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Environmental fate & pathways

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Description of key information

Biodegradation in water

Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test chemicalat a temperature of30°C and pH 7.0, respectively (Sikandar I. Mulla et. al.; 2016). Sphingomonassp. strain YL-JM2C (Bacteria) isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosanwas used as test inoculum.Stock solutions (5 g/L) were prepared with acetone and stored in brown bottles at-20°C before use.Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includesK2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. 0.04% yeast extract (sterilized by 0.45mm membrane) was added to this medium.ThepH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.40 ml bottle was used as a test vessel for the study.For biodegradation experiments, 1 mL of pure bacterial culture (mid-log period) was transferred into a 40 mL bottle (working volume of 10 mL). For control, 10 mL of sterile AMS medium containing test chemical (4 mg/l) was used. All these bottles were kept in shaker (150 rpm) at 30°C under dark condition. A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of triclocarban at a specific incubation period by high performance liquid chromatography (HPLC).Degradation of test chemical metabolites 3,4-dichloroaniline and 4-chloroaniline during growth ofSphingomonassp. strain YL-JM2C was determined at different intervals according to the total organic carbon (TOC) concentration by TOC analyser (Shimadzu TOC-V CPH, Japan). The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium withtest chemical by GC-MS revealed the presence of three compounds(Isolate I, Isolate II, and Isolate III). The mass spectra of isolatedcompound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively.The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days.In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions.In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the test bacterial inoculum Sphingomonassp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d.Thus, based on this,test chemicalis considered to be readily biodegradable in nature.

Biodegradation in water and sediment

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.

Biodegradation in soil

Biodegradation study in four different soils was conducted for evaluating the half-life value of test chemical (Qiuguo Fu et. al.; 2016). The study was performed under aerobic conditions at a temperature of 25°C.Soil samples of different textures (abbreviated herein as soil A, B, C, and D) were collected from the surface layer (0-10 cm) at two different locations. Soil A (sandy loam) was taken from the Experimental Station of University of California in Riverside, CA, while soils B, C, and D were taken fromfields at the University of California Research and Education Center in Irvine, CA. All soils were air dried and sieved using a 2-mm sieve before use.500 ml glass jar was used as a test vessel for the study. Initial test chemical conc. used for the study was 2 mg/kg.The stock solutions of these compounds were prepared in methanol and the working solution was prepared by diluting the stock solution with methanol. All stock and working solutions were stored in amber glass vials at-20°C. An aliquot of 200 g (dry weight) soil was placed in a 500-mL glass jar. The soil moisture was adjusted to 30% of the soil water holding capacity using deionized water. After a 7-d pre-incubation, 200mL of test chemical stock solution (2000 mg/l in methanol) was spiked to the soil to arrive at a nominal spiked concentration of 2 mg/kg. After spiking, the jars were left open in a fume hood until the solvent was evaporated. Deionized water was then added to adjust the soil moisture to 60% of the water holding capacity. The soil samples were mixed thoroughly with a stainless steel spatula. The soil jars were loosely covered with aluminum foil and kept in the dark.To maintain the aerobic conditions during the incubation experiment, each jar was opened every other day for aeration.The soil moisture was maintained by adding deionized water every two days. At 0, 5,11, 29, and 46 d after treatment, aliquots of 10 g soil (in triplicate) were removed from each jar and used for analyzing the test chemical concentration remaining in the soils.Soil samples collected at different time intervals were freeze dried, and 1.0 g soil subsamples (dry weight) were extracted three times. The extracts were condensed to near dryness under a gentle nitrogen stream, re-dissolved in 1.0mL methanol and thenfiltered into 2-mL amber glass vials through a 0.22-mmPTFEfilter membrane. Allfinal samples were stored at-20°C prior to instrumental analysis. Instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) combined with a Waters Micromass electrospray ionization tandem mass spectrometer (ESI-MS/MS) (Waters, Milford, MA). Separation was achieved with an ACQUITY UPLC BEH C18 column (2.1 mmX100 mm, 1.7mm particle size, Waters). Pure methanol and 5% methanol in water (containing 0.001% formic acid) were used as the mobile phases B and A, respectively, which was programmed (with respect to mobile phase A) as below: 0-5 min, 90%-0%; 5-6 min, 0-90%; and 6-8min, 90-10%, at aflow rate of 0.2 mL/min. The injection volume was 5mL and the column temperature was 40°C. The mass data was acquired under the multiple reactions monitoring (MRM) in the negative ESI mode. All data were expressed as the mean and standard deviation of triplicates. One-way ANOVA test was performed ata¼0.05 to evaluate the significance of difference between treatments. Statistical analyses were completed using the SPSS 19.0 software (IBM SPSS Statistics, Armonk, NY).The half-life value of test chemical in four different soils, i.e, soil A, B, C and D was determined to be 74±34, 82±20, 101±30 and 81±5 days, respectively, i.e, in the four soils,t1/2 ranged for the test chemical ranges from 74 to 101 d, respectively. Thus, considering all available data, the mean t1/2 value of test chemical in soil was determined to be 84 days.Based on this half-life value of test chemical, it is concluded that the test 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

Various experimental key and supporting studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Sikandar I. Mulla et. al.; 2016), biodegradation experiment was conducted for 6 days for evaluating the percentage biodegradability of test chemical at a temperature of30°C and pH 7.0, respectively. Sphingomonassp. strain YL-JM2C (Bacteria) isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosan was used as test inoculum. Stock solutions (5 g/L) were prepared with acetone and stored in brown bottles at-20°C before use. Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includesK2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. 0.04% yeast extract (sterilized by 0.45mm membrane) was added to this medium. The pH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.40 ml bottle was used as a test vessel for the study. For biodegradation experiments, 1 mL of pure bacterial culture (mid-log period) was transferred into a 40 mL bottle (working volume of 10 mL). For control, 10 mL of sterile AMS medium containing test chemical (4 mg/l) was used. All these bottles were kept in shaker (150 rpm) at 30°C under dark condition. A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of triclocarban at a specific incubation period by high performance liquid chromatography (HPLC).Degradation of test chemical metabolites 3,4-dichloroaniline and 4-chloroaniline during growth of Sphingomonassp. strain YL-JM2C was determined at different intervals according to the total organic carbon (TOC) concentration by TOC analyser (Shimadzu TOC-V CPH, Japan). The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium with test chemical by GC-MS revealed the presence of three compounds(Isolate I, Isolate II, and Isolate III). The mass spectra of isolated compound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively. The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days. In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions. In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the test bacterial inoculum Sphingomonassp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d. Thus, based on this, test chemical is considered to be readily biodegradable in nature.

 

Another biodegradation study was performed to determine biodegradability of test chemical (W. E. GLEDHILL et. al; 1975). In this the test chemical was radio labeled at parachloro aniline position and quantification of 14CO2 evolution was done by using Scintillation counting method , Counting was conducted on a Nuclear Chicago Isocap 300 counter with external standardization. Corrections for background and chemical quenching were made. Aqueous solutions, 100 ml, were incubated in 300 ml Bellco baffled Erlenmeyer flasks which were sealed with rubber stopper and an air inlet tube. The rest tube contained a 1 cm hole just below the rubber stopper and 3.0 ml of 0.5 N KOH. The test chemical concentration used in this study was 200 µg/L and temperature was 18-20oC. The total duration of study was 13 weeks. Percent degradation of test chemical was determined to be 70 % and 60 % by using activated sludge and raw sewage inoculums respectively in 28 days (4 weeks) and 88 % degradation in 23 weeks by using both inoculums by using CO2 evolution as parameter. On the basis of percent degradation value it is concluded that test chemical is readily biodegradable.

For the test chemical, an experiment was performed to determine biodegradability of test chemical (W. E. GLEDHILL et. al; 1975). In this the test chemical was radio labeled at parachloro aniline position and quantification of 14CO2 evolution was done by using Scintillation counting method , Counting was conducted on a Nuclear Chicago Isocap 300 counter with external standardization. Corrections for background and chemical quenching were made. Aqueous solutions, 100 ml, were incubated in 300 ml Bellco baffled Erlenmeyer flasks which were sealed with rubber stopper and an air inlet tube. The rest tube contained a 1 cm hole just below the rubber stopper and 3.0 ml of 0.5 N KOH. The test chemical concentration used in this study was 200 µg/L and temperature was 18-20 oC. The total duration of study was 13 weeks. Percent degradation of test chemical was determined to be 90 % , 3 % and 34% at 200, 2000 and 20µg/L test chemical concentration respectively in 28 days and 95%, 70% and 60% at 200, 2000 and 20µg/L test chemical concentration respectively in 13 weeks by using CO2 evolution as parameter. The percent degradation of test chemical at concentration 20 µg/L should be more but it is showing only 34 % degradation it may be due to binding of test chemical to activated sludge (inoculums used in this study ) .Thus on the basis of percent degradation value at concentration 200 µg/L it is concluded that test chemical is readily biodegradable.

 

Additional biodegradation study (from authoritative database, 2018) was performed to determine percent degradation of test chemical by using BOD and HPLC as parameter. Inoculum used in this study was activated sludge at concentration 30 mg/L and the initial concentration of test chemical used was 100 mg/L. Percent degradation of test chemical was determined to be 1.0 % by both parameters BOD and HPLC in 28 days. On the basis of percent degradation of test chemical, it is concluded that test chemical is not readily biodegradable.

 

On the basis of overall results of the test chemical (from peer reviewed journals and authoritative databaseJ-CHECK), it can be concluded that the test chemical can be considered to be readily biodegradable in water.

Biodegradation in water and sediment

In accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.

Biodegradation in soil

Various experimental key and supporting studies of the test chemical were reviewed for the biodegradation in soil end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Qiuguo Fu et. al.; 2016),biodegradation experiment in four different soils was conducted for evaluating the half-life value of test chemical. The study was performed under aerobic conditions at a temperature of 25°C.Soil samples of different textures (abbreviated herein as soil A, B, C, and D) were collected from the surface layer (0-10 cm) at two different locations. Soil A (sandy loam) was taken from the Experimental Station of University of California in Riverside, CA, while soils B, C, and D were taken from fields at the University of California Research and Education Center in Irvine, CA. All soils were air dried and sieved using a 2-mm sieve before use.500 ml glass jar was used as a test vessel for the study. Initial test chemical conc. used for the study was 2 mg/kg. The stock solutions of these compounds were prepared in methanol and the working solution was prepared by diluting the stock solution with methanol. All stock and working solutions were stored in amber glass vials at-20°C. An aliquot of 200 g (dry weight) soil was placed in a 500-mL glass jar. The soil moisture was adjusted to 30% of the soil water holding capacity using deionized water. After a 7-d pre-incubation, 200mL of test chemical stock solution (2000 mg/l in methanol) was spiked to the soil to arrive at a nominal spiked concentration of 2 mg/kg. After spiking, the jars were left open in a fume hood until the solvent was evaporated. Deionized water was then added to adjust the soil moisture to 60% of the water holding capacity. The soil samples were mixed thoroughly with a stainless steel spatula. The soil jars were loosely covered with aluminum foil and kept in the dark. To maintain the aerobic conditions during the incubation experiment, each jar was opened every other day for aeration. The soil moisture was maintained by adding deionized water every two days. At 0, 5,11, 29, and 46 d after treatment, aliquots of 10 g soil (in triplicate) were removed from each jar and used for analyzing the test chemical concentration remaining in the soils. Soil samples collected at different time intervals were freeze dried, and 1.0 g soil subsamples (dry weight) were extracted three times. The extracts were condensed to near dryness under a gentle nitrogen stream, re-dissolved in 1.0mL methanol and then filtered into 2-mL amber glass vials through a 0.22-mmPTFEfilter membrane. All final samples were stored at-20°C prior to instrumental analysis. Instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) combined with a Waters Micromass electrospray ionization tandem mass spectrometer (ESI-MS/MS) (Waters, Milford, MA). Separation was achieved with an ACQUITY UPLC BEH C18 column (2.1 mmX100 mm, 1.7mm particle size, Waters). Pure methanol and 5% methanol in water (containing 0.001% formic acid) were used as the mobile phases B and A, respectively, which was programmed (with respect to mobile phase A) as below: 0-5 min, 90%-0%; 5-6 min, 0-90%; and 6-8min, 90-10%, at a flow rate of 0.2 mL/min. The injection volume was 5mL and the column temperature was 40°C. The mass data was acquired under the multiple reactions monitoring (MRM) in the negative ESI mode. All data were expressed as the mean and standard deviation of triplicates. One-way ANOVA test was performed ata¼0.05 to evaluate the significance of difference between treatments. Statistical analyses were completed using the SPSS 19.0 software (IBM SPSS Statistics, Armonk, NY).The half-life value of test chemical in four different soils, i.e, soil A, B, C and D was determined to be 74±34, 82±20, 101±30 and 81±5 days, respectively, i.e, in the four soils,t1/2 ranged for the test chemical ranges from 74 to 101 d, respectively. Thus, considering all available data, the mean t1/2 value of test chemical in soil was determined to be 84 days. Based on this half-life value of test chemical, it is concluded that the test chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

Another biodegradation study in loam soil was conducted for evaluating the half-life value of test chemical (Guang-Guo Ying et. al., 2007). The study was performed under both aerobic and anaerobic conditions at a temperature of 22°C.A loam soil with pH value of 7.4 was collected from an agricultural land without sludge amendments and used in the laboratory biodegradation study. The soil contains52.1% of sand, 11.6% of silt, 34.7% of clay and 1.3% of organic carbon, respectively. For aerobic experiments, 5 g of soil was weighed into each scintillation vial (20 mL). The moisture level in each vial was adjusted using sterile water to 50% MWHC (maximum water holding capacity). Test chemical at a concentration of 1 mg/L in acetone was added into each vial to make 1 mg/kg in the soil. Lids were left open for 1 h to allow acetone to evaporate. Each vial was mixed well and incubated under darkness at 22°C in a constant temperature room. Half of the vials were sterilised by autoclaving at 120°C under 300 kPa chamber pressure for 30 min for three times within 3 days before adding the test chemical, and used as sterile controls. Each vial was opened weekly to let the air in to maintain its aerobic conditions during the incubation and for anaerobic experiments, preparation was carried in an anaerobic incubation chamber filled with nitrogen gas. Five grams of soil and 1 mL of sterile water was weighed into each Hungate anaerobic culture tube. Half of the tubes were taken out for autoclaving, and after sterilisation these tubes were placed back into the anaerobic chamber. Test chemical at a concentration of 1 mg/L in acetone was spiked into each tube to make 1 mg/kg in the soil. Lids were opened for some time to allow acetone to evaporate. Resazurin was added at a concentration of 0.0002% into two tubes as a redox indictor. Reducing conditions within the tubes were indicated by the disappearance of the red resazurin color. All Hungate tubes were incubated under darkness at 22°C.Concentrations of each compound in the samples were monitored at certain intervals (0, 1, 7, and weekly to 70 days) by utilising three samples from each treatment. Triplicate sterile controls were also monitored at the same time. For analytical analysis, test chemical in the soil samples was extracted twice using 20 mL of acetone. The extracts were blown to dryness by a gentle stream of nitrogen and re-dissolved in 0.5 mL of methanol. Test chemical was analysed on an Agilent 1100 series high performance liquid chromatograph (HPLC) fitted with a diode array detector and a SGE C18 RS column (100X4.6 mm, 5µm). Acetonitrile (ACN) and water were used as the mobile phase, which was programmed from 40% ACN at 0 min to 80% ACN at 10 min, 90% ACN at 12 min, 90% ACN at 20 min and back to 40% CAN at 25 min at a flow rate of 1 mL/min. The UV wavelength for detection was 265 nm. The retention time was 8.3 min. No changes in concentrations of test chemical were observed in the sterile soil within 70 days. This suggests that no degradation of test chemical by chemical processes occurred in the sterile soil. The half-life value of test chemical in loam soil was determined to be 108 days using a first order constant under both aerobic and anaerobic conditions. Based on this half-life value of test chemical, it is concluded that the test chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

On the basis of above results of the test chemical(from peer reviewed journals), it can be concluded that the half-life value of test chemical was determined to be ranges from 74 to 108 days, indicating that the test chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

Biodegradation in soil endpoint can also be considered for waiver as per in accordance with column 2 of Annex IX of the REACH regulation, testing for this end point is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable in water.