Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate

Endpoint summary

Description of key information

Biodegradation in water

Biodegradation study was conducted for 7 days for evaluating the percentage biodegradability of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate using Pseudomonas putida as a test inoculum (S. Sudheer Khan, et. al; 2015).Test inoculum Pseudomonas putida MTCC 4910 was obtained from Institute of Microbial Technology, Chandigarh, India.The bacterium was cultured in nutrient agar.Bacterial stock culture was maintained at 4 °C.P. putidawas inoculated in nutrient broth and kept at room temperature for 12–16 h. During exponential phase the cells were harvested. The bacterial suspension was centrifuged at 6000хgfor 10 min. The pellets were collected and the supernatant, discarded. The pellets were washed twice with 0.9% saline solution which were later suspended in 1mM NaCl solution and stored at 4°C.Test chemical conc. used for the study were 10, 25, 50, 100, 250 and 500 mg/l, respectively. Mineral salt medium (MSM) was used as a test medium for the study. Test medium contains10 g/l glucose, 1 g/l KH2PO4, 1g/l (NH4)2SO4, 500mg/l MgSO4 · 7 H2O and 1ml of trace metal solution containing 0.01 g/l ZnSO4 · 7 H2O, 0.1g/l MnCl2 · 4H2O, 0.392 g/l CuSO4 · 5H2O, 0.248g/l CoCl2 · 6H2O, 0.177 g/l NaB4O7 · 10H2O, and 0.02 g/l NiCl2 · 6 H2O.Erlenmeyer flask was used as a test vessel for the study.Erlenmeyerflasks containing MSM with BV and AB were inoculated with 2mL of freshly preparedP.putidacells (1х109 colony forming unit (CFU)/ml) separately. Flasks were incubated at room temperature in an orbital shaker for a week.Degradation rate was calculated periodically by measuring absorbance at 604nm for Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzene sulphonate using UV–Vis spectrophotometry. This was done by collecting and centrifuging aliquots (5 mL) of the culture at 10 000хgfor 10 min. The degradation experiments were performed in triplicate and six replicates were kept for each experiment.Uninoculated controls were used to compare abiotic color loss during the degradation studies.Similarly the effect of temperature & pH on the degradation of Acid blue 93 (50mg/l) was carried out at different temperatures (25, 30, 37, and 45°C) andpH range 4-10, respectively. Different concentrations of dyes were added to MSM to examine the effect of initial dye concentration on degradation. Degraded samples (after 24 h of bacterial degradation) were centrifuged at 10000хgfor 10 min. Extraction of metabolites was carried from the supernatant using equal volume of ethyl acetate. Extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of methanol and used for analysis. The identification of metabolites formed after degradation was carried out using a DSQ GC coupled with mass spectroscopy (Thermo GC ultra-version 5.0). GC was conducted in the temperature programming mode with a DB 35-MS capillary standard non-polar column (0.25mm, 30 m, 0.25mm). Initial column temperature was 80°C for 2 min, then increased linearly at 10°C/minto 280°C, and the GC–MS interface was maintained at 290°C. Helium gas was used as carrier and theflow rate was 1mL/min. Degradation products were identified by comparison of retention time and fragmentation pattern, as well as with mass spectra in the NIST spectral library stored in the computer software of the GC–MS.In order to determine the intermediate metabolites, GC–MS analysis was carried out for test substanceAcid blue 93after 16 h of incubation.Three peaks were observed in GC–MS analysis indicating the presence of intermediate metabolites, but it was unable to determine the possible products.The percentage degradation of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonatewas determined to be 100% after 76 hrs. Test organismP. putidawas able to degrade 100% of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonateat pH 7 and 6 & at different initial dye conc., respectively. The optimum temperature for decolorization of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was observed at 45°C in 24 hrs, respectively.Thus, based on percentage degradation, Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is considered to be readily biodegradable in nature.

Additional information

Biodegradation in water

Various experimental key and supporting studies for the target compound Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate (CAS No. 28983-56-4) 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 (S. Sudheer Khan, et. al; 2015), biodegradation experiment was conducted for 7 days for evaluating the percentage biodegradability of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate using Pseudomonas putida as a test inoculum. Test inoculum Pseudomonas putida MTCC 4910 was obtained from Institute of Microbial Technology, Chandigarh, India .The bacterium was cultured in nutrient agar. Bacterial stock culture was maintained at 4 °C.P. putida was inoculated in nutrient broth and kept at room temperature for 12–16 h. During exponential phase the cells were harvested. The bacterial suspension was centrifuged at 6000хgfor 10 min. The pellets were collected and the supernatant, discarded. The pellets were washed twice with 0.9% saline solution which were later suspended in 1mM NaCl solution and stored at 4°C.Test chemical conc. used for the study were 10, 25, 50, 100, 250 and 500 mg/l, respectively. Mineral salt medium (MSM) was used as a test medium for the study. Test medium contains10 g/l glucose, 1 g/l KH2PO4, 1g/l (NH4)2SO4, 500mg/l MgSO4 · 7 H2O and 1ml of trace metal solution containing 0.01 g/l ZnSO4 · 7 H2O, 0.1g/l MnCl2 · 4H2O, 0.392 g/l CuSO4 · 5H2O, 0.248g/l CoCl2 · 6H2O, 0.177 g/l NaB4O7 · 10H2O, and 0.02 g/l NiCl2 · 6 H2O.Erlenmeyer flask was used as a test vessel for the study. Erlenmeyerflasks containing MSM with BV and AB were inoculated with 2mL of freshly prepared P.putida cells (1х109 colony forming unit (CFU)/ml) separately. Flasks were incubated at room temperature in an orbital shaker for a week. Degradation rate was calculated periodically by measuring absorbance at 604nm for Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzene sulphonate using UV–Vis spectrophotometry. This was done by collecting and centrifuging aliquots (5 mL) of the culture at 10 000хgfor 10 min. The degradation experiments were performed in triplicate and six replicates were kept for each experiment. Uninoculated controls were used to compare abiotic color loss during the degradation studies. Similarly the effect of temperature & pH on the degradation of Acid blue 93 (50mg/l) was carried out at different temperatures (25, 30, 37, and 45°C) andpH range 4-10, respectively. Different concentrations of dyes were added to MSM to examine the effect of initial dye concentration on degradation. Degraded samples (after 24 h of bacterial degradation) were centrifuged at 10000хgfor 10 min. Extraction of metabolites was carried from the supernatant using equal volume of ethyl acetate. Extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of methanol and used for analysis. The identification of metabolites formed after degradation was carried out using a DSQ GC coupled with mass spectroscopy (Thermo GC ultra-version 5.0). GC was conducted in the temperature programming mode with a DB 35-MS capillary standard non-polar column (0.25mm, 30 m, 0.25mm). Initial column temperature was 80°C for 2 min, then increased linearly at 10°C/minto 280°C, and the GC–MS interface was maintained at 290°C. Helium gas was used as carrier and theflow rate was 1mL/min. Degradation products were identified by comparison of retention time and fragmentation pattern, as well as with mass spectra in the NIST spectral library stored in the computer software of the GC–MS. In order to determine the intermediate metabolites, GC–MS analysis was carried out for test substance Acid blue 93after 16 h of incubation. Three peaks were observed in GC–MS analysis indicating the presence of intermediate metabolites, but it was unable to determine the possible products. The percentage degradation of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonatewas determined to be 100% after 76 hrs. Test organism P. putida was able to degrade 100% of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonateat pH 7 and 6 & at different initial dye conc., respectively. The optimum temperature for decolorization of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was observed at 45°C in 24 hrs, respectively. Thus, based on percentage degradation, Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 24 hrs for evaluating the percentage biodegradability of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate (CAS no. 28983-56-4) using 12 different bacterial cultures as a test inoculum (Liaquat Ali, et. al; 2014). 12 Bacterial strains were isolated from petroleum sludge. These bacterial strains (H1-H12) were identified using partial 16S rRNA sequencing of the crude DNA on a 3500 Genetic Analyzer, Applied Biosystems, USA. The obtained DNA sequences were compiled in FASTA format and analyzed using BLAST (blastn) through NCBI.Aloopful of bacteria culture from glycerol stock was inoculated in a 50 mL sterile tube containing 15 ml nutrient broth and incubated at 37°C under shaking condition (200 rpm) for 24 h. Initial test chemical concentration used for the study was 20 mg/l, respectively.Media for culturing was obtained from Sigma-Aldrich. Nutrient Broth composition (Sigma) was as follows: 1 g/L D(+)-glucose, 15 g/L peptone, 6 g/L sodium chloride, 3 g/L yeast extract, 3 g/L, final pH 7.5 ± 0.2 (25°C). For sampling, all dyes from stock solution (2000ppm) were sterilized by filtering through 0.45 μm Nylon filter, before being added at 20 ppm in 15 mL nutrient broth and 0.15 mL freshly grown (overnight) bacterial culture, incubated for 24 h while shaking.Decolorization of dye was determined by measuring the absorbance of the cleared supernatant at the absorbance maxima of the respective dyes. All experiments were performed in triplicate. Test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate showed reasonable degradation when exposed to various bacterial strains, except H5 strain which failed to show any decolorization of the dyes under investigation.The percentage degradation of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was determined to be 41-60% after 24 hrs. Thus, based on percentage degradation, Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is considered to be readily biodegradable in nature.

 

In a supporting weight of evidence study from peer reviewed journal (D. Brown and B. Hamburger, 1987) for the read across chemical Disodium 4,4'-bis[(4-ethoxyphenyl)azo]stilbene-2,2'-disulphonate (CAS no. 2870-32-8),biodegradation experiment was carried out to determine the biodegradability rate of the read across substance Disodium 4,4'-bis[(4-ethoxyphenyl)azo]stilbene-2,2'-disulphonate. Activated sludge was used as an inoculum and the study was performed under anaerobic conditions at a temperature of 35°C for a period of 56 days. Samples of the aqueous phase were analyzed either qualitatively or quantitatively by an appropriate chromatographic method for the presence of certain of the expected aromatic amine metabolites. The percentage degradation of substance Disodium 4,4'-bis[(4-ethoxyphenyl)azo]stilbene-2,2'-disulphonate was determined to be 100% degradation by appropriate chromatography method in 7 days. The metabolites identified by the appropriate chromatographic method were 1-amino-4-ethoxybenzene and 4-amino-2-sulphobenzaldehyde, respectively. Thus, based on percentage degradation, chemical Disodium 4,4'-bis[(4-ethoxyphenyl)azo]stilbene-2,2'-disulphonate is considered to be readily biodegradable in nature.

 

On the basis of above results for target chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate(from peer reviewed journals) and for its read across substance (from peer reviewed journal), it can be concluded that the test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate can be expected to be readily biodegradable in nature.