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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data is from peer reviewed journal
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Biodegradation study was conducted for 5 days for evaluating the percentage biodegradability of test substance 2-Chloro-4-nitrophenol.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (IUPAC name): 2-Chloro-4-nitrophenol
- Molecular formula: C6H4ClNO3
- Molecular weight: 173.555 g/mol
- Smiles notation: c1(cc(c(O)cc1)Cl)[N+](=O)[O-]
- InChl: 1S/C6H4ClNO3/c7-5-3-4(8(10)11)1-2-6(5)9/h1-3,9H
- Substance type: Organic
- Physical state: Solid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sewage plant
- Laboratory culture: 1000ml volumetric cylinder
- Method of cultivation: Activated sludge taken from a sewage plant is cultivated in a 1000ml volumetric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentation ca. 600ml of the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume of ca. 800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated.
- Initial cell/biomass concentration: 100mg/l
Duration of test (contact time):
5 d
Initial conc.:
200 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
other: COD
Details on study design:
TEST CONDITIONS
- Composition of medium: In ca. 800 ml of distilled water solutions of calcium chloride (27.5 g CaCl2 in 1l Distilled water) magnesium sulphate (22.5 g MgSO4.7H20 in I 1. distilled water), and ferric chloride (0.25 g FeCI3.6 H20 in 11. distilled water) are added in 1 ml portions each. Then 5 ml ammonium sulphate solution [10g (NH4)2SO4 in 11. distilled water], 20ml of a phosphate buffer of pH 7.2 (8.5 g KH2PO4, 21.8g K2HPO4, and 44.7g Na2HPO4. 12 H2O in 1l distilled water), and 100ml of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000ml with distilled water.
- Test temperature: 20±3°C
- pH: 7.2
- pH adjusted: No
- Aeration of dilution water: The mixture is aerated with pressure air
- Continuous darkness: Yes , dark room
- Other: Beaker containing inoculums is placed on electromagnetic stirrer.

CONTROL AND BLANK SYSTEM
Inoculum blank: Yes
Key result
Parameter:
other: COD
Value:
71.5
Sampling time:
5 d
Remarks on result:
other: Other details not known
Details on results:
The percentage degradation of test substance 2-Chloro-4-nitrophenol was determined to be 71.5% after 5 days by using COD parameter.

Table: Biodegradability of the test compounds

 

Compound

Percent removed(based upon COD)

Rate of biodegradation (mg COD g-1h-1)

2-Chloro-4-nitrophenol

71.5

5.3

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
The percentage degradation of 2-Chloro-4-nitrophenol was determined to be 71.5% degradation by using COD parameter in 5 days. Thus, based on percentage degradation, test substance 2-Chloro-4-nitrophenol was considered to be readily biodegradable in water.
Executive summary:

A batch test in an open system was conducted for 5 days for evaluating the biodegradability of 2-Chloro-4-nitrophenol(CAS no. 619-08-9). Adapted activated sludge was used as a test inoculum obtained from a sewage plant is cultivated in a 1000ml volumetric cylinder.The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentationca.600mlof the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume ofca.800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. Test chemical conc. used for the study was 200 mg/l based on COD.To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200 mg/l. Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed tothe medium that the concentration of the dry matter is 100 mg/l.Simultaneously, a blank test is prepared.The beaker is placed in a dark room with a roughly 3 constant temperature of 20±3°C on an electromagnetic stirrer and a pH of 7.2 for 120 hrs. The initial value of COD or organic carbon of the liquid phase are determined.Samples filtered or centrifuged before analysis, are taken at suitable intervals. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The results are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a gramme of dry matter of the activated sludge per hour.The percentage degradation of 2-Chloro-4-nitrophenol was determined to be 71.5% degradation by using COD parameter in 5 days. Thus, based on percentage degradation, test substance 2-Chloro-4-nitrophenol was considered to be readily biodegradable in nature.

Description of key information

A batch test in an open system was conducted for 5 days for evaluating the biodegradability of 2-Chloro-4-nitrophenol (CAS no. 619-08-9) (P. Pitter, 1976). Adapted activated sludge was used as a test inoculum obtained from a sewage plant is cultivated in a 1000ml volumetric cylinder.The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentationca.600mlof the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume ofca.800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. Test chemical conc. used for the study was 200 mg/l based on COD.To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200 mg/l. Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed tothe medium that the concentration of the dry matter is 100 mg/l.Simultaneously, a blank test is prepared.The beaker is placed in a dark room with a roughly 3 constant temperature of 20±3°C on an electromagnetic stirrer and a pH of 7.2 for 120 hrs. The initial value of COD or organic carbon of the liquid phase are determined.Samples filtered or centrifuged before analysis, are taken at suitable intervals. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The results are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a gramme of dry matter of the activated sludge per hour.The percentage degradation of 2-Chloro-4-nitrophenol was determined to be 71.5% degradation by using COD parameter in 5 days. Thus, based on percentage degradation, test substance 2-Chloro-4-nitrophenol was considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Various experimental key and supporting studies for the target compound2-Chloro-4-nitrophenol(CAS No. 619-08-9) were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal, a batch test in an open system was conducted for 5 days for evaluating the biodegradability of target chemical 2-Chloro-4-nitrophenol (CAS no. 619-08-9) (P. Pitter, 1976). Adapted activated sludge was used as a test inoculum obtained from a sewage plant is cultivated in a 1000ml volumetric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentationca.600mlof the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume ofca.800 ml and 600 mg/l of starch or glucose, 600 mg/l of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. Test chemical conc. used for the study was 200 mg/l based on COD.To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200 mg/l. Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed to the medium that the concentration of the dry matter is 100 mg/l. Simultaneously, a blank test is prepared. The beaker is placed in a dark room with a roughly 3 constant temperature of 20±3°C on an electromagnetic stirrer and a pH of 7.2 for 120 hrs. The initial value of COD or organic carbon of the liquid phase are determined. Samples filtered or centrifuged before analysis, are taken at suitable intervals. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The results are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a gramme of dry matter of the activated sludge per hour. The percentage degradation of 2-Chloro-4-nitrophenol was determined to be 71.5% degradation by using COD parameter in 5 days. Thus, based on percentage degradation, test substance 2-Chloro-4-nitrophenol was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 1 hr (60 mins) for evaluating the percentage biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619-08-9) by Cupriavidus strain a3 (Jyoti Tiwar et. al, 2017).Cupriavidus strain a3 was used as a test inoculum isolated from soil sample which was collected from pesticide formulation industry in Nagpur, India and identified by 16S rRNA gene sequencing.. Test inoculum was prepared by growing the culture in minimal medium containing 0.3 mM of 2-chloro-4-nitrophenol as the sole source of carbon and nitrogen. After overnight growth, the culture was centrifuged, washed and resuspended in 25 mM phosphate buffer (pH 7.2). To study the effect of substrate concentration, 2-chloro-4-nitrophenol was added to the flasks in the concentration range of 0.1–0.5 mM (17.3555 to 88.2775 mg/l). In experiments with 2-chloro-4-nitrophenol as sole source of carbon and nitrogen, NH4Cl was omitted from the medium. Similarly, both NH4Cl and NaCl were removed during preparation of chloride free medium containing 2-chloro-4-nitrophenol as source of carbon and nitrogen. The inoculum was added at OD600 of 0.005 in the test medium, and flasks were shaken at 28⁰C and 150 rpm. The flasks were incubated on shaker, under static condition or overlaid with mineral oil to study the effect of oxygenation. All experiments were carried out in triplicate. Samples were collected at regular time intervals and analyzed for culture growth, 2-chloro-4-nitrophenol concentration and release of nitrite.Culture growth was monitored in terms of absorbance at 600nm (OD600). Degradation of 2-chloro-4-nitrophenol was followed spectrophotometrically at 400nm (OD400). Release of chloride was analyzed in chloride-free medium. Selected samples were also analyzed by HPLC and GC–MS to confirm the biodegradation of 2-chloro-4-nitrophenol and identify the metabolic intermediates. The degradation pattern followed Haldane substrate inhibition model with maximum specific degradation rate (qmax) of 0.13/h, half saturation constant (Ks) of 0.05 mM, and 2-chloro-4-nitrophenol inhibition constant (Ki) of 0.64 mM. Though, degradation of chemical was not observed under static or anoxic condition, biodegradation were observed only when flasks were shaken at 28⁰C under aerobic conditions. The degradation metabolites of test chemical2-chloro-4-nitrophenolwereidentified by GC-MS analysis were2-chlorobenzoquinone (CBQ) and 2-chlorohydroquinone (CHQ), respectively. However, HPLC analysis showed further biodegradation of 2-chlorohydroquinone (CHQ). Thus, based on this, 2 -chloro-4 -nitrophenol is considered to be biodegradable in nature.

 

In a supporting study from peer reviewed journal (Pankaj Kumar Arora and Rakesh Kumar Jain, 2011), biodegradation experiment was conducted for 50 hrs for evaluating the percentage biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619-08-9) by Arthrobacter sp. SJCon. Arthrobactersp. SJCon. isolated from a pesticide contaminated site of Punjab by enrichment method was used as a test inoculum. For enrichment, 1 g of soil sample was added to 500 ml flask containing 200 ml minimal media and 0.2 mM 2-chloro-4-nitrophenol as a sole source of carbon and energy. On decolorization of flask, culture were serially diluted and plated on minimal agar plates containing 0.2 mM 2-chloro-4-nitrophenol. Colonies were selected on the basis of decolorization. One bacterial strain designated as SJCon was selected from 2-chloro-4-nitrophenol agar plates and used for this study. Strain SJCon was ability to degrade 2-chloro-4-nitrophenol up to 0.3 mM concentration. Strain SJCon was identified by the 16S rRNA gene sequencing using universal primers 27f and 1492r. Strain SJCon was grown in 250 ml minimal media containing 0.3 mM 2-chloro-4-nitrophenol as sole source of carbon and energy. Samples were collected at regular interval and growth of microorganism was measured by taking optical density (O. D.) at 600 nm. For nitrite and chloride estimation, samples were centrifuged and supernatant was used for nitrite and chloride detection.Metabolites of test chemical2-chloro-4-nitrophenoldegradation were analyzed byTLC, HPLC, and GC–MS. The GC–MS analysis was carried out using a GC–MS-QP5000 Instrument. When strain SJCon was grown in 1L Erlenmeyer flask containing 250 ml minimal media and 0.3 mM 2-chloro-4-nitrophenol as sole source of carbon and energy, the yellow color of 2-chloro-4-nitrophenol changed to colorless indicating its utilization by the microorganisms. Microbial growth was measured by the increase in optical density (O.D.) at 600 nm and depletion of 2-chloro-4-nitrophenol was measured by the decrease in optical density at 420 nm. The microbial degradation of 2-chloro-4-nitrophenol was initiated with removal of nitro group and followed by chloride release.The percentage degradation of test substance2-chloro-4-nitrophenolwas determined to be 100% by using Test mat. analysis (absorbance O. D.), TLC, HPLC and GC-MS parameter after 48 hrs.. There was only one metabolite that was detected during the study of the degradation of 2-chloro-4-nitrophenol by TLC. The Rf value of this metabolite was 0.64 which was exactly matched with Rf value of the authentic chloro-hydroquinone (CHQ). HPLC results confirmed the complete depletion of 2-chloro-4-nitrophenol. In the sample of 12 h, only parent compound 2-chloro-4-nitrophenol was detected whereas metabolite along with parent compound was detected in the sample of 24 and 36 h of bacterial growth. In the sample of 48 h, neither metabolite nor 2-chloro-4-nitrophenol was detected because of complete degradation of 2-chloro-4-nitrophenol. The retention times of metabolite and 2-chloro-4-nitrophenol were 6.7 and 16.3 min, respectively. On the basis of retention time, chloro-hydroquinone was identified as a metabolite. GC–MS analysis was also carried out to confirm the presence of chloro-hydroquinone in the degradation pathway of 2-chloro-4-nitrophenol. Mass fragment of the metabolite was observed atm/z144 and exactly matched to that of the authentic chloro-hydroquinone (CHQ).Thus, based on percentage degradation, 2 -chloro-4 -nitrophenol is considered to be readily biodegradable in nature.

 

For the target compound 2-Chloro-4-nitrophenol(CAS No. 619-08-9), biodegradation study was conducted for 20 hrs for evaluating the percentage biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619 -08 -9) by Rhodococcus imtechensis Strain RKJ 300 under aerobic conditions at a temperature of 30⁰C (Anuradha Gosh et. al; 2010). Rhodococcusimtechensis strain RKJ300 was used as a test inoculum isolated on 4-NP using the enrichment culture technique from a pesticide-contaminated agricultural fieldof Punjab, India. Degradation experiments were also carried out with preinduced cells of strain RKJ300.Test chemical2-chloro-4-nitrophenol degradation was checkedby supplementing the minimal medium (MM; 13) with or without an additional carbon source. The organism was cultivated at 30°C under shaking conditions, and OD600 was measured using a UV-Vis spectrophotometer (Perkin-Elmer Lambda EZ 201). Release of chloride ions was detected and quantified following a colorimetric method. Briefly, to the diluted culture supernatant 200μL each of 250 mM ferric ammonium sulfate (prepared in 9 N nitric acid) and saturated solution of mercuric thiocyanate (prepared in ethanol) were added, mixed, and incubated at room temperature for 10-15 min. Quantification of the released chloride ions was performed by spectrophotometric measurement of absorbance at 460nm based on the standard curve for chloride ions generated with known concentrations of NaCl. Release of nitrite ions was detected spectrophotometrically in the culture supernatants. To the culture supernatant an equal volume of reagent A [0.1% (w/v)N-(1-naphthyl)ethylenediamine dihydrochloride in 30% (v/v) acetic acid] was added. After 2 min, an equal volume of reagent B [0.1% (w/v) sulfanilic acid in 30% (v/v) acetic acid] was added and incubated at room temperature for 25-30 min. The presence of nitrite ions in the sample was shown by the appearance of a purple color and quantified by calculating the absorbance at 540 nm. Concentration of nitrite ion released into the growth medium was determined with a standard calibration curve of NaNO2. The formation of metabolites in 2-chloro-4-nitrophenol was measured by analyzing samples of the supernatant collected at regular time intervals (10 min) using a high-performance liquid chromatography (HPLC) and GC-MS, respectively.The percentage degradation of test substance2-chloro-4-nitrophenolwas determined to be 100% by usingspectrophotometerparameter in within 10-12 hrs by induced cells as compared to 18-20 hrs by uninduced cells.The degradation metabolites identified by GC-MS analysis were 2-chlorohydroquinone (CHQ) and hydroquinone (HQ), respectively. Thus, based on percentage degradation, 2-chloro-4-nitrophenol is considered to be readily biodegradable in nature.

 

In a supporting study, biodegradation experiment was conducted (Henry H. Tabak et. al; 1964) for 3 to 6 days for evaluating the percentage biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619-08-9) by using the phenol-adapted bacteria as the test inoculums under aerobic conditions at a temperature of 30°C and pH of 7.2, respectively. Phenol-adapted bacteria i.e. Pseudomonas, Achromobacter, Flavobacterium and Xanthomonas were used as a test inoculum obtained from garden soil, compost, river mud, and sediment from a waste lagoon of a petroleum refinery catalytic cracking plant. Phenol-adapted bacteria were inoculated in liquid mineral salts medium containing 300 ppm of phenol as the only source of carbon, and incubated on a shaker at room temperature for 16 hr. The cells were then removed bv centrifugation, washed several times with buffered dilution water, stored overnight at 5°C in the same buffer, aerated 3 to 4 hr, removed by centrifugation, and resuspended in 0.067 M phosphate buffer at pH 7.2. Each Warburg flask contained an appropriate amount of 0.067 M buffer at pH 7.2 and 0.5 ml of cell suspension in the main compartment, 0.2 ml of 10% KOH solution in the center well, and an amount of stock solution of substrate in the side arm necessary to produce the desired test concentration. The total volume of reagents and cell suspension added to a flask was 3.2 ml. A flask containing substrate without cell suspension was included for each compound to control chemical oxidation, along with an endogenous control and a test with phenol plus cells to confirm that the organisms used in the test had a normally high capacity to utilize phenol. All flasks were incubated in a Warburg water bath at 30°C and shaken at a speed of 68 strokes per min. The gas phase was air. Results of 10-min observations of 02 uptake were averaged for each successive 30-min interval. Whenever the observed oxygen uptake appeared to be significant, the centrifuged supernatant fluid from the respirometric test was analyzed for residual substrate. It should be noted that during incubation of nitrophenol-containing culture, the yellow color, characteristic of nitrophenols and chloronitrophenols, progressively faded and finally disappeared from the medium. The color loss coincided with the appearance of abundant growth and disappearance of the substrate from the medium. The percentage degradation of test substance2-chloro-4-nitrophenolwas determined to be 95% after 6 days and O2 uptake of the chemical by the test organism was determined to be approx. 250 µl. Thus, based on percentage degradation, 2-chloro-4-nitrophenol is considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 230 mins for evaluating the percentage biodegradability of test substance 2-Chloro-4-nitrophenol (CAS no. 619-08-9) (from peer reviewed journal Cecil W. Chambers et. al; 1963) and secondary source Philip H. Howard et. al; 1976). Micro-organisms (mixture of several species with pseudomonads predominating) was used as a test inoculum for the study. The biodegradation study was performed at a temperature of 30°C. Test inoculum bacteria was isolated from different sources which includes soil, compost, or mud from a catalytic cracking plant waste lagoon. The test organism were grown on mineral salts medium with vitamin B12 added. The only source of carbon was 300 mg/l phenol. The cells were washed and centrifuged three times with the phosphate buffer dilution water and stored overnight at 5°C in the phosphate buffer dilution water. The next morning after storage they were aerated for 3 hr, removed by centrifugation, and resuspended in 0.067 M phosphate buffer at pH 7.2. The stock solution of substrates were prepared from chemical designated as highest purity. Test chemical concentration used for the study was 60 mg/l. Cell suspension was omitted from one flask to show whether any of the substrate was lost because of chemical oxidation. An endogenous control and a parallel test with phenol were always included in each experiment to show that the bacterial suspension has a normally high capacity to utilize phenol. A spectrophotometer was used to compare the concentration of substrate remaining in the centrifuged supernatant from the respirometric tests directly with the known concentration of the test compound. Test chemical was determined by the 4-amino-antipyridine method at 510 mµ. The oxygen uptake (O2 uptake) of the chemical by test microorganisms was determined to be 249 µl. Thus, based on this, chemical 2-Chloro-4-nitrophenol was considered to be biodegradable in nature.

 

Additional supporting biodegradation study from peer reviewed journal (Pankaj Kumar Arora et. al; 2012) was conducted for 48 hrs for evaluating the percentage biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619-08-9) by Burkholderia sp. RKJ 800.Test inoculum Burkholderia sp. RKJ 800 was isolated from the soil collected from a pesticide contaminated site, India by enrichment method. No specific permits were required for collection the sample from a pesticide contaminated site.For enrichment, 1 g of the soil sample was added to 250 ml Erlenmeyer flask containing 100 ml minimal media and 0.2 mM 2C4NP (a yellow coloured compound) as the sole source of carbon and energy. On the decolourization, culture was serially diluted, plated on 2C4NP agar plates and plates were incubated at room temperature for 2–5 days. One bacterial strain designated as RKJ 800 was selected from 2C4NP agar plates on the basis of decolorization and used for this study. Strain RKJ 800 was identified by the 16S rRNA gene sequencing using universal primers, 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’). For the biodegradation study, strain RKJ 800 was grown in 1L Erlenmeyer flask containing 300 ml minimal media and 0.3 mM 2C4NP as the sole source of carbon and energy and the samples were collected at regular intervals. The growth of strain RKJ 800 was measured by taking optical density at 600 nm. The depletion of 2C4NP was monitored by taking the optical density of the supernatant at 420 nm. Samples collected at different intervals were centrifuged and supernatants were extracted with ethyl acetate. Extracted samples were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). TLC results indicated depletion of 2C4NP with appearance of the two metabolites. No metabolite was detected in the sample of 0 h and 12 h. The presence of metabolite I was indicated in the sample of 24 h and 36 h whereas the presence of metabolite II was indicated only in the sample of 36 h. In the sample of 48 h, neither the presence of any metabolite nor parent compound was indicated. The Rf values of metabolite I, II and 2C4NP were 0.64, 0.48 and 0.72 respectively. The Rf values of metabolite I and II exactly matched to that of standard CHQ and HQ respectively. Furthermore, when TLC plates were sprayed with folin ciocalteu’s reagent, an immediate blue coloration was apparent in the case of suspected chlorohydroquinone and hydroquinone. HPLC confirmed complete depletion of 2C4NP by strain RKJ 800 within 48 h. GC-MS analysis was also carried out to confirm the presence of CHQ and HQ in the degradation pathway of 2C4NP.The percentage degradation of test substance 2-chloro-4-nitrophenol was determined to be 100% by using Test mat. analysis (absorbance O. D.), TLC, HPLC and GC-MS parameter after 48 hrs. Metabolite obtained during the degradation of the test chemical 2-chloro-4-nitrophenol were chloro-hydroquinone (CHQ) and hydroquinone (HQ), respectively. Thus, based on percentage degradation, 2 -chloro-4 -nitrophenol is considered to be readily biodegradable in nature.

 

For the target chemical from peer reviewed journal (Lu Guanghua et. al, 2001), the quantitative structure-biodegradability relationship studies were performed with the maximum specific removal rates (QTOD) for predicting the biodegradability of test substance 2-chloro-4-nitrophenol (CAS no. 619-08-9). The study was performed using through multiple linear regression analysis equation. The maximum specific removal rates of aromatic organic compounds were obtained using an adapted mixed culture (activated sludge).This microbial culture was cultivated semicontinuously on glucose and peptone and subsequently on the compound under study at a mean biomass retention time (MBRT) of approximately 5 days. This MBRT corresponds to biological treatment in the classical activated sludge wastewater treatment process. Test chemical was used as a sole source of organic carbon for the microbes of the culture. Initial chemical concentration used for the study was200 mg/l based on theoretical oxygen demand (TOD).MW, DH f and EHOMO of the substituted benzenes were calculated by the quantum chemical method MOPAC6.0-AM1 on energy-minimized structures. This method can automatically optimize the bond length, the bond angle and the twist angle, and yield a lot of structure information. Octanol-water partition coefficient (logP) was obtained from Biobyte package.All statistical analyses were carried out using SPSS package. The maximum specific removal rates are expressed as mg TOD of the compound removed per gram of the initial solids of the microbial culture per hour (QTOD). The maximum specific removal rate (QTOD) of the test chemical 2-chloro-4-nitrophenolusing through multiple linear regression analysis equation was evaluated to be 5mg/g.h (experimental value) and 12.4mg/g.h (predicted value), respectively. Thus, based on this value, chemical 2-chloro-4-nitrophenol can be considered to be not readily biodegradable in nature.

 

 

On the basis of above overall results for target chemical 2-Chloro-4-nitrophenol (from peer reviewed journals and secondary source), it can be concluded that the test substance 2-Chloro-4-nitrophenol can be expected to be readily biodegradable in nature.