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Biodegradation in water: screening tests

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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:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test chemical using Sphingomonas sp. strain YL-JM2C as a test inoculum.
GLP compliance:
not specified
Oxygen conditions:
not specified
Inoculum or test system:
other: Sphingomonas sp. strain YL-JM2C (Bacteria)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Strain YL-JM2C was isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosan.
Duration of test (contact time):
6 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: HPLC and GC-MS
Details on study design:
TEST CONDITIONS
- Composition of medium: Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includes K2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM.
- Additional substrate: 0.04% yeast extract (sterilized by 0.45 mm membrane) was added to this medium.
- Test temperature: 30°C
- pH: 7.0
- pH adjusted: yes, the pH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 40 ml bottle was used as a test vessel for the study.
- Measuring equipment: A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of test chemical at a specific incubation period by high performance liquid chromatography (HPLC).
- Other: All these bottles were kept in shaker (150 rpm) at 30°C under dark condition.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: Uninoculated controls were used to determine any transformation of 3,4-dichloroaniline and 4-chloroaniline affected by abiotic factors.
- Other: For control, 10 mL of sterile AMS medium containing test chemical (4 mg/L) was used.
Key result
Parameter:
other: HPLC
Value:
35
Sampling time:
5 d
Remarks on result:
other: Other details not known
Details on results:
Test substance undergoes 35% degradation by HPLC parameter in 5 days. The degradation product of test chemical was determined to be 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively. Further, TOC results revealed that the Sphingomonas sp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d.

The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium with test chemical by GC-MS revealed the presence of three compounds (Isolate I, Isolate II, and Isolate III). The mass spectra of isolated compound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively. Thus in strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions.In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol.

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days. In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions. In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the Sphingomonas sp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d. Thus, based on this, test chemical is considered to be readily biodegradable in water.
Executive summary:

Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test chemical at a temperature of30°C and pH 7.0, respectively.Sphingomonassp. strain YL-JM2C (Bacteria) isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosanwas used as test inoculum.Stock solutions (5 g/L) were prepared with acetone and stored in brown bottles at-20°C before use.Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includesK2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. 0.04% yeast extract (sterilized by 0.45mm membrane) was added to this medium.ThepH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.40 ml bottle was used as a test vessel for the study.For biodegradation experiments, 1 mL of pure bacterial culture (mid-log period) was transferred into a 40 mL bottle (working volume of 10 mL). For control, 10 mL of sterile AMS medium containing test chemical (4 mg/l) was used. All these bottles were kept in shaker (150 rpm) at 30°C under dark condition. A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of triclocarban at a specific incubation period by high performance liquid chromatography (HPLC).Degradation of test chemical metabolites 3,4-dichloroaniline and 4-chloroaniline during growth ofSphingomonassp. strain YL-JM2C was determined at different intervals according to the total organic carbon (TOC) concentration by TOC analyser (Shimadzu TOC-V CPH, Japan). The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium with test chemical by GC-MS revealed the presence of three compounds(Isolate I, Isolate II, and Isolate III). The mass spectra of isolatedcompound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively.The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days.In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions.In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the test bacterial inoculum Sphingomonassp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d.Thus, based on this,test chemical is considered to be readily biodegradable in nature.

Description of key information

Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test chemicalat a temperature of30°C and pH 7.0, respectively (Sikandar I. Mulla et. al.; 2016). Sphingomonassp. strain YL-JM2C (Bacteria) isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosanwas used as test inoculum.Stock solutions (5 g/L) were prepared with acetone and stored in brown bottles at-20°C before use.Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includesK2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. 0.04% yeast extract (sterilized by 0.45mm membrane) was added to this medium.ThepH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.40 ml bottle was used as a test vessel for the study.For biodegradation experiments, 1 mL of pure bacterial culture (mid-log period) was transferred into a 40 mL bottle (working volume of 10 mL). For control, 10 mL of sterile AMS medium containing test chemical (4 mg/l) was used. All these bottles were kept in shaker (150 rpm) at 30°C under dark condition. A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of triclocarban at a specific incubation period by high performance liquid chromatography (HPLC).Degradation of test chemical metabolites 3,4-dichloroaniline and 4-chloroaniline during growth ofSphingomonassp. strain YL-JM2C was determined at different intervals according to the total organic carbon (TOC) concentration by TOC analyser (Shimadzu TOC-V CPH, Japan). The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium withtest chemical by GC-MS revealed the presence of three compounds(Isolate I, Isolate II, and Isolate III). The mass spectra of isolatedcompound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively.The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days.In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions.In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the test bacterial inoculum Sphingomonassp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d.Thus, based on this,test chemicalis considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

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 (Sikandar I. Mulla et. al.; 2016), biodegradation experiment was conducted for 6 days for evaluating the percentage biodegradability of test chemical at a temperature of30°C and pH 7.0, respectively. Sphingomonassp. strain YL-JM2C (Bacteria) isolated from activated sludge of a wastewater treatment plant in Xiamen, China by enrichment on triclosan was used as test inoculum. Stock solutions (5 g/L) were prepared with acetone and stored in brown bottles at-20°C before use. Ammonium mineral salts (AMS) medium was used as a test medium. The composition of the medium includesK2SO4, 0.98 mM; KH2PO4, 3.9 mM; Na2HPO4.12H2O, 6.1 mM; (NH4)2SO4, 5.88 mM; MgSO4.7H2O, 0.15 mM; CaSO4.2H2O, 0.07 mM; CoMoO4, 0.004 mM; KI, 0.001 mM; ZnSO4.7H2O, 0.002 mM; MnSO4.H2O, 0.002 mM; H3BO3, 0.002 mM; FeSO4.H2O, 0.08 mM; H2SO4, 0.1 mM. 0.04% yeast extract (sterilized by 0.45mm membrane) was added to this medium. The pH of AMS medium was adjusted to 7.00 (using 1 M NaOH or 1 M H2SO4) and sterilized by autoclaving.40 ml bottle was used as a test vessel for the study. For biodegradation experiments, 1 mL of pure bacterial culture (mid-log period) was transferred into a 40 mL bottle (working volume of 10 mL). For control, 10 mL of sterile AMS medium containing test chemical (4 mg/l) was used. All these bottles were kept in shaker (150 rpm) at 30°C under dark condition. A set of bottles (inoculated and uninoculated) were sacrificed to determine degradation of triclocarban at a specific incubation period by high performance liquid chromatography (HPLC).Degradation of test chemical metabolites 3,4-dichloroaniline and 4-chloroaniline during growth of Sphingomonassp. strain YL-JM2C was determined at different intervals according to the total organic carbon (TOC) concentration by TOC analyser (Shimadzu TOC-V CPH, Japan). The analysis of 24 and 72 h-old culture supernatant of Sphingomonassp. strain YL-JM2C grown in the AMS medium with test chemical by GC-MS revealed the presence of three compounds(Isolate I, Isolate II, and Isolate III). The mass spectra of isolated compound I, compound II and compound III were identical to that of authentic 3,4-dichloroaniline, 4-chloroaniline and 4-chlorocatechol, respectively. The percentage degradation of test chemical was determined to be 37% by HPLC parameter within 5 days. In strain YL-JM2C, test chemical was transformed into 3,4-dichloroaniline and 4-chloroaniline and 3,4-dichloroaniline was further transformed into 4-chloroaniline with the release of chloride ions. In the third step, in strain YL-JM2C, 4-chloroaniline was transformed into 4-chlorocatechol. Of these metabolites, TOC results revealed that the test bacterial inoculum Sphingomonassp. strain YLJM2C degraded up to 77% of 3,4-dichloroaniline and 80% of 4- chloroaniline within 5 d. Thus, based on this, test chemical is considered to be readily biodegradable in nature.

 

Another biodegradation study was performed to determine biodegradability of test chemical (W. E. GLEDHILL et. al; 1975). In this the test chemical was radio labeled at parachloro aniline position and quantification of 14CO2 evolution was done by using Scintillation counting method , Counting was conducted on a Nuclear Chicago Isocap 300 counter with external standardization. Corrections for background and chemical quenching were made. Aqueous solutions, 100 ml, were incubated in 300 ml Bellco baffled Erlenmeyer flasks which were sealed with rubber stopper and an air inlet tube. The rest tube contained a 1 cm hole just below the rubber stopper and 3.0 ml of 0.5 N KOH. The test chemical concentration used in this study was 200 µg/L and temperature was 18-20oC. The total duration of study was 13 weeks. Percent degradation of test chemical was determined to be 70 % and 60 % by using activated sludge and raw sewage inoculums respectively in 28 days (4 weeks) and 88 % degradation in 23 weeks by using both inoculums by using CO2 evolution as parameter. On the basis of percent degradation value it is concluded that test chemical is readily biodegradable.

For the test chemical, an experiment was performed to determine biodegradability of test chemical (W. E. GLEDHILL et. al; 1975). In this the test chemical was radio labeled at parachloro aniline position and quantification of 14CO2 evolution was done by using Scintillation counting method , Counting was conducted on a Nuclear Chicago Isocap 300 counter with external standardization. Corrections for background and chemical quenching were made. Aqueous solutions, 100 ml, were incubated in 300 ml Bellco baffled Erlenmeyer flasks which were sealed with rubber stopper and an air inlet tube. The rest tube contained a 1 cm hole just below the rubber stopper and 3.0 ml of 0.5 N KOH. The test chemical concentration used in this study was 200 µg/L and temperature was 18-20 oC. The total duration of study was 13 weeks. Percent degradation of test chemical was determined to be 90 % , 3 % and 34% at 200, 2000 and 20µg/L test chemical concentration respectively in 28 days and 95%, 70% and 60% at 200, 2000 and 20µg/L test chemical concentration respectively in 13 weeks by using CO2 evolution as parameter. The percent degradation of test chemical at concentration 20 µg/L should be more but it is showing only 34 % degradation it may be due to binding of test chemical to activated sludge (inoculums used in this study ) .Thus on the basis of percent degradation value at concentration 200 µg/L it is concluded that test chemical is readily biodegradable.

 

Additional biodegradation study (from authoritative database, 2018) was performed to determine percent degradation of test chemical by using BOD and HPLC as parameter. Inoculum used in this study was activated sludge at concentration 30 mg/L and the initial concentration of test chemical used was 100 mg/L. Percent degradation of test chemical was determined to be 1.0 % by both parameters BOD and HPLC in 28 days. On the basis of percent degradation of test chemical, it is concluded that test chemical is not readily biodegradable.

 

On the basis of overall results of the test chemical (from peer reviewed journals and authoritative databaseJ-CHECK), it can be concluded that the test chemical can be considered to be readily biodegradable in water.