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EC number: 202-336-5 | CAS number: 94-47-3
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- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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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). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 ± 0.2 cm and average weight of 0.09 ± 0.01 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test conditions during the housing of the test organisms were oxygen content of 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes with biomass of 0.14g/L were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.3 & 7.9 and DO of control at 0 and 96 hr was 6.8 & 4.8 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined at 0 our and 96 hours of the exposure durations which were maintained with in range of 95.92 - 111.12%, 104.16 - 92.48%, 83.2 - 85.12%, 104.74 - 104.04%, 87.2 - 90.23% at 6.25, 12.5, 25, 50 and 100 mg/L concentrations. 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 mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be in the range of > 25 to < 50 mg/L, 37.5 mg/L (based on average of LC0 and LC100) . Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.
Short term toxicity to aquatic invertebrates
This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (using a spacing factor of 1.5) was conducted using 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and 21.72 mg/L but 5%, 20%, 60% and 90% immobilisation were observed in the test concentrations of 32.58, 48.87, 73.30, 110 mg/L, respectively. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 32.58, 48.87, 73.30, 110 mg/L, conc were 97.42%, 102.76%, 95.58%, 105.38%, 102.22%, and 102.36%, respectively, after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.0 -7.2), temperature (20-21 °C), dissolve oxygen (8.6 - 8.1 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 µS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to daphnid, Daphnia magna are 66.46. The 48-h EC50of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT plus Software, version 8. Hence, as per CLP classification category the test chemical can be categorized as Chronic Category 3.
Toxicity to aquatic algae and cyanobacteria
A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Experimental study report, 2020). 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. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 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. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. 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 growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the 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 and by evaluating the effect of test chemical on test organism, the EC50 value can be expected to be in the range 0.11 to 5.7 mg/l and LOEC value was expected to be in the range 0.02 to 0.48 mg/l, respectively during the different exposure study duration.
Additional information
Short term toxicity to fish
Various experimental studies of the test chemical and 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:
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 1.5 ± 0.2 cm and average weight of 0.09 ± 0.01 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test conditions during the housing of the test organisms were oxygen content of 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes with biomass of 0.14g/L were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.3 & 7.9 and DO of control at 0 and 96 hr was 6.8 & 4.8 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined at 0 our and 96 hours of the exposure durations which were maintained with in range of 95.92 - 111.12%, 104.16 - 92.48%, 83.2 - 85.12%, 104.74 - 104.04%, 87.2 - 90.23% at 6.25, 12.5, 25, 50 and 100 mg/L concentrations. 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 mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be in the range of > 25 to < 50 mg/L, 37.5 mg/L (based on average of LC0 and LC100) . Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.
In a prediction done using EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to fish was predicted. On the basis of effect of chemical observed on the mortality of the test organism, the 96 hr lethal effect concentration (LC50) was estimated to be 2.81 mg/l. Thus, based on the LC50 value, chemical can be considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.
Another short term fish toxicity study was conducted for 120 hrs for assessing the effect of test chemical. Cyprinus carpio (Common carp) of weight 1 -10 pounds (average weight of around 3 pounds) captured with an alternating-current electric boat shocker in the New York State Barge Canal was used as a test organism for the study. After transportation of test organism to the laboratory, oxygen was supplied. Test fishes were acclimatized for weeks at spring temperature of 47˜F. Test chemical concentrations were not verified analytically. Test chemical concentrations used for the study were 66, 132 and 155 mg/Kg (nominal concentrations), respectively. Study was performed using fishes in a flow through system at 18.33°C (65°F) temperature, 6.7 pH and alkalinity of 10 mg/l CaCO3. Test fishes were exposed to test chemical in 350 and 550 gallons glass-fronted, fiber glass tank for 120 hr. After exposure period of 120 hr, mortality and other visual symptoms of the test organism was noted. No abnormal responses of the test fish were observed. On the basis of the effect on mortality of the test organism Cyprinus carpio, the 120 hr NOEC was determined to be 66-155 mg/Kg (nominal concentration).
In a supporting weight of evidence study from peer reviewed journals, authoritative database and secondary source, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The study was performed following the OECD Guideline 203 (Fish, Acute Toxicity Test) under flow through conditions. Oryzias latipes (Japanese Medaka) of 28 -43 days old and 18 -71 mg weight obtained from the Environmental Research Laboratory-Duluth (ERL-D) culture unit was used as a test organism. Test fishes were nurtured in tank at 25°C and fed with live Biomaine brand brine shrimp. Before 24 hr or during the study, test organism was not fed. 5 different concentrations of test chemical along with the control were taken for the study. Stock solutions of test chemical was prepared by dissolving the test chemical in Lake Superior water, using a high speed stirrer. Stock solutions were then transferred to a glass stock bottle inside the vented diluter enclosure using Teflon tubing and air pressure. During each test, a predetermined volume (ml/min) of stock solution was continuously pumped from the stock bottle into the mixing cell of the diluter system. Test chemical concentrations were verified analytically and analysis was carried out bya Hewlett Packard 5730A gas chromatograph (GC) equipped with a flame ionization detector (FID) linked to an HP 3350 lab automation system. All test analyses were accomplished using direct aqueous injection. GC column consists of a wall-coated open tubular silica column, 0.53 mm I.D. x 15 cm, with a 2.5 µ phase of bonded polyethlene glycol at isothermal oven temperature of 85, 120 and 110°C, respectively. Total 20 fishes/conc (10 organisms/replicate) were exposed to test chemical in a2.0 lit glass aquaria tank.2.0 l glass aquaria tank has a dimension of 18.5 X 14.0 X 13.0 cm deep. It has a 8.6 cm standpipe which resulted in a total volume of 2.0 lit. Continuous-flow mini diluter exposure system with vented enclosures was used for the study. Flow rate during the study was 25 ml/min with 90% replacement times of 2.8 hr. Lake superior water was used. It was filtered before use through sand, a 50-micron filter; a 5-micron filter; and then exposed to ultraviolet light before heating to the test temperature of 25±1°C.The test vessels were placed in a room at a temperature of 25±1°C, pH 7.88 ± 0.18 (7.31 to 8.85), dissolve oxygen (D. O) 6.8 ± 0.7 (5.0 to 8.5), hardness of water 45.8 (38.0 to 52.0) mg/l as CaCO3, alkalinity 45.9 (35.0 to 58.5 mg/l) as CaCO3 and under a 16 hr photoperiod with a light intensity of 12 to 25 lumens provided by fluorescent lamps for 96 hrs. All experiments were performed in replicate.95% confidence intervals were calculated using the binomial tests. Dissolved oxygen (D.O.) was measured by a dissolved oxygen meter. pH was determined on one set of replicate tanks atleast once and often twice during the test. On the other hand, hardness and alkalinity determinations were done at a minimum on a control, one intermediate and one at high test concentration tank; it was carried out once or twice during the study. Mortality was noted after an exposure period of 96 hrs. No mortalities were observed in the control vessel, the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 82.3%) of the air saturation value in test vessels throughout the study period and analytical monitoring of test concentrations has been carried out, thus fulfilling the validity criteria of the study. As the test concentrations were maintained within ±20% of the initial measured concentrations throughout the study, all results will be reported in nominal concentrations. On the basis of the effect on mortality of the test organismOryzias latipes(Japanese Medaka), the 96 hr LC50 was determined to be 4.0 mg/l (95% C. I. = 3.48 to 4.60 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria.
For the test chemical, 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 weight 0.473 g and average length of 1.76 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions 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 7.8 mg/l, pH 7.65, water temperature 24.5°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test chemical conentrations were not verified analytically. Nominal test chemical concentrations selected for the study were 0, 0.625,1.25, 2.5, 5 and 10 mg/L. respectively. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24.4°C, pH 7.03, hardness of water 152.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. The fishes were moving slowly in the test chemical conc. as compared to the control. No mortalities were observed in the control vessel and the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 90.96%) of the air saturation value in test vessels throughout the study period. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 2.5 to < 5.0 mg/L. Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
On the basis of the above results, it can be concluded that the test chemicalwas considered as toxic to aquatic fishes and hence, considered to be classified in ‘aquatic chronic category 2’ 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:
This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study (using a spacing factor of 1.5) was conducted using 0 (control), 21.72,32.58, 48.87, 73.30, 110 mg/L concentrations. 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups and 21.72 mg/L but 5%, 20%, 60% and 90% immobilisation were observed in the test concentrations of 32.58, 48.87, 73.30, 110 mg/L, respectively. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 32.58, 48.87, 73.30, 110 mg/L, conc were 97.42%, 102.76%, 95.58%, 105.38%, 102.22%, and 102.36%, respectively, after 48 hours of exposure, which were found in acceptable range. Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.0 -7.2), temperature (20-21 °C), dissolve oxygen (8.6 - 8.1 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 µS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to daphnid, Daphnia magna are 66.46. The 48-h EC50of reference item (Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. 48h EC50 value with 95% confidence limits (upper limit, lower limit) were calculated by probit analysis using STAT plus Software, version 8. Hence, as per CLP classification category the test chemical can be categorized as Chronic Category 3.
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 saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 162.82 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 6.25, 13.12, 27.55, 57.85 and 121.48 mg/l, respectively was from the saturated test concentration. 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 20°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 63.86 mg/L. Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per CLP classification criteria.
Another acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2017). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 100 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 1.2, 2.0, 3.5, 5.8 and 10.0 mg/l, respectively. Study was performed using 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. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. EC50 was calculated using non-linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. The dissolved oxygen concentration at the end of the test both in the control and test vessels was evaluated to be ≥ 3 mg/l (i.e, reported as > 8.6 mg/l), indicating that the validity criteria has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 3.6 mg/l (95 % C. I. - 2.7 to 4.8 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
In a supporting weight of evidence study, short term toxicity to aq. Invertebrate study was conducted for 24 hrs for assessing the effect of test chemical (Cecilia Labbe et. al; 2005 and authoritative database, 2017). Artemia salina (Brine shrimp) (one-day nauplii) was used as a test organism. Study was performed using 10 test organisms alongwith the test chemical in 10 ml of seawater solution in a static system. Test organism was exposed to different nominal test chemical concentrations (0, 2.5, 4, 10, 25 and 50 mg/l) for a period of 24 hrs. All experiments were performed in triplicates. Lindane was used as a reference substance for the study. Test organism survivors were counted after an exposure period of 24 hrs. The LC50 values were calculated using Finney’s Probit Analysis program. The 24 hr LC50 value of the reference substance lindane was determined to be 7.2 mg/l (95% C. I. = 4.9 to 9.7). On the basis of effect on the mortality of the test organism, the 24 hr LC50 value was determined to be < 2.5 mg/l. Thus, based on the LC50 value, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations.
For the test chemical from study report (2017), short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed following the OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 10 g/l was prepared by dissolving test chemical in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0, 1.0, 1.5, 2.2, 3.4 and 5.0 mg/l, respectively. Study was performed using total 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. Control solution vessel containing reconstituted water without the test chemical and other control vessel containing reconstituted water plus acetone without test chemical were also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. The dissolved oxygen concentration at the end of the test both in the control and test vessels was evaluated to be ≥ 3 mg/l (i.e, reported as > 7.4 mg/l), indicating that the validity criteria has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 1.7 mg/l (1.4 to 2.0 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
On the basis of the overall results, it can be concluded that the test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
Toxicity to aquatic 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 (2020),a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. 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. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 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. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. 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 growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis).
Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2017). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 50.0 g/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 0.7, 1.2, 2.0, 3.5, 5.8 and 10 mg/l, respectively. 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. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.0% i.e, not exceeded 7%, the biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 1.6 mg/l (95 % CI 1.3 - 2.1 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (2017). 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. 5000 cells /ml was used as a test organism. The stock solution 50 g/l was prepared by dissolving test chemical in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium. Test solutions were kept in ultrasonic bath for 20 min and then were inoculated by algae. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 0, 2.5, 3.8, 5.6, 8.4 and 12.7 mg/l, respectively. Study was performed in a static fresh water system for 72 hrs. 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. Control solution vessel containing OECD medium without the test chemical and other control vessel containing OECD medium plus acetone without test chemical were also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 0.7% in control and 1.1% in control plus acetone i.e, not exceeded 7%, the biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 5.5 mg/l (95% C. I. = 4.6 to 6.8 mg/l) (nominal concentration).
On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
Toxicity to microorganisms
Data available of the read across chemicals has been reviewed to determine the effect of the test chemical on toxicity to microorganisms. The studies are as mentioned below:
Toxicity to micro-organisms study was conducted on three different test organism, i.e, Vibrio fischeri, Photobacterium leiognathi SB strain and Tetrahymena thermophila. Studies were performed on these organisms using different bioassay test i.e, Microtox bioassay, Toxscreen bioassay and Protoxkit bioassay test. All stock solutions were prepared in methanol and serially diluted in distilled water to obtain the target concentrations. The methanol concentration in the exposure solutions, including controls, was 0.01% (v/v) in distillate water in the tested solutions. 2, 4 -dichloro phenol (DCP) was used as control solution for both Microtox bioassay and Toxscreen bioassay, whereas in Protoxkit bioassay test, Potassium dichromate (K2Cr2O7) was used as control substance. Test bacteria were exposed to the test chemical with a total exposure duration of 30 mins & 28 hrs in different bioassay. Bacterial luminescence inhibition was measured after 15 and 30 mins and inhibition in bacterial growth was measured after 24 and 28 hrs. On the basis of these effects on different test organism, the EC50 value can be determined to be in the range 0.11 to 5.7 mg/l and LOEC value was determined to be in the range 0.02 to 0.48 mg/l, respectively during the different exposure study duration.
In a supporting weight of evidence study, toxicity study of micro-organism study was carried out for assessing the effect of the test chemical. Study was performed using Photobacterium phosphoreum, strain NRRL-B-11177 (also referred to as Vibrio fischerii, strain NRRL-B-11177) as a test organism at a temperature of 15°C and pH range 5 to 9. Recommneded reference substance that can be used for the study were Phenol and Sodium pentachlorophenate, respectively. When the test bacterium was exposed to the test chemical, reduction in light output was observed. Thus, based on this effect, the EC50 value during 5, 15 and 30 min exposure period was determined to be 3.273, 3.859 and 4.52 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 and by evaluating the effect of test chemical on test organism, the EC50 value can be expected to be in the range 0.11 to 5.7 mg/l and LOEC value was expected to be in the range 0.02 to 0.48 mg/l, respectively during the different exposure study duration.
On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as toxic to aquatic organisms at environmental relevant concentrations and considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.
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