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

Endpoint summary

Description of key information

Biodegradation in water

Estimation Programs Interface Suite was run to predict the biodegradation potential of the test chemical in the presence of mixed populations of environmental microorganisms. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that test chemical is expected to be not readily biodegradable.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 78.1% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be 120 days (2880 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

Additional information

Biodegradation in water

Predicted data for the test chemical and various supporting weight of evidence studies for its structurally and functionally similar read across substance were reviewed for the biodegradation end point which are summarized as below:

 

In a prediction using the Estimation Programs Interface Suite (2017), the biodegradation potential of the test chemical in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that test chemical is expected to be not readily biodegradable.

 

In a supporting weight of evidence study from peer reviewed journal (U. Pagga, et. al., 1986) for the test chemical,the aerobic biodegradation experiment was performed for test chemical using activated sludge at concentration 0.5 g/L dry material as inoculums and initial concentration of chemical taken was 100mg/L for 42 days. By considering DOC removal parameter test chemical showed 29 % degradation in 42 days. The test chemical belongs to B category according to table 1 and 2 as its limit value falls in that range. On the basis percentage value it is concluded that test chemical is not readily biodegradable.

 

Another biodegradation study was conducted for 5 days for evaluating the percentage biodegradability of test chemical using 5 day BOD test under aerobic conditions (authoritative database HSDB, 2017). Sewage sludge was used as a test inoculum. Initial test substance conc. used in the study was 2.5 mg/l. The percentage degradation of test substance was determined to be 0% by BOD parameter in 5 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

For the test chemical,biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test substance (authoritative database J-CHECK and EnviChem, 2017). The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I). Activated sludge was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test substance was determined to be 1 and 3% by BOD and HPLC parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

In an additional study from authoritative databases (2017), biodegradation experiment was conducted for 14 days for evaluating the percentage biodegradability of test substance. The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)) under aerobic conditions. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test substance was determined to be 0.2 and 0.3% by BOD and GC parameter in 14 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

On the basis of above results of test chemical, it can be concluded that the test chemical can be expected to be not readily biodegradable in nature.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 78.1% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be 120 days (2880 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.