Methyl anthranilate

Endpoint summary

Description of key information

Hydrolysis:

A 38 days study was conducted to determine the rate of hydrolysis of test chemical at the temperature of 25°C. Test solution was prepared by adding 3ml of test chemical to 300ml of deionized water which was stirred for 12h. Phosphate buffer media was used at pH 5, 7 and 9. Hence, after 38 days the percent recovery of test chemical at pH 5, 7 and 9 was determined to be 100-96%, 97-93% and 97-91%, respectively. Therefore, from the above value, it can be said that the test chemical was non-hydrolysable.

Biodegradation in water:

Study was to determine biodegradation rate of test chemical in water. Study was carried out for 20 days under the condition of optimal bacterial growth. Test chemical was purchased from Fluka Chemical Company. The initial concentration of test chemical used in the study was 50 mg/L. Test chemical concentration was determined by High performance liquid chromatography (HPLC). The HPLC system consisted of a Rainin HPXL solvent delivery system, Dynamax A1-2 autosampler equipped with a 2 0 4 fixed sample loop and a Dynamax UV-M ultraviolet detector. Quantitative analysis of test chemical was done by using Hewlett Packard 3390A recording integrator. Brucker MSL-400 (400 MHz) NMR spectrometer, Finnegan GC/MS spectrometer equipped with a Data General Nova computer system, a Shimadzu IR-435 infrared spectrophotometer and a Gilford 2600 ultraviolet spectrophotometer were used for the spectral analysis. Approximately 50 mg liter of two solution of test chemical were prepared in dechlorinated, charcoal-filtered Vessels which were stored open to natural inoculation in a water bath maintained at 13-15°C for seven days prior to initiation of the test. One tank received 1 g of sodium azide, a metabolic poison after seven days resulting in a final concentration of 0.1 g liter. There is no further treatment given to control tank. 100 ml of samples were drawn, sealed (to prevent further inoculation) and stored at 23°C in sterilized, sealed amber vials. Vials were stored under dark or light: dark (12: 12 h) conditions. Aliquots from all treatments were analyzed for test chemical content at intervals of 0, 0.2, 1, 2, 5, 6, 7, 8, 9 and 20 days post-treatment. Under conditions of optimal bacterial growth (warmth and darkness), biodegradation of test chemical was determined to be 100% in 20 days. Based on the test chemical analysis, test chemical determined to be readily biodegradable in water.

Biodegradation in water and sediments:

Estimation Programs Interface (EPI Suite) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 32.9 % of the test 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 15days (360 hrs). The half-life (15 days estimated by EPI suite) indicates that the chemical was 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 was 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 0.105%), indicates that test chemical is not persistent in sediment.

Biodegradation in soil:

Test was carried out to determine degradation of test chemical in environment. Study was carried out for 64 days using a 1% suspension of Niagara silt loam soil. Test chemical observed to be completely degraded in 64 days.

Bioaccumulation:

Aim of the study was to determine bioconcentration factor (BCF) of test chemical in test organism. Study was performed for 6 weeks of depuration time. Test was conducted on juvenile hybrid striped bass (Morone chrysops and M. saxatilis) with an average weight of 10 g. Test organisms were purchased from Delmarva Aquatics and acclimatized to laboratory conditions over a period of 2 weeks. Test organisms were kept in round fibre glass 170 L tanks fitted with a drain at the centre and a flow-through system in which the water flow rate was 2 L/min. Aeration was supplied through large air stone present in each tank. A total of six tanks were used, three for the control and three for the experimental group. The water temperature fluctuated between 19 and 22°C throughout the experimental period. A total of 22 fish were stocked in each tank. Fish were fed with the pelleted feed (ASD2-30) containing 56.6% protein, 14.6% fat and 8.9% ash twice a day (early morning and late afternoon) for 5 days, and once a day during the remaining 2 days of the week. Food was stored in sealed plastic containers in a freezer (-20°C) and the daily ration was removed from the freezer and left to thaw fully before feeding the fish. Test was carried out under flow-through condition at pH of 6.6-7.2 and 19-22°C temperature. During the test dissolved oxygen was fluctuated between 5.3 and 7.2mg/L and the photoperiod were maintained at 12L: 12D with artificial fluorescent illumination. The dissolved oxygen monitored with the aid of an YSI (Yellow Springs Instruments oxygen meter. The fish were food deprived for1day at the end of 6-week growth period and a sample of fish from each tank was removed. The fish were decapitated their digestive tracts were dissected and the different tissues were placed in test tubes. 2mL solution of 0.1% sodium azide (NaN3) was added in test tubes to prevent microbial degradation. Different body parts of fish, scales and outer surface mucus, liver, spleen, fat bodies and the muscle near the dorsal fin. Samples were then sealed and kept frozen at -20°C until analysis. 6 weeks chemical exposure period was provided to test fishes. The experiment was further extended for additional 6 weeks and the fish were assayed following a depuration period in which previously test chemical-fed fish were given the control diet (containing no additional test chemical) and after the exposure period, the fishes were observed for the presence of residual test chemical. Tissue and food samples were extracted by using A SpeedVac Savant Concentrator, a 220 ultrasonic bath and an EIC HN-S II centrifuge. HPLC was performed using a Raining HPXL two-pump solvent delivery system, Rheodyne 7161 sample injector equipped with a 20mL sample loop. The detection was carried out by a Dynamax FL-1 fluorescent detector. Chromatograms were recorded and integrated using a Hewlett Packard 3390A integrator. The results were subjected to ANOVA followed by Tukey ’s pair test. The BCF of test chemical was determined to be 6.7 ±1.8, 8.9±2.1, 11.2±2.6, 0.1±0.05 µg/g in spleen, liver, abdominal fat and in muscle, respectively while traces of test chemical were found in scales and mucus. The deposition rate of test chemical in the hybrid striped bass fed feed adulterated with 100 mg/kg of test chemical corresponded to 0.7%. The experimental BCF value does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

 

Adsorption:

Adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical. The adsorption coefficient (Koc) value was calculated using a logKow of 1.88 and a regression derived equation. The adsorption coefficient (Koc) value of test chemical was estimated to be 75 (Log Koc = 1.87). This Koc value indicates that the test chemical has low sorption to soil and sediment and therefore have moderate migration potential to ground water.

Additional information

Hydrolysis:

Following different studies includes experimental and estimated studies for the test chemical to observe the hydrolysis of test chemical in water.

 

A 38 days study was conducted to determine the rate of hydrolysis of test chemical at the temperature of 25°C. Test solution was prepared by adding 3ml of test chemical to 300ml of deionized water which was stirred for 12h. Phosphate buffer media was used at pH 5, 7 and 9. Hence, after 38 days the percent recovery of test chemical at pH 5, 7 and 9 was determined to be 100-96%, 97-93% and 97-91%, respectively. Therefore, from the above value, it can be said that the test chemical was non-hydrolysable.

 

In next study, the base catalyzed second order hydrolysis rate constant of test chemical was determined using a structure estimation method. The second order hydrolysis rate constant of test chemical was determined to be 0.042 L/mol-sec with a half-life value of 5.5 yrs and 200 days at pH 7.0 and 8.0, respectively. Based on the half-life values, it is concluded that the test chemical is not hydrolysable.

 

Based on the above value, the test chemical determined to be non-hydrolysable.

 

Biodegradation in water:

Following different studies includes experimental study for the test chemical and for read-across analogues which is extracted by using mechanistic approach and functionally and structurally similar to the test chemical to observe the biodegradation rate of test chemical in water.

 

The principle of the first study was to determine biodegradation rate of test chemical in water. Study was carried out for 20 days under the condition of optimal bacterial growth. Test chemical was purchased from Fluka Chemical Company. The initial concentration of test chemical used in the study was 50 mg/L. Test chemical concentration was determined by High performance liquid chromatography (HPLC). The HPLC system consisted of a Rainin HPXL solvent delivery system, Dynamax A1-2 autosampler equipped with a 2 0 4 fixed sample loop and a Dynamax UV-M ultraviolet detector. Quantitative analysis of test chemical was done by using Hewlett Packard 3390A recording integrator. Brucker MSL-400 (400 MHz) NMR spectrometer, Finnegan GC/MS spectrometer equipped with a Data General Nova computer system, a Shimadzu IR-435 infrared spectrophotometer and a Gilford 2600 ultraviolet spectrophotometer were used for the spectral analysis. Approximately 50 mg liter of two solution of test chemical were prepared in dechlorinated, charcoal-filtered Vessels which were stored open to natural inoculation in a water bath maintained at 13-15°C for seven days prior to initiation of the test. One tank received 1 g of sodium azide, a metabolic poison after seven days resulting in a final concentration of 0.1 g liter. There is no further treatment given to control tank. 100 ml of samples were drawn, sealed (to prevent further inoculation) and stored at 23°C in sterilized, sealed amber vials. Vials were stored under dark or light: dark (12: 12 h) conditions. Aliquots from all treatments were analyzed for test chemical content at intervals of 0, 0.2, 1, 2, 5, 6, 7, 8, 9 and 20 days post-treatment. Under conditions of optimal bacterial growth (warmth and darkness), biodegradation of test chemical was determined to be 100% in 20 days. Based on the test chemical analysis, test chemical determined to be readily biodegradable in water.

 

Supporting study was carried out to determine biodegradation of test chemical in water. Test was conducted in accordance with EPA OTS 796.3260 (Ready Biodegradability: Modified Sturm Test) for 29 days. Activated sewage sludge was used as test inoculums for the study. The initial concentration of test chemical used in the study was 28 mg/L. Based on the carbon dioxide evolution, the percent degradation of test chemical was determined to be 62% in 29 days. Thus on the basis of determined percent degradation value, test chemical is considered to be readily biodegradable in water.

 

The next biodegradation study was conducted for 14 days for evaluating the percentage biodegradability of test chemical. Activated sludge was used as test inoculums for the study. Concentration of inoculum i.e., sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test chemical was determined to be 85, 99 and 100% degradation by O2 consumption (BOD (NH3)), TOC removal andtest material analysis byHPLC parameter in 14 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in water.

 

 

On the basis of above determined values, test chemical is considered to be readily biodegradable in water.

 

Biodegradation in water and sediment:

Estimation Programs Interface (EPI Suite) prediction model was run to predict the half-life in water and sediment for the test chemical. If released in to the environment, 32.9 % of the test 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 15days (360 hrs). The half-life (15 days estimated by EPI suite) indicates that the chemical was 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 was 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 0.105%), indicates that test chemical is not persistent in sediment.

 

Biodegradation in soil:

Following different studies includes experimental and estimated study for the test chemical to observe the biodegradation rate of test chemical in soil.

 

Test was carried out to determine degradation of test chemical in environment. Study was carried out for 64 days using a 1% suspension of Niagara silt loam soil. Test chemical observed to be completely degraded in 64 days.

 

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (EPI suite). If released into the environment, 66.2% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil was estimated to be 30 days (720hrs). Based on this half-life value of test chemical, it can be concluded that the test chemical was not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

Based on this half-life value of test chemical, it can be concluded that the test chemical was not persistent in the soil environment.

 

Bioaccumulation:

Following different studies includes experimental study for the test chemical to observe the bioaccumulation rate of test chemical in test organism.

 

Aim of the first study was to determine bioconcentration factor (BCF) of test chemical in test organism. Study was performed for 6 weeks of depuration time. Test was conducted on juvenile hybrid striped bass (Morone chrysops and M. saxatilis) with an average weight of 10 g. Test organisms were purchased from Delmarva Aquatics and acclimatized to laboratory conditions over a period of 2 weeks. Test organisms were kept in round fibre glass 170 L tanks fitted with a drain at the centre and a flow-through system in which the water flow rate was 2 L/min. Aeration was supplied through large air stone present in each tank. A total of six tanks were used, three for the control and three for the experimental group. The water temperature fluctuated between 19 and 22°C throughout the experimental period. A total of 22 fish were stocked in each tank. Fish were fed with the pelleted feed (ASD2-30) containing 56.6% protein, 14.6% fat and 8.9% ash twice a day (early morning and late afternoon) for 5 days, and once a day during the remaining 2 days of the week. Food was stored in sealed plastic containers in a freezer (-20°C) and the daily ration was removed from the freezer and left to thaw fully before feeding the fish. Test was carried out under flow-through condition at pH of 6.6-7.2 and 19-22°C temperature. During the test dissolved oxygen was fluctuated between 5.3 and 7.2mg/L and the photoperiod were maintained at 12L: 12D with artificial fluorescent illumination. The dissolved oxygen monitored with the aid of an YSI (Yellow Springs Instruments oxygen meter. The fish were food deprived for1day at the end of 6-week growth period and a sample of fish from each tank was removed. The fish were decapitated their digestive tracts were dissected and the different tissues were placed in test tubes. 2mL solution of 0.1% sodium azide (NaN3) was added in test tubes to prevent microbial degradation. Different body parts of fish, scales and outer surface mucus, liver, spleen, fat bodies and the muscle near the dorsal fin. Samples were then sealed and kept frozen at -20°C until analysis. 6 weeks chemical exposure period was provided to test fishes. The experiment was further extended for additional 6 weeks and the fish were assayed following a depuration period in which previously test chemical-fed fish were given the control diet (containing no additional test chemical) and after the exposure period, the fishes were observed for the presence of residual test chemical. Tissue and food samples were extracted by using A SpeedVac Savant Concentrator, a 220 ultrasonic bath and an EIC HN-S II centrifuge. HPLC was performed using a Raining HPXL two-pump solvent delivery system, Rheodyne 7161 sample injector equipped with a 20mL sample loop. The detection was carried out by a Dynamax FL-1 fluorescent detector. Chromatograms were recorded and integrated using a Hewlett Packard 3390A integrator. The results were subjected to ANOVA followed by Tukey ’s pair test. The BCF of test chemical was determined to be 6.7 ±1.8, 8.9±2.1, 11.2±2.6, 0.1±0.05 µg/g in spleen, liver, abdominal fat and in muscle, respectively while traces of test chemical were found in scales and mucus. The deposition rate of test chemical in the hybrid striped bass fed feed adulterated with 100 mg/kg of test chemical corresponded to 0.7%. The experimental BCF value does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

 

Second study was conducted to determine bioconcentration factor (BCF) of test chemical in test organism. Study was performed for 6 weeks of depuration time. Test was conducted on African cichlid fish Aulonocara jacobfreibergi with an average weight of 1 g. Test organisms were purchased from Schmalbach farm and acclimatized to laboratory conditions over a period of 10 days. Fishes were maintained in glass aquariums containing 35L of water. Each aquarium was equipped with an internal biofilter through which air was bubbled at a constant rate. The total no. of test vessel used during study was twelve, six for the control-fed fish and six for the experimental group. The temperature was maintained at 25±2°C by heating the room in which the experiment was run. Fishes were fed with the pelleted feed (ASD2-30) containing 56.6% protein, 14.6% fat and 8.9% ash twice a day (early morning and late afternoon) for 5 days, and once a day during the remaining 2 days of the week. Food was stored in sealed plastic containers in a freezer (-20°C) and the daily ration was removed from the freezer and left to thaw fully before feeding the fish. Test was carried out under flow-through condition at pH of 6.6-7.2. During the test dissolved oxygen was fluctuated between 5.3 and 7.2mg/L and the photoperiod were maintained at 12L: 12D with artificial fluorescent illumination. The dissolved oxygen monitored with the aid of an YSI (Yellow Springs Instruments oxygen meter. The fish were food deprived for 1 day at the end of 6-week growth period and a sample of fish from each tank was removed. The fish were decapitated their digestive tracts were dissected and the different tissues were placed in test tubes. 2mL solution of 0.1% sodium azide (NaN3) was added in test tubes to prevent microbial degradation. Tissue and food samples were extracted by using A SpeedVac Savant Concentrator, a 220 ultrasonic bath and an EIC HN-S II centrifuge. HPLC was performed using a Raining HPXL two-pump solvent delivery system, Rheodyne 7161 sample injector equipped with a 20mL sample loop. The detection was carried out by a Dynamax FL-1 fluorescent detector. Chromatograms were recorded and integrated using a Hewlett Packard 3390A integrator. The results were subjected to ANOVA followed by Tukey ’s pair test. The BCF of test chemical was determined to be 12.7±2.1 µg/g based on the whole body accumulation or food intake level. Deposition rate of test chemical in juvenile African cichlid fish Aulonocara jacobfreibergi (whole-body accumulation/food intake level) was observed to be of 0.3%. The experimental BCF value does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

 

The last bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of test chemical. The bioaccumulation factor (BCF) value was calculated using a log Kow of 1.88 and a regression-derived equation. The BCF value of test chemical was estimated to be 8 dimensionless, which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

 

Based on the above experimental BCF value does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is considered to be non-accumulative in aquatic organisms.

 

Adsorption:

Adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical. The adsorption coefficient (Koc) value was calculated using a logKow of 1.88 and a regression derived equation. The adsorption coefficient (Koc) value of test chemical was estimated to be 75 (Log Koc = 1.87). This Koc value indicates that the test chemical has low sorption to soil and sediment and therefore have moderate migration potential to ground water.