Sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

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 evaluating the percentage biodegradability of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (IUPAC Name): sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate
- Common name: C. I. Acid Red 266
- Molecular formula: C17H10ClF3N3NaO4S
- Molecular weight: 467.786 g/mol
- Smiles notation:
c12c(cc(S(=O)(=O)[O-])cc2O)ccc(c1\N=N/c1c(cc(Cl)cc1)C(F)(F)F)N.[Na+]
- InChl: 1S/C17H11ClF3N3O4S.Na/c18-9-2-4-13(11(6-9)17(19,20)21)23-24-16-12(22)3-1-8-5-10(29(26,27)28)7-14(25)15(8)16;/h1-7,25H,22H2,(H,26,27,28);/q;+1/p-1/b24-23-;
- Substance type: Organic
- Physical state: Solid

Oxygen conditions:

not specified

Inoculum or test system:

other: Anaerobic granular sludge (non-adapted)

Details on inoculum:

- Concentration of sludge: 1500 mg/l (1.5 g/l) volatile suspended solids

Initial conc.:

> 100 - < 300 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

(%decolourisation)

Details on study design:

TEST CONDITIONS
- Composition of medium: Basal medium was used as a test medium for the study. The composition of medium includes 2.8 g NH4Cl/l, 0.057 g 0CaCl2 /l, 2.5 g KH2PO4/l and 1 g MgSO4.7H2O/l and the medium was buffered at a pH of 7.3 ± 0.2 with NaHCO3 (5 g/l).
- Test temperature: 30°C
- pH: 7.3 ± 0.2
- pH adjusted: yes, pH was adjusted with NaHCO3 (5 g/l)

TEST SYSTEM
- Culturing apparatus: 120 ml serum bottles were used as a test vessel for the study.
- Measuring equipment: At selected intervals, colour was measured spectrophotometrically at the dye’s wavelength of maximum absorbance (ƛ max).
- Test performed in closed vessels due to significant volatility of test substance: The decolourisation assay was performed in a serum bottles containing medium and an overlying headspace composed of N2/CO2 (80%/20%) which was sealed with a butyl rubber stopper.
- Other: The serum bottles were incubated at 30°C in a rotary shaker at 50 rpm.

Key result

Parameter:

other: %decolourisation (Test material analysis)

Value:

95

Remarks on result:

other: Other details not known

Details on results:

Test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate (Acid Red 266) undergoes 95% decolourisation.

Table: Overall results of azo dye decolourisation by anaerobic granular sludge.

 

 

Dye	Purity (%)	ƛmax (nm)	Decolourisation (%max)	K(d-1)^a
Acid Red 266	NA^b	492	95	0.20 ± 0.07

 

 

Where,

a = k-values (first order rate constants) were obtained from fitting equation to the complete decolourisation curve (monoazo dye) or to the first part of the decolourisation curve (disazo and polyazo dyes); for experiments which were replicated, standard deviations are mentioned behind the ± sign.

NA^b= information not available

 

The decolourisation of Acid Red 266 by anaerobic granular sludge was measured as the decrease of visible light absorbance at the previously assessed wavelength of maxium absorbance (ƛmax). The reactions proceeded without lag phase.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Conclusions:

The percentage decolourisation of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was determined to 95%. Thus, based on percentage decolourisation, sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate is considered to be readily biodegradable in water.

Executive summary:

Biodegradation study was conducted for evaluating the percentage biodegradability of test substancesodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4 -hydroxynaphtha lene-2 -sulphonate (CAS no. 57741 -47 -6) at a temperature of 30°C and pH of 7.3 ± 0.2.Anaerobic granular sludge (non-adapted) was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 1500 mg/l (1.5 g/l) volatile suspended solids and initial test substance conc. used in the study was 100-300 mg/l, respectively. Basal medium was used as a test medium for the study. The composition of medium includes 2.8 g NH4Cl/l, 0.057 g 0CaCl2 /l, 2.5 g KH2PO4/l and 1 g MgSO4.7H2O/l and the medium was buffered at a pH of 7.3 ± 0.2 with NaHCO3 (5 g/l). The biological dye decolourisation assay was performed in 120 ml serum bottles containing 50 ml of medium and an overlying headspace composed of N2/CO2 (80%/20%) which was sealed with a butyl rubber stopper. The primary electron donating substrate of the medium was composed of 2 g/l chemical oxygen demand (COD) of an NaOH-neutralised volatile fatty acids (VFA) mixture containing acetate, propionate and butyrate in a COD-based ratio of 1:10:10. Non-adapted anaerobic granular sludge was added to the medium at a concentration of 1500 mg/l volatile suspended solids (VSS). The medium was flushed with the N2/CO2 (80%/20%) and preincubated with the sludge for 2-3 days. The background level of sulphide in the medium was 0.7 ± 0.02 mM. The test chemical acid red 266 was added to a final conc. of approx. 0.3 mM (100-300 mg/l) with a syringe from a concentrated stock solution. The serum bottles were incubated at 30°C in a rotary shaker at 50 rpm.At selected intervals, colour was measured spectrophotometrically at the dye’s wavelength of maximum absorbance (ƛ max). For this purpose, samples were centrifuged after dilution to less than 1 absorbance unit (AU) in a phosphate buffer (10.86 g/l Na2HPO4.2H2O; 5.38 g/l Na2HPO4.H2O) that contained ascorbic acid (200 mg/l) to effectively prevent auto-oxidation. The background light absorbance of the control medium in the buffer was less than 0.5% of the absorbance due to dye containing medium in the buffer and could therefore be neglected. The percentage decolourisation of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was determined to 95%. Thus, based on percentage decolourisation,sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate is considered to be readily biodegradable in nature.

Description of key information

Biodegradation study was conducted for evaluating the percentage biodegradability of test substancesodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4 -hydroxynaphtha lene-2 -sulphonate (CAS no. 57741 -47 -6) at a temperature of 30°C and pH of 7.3 ± 0.2 (Frank P. van der Zee, et. al; 2001). Anaerobic granular sludge (non-adapted) was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 1500 mg/l (1.5 g/l) volatile suspended solids and initial test substance conc. used in the study was 100-300 mg/l, respectively. Basal medium was used as a test medium for the study. The composition of medium includes 2.8 g NH4Cl/l, 0.057 g 0CaCl2 /l, 2.5 g KH2PO4/l and 1 g MgSO4.7H2O/l and the medium was buffered at a pH of 7.3 ± 0.2 with NaHCO3 (5 g/l). The biological dye decolourisation assay was performed in 120 ml serum bottles containing 50 ml of medium and an overlying headspace composed of N2/CO2 (80%/20%) which was sealed with a butyl rubber stopper. The primary electron donating substrate of the medium was composed of 2 g/l chemical oxygen demand (COD) of an NaOH-neutralised volatile fatty acids (VFA) mixture containing acetate, propionate and butyrate in a COD-based ratio of 1:10:10. Non-adapted anaerobic granular sludge was added to the medium at a concentration of 1500 mg/l volatile suspended solids (VSS). The medium was flushed with the N2/CO2 (80%/20%) and preincubated with the sludge for 2-3 days. The background level of sulphide in the medium was 0.7 ± 0.02 mM. The test chemical acid red 266 was added to a final conc. of approx. 0.3 mM (100-300 mg/l) with a syringe from a concentrated stock solution. The serum bottles were incubated at 30°C in a rotary shaker at 50 rpm.At selected intervals, colour was measured spectrophotometrically at the dye’s wavelength of maximum absorbance (ƛ max). For this purpose, samples were centrifuged after dilution to less than 1 absorbance unit (AU) in a phosphate buffer (10.86 g/l Na2HPO4.2H2O; 5.38 g/l Na2HPO4.H2O) that contained ascorbic acid (200 mg/l) to effectively prevent auto-oxidation. The background light absorbance of the control medium in the buffer was less than 0.5% of the absorbance due to dye containing medium in the buffer and could therefore be neglected. The percentage decolourisation of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was determined to 95%. Thus, based on percentage decolourisation,sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate is considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Experimental study for the target compound sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate (CAS No. 57741-47-6) and supporting study for its structurally similar read across substance were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Frank P. van der Zee, et. al; 2001), biodegradation experiment was conducted for evaluating the percentage biodegradability of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4 -hydroxynaphtha lene-2 -sulphonate (CAS no. 57741 -47 -6) at a temperature of 30°C and pH of 7.3 ± 0.2.Anaerobic granular sludge (non-adapted) was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 1500 mg/l (1.5 g/l) volatile suspended solids and initial test substance conc. used in the study was 100-300 mg/l, respectively. Basal medium was used as a test medium for the study. The composition of medium includes 2.8 g NH4Cl/l, 0.057 g 0CaCl2 /l, 2.5 g KH2PO4/l and 1 g MgSO4.7H2O/l and the medium was buffered at a pH of 7.3 ± 0.2 with NaHCO3 (5 g/l). The biological dye decolourisation assay was performed in 120 ml serum bottles containing 50 ml of medium and an overlying headspace composed of N2/CO2 (80%/20%) which was sealed with a butyl rubber stopper. The primary electron donating substrate of the medium was composed of 2 g/l chemical oxygen demand (COD) of an NaOH-neutralised volatile fatty acids (VFA) mixture containing acetate, propionate and butyrate in a COD-based ratio of 1:10:10. Non-adapted anaerobic granular sludge was added to the medium at a concentration of 1500 mg/l volatile suspended solids (VSS). The medium was flushed with the N2/CO2 (80%/20%) and preincubated with the sludge for 2-3 days. The background level of sulphide in the medium was 0.7 ± 0.02 mM. The test chemical acid red 266 was added to a final conc. of approx. 0.3 mM (100-300 mg/l) with a syringe from a concentrated stock solution. The serum bottles were incubated at 30°C in a rotary shaker at 50 rpm. At selected intervals, colour was measured spectrophotometrically at the dye’s wavelength of maximum absorbance (ƛ max). For this purpose, samples were centrifuged after dilution to less than 1 absorbance unit (AU) in a phosphate buffer (10.86 g/l Na2HPO4.2H2O; 5.38 g/l Na2HPO4.H2O) that contained ascorbic acid (200 mg/l) to effectively prevent auto-oxidation. The background light absorbance of the control medium in the buffer was less than 0.5% of the absorbance due to dye containing medium in the buffer and could therefore be neglected. The percentage decolourisation of test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate was determined to 95%. Thus, based on percentage decolourisation, sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate is considered to be readily biodegradable in nature.

 

For the read across chemical disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate (CAS no. 3734-67-6) from peer reviewed journal (Lata Kumari, et. al; 2016),biodegradation study was conducted for evaluating the percentage biodegradability of read across chemical Disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate by indigenous bacteria Stenotrophomonassp. BHUSSp X2. Stenotrophomonassp. BHUSSp X2 was used as a test inoculum isolated from soil collected from the carpet dyeing industries located in Bhadohi, India. Soil and effluents were collected from the carpet dyeing industries located in Bhadohi, India. One hundred milliliters of 1 % soil solution was incubated with 500-mg/l RED G dye in nutrient broth maintained at 35±2 °C for 1 week under static and shaking conditions. The resultant broth sample was streaked on nutrient agar plate having 500-mg/l RED G dye, and this was again incubated at 35±2 °C for a week. Morphologically distinct bacteria were isolated from this agar plate and were again streaked on fresh nutrient agar plate for isolation of pure bacteria. The most promising isolates selected in this manner were tested for decolorization of dye in submerged condition. The dye degrading bacteria were identified on the basis of morphological colony; Gram staining and biochemical test of isolated strain were performed according to the Bergey’s manual and on the basis of 16S rRNA gene sequence analysis. The sequence was compared using BLAST programmed at NCBI server to identify bacteria. The 16S rRNA sequence of isolated bacterial strain and related sequences of NCBI were aligned using Cluster W, and phylogenetic tree was made using neighbor-joining methods of MEGA (Version 6).The decolorization experiments were carried out in 250-ml Erlenmeyer flasks containing 100-ml nutrient broth supplemented with RED G dye (100 mg/l). The media were inoculated with respective bacterial strains by addition of inoculums with uniform cell density (O.D. 0.5). Decolorization studies were carried out under static and shaking conditions in 100-ml nutrient broth having dye concentration 200 mg/l dye. All the flasks were incubated under static and shaking (100 rpm) conditions at 35±2 °C for 24 h. Samples were periodically withdrawn after every 2 h, centrifuged (10,000 rpm) to estimate the extent of decolorization using UV-Vis spectrophotometer. The degraded metabolites were extracted with equal volumes of ethyl acetate, and this was evaporated to dryness in rotatory evaporator. The extracted metabolites were mixed with HPLC grade potassiumbromide (KBr) in the ratio of 5:95 and analyzed at mid IR region (400–4000 cm−1) by using FTIR Perkin Elmer, Spectrophotometer. The intermediates produced after degradation were analyzed by GC-MS. The presence of various groups in the GC-MS spectra of dye sample before degradation confirms the characteristics of ACID RED 1 dye, whereas GC-MS spectra of degraded samples confirms the presence of aniline (M.W. 93 peak at 93 m/z), benzene with M.W. 78 m/z 74, sodium7-amino-6-hydroxynapthalene-2-sulfonate (M.W. 261, m/z 263), 3- hydroxyphthalic acid (M.W. 182, m/z 184), and pyrocatechol (M.W.110, m/z 110).The percentage decolorization of test chemical Disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate was determined to be 95 and 45% under static and shaking conditions after 28 hrs, respectively. Thus, based on percentage decolorization, chemical disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate can be considered to be readily biodegradable in water.

 

On the basis of above results for target chemical sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonate(from peer reviewed journal) and for its read across substance (from peer reviewed journal), it can be concluded that the test substance sodium 6-amino-5-[[4-chloro-2-(trifluoromethyl)phenyl]azo]-4-hydroxynaphthalene-2-sulphonatecan be expected to be readily biodegradable in nature.