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Classification & Labelling & PBT assessment

PBT assessment

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PBT assessment: overall result

PBT status:
the substance is not PBT / vPvB
Justification:

Classification of Trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate for effects in the environment:

 

The chemical Trisodium 4-[[4-chloro-6-[(4-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-[[1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]benzenesulphonate (Reactive yellow 2; CAS no. 50662-99-2) is used as an intermediate in dyestuff industry and for chemical synthesis. The aim was to assess whether the PBT criterion within Annex XIII was fulfilled for Reactive yellow 2. The PBT criterion was herein assessed based on experimental data in conjunction with standardized environmental fate models. Here follows a description of the PBT assessment.

 

 

Persistence assessment

The tested substance does not fulfil the P criterion within Annex XIII based on the assessment that here follows:

 

Biotic degradation

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). Viable cells were used for the immobilization and suspended free cell studies.

Suspended free cell study:

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 At zero time and at 48 hr of incubation, 5 ml of sample was withdrawn from each flask for determination of percentage of dye loss.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.

 

Immobilised cell study:

A 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) with immobiilised cells. 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). Viable cells were used for the immobilization and suspended free cell studies.

For immobilization of test inoculum Pseudomonas 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 can be expected to be readily biodegradable in nature..

 

Hence it has been concluded that Reactive yellow 2 is not persistent in nature.  

 

 

Bioaccumulation assessment

The tested substance fulfils the B criterion within Annex XIII based on the assessment that here follows:

 

The estimated BCF value from was determined to be in the range 3.2L/kg wet wt. If this chemical is released into the aquatic environment, there should be a low risk for the chemical to bioaccumulate in fish and food chains.

 

Toxicity assessment

The tested substance does not fulfil the T criterion within Annex XIII based on the assessment that here follows:

 

Mammals

The tested chemical is regarded to be not classified for carcinogenicity, mutagenicity and reprotoxicity, Further, there is no evidence of chronic toxicity, as identified by the classifications STOT (repeated exposure), category 1(oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume) or category 2 (oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume).

 

Aquatic organisms

All of the available short-term eco-toxicity estimation for fish, invertebrates and algae for the substance indicates the LC50/EC50 value to be >100 mg/L. These value suggest that the substance is likely to be non-hazardous to aquatic organisms at environmentally relevant concentrations and can be considered to be not classified as per the CLP regulation.

 

There are no available long-term toxicity evaluations for Reactive yellow 2. By speculation, long-term NOEC for aquatic organisms were not expected for Reactive yellow 2 at concentration below 0.01 mg/L based on the data mentioned above.

 

The chemical was therefore not considered as hazardous to aquatic environments as per the criteria set out in Annex XIII.

 

Conclusion

Based on critical, independent and collective evaluation of information summarized herein, the tested compound does not fulfil the P, B and T criterion and has therefore not been classified as a PBT compound within Annex XIII.