N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Endpoint summary

Description of key information

Short term toxicity to fish

In an experimental study from study report (2020), an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.87 cm was used as a test organism. Test fishes were kept in the test water under natural conditions along with proper feed and aeration for seven days. Test chemical solution was prepared by dissolving 5000 mg of test chemical in 5000 ml of RO water to get the final concentration of 1000 mg/L. which was then analytically determined. The final solubility value obtained after analytical detection was 0.16 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. Limit test was performed at a test chemical concentration of 0.16 mg/l. The analytical determinations were performed by UV-VIS spectrophotometer for selected test concentration at 0 hour and 96 hours. The concentration of the test chemical being tested and was not within the range of ± 20 %. Therefore, the analysis of the results was based on measured concentration. Test fishes were exposed to test chemical in a 7 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed at a temperature of 21-25°C, pH 7.6, dissolved oxygen 8.3 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. The pH and oxygen values were measured at the beginning of the test and every 24 hours thereafter. The lethal concentration was calculated using the concentration/ percentage response curve. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 93% of the air saturation value, thus meeting the validity criteria. The median lethal concentrations [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 0.16 mg/L. 

Long term Toxicity to Fish

The aim of the study was todetermine the EC₅₀, LOEC and NOECof test chemical on the growth and development of embryos and larvae of the freshwater fish species zebrafish (Danio rerio) in a Fish Early-Life Stage (ELS) Toxicity Test. The study was conducted according to the OECD guideline No. 210, Adopted 26^thJuly, 2013.

The test was conducted with a semi-static design with renewal once 48 h.

Concentrated stock solutions and test media were analysed during the ELS test. Based on the range finding result the definitive test was conducted with five test concentrations of 0.01, 0.03, 0.10, 0.30 and 1.00 mg/L in terms of test chemical with a conversion factor of 3.2. Both control and solvent control were included in the test design. Each concentration was divided into four replicate with 20 eggs each and including the controls.

The cumulative mortality of the control and solvent control group at 30 days post-hatch was 11.25 and 11.25 %, whereas corrected cumulative mortality of embryos, larvae and juvenile fish after at the end of exposure to test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg /Lwas 12.68, 28.45, 49.30, 67.61and 92.96%,respectively.

Hatching success in the control and solvent control group was 93.42 and 94.67% respectively, satisfying the validation criterion for hatching success (70%),whereas in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas 91.18, 87.93, 67.92, 53.49and 25.00%,respectively. Fertility reduction in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas3.69, 7.12, 28.26, 43.50 and 73.59%,respectively in comparison with the solvent control group.The NOEC and LOEC for hatching success were determined as 0.01 and 0.03 mg/L, respectively.

Larval survival until Day 30 post-hatch in the control and solvent control group was 95.0 and 93.75% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas85.0, 72.50, 66.25, 53.75 and 25.0%,respectively. Larval survival reduction in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas 9.33, 22.67, 29.33, 42.67 and 73.33%,respectively in comparison with the solvent control group.In terms of concentrations, the NOEC and LOEC for post-hatch larval survival untilDay 30 were both considered to be equal to or less than 0.01 mg/L.

For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.01 and 0.03mg/L, respectively. For fish dry weights, the NOEC and LOEC determined on Day 30 post-hatch were 0.01 and 0.03 mg/L, respectively.

The concentrations of test chemical was determined using a validated liquid chromatographic method. Samples of each fresh test substance concentration, control and solvent control collected at exposure initiation and at renewals, and samples of each spent test substance concentration, control and solvent control collected at each renewal and at exposure termination were chemically determined.

In fresh samples of the test substance concentrations, the determined concentrations of test chemical was between 99.3 and 101.7%, concentrations, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was between 96.1 and 96.9% of the concentrations, respectively.

All validity criteria were satisfied during the test, therefore the test was considered to be valid.

Results:The effects of test chemical on mortality, hatching success and fish larvae survivalDanio rerioin the definitive test are summarized in Table below.

LC50mortality

0.064±0.01

EC50fertility

0.34±0.047

EC50surviving

0.31±0.06

NOECmortality

<0.01

NOECfertility

0.01

NOECsurviving

<0.01

LOECmortality

<0.01

LOECfertility

0.03

LOECsurviving

<0.01

Thus based on the results obtained chemical can be categorised as into chronic category 1 as per CLP classification criteria.

Short term toxicity to aquatic invertebrate

Acute Immobilisation Test for 48 h in static method to Daphnia magna STRAUS was performed according to OECD-Guideline 202 for Testing of Chemicals (2004) EC Directive 92/69/EC Method C.2 (1992) (Experimental study report, 2005). The solution (100 mg/L test chemical was weighed out) was prepared with dilution water one day prior to application in the test medium. The solubility of test substance is < 1 mg/L (related to the pigment). The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 μm .The effect of the saturated solution (100 mg/L) as limit concentration of the test chemical was determined. The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. No test chemical analysis was carried out. A reference test was carried out with potassium dichromate to determine the toxicity of the reference substance. EC10 and EC50 values were only calculated for the reference chemical via probit according to WEBER (1986). Calculation of the confidence intervals were carried out using standard procedures according to BREITIG & TÜMPLING (1982). The median effect concentration (EC50) of saturated solution of test chemical was observed to be >100 mg/l after 48 hrs of exposure period.

Long term toxicity to aquatic invertebrate:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Daphnia magna of length 0.45 cm was used as a test organism. Eggs of Daphnia magna were obtained from Denmark Technical University. Test daphnids were fed with living cells of Selenestrum capricornutum and it was done thrice a week. DMSO or acetone was used as a vehicle. The in-house water solubility for the test chemical is 0.22 mg/L. Therefore, the test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution will be filtered by using whatman filter paper no. 42, which will then analytically determine. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions ranging from concentrations, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. The concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. Test chemical conc. used in the defiinite study was 0.1 mg/l. Thus, limit test was performed at 0.1 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 18-22°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 0.1 mg/l. Thus based on the outcomes chemical could be classified into aquatic chronic category 1 as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

In an experimental study from study report (2020), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical ( 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 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 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l , respectively.

Toxicity to microorganism

The microbial respiration study was conducted by following the OECD 209 guidelines, in which inhibition of respiration rate of inoculum was determined when exposed to test chemical for 3 hours. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 1 hours from collection and kept it aerobic during transport.  Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed by mineral media followed by aerating for 1 day at the 20º C test temperature. the test chemical and reference concentrations were prepared in 1000 ml glass beakers. The number of replicates in the control and abiotic control was 6 , and for test concentrations and reference compound the number of replicate were 5. the respective amount chemical was taken for preparing for concentrations ranging form 0.156 to 2.5 mg/l and reference compound of 25 mg/l were aerated (0.5 to 1 L/min) and incubated for a period of 3 hours at 20° C by adding a concentration of 32 mL/400 mL of activated sludge solution into every flask. This concentration gave suspended solid of 1.0 g/L. Later sample of each mixture was placed in a measuring (BOD) bottle (volume 125 mL) and the decrease in the oxygen concentration in each mixture was recorded with an oxygen electrode during the period of 5 minutes. The sensitivity of each batch of inoculum was checked with a positive control.

The effects on micro-organisms from inoculum were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. The respiration rates of samples of inoculum fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours. Based outcomes on respiration inhibition the EC10 was reported to be 0.15 mg/L

Additional information

Short term toxicity to fish

Experimental studyand predicted data of the target chemical and various supporting weight of evidence studies for its read across substance was reviewed for the short term toxicity to fish end point which is summarized as below:

 

In an experimental study from study report (2020), an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.87 cm was used as a test organism. Test fishes were kept in the test water under natural conditions along with proper feed and aeration for seven days. Test chemical solution was prepared by dissolving 5000 mg of test chemical in 5000 ml of RO water to get the final concentration of 1000 mg/L. which was then analytically determined. The final solubility value obtained after analytical detection was 0.16 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. Limit test was performed at a test chemical concentration of 0.16 mg/l. The analytical determinations were performed by UV-VIS spectrophotometer for selected test concentration at 0 hour and 96 hours. The concentration of the test chemical being tested and was not within the range of ± 20 %. Therefore, the analysis of the results was based on measured concentration. Test fishes were exposed to test chemical in a 7 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed at a temperature of 21-25°C, pH 7.6, dissolved oxygen 8.3 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. The pH and oxygen values were measured at the beginning of the test and every 24 hours thereafter. The lethal concentration was calculated using the concentration/ percentage response curve. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 93% of the air saturation value, thus meeting the validity criteria. The median lethal concentrations [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 0.16 mg/L. 

 

In a prediction done using the EPI Suite ECOSAR version 1.10 (2017), the 96 hours LC50 was estimated to be 0.945 mg/l on fish for substance test substance with mortality effects.

 

Another Prediction done using Danish QSAR database (2018) in which the average value of both models i.e Leadscope and SciMatics SciQSAR model were presented in Battery model. On the basis of this, 96 hours LC50 was estimated to be 0.44 mg/l on Pimephales promelas for test substance with mortality effects.

 

In a supporting weight of evidence study from peer reviewed journal (Kuo-HsiungL et al, 2002), short term toxicity test was performed to evaluate the effect of test material to fish Cyprinus carpio. The test animals were fed with yeast in a 224 (diameter), 46 (height)-cm circular plastic pool. Before the experiment, the test organisms were acclimatized in aquaria for 2 weeks under conditions similar to those under which the tests were performed. Four carps of 2 to 6 cm in size, were introduced to each 10-L beaker containing 5 L of different concentrations of test chemical i.e, 10, 50, 100, 200, and 500 mg/l. After 48 h of exposure period, median lethal concentration (LC50) values were calculated by using trimmed Spearman Karber method. The median lethal concentration (LC50) of test material on Cyprinus carpio after 48 h was observed to be 0.022 mg/l.

 

For the test chemical, another short term toxicity test was performed to evaluate the effect of test material to fish Cyprinus carpio (Kuo-HsiungL et al, 2002). The test procedure and conditions were similar as mentioned above. The median lethal concentration (LC50) of test material on Cyprinus carpio after 48 h was observed to be 0.045 mg/l.

 

Thus, based on the EC50 value, test chemical can be considered as toxic to aq. algae and thus can be considered to be classified in ‘Aquatic acute/chronic 1 category’ as per CLP classification criteria.

Long term toxicity to fish:

The aim of the study was todetermine the EC₅₀, LOEC and NOECof test chemical on the growth and development of embryos and larvae of the freshwater fish species zebrafish (Danio rerio) in a Fish Early-Life Stage (ELS) Toxicity Test. The study was conducted according to the OECD guideline No. 210, Adopted 26^thJuly, 2013.

The test was conducted with a semi-static design with renewal once 48 h.

Concentrated stock solutions and test media were analysed during the ELS test. Based on the range finding result the definitive test was conducted with five test concentrations of 0.01, 0.03, 0.10, 0.30 and 1.00 mg/L in terms of test chemical with a conversion factor of 3.2. Both control and solvent control were included in the test design. Each concentration was divided into four replicate with 20 eggs each and including the controls.

The cumulative mortality of the control and solvent control group at 30 days post-hatch was 11.25 and 11.25 %, whereas corrected cumulative mortality of embryos, larvae and juvenile fish after at the end of exposure to test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg /Lwas 12.68, 28.45, 49.30, 67.61and 92.96%,respectively.

Hatching success in the control and solvent control group was 93.42 and 94.67% respectively, satisfying the validation criterion for hatching success (70%),whereas in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas 91.18, 87.93, 67.92, 53.49and 25.00%,respectively. Fertility reduction in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas3.69, 7.12, 28.26, 43.50 and 73.59%,respectively in comparison with the solvent control group.The NOEC and LOEC for hatching success were determined as 0.01 and 0.03 mg/L, respectively.

Larval survival until Day 30 post-hatch in the control and solvent control group was 95.0 and 93.75% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas85.0, 72.50, 66.25, 53.75 and 25.0%,respectively. Larval survival reduction in the group treated with test chemical at concentrations of 0.01, 0.03, 0.10, 0.30and 1.00 mg/Lwas 9.33, 22.67, 29.33, 42.67 and 73.33%,respectively in comparison with the solvent control group.In terms of concentrations, the NOEC and LOEC for post-hatch larval survival untilDay 30 were both considered to be equal to or less than 0.01 mg/L.

For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.01 and 0.03mg/L, respectively. For fish dry weights, the NOEC and LOEC determined on Day 30 post-hatch were 0.01 and 0.03 mg/L, respectively.

The concentrations of test chemical was determined using a validated liquid chromatographic method. Samples of each fresh test substance concentration, control and solvent control collected at exposure initiation and at renewals, and samples of each spent test substance concentration, control and solvent control collected at each renewal and at exposure termination were chemically determined.

In fresh samples of the test substance concentrations, the determined concentrations of test chemical was between 99.3 and 101.7%, concentrations, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was between 96.1 and 96.9% of the concentrations, respectively.

All validity criteria were satisfied during the test, therefore the test was considered to be valid.

Results:The effects of test chemical on mortality, hatching success and fish larvae survivalDanio rerioin the definitive test are summarized in Table below.

LC50mortality

0.064±0.01

EC50fertility

0.34±0.047

EC50surviving

0.31±0.06

NOECmortality

<0.01

NOECfertility

0.01

NOECsurviving

<0.01

LOECmortality

<0.01

LOECfertility

0.03

LOECsurviving

<0.01

Thus based on the results obtained chemical can be categorised as into chronic category 1 as per CLP classification criteria.

Short term toxicity to aquatic invertebrate

Acute Immobilisation Test for 48 h in static method to Daphnia magna STRAUS was performed according to OECD-Guideline 202 for Testing of Chemicals (2004) EC Directive 92/69/EC Method C.2 (1992) (Experimental study report, 2005). The solution (100 mg/L test chemical was weighed out) was prepared with dilution water one day prior to application in the test medium. The solubility of test substance is < 1 mg/L (related to the pigment). The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 μm .The effect of the saturated solution (100 mg/L) as limit concentration of the test chemical was determined. The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. No test chemical analysis was carried out. A reference test was carried out with potassium dichromate to determine the toxicity of the reference substance. EC10 and EC50 values were only calculated for the reference chemical via probit according to WEBER (1986). Calculation of the confidence intervals were carried out using standard procedures according to BREITIG & TÜMPLING (1982). The median effect concentration (EC50) of saturated solution of test chemical was observed to be >100 mg/l after 48 hrs of exposure period.

Long term toxicity to aquatic invertebrate:

A chronic study was conducted for assesing the effect of test chemical. The study was performed in accordance with the principles of the OECD Guideline 211 (Daphnia magna Reproduction Test). Daphnia magna of length 0.45 cm was used as a test organism. Eggs of Daphnia magna were obtained from Denmark Technical University. Test daphnids were fed with living cells of Selenestrum capricornutum and it was done thrice a week. DMSO or acetone was used as a vehicle. The in-house water solubility for the test chemical is 0.22 mg/L. Therefore, the test chemical will be prepared by dissolving 1000 mg of test chemical in 1000 mL of M7 media with 48 hours stirring to get the final concentration of 1000 mg/L. This stock solution will be filtered by using whatman filter paper no. 42, which will then analytically determine. The final solubility value obtained in media was used to prepare the remaining test concentrations from the above stock solution. The analytical determinations were performed by UV-VIS spectrophotometer. The pre-treated stock solution was then diluted with media in order to get the required test solutions ranging from concentrations, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mg/L. The absorbance of resulting solution was measured using UV-VIS spectrophotometer against corresponding blank at lambda max (λmax). Standard curve was plotted against concentration verses absorbance and the maximum solubility was determined from the below standard curve. Analytical assessments were performed for selected test concentration at 1st, 2nd and 3rd week. The concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. Test chemical conc. used in the defiinite study was 0.1 mg/l. Thus, limit test was performed at 0.1 mg/l conc. Test daphnids were exposed to test chemical conc. in a glass beaker for an exposure period of 21 days. Vessels were not aerated during the study. M7 medium was used as a test medium. Test conditions involve a temperature range of 18-22°C, 16:8 light:dark conditions and light intensity not exceeding 1000-1500 lux, respectively. The evaluation of the NOEC was determined based on the number of offspring’s produced per living parent Daphnia. Defined concentrations of the test chemical led to a certain percentage reduction of the parthenogenetic reproduction rate at the end of the 21 day study period. The living offspring was counted daily along with the renewal of the test medium. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. On the basis of the effect on reproduction of the test daphnids, the 21 d NOEC value was determined to be 0.1 mg/l. Thus based on the outcomes chemical could be classified into aquatic chronic category 1 as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the target chemical and structurally & functionally similar read across chemicals has been reviewed to determine the toxic effect of the test chemical on aquatic algae and cyanobacteria.The studies are as mentioned below:

 

Experimental study and predicted data of the target chemical and various supporting weight of evidence studies for its read across substance was reviewed for toxicity to aquatic algae end point which are summarized as below:

 

In an experimental study from study report (2020), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 500 mg of test chemical in 500 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical ( 0, 0.15, 0.19, 0.24, 0.31, 0.40 and 0.52 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 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr EC10 and median effect concentration (EC50) was determined to be 0.17 and > 0.52 mg/l , respectively.

In a prediction done using EPI suite ECOSAR version 1.11, toxicity of the test chemical to green algae was evaluated. 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 1.503 mg/l.

 

Another toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Selenastrum capricornutum (Secondary source, 1996). The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test) in a static fresh water system. Selenastrum capricornutum ATCC 22662 (Green algae) was used as test organism. 8 different nominal concentrations of test chemical were used. Test chemical conc. ranges from 0.058 to 3.2 mg/l, respectively. Stock solution of test chemical was prepared with DMSO. No analytical monitoring of test chemical concentration were done. On the basis of effect of the test chemical on biomass of the test organism Selenastrum capricornutum, the 72 hr NOEC and EC50 value was determined to be 0.10 and 0.25 mg/l, respectively.

 

For the test chemical from secondary source (2017), toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Scenedesmus subspicatus. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test) in a static fresh water system. Scenedesmus subspicatus (Green algae) was used as test organism. On the basis of effect of the test chemical on growth rate of the test organism Scenedesmus subspicatus, the 72 hr ErC50 value was determined to be 0.6 mg/l.

Thus, based on the EC50 value, test chemical can be considered as toxic to aq. algae and thus can be considered to be classified in ‘Aquatic acute/chronic 1 category’ as per CLP classification criteria.

Toxicity to microorganism

Data available for the target chemical and structurally & functionally similar read across chemicals has been reviewed to determine the toxic effect of the test chemical on micro-organisms. The studies are as mentioned below:

 

The microbial respiration study was conducted by following the OECD 209 guidelines, in which inhibition of respiration rate of inoculum was determined when exposed to test chemical for 3 hours. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 1 hours from collection and kept it aerobic during transport.  Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed by mineral media followed by aerating for 1 day at the 20º C test temperature. the test chemical and reference concentrations were prepared in 1000 ml glass beakers. The number of replicates in the control and abiotic control was 6 , and for test concentrations and reference compound the number of replicate were 5. the respective amount chemical was taken for preparing for concentrations ranging form 0.156 to 2.5 mg/l and reference compound of 25 mg/l were aerated (0.5 to 1 L/min) and incubated for a period of 3 hours at 20° C by adding a concentration of 32 mL/400 mL of activated sludge solution into every flask. This concentration gave suspended solid of 1.0 g/L. Later sample of each mixture was placed in a measuring (BOD) bottle (volume 125 mL) and the decrease in the oxygen concentration in each mixture was recorded with an oxygen electrode during the period of 5 minutes. The sensitivity of each batch of inoculum was checked with a positive control.

The effects on micro-organisms from inoculum were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. The respiration rates of samples of inoculum fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours. Based outcomes on respiration inhibition the EC10 was reported to be 0.15 mg/L

 

For the test chemical, another short term toxicity to Vibrio fischeri study was carried out.The study was performed in accordance with the standard method ‘DIN 38412 Part 34’. On the basis of the effect of test chemical on luminescence of the test organism Vibrio fischeri, the EC20 value was determined to be 0.5 mg/l.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, both the IC50 and EC20 value the test chemical on the test organism was observed to be 0.5 mg/l.