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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
other: as mentioned below
Principles of method if other than guideline:
The microbial biodegradation of benzimidazole by pure and mixed cultures.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report):Benzimidazole
- Molecular formula :C7H6N2
- Molecular weight: 118.1384 g/mol
- Substance type:Organic
- Physical state:Solid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Samples of river water were collected aseptically from the River Aire in central Leeds and also at Beal Weir (near Castleford, West Yorkshire, UK). The main source of activated sludge was from an industrial effluent treatment plant treating effluents from the manufacture of rubber additives. Activated sludge was also obtained from Knostrop and Owl- wood Sewage works, near Leeds ; the former treats a mixture of domestic and industrial effluents while the latter only domestic sewage.

- Laboratory culture: For the isolation of pure cultures capable of degrading BTH, culture M was plated out onto BTH- mineral salts agar using a spread plate technique with several dilutions of the culture so as to obtain well separated colonies.

- Method of cultivation: Liquid cultures were grown in 100ml of medium in 250ml Erlenmeyer flasks which were incubated at 27°Cin an orbital incubator and agitated at 200 rpm

- Preparation of inoculum for exposure: Cultures were grown in mineral salts media containing (g/l of distilled water): KH2PO4, 1; K2HPO4, 1; FeCl3.6H20, 0.004; MgSO4.7H20, 0.04. Carbon and nitrogen sources were added to phosphate before autoclaving.

- Concentration of sludge: No data

- Water filtered: yes/no: No

- Type and size of filter used, if any: No
Duration of test (contact time):
5 h
Initial conc.:
0.11 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Phosphate buffer (1 ml KH2PO4, 2g/l, pH= 7.0) was placed in the main compartment of the differential respirometer together with the substrate (1µmol) and water to a volume of 2.5ml. Concentrated washed cells suspensions (0.5 ml; about 0.1 – 0.2 mg dry weight of bacterial cells/flask) was placed in the side arm and the centre well contained 0.1ml 40% w/v KOH together with a small piece of fluted filter paper

- Test temperature: 30°C, in air
- pH: 7.0
- pH adjusted: yes/no: No

TEST SYSTEM
- Culturing apparatus: Gilson differential respirometer
- Number of culture flasks/concentration: 1
- Measuring equipment: Benzimidazole was assayed by measurement of the U.V light absorption spectra at λmax after appropriate dilution in phosphate buffer (KH2PO4, 2g/l, adjusted to pH= 7.0)

CONTROL AND BLANK SYSTEM
Inoculum blank: endogenous controls used, but number and type not specified
Key result
Parameter:
% degradation (O2 consumption)
Value:
41
Sampling time:
5 h
Remarks on result:
other: Culture M was immediately able to oxidize benzimidazole, although a little of (ca 25%) of nitrogen was released as ammonia
Details on results:
Examination of the absorption spectra of the culture fluids revealed small decrease in the characteristic light absorption peak for benzimidazole, although the O2 uptake was higher (37% of theoretical), and the yellow color much more intense (A400nm was about 2).
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Biodegradation study of benzimidazole by pure and mixed cultures was carrried out. For estimation of the microbial degradation of benzimidazole, oxygen uptake measurements were done. Culture M was immediately able to oxidize benzimidazole (41% degradation by O2 uptake), although a little of (ca 25%) of nitrogen was released as ammonia. Examination of the absorption spectra of the culture fluids revealed small decrease in the characteristic light absorption peak for benzimidazole, although the O2 uptake was higher(37% of theoretical), and the yellow color much more intense (A400nm was about 2). Thus from the study conditions, it can be concluded that benzimidazole is readily biodegradable.
Executive summary:

Biodegradation study of benzimidazole by pure and mixed cultures was carrried out. For estimation of the microbial degradation of benzimidazole, oxygen uptake measurements were done. Samples of river water were collected aseptically from the River Aire in central Leeds and also at Beal Weir (near Castleford, West Yorkshire, UK). The main source of activated sludge was from an industrial effluent treatment plant treating effluents from the manufacture of rubber additives. Activated sludge was also obtained from Knostrop and Owl- wood Sewage works, near Leeds ; the former treats a mixture of domestic and industrial effluents while the latter only domestic sewage.

For the isolation of pure cultures capable of degrading BTH, culture M was plated out onto BTH- mineral salts agar using a spread plate technique with several dilutions of the culture so as to obtain well separated colonies.Representative colonies of each colonial type were chosen and rigorously purified by streaking out alternately n the BTH mineral salts agar and nutrient agar. Seven colonial types were obtained in pure culture on solid media; all of these were then inoculated into 1mmol/l BTH liquid mineral salts medium. Growth and BTH concentrations were regularly monitored over a period of 42 days.Cultures were grown in mineral salts media containing (g/l of distilled water): KH2PO4, 1; K2HPO4, 1; FeCl3.6H20, 0.004; MgSO4.7H20, 0.04. Carbon and nitrogen sources were added to phosphate before autoclaving.

Oxygen uptake experiments were carried out using a Gilson differential respirometer. Phosphate buffer (1 ml KH2PO4, 2g/l, pH= 7.0) was placed in the main compartment of the differential respirometer together with the substrate (1µmol) and water to a volume of 2.5ml. Concentrated washed cells suspensions (0.5 ml; about 0.1 – 0.2 mg dry weight of bacterial cells/flask) was placed in the side arm and the centre well contained 0.1ml 40% w/v KOH together with a small piece of fluted filter paper. All experiments were done at 30°C in air. Cells for these experiments were grown on the BTH-mineral salts media in which the carbon source was present at 1mmol/l; after harvesting and washing by centrifugation the cells were resuspended in phosphate buffer to give ca a 75 fold concentrate. Typically O2uptake experiments were for 4-5 hours duration. Benzimidazole was assayed by measurement of the U.V light absorption spectra atλmax after appropriate dilution in phosphate buffer (KH2PO4, 2g/l, adjusted to pH= 7.0). Culture M was immediately able to oxidize benzimidazole (41% O2uptake), although a little of (ca 25%) of nitrogen was released as ammonia. Examination of the absorption spectra of the culture fluids revealed small decrease in the characteristic light absorption peak for benzimidazole, although the O2uptake was higher(37% of theoretical), and the yellow color much more intense (A400nmwas about 2). Thus from the study results, it can be concluded that benzimidazole is considered to be readily biodegradable.

Description of key information

Biodegradation study of benzimidazole by pure and mixed cultures was carrried out. For estimation of the microbial degradation of benzimidazole, oxygen uptake measurements were done. Samples of river water were collected aseptically from the River Aire in central Leeds and also at Beal Weir (near Castleford, West Yorkshire, UK). The main source of activated sludge was from an industrial effluent treatment plant treating effluents from the manufacture of rubber additives. Activated sludge was also obtained from Knostrop and Owl- wood Sewage works, near Leeds ; the former treats a mixture of domestic and industrial effluents while the latter only domestic sewage.

For the isolation of pure cultures capable of degrading BTH, culture M was plated out onto BTH- mineral salts agar using a spread plate technique with several dilutions of the culture so as to obtain well separated colonies.Representative colonies of each colonial type were chosen and rigorously purified by streaking out alternately n the BTH mineral salts agar and nutrient agar. Seven colonial types were obtained in pure culture on solid media; all of these were then inoculated into 1mmol/l BTH liquid mineral salts medium. Growth and BTH concentrations were regularly monitored over a period of 42 days.Cultures were grown in mineral salts media containing (g/l of distilled water): KH2PO4, 1; K2HPO4, 1; FeCl3.6H20, 0.004; MgSO4.7H20, 0.04. Carbon and nitrogen sources were added to phosphate before autoclaving.

Oxygen uptake experiments were carried out using a Gilson differential respirometer. Phosphate buffer (1 ml KH2PO4, 2g/l, pH= 7.0) was placed in the main compartment of the differential respirometer together with the substrate (1µmol) and water to a volume of 2.5ml. Concentrated washed cells suspensions (0.5 ml; about 0.1 – 0.2 mg dry weight of bacterial cells/flask) was placed in the side arm and the centre well contained 0.1ml 40% w/v KOH together with a small piece of fluted filter paper. All experiments were done at 30°C in air. Cells for these experiments were grown on the BTH-mineral salts media in which the carbon source was present at 1mmol/l; after harvesting and washing by centrifugation the cells were resuspended in phosphate buffer to give ca a 75 fold concentrate. Typically O2uptake experiments were for 4-5 hours duration. Benzimidazole was assayed by measurement of the U.V light absorption spectra atλmax after appropriate dilution in phosphate buffer (KH2PO4, 2g/l, adjusted to pH= 7.0). Culture M was immediately able to oxidize benzimidazole (41% O2uptake), although a little of (ca 25%) of nitrogen was released as ammonia. Examination of the absorption spectra of the culture fluids revealed small decrease in the characteristic light absorption peak for benzimidazole, although the O2uptake was higher(37% of theoretical), and the yellow color much more intense (A400nmwas about 2). Thus from the study results, it can be concluded that benzimidazole is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Two experimental study from peer reviewed journal and one experimental study from study report for the target compound Benzimidazole(CAS no. 51-17-2) were reviewed for the biodegradation end point which are summarized as below:

 

In a key study from peer reviewed Journal of Applied Microbiology (1997) for target chemical 4-Benzimidazole (CAS no. 51-17-2), biodegradation study of benzimidazole by pure and mixed cultures was carried out. For estimation of the microbial degradation of benzimidazole, oxygen uptake measurements were done. Samples of river water were collected aseptically from the River Aire in central Leeds and also at Beal Weir (near Castleford, West Yorkshire, UK). The main source of activated sludge was from an industrial effluent treatment plant treating effluents from the manufacture of rubber additives. Activated sludge was also obtained from Knostrop and Owl- wood Sewage works, near Leeds ; the former treats a mixture of domestic and industrial effluents while the latter only domestic sewage.

For the isolation of pure cultures capable of degrading BTH, culture M was plated out onto BTH- mineral salts agar using a spread plate technique with several dilutions of the culture so as to obtain well separated colonies. Representative colonies of each colonial type were chosen and rigorously purified by streaking out alternately n the BTH mineral salts agar and nutrient agar. Seven colonial types were obtained in pure culture on solid media; all of these were then inoculated into 1mmol/l BTH liquid mineral salts medium. Growth and BTH concentrations were regularly monitored over a period of 42 days. Cultures were grown in mineral salts media containing (g/l of distilled water): KH2PO4, 1; K2HPO4, 1; FeCl3.6H20, 0.004; MgSO4.7H20, 0.04. Carbon and nitrogen sources were added to phosphate before autoclaving.

Oxygen uptake experiments were carried out using a Gilson differential respirometer. Phosphate buffer (1 ml KH2PO4, 2g/l, pH= 7.0) was placed in the main compartment of the differential respirometer together with the substrate (1µmol) and water to a volume of 2.5ml. Concentrated washed cells suspensions (0.5 ml; about 0.1 – 0.2 mg dry weight of bacterial cells/flask) was placed in the side arm and the centre well contained 0.1ml 40% w/v KOH together with a small piece of fluted filter paper. All experiments were done at 30°C in air. Cells for these experiments were grown on the BTH-mineral salts media in which the carbon source was present at 1mmol/l; after harvesting and washing by centrifugation the cells were resuspended in phosphate buffer to give ca a 75 fold concentrate. Typically O2uptake experiments were for 4-5 hours duration. Benzimidazole was assayed by measurement of the U.V light absorption spectra atλmax after appropriate dilution in phosphate buffer (KH2PO4, 2g/l, adjusted to pH= 7.0). Culture M was immediately able to oxidize benzimidazole (41% O2uptake), although a little of (ca 25%) of nitrogen was released as ammonia. Examination of the absorption spectra of the culture fluids revealed small decrease in the characteristic light absorption peak for benzimidazole, although the O2uptake was higher (37% of theoretical), and the yellow color much more intense (A400nmwas about 2). Thus from the study results, it can be concluded that benzimidazole is considered to be readily biodegradable.

 

Another supporting study of biodegradation from peer reviewed journal for target chemical Benzimidazole(CAS no. 51-17-2) was carried out by using a Rhodococcus PA. The isolation of a pure bacterial culture PA, thought to be a strain of Rhodococcus, capable of growing on BTH itself as a sole carbon, nitrogen and energy. For the isolation of pure cultures capable of degrading BTH, culture M was plated out onto BTH- mineral salts agar using a spread plate technique with several dilutions of the culture so as to obtain well separated colonies. Representative colonies of each colonial type were chosen and rigorously purified by streaking out alternately n the BTH mineral salts agar and nutrient agar. Seven colonial types were obtained in pure culture on solid media; all of these were then inoculated into 1mmol/l BTH liquid mineral salts medium. Growth and BTH concentrations were regularly monitored over a period of 42 days. Cultures were grown in mineral salts media containing (g/l of distilled water): KH2PO4, 1; K2HPO4, 1; FeCl3.6H20, 0.004; MgSO4.7H20, 0.04. Carbon and nitrogen sources were added to phosphate before autoclaving. Cell suspensions of organism PA were harvested by centrifugation and washed twice with distilled water by centrifugation before being suspended in phosphate buffer (2g/l, pH =7.0). Cells were broken by ultrasonic disintegration for 3-5 min, each 0.5 min burst of sonication being followed by 2 min cooling on ice. Unbroken cells and cell fragments were removed by centrifugation for 10min at 30000g at 4°C; the supernatant fluid was then kept on ice until assayed. Oxygen uptake experiments were carried out using a Gilson differential respirometer. Phosphate buffer (1 ml KH2PO4, 2g/l, pH= 7.0) was placed in the main compartment of the differential respirometer together with the substrate (1µmol) and water to a volume of 2.5ml. Concentrated washed cells suspensions (0.5 ml; about 0.1 – 0.2 mg dry weight of bacterial cells/flask) was placed in the side arm and the centre well contained 0.1ml 40% w/v KOH together with a small piece of fluted filter paper. All experiments were done at 30°C in air. Cells for these experiments were grown on the BTH-mineral salts media in which the carbon source was present at 1mmol/l; after harvesting and washing by centrifugation the cells were resuspended in phosphate buffer to give ca a 75 fold concentrate. Typically O2uptake experiments were for 4-5 hours duration. In the respirometric tests PA, there was immediate but limited O2 uptake (25% of the theoretical maximum) with benzimidazole and the development of a yellow coloration. Under the test conditions, the test substance is readily biodegradable.

Last study was experimental study in this study 28-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test item Benzimidazole (CAS no. 51 -17 -2). The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. 1 polyseed capsule were added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism.The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was  32 ml/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be  74.69 %. Degradation of Sodium Benzoate exceeds 79.15  % on 7 days & 52.4 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 0.43 mgO2/mg. ThOD was calculated as 1.89 mgO2/mg. Accordingly, the % degradation of the test chemical Benzimidazole after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be  22.75 %. Based on the results, the test chemical Benzimidazole, under the test conditions, was considered to be inherently biodegradable in nature.

 

Although last study was showing that test chemical is inherently biodegradable but other two details study showing that test chemical is readily biodegradable therefore on the basis of this target chemical Benzimidazole (from peer reviewed journal), it can be concluded that the test substance Benzimidazole is expected to be readily biodegradable in nature.