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

Endpoint summary

Description of key information

Hydrolysis

In accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is readily biodegradable.

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.

Bioaccumulation: aquatic / sediment

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical), the bioaccumulation i.e BCF for test substance Disodium [[4 -[bis[4 -[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5 -dien-1 -ylidene]amino]benzenesulphonate (CAS no. 28983 -56 -4) was estimated to be 4.84 dimensionless . The predicted BCF result based on the 5 OECD principles. Thus based on the result it is concluded that the test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is non-bioaccumulative in nature.

Adsorption / desorption

Adsorption study was conducted for evaluating the adsorption capacity of test chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate (Acid blue 93) (CAS no.28983-56-4) from aqueous solution by using Pseudomonas putida MTCC 4910 (A. Arunarani, et. al; 2013)

. The study was performed using thebatch equilibrium method.P. putidaMTCC 4910 strain used in present study was obtained from Institute of Microbial Technology, Chandigarh, India. The bacterium was cultured in a nutrient broth medium and incubated at 37◦C in a rotary shaker at 150 rpm overnight. The culture was maintained on agar slants at 4◦C and subcultured on nutrient agar medium at monthly intervals.Test chemical concentrations used for the study were in the range from 50-300 mg/l, respectively.The adsorption isotherm experiment was performed by interacting varying concentrations of dye (50–300 mg/L) solutions withP. putida(1×108 CFU/mL) in a rotary shaker at 150 rpm for 4 h. The medium used for the interaction contains 1 mM NaCl prepared in distilled water. The interaction was followed by centrifugation at 10,000×gfor 10 min. The supernatants were carefully collected and the amounts of dye left in the supernatants were calculated by using UV–visible spectrophotometer by measuring the absorbance at 604 nm. The dye without bacteria was used as control. For determined the effect of pH on adsorption, the experiment procedure used was same as described but at different pH (4–9).The bacterial species exhibited almost 90–100% removal of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate at an initial concentration of 250 mg/L in all the pH (4–9) tested.The amount of the dye adsorbed was determined to be constant at different pH 4-9 and ranges from 2.3 to 2.4, respectively and the Freundlich adsorption isotherm of the chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was determined to be 0.9912 (R²). The high ‘R2’ values (linear regression coefficients) indicate that the Freundlich model predicts well the adsorption behaviour of dye Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate on P.putida than Langmuir model and thus chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate can be considered to have strong sorption on sewage sludge.

Additional information

Hydrolysis

In accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is readily biodegradable.

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.

Bioaccumulation: aquatic / sediment

Predicted data 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 various supporting weight of evidence studies for its structurally similar read across substance were reviewed for the bioaccumulation end point which are summarized as below:

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical), the bioaccumulation i.e BCF for test substance Disodium [[4 -[bis[4 -[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5 -dien-1 -ylidene]amino]benzenesulphonate (CAS no. 28983 -56 -4) was estimated to be 4.84 dimensionless . The predicted BCF result based on the 5 OECD principles. Thus based on the result it is concluded that the test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is non-bioaccumulative in nature.

 

In a supporting weight of evidence study from authoritative database (J-CHECK, 2017) for the read across chemical 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt (CAS no. 2611-82-7),bioaccumulation experiment was conducted on test organism Cyprinus carpio for 28 days for evaluating the bioconcentration factor (BCF value) of read across chemical 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt. The study was performed according to other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances". Cyprinus carpio was used as a test organism for the study. Details on analytical methods involve the recovery ratio: Test water: 100% (The test solution were input analytical equipment directly), Fish: 82.4%, - Limit of quantitation: Test water: 1st concentration area : 21µg/l, 2nd concentration area : 2.1µg/l, Fish: 260 ng/g. Test chemical nominal conc. used for the study were 0.474mg/land 0.0474 mg/l, respectively. Range finding study involve the LC50 (96 hr) > 200 mg/l onRice fish (Oryzias latipes).The bioconcentration factor (BCF value) of substance 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-((4-sulfo-1-naphthalenyl)azo)-, trisodium salt on Cyprinus carpio was determined to be ≤ 0.55 L/Kg at a conc. of 0.474 mg/l and ≤ 5.6 L/Kg at a conc. of 0.0474 mg/l, respectively.

 

Another bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of read across chemical 1,3 -Naphthalenedisulfonic acid, 7 -hydroxy-8 -((4 -sulfo-1 -naphthalenyl)azo)-, trisodium salt (CAS no. 2611 -82 -7) (HSDB, 2017). The bioaccumulation factor (BCF) value was calculated using a measured water solubility of 80,000 mg/l and a recommended regression-derived equation. The estimated BCF (bioaccumulation factor) valueof 1,3 -Naphthalenedisulfonic acid, 7 -hydroxy-8 -((4 -sulfo-1 -naphthalenyl)azo)-, trisodium salt was determined to be 1.0 dimensionless.

 

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 CompTox Chemistry Dashboard,  2017) and for its read across substance (from authoritative database J-CHECK and HSDB, 2017), it can be concluded that the BCF value of test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was estimated to be 4.84 dimensionless,which does not exceed the bioconcentration threshold of 2000, indicating that the chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate is not expected to bioaccumulate in the food chain.

Adsorption / desorption

Various experimental 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) were reviewed for the adsorption end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (A. Arunarani, et. al; 2013), adsorption experiment was conducted for evaluating the adsorption capacity of test chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate (Acid blue 93) (CAS no.28983-56-4) from aqueous solution by using Pseudomonas putida MTCC 4910. The study was performed using the batch equilibrium method. P. putida MTCC 4910 strain used in present study was obtained from Institute of Microbial Technology, Chandigarh, India. The bacterium was cultured in a nutrient broth medium and incubated at 37◦C in a rotary shaker at 150 rpm overnight. The culture was maintained on agar slants at 4◦C and subcultured on nutrient agar medium at monthly intervals. Test chemical concentrations used for the study were in the range from 50-300 mg/l, respectively. The adsorption isotherm experiment was performed by interacting varying concentrations of dye (50–300 mg/L) solutions with P. putida(1×108 CFU/mL) in a rotary shaker at 150 rpm for 4 h. The medium used for the interaction contains 1 mM NaCl prepared in distilled water. The interaction was followed by centrifugation at 10,000×gfor 10 min. The supernatants were carefully collected and the amounts of dye left in the supernatants were calculated by using UV–visible spectrophotometer by measuring the absorbance at 604 nm. The dye without bacteria was used as control. For determined the effect of pH on adsorption, the experiment procedure used was same as described but at different pH (4–9).The bacterial species exhibited almost 90–100% removal of Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate at an initial concentration of 250 mg/L in all the pH (4–9) tested. The amount of the dye adsorbed was determined to be constant at different pH 4-9 and ranges from 2.3 to 2.4, respectively and the Freundlich adsorption isotherm of the chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate was determined to be 0.9912 (R2). The high ‘R2’ values (linear regression coefficients) indicate that the Freundlich model predicts well the adsorption behaviour of dye Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate on P.putida than Langmuir model and thus chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate can be considered to have strong sorption on sewage sludge.

 

Another adsorption study was conducted for evaluating the adsorption capacity of test chemical Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate (CAS no. 28983-56-4) on As-synthesized hollow silica microspheres (Shiquan Liu, et. al; 2017). Hollow silica microspheres with the same size and hollow structure but with micro- or mesoporous walls were used to adsorb methyl blue (MB).The pH values of the Methyl Blue solutions used in the tests was 6.57, respectively. Hollow SiO2 microspheres were synthesized in a medium with molar ratios of 1TEOS:0.17HCl:37.224H2O:0.451C8H19N. The synthesis procedures were as follows: TEOS and octylamine were mixed under stirring for 3 min. Then, mixed solution of water and hydrochloric acid was added into the above mixture. After a reaction of 60 min, the product was filtered and collected and fully dried at room temperature. 1 g of the dried sample was subjected to hydrothermal treatment in an autoclave at 100°C for 72 h. The dried sample was calcined at 550°C for 6 h. The ramping rate was 1°C/min. The as-synthesized and thermally treated samples were designated as O60 and O60T, respectively. The dispersion of the sample is good, but the uniformity is poor. The sizes of the microspheres are mostly about 75 and 145lm, and the wall thickness of the hollow spheres is estimated to be about 18–42lm, based on the measurements on different broken spheres in different images.The adsorption kinetics of methyl blue on the samples shows the opposite trend with adsorption capacities of 3.96 and 7.94 mg/g, respectively. It can be seen that the adsorption by the as-synthesized sample increases within 5–30 min; however, it only slightly increases with the adsorption time and the total adsorption at 180 min is only about 20%. In contrast, the adsorption of MB by the hydrothermally treated sample can be divided into two stages. During the initial 60 min, the adsorption rate of the sample is fast, which can be ascribed to the adsorption on the surfaces of the spheres and the pores. With further prolonging the adsorption time, the adsorption slowly increases, which suggests a slow diffusion of the dye molecules into the hollows of the microspheres. The final adsorption efficiency is 45%, more than double the adsorption by the as-synthesized sample. The total adsorption of dye methyl blue on the hydrothermally treated sample is 43.4%.Thus, based on this, it can be concluded that the chemical has a moderate sorption on silica microsphere.

 

On the basis of above overall results for target chemical Disodium [[4 -[bis[4 -[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5 -dien-1 -ylidene]amino]benzenesulphonate (methyl blue) (from peer reviewed journals), it can be concluded that the test substance Disodium [[4-[bis[4-[(sulphonatophenyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]amino]benzenesulphonate has a moderate to strong sorption.