undecan-2-one; methyl nonyl ketone

Endpoint summary

Description of key information

Short term toxicity to fish

An acute toxicity to fish study was carried out for 96 hrs (from authoritative databases and secondary sources). Pimephales promelas (Fathead Minnow) of 31 d old was used as a test fishes. It has a length of 21.9 mm and weight of 0.157 gm, respectively. Test fishes were cultured at the U. S. EPA Environmental Research Laboratory-Duluth and the University of the Wisconsin-Superior campus. Adults were held in flowing water with a 16 hr light controlled photoperiod and fed frozen adult brine shrimp. They were provided with asbestos pipes (cut in half-longitudinally) as spawning substrates. Test fishes were not fed 24 hr before or during the test. Study was performed under flow through conditions at a temperature of 25.1°C, hardness of 48.8 mg/L CaCO3, dissolved oxygen of 6.6 mg/l and pH of 7.7 mg/l, respectively. Test chemical analysis was carred out either using gas-liquid chromatography, high performance liquid chromatography and UV-visible spectrophotometry. Lake Superior water or water from the municipal suplly for the City of Superior, Wisconsin, which is derived from wells beneath Lake Superior and it is very similar in its natural chemical characteristics to Lake Superior water. A continuous flow mini-diluter, an ABC solenoid-operated electronic diluter, or a single cell/glass column system was used as a test vessel. Prior to initiating a test, prospective test fish were ''pooled'' if reared in more than one tank. At the start of the test, individuals were removed from the ''common pool'' of fish with a net and distributed at random among the exposure chambers. The tests were intiated by adding 20, 10 or 5 fish per treatment and control for the mini, electronic, and single cell/glass column systems, respectively. Fish loading did not exceed 0.5 g/l/day in flow through systems. All experiments were performed in duplicates. All test exposure chambers were sampled at 0, 24, 48, 72 and 96 hr. It was analyzed immediately or adequately preserved for later analysis. The number of dead fish was noted after every 24 hr after the beginning of a test, at which time they were also removed. Observations of fish behavior and toxic signs were noted. Unique behavior was also recorded using a color video camera and 0.5 inch video tape recorder. The LC50 value with corresponding 95% C. I. were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber method. On the basis of the effect on mortality of the test fishes, the 96 hr LC50 value was determined to be 1.5 mg/l (95% C. I. = 1.39 to 1.62 mg/l). Thus, test chemical was considered as toxic to fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

Short term toxicity to aquatic invertebrates

An acute toxicity to aquatic invertebrate was carried out for assessing the effect of test chemical. Study was performed following the principles of the OECD TG 202 (Dapnia sp. Acute immobilization test). Daphnia magna (<24 hr old) obtained from Shell Research Laboratories was used as a test organism. Daphnids were cultured a temperature of 20°C with a dissolved oxygen of >60% ASV, photoperiod of 16:8 light:dark conditions. Each working day, a suspension of Chlorella vulgaris at a rate of l mg organic carbon per litre of culture water. The Daphnia magna for the test were cultured under semi-static conditions and were not fed during the test. 10 mg/l stock solution of test chemical was prepared by initially dissolving the substance in dimethyl formamide (DMF) prior to dilution in dechlorinated water. The test concentrations were prepared by addition of the appropriate volume of this stock solution to the dilution water to give the required concentrations for the test. Test chemical conc. taken for the study were 0 (control), 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0mg/l, respectively. Samples of the test solutions in which the organisms were exposed were taken for analytical verification at the start of the definitive study, before and after renewal of solutions at 24 hours and at the end of the 48 hour exposure period. Test substance specific analysis of each test concentration was carried out as soon as possible after sampling. Analytical measurements were carried out GC. A further control solution was prepared containing dimethyl formamide at a concentration of 1 ml DMF/l. Daphnids (total 20 daphnids) were exposed to different test chemical conc. in each test vessel. 5 Daphnia were placed at random in each dish containing 25 ml of prepared test medium, diluent water only or diluent water plus 1 ml DMF per litre, as appropriate. The dishes were covered with a transparent perspex sheet to limit aerial contamination of the test vessels and reduce evaporative losses. The test vessels were placed into an incubator at a temperature of 20 ± 1°C, hardness of 240 mg/l as CaCO3, DO of 98-100% ASV with a light cycle of 16 hr light and 8 hr dark and without supplementary aeration or feeding during the 48 hour exposure period. The 48 hour EC50 value of test chemical to Daphnia magna and the highest no observed effect concentration (NOEC) after 48 hours were noted. Temperature, pH values and dissolved oxygen concentration were also recorded at the start of the study, before and after renewal of solutions at 24 hours and at the end of the 48-hour exposure period. Reference substance were also run simultaneously during the study. All experiments were performed in 5 replicates. The EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software". The analytical data clearly demonstrated that recovery of test chemical from solution varies with typical recovery rates at 0 hours of 47 to 80%. After 24 hours recovery is shown to have dropped significantly to only 0.5 to 7.0%. Similar values were obtained for the replacement solutions at 24 hours, with initial recovery of 38-to 91 % of the nominal value, which dropped to 2 to 5% after the solutions had aged for a further 24 hours. The water quality measurements of the test solutions were within acceptable limits. Specifically, the dissolved oxygen concentration remained greater than 60% of the air saturation value, which fulfils the validity criteria of the study. None (0%) of the twenty control Daphnia, maintained in dilution water, or in lml/l DMF were immobilised during the study, and no Daphnia were trapped at the surface of the water in the control vessels, this fulfils the validity criteria of the study which states that control immobilisation must not exceed 10% at the end of the test. The 48 hr EC50 value of the reference substance was determined to be 0.9 mg/l (95% C. I. 0.8 to 1.1 mg/l). On the basis of the effect on immobilization of test daphnids, the 48 hr EC50 value was determined to be 0.23 mg/l (based on actual measured (arithmetic mean) concentrations). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Toxicity to aquatic algae study was carried out for 72 hrs (Secondary source, 2008). Study was performed following the principles of the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) was used as a test algae. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 38 – 91 % and ranged from 0.5 – 7.0 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively. Initial cell density of test algae was 10000 cells/ml. Study was performed under static conditions. Test conditions involve a pH of 8.0 – 8.1 (at start), 7.1 – 9.5 (at end); temperature of 22°C under a continuous photoperiod and a light intensity of 7000 lux. All test treatment and vehicle control system were performed in triplicates whereas control system were performed in 6 replcates. At all test concentrations, growth of the algae was delayed. Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively. Thus, based on the EC50 value, 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 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity to microorganisms study was carried out for 3 hrs (Secondary source, 2008). Study was performed in accordance with the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test. Activated sludge, domestic was used as a test organism. Test inoculum were exposed to different nominal test chemical conc. (i.e., 0, 60, 120, 240, 480, 960 mg/l (nominal conc.)) for a period of 3 hrs. Test trestment was performed in 1 replicate and control system was performed in duplicates. Test conditions involve a temperature of 20 ± 2°C and a pH of 7.88 (at the start), respectively. 3,5-dichlorophenol was used as a reference substance. The 3 hr EC50 value of the reference substance was determined to be 6.75 mg/l. After 3 hours of contact time the inhibition of respiration by the test item was – 2 %, 22 %, 37%, 65 % and 68 % hours for the nominal test concentrations of 60, 120, 240, 480, 960 mg/L respectively. Respiration rates for the two control vessels were comparable. Based on the effect of test chemical on respiration inhibition of the test bacteria, the 3 hr EC50 value was determined to be 379.49 mg/l.

Additional information

Short term toxicity to fish

Various experimental studies and predicted data of the test chemical and its read across chemical were reviewed for short term toxicity to fish end point which are summarized as below:

 

In an experimental study from authoritative databases and secondary sources, an acute toxicity to fish study was carried out for 96 hrs. Pimephales promelas (Fathead Minnow) of 31 d old was used as a test fishes. It has a length of 21.9 mm and weight of 0.157 gm, respectively. Test fishes were cultured at the U. S. EPA Environmental Research Laboratory-Duluth and the University of the Wisconsin-Superior campus. Adults were held in flowing water with a 16 hr light controlled photoperiod and fed frozen adult brine shrimp. They were provided with asbestos pipes (cut in half-longitudinally) as spawning substrates. Test fishes were not fed 24 hr before or during the test. Study was performed under flow through conditions at a temperature of 25.1°C, hardness of 48.8 mg/L CaCO3, dissolved oxygen of 6.6 mg/l and pH of 7.7 mg/l, respectively. Test chemical analysis was carred out either using gas-liquid chromatography, high performance liquid chromatography and UV-visible spectrophotometry. Lake Superior water or water from the municipal suplly for the City of Superior, Wisconsin, which is derived from wells beneath Lake Superior and it is very similar in its natural chemical characteristics to Lake Superior water. A continuous flow mini-diluter, an ABC solenoid-operated electronic diluter, or a single cell/glass column system was used as a test vessel. Prior to initiating a test, prospective test fish were ''pooled'' if reared in more than one tank. At the start of the test, individuals were removed from the ''common pool'' of fish with a net and distributed at random among the exposure chambers. The tests were intiated by adding 20, 10 or 5 fish per treatment and control for the mini, electronic, and single cell/glass column systems, respectively. Fish loading did not exceed 0.5 g/l/day in flow through systems. All experiments were performed in duplicates. All test exposure chambers were sampled at 0, 24, 48, 72 and 96 hr. It was analyzed immediately or adequately preserved for later analysis. The number of dead fish was noted after every 24 hr after the beginning of a test, at which time they were also removed. Observations of fish behavior and toxic signs were noted. Unique behavior was also recorded using a color video camera and 0.5 inch video tape recorder. The LC50 value with corresponding 95% C. I. were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber method. On the basis of the effect on mortality of the test fishes, the 96 hr LC50 value was determined to be 1.5 mg/l (95% C. I. = 1.39 to 1.62 mg/l). Thus, test chemical was considered as toxic to fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

Another short term toxicity to fish study was carried out (from authoritative databases, secondary sources and PPDB database). Oncorhynchus mykiss (Rainbow trout) of 1.2 gm was used as a test fishes. Study was performed under static conditions for a period of 96 hrs. On the basis of the effect on mortality of the test fishes, the 96 hr LC50 value was determined to be 3.0 mg/l (95% C. I. = 2.2 to 4.4 mg/l). Thus, test chemical was considered as toxic to fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

In a supporting study from authoritative databases and secondary sources, short term toxicity to fish study was carried out. Lepomis macrochirus (Bluegill) of 1.0 gm in weight was used as a test fishes. Study was performed under static conditions for a period of 96 hrs. On the basis of the effect on mortality of the test fishes, the 96 hr LC50 value was determined to be 2.1 mg/l (95% C. I. = 1.6 to 3.1 mg/l). Thus, test chemical was considered as toxic to fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

In a prediction done using the EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to aquatic fish was predicted. On the basis of effect of test chemical observed in a static system on the mortality of the test organism during the 96 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 4.213 mg/l. Thus, based on the LC50 value, 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 and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

For the test chemical, short term toxicity to fish study was carried out. Study was performed as per the OECD Guideline 203 (Fish, Acute Toxicity Test). Oryzias latipes (Japanese rice fish (Medaka)) was used as a test fishes. Conc. of vehicle used in the test solution was 0.1 ml/l. Test fishes (10 fishes/conc.) were exposed to different test chemical conc. (i.e., 0 (control), 0 (vehicle control), 1.0, 1.8, 3.2, 5.6, 10 mg/L) for a period of 96 hrs under semi-static conditions. Total amount of the test solution was renewed every 24 hours. Aeration was not provided in the test vessel during the study. Test conditions involve a temperature of 24 ± 1°C under a photoperiod of 16 hours light / 8 hours dark in a room light. On the basis of the effect on mortality of the test fishes, the 96 hr LC50 value was determined to be 1.7 mg/l. Thus, test chemical was considered as toxic to fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

On the basis of the above results, it can be concluded that the 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 and hence, considered to be ‘not classified’ as per CLP classification criteria

Short term toxicity to aquatic invertebrates

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

In an experimental study, an acute toxicity to aquatic invertebrate was carried out for assessing the effect of test chemical. Study was performed following the principles of the OECD TG 202 (Dapnia sp. Acute immobilization test). Daphnia magna (<24 hr old) obtained from Shell Research Laboratories was used as a test organism. Daphnids were cultured a temperature of 20°C with a dissolved oxygen of >60% ASV, photoperiod of 16:8 light:dark conditions. Each working day, a suspension of Chlorella vulgaris at a rate of l mg organic carbon per litre of culture water. The Daphnia magna for the test were cultured under semi-static conditions and were not fed during the test. 10 mg/l stock solution of test chemical was prepared by initially dissolving the substance in dimethyl formamide (DMF) prior to dilution in dechlorinated water. The test concentrations were prepared by addition of the appropriate volume of this stock solution to the dilution water to give the required concentrations for the test. Test chemical conc. taken for the study were 0 (control), 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0mg/l, respectively. Samples of the test solutions in which the organisms were exposed were taken for analytical verification at the start of the definitive study, before and after renewal of solutions at 24 hours and at the end of the 48 hour exposure period. Test substance specific analysis of each test concentration was carried out as soon as possible after sampling. Analytical measurements were carried out GC. A further control solution was prepared containing dimethyl formamide at a concentration of 1 ml DMF/l. Daphnids (total 20 daphnids) were exposed to different test chemical conc. in each test vessel. 5 Daphnia were placed at random in each dish containing 25 ml of prepared test medium, diluent water only or diluent water plus 1 ml DMF per litre, as appropriate. The dishes were covered with a transparent perspex sheet to limit aerial contamination of the test vessels and reduce evaporative losses. The test vessels were placed into an incubator at a temperature of 20 ± 1°C, hardness of 240 mg/l as CaCO3, DO of 98-100% ASV with a light cycle of 16 hr light and 8 hr dark and without supplementary aeration or feeding during the 48 hour exposure period. The 48 hour EC50 value of test chemical to Daphnia magna and the highest no observed effect concentration (NOEC) after 48 hours were noted. Temperature, pH values and dissolved oxygen concentration were also recorded at the start of the study, before and after renewal of solutions at 24 hours and at the end of the 48-hour exposure period. Reference substance were also run simultaneously during the study. All experiments were performed in 5 replicates. The EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software". The analytical data clearly demonstrated that recovery of test chemical from solution varies with typical recovery rates at 0 hours of 47 to 80%. After 24 hours recovery is shown to have dropped significantly to only 0.5 to 7.0%. Similar values were obtained for the replacement solutions at 24 hours, with initial recovery of 38-to 91 % of the nominal value, which dropped to 2 to 5% after the solutions had aged for a further 24 hours. The water quality measurements of the test solutions were within acceptable limits. Specifically, the dissolved oxygen concentration remained greater than 60% of the air saturation value, which fulfils the validity criteria of the study. None (0%) of the twenty control Daphnia, maintained in dilution water, or in lml/l DMF were immobilised during the study, and no Daphnia were trapped at the surface of the water in the control vessels, this fulfils the validity criteria of the study which states that control immobilisation must not exceed 10% at the end of the test. The 48 hr EC50 value of the reference substance was determined to be 0.9 mg/l (95% C. I. 0.8 to 1.1 mg/l). On the basis of the effect on immobilization of test daphnids, the 48 hr EC50 value was determined to be 0.23 mg/l (based on actual measured (arithmetic mean) concentrations). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

 

In an experimental study from secondary source, an acute toxicity to aquatic invertebrate study was carried out for 48 hrs. Study was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of <24 hr old was used as a test organism. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 47 – 80 % and ranged from 0.5 – 7 % after 24 hours. After renewal, initial recovery (0 hours) ranged from 38 – 91 % and ranged from 2 – 5 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control), 13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l, respectively. Test daphnids (5 daphnids/test vessel) were exposed to different test chemical conc. in a semi-static system. Test conditions involve a pH 7.7 to 8.0, temperature of 20°C, total hardness of 240 mg/L CaCO3 and dissolved oxygen of 98 –100 % O2 saturation, respectively with a photoperiod of 16: 8 light:dark cycle and light intensity of 350 lux. All experiments were performed in 4 replicates. No immobilization occurred in the control or in the solvent control group. No immobilization was noted after 48 hours up to the treatment level of 0.25 mg/L.  After 48 hours immobilization in the treatment groups of 0.50, 1.0, 2.0 and 4.0 mg/L was 15 %, 30 %, 30 % and 75 % respectively. On the basis of the effect of test chemical on mobility of test daphnids, the 48 hr EC50 value was determined to be 2.3 mg/l. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates. 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 short term toxicity to aquatic invertebrate study was carried out for 48 hrs (J-CHECK, 2021). Study was performed in accordance with the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Test was carried out under static system using Daphnia magna (Water flea) as a test organism. On the basis of the effect of test chemical on mobility of test daphnids, the 48 hr EC50 value was determined to be 1.3 mg/l. Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates. 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.

 

In a supporting weight of evidence study from peer reviewed journals, short term toxicity to aquatic invertebrate study was carried out for 48 hrs. Study was performed in accordance with the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Test was carried out under static system using Daphnia magna (Water flea) as a test organism. On the basis of the effect of test chemical on mobility of test daphnids, the 48 hr EC50 value was determined to be 0.23 mg/l (measured conc.). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

 

For the test chemical, short term toxicity to aquatic invertebrate study was carried out for 48 hrs (from authoritative databases and secondary sources). Test was carried out under static system. Daphnia magna (Water flea) of <24 hr old was used as a test organism. On the basis of the effect of test chemical on mobility of test daphnids, the 48 hr EC50 value was determined to be 0.54 mg/l (95% C. I. = 0.3 to 2.1 mg/l). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

 

In a prediction done using the EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to aquatic invertebrates was predicted. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 2.797 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic invertebrates. 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.

 

Additional short term toxicity to aquatic invertebrate study was carried out for 48 hrs. Study was performed in accordance with the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Conc. of vehicle used in the test solution was 100 microL/L. Test was carried out under semi-static system using Daphnia magna (Water flea) as a test organism. Total amount of the test solution was renewed every 24 hours. Test daphnids (20 daphnids per concentration) were exposed to different test chemical conc. (i.e., 0 (control), 0 (vehicle control), 0.10, 0.22, 0.46, 1.0, 2.2 mg/L) for a period of 48 hrs. Test conditions involve a temperature of 24 ± 1° C under a photoperiod of 16 hours light / 8 hours dark in a room light. All experiments were performed in 4 replicates. On the basis of the effect of test chemical on mobility of test daphnids, the 48 hr EC50 value was determined to be 0.61 mg/l (95% C. I. = 0.49 to 0.75 mg/l). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' 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 invertebrates and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Various experimental studies and predicted data of the test chemical and its read across chemical were reviewed for toxicity to aquatic algae end point which are summarized as below:

 

In an experimental study from secondary source (2008), toxicity to aquatic algae study was carried out for 72 hrs. Study was performed following the principles of the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) was used as a test algae. Test chemical analysis was carried out using gas chromatography with flame ionization detection. Initial recovery (0 hours) ranged from 38 – 91 % and ranged from 0.5 – 7.0 % after 24 hours Dimethyl formamide was used as a vehicle. Test chemical conc. used for the study were 0 (control); 0 (solvent control (dimethyl formamide)); 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 mg/l, respectively. Initial cell density of test algae was 10000 cells/ml. Study was performed under static conditions. Test conditions involve a pH of 8.0 – 8.1 (at start), 7.1 – 9.5 (at end); temperature of 22°C under a continuous photoperiod and a light intensity of 7000 lux. All test treatment and vehicle control system were performed in triplicates whereas control system were performed in 6 replcates. At all test concentrations, growth of the algae was delayed. Based on the effect of test chemical on growth rate of test algae, the 48 & 72 hr EC50 value was determined to be 7.3 and 14.3 mg/l, respectively. On the basis of the effect on biomass, the 48 & 72 hr EC50 value was determined to be <2.5 and 2.9 mg/l, respectively. Thus, based on the EC50 value, 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 'not classified' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was carried out for 72 hrs. Study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 72 hr NOEC and EC50 value was determined to be 0.33 and 1.9 mg/l, respectively. Thus, based on the EC50 value, 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 'not classified' as per the CLP classification criteria.

 

Another acute toxicity to aquatic algae was carried out for assessing the effect of test chemical. Study was performed following the principles of the OECD TG 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test).  Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany) was used as a test algae. The algae were cultivated in RCC’s laboratories under standardized conditions according to the test guidelines. Due to the limited solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared by mixing 109.2 mg of the test item into 1090 mL of test water using intense stirring. The stirring vessel was completely filled and sealed by a glass stopper. The dispersion was stirred for 24 hours at room temperature in the dark in order to dissolve a maximum amount of the test item in the dispersion. After the stirring period, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. The negative pressure of the filtration unit was reduced as much as possible to avoid losses of the test item during filtration. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test. Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing with modifications according to the International Standard ISO 14442 [2]. The modifications were made to improve the growth conditions for the algae in a closed test system.The concentration of NaHCO3 in the test water was increased by 200 mg/L to 250 mg/L (as carbon source for the algal growth), and 6 mmol/L HEPES-buffer were added to keep the pH in the test media during the test period as constant as possible.Analytical measurements of test chemical conc. were done by using HPLC analysis with UV/Vis detection. For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. Additionally, one flask of test medium of the dilution 1:10 was incubated without algae under test conditions in order to document possible adsorption of the test item onto the algae. Immediately after sampling, the samples were diluted 1:1 (v/v) with acetonitrile in order to stabilize the samples during the storage period. All samples were stored at about -20 °C until analysis. In pre-experiments (non-GLP), the test item proved to be stable in the test water under these storage conditions.The concentrations of the test chemical were determined in the duplicate test medium samples from the dilutions of 1:32, 1:10, 1:3.2 and the undiluted filtrate. The samples from the dilution of 1:100 were not analyzed, since this concentration was below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times. A saturated solution (filtrate) and the dilutions 1:3.2, 1:10, 1:32 and 1:100 of the saturated solution were tested. Additionally, a control was tested in parallel (test water without test item). The enlarged spacing factor of 3.2 between the test concentrations was chosen, as the concentration-effect relationship was flat according to the results of the range-finding test. Thus, a wide concentration range had to be tested. 50-mL Erlenmeyer flasks were used per replicate. Each test flask was filled with approximately 60 mL algal suspension and sealed with a glass stopper to prevent loss of the volatile test item. The test flasks were labeled with the RCC study number and all necessary additional information to ensure unique identification. During the test, the test solutions were continuously stirred by magnetic stirrers. The test design included three replicates per test concentration and six replicates of the control. The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM). The initial cell density corresponded to 1.1 x 10E3 relative fluorescence units. The conversion factor between algal cell density and fluorescence signal was therefore 9.0 cells/mL per fluorescence unit. A static test design in a closed test system was applied. The duration of the test was 72 hours. Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test condition. The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from a test concentration with reduced algal growth (dilution 1:3.2). The shape and size of the algal cells were visually inspected. This test concentration was chosen, since the algal cell density was too low for a reliable examination at the highest test concentration. During the test period of 72 hours, the highest inhibitory effect on the growth of the algae was determined during the first day of the test. A lag phase was determined in the algal cultures exposed to the test item. During the second and third day of the test, a recovery of the algal growth was determined in the test item treatments with the exception of the highest test concentration in which the algal growth was completely inhibited. The lag phase in the exposed algal cultures may indicate recovery after initial toxic stress or reduced exposure due to loss of the test item (although the test was performed in a closed test system). Therefore, the algal growth inhibition determined during the first day of the test was taken into account for the evaluation of the study and the biological results were related to the initial measured concentrations of the test item in the test media.The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the dilution of 1:3.2 and the algal cells in the control. The test item did not affect the shape and size of the algal cells up to at least this concentration. In the control the biomass increased by a factor of 242 over 72 hours (>16). The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, during 72 hours was 17%. (< 35%). The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.3%. (< 7%). The EC10, EC20 and EC50 values for the inhibition of growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis. For the determination of the LOEC and NOEC, growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests. Only effect concentration at 24h were derived due to technical impossibilities and lack of detectable levels of test substance at 72h. The 72 hr EC50 value of the reference substance was determined to be 1.2 mg/l. The 24 hr EC50 value of the test chemical was determined to be 1.9 mg/l. Thus, test chemical was considered as toxic to aquatic algae.

 

In a supporting weight of evidence study frompeer reviewed journal (A.M. Apia et. al., 2019), toxicity to aquatic algae study was carried out for 72 hrs. Study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 24 hr EC10 value was determined to be 0.79 mg/l. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae.

 

For the test chemical, toxicity to aquatic algae study was carried out for 72 hrs (PPDB database, 2021). Pseudokirchneriella subcapitata (green algae) was used as a test organism. Test was performed under static conditions. On the basis of the effect of test chemical on growth rate of test algae, the 72 hr EC50 value was determined to be >0.25 mg/l. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae.

 

In a prediction done using the EPI Suite ECOSAR version 1.11, the toxicity of the test chemical to aquatic algae was predicted. On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 3.978 mg/l. Thus, based on the EC50 value, 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 'not classified' as per the CLP classification criteria.

 

Additional toxicity to aquatic algae study was carried out for 72 hrs. Study was performed following the OECD Guideline 201 (Alga, Growth Inhibition Test). Test was performed under static conditions. Based on the effect of test chemical on growth rate of test algae, the 72 hr NOEC and EC50 value was determined to be 0.56 and 0.028 mg/l, respectively. On the basis of the effect on area under the growth curve (AUG), the 72 hr NOEC and EC50 value was determined to be 0.72 and 0.09 mg/l, respectively. Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae.

Toxicity to microorganisms

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

 

In an experimental study from secondary source, toxicity to microorganisms study was carried out for 3 hrs. Study was performed in accordance with the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test. Activated sludge, domestic was used as a test organism. Test inoculum were exposed to different nominal test chemical conc. (i.e., 0, 60, 120, 240, 480, 960 mg/l (nominal conc.)) for a period of 3 hrs. Test trestment was performed in 1 replicate and control system was performed in duplicates. Test conditions involve a temperature of 20 ± 2°C and a pH of 7.88 (at the start), respectively. 3,5-dichlorophenol was used as a reference substance. The 3 hr EC50 value of the reference substance was determined to be 6.75 mg/l. After 3 hours of contact time the inhibition of respiration by the test item was – 2 %, 22 %, 37%, 65 % and 68 % hours for the nominal test concentrations of 60, 120, 240, 480, 960 mg/L respectively. Respiration rates for the two control vessels were comparable. Based on the effect of test chemical on respiration inhibition of the test bacteria, the 3 hr EC50 value was determined to be 379.49 mg/l.

 

Another toxicity to microorganisms study was carried out. Tetrahymena pyriformis (ciliate) was used as a test bacteria. Stock solutions of each ketone were prepared in dimethyl sulfoxide (DMSO) at concentrations of 2.5, 5, 10, 25, or 50 parts per million. In all cases, the volume of stock solution added to each test flask did not exceed 0.35 ml, an amount that does not alter Tetrahymena population growth. Chemical was tested in a range-finder prior to testing in duplicate for three additional replicates. Only replicates with control-absorbency values from 0.6 to 0.9 were used in the analyses. Test bacteria were exposed to different test chemical conc. for a period of 2 days. All experiments were performed in duplicates. Population density was measured spectrophotometrically at 540 nm. The 50 percent growth inhibitory concentration, IGC50, was determined for each ketone using Probit Analysis of Statistical Analysis System (SAS) softwareI6 with Y as the absorbency normalized as percentage of control and X as the toxicant concentration in parts per million. Based on the effect on growth inhibition of the test bacteria, the 2 d IC50 value was determined to be 3652.8 mg/l (IC50 = 21.45 mM).

 

In a supporting study from peer reviewed journal and REAXY’s database,toxicity to microorganisms study was carried out. Vibrio fisheri was used as a test bacteria. It was bacteria were cultured to late log-phase growth and harvested. Bacteria was being frozen and maintained at -70°C in solution containing 15% dimethyl sulfoxide (DMSO). Test bacteria was thawed prior to testing and prepared in a 1:10 dilution in 2% NaCl. Stock solutions of each chemical were prepared in DMSO. Two-to-one serial dilutions of test chemical was made using 2% saline solution. Light emission was monitored using a Model 500 Analyzer (Azur Corp., Carlsbad, CA). All experiments were performed in duplicates. The 15-min EC50 values were determined using the Microtox Data Capture and Reporting Program, Version 7.82 (Azur Corp.). On the basis of the effect on growth inhibition of the test bacteria, the 15 mins EC50 value was determined to be 199.52 mg/l and the logarithm of the inverse of 15-min toxicity (pT15) was evaluated to be 2.3, respectively.

 

Additional toxicity to microorganisms study was carried out (Mark T. D. Cronin et. al., 1998). Tetrahymena pyriformis (ciliate) was used as a test bacteria. Study was performed under static conditions for 40 hrs. Population density of test organism Tetrahymena pyriformis was quantified spectrophotometrically. The reported endpoint is the 50% growth inhibitory concentration, IGC50. On the basis of the effect of test chemical on growth inhibition of the test bacteria, the 40 hr IC50 value was determined to be 31.62 mg/l (log(1/IG50) = 1.5).

 

For the test chemical, toxicity to microorganisms study was carried out. Tetrahymena pyriformis (ciliate) was used as a test bacteria. Study was performed under static conditions for 40 hrs. On the basis of the effect of test chemical on growth inhibition of the test bacteria, the 40 hr EC50 value was determined to be 5.76 mg/l.

 

On the basis of the above results, EC50 value of the test chemical was evaluated to be 5.76 to 3652.8 mg/l, respectively.

On the basis of the available information of aquatic toxicity studies, test chemical was considered as toxic to aquatic organisms at environmental relevant concentrations and hence,considered to be classified in 'aquatic acute category 1/chronic category 1' as per the CLP classification criteria.