2-amino-4-nitrophenol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Biodegradation study was conducted for 150 days for evaluating the percentage biodegradability of test chemical under anaerobic conditions.

GLP compliance:

not specified

Specific details on test material used for the study:

- Other: All Chemicals were purchased from Acros Chimica (Geel, Belgium), Merck (Darmstadt, Germany) and Sigma (Bornem, Belgium) and were used without further purification.

Oxygen conditions:

anaerobic

Inoculum or test system:

other: Methanogenic granular sludge

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The methanogenic granular sludge obtained from a full-scale upward-flow anaerobic sludge bed reactor (UASB) treating a petrochemical wastewater containing benzoate and acetate as primary substrates was used as a test inoculum for the study.
- Pretreatment: The sludge was elutriated to remove the fines and predigested at 30°C during a 30 days period in order to deplete all endogenous substrate in the sludge.
- Other: The sludge contained 10.5% TSS and 8.5% VSS.

Duration of test (contact time):

150 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: Methane production

Details on study design:

TEST CONDITIONS
- Composition of medium: Basal medium was used as a test medium for the study, with the exception of NaHCO3 supplied at 5 g/l.
- Test temperature: 30°C
- Other: Serum flasks were incubated with shaking (50 rpm) in a temperature controlled room at 30°C

TEST SYSTEM
- Culturing apparatus: 120 ml glass serum flask was used as a test vessel for the study.
- Number of culture flasks/concentration: Triplicates, except the sludge blanks which utilized six serum flasks to assess the methane production.
- Method used to create anaerobic conditions: The serum flasks were sealed with 12 mm thick butyl rubber stoppers and flushed with 70% N2-30% CO2 gas for 5 minutes and incubated overnight at 30°C to allow for biological consumption of residual O2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Sludge blank which contains no test chemical was setup to correct for background gas production from the sludge.

Reference substance:

other: Both Benzoate and phenol were used as reference compounds in the study. The concentrations of benzoate and phenol used were 250 mg/L.

Key result

Parameter:

other: Methane production

Value:

0

Sampling time:

150 d

Remarks on result:

other: Other details not known

Details on results:

Test chemical undergoes 0% degradation after a period of 150 days.

Results with reference substance:

The benzoate was completely degraded in 20 days and the phenol in 45 days. ultimate conversion of the substrate COD to methane was equal to 85.5% ± 1.82 and 82.8% ± 2.32 for benzoate and phenol respectively.

Reproducibility of the methane production among replicate sludge blank serum flasks was satisfactory, with standard deviations accounting for less than 2% of the mean.

Validity criteria fulfilled:

not specified

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The percentage degradation of test chemical was determined to be 0% after 150 days. Thus, based on percentage degradation, test chemical is considered to be not biodegradable in water.

Executive summary:

Biodegradation study was conducted for 150 days for evaluating the percentage biodegradability of test chemical. The study was performed under anaerobic conditions at a temperature of 30°C.The methanogenic granular sludge obtained from a full-scale upward-flow anaerobic sludge bed reactor (UASB) treating a petrochemical wastewater containing benzoate and acetate as primary substrates was used as a test inoculum for the study.The sludge was elutriated to remove the fines and predigested at 30°C during a 30 days period in order to deplete all endogenous substrate in the sludge.The sludge contained 10.5% TSS and 8.5% VSS. Initial test substance conc. used in the study was 100 mg/l, respectively. 120 ml glass serum flask was used as a test vessel for the study. Basal medium was used as a test medium for the study, with the exception of NaHCO3 supplied at 5 g/l.Predigested granular sludge (1 g VSS/L) was transferred to serum flasks containing 24 mL of the basal medium and acetate from a neutralized stock to yield a final concentration of 50 mg of chemical oxygen demand (COD)/L. The serum flasks were sealed with 12 mm thick butyl rubber stoppers and flushed with 70% N2-30% CO2 gas for 5 minutes and incubated overnight at 30°C to allow for biological consumption of residual O2. The desired amount of test chemical was then added to triplicate serum flasks using concentrated stock solutions. Later serum flasks were incubated with shaking (50 rpm) in a temperature controlled room at 30°C over a 150 day period.The methane composition in the headspace of each serum flask was monitored periodically during the assays. The serum flasks were shaken vigorously before gas measurements were taken. Methane production was calculated from the volume of the headspace and the methane composition in the gas. Net methane production was calculated by subtracting background methane production in the controls from that in the test vials. The corrected methane production (M) was expressed as a percentage of the theoretical methane production (TMP) expected from the test chemical mineralization. Sludge blank which contains no test chemical was setup to correct for background gas production from the sludge. Both Benzoate and phenol were used as reference compounds in the study.The concentrations of benzoate and phenol used were 250 mg/L. The benzoate was completely degraded in 20 days and the phenol in 45 days. ultimate conversion of the substrate COD to methane was equal to 85.5% ± 1.82 and 82.8% ± 2.32 for benzoate and phenol respectively.The percentage degradation of test chemical was determined to be 0% after 150 days. Thus, based on percentage degradation, test chemical is considered to be not biodegradable in water.

Description of key information

Biodegradation study was conducted for 150 days for evaluating the percentage biodegradability of test chemical (Elias Razo Flores et. al., 1996). The study was performed under anaerobic conditions at a temperature of 30°C.The methanogenic granular sludge obtained from a full-scale upward-flow anaerobic sludge bed reactor (UASB) treating a petrochemical wastewater containing benzoate and acetate as primary substrates was used as a test inoculum for the study.The sludge was elutriated to remove the fines and predigested at 30°C during a 30 days period in order to deplete all endogenous substrate in the sludge.The sludge contained 10.5% TSS and 8.5% VSS. Initial test substance conc. used in the study was 100 mg/l, respectively. 120 ml glass serum flask was used as a test vessel for the study. Basal medium was used as a test medium for the study, with the exception of NaHCO3 supplied at 5 g/l.Predigested granular sludge (1 g VSS/L) was transferred to serum flasks containing 24 mL of the basal medium and acetate from a neutralized stock to yield a final concentration of 50 mg of chemical oxygen demand (COD)/L. The serum flasks were sealed with 12 mm thick butyl rubber stoppers and flushed with 70% N2-30% CO2 gas for 5 minutes and incubated overnight at 30°C to allow for biological consumption of residual O2. The desired amount of test chemical was then added to triplicate serum flasks using concentrated stock solutions. Later serum flasks were incubated with shaking (50 rpm) in a temperature controlled room at 30°C over a 150 day period.The methane composition in the headspace of each serum flask was monitored periodically during the assays. The serum flasks were shaken vigorously before gas measurements were taken. Methane production was calculated from the volume of the headspace and the methane composition in the gas. Net methane production was calculated by subtracting background methane production in the controls from that in the test vials. The corrected methane production (M) was expressed as a percentage of the theoretical methane production (TMP) expected from the test chemical mineralization.Sludge blank which contains no test chemical was setup to correct for background gas production from the sludge.Both Benzoate and phenol were used as reference compounds in the study.The concentrations of benzoate and phenol used were 250 mg/L. The benzoate was completely degraded in 20 days and the phenol in 45 days. ultimate conversion of the substrate COD to methane was equal to 85.5% ± 1.82 and 82.8% ± 2.32 for benzoate and phenol respectively.The percentage degradation of test chemical was determined to be 0% after 150 days. Thus, based on percentage degradation, test chemicalis considered to be not biodegradable in water.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Experimental studies and predicted data of the test chemical and various supporting studies for its structurally similar read across substance were reviewed for the biodegradation in water end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Elias Razo Flores et. al., 1996),biodegradation study was conducted for 150 days for evaluating the percentage biodegradability of test chemical. The study was performed under anaerobic conditions at a temperature of 30°C.The methanogenic granular sludge obtained from a full-scale upward-flow anaerobic sludge bed reactor (UASB) treating a petrochemical wastewater containing benzoate and acetate as primary substrates was used as a test inoculum for the study. The sludge was elutriated to remove the fines and predigested at 30°C during a 30 days period in order to deplete all endogenous substrate in the sludge. The sludge contained 10.5% TSS and 8.5% VSS. Initial test substance conc. used in the study was 100 mg/l, respectively. 120 ml glass serum flask was used as a test vessel for the study. Basal medium was used as a test medium for the study, with the exception of NaHCO3 supplied at 5 g/l. Predigested granular sludge (1 g VSS/L) was transferred to serum flasks containing 24 mL of the basal medium and acetate from a neutralized stock to yield a final concentration of 50 mg of chemical oxygen demand (COD)/L. The serum flasks were sealed with 12 mm thick butyl rubber stoppers and flushed with 70% N2-30% CO2 gas for 5 minutes and incubated overnight at 30°C to allow for biological consumption of residual O2. The desired amount of test chemical was then added to triplicate serum flasks using concentrated stock solutions. Later serum flasks were incubated with shaking (50 rpm) in a temperature controlled room at 30°C over a 150 day period. The methane composition in the headspace of each serum flask was monitored periodically during the assays. The serum flasks were shaken vigorously before gas measurements were taken. Methane production was calculated from the volume of the headspace and the methane composition in the gas. Net methane production was calculated by subtracting background methane production in the controls from that in the test vials. The corrected methane production (M) was expressed as a percentage of the theoretical methane production (TMP) expected from the test chemical mineralization. Sludge blank which contains no test chemical was setup to correct for background gas production from the sludge. Both Benzoate and phenol were used as reference compounds in the study. The concentrations of benzoate and phenol used were 250 mg/L. The benzoate was completely degraded in 20 days and the phenol in 45 days. ultimate conversion of the substrate COD to methane was equal to 85.5% ± 1.82 and 82.8% ± 2.32 for benzoate and phenol respectively. The percentage degradation of test chemical was determined to be 0% after 150 days. Thus, based on percentage degradation, test chemical is considered to be not biodegradable in water.

 

Another biodegradation study was conducted for 16 days for evaluating the percentage biodegradability of test chemical (authoritative database HSDB and PubChem, 2017). The study was performed at a temperature of 30°C.Nocardia sp. (bacteria) was used as a test inoculums for the study. Initial test substance conc. used in the study was 250 mg/l (0.025%), respectively. Test chemical was not utilized as a carbon source by test inoculum which was measured by visible growth of the test inoculum. The percentage degradation of test chemical was determined to be 0% in 16 days. Thus, based on percentage degradation, test chemical is considered to be not biodegradable in water.

 

In a prediction using the Estimation Programs Interface Suite (2018), the biodegradation potential of the test chemical in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that test chemical is expected to be not readily biodegradable.

 

For the test chemical, manometric respirometry test was carried out for a period of 28 days according to the OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992 and the Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008) for determining the percentage biodegradability of test chemical (Experimental study report, 2014). The biodegradation was followed by the oxygen uptake of the microorganisms during exposure with a test item loading rate of 102 mg/L corresponding to an oxygen demand of about 195 mg/L (ThODNH4) and 255 mg/L (ThODNO3). As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The reference item sodium benzoate was sufficiently degraded to 83% after 14 days and to 86% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. In the toxicity control containing both, the test item and the reference item sodium benzoate, 45% biodegradation was noted within 14 days and 56% biodegradation after 28 days of incubation. Thus, it can be assumed that the test item is not inhibitory to the aerobic activated sludge microorganisms. In the test flasks, containing the test item and activated sludge (inoculum), the mean biodegradation after 28 days of test item was 0% (ThODNO3); the 10 day window criterion was not passed. Therefore, the test item was considered to be not readily biodegradable.

 

On the basis of above overall results of test chemical, it can be concluded that the test chemical can be considered to be not biodegradable in water.