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Biodegradation in water

Biodegradation study was conducted for 28-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical, toxicity control and referencesubstance. Polyseed capsule (mixed culture) was used as a test inoculum for the study. Test inoculum polyseed capsule was composed of blend of specialized microbial cultures and food grade gelatin made by International Laboratory Supply (InterLab), LTD. No pretreatment / preconditioning was given to the test inoculum as the polyseed capsule requires only one hour of stirring to activate it. Polyseed capsule is a blend of broad spectrum bacteria designed specifically as seed inoculums for the BOD test. Polyseed is an EPA approved BOD seed inoculum. 38.4 mg of polyseed from the capsule was weighed and added in 100 mL mineral media water and then stirred for 1 h. This gave the bacterial count as 104to 106CFU/L. A further concentration of 1 mL/L of polyseed solution is prepared and from this working inoculum 0.125 mL solution is added to every bottles of 125 mL.The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 100 %. Degradation of Sodium Benzoate exceeds 68.86 % on 7 days & 77.84 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.The BOD28 value of test chemical was observed to be 1.75 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 66.79 %. Based on the results, the test chemical under the test conditions, was considered to be readily biodegradable in nature

Biodegradation in water and sediment

Estimation Programs Interface prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 25.1% 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 0.496%), 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. If released into the environment, 74.1% 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 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

From authorative database HSDB, an estimated BCF of 57 was calculated for test chemical, using an estimated log Kow of 2.61and a regression derived equation.This BCF suggests the potential for bioconcentration in aquatic organisms is moderate.

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 solventsA test item solution was prepared by accurately weighing 5 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 500 mg/l. The pH of test substance was 6.6.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. 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 Xylene, Ethylbenzene, Toluene, Naphthalene, Phenol, phenanthrene were chosen having Koc value range from 1.32 to 4.09.The Log Koc value of test chemical was determined to be 1.759 ± 0.001 dimensionless 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

Biodegradationin water

Various experimental studies of the target chemical were reviewed for biodegradation endpoint which are summarized as below:

 

Biodegradation study was conducted for 28-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical, toxicity control and referencesubstance. Polyseed capsule (mixed culture) was used as a test inoculum for the study. Test inoculum polyseed capsule was composed of blend of specialized microbial cultures and food grade gelatin made by International Laboratory Supply (InterLab), LTD. No pretreatment / preconditioning was given to the test inoculum as the polyseed capsule requires only one hour of stirring to activate it. Polyseed capsule is a blend of broad spectrum bacteria designed specifically as seed inoculums for the BOD test. Polyseed is an EPA approved BOD seed inoculum. 38.4 mg of polyseed from the capsule was weighed and added in 100 mL mineral media water and then stirred for 1 h. This gave the bacterial count as 104to 106CFU/L. A further concentration of 1 mL/L of polyseed solution is prepared and from this working inoculum 0.125 mL solution is added to every bottles of 125 mL.The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 100 %. Degradation of Sodium Benzoate exceeds 68.86 % on 7 days & 77.84 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.The BOD28 value of test chemical was observed to be 1.75 mgO2/mg. ThOD was calculated as 2.62 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 66.79 %. Based on the results, the test chemical under the test conditions, was considered to be readily biodegradable in nature

In an experimental study from peer reviewed journal (E. H. Snider and F. S. Manning, 1982), biodegradation experiment was conducted for evaluating the percentage biodegradability of test chemical under aerobic conditions. Activated sludge was used as a test inoculum. The organic analyses in the study were performed by gas chromatography / mass spectrometry (GC/MS) on methylene chloride extracts of the wastewater samples. Three extracts were collected--a neutral extract, an acid extract, and a base extract. Water samples extracted and analyzed included the raw wastewater after dissolved air flotation (DAF) treatment, effluent from activated sludge (AS) treatment, and effluent from activated carbon (AC) columns. The percentage degradation of test substance was determined to be 99.98% in 4 days. Thus, based on percentage degradation, test chemical was considered to be readily biodegradable in nature.

 

In a supporting weight of evidence study, a batch test in an open system was conducted for 5 days (120 hr) for evaluating the biodegradability of test chemical (from peer reviewed journal, authoritative database and secondary source). Adapted activated sludge was used as a test inoculum obtained from a sewage plant is cultivated in a1000ml volumetric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentationca.600mlof the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume ofca.800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. Test chemical conc. used for the study was 200 mg/l based on COD. To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200 mg/l. Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed to the medium that the concentration of the dry matter is 100 mg/l. Simultaneously, a blank test is prepared. The beaker is placed in a dark room with a roughly 3 constant temperature of 20±3°C on an electromagnetic stirrer and a pH of 7.2 for 120 hrs. The initial value of COD or organic carbon of the liquid phase are determined. Samples filtered or centrifuged before analysis, are taken at suitable intervals. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The results are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a gram of dry matter of the activated sludge per hour. The percentage degradation of test chemical was determined to be 95.5% by using COD removal parameter in 5 days. Thus, based on percentage degradation, test chemical was considered to be readily biodegradable in nature.

 

Another biodegradation study (JAMES G. MUELLER et. al., 1991) was carried out for 14 days for evaluating the percentage biodegradability of test chemical. Micro-organism was used as a test inoculum isolated from surface soil which was freshly obtained from the American Creosote Works site. Microbial inoculum was prepared by mixing 25 g of creosote-contaminated surface soil (17) freshly obtained from the American Creosote Works site with 100 ml of 2.5 mM phosphate buffer (pH 7). After being mixed well, the suspension was centrifuged (2,500 rpm, 10 min) to remove larger soil particles. The resultant supernatant was decanted and used as a source of indigenous, "creosote-adapted" microorganisms for the ground water medium (GWM).Ground water medium (GWM) was used for the study. For the biodegradation study, approximately 400 liters of groundwater contaminated with creosote and PCP was recovered from an on-site sampling well. Groundwater was removed from a depth of 7 m through Teflon-coated Bev-a-line tubing (15 mm inner diameter) by means of an electric pump. Groundwater was delivered directly into two freshly rinsed, 208-liter steel drums (DOT-17E) and stored on site for ancillary testing. Five subsamples (1.0 liter) were collected in clean, sterile, 1.0-liter Wheaton bottles fitted with Teflon-lined screw caps and stored on ice for transport to the laboratory. Upon arrival at the laboratory, subsamples were stored in darkness at 2°C for subsequent biodegradation studies, toxicity and teratogenicity testing, and chemical analyses. It contains 2.5 ml of filtered groundwater (passed through a plug of silanized glass wool to remove undissolved solids) plus 12.5 ml of modified Bushnell-Haas medium. Additionally, two clean, sterile, 1.0-liter Wheaton bottles fitted with Teflon-lined screw caps received 200 ml of the same medium.Ground water contains the test chemical2,3,5-trimethylphenol. Wheaton bottles consist of 200 ml of ground water medium (which contain ground water and modified Bushnell-Haas medium. Duplicate 25 ml samples were immediately extracted for time zero analysis. Flasks were incubated at 30°C with shaking (200 rpm) in the dark for 14 days. Killed-cell controls were prepared for each sampling time point by adding 2.5 ml of a 37% formaldehyde solution to five of the shake flasks containing 25 ml of GWM. After 1, 3, 5, 8, and 14 days of incubation, the entire contents of two active flasks and one killed-cell control flask were separately extracted and analyzed by GC for the presence of creosote constituents. These data were compared with those obtained from untreated (non-inoculated) GWM that had been stored at 2°C during the 14-day incubation period. As the limit of detection of test chemical by GC is very low during a period 14 days, test chemical was considered to be readily biodegradable in water.

 

For the test chemical, preliminary biodegradability screening test was conducted for 20 days for evaluating the percentage biodegradability of test chemical (1978). Domestic sewage was used as a test inoculum was obtained from the Chapel Hill sewage treatment plant. The test compound were added to duplicate, acid-washed, 300 ml BOD bottles at concentration of approximately 5 mg/l. Dilution water was prepared from water which had been passed through activated carbon and ion exchange columns and then glass-distilled. Standard nutrients were added to the water as was 0.5 mg/l allylthiourea for control of nitrification. The dilution water was seeded with 1.5 mg/l of domestic sewage. The BOD bottles were filled, stoppered, and incubated in the dark at 65°F for 20 days. Oxygen uptake was measured at various intervals over the 20 day period by means of a Weston and Stack dissolved oxygen meter. The percentage degradation of test chemicals was determined to be 91% by O₂uptake parameter in 20 days. Thus, based on percentage degradation, test chemical was considered to be readily biodegradable in nature.

 

In a supporting weight of evidence study, biodegradation study was conducted according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)) for evaluating the percentage biodegradability of test chemical (from Kondo, M et. al., 1988 and secondary source). Initial test substance conc. used in the study was 20 mg/l. Namely, a water, acetone or DMSO solution (0.1 ml) of the test chemicals was added to a mixture of river/sea water (4.9 ml) from an unpolluted area and an autoclaved solution (5.0ml) of 0.2% peptone in a sterile test tube with a tight plug. After sealed with film and fixed at an angle of 30°in a dark box, the test tubes were incubated at 30°C and shaked at 120rpm. Inoculum used for the study was mixed culture obtained from different sources (Sea water from Enoshima Beach and River water from Tama River). The percentage degradation of test chemical in river and sea water was determined to be 100 and 54% by BOD parameter in 3 days. Thus, based on percentage degradation, test chemical was considered to be readily biodegradable in water.

 

On the basis of the above results, test chemical was considered to be readily biodegradable in water.

Biodegradation in water and sediment

Estimation Programs Interface prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 25.1% 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 0.496%), 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. If released into the environment, 74.1% 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 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

Experimental study and predicted data of the target chemical were reviewed for the bioaccumulation end point which are summarized as below:

 

In an experimental study from authoritative database, an estimated BCF of 57 was calculated for test chemical, using an estimated log Kow of 2.61and a regression derived equation.

 

In a prediction done using the BCFBAF (v3.00) model of EPI suite, the estimated bio concentration factor (BCF) for test chemical is 20.1 L/kg wet-wt.

 

From Scifinder database, the bioconcentration factor (BCF) for target chemical was predicted to be 38.0 at pH range 1-8, 36.6 at pH 9 and 27.6 at pH 10 at temperature 25 deg.C.

 

Using the Chemspider - ACD/PhysChem Suite prediction model the Bioconcentration factor (BCF) for target chemical was estimated to be 36.5 at pH 5.5 and 7.4 at temperature 25°C.

 

On the basis of above results, it can be concluded that the BCF value of test chemical was evaluated to be ranges from 20.1 to 57, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

Adsorption / desorption

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

 

In an experimental 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 Acetonitrile up to 10 ml. Thus, the test solution concentration was 500 mg/l. The pH of test substance was 6.6.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. 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 Xylene, Ethylbenzene, Toluene, Naphthalene, Phenol, phenanthrene were chosen having Koc value range from 1.32 to 4.09.The Log Koc value of test chemical was determined to be 1.759 ± 0.001 dimensionless 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.

 

For the test chemical, the Koc of test chemical was estimated to be 630, using a structure estimation method based on molecular connectivity indices. This estimated Koc value suggests that test chemical was expected to have low mobility in soil.

 

Thus, on the basis of above information, the logKoc value of the test chemical was determined to be ranges from 1.75 to 2.79, respectively, indicating the substance has a low to moderate sorption to soil and sediment and therefore have moderate to slow migration potential to ground water.

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