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Biodegradation in water: screening tests

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Dye decolourisation was used as a measure of biodegradation in the test for biodegradability of Brilliant Black BN. D. abyssi MT1.1T bacteria isolated from the Challenger Deep sediment of the Mariana Trench for dye decolorization. The maximal rates for dye decolorization for the substance occurs at 50°C, exhibiting thermal tolerance for up to 1 h, with complete degradation of 250 mg/L of BBN in reaction conditions up to 5.0% NaCl. The substance can be considered to be readily biodegradable considering that nearly 100% decolorisation was observed in 3 hours. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

From publication data and data from read across were used to estimate the biodegradation in water for the test compound Brilliant Black BN. The summary is as below:

 

Dye decolourisation was used as a measure of biodegradation in the test for biodegradability of Brilliant Black BN. D. abyssi MT1.1T bacteria isolated from the Challenger Deep sediment of the Mariana Trench for dye decolorization. The maximal rates for dye decolorization for the substance occurs at 50°C, exhibiting thermal tolerance for up to 1 h, with complete degradation of 250 mg/L of BBN in reaction conditions up to 5.0% NaCl. The substance can be considered to be readily biodegradable considering that nearly 100% decolorisation was observed in 3 hours.

 

Short term aerobic biodegradation study (Lata Kumari and et. al, 2016) the study deals with the isolation and identification of bacterial strain BHUSSp X2 and its subsequent application for the bioremediation of carpet dyeing effluent of carpet industries (Bhadohi) Uttar Pradesh, India. Contaminated soil sample from the site was used for isolation of bacterial strain, and their identification was done using 16S rRNA gene sequence technique. Important operating parameters were optimized through aerobic degradation in batch mode. GC-MS studies were performed for the detection of biotransformation pathway of RED G dye. Commercial grade Acid Red G was procured from Local Carpet Industries, Bhadohi (Uttar Pradesh), India. Nutrient broth, nutrient agar, and mineral salt media (MSM media) were obtained from Hi-Media, 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 staticconditions. 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). 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). Initial test concentration was 200mg/l. All the flasks were incubated under static conditions at 35±2°C for 24 hours at pH = 7. Samples were periodically withdrawn after every 2 hours, centrifuged 10000 rpm to estimate extent of decolorization. The UV-Vis spectrophotometer (Systronics 2202) was used to determine the extent of decolorization. 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 potassium bromide (KBr) in the ratio of 5:95 and analyzed at mid IR region (400–4000 cm−1) by using FTIR Perkin Elmer, Spectrophotometer (Perkin Elmer, USA). The intermediates produced after degradation were analyzed by GC-MS. To understand the degradation pathway, GC-MS analysis of dye samples prior to and after degradation was carried out. 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). Aniline further breaks into benzene and the unknown intermediate into sodium7-amino-6- hydroxynapthalene-2 possibly through desulphonation and deamination which finally converts to pyrocatechol. Upto 97 % decolorization could be achieved at pH 8, temperature 35 °C under static condition, and initial concentration of 200 mg/l. It can be concluded that under the test conditions RED 2G was found to be readily biodegradable.

 

Short term aerobic biodegradation study (Lata Kumari and et. al, 2016) BHUSSp X2 microbial strain was isolated from contaminated sites of carpet cluster Bhadhoi. The bacteria were used for the decolorization of effluent from dying plants of the cluster in Uttar Pradesh, India. Upto 45 % decolorization could be achieved at pH 8, temperature 35 °C under shaking condition (100 rpm) and initial concentration of 200 mg/l.

 

Short term aerobic biodegradation study (Lata Kumari and et. al, 2016), Halophiles are exteremophiles able to survive in extreme conditions of salt concentration, and are gaining interest in areas such as industrial waste treatment .A moderately halotolerant microbe (0.5-2.5 M NaCl sustaining) was isolated from water sample from Marina beach and was identified as Bacillus megaterium. For the isolation of halophilic bacteria water sample was collected in sterile plastic bottles from the Marina beach, Chennai. 1ml of the sample was serially diluted and the dilutions 10-1–10-6were inoculated on the nutrient agar plates (10% NaCl) by pour plate method, Colonies got after incubation were purified on quadrant streaking, purity of colony was checked by Grams staining procedure, the purified colonies were than streaked on the modified DSC plates (10–12% NaCl) [for halophiles] in order to countercheck the halophilic nature. The media used for the screening was the mineral salts basal media modified in order to give carbon source, had deficiency of nitrogen source so that the dye can be utilized as a nitrogen source by microorganisms. All the six purified colonies named as S1, S2, S3, S4,S5,S6were streaked on the screening media and were kept for incubation for one week. A control plate was also maintained for comparison. After one week of incubation the plates were observed for decolorization of the dye at and around the culture position. After incubation the dye decolorization was ranked on the basis of visual identification as Intense (+++), Moderate (++), Slight (+), No decolorization (-). The isolate S4 showed maximum decolorization and therefore it was selected for further studying the dye decolorization. The standard inoculum of the isolated strain S4 giving intense decolourization of azo dye was prepared by inoculating it in 25 ml nutrient broth containing high salt concentration. The standard inoculums (25 ml) was then used to inoculate 225 ml mineral salts basal media in which 15 ml of the dye solution has been added after autoclaving. Accontrol flask was also maintained. After inoculation 5 ml of the sample was taken out in sterile condition and centrifuged at 6000 rpm for 10 min.c Supernatant was taken and its optical density was determined spectrophotometrically at 532 nm the absorbance maxima of azo dye Red 2G being studied for decolourization. Thereafter the percent of dye decolourization on zero day was calculated. The inoculated media was then incubated in shaking incubator at 150 rpm. Percentage dye degradation was calculated every alternate day for twenty days and the above described method was used every time. There was a continuous increase in the degradation percentage of dye degradation it shows that with the increase in the number of colonies the in the culture flask the dye was readily degraded by the isolate in order to get the nitrogen source from the dye. Percentage dye degradation by the isolated Bacillus megaterium was found to be 64.89% in 20 days. Under the test conditions, we can conclude that Red 2G is biodegradable

 

Based on the available studies from test compound and read across substance data, the test compound is considered to be readily biodegradable in water.