Decan-5-olide

Endpoint summary

Description of key information

Short tertm toxicity to fish:

The objective of this study was to assess the acute toxicity of test chemical in fresh water Fish (Danio rerio). The study was performed in compliance with OECD 203 Guideline for the testing of chemicals; Fish, Acute Toxicity Testing, (18th June, 2019). Solubility of the test item was performed by weighed 25.7 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark using natural water and the resulting concentration was 102.8 mg/L. Stability of the test item in natural water determined by analyzing the test concentrations of 0.1 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.1 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. Range finding test was conducted with test concentration of 0.48 and 50.00 mg/L along with a control groups without test item by static method. Each concentration contained seven fish. Test item was formulated in dilution water. The treated fish was maintained in a test condition with pH value within 6.0 -8.5, hardness between 40 -250 caco3 and conductivity 0.417 mS/cm. No cumulative mortality (percent) observed in control group and 0.48, 1.53, 4.88 mg/L whereas, 14.29% and 100% in the tested concentration of 15.63 and 50.00 mg/L for a period of 96 hours. No Fish exhibited any abnormal behaviour in control group and 0.48, 1.53, 4.88 mg/L whereas, Loss of buoyancy control and Hypoactivity behaviours were observed in the tested concentration of 15.63 and 50.00 mg/L for a period of 96 hours.

Based on the results of range finding test, the definitive test was conducted with test concentration of 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L, in a geometric series with a sacing factor of 1.8 along with a control group without test item by static method. Each concentration contained seven fish. Test item was formulated in dilution water. The treated fish was maintained in a test condition. No cumulative mortality (percent) observed in control group and 4.8 and 8.6 mg/L whereas, 14.29%, 100% and 100% in the tested concentration of 15.4, 27.8 and 50.0 mg/L for a period of 96 hours. No Fish exhibited any abnormal behaviour in control group and 4.8, 8.6, 15.4 mg/L whereas, Loss of buoyancy control and abnormal vertical orientation behaviours were observed in the tested concentration of 27.8 and 50.0 mg/L on day 0. The test item available in the test medium natural water was determined by a validated GC method. The test item concentration of test item in the test medium at the initiation (0 hour) and 96 hours was 99.14% to 100.01 % and 98.80 % to 99.32 % at 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L, of the nominal test concentrations. The concentration of the test item has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the LC50 value for 96 hours of 6-pentyloxan-2-one was found to be 18.70 mg/L with 95% confidence limits between 17.65mg/L and 19.75 mg/L. The results observed in the present study meets all the validity criteria as per OECD 203 Test guideline. Based on the median lethal concentration test chemical can be classified into aquatic chronic category 3 as per CLP classication 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 (2018). 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 4.5 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations

Short term toxicity to aquatic invertebrates

The objective of this study was to evaluate the Acute toxicity of test chemical to the Daphnia sp., (Daphnia magnaStraus). The study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 202, Adopted by the Council on 13 April 2004, Daphnia sp. Acute Immobilization Test. Solubility of the test item was performed by weighing 25.7 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark using natural water and the resulting concentration is 102.8 mg/L. Stability of the test item in natural water determined by analyzing the test concentrations of 0.1 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.1 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The brood daphnids were acclimatized 48 hours prior to the test item exposure in natural water. Less than 24 hours 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 hours. Range finding test was conducted at test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L in a geometric factor of 2 along with control groups without test item by static method. Each concentration contained two replicates and five Daphnids per replicate. Test item was formulated in natural water. No immobilization observed in control group whereas, 10.0%, 20.0% , 30.0% , 70.0% and 90% in 6.25, 12.5, 25, 50 and 100 mg/L for a period of 48 hours. No abnormal behaviour observed in control groups and in all the tested concentrations for a period of 48 hours. Based on the results of range finding test, the definitive test was conducted at test concentrations of 3.5, 7.7, 16.94, 37.27 and 81.99 mg/L in a geometric factor of 2.2 along with control group without test item by static method. Each concentration contained four replicates and five Daphnids per replicate. Test item was formulated in natural water. No immobilization observed in control group whereas, 5.0%, 10.0% , 20.0% , 55.0% and 85.0% in 3.5, 7.7, 16.94, 37.27 and 81.99 mg/L for a period of 48 hours. No abnormal behaviour observed in control group and in all the tested concentrations for a period of 48 hours. During the Definitive test period, all the beakers were incubated in the room under test condition. The pH of the control at the test start was 7.5 and at termination of the test was 7.8 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.0 to 7.6 at the beginning of the test and 7.8 to 7.9 at test termination. The temperature of the control and test concentration at the beginning and at test termination was21.0°C to 21.8°C. The Dissolved oxygen of the concentrations was 7.6 to 9.5 mg/ L at the beginning of the test and 6.7 to 7.4 mg/L at test termination. The mean intensity of light ranged from 1334 to 1349 Lux. Based on the results of this study, the EC₅₀value for 48 hours of test chemical was found to be 31.81mg/L with 95% confidence limits between 26.97mg/L and 36.65 mg/L.The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline. Thus chemical can be classified into aquatic chroinc category 3 as per 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 solution of test chemical was prepared by dissolving 100 mg of test chemical in 100 ml of Adams medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-Vis Spectrophotometer and the final solubility value obtained after analytical detection was 296.79 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.5, 1, 2, 4 and 8 mg/L, respectively. Daphnia magna (water flea) of length 0.37 cm was used as a test organism. A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (Adams medium') was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum) thrice a week. Total 50 test organisms were exposed to five test chemical concentrations (n = 10) 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 200 mg/l as CaCO3, pH 7.8, dissolved oxygen 6.3 to 8.3 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 at least three times a week. The test concentrations were measured and was observed be maintained within 80-120% of nominal concentration, thus, effect concentrations (EC) would be evaluated as the nominal concentrations. 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 and EC 10 value was determined to be 0.5581 mg/l and 0.198 mg/L respectively ( nominal concentration). Thus, 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

Toxicity to aquatic algae study was carried out. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) obtained from the Denmark Technical University, Denmark was used as a test algae. The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201. Test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L). Test chemical conc. used for the study were 2.5, 5.0, 10, 20, 40, and 80 mg/l, respectively. Test samples were analysed immediately after sampling by using GC-MS. Test algae were exposed to different test chemical conc. in a conical flasks. Conical flasks of 100 ml was filled with 60 ml which was loosely closed and it has with 40 ml head space. Aeration was not proviided not during the study. Intial cell density of the test algae was 10000 cells/ml. OECD medium was used as a standard test medium during the study. Test conditions involve a temperature of 21°C to 22.2°C, pH of 6.9 to 7.4 with a continuous, uniform fluorescent illumination(3000-4000 Lux) under a photoperiod of 16 Hour Light Period : 8 Hour Dark Period.  The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer. All test experiments were performed in duplicates and control experiments were performed in triplicates, respectively. K2Cr2O7 (Potassium Dichromate) was used as a reference substance. The EC50 value of the reference substance Potassium Dichromate was determined to be 1.732 mg/L. Quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values. Test validity criterion has been fulfilled during the study. On the basis of the effect on growth rate of the test algae, the 96 hr EC50 value was determined to be 26.54 mg/L (95% CI  is 21.90 to 32.66 mg/L). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in ''aquatic chronic category 3'' as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity to microorganism study was performed using Tetrahymena pyriformis as a test organism (T. W. Schultz et. al., 1999 and authoritative database, 2012). The study was carried out under static conditions. Stock solutions of test chemical were prepared in dimethyl sulfoxide. Exact test chemical concentrations used for the study was not known, but each test replicate consists of six to eight different concentrations. Range finding study was performed in duplicates prior to the initiation of study. Flasks were used as a test vessel. To each test flask were added test organism and a solution of test chemical. Two control vessels, one control with no test chemical but inoculated with T. pyriformis, and the other, a blank control, which has neither test chemical nor test organism were run simultaneously during the study. All test concentrations experiments were performed in duplicates. Test conditions allow for 8-9 cell cycles in control cultures. Only replicates with control-absorbency values > 0.6 but < 0.75 were used for the analysis. Population density of test organism was measured spectrophotometrically at 540 nm. The 50% growth inhibitory concentrations, IGC50, were determined by Probit Analysis of Statistical Analysis System (SAS) software. On the basis of the effect on growth inhibition of the test organism Tetrahymena pyriformis, the 40 hr IGC50 value was determined to be 204.64 mg/l.

Additional information

Short term toxicity to fish

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

 

Short tertm toxicity to fish:

The objective of this study was to assess the acute toxicity of test chemical in fresh water Fish (Danio rerio). The study was performed in compliance with OECD 203 Guideline for the testing of chemicals; Fish, Acute Toxicity Testing, (18th June, 2019). Solubility of the test item was performed by weighed 25.7 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark using natural water and the resulting concentration was 102.8 mg/L. Stability of the test item in natural water determined by analyzing the test concentrations of 0.1 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.1 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. Range finding test was conducted with test concentration of 0.48 and 50.00 mg/L along with a control groups without test item by static method. Each concentration contained seven fish. Test item was formulated in dilution water. The treated fish was maintained in a test condition with pH value within 6.0 -8.5, hardness between 40 -250 caco3 and conductivity 0.417 mS/cm. No cumulative mortality (percent) observed in control group and 0.48, 1.53, 4.88 mg/L whereas, 14.29% and 100% in the tested concentration of 15.63 and 50.00 mg/L for a period of 96 hours. No Fish exhibited any abnormal behaviour in control group and 0.48, 1.53, 4.88 mg/L whereas, Loss of buoyancy control and Hypoactivity behaviours were observed in the tested concentration of 15.63 and 50.00 mg/L for a period of 96 hours.

Based on the results of range finding test, the definitive test was conducted with test concentration of 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L, in a geometric series with a sacing factor of 1.8 along with a control group without test item by static method. Each concentration contained seven fish. Test item was formulated in dilution water. The treated fish was maintained in a test condition. No cumulative mortality (percent) observed in control group and 4.8 and 8.6 mg/L whereas, 14.29%, 100% and 100% in the tested concentration of 15.4, 27.8 and 50.0 mg/L for a period of 96 hours. No Fish exhibited any abnormal behaviour in control group and 4.8, 8.6, 15.4 mg/L whereas, Loss of buoyancy control and abnormal vertical orientation behaviours were observed in the tested concentration of 27.8 and 50.0 mg/L on day 0. The test item available in the test medium natural water was determined by a validated GC method. The test item concentration of test item in the test medium at the initiation (0 hour) and 96 hours was 99.14% to 100.01 % and 98.80 % to 99.32 % at 4.8, 8.6, 15.4, 27.8 and 50.0 mg/L, of the nominal test concentrations. The concentration of the test item has been satisfactorily maintained within 80 to 120% of the nominal concentration during the exposure period. Based on the results of this study, the LC50 value for 96 hours of 6-pentyloxan-2-one was found to be 18.70 mg/L with 95% confidence limits between 17.65mg/L and 19.75 mg/L. The results observed in the present study meets all the validity criteria as per OECD 203 Test guideline. Based on the median lethal concentration test chemical can be classified into aquatic chronic category 3 as per CLP classication criteria.

In an experimental study from study report (2018), an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on fish. The test was performed in accordance with the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.0792 g and average length of 1.88 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 7.0 mg/l, pH 7.1, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test chemical concentrations were not verified analytically. Nominal concentrations of test chemical used for the study were 0, 6.25, 12.5, 25, 50 and 100mg/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°C, pH 6.9, hardness of water 200 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. No mortalities were observed in the control vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) and LC100 value was determined to be > 6.25 and 12.5 mg/l.

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to fish and considered to be classified in ‘aquatic chronic category 3’ as per 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 (2018). 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 4.5 mg/l for fish for 28 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as non-toxic to fish at environmentally relevant concentrations

Short term toxicity to aquatic invertebrates

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

The objective of this study was to evaluate the Acute toxicity of test chemical to the Daphnia sp., (Daphnia magnaStraus). The study was performed in compliance with OECD Guideline for Testing of chemicals OECD NO. 202, Adopted by the Council on 13 April 2004, Daphnia sp. Acute Immobilization Test. Solubility of the test item was performed by weighing 25.7 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark using natural water and the resulting concentration is 102.8 mg/L. Stability of the test item in natural water determined by analyzing the test concentrations of 0.1 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.1 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test. The brood daphnids were acclimatized 48 hours prior to the test item exposure in natural water. Less than 24 hours 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 hours. Range finding test was conducted at test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L in a geometric factor of 2 along with control groups without test item by static method. Each concentration contained two replicates and five Daphnids per replicate. Test item was formulated in natural water. No immobilization observed in control group whereas, 10.0%, 20.0% , 30.0% , 70.0% and 90% in 6.25, 12.5, 25, 50 and 100 mg/L for a period of 48 hours. No abnormal behaviour observed in control groups and in all the tested concentrations for a period of 48 hours. Based on the results of range finding test, the definitive test was conducted at test concentrations of 3.5, 7.7, 16.94, 37.27 and 81.99 mg/L in a geometric factor of 2.2 along with control group without test item by static method. Each concentration contained four replicates and five Daphnids per replicate. Test item was formulated in natural water. No immobilization observed in control group whereas, 5.0%, 10.0% , 20.0% , 55.0% and 85.0% in 3.5, 7.7, 16.94, 37.27 and 81.99 mg/L for a period of 48 hours. No abnormal behaviour observed in control group and in all the tested concentrations for a period of 48 hours. During the Definitive test period, all the beakers were incubated in the room under test condition. The pH of the control at the test start was 7.5 and at termination of the test was 7.8 and therefore did not vary more than 1.5 units during the study. The pH of all the tested concentrations was 7.0 to 7.6 at the beginning of the test and 7.8 to 7.9 at test termination. The temperature of the control and test concentration at the beginning and at test termination was21.0°C to 21.8°C. The Dissolved oxygen of the concentrations was 7.6 to 9.5 mg/ L at the beginning of the test and 6.7 to 7.4 mg/L at test termination. The mean intensity of light ranged from 1334 to 1349 Lux. Based on the results of this study, the EC₅₀value for 48 hours of test chemical was found to be 31.81mg/L with 95% confidence limits between 26.97mg/L and 36.65 mg/L.The results observed in the present study meets all the validity criteria as per OECD 202 Test guideline. Thus chemical can be classified into aquatic chroinc category 3 as per CLP classification criteria.

 

In an experimental study from study report (2018), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. 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. The 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, 10, 20, 40 and 80 mg/l, respectively. Study was performed using total 5 organisms per vessel/replicates in a static 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 run simultaneously. The 24 hr EC50 value of the reference substance was determined to be 0.73 mg/l. 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 and the dissolved oxygen concentration at the end of the test was evaluated to be ≥ 3 mg/l (i.e, reported as > 7 mg/l) both in the control and test vessels. Thus, the validity criterion of the test 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 30.1 mg/l (95 % C. I. - 23.9 to 37.9 mg/l).

 

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic invertebrates at environmental related concentrations and considered to be classified in 'aquatic chronic category 3' 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 solution of test chemical was prepared by dissolving 100 mg of test chemical in 100 ml of Adams medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-Vis Spectrophotometer and the final solubility value obtained after analytical detection was 296.79 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Thus, test chemical concentrations used for the study were 0 (control), 0.5, 1, 2, 4 and 8 mg/L, respectively. Daphnia magna (water flea) of length 0.37 cm was used as a test organism. A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (Adams medium') was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Selenestrum capricornutum) thrice a week. Total 50 test organisms were exposed to five test chemical concentrations (n = 10) 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 200 mg/l as CaCO3, pH 7.8, dissolved oxygen 6.3 to 8.3 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 at least three times a week. The test concentrations were measured and was observed be maintained within 80-120% of nominal concentration, thus, effect concentrations (EC) would be evaluated as the nominal concentrations. 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 and EC 10 value was determined to be 0.5581 mg/l and 0.198 mg/L respectively ( nominal concentration). Thus, 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

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

 

Toxicity to aquatic algae study was carried out. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) obtained from the Denmark Technical University, Denmark was used as a test algae. The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. Algae was maintained in the OECD medium in the orbital shaker, at temperature 22 ± 2°C, as per the recomeded light intesity as per OECD 201. Test chemical was directly dissolved in the OECD medium (500mg of test chemical was dissolved in 500 ml of OECD medium to get final concenttration of 1g/L). Test chemical conc. used for the study were 2.5, 5.0, 10, 20, 40, and 80 mg/l, respectively. Test samples were analysed immediately after sampling by using GC-MS. Test algae were exposed to different test chemical conc. in a conical flasks. Conical flasks of 100 ml was filled with 60 ml which was loosely closed and it has with 40 ml head space. Aeration was not proviided not during the study. Intial cell density of the test algae was 10000 cells/ml. OECD medium was used as a standard test medium during the study. Test conditions involve a temperature of 21°C to 22.2°C, pH of 6.9 to 7.4 with a continuous, uniform fluorescent illumination(3000-4000 Lux) under a photoperiod of 16 Hour Light Period : 8 Hour Dark Period.  The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae with the help of heamocytometer. All test experiments were performed in duplicates and control experiments were performed in triplicates, respectively. K2Cr2O7 (Potassium Dichromate) was used as a reference substance. The EC50 value of the reference substance Potassium Dichromate was determined to be 1.732 mg/L. Quantitative concentration-response relationship by regression analysis, was done using SysSTAT Pro software to obtain EC50 values. Test validity criterion has been fulfilled during the study. On the basis of the effect on growth rate of the test algae, the 96 hr EC50 value was determined to be 26.54 mg/L (95% CI  is 21.90 to 32.66 mg/L). Thus, test chemical was considered as toxic to aquatic algae at environmental relevant concentrations and hence, considered to be classified in ''aquatic chronic category 3'' as per the CLP classification criteria.

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report. 2018). 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 100 mg/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, 2.5, 5.0, 10, 20, 40 and 80 mg/l, respectively. Study was performed in a static 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 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. The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. 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 4.5% and the specific growth rate in the control was 1.77 per day, indicating that the biomass in the control cultures have increased exponentially by a factor of more than 16 within the 72 hr exposure duration. Thus, the validity criterion of the test 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 20.5 mg/l (95% CL: 10.7 to 39.3 mg/l). 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 3' 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 green algae. The test was performed in accordance with the Scenedesmus cell proliferation inhibition test, DIN 38412 part 9.Test chemical concentrations were not verified analytically. Test chemical conc. used for the study were 0 (control), 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 mg/l (nominal conc.). Study was performed using Scenedesmus subspicatus as a test organism in a static system in quadruplicates (for test conc.). Initial cell concentration used for the study was 10000 cells/ml. Test organisms were exposed to test chemical in 250 mL Erlenmeyer flask. The test vessels were placed at a temperature of 19.85°C under continuous illumination with a light intensity of 6.2 miS/cm. Cell growth was measured fluorometrically in all flasks at 24, 48, 72 and 96 hours of incubation period. On the basis of effect on growth rate of the test organism Scenedesmus subspicatus, the 96 hr EC20, ErC50 and EC90 was determined to be 20, 79 and > 500 mg/l (nominal conc.), respectively. Thus, based on this, chemical was considered as toxic to aquatic algae and hence, considered to be classified in ‘aquatic chronic category 3’ as per the 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 algae and considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity to microorganism study was performed using Tetrahymena pyriformis as a test organism (T. W. Schultz et. al., 1999 and authoritative database, 2012). The study was carried out under static conditions. Stock solutions of test chemical were prepared in dimethyl sulfoxide. Exact test chemical concentrations used for the study was not known, but each test replicate consists of six to eight different concentrations. Range finding study was performed in duplicates prior to the initiation of study. Flasks were used as a test vessel. To each test flask were added test organism and a solution of test chemical. Two control vessels, one control with no test chemical but inoculated with T. pyriformis, and the other, a blank control, which has neither test chemical nor test organism were run simultaneously during the study. All test concentrations experiments were performed in duplicates. Test conditions allow for 8-9 cell cycles in control cultures. Only replicates with control-absorbency values > 0.6 but < 0.75 were used for the analysis. Population density of test organism was measured spectrophotometrically at 540 nm. The 50% growth inhibitory concentrations, IGC50, were determined by Probit Analysis of Statistical Analysis System (SAS) software. On the basis of the effect on growth inhibition of the test organism Tetrahymena pyriformis, the 40 hr IGC50 value was determined to be 204.64 mg/l.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical can be considered as toxicto aquatic organisms at environmental relevant concentrations and considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.