Cinnamaldehyde

Endpoint summary

Description of key information

Short terrm toxicity to fish

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical in fish. Study was performed following the EU Method C.1 (Acute Toxicity for Fish) (Council Directive 92/69/EEC C.1 (1992)). Danio rerio (Zebra fish) obtained from west-aquarium of 2.5 to 3.5 cm length was used as a test organism for the study. Test chemical concentrations were verified analytically by HPLC at the start and after 24 hr of the experiment. Nominal test chemical concentrations used for the study were 0, 2.8, 3.9, 5.5 and 7.8 mg/l, respectively. The test solution was preared by dissolved the test chemical into synthetic water and treated for 60 seconds at 8000 rpm with an ultra-turrax. Test fishes were exposed with the test chemical in a close ventilated test vessel. Study was performed using test fishes in a semi-static system at 20.7-22.8°C temperature, 7.5 - 8.2 pH, 8.2 - 9.6 (mg/l) dissolved oxygen and hardness 14.5 [°dH] under a photoperiod of 12:12 light:dark conditions for 96 hrs. Daily renewal of the test medium was done during the study. The LC0 and the LC100 are determined directly from the study, while the LC50 is calculated or read from the dose-response curve. On the basis of the effect of test chemical on the mortality of the test organism, the 24hr LC0, LC50 and LC100 value was determined to be 1.41 mg/l (measured conc.), 2.35 mg/l (measured conc.) & 3.93 mg/l (measured conc.) and the 96 hr LC50 & LC100 value was determined to be in the range of > 3.9 to < 5.5 mg/l (nominal conc.) and 5.5 mg/l (nominal conc.), respectively. Thus, based on the LC50 value, chemical was considered as toxic to aquatic fish. Since, the test chemical is readily biodegradable in water, chemical was considered to be non-toxic to aquatic fish at environmental relevant concentrations and thus considered to be 'not classified' as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.11, the long term toxicity on fish was predicted for test chemical. On the basis of effects observed in a flow through freshwater system on test organism, the NOEC value for the substance was estimated to be 15.159 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical was considered as non-toxic to fish at environmentally relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2016). 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 (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0.5, 1, 2, 3, 4 and 8 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. 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 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Chronic toxicity to aquatic invertebrate study was conducted for 21 days for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2019). The study was performed following the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of Adams media and stock solution was analytically detected and then the test solutions were prepared for achieving the test concentrations of 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/l, respectively. The solution of test chemical was prepared by dissolving 200 mg of test chemical in 200 ml of Adams medium. Test chemical concentrations were not verified analytically. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L, respectively. Daphnia magna (water flea) was used as a test organism. Test organisms were exposed to five test chemical concentrations in 100 ml glass beaker. In addition to this, control test vessel was also setup during the study. Test vessels were then placed at temperature of 18 – 22°C, hardness 155 mg/l as CaCO3, pH 7.4 (Range 6 to 9 pH), dissolved oxygen greater than 3 mg/l and under a photoperiod of 16 hour light and 8 hour dark with 1000 – 1500 lux light intensity. Reproduction rate and the mobility behaviour / mortality rate of parent Daphnia was assessed. The mortality in the control groups did not exceeded 20% percent. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the reproduction of the test organism Daphnia magna, the 21 days EC50 value was determined to be 0.402 mg/l (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2016). 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 (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration 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, 3, 6, 12, 24 and 50 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 100 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. 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 31.6 mg/l (95 % CI 25.9 - 38.6 mg/l) (nominal concentration). Thus, based on the EC50 value, 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 and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity to microorganism study was carried out for 3 hrs (Experimental study report, 1992). The study was performed in accordance with the ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation). Activated sludge obtained from aeration tank of a waste water treatment plant treating predominantly domestic sewage was used as a test organism. Pretreatment to sludge was given by providing the aeration and feeded with the synthetic medium daily. Nominal test chemical concentrations used for the study were 10, 18, 32, 56 and 100 mg/l, respectively. These chemical concentrations were not verified analytically. Study was performed in a static system for 3 hrs. 3.5 –dichlorophenol in the concentration range of 5 to 30 mg/l was used as a reference substance for the study. The respiration rate of an activated sludge was measured after a contact time of 3 hours. The EC values were determined by Probit analysis. On the basis of the effect of test chemical on respiration rate of the test organism activated sludge, the 3 hr EC50 value was determined to be 71 mg/l.

Additional information

Short terrm toxicity to fish

Various experimental studies of the test chemical were reviewed for the short term toxicity to fish end point which are summarized as below:

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical in fish. Study was performed following the EU Method C.1 (Acute Toxicity for Fish) (Council Directive 92/69/EEC C.1 (1992)). Danio rerio (Zebra fish) obtained from west-aquarium of 2.5 to 3.5 cm length was used as a test organism for the study. Test chemical concentrations were verified analytically by HPLC at the start and after 24 hr of the experiment. Nominal test chemical concentrations used for the study were 0, 2.8, 3.9, 5.5 and 7.8 mg/l, respectively. The test solution was preared by dissolved the test chemical into synthetic water and treated for 60 seconds at 8000 rpm with an ultra-turrax. Test fishes were exposed with the test chemical in a close ventilated test vessel. Study was performed using test fishes in a semi-static system at 20.7-22.8°C temperature, 7.5 - 8.2 pH, 8.2 - 9.6 (mg/l) dissolved oxygen and hardness 14.5 [°dH] under a photoperiod of 12:12 light:dark conditions for 96 hrs. Daily renewal of the test medium was done during the study. The LC0 and the LC100 are determined directly from the study, while the LC50 is calculated or read from the dose-response curve. On the basis of the effect of test chemical on the mortality of the test organism, the 24hr LC0, LC50 and LC100 value was determined to be 1.41 mg/l (measured conc.), 2.35 mg/l (measured conc.) & 3.93 mg/l (measured conc.) and the 96 hr LC50 & LC100 value was determined to be in the range of > 3.9 to < 5.5 mg/l (nominal conc.) and 5.5 mg/l (nominal conc.), respectively. Thus, based on the LC50 value, chemical was considered as toxic to aquatic fish. Since, the test chemical is readily biodegradable in water, chemical was considered to be non-toxic to aquatic fish at environmental relevant concentrations and thus considered to be 'not classified' as per the CLP classification criteria.

Another short term toxicity to fish study was conducted for 96 hrs for assessing the effect of test chemical (Experimental study report, 2013). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Poecilia reticulata (Guppy fish) of age 3 -4 months old, average weight 0.25 g and average length of 2.0 to 2.5 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 7.4 mg/l, pH 7 -8, water temperature 25°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. The test solution was prepared by dissolving 15mg, 25mg and 35mg of the test substance in 10 liters deionized water separately with continuous stirring for achieving the test concentration of 1.5 mg/L, 2.5 mg/L & 3.5 mg/L, respectively. Range finding test was conducted with the several test concentrations, i.e., 1.5 mg/L, 2.5 mg/L, 3.5 mg/L, 6.5 mg/L, 25 mg/L & 50 mg/L. But at 6.5 mg/L, 25 mg/L & 50 mg/L 100% mortality was observed within 24 hrs. Therefore, the experiment was continued only for three concentrations, i.e., 1.5 mg/L, 2.5 mg/L, 3.5 mg/L in which no mortality was found. In definitive study, limit test was conducted using a 100 mg/l test chemical concentration. 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 under a photoperiod of 12:12 hr light: dark conditions for 96 hrs. 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 and test vessel during the range finding study (at all three test concentrations i.e; 1.5, 2.5 and 3.5 mg/l). On the basis of this, the 96 hr LC0, median lethal concentrations (LC50) and LC100 value for test chemical on Poecilia reticulata (Guppy fish) was determined to be 3.5 mg/l, > 3.5 to < 6.5 mg/L and 6.5 mg/l, respectively.

 

For the test chemical, short term toxicity to fish study was conducted for 96 hrs for assessing the effect of test chemical (Secondary source, 2019). Lepomis macrochirus (Bluegillfish) was used as a test organism. Study was performed in a static system. On the basis of the effect on mortality of the test organism, the 96 hr NOEL and LC50 was determined to be 1.97 and > 20 mg/l. Thus, test chemical was considered as non-toxic to aquatic fish.

 

Additional short term toxicity to fish study from secondary source (2019) was conducted for 96 hrs for assessing the effect of test chemical. Lepomis macrochirus (Bluegillfish) was used as a test organism. Study was performed in a static system. On the basis of the effect on mortality of the test organism, the 96 hr NOEL and LC50 was determined to be 0.7 and 2.99 mg/l (95% C. I. = 2.24 to 4.0 mg/l), respectively.

 

Another short term toxicity to fish study was conducted for 96 hrs for assessing the effect of test chemical (2019). Oncorhynchus mykiss (Rainbow Trout) was used as a test organism. Study was performed in a static system. On the basis of the effect on mortality of the test organism, the 96 hr NOEL and LC50 was determined to be 1.25 and 1.67 mg/l (95% C. I. = 1.25 to 2.24 mg/l), respectively..

 

In a supporting weight of evidence study from review article and secondary source, short term fish toxicity was conducted for 47 hrs for assessing the effect of test chemical. Cyprinus carpio (Common carp) captured with an alternating-current electric boat shocker in the New York State Barge Canal of weight 1-10 pounds (average weight of around 3 pounds) 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 81 and 163 mg/l (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, fiberglass tank for 47 hr. After exposure period of 47 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 47 hr NOEC was determined to be in the range of 81-163 mg/l (nominal concentration).

 

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

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.11, the long term toxicity on fish was predicted for test chemical. On the basis of effects observed in a flow through freshwater system on test organism, the NOEC value for the substance was estimated to be 15.159 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical was considered as non-toxic to fish at environmentally relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

Various experimental studies of the test chemical were reviewed for the short term toxicity to aquatic invertebrate end point which are summarized as below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2016). 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 (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0.5, 1, 2, 3, 4 and 8 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. 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 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrates study was carried out for 48 hrs (from peer reviewed journal, authoritative database and secondary sources, 2019). The study was performed following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of< 24 hour old (Juvenile daphnids) produced from an in-house culture of adults was used as a test organism. Prior to the 48 hr study, the daphnid culture were maintained in 100% dilution water for 48 hrs. 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 7 days prior to the beginning of the test. Test chemical concentrations were verified analytically by HPLC/UV detector. Test chemical concentrations used for the study were 0, 2.0, 3.3, 5.5, 9.0, 15 and 25 mg/l (nominal concentrations) and 0.00452 (control), 1.91, 3.34, 5.30, 9.57, 13.9 and 25.5 mg/l (mean measured concentrations), respectively. Renewal of test solutions was carried every 24 hours. Study was performed using 10 daphnids/replicate for 48 hrs in a semi-static system in duplicates. Test vessels were placed at a temperature of19.5 to 20.7°C, pH6.8 to 7.1, dissolved oxygen of 8.2 to 9.1 mg/lunder a photoperiod of 16: 8 hr light : dark conditions. Mortality, immobility, and sub-lethal effects were determined visually at 0, 24, and 48 hours. Statistical analysis involve the use of Probit method. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration (i.e., maintained within 93-106% of nominal concentrations) throughout the test. Therefore, the analysis of the results were based on nominal concentration. On the basis of the effect on mobility and mortality of the test organism Daphnia magna, the 48 hr NOEC, EC50 and LC50 value was determined to be 1.91 mg/l, 3.86 mg/l and 4.22 mg/l (nominal concentrations), respectively.

 

For the test chemical, short term toxicity to aquatic invertebrates study was carried out for 48 hrs (authoritative database, 2019 and secondary sources). The study was performed following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of2-24 hour old was used as a test organism. Test chemical concentrations were verified analytically by GLC. Test chemical solutions were prepared by emulsification of the test substance in water with acetone. Test chemical concentrations used for the study were 0, 3.8, 7.5, 15.0, 30.0, and 60 mg/L (nominal concentrations) and 0, 3.8, 7.5, 15, 30 and 60 mg/l (mean measured concentrations), respectively. Study was performed using 5 daphnids/vessel for 48 hrs in a static system. Test vessels (25 ml glass beaker) were placed at a temperature of20.2 to 20.5°C, pH7.75 to 8.19, dissolved oxygen of 8.4 to 8.6 mg/l under a photoperiod of 16: 8 hr light : dark conditions with light intensity of 200 lx. After a period of 24 and 48 hrs, mobility of the test organisms were determined. Daphnia which showed no reaction after 15 seconds were considered immobile. The pH, oxygen content and temperature were measured at the beginning and end of the test. Probit analysis was performed for determining the EC50 value. On the basis of the effect on mobility of the test organism Daphnia magna, the 48 hr EC50 and EC100 value was determined to be 11.5 mg/l and 30 mg/l (nominal concentrations), respectively.

 

On the basis of the above results, it can be concluded that the test chemical was consideredastoxic to aquatic invertebrates at environmental related concentrations.Since, the test chemical is readily biodegradable in water, chemical was considered to be non-toxic to aquatic invertebrate and hence, considered to be 'not classified' as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates

Chronic toxicity to aquatic invertebrate study was conducted for 21 days for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2019). The study was performed following the OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of Adams media and stock solution was analytically detected and then the test solutions were prepared for achieving the test concentrations of 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/l, respectively. The solution of test chemical was prepared by dissolving 200 mg of test chemical in 200 ml of Adams medium. Test chemical concentrations were not verified analytically. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L, respectively. Daphnia magna (water flea) was used as a test organism. Test organisms were exposed to five test chemical concentrations in 100 ml glass beaker. In addition to this, control test vessel was also setup during the study. Test vessels were then placed at temperature of 18 – 22°C, hardness 155 mg/l as CaCO3, pH 7.4 (Range 6 to 9 pH), dissolved oxygen greater than 3 mg/l and under a photoperiod of 16 hour light and 8 hour dark with 1000 – 1500 lux light intensity. Reproduction rate and the mobility behaviour / mortality rate of parent Daphnia was assessed. The mortality in the control groups did not exceeded 20% percent. Thus, the validity criterion of the test has been fulfilled. On the basis of the effect of test chemical on the reproduction of the test organism Daphnia magna, the 21 days EC50 value was determined to be 0.402 mg/l (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Various experimental studies of the test chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2016),toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. 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 (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration 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, 3, 6, 12, 24 and 50 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 100 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. 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 31.6 mg/l (95 % CI 25.9 - 38.6 mg/l) (nominal concentration).

 

Another algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2015) 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 1 х 10E4 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 50 mg of test chemical in 100 ml of BBM to get the final concentration of 500 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Green algae were exposed to nominal concentration of test chemical (1.8 mg/L,3.6 mg/L, 7.2 mg/L, 14.4 mg/L,28.8 mg/L and 57.6 mg/L) in 100 ml conical flasks. 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. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (EC50) value was determined to be 16.09 mg/l.

 

For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (from A.M. Api et. al. (2019), authoritative database and secondary sources, 2019). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Selenastrum capricornutum (green algae) of strain UTEX 1648 with a control cell density of 366000 cells/ml was used as a test organism. Green algae, U. of Texas was maintained at test conditions for 14 days prior to the test. The culture was growing in at least 2 subcultures prior to the initiation of the test. Test chemical concentrations were verified analytically by HPLC/UV detector. Range finding study was performed. Test chemical concentrations used during the range finding study were 0.1, 1.0, and 100 mg/l. In a range finding test, the number of cells/mL was >100 % of controls at 0.10 mg/L, 80% at 1.0 mg/L, and <1% at 100 mg/L after three days. Test chemical concentrations used for the definitive study were 0, 0.50, 1.0, 2.0, 4.0 and 8.0 mg/l (nominal concentrations) and 0, 0.523, 1.04, 2.00, 3.80, and 7.03 mg/l (initial mean measured concentrations), respectively. Test organisms were exposed to test chemical for a period of 72 hrs at 23.2 to 24.0°C temperature, adjusted pH of 7.5 under a photoperiod of 16: 24 light: dark with light intensity provided using 400-410 candles. All test experiments were carried out in triplicates and controls were setup in 6 replicate. After an exposure period of 72 hrs, the number of algal cells/ml, relative size, cell shapes, color, adherence and aggregation of cells was determined. At 24, 48, and 72 hours treatment and control vessels were sacrificed to determine the number of algal cells/ml.EC50 values determined by weighted least squares non-linear regression. NOEC was determined using a one-way analysis of variance (ANOVA) and Bonferroni's test. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration (i.e., maintained within 88-105% of nominal concentrations) throughout the test. Therefore, the analysis of the results were based on nominal concentration. On the basis of the effect on number of cells/ml of the test organism Selenastrum capricornutum, the 72 hrs NOEC was determined to be 2.0 mg/l and based on the effect on growth rate, number of cells/ml & area under the growth curve, theEC50 value was determined to be 6.87 mg/l, 4.56 mg/l and 4.07 mg/l, respectively.

 

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

Toxicity to microorganisms

Various experimental studies of the test chemical were reviewed for toxicity to microorganism end point which are summarized as below:

 

In an experimental key study, toxicity to microorganism study was carried out for 3 hrs (Experimental study report, 1992). The study was performed in accordance with the ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation). Activated sludge obtained from aeration tank of a waste water treatment plant treating predominantly domestic sewage was used as a test organism. Pretreatment to sludge was given by providing the aeration and feeded with the synthetic medium daily. Nominal test chemical concentrations used for the study were 10, 18, 32, 56 and 100 mg/l, respectively. These chemical concentrations were not verified analytically. Study was performed in a static system for 3 hrs. 3.5 –dichlorophenol in the concentration range of 5 to 30 mg/l was used as a reference substance for the study. The respiration rate of an activated sludge was measured after a contact time of 3 hours. The EC values were determined by Probit analysis. On the basis of the effect of test chemical on respiration rate of the test organism activated sludge, the 3 hr EC50 value was determined to be 71 mg/l.

 

Another toxicity to micro-organism study was carried out for 120 minutes (Secondary source, 2000). Bacteria was used as a test organism. On the basis of effect on growth inhibition of the test organism bacteria, the 120 min EC100 value was determined to be in the range of 1000 to 20000 mg/l.

On the basis of the above results, the 3 hr EC50 value was evaluated to be 71 mg/l.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as toxicto aquatic organisms at environmental relevant concentrations and considered to be classified in 'aquatic chronic category 3' as per CLP classification criteria.