mg/l. 50% mortality was observed at range concentration of 53-78 mg/l. 100% mortality was observed at range concentration of 87-131 mg/l.As available lethal concentration indicate the test substance classified in aquatic chronic 3; since the test chemical readily biodegradable in nature thus not consider for the aquatic classification as per the CLP criteria.

 

Since, varied results on different fishes were observed the experimental data conducted occording to OECD guideline is considered for the classification of test substance. The lethal concentrations LC50 was found to be >100 mg/l. Based on the LC50, it can be consider that the chemical was non toxic and can be consider to be not classified as per the CLP classification criteria.

Long term toxicity to fish:

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test substance . On the basis of effects observed in a static freshwater system, the NOEC value for the substance is estimated to be1.483mg/l for fish for 28 days of exposure duration.Since, , it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations and can be considered not-classified as per the CLP classification criteria. 

Short term toxicity to aquatic invertebrate:

Toxicity of aquatic invertebrate was evaluated based on the two experimental data which were performed according to OECD guideline and along with that data from authoritive data base and secondary sources were also discussed as below:

Determination of the inhibition of the mobility of daphnids was carried out with the test substance according to OECD Guideline 202. The stock solution 200 mg/l was prepared by dissolving colorless liquid in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. The test substance was tested at the concentrations 0, 0, 10, 20, 40, 80, 160 mg/L. Potassium dichromate (K2Cr2O7) was used as a positive reference substance. Effects on immobilization were observed for 48 hours. EC50 was calculated using non linear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance ethyl butyrate in Daphnia magna was determined to be 116.6 mg/L for immobilization effects. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

 

First study was supported by the second. Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for test material. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 500 mg of the test substance in 500 ml of ADaM’s media. Achieving test concentrations of 1 g/L, respectively. The nominal concentration selected for the experiment were 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/L and test Daphnia magna were exposed to these concentration for 48 hours. The median lethal concentration (EC50) for test material on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be >100 mg/l. Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the substance, does not exhibit short term toxicity to Daphnia. EC50 (48 hours) Experimental is > 100 mg/l . After 48 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be > 100 mg/l . Based on the EC50, it can be consider that the chemical was non toxic and can be consider to be not classified as per the CLP classification criteria.

 

Similar short term toxicity study of test material was carried out on the Daphnia magna and was observed for 24 hrs. Less than 24 hrs 5 daphnia were used in fresh water and static condition at 20 -22 degree C temperatures. 50% mortality (LC 50) was observed at range concentration of 755 mg/l. Based on the value, ethyl butyrate was considered to be non-toxic to aquatic invertebrates and can be considered to be not classified as per the CLP regulations.

 

In short term toxicity to aquatic invertebrate study for test chemical in Daphnia magna was observed for 24 hrs.Less than 24 hrs 5 daphnia were used in fresh water and static condition.EC 50 was observed at range concentration of 750 mg/l on the basis of behaviour. Based on the value, ethyl butyrate was considered to be non-toxic to aquatic invertebrates and can be considered to be not classified as per the CLP regulations.

 

Based on the above data for test chemical it can be concluded that test chemical is non toxic to aquatic invertebrate and can be considered to be not classified as per CLP criteria.

Toxicity of aquatic algae and cyanobacteria:

Experimental study to evaluate toxicity of aquatic algae and cyanobcateria for the test material was performed according to OECD guideline, and along with the experimental report description of few peer reviewed journals and secondary sources were also described as mentioned below:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The solution 100 mg/l was prepared by dissolving colourless liquid in OECD growth medium .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. Effect on the growth of algae was determine after an exposure period of 72 hrs. The inhibition percentage for the test substance in algae was determined to be 1.5% on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as aquatic acute as per the CLP criteria.

 

Similar short term toxicity to Chlorococcales (green algae) study was carried out for 24 hrs. The study was based on the effects of the test compound on green algae in a static fresh water system. Based on effect on physiology of the test organism Chlorococcales(green algae), the 24 hr EC10 and EC50 value was determined to be 340 and 1000 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance can be considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

 

In the third study toxicity of aquatic algae and cyano bacteria was evaluated for the test material . The test material was tested for 8 days on green algae Microcystis aeruginosa. The effect concentration (EC0) was observed to be 700 mg/l after 8 days. No toxic effects were observed on the test organism. Based on the above effect concentration it can be considered that test material is non toxic to aquatic algae and can not be classified as per CLP criteria.

 

Similarly in toxicity to green algae study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. Prepare dilutions with varying volume ratios using sterile double-distilled water. These dilutions each contain 1 part v/v of pollutant solution in 2 o to 214 parts v/v mixture. When preparing the two parallel dilution series in 300-ml Erlenmeyer flasks proceed as follows: the first flask of each dilution series contains 80 ml of pollutant solution at the start. Starting from this flask, prepare subsequent dilutions using a constant dilution ratio of 40 ml preliminary pollutant dilution + 40 ml double-distilled water. So each flask will first contain 40 ml of liquid. Then, complete each flask from the dilution series to be inoculated to the rated value of 50 ml by adding 5 ml each of stock solution I and 5 ml each of the algal suspension of the preliminary culture having a known adjusted extinction value. Scenedesmus quadricauda was used as test organism. Store stock cultures of the test strain Scenedesmus quadricauda in 20ml nutrient solution I in 100-ml Erlenmeyer flasks stoppered with metal caps, on a white surface protected against daylight and exposed to constant lighting by Luminescent worm white tubes at 60cm distance from each other, at 27°C and a relative humidity of 50%. For maintenance of the test strain, prepare fresh stock cultures continuously at 10days' intervals. The toxicity threshold (TT) of test material was determined to be 47mg/l for Scenedesmus quadricauda. Therefore LOEC was considered to be 47 mg/l for test material for 7 days in Scenedesmus quadricauda.

 

Long term toxicity to Scenedesmus quadricauda (green algae) study was carried out for 8 days. The study was based on the effects of the test compound on green algae in a static fresh water system at a temperature of 27°C and pH 7.0 respectively. Scenedesmus quadricauda was used as a test organism. Test substance was in double – distilled water, and the stock solution subsequently neutralized. Two parallel concentration series were tested, which is obtained by stepwise diluting (factor: 0.5) from one stock solutions. Start of test by adding aldehyde suspension with defined extinction value at 578 nm (water at unvaccinated Controls) and nutrient medium to the dilution series solutions. From each inoculated mixture, three cultured tubes of 10 ml solution (= test volume), one tube per tube control approach. 1 x daily shake the test cultures. Determination of the cell density at the photometric test at 578 nm (436 and 691 nm, respectively, in chemical-chemical cases conditional strong color of the culture medium) against algae-free blank. For the evaluation of the measurement results were cell density values ​​against the concentration of the test substance semi-logarithmic. The inhibition commencement was graphical determined from the cell density values ​​of the cultures with the highest non-toxic and the lowest toxic pollutant concentration. The TGK is maintained at 3% (standard deviation) below the cell density of cultures. Based on effect on cell density of the test organism Scenedesmus quadricauda, the LOEC value was determined to be > 47 mg/l, respectively.

 

Eventhough , some studies suggest that the test material has the potential to cause growth inhibition , but according to the effect values of experimental report and other authoratives databases and peer reviewed journal it can be considered that the test material has no negative impact on the growth rate of algae and can not be classified as per CLP criteria.

Long term toxicity to aquatic invertebrate:

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on aquatic invertebrate was predicted for test substance . On the basis of effects observed in a static freshwater system, the NOEC value for the test substance is estimated to be 28.833mg/l for aquatic invertebrate for 21 days of exposure, it can be concluded that the test chemical can be considered as non-toxic to aquatic invertebrate at environmentally relevant concentrations and can be considered not-classified as per the CLP classification criteria.

Toxicity to microorganism:

Two experimental studies was used to explain the effect of test material on microorganisms:

In the first study toxicity to microorganism study for test material the biological effect was evaluated by using analogous methods of the cell multiplication inhibition test. For maintenance of the test strain of Entosiphon sulcatum continuously inoculate the expected required number of stock cultures at 72 h or 96 h intervals. For this, mix 8 ml of stock solution I and 8 ml of sterile double-distilled water in 300-ml Erlenmeyer flasks stoppered with metal caps and add 2 ml of a stock culture and 2 ml of the adjusted bacterial suspension for Entosiphon stock cultures. Store stock cultures at 25°C. Before inoculation control the flasks containing the stock cultures by means of an inverse microscope. Pretreatment of the bacteria used for feeding preliminary cultures of Entosiphon sulcatum does not differ from that of bacteria used for feeding the stock cultures. Keep preliminary cultures at 25°C over 72 h before using them for inoculation of the test cultures. Before inoculation control each flask containing the preliminary cultures by means of an inverse microscope. The toxicity threshold (TT) of test material was determined to be 236 mg/l for Entosiphon sulcatum. Therefore LOEC was considered to be 236mg/l for 72h in Entosiphon sulcatum.

Above study was supported by the second study. In Toxicity to microorganisms study for test chemical the biological effect was evaluated. By using analogous methods of the cell multiplication inhibition test.

Prior to the preparation of test cultures neutralize the test chemical of a known content in sterile double-distilled water to be tested, select the concentration of the acid or alkaline solution to be added in such a way that the volume added is kept as small as possible. Do not adjust pH where the eft'oct of the pH of the pollutant solution to be studied is to be included in the test. Prepare from the pollutant solution prepared as described or the sewage and sterile double-distilled water, four parallel dilution series in 300-ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps. Each of the dilutions contains 1 part r/v of the pollutant solution in 20 to 214 parts 0/v of mixture. Prepare the dilution series as follows: the first flask of each series contains 160ml of pollutant solution at the start. Starting from this flask prepare the subsequent dilution stein at a constant dilution ratio by consistently mixing 80ml of preliminary pollutant dilution and 80ml double distilled water. Consequently, each flask contains 80 ml of culture liquid at the start. Make up each flask of the three dilution series to be inoculated to 100ml by adding 5 ml each of stock solution I, 5 ml each of stock solution II and I0 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value

Keep stock cultures of the test strain, Pseudoraonas putida, on the nutrient for stock and preliminary cultures in agar slant tubes. Prepare, for onward culturing of the test strain, new stock cultures at intervals of 1 week each. Incubate the inoculated stock cultures at 25"C for 24 h and keep in stock. If needed, prepare preliminary cultures from stock cultures on the above mentioned nutrient medium in agar slant tubes and incubate at 25°C for 24 h. Then, wash off the cell material with sterile saline. Determine the extinction of the monochromatic radiation at 436 nm for a 10 ram layer of the bacterial suspension by photoelectric measurement. On the basis of the values thus measured, adjust the final turbidity value of the bacterial suspension by means of sterile saline in such a way that the extinction value for a measuring sample " that has been subject to onward dilution 1 + 9 with saline will correspond to the extinction value of a Formazin standard suspension TE/F/436 nm= 10.*

The toxicity threshold (TT) of test chemical was determined to be 140mg/l in  bacteria Pseudomonas putida. Therefore LOEC was considered to be 140mg/l for 16 h for Pseudomonas putida.