Registration Dossier

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Short term toxicity to fish:

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio) (Experimental study report, 2013). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 2.3 cm and average weight of 0.116 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water under natural conditions for 15 days along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.4 mg/l, pH 6 -7, water temperature 29°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. Test chemical 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. Range finding test was conducted with these two test concentrations, i.e., 5 mg/L and 7.5 mg/L. Since, at 7.5 mg/L one mortality was observed within 24 hrs. Therefore, the final experiment was continued for both these test chemical concentration, in which no mortality was found. Observations (mortality, visible symptoms, pH, Temperature, dissolved oxygen content) were recorded after 24 hours, 48 hours, 72 hours and 96 hours of the start of the experiment. Test fishes were exposed to test chemical in a glass bowl aquaria. The test vessels were placed in a room at a temperature of 29°C, pH 6 -7 under a photoperiod of 12:12 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. Mortality in the control vessel was observed to be 0%. On the basis of effect of test chemical on mortality of the test organism, the 96 hr LC0 and median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be 7.5 and > 7.5 mg/l, respectively. Thus, test chemical can be considered as toxic to aquatic fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to fish and hence, considered to be '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:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna (Experimental study report, 2019). The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 200mg of test chemical in 200ml of Adams medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 5 mg/L. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per CLP classification criteria.

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 and thus classified as aquatic chronic 3 category as per the CLP classification criteria.

Toxicity to algae and cyanobacteria:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris (Experimental study report, 2014). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. BBM medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance solution was prepared by dissolving 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask following changes were observed with increase in test substance concentration like decrease in cell count and discoloration of algal cells. On the basis of the effect of test chemical on growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 50.26 mg/l and 50.12 (calculated from equation and graphically through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classifed' 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:

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

 

In an experimental study from study report (2013),an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 2.3 cm and average weight of 0.116 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water under natural conditions for 15 days along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.4 mg/l, pH 6 -7, water temperature 29°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. Test chemical 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. Range finding test was conducted with these two test concentrations, i.e., 5 mg/L and 7.5 mg/L. Since, at 7.5 mg/L one mortality was observed within 24 hrs. Therefore, the final experiment was continued for both these test chemical concentration, in which no mortality was found. Observations (mortality, visible symptoms, pH, Temperature, dissolved oxygen content) were recorded after 24 hours, 48 hours, 72 hours and 96 hours of the start of the experiment. Test fishes were exposed to test chemical in a glass bowl aquaria. The test vessels were placed in a room at a temperature of 29°C, pH 6 -7 under a photoperiod of 12:12 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. Mortality in the control vessel was observed to be 0%. On the basis of effect of test chemical on mortality of the test organism, the 96 hr LC0 and median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be 7.5 and > 7.5 mg/l, respectively. Thus, test chemical can be considered as toxic to aquatic fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to fish and hence, considered to be 'not classified' as per the CLP classification criteria.

 

In a supporting weight of evidence study, short term toxicity to fish study was conducted for 96 hr for assessing the effect of the test chemical (Geiger D.L. et. al, 1988). Pimephales promelas (Fathead minnows) of 29 days old with length of 20 mm +/- 1.91 mm and weight of 0.122 +/- 0.035 g was used as a test organism. Stock solution test chemical (42.3 mg/l) were prepared by blending. Test chemical concentrations were verified analytically using gas liquid chromatography. Chemical concentrations used for the study were 0, 1.47, 2.26, 3.47, 5.34 and 8.22 mg/l (nominal conc.) and 0, 1.42, 1.75, 2.54, 4.10 and 6.98 mg/l (measured conc.), respectively. Total 20 fishes were exposed with the test chemical in a 2.0 lit tank for 96 hrs. Renewal rate of the test solution was 18 chamber additions/day and biomass loading rate was 1.22 g/l. Study was performed in a flow through system under test conditions like hardness of 44.0 +/- 0.41 mg/l CaCO3, temperature of 25.5 +/- 0.44 °C, pH 7.8 +/- 0.05 and dissolved oxygen of 6.8 +/- 0.31 mg/l. All test experiments were performed in 1 replicate. After an exposure period of 96 hrs, mortality and symptoms of intoxications was noted. On the basis of the effect of test chemical on growrh rate and behaviour of the test organism Pimephales promelas, the 96 hr EC50 and LC50 value was determined to be 4.71 and 5.09 mg/l, respectively. Thus, chemical can be considered as toxic to fish. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classificatiins criteria.

 

Another acute fish toxicity study from the peer reviewed journal (A. M. Api et. al, 2016) was conducted for 96 hr for assessing the effect of the test chemical. The study was performed following the german standard guideline DIN 38412 part 15 under static system. Leuciscus idus (Goldn orfe) was used as a test organism. On the basis of the effect of test chemical on mortality of the test organism Leuciscus idus, the 96 hr LC50 value was determined to be 6.81 mg/l. Thus, test chemical can be considered as toxic to aquatic fish. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be non-classified as per the CLP classification criteria.

 

For the test chemical, short term fish toxicity study was conducted for 96 hr for assessing the effect of the test chemical (J-CHECK, 2018). The study was performed in accordance with the OECD Guideline 203 (Fish, Acute Toxicity Test) in a static system. On the basis of the effect of test chemical on mortality of the test organism, the 96 hr LC50 value was determined to be 3.0 mg/l. Thus, test chemical can be considered as toxic to aquatic fish. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be non classified as per the CLP classification criteria.

 

On the basis of the overall results, it can be concluded that the test chemicalwas considered as toxic to fish. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes at environmental concentrations and hence, considered to be'not classified' as per 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:

Various experimental studies of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across chemical were reviewed for short term toxicity to aquatic invertebrate end point which are summarized as below:

 

In an experimental study from study report (2019),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”. The test solution was prepared by dissolving 200mg of test chemical in 200ml of Adams medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 5 mg/L. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per CLP classification criteria.

 

Another acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna (A.M. Api, et.al, 2016). The test was performed in accordance to EU Method C.2 (Acute Toxicity for Daphnia), Directive 92/69/EEC in a static system. On the basis of the effect of test chemical on mobility of the test organism Daphnia magna, the 48hr median effect concentration (EC50) value was determined to be 5.7 mg/l. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

In a supporting weight of evidence study, short term toxicity study was conducted to assess the effect of test chemical on the immobilisation of test organism daphnia magna (authoritative database, 2018 and secondary source, 2018). Test was performed in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample was carried out by using HPLC/UV detector. Test organism, 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. Test chemical concentrations used for the study were 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L (nominal concentrations) and 0.621, 1.14, 1.82, 2.99, and 5.32 (mean measured concentration), respectively. 10 daphnia per vessel added and tests were conductedl 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. Based on these effects, the 48 hr NOEC, EC50 and LC50 value was determine to be 1.14 mg/l, 2.65 mg/l and 3.11 mg/l, respectively. The measured concentrations after 24 and 48 hours were 80-89% (which is within the range of ± 20%) of the nominal concentrations, with the concentration being held steady throughout the test period. Therefore, the analysis of the results was based on nominal concentration. On the basis of result, test chemical was considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

For the test chemical from secondary source (2004), short term toxicty to aquatic invertebrates test was carried out following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of 2 -24 hr old was used as a test organism. Age of the stock animals was 2 -4 weeks old. Test organism was not fed during the study. Nominal test chemical concentration used for the study were 0, 1, 2, 4, 8 and 16 mg/l, respectively. The dilution factor was 2. Test chemical concentrations were verified analytically by HPLC. Study was performed using daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 20 ml test vessel in a volume of 10 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, pH 7.5 to 8.5, DO of 8.8 to 9.1 mg/l, hardness of water 2.20 - 3.20 mM/l and under a photoperiod of 16:8 hr light: dark conditions with light intensity 550 – 650 µS/cm, respectively. One control vessel was also run simultaneously during the study. After a period of 0, 24 and 48 hrs, number of mobile daphnids were measured. The EC50 (48 h) was calculated using the Probit analyses. On the basis of the effect of test chemical on mobility of the test organism Daphnia magna, the 48hr median effect concentration (EC50) value was determined to be 3.7 mg/l (initial measured conc.). Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

On the basis of the overall results, it can be concluded that the test chemicalwas considered as toxic to aquatic invertebrates. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates at environmental concentrations and hence, considered to be'not classified' as per 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:

Various experimental studies of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2014),a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. BBM medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance solution was prepared by dissolving 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask following changes were observed with increase in test substance concentration like decrease in cell count and discoloration of algal cells. On the basis of the effect of test chemical on growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 50.26 mg/l and 50.12 (calculated from equation and graphically through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was conducted for 96 hrs for assessing the effect of test chemical (A.M. Api, et.al, 2016). The study was performed following the german standard guideline DIN 38412 part 9 under static system. On the basis of the effect of test chemical on growth rate and biomass of the test organism Desmodesmus subspicatus, the 72 hr EC50 value was determined to be 22.9 and 20.9 mg/l and the 96 hr EC50 value was determined to be 21.9 and 12.2 mg/l, respectively. Thus, test chemical can be considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be non classified as per the CLP classification criteria.

 

For the test chemical from peer reviewed journal (Jihai Shaoa, et.al, 2011), 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. 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. On the basis of the effect of test chemical on growth rate of the test organism, the 48 hr EC50 value was determined to be 21.23±1.87 mg/l.

 

On the basis of the overall results, it can be concluded that the test chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae at environmental concentrations and hence, considered to be'not classified' as per 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.