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Biodegradation in water

Biodegradation study was conducted for 6 days for evaluating the percentage biodegradability of test substance trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS no. 50662 -99 -2) using the fed-batch cultivation method (C. J. Ogugbue and N. A. Oranusi, 2006).Pseudomonas spp.was used as a test organism obtained from a dye house effluent and adapted to growth on azo dyes (Orange II and Direct Blue 71). Stock cultures were preserved on nutrient agar (Oxoid) slants at 4°C in a refrigerator. Cells from stock cultures were inoculated in 50 ml nutrient broth (Oxoid) contained in replicate 250 ml Erlenmeyer flasks. Cells are harvested by centrifugation at 6,000 rpm for 15 min in a refrigerated centrifuge at 10°C. Cell pellets are suspended in 20 ml phosphate – saline buffer (PSB) at pH 7.0 and recentrifuged and later suspended in duplicate 150 ml flasks each containing 20 ml PSB. Flasks were coded FA and FB. Loopful of cultures from each flask was inoculated into 10 ml nutrient broth and incubated at 28 ± 2°C for 24 hr. There was increased turbidity of the medium. This confirmed that the cells were viable and were used for the immobilization and suspended free cell studies. Initial test substance conc. used in the study was 0.02 mg/l. Fed batch cultivation was carried out in three cycles.

In 1stcycle, 10 ml of standard inoculum (flask B) was inoculated into each of triplicate set of 250 ml Erlenmeyer flasks which contained 100 ml of medium A. Then the flasks were incubated at 28 ± 2°C with shaking at 150 rpm. At zero time and at 48 hr of incubation, 5 ml of sample was withdrawn from each flask for determination of percentage of dye loss. Samples were centrifuged at 6,000 rpm for 30 min in a bench centrifuge. The optical density of the resulting supernatant was determined spectrophotometrically in a spectrophotometer 6110 at λmax 404 nm. Dye concentration was obtained from the calibration curve of Od against concentration of dye.

In 2ndcycle, after 48 hr of incubation, the culture broth from the 1stcycle was withdrawn. The cells were washed twice with physiological saline. After washing, 100 ml of fresh medium A was added into each flasks (containing free cells). Cultures and control were treated as for the first cycle. For the 3rdcycle, at the end of 48 hr of incubation, the culture broth from the 2ndcycle was treated as for the second cycle.The percentage degradation of test substance was determined to be77.50% by spectrophotometer parameter in 2 days.Thus, based on percentage degradation,trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonateis considered to be readily biodegradable in nature.

Additional information

Biodegradation in water

Various studies for the test compound trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS no. 50662-99-2) were reviewed for the biodegradation end point which are summarized as below:

 

In a key study from peer reviewed journal (C. J. Ogugbue and N. A. Oranusi, 2006), biodegradation experiment was conducted for 6 days for evaluating the percentage biodegradability of test substance trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS no. 50662-99-2) using the fed-batch cultivation method.Pseudomonas spp.was used as a test organism obtained from a dye house effluent and adapted to growth on azo dyes (Orange II and Direct Blue 71). Stock cultures were preserved on nutrient agar (Oxoid) slants at 4°C in a refrigerator. Cells from stock cultures were inoculated in 50 ml nutrient broth (Oxoid) contained in replicate 250 ml Erlenmeyer flasks. Cells are harvested by centrifugation at 6,000 rpm for 15 min in a refrigerated centrifuge at 10°C. Cell pellets are suspended in 20 ml phosphate – saline buffer (PSB) at pH 7.0 and recentrifuged and later suspended in duplicate 150 ml flasks each containing 20 ml PSB. Flasks were coded FA and FB. Loopful of cultures from each flask was inoculated into 10 ml nutrient broth and incubated at 28 ± 2°C for 24 hr. There was increased turbidity of the medium. This confirmed that the cells were viable and were used for the immobilization and suspended free cell studies. Initial test substance conc. used in the study was 0.02 mg/l. Fed batch cultivation was carried out in three cycles.

In 1stcycle, 10 ml of standard inoculum (flask B) was inoculated into each of triplicate set of 250 ml Erlenmeyer flasks which contained 100 ml of medium A. Then the flasks were incubated at 28 ± 2°C with shaking at 150 rpm. At zero time and at 48 hr of incubation, 5 ml of sample was withdrawn from each flask for determination of percentage of dye loss. Samples were centrifuged at 6,000 rpm for 30 min in a bench centrifuge. The optical density of the resulting supernatant was determined spectrophotometrically in a spectrophotometer 6110 at λmax 404 nm. Dye concentration was obtained from the calibration curve of OD against concentration of dye.

In 2ndcycle, after 48 hr of incubation, the culture broth from the 1stcycle was withdrawn. The cells were washed twice with physiological saline. After washing, 100 ml of fresh medium A was added into each flasks (containing free cells). Cultures and control were treated as for the first cycle. For the 3rdcycle, at the end of 48 hr of incubation, the culture broth from the 2ndcycle was treated as for the second cycle.The percentage degradation of test substance was determined to be77.50% by spectrophotometer parameter in 2 days.Thus, based on percentage degradation,trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonateis considered to be readily biodegradable in nature.

 

Another supporting study of biodegradation for the test substance trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS no. 50662-99-2) was conducted for 6 days using the fed-batch cultivation method (C. J. Ogugbue and N. A. Oranusi, 2006).Pseudomonas spp.was used as a test organism obtained from a dye house effluent and adapted to growth on azo dyes (Orange II and Direct Blue 71). Stock cultures were preserved on nutrient agar (Oxoid) slants at 4°C in a refrigerator. Cells from stock cultures were inoculated in 50 ml nutrient broth (Oxoid) contained in replicate 250 ml Erlenmeyer flasks. Cells are harvested by centrifugation at 6,000 rpm for 15 min in a refrigerated centrifuge at 10°C. Cell pellets are suspended in 20 ml phosphate – saline buffer (PSB) at pH 7.0 and recentrifuged and later suspended in duplicate 150 ml flasks each containing 20 ml PSB. Flasks were coded FA and FB. Loopful of cultures from each flask was inoculated into 10 ml nutrient broth and incubated at 28 ± 2°C for 24 hr. There was increased turbidity of the medium. This confirmed that the cells were viable and were used for the immobilization and suspended free cell studies. For immobilization of test inoculumPseudomonas spp., 4 grams of agar no. 1 (Oxoid) was added in 200 ml phosphate buffer at pH 7.0 contained in 2 l Erlenmeyer flask. Sterilization was by autoclaving. After sterilization, the agar solution was kept molten (45-50°C) in a water bath. 10 ml of cell suspension from FA flask was mixed with 10 ml of molten agar by gentle stirring with glass rod for 15 min to obtain 1% (w/v) agar solution. The resulting agar/cell suspension was extruded with a hypodermic syringe (diameter 1.0 mm) into sterile 600 ml vegetable oil contained in 2 l flasks maintained at 45-50°C in a water bath and mixed with gentle stirring for 10 min. the macroparticle agar gel beads (approx.. 2mm diameter) formed were hardened in a refrigerator at 10°C for 24 hr. Excell oil was decanted. Residual oil on the beads was removed by repeated gentle washing with a mixture of phosphate buffer solution/Tween 80 until no oil sheen was visible on the surface of the supernatant. Tween 80 was washed off with phosphate buffer. The immobilized cells were suspended in the growth medium A until used. Prior to use, a loopful of culture from medium A was streaked onto nutrient agar plates. Plates were incubated in deionized water and sterilized by membrane filtration (membrane filter 0.2µm pore size).

Initial test substance conc. used in the study was 0.02 mg/l. Fed batch cultivation was carried out in three cycles.

In 1stcycle, 10 ml of standard inoculum (flask B) was inoculated into each of triplicate set of 250 ml Erlenmeyer flasks which contained 100 ml of medium A. Then the flasks were incubated at 28 ± 2°C with shaking at 150 rpm. At zero time and at 48 hr of incubation, 5 ml of sample was withdrawn from each flask for determination of percentage of dye loss. Samples were centrifuged at 6,000 rpm for 30 min in a bench centrifuge. The optical density of the resulting supernatant was determined spectrophotometrically in a spectrophotometer 6110 at λmax 404 nm. Dye concentration was obtained from the calibration curve of OD against concentration of dye.

In 2ndcycle, after 48 hr of incubation, the culture broth from the 1stcycle was withdrawn. The cells were washed twice with physiological saline. After washing, 100 ml of fresh medium A was added into each flasks (containing free cells). Cultures and control were treated as for the first cycle. For the 3rdcycle, at the end of 48 hr of incubation, the culture broth from the 2ndcycle was treated as for the second cycle. The percentage degradation of test substance was determined to be 78.62% by spectrophotometer parameter in 6 days.Thus, based on percentage degradation, trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate is considered to be readily biodegradable in nature.

 

On the basis of above results for target chemical trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (from peer reviewed journal), it can be concluded that the test substance trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate can be expected to be readily biodegradable in nature.