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Short term toxicity to fish

Estimated 96 hrs LC50 value of test chemical on the test organismFathead minnowwas evaluated by three different models i.e, Battery, Leadscope and SciQSAR used within Danish QSAR database (2017). On the basis of effect of test chemical on the mortality of the test organismFathead minnow, the 96 hrs LC50 was estimated to be 2.33 mg/l.Thus, based on the LC50 value, chemical can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aq. organisms and thus can be considered to be not classified as per CLP classification criteria.

Short term toxicity to aquatic invertebrates

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, the EC50 value the test chemical on the mobility of the test organism Daphnia magna can be expected to be ranges from 18.7 to 24.6 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aq. invertebrate. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aq. invertebrate and thus can be considered to be not classified as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

The 96 hrs EC50 value of test chemical on green algae was estimated using the ECOSAR model (from peer reviewed journal A.M. Api et. al., 2017 and modelling databases, 2018). On the basis of the effect of test chemical on the growth rate of the test organism green algae, the 96 hrs EC50 was estimated to be 1.026 mg/l.Thus, based on the EC50 value, chemical can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aq. organisms and thus can be considered to be not classified as per CLP classification criteria.

Toxicity to microorganisms

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, the EC0 value the test chemical on the growth rate, i.e, multiplication of the test organism Pseudomonas putida can be expected to be ranges from 83 to 115 mg/l, respectively.

Additional information

Short term toxicity to fish

Predicted data of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for the short term toxicity to fish end point which are summarized as below:

 

In a prediction done usingthree different models i.e, Battery, Leadscope and SciQSAR used within Danish QSAR database(2017), theEstimated 96 hrs LC50 value of test chemical on the test organism Fathead minnow was evaluated. On the basis of effect of test chemical on the mortality of the test organism Fathead minnow, the 96 hrs LC50 was estimated to be 2.33 mg/l.

 

In a supporting weight of evidence study from authoritative database (J-CHECK, 2018),an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Oryzias latipes (Japanese rice fish). The test was performed in accordance to OECD guideline No. 203“Fish Acute Toxicity Test”. Oryzias latipes (Japanese rice fish) was used as a test organism for the study. Test chemical conc. used for the study were 35.0, 26.9, 20.7, 15.9, 12.3 mg/L (geometric ratio1.3), control. Analytical monitoring involve the use of HS-GC. Study was performed using 10 per conc. (5 per vessel) Oryzias latipes in a semi-static fresh water system. Total 50 fishes were exposed to test chemical in a closed vessel. The test vessels were placed under a photoperiod of room light, 16:8 hr light: dark conditions, respectively. On the basis of mortality of the test organism Oryzias latipes (Japanese rice fish), the 96 hrs LC50 value was determined to be 16.6 mg/l (95% C. I. – 12.3 to 23.8 mg/l), respectively. 

 

For the test chemical, Short term toxicity to fish study was carried out for 96 hrs (Handbook, 2009). The study was based on the effects of the test chemical on Pimephales promelas in a static fresh water system. Pimephales promelas (Fathead minnow) was used as a test organism. On the basis of mortality of the test organism Pimephales promelas, the 96 hr LC50 was determined to be 18 mg/l.

 

On the basis of the above results, it can be concluded that the test chemical can be considered astoxic to fish. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to fish and thus can be considered to be not classified as per CLP classification criteria.

Short term toxicity to aquatic invertebrates

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxic effect 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 effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Own breeding stock at University of Chemistry and Technology, Prague of Daphnia magna was used as a test organism for the study. The stock solution 100 mg/l was prepared by dissolving colourless liquid in reconstituted water. Test solutions of required concentrations, i.e, 6, 10, 17, 29 and 50 mg/l were prepared by mixing the stock solution of the test sample with reconstituted test water. Study was performed using total 100 daphnids (5 organisms per vessel/replicates) in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±1°C, pH 7.9 in dark conditions, respectively. 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. On the basis of the effect of test chemical on the mobility of the test organism Daphnia magna, the 48 hr EC50 value was determined to be 18.7 mg/l with a 95% confidence interval value ranging from 16.4 to 21.3 mg/l, respectively.

 

In an another study, short term toxicity test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used as a test organism for the study. Test chemical conc. used for the study were 19.1, 34.3, 61.7, 111 and 200 mg/L (geometric ratio1.8), control. Analytical monitoring involve the use of HS-GC. Study was performed using 20 per conc. (5 per vessel) Daphnia magna in a semi-static fresh water system. Total 100 fishes were exposed to test chemical in a closed vessel. The test vessels were placed in a room at a temperature of 20±1°C under a photoperiod of room light, 16:8 hr light: dark conditions, respectively. On the basis of mobility of the test organism Daphnia magna, the 48 hrs LC50 value was determined to be 24.6 mg/l (95% C. I. – 15.5 to 29.2 mg/l), respectively. 

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, the EC50 value the test chemical on the mobility of the test organism Daphnia magna can be expected to be ranges from 18.7 to 24.6 mg/l, respectively. Thus, based on the EC50 value, test chemical can be considered as toxic to aq. invertebrate. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aq. invertebrate and thus can be considered to be not classified as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Predicted data of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for the toxicity to aquatic algae end point which are summarized as below:

 

In a weight of evidence study from peer reviewed journal (A.M. Api et. al., 2017) and modelling database (2018), the 96 hrs EC50 value of test chemical on green algae was estimated using the ECOSAR model. On the basis of the effect of test chemical on the growth rate of the test organism green algae, the 96 hrs EC50 was estimated to be 1.026 mg/l.

 

In a supporting weight of evidence study from study report (2017), an acute test was conducted for 48 hrs for assessing the effect of test chemical on Desmodesmus subspicatus. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5x10(3) cells /ml was used as a test organism for the study. The stock solution 100 mg/l was prepared by dissolving colourless liquid in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with the OECD growth medium and inoculum culture. Nominal test chemical conc. used for the study were 12, 20, 34, 58 and 100 mg/l, respectively. Study was performed using Desmodesmus subspicatus as a test organism in a static fresh water system. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 23±2°C, pH 7.9 with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, 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. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr ErC50 value was determined to be 53.5 mg/l with a 95% confidence interval value ranging from 46.9 to 61.1 mg/l, respectively.

 

For the test chemical from peer reviewed journal (G. BRINGMANN et. al., 1980), short term toxicity to Scenedesmus quadricauda study was carried out for 7 days. The study was based on the effects of the test chemical on Scenedesmus quadricauda in a static fresh water system at a temperature of 27ᵒC and under a constant lightning by luminescent worm white tubes at 60 cm distance from each other. Test chemical of known concentration was prepared in sterile double distilled water. Composition of test medium contains sodium nitrate (496 mg), dipotassium hydrogen phosphate (39 mg), magnesium chloride (75 mg), sodium metasilicate (40 mg), sodium carbonate (58 mg), citric acid (3 mg), iron (III) citrate (3 mg) and disodium salt of ethylene diamine tetra acetic acid (10 mg), respectively. For maintenance of the test strain of Scenedesmus quadricauda, prepare fresh stock cultures continuously at 10days intervals. For this purpose sterilize the required number of Erlenmeyer flasks* containing 20 ml of nutrient solution I and stoppered with metal caps in a steam sterilizer for 30 min each on two consecutive days. When cool inoculate the contents of each flask with 2mi cell suspension from a 10-day stock culture. Prepare dilution series in 100-ml Erlenmeyer flasks stoppered with metal caps. The dilutions will contain 1 part v/v each of pollutant solution in 20 to 214 parts v/v of mixture. Test vessel contain the known conc. of chemical and test organism. Following inoculation, at the end of 7 day, measure the extinction value of the monochromatic radiation at 578 nm in a 10 mm layer of the cell suspension from each test culture. Pollutant concentrations at which the onset of inhibitory action was observed have been obtained by mathematical evaluation. For mathematical evaluation by means of a suitable electronic calculator (a) (highest non-toxic pollutant concentration) is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After entering (A - 3%), the pollutant concentration at which the inhibitory action (c) begins may be obtained from the regression line between (a;A) and(b;B)if a negative deviation of the mean extinction by a 3% difference against the mean extinction value for all test cultures having a non-toxic and non-stimulating pollutant concentration is used as an indicator of the beginning of inhibitory action. For test chemical, based on effect on growth rate of the test organism Scenedesmus quadricauda, the 7 d EC50 value was determined to be > 21 mg/l.

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic algae and cyanobacteria. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aquatic algae and thus can be considered to be not classified as per CLP classification criteria.

Toxicity to microorganisms

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxic effect of the test chemical on micro-organisms. The studies are as mentioned below:

 

Short term toxicity to Pseudomonas putida study was carried out for 16 hr. The study was based on the effects of the test chemical on Pseudomonas putida at a temperature of 25ᵒC. Test chemical of known concentration was prepared in sterile double distilled water. Composition of test medium contains sodium nitrate (1.06 g), dipotassium hydrogen phosphate (0.6 g), magnesium sulphate (0.2 g), D (+) glucose (10 g), Difco bacto agar (18 g), ferrous sulphate (0.001 g), and 1.5 ml trace element solution. Pseudomonas putida was used as a test organism. Keep stock cultures of the test strain, Pseudomonas 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 of1 week each. Incubate the inoculated stock cultures at 25"C for 24 h and keep in stock. 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 436nm for a 10 mm layer of the bacterial suspension by photoelectric measurement. Erlenmeyer flask of 300 ml stoppered with cottoned lined plastic caps was used for the study. Prepare dilution series in the test vessel. Each of the dilutions contains 1 part v/v of the pollutant solution in 20 to 214 parts v/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 8O ml 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 100 ml by adding 5 ml each of stock solution I, 5 ml each of stock solution II and 10 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value. Leave both inoculated and non-inoculated dilution series at 25°C for 16 h. After termination of the test period measure the extinction of the monochromatic radiation at 436 nm in a 10-mm layer in the inoculated dilution series. On the basis of the effect of test chemical on growth rate, i.e, multiplication of the test organism Pseudomonas putida, the 16 h EC0 value was determined to be 115 mg/l.

 

For the test chemical, another toxicity study to micro-organisms was carried out according to the same procedure as mentioned above. In this study, on the basis of the effect of test chemical on growth rate, i.e, multiplication of the test organism Pseudomonas putida, the 16 h EC0 value was determined to be 83 mg/l.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, the EC0 value the test chemical on the growth rate, i.e, multiplication of the test organism Pseudomonas putida can be expected to be ranges from 83 to 115 mg/l, respectively.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemicalcan be considered as toxic to aquatic organisms at environmental relevant concentrations. Since the test chemical is readily biodegradable in water, test chemical can be considered as non-toxic to aquatic organisms and thus can be considered to be not classified as per CLP classification criteria.