Phenylacetic acid

Endpoint summary

Description of key information

Hydrolysis

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 in water

Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test chemical (from peer reviewed journal (H. Leidner et. al, 1980), authoritative databases (2017) and secondary source). The study was performed according to OECD Screening test.The study was performedunder aerobic conditions at a temperature of 20°C.Mixed culture isolated from soil was used as a test inoculum for the study. Initial test substance conc. used in the study was 8 mg/l. Erlenmeyer flasks were used as a test vessel for the study. Test inoculum i.e, mixed microbial biocenosis(a community of various species of organotrophic microorganisms) was prepared by diluting a suspension of garden soil. 2 l of mineral salt medium containing the test chemical as a sole source of carbon were inoculated with 2ml of a mixed culture. The test vessels were incubated at 20°C with continuous stirring. Degradation of test chemical was determined by DOC, GC and HPLC analysis. For Gas chromatography analysis, sample preparation involve 50ml of the bioassay mixture were centrifuged for 15 min at 30,000 g to remove the biomass from the liquid. For GLC analysis, test chemical was esterified via their silver salts. The FID signal was electronically integrated (CRS-100AInfotronics, Ireland). n-Tridecane served as an internal standard in quantitative determinations.HPLC methods were also used to follow the degradation. ion pair partition chromatography on a bonded phasecolumn(250x4mm, packed with 5µsilica, chemically modified with dichlorodimethylsilane) was used as a separation column.A0.01Mtributylamine solution in methanol-water (1:2, v/v), titrated with perchloric acid to pH 2, served as the eluent. Varian 850 dual syringe pump instrument equipped with a variable wavelength detector (Varichrom, Varian) was used. The decreases in peak heights in proportion to the initial heights served as a relative quantitative measure for degradation.In additional to GC and HPLC,Dissolved organic carbon was also determined of the test chemical. For this,bacteria-free 50 ml samples were acidified with HCI. Dissolved organic carbon (dissolved organic compounds expressed as carbon) was determined with a carbon analyser 'Unor' (Maihak, Hamburg, FR Germany). Detection limit: -0.1 p.p.m. above background.The percentage degradation of test chemical was determined to be 100% by DOC, GC and HPLC parameter after a period of 3 days. Thus, based on percentage degradation,test chemical is considered to be readily biodegradable in nature.

Biodegradation in water and sediment

Estimation Programs Interface (2018) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 26% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical in water is estimated to be 15 days (360 hrs). The half-life (15 days estimated by EPI suite) indicates that the chemical is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.0796%), indicates that test chemical is not persistent in sediment.

 

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, 73.3% 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 30 days (720 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.

Bioaccumulation: aquatic / sediment

The bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of test chemical (authoritative databases, 2017). The bioaccumulation factor (BCF) value was calculated using a logKow of 1.41 and a regression-derived equation. The estimated BCF (bioaccumulation factor) valueof test chemical was determined to be 3.0 dimensionless, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

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 (Experimental study report, 2017). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 5 mg of test item and diluted with ACN up to 10 ml. Thus, the test solution concentration was 500 mg/l. The pH of test substance was 5.6. 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 estimated Koc range of the test substance and generalized calibration graph was prepared. The reference substances were Acetanilide, 4-chloroaniline, 4-methylaniline(p-Tolouidine), N-methylaniline, p-toluamide, Aniline, 2,5- Dichloroaniline, 4 -nitrophenol, 2 - nitrophenol, 2-nitrobenzamide, 3-nitrobenzamide, Nitrobenzene, 4 - Nitrobenzamide, 1-naphthylamine, 1-naphtol, Direct Red 81, Benzoic acid methylester, Carbendazim, Xylene, Ethylbenzene, Toluene, Naphthalene, 1,2,3 -trichlorobenzene, Pentachlorophenol, Phenol, N,Ndimethylbenzamide, 3,5-dinitrobenzamide, N-methylbenzamide, Benzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 1.932± 0.001dimensionless at 25°C.This log Koc value indicates that the substance has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

Additional information

Hydrolysis

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 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 (H. Leidner et. al, 1980), authoritative databases (2017) and secondary source,biodegradation experiment was conducted for 6 days for evaluating the percentage biodegradability of test chemical. The study was performed according to OECD Screening test. The study was performed under aerobic conditions at a temperature of 20°C.Mixed culture isolated from soil was used as a test inoculum for the study. Initial test substance conc. used in the study was 8 mg/l. Erlenmeyer flasks were used as a test vessel for the study. Test inoculum i.e, mixed microbial biocenosis(a community of various species of organotrophic microorganisms) was prepared by diluting a suspension of garden soil. 2 l of mineral salt medium containing the test chemical as a sole source of carbon were inoculated with 2ml of a mixed culture. The test vessels were incubated at 20°C with continuous stirring. Degradation of test chemical was determined by DOC, GC and HPLC analysis. For Gas chromatography analysis, sample preparation involve 50ml of the bioassay mixture were centrifuged for 15 min at 30,000 g to remove the biomass from the liquid. For GLC analysis, test chemical was esterified via their silver salts. The FID signal was electronically integrated (CRS-100AInfotronics, Ireland). n-Tridecane served as an internal standard in quantitative determinations. HPLC methods were also used to follow the degradation. ion pair partition chromatography on a bonded phase column(250x4mm, packed with 5µsilica, chemically modified with dichlorodimethylsilane) was used as a separation column.A0.01Mtributylamine solution in methanol-water (1:2, v/v), titrated with perchloric acid to pH 2, served as the eluent. Varian 850 dual syringe pump instrument equipped with a variable wavelength detector (Varichrom, Varian) was used. The decreases in peak heights in proportion to the initial heights served as a relative quantitative measure for degradation. In additional to GC and HPLC, Dissolved organic carbon was also determined of the test chemical. For this, bacteria-free 50 ml samples were acidified with HCI. Dissolved organic carbon (dissolved organic compounds expressed as carbon) was determined with a carbon analyser 'Unor' (Maihak, Hamburg, FR Germany). Detection limit: -0.1 p.p.m. above background. The percentage degradation of test chemical was determined to be 100% by DOC, GC and HPLC parameter after a period of 3 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 20 days for evaluating the percentage biodegradability of test chemical (R. V. Subba-Rao et. al., 1977). The study was performed under aerobic conditions at a temperature of25°C.Bacteria obtained from Hudson Collamer silt loam was used as a test inoculum for the study. The chemical was introduced into the BOD bottles as a source of carbon at a concentration of 2 mg of carbon per bottle. Test chemical was added in the acetone solutions, and the acetone was evaporated prior to the addition of O2-saturated water. Each bottles received 5 mg of Hudson Collamer silt loam as a source of the microbial inoculum. The bottles were filled with the air-saturated salts solution and closed with glass stoppers. Bottles containing 02-saturated water inoculated with soil but no carbon source were also included in the study to account for theO2depletion resulting from microbial oxidation of organic matter and ammonium. Each compound was also tested in combination with glucose (both at a concentration of 2 mg of carbon per bottle) to test whether the possible lack of biodegradation was a result of toxicity of the test chemical. The bottles were incubated in the dark at 25°C.Dissolved O2 in the bottles was measured at regular intervals using a Yellow Spring Instrument Co. (Yellow Springs, Ohio) oxygen analyzer, Model 53. The instrument was calibrated with the salts solution, theO2content of which was determined by the Alsterberg modification of the Winkler method (American Public Health Association, 1971). At regular intervals, the dissolved O2 in the samples was measured after calibrating the instrument with a BOD bottle containing inoculated 02-saturated water supplemented with 0.1% KCN. The solutions in bottles showing O2depletion were used to obtain microorganisms capable of utilizing the substrate. The BOD5 of the test chemical was determined to be approx. 5.4 O2 consumed µg/ml. Thus, based on this, test chemical is considered to be readily biodegradable in nature.

 

In a supporting study from peer reviewed journal (W. R. Mayberry et. al., 1967),biodegradation experiment was conducted for evaluating the percentage biodegradability of test chemical. The study was performed at a temperature of 30°C. Test bacterial inoculum (Pseudomonas C12B and TEG-5) obtained from soil was used as a test inoculum for the study. Cells were adapted to the test substrates by three transfers into a basal salts medium containing the compound of interest at a concentration of 0.25%, w/v, with incubation at 30°C on a rotary shaker. Adapted cells were harvested by centrifugation (37,000 X g for 30 min) of late-log-phase cultures, and washed twice with sterile basal salts medium to remove soluble substrates. The substrate-free cells were then reharvested and washed twice with sterile basal salts. Washed adapted cells from the various cultures were resuspended in the sterile mineral medium to an optical density of 0.65 at 420 ml,. Portions of 0.5 ml each were used as inocula. Test chemical conc. used for the study were 20, 40, 60 and 80 µM per 30 ml, respectively. Basal salt medium was used as a test medium for the study. Warburg flasks were used as a test vessel. Test was performed in 4 replicates. Replicate sets of four flasks containing a fourfold range of substrate concentrations, approximately 20, 40, 60, and 80 µmoles per 30 ml, were prepared with 0.5 ml of 50% KOH in the center wells to trap carbon dioxide. Inocula were placed in the side arms, bringing the total fluid volume to 31 ml and the total culture volume to 30.5 ml. After equilibration at 30 C, the cells were tipped in and oxygen uptake was followed manometrically as the bacteria multiplied until semilogarithmic plots of oxygen uptake versus time were no longer linear. Flasks were removed from the apparatus, cell crops determined, and the media analyzed for residual substrate and possible end products of metabolism. Concentration of the test chemical was determined in all cases by calculation of the ratio of the peak area for the compound of interest to the peak area for the internal standard. For each substrate, a standard curve was established relating the peak area ratios to concentrations encompassing a range of 0.5 to 20 µmoles per 30 ml. In all cases, substrate utilized was considered to be the difference between original and residual concentrations. The 95% confidence intervals were calculated by Student's t formula. The percentage degradation of test chemical was determined to be 82.21% by using Test material analysis (% degradation) parameter. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

For the test chemical, biodegradability of test chemical was estimated using the BIODEG linear and non-linear model (E. Rorije et. al., 1997). The models have been applied are Quantitative Structure-Activity Relationships. Two models are fitted to the judgement of an evaluation of various different biodegradation rates of chemical. This include the one using linear regression and other using a form of non-linear fitting. These models used 36 fragments as descriptors with the molecular weight of the molecule as an added descriptor, and are fitted on the evaluated biodegradation data of a series of 295 compounds. The output of the models, x, is the number that should be interpreted as x < 0.5 (=0) (Chemical biodegrades slowly or not at all) or x > 0.5 (=1) (Chemical biodegrades fast), respectively. These tests typically produce ‘biodegradable’ (1) or ‘non-biodegradable’ (0) as a result. The application of the models has been automated, the BIODEG models are incorporated in the PC-based program. As the biodegradability prediction of test chemical by the BIODEG models comes out to be 1, test chemical is estimated to be readily biodegradable in nature.

 

On the basis of above results for test chemical, it can be concluded that the test chemical can be considered to be readily biodegradable in nature.

Biodegradation in water and sediment

Estimation Programs Interface (2018) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 26% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical in water is estimated to be 15 days (360 hrs). The half-life (15 days estimated by EPI suite) indicates that the chemical is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.0796%), indicates that test chemical is not persistent in sediment.

 

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, 73.3% 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 30 days (720 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.

On the basis of available information, the test chemicalcan be considered to be readily biodegradable in nature.

Bioaccumulation: aquatic / sediment

Various predicted data of the test chemical were reviewed for the bioaccumulation end point which are summarized as below:

 

In a weight of evidence study from authoritative databases (2017), the bioaccumulation experiment was conducted for estimating the BCF (bioaccumulation factor) value of test chemical. The bioaccumulation factor (BCF) value was calculated using a logKow of 1.41 and a regression-derived equation. The estimated BCF (bioaccumulation factor) valueoftest chemicalwas determined to be 3.0 dimensionless.

 

In a prediction done using the BCFBAF Program of Estimation Programs Interface was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 3.162 L/kg whole body w.w (at 25 deg C).

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical), the bioaccumulation i.e BCF for test chemical was estimated to be 3.54 dimensionless . The predicted BCF result based on the 5 OECD principles.

 

On the basis of above results for test chemical, it can be concluded that the BCF value of test chemical was estimated to be ranges from 3.0 to 3.54 respectively,which does not exceeds the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

Bioaccumulation endpoint can also be considered for waiver as per inaccordance with column 2 of Annex IX of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical has a low potential for bioaccumulation based on logKow ≤ 3.

Adsorption / desorption

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

 

In an experimental key study from study report (2017),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 5 mg of test item and diluted with ACN up to 10 ml. Thus, the test solution concentration was 500 mg/l. The pH of test substance was 5.6. 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 estimated Koc range of the test substance and generalized calibration graph was prepared. The reference substances were Acetanilide, 4-chloroaniline, 4-methylaniline(p-Tolouidine), N-methylaniline, p-toluamide, Aniline, 2,5- Dichloroaniline, 4 -nitrophenol, 2 - nitrophenol, 2-nitrobenzamide, 3-nitrobenzamide, Nitrobenzene, 4 - Nitrobenzamide, 1-naphthylamine, 1-naphtol, Direct Red 81, Benzoic acid methylester, Carbendazim, Xylene, Ethylbenzene, Toluene, Naphthalene, 1,2,3 -trichlorobenzene, Pentachlorophenol, Phenol, N,Ndimethylbenzamide, 3,5-dinitrobenzamide, N-methylbenzamide, Benzamide, phenanthrene, DDT having Koc value ranging from 1.25 to 5.63. The Log Koc value of test chemical was determined to be 1.932± 0.001dimensionless at 25°C.This log Koc value indicates that the substance has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

 

In a supporting study,adsorption experiment was conducted for evaluating the adsorption capacity of test chemical onto three different soils (from peer reviewed journal (B. von Oepen et. al., 1991) and authoritative databases, 2017). The study was performed in accordance with the Modified OECD-Guideline 106 Adsorption/Desorption using the batch equilibrium method. The soils used for testing covers a broad spectrum of sorption relevant properties e.g. organic carbon content (% oc), clay content, pH value, Cation Exchange Capacity etc. The soils used for testing were an acidic forest soil, a Podzol and an agricultural soil (Alfisol). Additionally the sorption behaviour of a sublimnic soil, a sediment of the Lake Constance, was investigated .Initial test chemical concentrations used for the study were about 0.15, 0.5, 5 and 15 mg/l, respectively. For sorption experiments,50 ml of the test solution were added to 10 g (dry weight) of the specific soils. The soil samples were shaken for 0.5, 1, 1.5, 5, 24 respectively 72 hours. Then an aliquot of 1 ml of the water-phase was removed and the concentration of the test chemical in the aqueous phase was determined. As for test chemical, the sorption equilibrium was reached within 16 hours, the Freundlich isotherms were determined after an incubation period of 16 hours. The initial concentrations used were about 15 mg/l, 5 mg/1, 0.5 and 0.15 mg/l. After reaching the equilibrium the soil samples were centrifuged, decanted, and the concentrations in the supernatant were determined by means of GC, HPLC or scintillation measurements. All samples were determined in parallel. One control and one blank were investigated additionally. Alter the adsorption step, a two step desorption test was performed with an equilibrium time of 8 hours followed by a desorption period of 16 hours. The Freundlich constants and Koc values were calculated. For the test chemical, the sorption equilibrium was reached within 16 hrs. The soil with the highest organic carbon content, the Podzol, has the highest sorption capacity towards the test chemical as compared to the Alfisolsoil and Sublimnic soil. Consequently the sorption coefficients determined for Alfisol and the sediment are lower than those determined for the Podzol. The desorption data revealed that sorption was reversible to a great extent for all substances under study. The mass balance resulted in a recovery of ˃ 80%. The adsorption coefficient (Koc) value of test chemical onto three different soils, a Podzol, an alfisol and sediment was determined to be 31 (Log Koc= 1.491), 26 (Log Koc= 1.414) and 28 (Log Koc= 1.447) dimensionless with a corresponding Freundlich constant (Kf) value of 1.48, 0.33 and 0.44, respectively. This logKoc value indicates that the test chemical has a negligible sorption to soil and sediment and therefore has rapid migration potential to ground water.

 

For the test chemical from peer reviewed journal (S. Bintein et. al., 1994), adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical. The adsorption coefficient (Koc) value was calculated using the QSAR model. This model involves a variety of abiotic and biotic parameters as inputs, among which one such parameter is the soil or sediment sorption coefficient of test chemical (Kp) in models used to estimate the environmental mobility and fate of the test chemical. The QSAR model involves the use of quantitative structure activity relationship (QSAR) equation. Equations used in developing QSAR model for describing the sorption behavior of test chemical in soil or sediments were Koc = (Kp X 100)/%OC and logKp = 0.93 logKow + 1.09 log fOC+ 0.32 CFa – 0.55 CFb’ + 0.25, respectively, in which, the Kp, logKow and pKa value were obtained from the different literatures. Thus, the adsorption coefficient (Koc) value of test chemical was estimated to be ranges from 29.85 to 71.42 (Log Koc= 1.47 to 1.85), respectively, calculated using a QSAR equation. This Koc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

 

On the basis of above overall results for test chemical (from study report, peer reviewed journals, and authoritative databases), it can be concluded that the logKoc value of test chemical ranges from 1.41–1.9, respectively, indicating that the test chemical has a negligible to low sorption to soil and sediment and therefore have rapid to moderate migration potential to ground water.

 

In addition to the above information, adsorption endpoint can also be considered for waiver as per in accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpointis scientifically not necessary and does not need to be conducted since the test chemical has a low octanol water partition coefficient and the adsorption potential of this substance is related to this parameter.