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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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: refer below principle
Principles of method if other than guideline:
Biodegradation study of the test substance 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol was carried out to determine the biodegradability rate of the 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol.
GLP compliance:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol
- Common name: Sudan II
- Molecular formula: C18H16N2O
- Molecular weight: 276.337 g/mol
- Smiles notation: c12c(\N=N/c3c(cc(C)cc3)C)c(ccc1cccc2)O
- InChl:1S/C18H16N2O/c1-12-7-9-16(13(2)11-12)19-20-18-15-6-4-3-5-14(15)8-10-17(18)21/h3-11,21H,1-2H3
- Substance type: Organic
- Physical state: Solid
Oxygen conditions:
anaerobic
Inoculum or test system:
other: Bacteria
Details on inoculum:
- Laboratory culture: The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).
- Method of cultivation: The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10 µg/ml, the cultures were incubated at 37 ᵒC in an anaerobic chamber for 2 days without agitation.
- Storage conditions: All strains were preserved at -80 ᵒC in 10 to 15% glycerol stocks and revived as needed.
- Preparation of inoculum for exposure: The strains, except for Lactobacillus species, which were routinely cultured on deMann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37 ᵒC under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide.
- Pretreatment: : The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture.
- Concentration of sludge: 10 µg/ml (10 mg/l)
Duration of test (contact time):
2 d
Initial conc.:
10 mg/L
Based on:
not specified
Parameter followed for biodegradation estimation:
other: %degradation
Details on study design:
TEST CONDITIONS
- Composition of medium: Brain Heart Infusion (BHI) broth or deMann-Rogosa-Sharpe (MRS) broth or agar was used.
- Additional substrate: vitamin k, hemin.
- Test temperature: 37 ᵒC
- Other: the cultures were incubated in an anaerobic chamber for 2 days without agitation.

TEST SYSTEM
- Culturing apparatus: Erlenmeyer flask

CONTROL AND BLANK SYSTEM
- Inoculum blank: Three control consisted of sterile liquid medium, sterile liquid medium with bacteria, and one of sterile liquid medium with dyes were used in the study.
Reference substance:
not specified
Preliminary study:
No data available
Test performance:
No data available
Key result
Parameter:
other: Average percentage degradation
Value:
100
Sampling time:
2 d
Remarks on result:
other: degradation of test substance by bacterial strains such as B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum.
Details on results:
Test substance undergoes 100% degradation in 2 days by bacterial strains such as B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum, respectively.

Table: Sudan II reduction by thirty five prevalent human intestinal bacterial species.

Human intestinal bacterial species

Biodegradation (%)

Bacteroides vulgatusATCC 8482

-

Bacteroides ovatusATCC 8483

60

Bacteroides uniformisATCC

-

Bacteroides distasonisATCC 8503

40

Bacteroides fragilisATCC 23745

-

Bacteroides thetaiotaomicronATCC 29148

40

Bacteroides caccaeATCC 43185

40

Bifidobacterium longumATCC 15707

-

Bifidobacterium adolesecentisATCC 15703

-

Bifidobacterium infantisATCC 15697

100

Bifidobacterium catenulatumATCC 27539

-

Bifidobacterium angulatumATCC 27535

-

Clostridium perfringensATCC 13124

-

Clostridium butyricumATCC 19398

-

Clostridium ramosumATCC 25582

-

Clostridium difficle ATCC 9689

-

Clostridium indolisATCC 25771

100

Clostridium leptumATCC 29065

-

Clostridium clostridioformeATCC 29084

60

Eubacterium aerofaciensATCC 25986

-

Eubacterium limosumATCC 8486

-

Eubacterium tenueATCC 25553

-

Enterococcus faecalisATCC 27274

100

Enterococcus faeciumATCC 19434

40

Escherichia coliATCC 25922

-

Fusobacterium russiATCC 25533

40

Fusobacterium nucleatumATCC 25586

-

Lactobacillus bifidus ATCC 11146

-

Lactobacillus paracaseiATCC 27092

20

Lactobacillus reuteriATCC 23272

-

Lactobacillus rhamnosusATCC 53103

100

Lactobacillus ruminisATCC 25644

-

Peptostreptococcus magnusATCC 14955

-

Ruminococcus obeumATCC 29174

100

Ruminococcus gnavusATCC 29149

-

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum were able to completely degrade (100%) the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol. Thus, the substance 1- (2, 4-dimethylphenylazo)-2-naphthol is readily biodegradable in water. The metabolite produced from 1- (2, 4-dimethylphenylazo)-2-naphthol by E. faecalis was identified as 2,4-dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z 122 [MH+] and 163 [MH+ + acetonitrile]. 1-Amino-2-naphthol from 1- (2, 4-dimethylphenylazo)-2-naphthol dye could not be detected in the extracted samples. No metabolites of 1- (2, 4-dimethylphenylazo)-2-naphthol produced by E. coli was detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium.
Executive summary:

Biodegradation study of 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6) was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days under anaerobic conditions using bacteria as a test inoculum.The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Three control consisted of sterile liquid medium, sterile liquid medium with bacteria, and one of sterile liquid medium with dyes were used in the study. All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The strains, except forLactobacillusspecies, which were routinely cultured on deMann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37C under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. Initial test subs. concentrations and inoculum conc. used for the study was 10 mg/l.Metabolites of the reduction of the test compound 1- (2, 4-dimethylphenylazo)-2-naphthol were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS). After extraction of samples and filtration, 40 µl of each sample was analyzed with a Helwet-Packard 1050 Series high performance liquid chromatography (HPLC) equipped with awith a variable wavelength detector (detection wavelengths were 250 and 500 nm), an auto sampler, and a Luna C18 column (150×3.0 mm, particle size, 5µm, Phenomenex) with a guard column (40 × 3.0 mm, Phenomenex). The mobile phase was composed of a linear gradient of acetonitrile: water containing 0.1% formic acid from 30:70 to 95:5 for 40 min. The peak area was used with a standard curve to calculate the concentration of Sudan dyes.

Identification of the metabolites was also performed using a similar procedure as detailed above with LC/ESI-MS. LC/ESI-MS data were acquired on a ThermoFinnigan Quantum Ultra mass spectrometer equipped with an Agilent 1100 Series HPLC system and a Prodigy ODS 2.0×250 mm 5µm 100 A HPLC column (Phenomenex). The mass spectrometer was operated in the positive-ion electrospray ionization (ESI) mode with an in-source collision-induced dissociation (CID) offset of 0 V. Other ESI conditions were spray voltage 4.0 kV, capillary temperature 350ᵒC, sheath gas 40 psi, ion sweep gas 0 and auxiliary gas 25. Standards and samples were extracted with ethyl acetate, which was dried and then redissolved in a starting buffer (acetonitrile: water containing 0.1% formic acid- 5:95). Much of the dried sample was insoluble, so samples were taken without including the precipitate. The starting buffer was held 20 min, ramped in 1 min to acetonitrile: water containing 0.1% formic acid-95:5 at 21 min and held until 40 min. Reduction of the1- (2, 4-dimethylphenylazo)-2-naphtholwas determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations.

Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus andR. obeum were able to completely degrade (100%) the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol whereas partial degradation of the 1- (2, 4-dimethylphenylazo)-2-naphthol occurred by the bacterial strains such as B. ovatus, B. distasonis, B. thetaiotaomicron, B. caccae,C. clostridioforme, E. faecium, F. russi and L. paracasei. Thus, the test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is readily biodegradable in water.The bacteria which are unable to degrade the 1 - (2, 4 -dimethylphenylazo)-2 -naphthol are B. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnus and R. gnavus.The metabolite produced from 1 - (2, 4 -dimethylphenylazo)-2 -naphthol by E. faecalis was identified as 2,4 -dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z122 [MH+] and 163 [MH++acetonitrile]. 1 -Amino-2 -naphthol from1 - (2, 4 -dimethylphenylazo)-2 -naphthol could not be detected in the extracted samples. No metabolites of 1 - (2, 4 -dimethylphenylazo)-2 -naphthol produced by E. coli was detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium. Thus, based on percentage degradation of test chemical,1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be readily biodegradable in nature.

Description of key information

Biodegradation study of 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6) was performed (Haiyan Xu et. al; 2010). The test is carried at a temperature of 37ᵒCwith a duration period of 2 days under anaerobic conditions using bacteria as a test inoculum.The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Three control consisted of sterile liquid medium, sterile liquid medium with bacteria, and one of sterile liquid medium with dyes were used in the study. All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The strains, except forLactobacillusspecies, which were routinely cultured on deMann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37C under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. Initial test subs. concentrations and inoculum conc. used for the study was 10 mg/l.Metabolites of the reduction of the test compound 1- (2, 4-dimethylphenylazo)-2-naphthol were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS). After extraction of samples and filtration, 40 µl of each sample was analyzed with a Helwet-Packard 1050 Series high performance liquid chromatography (HPLC) equipped with awith a variable wavelength detector (detection wavelengths were 250 and 500 nm), an auto sampler, and a Luna C18 column (150×3.0 mm, particle size, 5µm, Phenomenex) with a guard column (40 × 3.0 mm, Phenomenex). The mobile phase was composed of a linear gradient of acetonitrile: water containing 0.1% formic acid from 30:70 to 95:5 for 40 min. The peak area was used with a standard curve to calculate the concentration of Sudan dyes.

Identification of the metabolites was also performed using a similar procedure as detailed above with LC/ESI-MS. LC/ESI-MS data were acquired on a ThermoFinnigan Quantum Ultra mass spectrometer equipped with an Agilent 1100 Series HPLC system and a Prodigy ODS 2.0×250 mm 5µm 100 A HPLC column (Phenomenex). The mass spectrometer was operated in the positive-ion electrospray ionization (ESI) mode with an in-source collision-induced dissociation (CID) offset of 0 V. Other ESI conditions were spray voltage 4.0 kV, capillary temperature 350ᵒC, sheath gas 40 psi, ion sweep gas 0 and auxiliary gas 25. Standards and samples were extracted with ethyl acetate, which was dried and then redissolved in a starting buffer (acetonitrile: water containing 0.1% formic acid- 5:95). Much of the dried sample was insoluble, so samples were taken without including the precipitate. The starting buffer was held 20 min, ramped in 1 min to acetonitrile: water containing 0.1% formic acid-95:5 at 21 min and held until 40 min. Reduction of the1- (2, 4-dimethylphenylazo)-2-naphtholwas determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations.

Among the tested bacterial strains,B. infantis,C. indolis,E. faecalis,L. rhamnosusandR. obeum were able to completely degrade (100%) the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol whereas partial degradation of the 1- (2, 4-dimethylphenylazo)-2-naphthol occurred by the bacterial strains such asB. ovatus,B. distasonis,B. thetaiotaomicron,B. caccae,C. clostridioforme,E. faecium,F. russiandL. paracasei. Thus, the test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is readily biodegradable in water.The bacteria which are unable to degrade the 1 - (2, 4 -dimethylphenylazo)-2 -naphthol areB. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnusandR. gnavus.The metabolite produced from 1 - (2, 4 -dimethylphenylazo)-2 -naphthol byE. faecaliswas identified as 2,4 -dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z122 [MH+] and 163 [MH++acetonitrile]. 1 -Amino-2 -naphthol from1 - (2, 4 -dimethylphenylazo)-2 -naphthol could not be detected in the extracted samples. No metabolites of 1 - (2, 4 -dimethylphenylazo)-2 -naphthol produced byE. coliwas detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium. Thus, based on percentage degradation of test chemical,1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Experimental key study and various supporting studies for the target compound1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol(CAS No. 3118-97-6) were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Haiyan Xu et. al; 2010), biodegradation experiment of 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6) was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days under anaerobic conditions using bacteria as a test inoculum. The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Three control consisted of sterile liquid medium, sterile liquid medium with bacteria, and one of sterile liquid medium with dyes were used in the study. All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed. The strains, except for Lactobacillus species, which were routinely cultured on de Mann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37C under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide. The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. Initial test subs. concentrations and inoculum conc. used for the study was 10 mg/l. Metabolites of the reduction of the test compound 1- (2, 4-dimethylphenylazo)-2-naphthol were isolated and identified by HPLC and Liquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS). After extraction of samples and filtration, 40 µl of each sample was analyzed with a Helwet-Packard 1050 Series high performance liquid chromatography (HPLC) equipped with a with a variable wavelength detector (detection wavelengths were 250 and 500 nm), an auto sampler, and a Luna C18 column (150×3.0 mm, particle size, 5µm, Phenomenex) with a guard column (40 × 3.0 mm, Phenomenex). The mobile phase was composed of a linear gradient of acetonitrile: water containing 0.1% formic acid from 30:70 to 95:5 for 40 min. The peak area was used with a standard curve to calculate the concentration of Sudan dyes.

Identification of the metabolites was also performed using a similar procedure as detailed above with LC/ESI-MS. LC/ESI-MS data were acquired on a Thermo Finnigan Quantum Ultra mass spectrometer equipped with an Agilent 1100 Series HPLC system and a Prodigy ODS 2.0×250 mm 5µm 100 A HPLC column (Phenomenex). The mass spectrometer was operated in the positive-ion electrospray ionization (ESI) mode with an in-source collision-induced dissociation (CID) offset of 0 V. Other ESI conditions were spray voltage 4.0 kV, capillary temperature 350ᵒC, sheath gas 40 psi, ion sweep gas 0 and auxiliary gas 25. Standards and samples were extracted with ethyl acetate, which was dried and then redissolved in a starting buffer (acetonitrile: water containing 0.1% formic acid- 5:95). Much of the dried sample was insoluble, so samples were taken without including the precipitate. The starting buffer was held 20 min, ramped in 1 min to acetonitrile: water containing 0.1% formic acid-95:5 at 21 min and held until 40 min. Reduction of the1- (2, 4-dimethylphenylazo)-2-naphtholwas determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations.

Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum were able to completely degrade (100%) the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol whereas partial degradation of the 1- (2, 4-dimethylphenylazo)-2-naphthol occurred by the bacterial strains such as B. ovatus, B. distasonis, B. thetaiotaomicron, B. caccae, C. clostridioforme, E. faecium, F. russi and L. paracasei. Thus, the test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is readily biodegradable in water.The bacteria which are unable to degrade the 1 - (2, 4 -dimethylphenylazo)-2 -naphthol are B. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnus and R. gnavus. The metabolite produced from 1 - (2, 4 -dimethylphenylazo)-2 -naphthol by E. faecalis was identified as 2,4 -dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z122 [MH+] and 163 [MH++acetonitrile]. 1 -Amino-2 -naphthol from1 - (2, 4 -dimethylphenylazo)-2 -naphthol could not be detected in the extracted samples. No metabolites of 1 - (2, 4 -dimethylphenylazo)-2 -naphthol produced by E. coli was detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium. Thus, based on percentage degradation of test chemical,1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be readily biodegradable in nature.

 

In an another study from peer reviewed journal (Haiyan Xu et. al; 2007), biodegradation experiment of 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol (CAS no. 3118-97-6) was performed. The test is carried for 30 hrunder anaerobic conditions using microorganismsas a test inoculum. Test microorganisms were obtained from human intestine. Test chemical conc. used for the study was 10 mg/l. Stock solutions of test chemical Sudan II was prepared by dissolving in 100% ethanol (1 mg/ml). Fresh diluted human fecal suspensions (6 ml; 10%, wt/vol) were transferred under anaerobic conditions into flasks containing 300 ml brain heart infusion broth to observe the effect of the microflora on decolorization of the 1 -[(2,4 -dimethylphenyl)diazenyl]-2 -naphthol. Samples (supernatants, cell extracts, and debris) were extracted with ethyl acetate to ensure that dye bound to bacterial cells could be released from the cells as well. Each residue was dissolved in 1 ml acetonitrile, and 40µl of each sample was analyzed by high-performance liquid chromatography with a Hewlett-Packard 1050 equipped with a variable-wavelength detector (the detection wavelengths used were 250 and 500 nm) and a reversed-phase Luna C18 column. The peak area was used to calculate the concentration of Sudan dye. Reduction of Sudan dyes was determined by monitoring the disappearance of the absorption peak for each dye at 500 nm and by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESIMS/ MS) when the cultures were extracted with ethyl acetate as well. To identify the Sudan dye metabolites, separate experiments were conducted in which the human intestinal microflora was incubated with Sudan II for 30 hr. Ethyl acetate extracts of incubation cultures were dried, and the residues were extracted with starting buffer (5% acetonitrile, 94.9% water, 0.1% formic acid). Much of the dried sample was insoluble, and soluble metabolites were analysed by LC/ESI-MS/MS. For analyses of Sudan dye metabolites, the starting buffer composition was held for 20 min, ramped quickly (in 1 min) to 95% acetonitrile–4.9% water–0.1% formic acid at 21 min, and held to 40 min.Product ion spectra, retention times (Rts), and UV data for metabolites were compared to those for authentic compounds for identification.The metabolites from Sudan dyes were detected by LC/ESI-MS/MS after 4 h incubation, and the amounts of the metabolites increased with the incubation time. No reduction of the tested Sudan dyes was observed in uninoculated controls.The percentage degradation of test substance 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol was determined to be 100% degradation after 30 hrs.The metabolite with an Rt of 18.7 min from1-[(2,4-dimethylphenyl)diazenyl]-2-naphtholwas identified as 2.3µg/ml 2,4-dimethylaniline (52.8%) on the basis of its Rt and product ion mass spectrum. Thus, based on percentage degradation,1 -[(2,4 -dimethylphenyl)diazenyl]-2 -naphthol was considered to be readily biodegradable in nature.

 

In an additional supporting study from authoritative database (SRC PhysProp Database, 2017), biodegradation experiment was carried out for 30 days for determining the percentage degradation of the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6). The test is carried under aerobic condition at a temperature of 27°C and pH 7. Sewage was used as a test inoculum for the study.Initial cell/ biomass concentration were1350, 1450, 1500, 1450, 1390 and 1500 mg/l, respectively. Conc. of test chemical used for the study were40, 60, 80, 100, 120 and 140 mg/l, respectively. Analytical methods used for the study were spectrophotometer and TLC. The percentage degradation of test substance 1- (2, 4-dimethylphenylazo)-2-naphthol was determined to be 100% in 30 days. Thus, based on percentage degradation, 1- (2, 4-dimethylphenylazo)-2-naphthol is considered to be readily biodegradable in nature.

 

Another biodegradation study was carried out for 60 days for determining the percentage degradation of the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6)(SRC PhysProp Database, 2017). The test is carried under aerobic condition at a temperature of 27°C and pH 7. Sewage was used as a test inoculum for the study. Initial cell/ biomass concentration were 1720; 1050 mg/l and1540; 920mg/l, respectively. Conc. of test chemical used for the study was 20mg/l, respectively. Analytical methods used for the study were spectrophotometer and TLC. The percentage degradation of test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol was determined to be 100% after 60 days. Thus, based on percentage degradation, 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be inherently biodegradable in nature.

 

Although one study from authoritative SRC PhysProp Database (2017) indicate that the chemical1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol is inherently biodegradable in nature, but based on the results of the experimental studies from peer reviewed journals,it can be concluded that the test substance1-[(2,4-dimethylphenyl)diazenyl]-2-naphtholcan be expected to be readily biodegradable in nature.