Registration Dossier

Environmental fate & pathways

Endpoint summary

Administrative data

Description of key information

Stability:

Hydrolysis

HYDROWIN v2.00 program of Estimation Programs Interface (2018) prediction model was used to predict the hydrolysis half-life of test chemical (Modelling database, 2018 and secondary source, 2002). The estimated half-life of test chemical was evaluated to be > 1 yr (at 25ᵒC) respectively, indicating that it is not hydrolysable in water

Biodegradation:

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.

Bioaccumulation:

Bioaccumulation: aquatic/sediments:

The bioaccumulation test was conducted for 28 days for determination the bioconcentration factor (BCF) of chemical on test organism Cyprinus carpio. Nominal concentrations used in the study are Nominal concentrations 1st Concentration area : 0.020 mg/L, 2nd Concentration area : 0.0020 mg/Land Range finding study was carried out on Rice fish (Oryzias latipes) LC50(96h) > 5.0 mg/L,. The vehicle used in this study was HCO-40.The bioconcentration factor (BCF) for test chemical was determined in Cyprinus carpio . The lipid concentration was 3.6% at start of exposure and 3.4 % at end of exposure. The bioconcentration factor (BCF) for test chemical was determined in Cyprinus carpio. The BCF value was observed to be in range 63-93 L/kg and 80 L/kg at steady state at dose concentration 0.02 mg/L and in range 61-98 L/kg and 81 L/kg at steady state at dose concentration 0.002 mg/L. On the basis of total lipid content on test organism Cyprinus carpio during 28 days period. Therefore it is concluded that this test chemical is nonbioaccumulative.

Transport and distribution:

Adsorption/desorption:

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the Reverse Phase High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals. The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 4 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.5. Each of the reference substance and test substance were analysed by HPLC at 210 nm. After equilibration of the HPLC system, Urea was injected first, the reference substances were injected in duplicate, followed by the test chemical solution in duplicate. Reference substances were injected again after test sample, no change in retention time of reference substances was observed. Retention time tR were measured, averaged and the decimal logarithms of the capacity factors k were calculated. The graph was plotted between log Koc versus log k(Annex - 2).The linear regression parameter of the relationship log Koc vs log k were also calculated from the data obtained with calibration samples and therewith, log Koc of the test substance was determined from its measured capacity factor. The reference substances were chosen according to functional similarity with the test substance and calibration graph prepared. The reference substances were 4-chloroaniline, Aniline, 2,5 -Dichloroaniline, Naphthalene, 1,2,3-trichlorobenzene, phenanthrene having Koc value ranging from 1.96 to 4.09. The Log Koc value of test chemical was determined to be 3.486 ± 0.004 dimensionless at 25°C.This log Koc value indicates that the substance has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

Additional information

Stability:

Hydrolysis

HYDROWIN v2.00 program of Estimation Programs Interface (2018) prediction model was used to predict the hydrolysis half-life of test chemical (Modelling database, 2018 and secondary source, 2002). The estimated half-life of test chemical was evaluated to be > 1 yr (at 25ᵒC) respectively, indicating that it is not hydrolysable in water.

Hydrolysis endpoint can also be considered for waiveras per in accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable.

Biodegradation:

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.

Bioaccumulation:

Bioaccumulation: aquatic/sediments:

Two experimental studies have been reviewed to determine bioconcentration factor of test chemical from different sources and their results are summarized below.

In first study the bioaccumulation test was conducted for 28 days for determination the bioconcentration factor (BCF) of chemical on test organism Cyprinus carpio. Nominal concentrations used in the study are Nominal concentrations 1st Concentration area : 0.020 mg/L, 2nd Concentration area : 0.0020 mg/Land Range finding study was carried out on Rice fish (Oryzias latipes) LC50(96h) > 5.0 mg/L,. The vehicle used in this study was HCO-40.The bioconcentration factor (BCF) for test chemical was determined in Cyprinus carpio . The lipid concentration was 3.6% at start of exposure and 3.4 % at end of exposure. The bioconcentration factor (BCF) for test chemical was determined in Cyprinus carpio. The BCF value was observed to be in range 63-93 L/kg and 80 L/kg at steady state at dose concentration 0.02 mg/L and in range 61-98 L/kg and 81 L/kg at steady state at dose concentration 0.002 mg/L. On the basis of total lipid content on test organism Cyprinus carpio during 28 days period. Therefore it is concluded that this test chemical is nonbioaccumulative.

In another study the Bioconcentration factor of test chemical was determined in Blue gill fish by flow through system for 30 days. Test chemical concentration used in the study was 0.72µg/L. The depuration duration of this study was 8 days. In this study population of approximately 80 bluegill (Lepomis macroiborius), 5 cm length, were exposed to a nominal test chemical concentration of 1.0µg/L.ina flow through system .The test chemical was 3.4 ring labelled HC with a specific activity of 4.54 mCi/mM (Monsanto Code 103). The bluegills were exposed for 30 days of uptake and 8 days of depuration. Fish were sampled on days 2, 7, 14, 22, 24, and 30 of uptake and on days of depuration. On each sampling day some fish were left whole and others were dissected into muscle, gills, intestines, liver, gall bladder, spleen, and remains. Dissected fish were pooled, two fish per observation with two observations in each mean estimate. All fish were dried, weighed combusted to carbon dioxide, and counted for 1C activity. The BCF value was determined to be 887 wt/wt. in whole fish. By considering this BCF value it is concluded that test chemical is non-bioaccumulative in blue gill fish.

By considering results of both the studies mentioned above it is observed that bioaccumulation concentration factor (BCF) in blue gill and cyprinus carpio fish was in range of 61- 887 L/Kg which is less than 2000. On the basis this range BCF value it is concluded that test chemical is non bioaccumulative in nature according to CLP classification criteria.

Transport and distribution:

Adsorption/desorption:

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

 

In an experimental study from study report (2018),the adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the Reverse Phase High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals. The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 4 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.5. Each of the reference substance and test substance were analysed by HPLC at 210 nm. After equilibration of the HPLC system, Urea was injected first, the reference substances were injected in duplicate, followed by the test chemical solution in duplicate. Reference substances were injected again after test sample, no change in retention time of reference substances was observed. Retention time tR were measured, averaged and the decimal logarithms of the capacity factors k were calculated. The graph was plotted between log Koc versus log k(Annex - 2).The linear regression parameter of the relationship log Koc vs log k were also calculated from the data obtained with calibration samples and therewith, log Koc of the test substance was determined from its measured capacity factor. The reference substances were chosen according to functional similarity with the test substance and calibration graph prepared. The reference substances were 4-chloroaniline, Aniline, 2,5 -Dichloroaniline, Naphthalene, 1,2,3-trichlorobenzene, phenanthrene having Koc value ranging from 1.96 to 4.09. The Log Koc value of test chemical was determined to be 3.486 ± 0.004 dimensionless at 25°C.This log Koc value indicates that the substance has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

Another adsorption study was conducted for evaluating the adsorption capacity of test chemical onto two different soils, i.e, silt clay soil (BC) and sandy loam soil (DL) (CHENXI WU et. al., 2009). The study was performed using the batch equilibrium method in accordance with OECD Guideline 106 (Adsorption - Desorption Using a Batch Equilibrium Method). Test chemical was purchased from Sigma-Aldrich (St. Louis, MO). Test chemical conc. used for the study were0.1, 0.4, 0.8, 1.4, and 2.0 mg/L, respectively. Two soils were used during the study. One was silt clay soil (BC)and the other was sandy loam soil (DL).A silt clay soil (BC) and a sandy loam soil (DL) were collected from the top layer (0-20 cm) in two fields with no biosolids application history in Lucas county, northwestern Ohio. The soil samples were air-dried, gently disaggregated, and sieved to a particle size of≤2 mm. Biosolids were aerobically digested sludge generated at a local wastewater treatment plant (Oregon, OH), which uses activated sludge treatment techniques. Samples were taken directly from a field at the time of biosolids application. Biosolids contained 37.2 g L-1 total suspended solids and 20.5 g L-1 volatile suspended solids and had a pH of 7.9. locally. The biosolids-amended soils (BCB and DLB) were air-dried and passed through a 2 mm sieve. Soil pH was measured in 0.01 M CaCl2 at a ratio of 1:2 (w/v) and texture was determined using the pipet method. Soil organic matter (SOM) content was measured by loss on ignition at 450°C for 4 h. Total organic carbon (TOC) content and cation exchange capacity (CEC) were determined by Spectrum Analytic Inc. (Washington Court House, OH).All soil samples were stored in plastic bags in dark conditions at room temperature (23± 3°C) for less than a month before use.50 ml glass centrifuge tubes, sealed withTeflon-lined screw caps were used as a test vessel for the study. In a preliminary experiment, sorption kinetics were investigated by determining the liquid phase concentration at 2, 4, 8, 24, 48, 72, and 120 h. After 48 h, concentration varied by <5%; thus, 48 h was chosen as the apparent equilibrium time for the sorption experiments. In a spiked control experiment, sorption on test vessel surfaces was observed for test chemical; therefore, both aqueous phase and sorbed phase concentrations were analysed as recommended in the OECD Guidelines. In the sorption experiment, soil samples (1 g, dry weight) were mixed with 40 mL of 0.01 M CaCl2 solution containing 0.1% NaN3 (w/v) in 50 mL glass centrifuge tubes, sealed with Teflon-lined screw caps, and agitated for 24 h using a reciprocal shaker. NaN3 was used to inhibit the activity of microbes. The soil slurry was then spiked with 0.04 mL of standard solution prepared in acetone (final acetone content=0.1%) to achieve concentrations of 0.1, 0.4, 0.8, 1.4, and 2.0 mg/L. After agitation for 48 h, samples were withdrawn and centrifuged at 1500gfor 20 min. Aliquots of 0.5 mL of supernatant were transferred into 2 mL amber glass vials and diluted with 0.5 mL of methanol to reduce sorption to the vials. The remaining supernatant was decanted, and soil samples were then freeze-dried and extracted using accelerated solvent extraction (ASE) with a Dionex ASE 200 system. Briefly, freeze-dried samples were mixed with Ottawa sand and transferred into 11 mL extraction cells. The cells were sealed at the bottom with glass fiber filters, filled with Ottawa sand to the top, and tightened with screw caps. The prepared cells were extracted with methanol using the following conditions: preheat, 0 min; static, 5 min; flush, 60%; cycles, 2; purge, 120 s; oven temperature, 80°C; pressure, 1500 psi. The extracted samples were collected in 60 mL glass vials. Final volume was about 20 mL. Aliquots of 1 mL extracted samples were transferred into amber glass vials. Extraction recoveries were >80% for both compounds with<10%relative standard deviation. Test vessel was agitated for 24 h using a reciprocal shaker. Blank samples without spiking were also included. All experiments were peformed in duplicate. Prepared samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).The Freundlich Kf values for the test chemical in the two soils were different. For the test chemical, the KF value of test chemical on two soil ,i.e, silt clay soil (BC) and sandy loam soil (DL) was determined to be 799 µg1-nLn/Kg (457-1140) and 2624 µg1-nLn/Kg (1381-3867), respectively. The adsorption coefficient (Koc) value of test chemical onto two different soils, i.e, silt clay soil (BC) and sandy loam soil (DL)were 48865 (logKoc = 4.688) and 64037 (logKoc = 4.8), respectively. This Koc value indicates that the test chemical has a very strong sorption to soil and sediment and therefore have negligible migration potential to ground water.

 

In an supporting study,the adsorption coefficient of test chemical was determined in five different types of soils ray, drummer, Spinks , Lintonia and sediment lake 24 by using batch equilibrium method (secondary source NTRL report, 1980). The initial concentration of test chemical was 5 10, 20, 35 and 50 ppb. The tubes were capped, mixed on a Vortex, and placed on a gyratory shaker in the absence of light at 25oC. Three tubes from each of the five test chemical solution concentrations were removed from the shaker at 24 and 48 hours. The tubes were agitated on a Vortex shaker and centrifuged at 11,000 rpm for 30 minutes. Duplicate 1.0 mL samples of the supernatant were analyzed for 14 C activity by liquid scintillation counting method .Soil/sediment samples were extracted twice using 2 mL Burdick and Jackaon UV grade methanol. Methanol extracts were evaporated under nitrogen at 85 oC. Extracted test chemical was resolubilized in 3 mL methanol and counted by liquid scintillation methods. The adsorption coefficent koc value of test chemical was determined to be 53000,210000, 56000, 88000 and 210000 (log koc = 4.724, 5.322, 4.748, 4.944 and 5.322 respectively)  in different soil samples namely ray, drummer, Spinks , Lintonia and sediment lake 24 respectively by using batch equilibrium method. On the basis of log koc values it is concluded that test chemical very strong sorption to soils and negligible migration potential to ground water.

 

For the test chemical from authoritative databases (2017), adsorption study was conducted for evaluating the adsorption capacity of test chemical onto agricultural soil. Agricultural soil (characteristics 20% clay, 69% silt and 11% sand) was obtained from Corrstown, Co. Dublin, Ireland. The adsorption coefficient (Koc) value of test chemical onto agricultural soil was determined to be 12,000 L/Kg (logKoc = 4.08). This Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

In an additional supporting study from peer reviewed journal (W. E. GLEDHILL, 1975), percent adsorption of test chemical was determined by batch equilibration method in this study the test chemical used was radio-labelled at parachloro aniline ring of test chemical. Adsorption experiment was carried out in flasks containing activated sludge and test chemical at concentration 200 µg/L and 20 µg/L and were shaken on a rotary shaker for 2 h at room temperature after which the suspensions were centrifuged and the residual dissolved test chemical in the supernatant assayed by using Scintillation counting. The percent adsorption of test chemical of equilibration at 18-20oC temperature activated sludge was 61-96 in 2 hrs. On the basis of percent adsorption value it is concluded that test chemical has strong sorption to sludge and therefore has negligible to slow migration potential to ground water.

 

On the basis of above results of test chemical (from study report, peer reviewed journals, secondary source and authoritative databases), it can be concluded that the logKoc value of test chemicalwas evaluated to be ranges from3.486 to 5.32,indicating that the test chemicalhas a strong to very sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.