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Biodegradation in water

Biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions(K. R. Girija et. al; 2002). Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, onlyPseudomonas aeruginosastrain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

Biodegradation in water & sediment

Estimation Programs Interface (EPI Suite, 2017) prediction model was run to predict the half-life in water and sediment for the test compound Pyrazine-2-carboxylic acid (CAS No: 98-97-5). If released in to the environment, 34.5% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical Pyrazine-2-carboxylic acid in water is estimated to be 15 days (360 hrs). The half-life (15 days) estimated by EPI suite indicates that the chemical Pyrazine-2- carboxylic acid is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.0692%), indicates that test chemical Pyrazine-2-carboxylic acid is not persistent in sediment.

Biodegradation in soil

The half-life period of Pyrazine-2-carboxylic acid (CAS No: 98-97-5) in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (EPI suite, 2017). If released into the environment, 65.4 % of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of Pyrazine-2-carboxylic acid in soil is estimated to be 30 days (720 hrs). Based on this half-life value of Pyrazine-2-carboxylic acid, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

Bioaccumulation: aquatic/sediment

BCFBAF model (v3.01) of Estimation Programs Interface (EPI Suite, 2017) was used to predict the bioconcentration factor (BCF) of test chemical Pyrazine-2-carboxylic acid (CAS No. 98 -97 -5). The bioconcentration factor (BCF) of Pyrazine-2-carboxylic acid was estimated to be 3.162 L/kg whole body w.w (at 25 deg C) which does not exceed the bioconcentration threshold of 2000, indicating that the chemical  Pyrazine-2-carboxylic acid is not expected to bioaccumulate in the food chain.

Adsorption/desorption

KOCWIN model (v2.00) of Estimation Programs Interface (EPI Suite, 2017) was used to predict the soil adsorption coefficient i.e Koc value of test chemical Pyrazine-2-carboxylic acid (CAS No. 98 -97 -5). The soil adsorption coefficient i.e Koc value of Pyrazine-2-carboxylic acid was estimated to be 4.192 L/kg (log Koc=0.6225)  by means of MCI method (at 25 deg C). This Koc value indicates that the substance Pyrazine-2-carboxylic acid has a negligible sorption to soil and sediment and therefore have rapid migration potential to ground water.

Additional information

Biodegradation in water

Various experimental studies and predicted data for the target compound Pyrazine-2-carboxylic acid (CAS No. 98-97-5) and supporting weight of evidence studies for its closest read across substance with logKow as the primary descriptor were reviewed for the biodegradation end point which are summarized as below:

 

In a weight of evidence study from review article (K. R. Girija et. al; 2002), biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions. Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, only Pseudomonas aeruginosa strain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for evaluating the percentage biodegradability of test substance Pyrazine-2-carboxylic acid (CAS no. 98-97-5) (K. S. Rajini, Ch. Sasikala, Ch. V. Ramana, 2010). Stenotrophomonas sp. HCU1 was used as a test inoculum. Strain HCU1 was serendipitously identified from a pyrazine-2-carboxylate contaminated broth growing in lab. Conc. of test chemical used for the study was24.81 mg/l (200µM).Pyrazine-2-carboxylate (used as an additional supplement) adapted strain HCU1 was harvested by centrifugation (16,0009gfor 10 min) and the pellet was washed (twice) with 0.05 M Tris–HCl buffer (pH 7.4) and resuspended in 1 ml of the same buffer. Cells were sonicated with MS-72 probe (Bandelin, model-UW 2070) to complete cell lysis after 8–9 cycles. The sonicated cell lysate was used for assay of pyrazine-2-carboxylate reduction and degradation. The assay mixture (1 ml) contained; 200µM of pyrazine-2-carboxylate, 200µM of NADPH, 5 mM MgCl2 and 40µl (130–140µg ml-1 protein) of the cell free extract in Tris HCl (pH 7.4). The reaction was stopped at an appropriate time by adding 10µl of 5 N HCl and filtered through 0.22µm polyvinyldienefluoride membrane. The samples were stored at 4°C and were analyzed in HPLC. Ring reduction of pyrazine-2-carboxylate was enzymatically assayed using 1 mM PCA, 200µM NADPH and increase in absorbance of PCA was calculated in terms of % peak height increase/microgram gram protein/minute. Km and Vmax of the enzyme were calculated using the software graph pad prism 5.0.

Mid-logarithmic phase culture (8 h) of strain HCU1 was harvested by centrifugation (12,0009gfor 20 min) and the supernatant was directly concentrated in flash evaporator to dryness. The concentrate was extracted with 50 ml methanol and the precipitated impurities were removed by centrifugation (12,0009gfor 15 min). The methanolic supernatant was diluted with methanol, water (1:0.2) where further precipitation occurred, which was separated by centrifugation (12,0009gfor 15 min). The precipitate was redissolved in methanol (fraction A) and the methanolic water supernatant was concentrated to dryness (fraction B). Both fractions were used for further metabolite purification using semi-preparative HPLC. Analysis of substrates and products was performed in HPLC at room temperature using a Shimadzu SPD-10AT isocratic or gradient system. HPLC instrument details are: solvent flow rate—1.5 ml min-1, Luna 5µC18 100A column (250×4.6 mm) and the heterocyclic compounds were detected using PDA detector at 268 nm. Pyrazine-2-carboxylate, (tR=1.77 min) were identified using standards. Increase in the absorbance (m AU) at 268 nm in HPLC was attributed to aromatic ring reduction of the compounds and loss as its utilization. Purity of the metabolites were confirmed using HPLC with three different solvent systems (methanol: water [1:1]; methanol: water: acetonitrile [1:1: 0.25]; water: acetonitrile [7:3]) at 230, 268, 280 and 350 nm for independent analysis. In addition to this, LC-MS, NMR and Gel exclusion chromatography in FPLC (Fast Protein Liquid Chromatography) was also carried out.  

One metabolite was isolated through semi-preparative HPLC from fraction A. This metabolite had a tR of 1.55 min. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. The metabolite is a white amorphous solid and has absorption maximum at 268 nm. Based on the spectral data the metabolite is proposed as 1,2,5,6-tetrahydropyrazine-2-carboxylate. Eight chromatographically (HPLC) distinct peaks were observed from fraction B. This metabolite had a tR of 1.55 min. The metabolite is an orangish sticky compound and has an absorption maximum at 400 nm. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. Based on the spectral data analyses, the metabolite is proposed as 2-amino-2-hydroxy-3-(methylamino)propanoic acid. The percentage degradation of test substance Pyrazine-2-carboxylic acid was determined to be 86% by HPLC parameter. Thus, based on percentage degradation, Pyrazine-2-carboxylic acid is considered to be readily biodegradable in nature.

 

In a prediction using the Estimation Programs Interface Suite (EPI suite, 2017), the biodegradation potential of the test compoundPyrazine-2-carboxylic acid(CAS No. 98-97-5) in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that chemical Pyrazine-2-carboxylic acid is expected to be readily biodegradable.

 

In a supporting weight of evidence study from authoritative database (HSDB, 2017) and secondary source (2003) for the read across chemical 3-methylpyridine (CAS no. 108-99-6),an aerobic river die-away test was performed for test chemical 3-methylpyridine (CAS No: 108-99-6) to determine its percentage biodegradability for 18 days. Sewage inoculum and polluted river water were used as a test inoculum obtained from Ohio and Little Miami River waters. Initial test substance conc. used for the study was 1 mg/l. Experiment consisted of adding test substance to carboys (size/volume not stated) containing Ohio River water and stored at 20 °C. Samples were removed periodically and analyzed for test substance. After the test substance disappeared, additional test substance was added and disappearance of that portion was monitored. After an initial lag phase of approximately 9 days, biodegradation was rapid. Subsequent additions of test compound resulted in rapid degradation of 100% with no or little lag phase. Degradation attained approximately 100% after 14 days. Additions of test substance after the disappearance of the initial dose were rapidly biodegraded within two days. Thus, based on percentage degradation, chemical 3 -methylpyridine can be considered to be readily biodegradable in nature.

 

For the same read across chemical 3-methylpyridine (CAS no. 108-99-6) from secondary source (2003), a standard BOD5 test of 3-methylpyridine was conducted to evaluate its biodegradability in wastewater. Activated sludge organisms was used as a test inoculum obtained from wastewater. In addition, removal during a pilot plant biological treatment system consisting of a recirculating trickling filter, followed by activated sludge aeration was evaluated. Evaluations were performed at 163, 413 and 746 mg/l of 3-methylpyridine and oxygen demand by BOD5 and test substance removal was calculated. The calculated oxygen demand by BOD5 was 31.0% and 93.8% removal of the read across substance 3-methylpyridine was observed under continuous flow conditions with retention times of up to 312 hours (13 days). Thus, based on percentage degradation, chemical 3-methylpyridine can be considered to be readily biodegradable in nature.

 

For the another read across chemical Benzoic acid (CAS no. 65-85-0) from authoritative database (J-CHECK, 2017), biodegradation experiment was conducted for 14 days for evaluating the percentage biodegradability of read across substance Benzoic acid (CAS no. 65-85-0). Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of substance Benzoic acid was determined to be 85, 98 and 100% by BOD, TOC removal and UV-Vis parameter in 14 days. Thus, based on percentage degradation, Benzoic acid is considered to be readily biodegradable in nature.

 

On the basis of above results for target chemicalPyrazine-2-carboxylic acid(fromreview article, peer reviewed journaland EPI suite, 2017) and for its read across substance (from authoritative database J-CHECK, HSDB and secondary source), it can be concluded that the test substancePyrazine-2-carboxylic acidcan be expected to be readily biodegradable in nature.

Biodegradation in water & sediment

Estimation Programs Interface (EPI Suite, 2017) prediction model was run to predict the half-life in water and sediment for the test compound Pyrazine-2-carboxylic acid (CAS No: 98-97-5). If released in to the environment, 34.5% of the chemical will partition into water according to the Mackay fugacity model level III and the half-life period of test chemical Pyrazine-2-carboxylic acid in water is estimated to be 15 days (360 hrs). The half-life (15 days) estimated by EPI suite indicates that the chemical Pyrazine-2- carboxylic acid is not persistent in water and the exposure risk to aquatic animals is moderate to low whereas the half-life period of test chemical in sediment is estimated to be 135 days (3240 hrs). However, as the percentage release of test chemical into the sediment is less than 1% (i.e, reported as 0.0692%), indicates that test chemical Pyrazine-2-carboxylic acid is not persistent in sediment.

Biodegradation in soil

The half-life period of Pyrazine-2-carboxylic acid (CAS No: 98-97-5) in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (EPI suite, 2017). If released into the environment, 65.4 % of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of Pyrazine-2-carboxylic acid in soil is estimated to be 30 days (720 hrs). Based on this half-life value of Pyrazine-2-carboxylic acid, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

On the basis of available information, the test substance Pyrazine-2 -carboxylic acid can be considered to be readily biodegradable in nature.

Bioaccumulation: aquatic/sediment

Various predicted data for the target compound Pyrazine-2-carboxylic acid (CAS No. 98-97-5) and supporting weight of evidence study for its read across substance were reviewed for the bioaccumulation end point which are summarized as below:

 

In a prediction done using theBCFBAF Program(v3.01) of Estimation Programs Interface (EPI Suite, 2017), the bioconcentration factor (BCF) of Pyrazine-2-carboxylic acid was estimated to be 3.162 L/kg whole body w.w (at 25 deg C) which does not exceed the bioconcentration threshold of 2000, indicating that the chemical Pyrazine-2-carboxylic acid is not expected to bioaccumulate in the food chain.

 

In an another prediction done by using Bio-concentration Factor (v12.1.0.50374) module Bio-concentration Factor over the entire pH scale (pH 0 -14) of the test substance Pyrazine-2-carboxylic acid (CAS no. 98 -97 -5) was estimated to be 1. This value indicates that the test substance Pyrazine-2-carboxylic acid was considered to be non-accumulative in aquatic organisms.

 

Bioconcentration Factor (BCF) of test chemical Pyrazine-2-carboxylic acid was estimated using Chemspider database(ChemSpider, 2017). The bioconcentration factor of test substance Pyrazine-2-carboxylic acid was estimated to be 1 at both pH 5.5 and 7.4, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the chemical Pyrazine-2-carboxylic acid is not expected to bioaccumulate in the food chain.

 

SciFinder database (American Chemical Society (ACS), 2017) was used for predicting the bioconcentration factor (BCF) of test chemical Pyrazine-2-carboxylic acid (CAS No. 98 -97 -5). The bioconcentration factor (BCF) of Pyrazine-2-carboxylic acid was estimated to be 1 (at 25 deg C) which does not exceed the bio concentration threshold of 2000, indicating that the chemical Pyrazine-2-carboxylic acid is not expected to bioaccumulate in the food chain

 

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 Pyrazine-2-carboxylic acid was estimated to be 4.28 dimensionless . The predicted BCF result based on the 5 OECD principles. Thus based on the result it is concluded that the test substance Pyrazine-2-carboxylic acid is non-bioaccumulative in nature.

 

In a supporting weight of evidence study from authoritative database (HSDB, 2017) for the read across chemical Nicotinic acid (CAS no. 59-67-6), the bioaccumulation study on fish was conducted for estimating the BCF (bioaccumulation factor) value of read across chemical Nicotinic acid. The bioaccumulation factor (BCF) value was calculated using an estimated log Kow of 0.36 and a regression derived equation. The BCF (bioaccumulation factor) value of Nicotinic acid was determined to be 3 dimensionless, which does not exceeds the bioconcentration threshold of 2000, indicating that the chemical Nicotinic acid is considered to be non-bioaccumulative in aquatic organisms.

 

For the another read across chemical pyridine-2,5-dicarboxylic acid (CAS no. 100-26-5) from authoritative database (J-CHECK, 2017), bioaccumulation experiment was conducted on test organism Cyprinus carpiofor 6 weeks for evaluating the bioconcentration factor (BCF value) of substance pyridine-2,5-dicarboxylic acid (CAS no. 100-26-5) under static conditions. Test chemical nominal conc. used for the study were 2 mg/l and 0.2 mg/l (w/v), respectively. Range finding study involve the TLm (48 hr) >1000 mg/l on Rice fish (Oryzias latipes). Lipid content of the test organism was determined to be 4.1%.The bioconcentration factor (BCF value) of substance pyridine-2,5 -dicarboxylic acid on Cyprinus carpio was determined to be < 0.3 – 0.9 L/Kg at a conc. of 2 mg/l and < 2.9 – 8.6 L/Kg at a conc. of 0.2 mg/l, respectively, which does not exceed the bioconcentration threshold of 2000, indicating that the chemical pyridine-2,5 -dicarboxylic acid is not expected to bioaccumulate in the food chain.

 

On the basis of above results for target chemical Pyrazine-2 -carboxylic acid (from EPI suite, ACD labs,ChemSpider, SciFinder database and CompTox Chemistry Dashboard,  2017) and for its read across substance (from authoritative database HSDB and J-CHECK, 2107), it can be concluded that the BCF value of test substance Pyrazine-2 -carboxylic acid ranges from 1 – 4.28 which does not exceed the bioconcentration threshold of 2000, indicating that the chemical Pyrazine-2-carboxylic acid is not expected to bioaccumulate in the food chain.

Adsorption/desorption

Various predicted data for the target compound Pyrazine-2-carboxylic acid (CAS No. 98-97-5) were reviewed for the adsorption end point which are summarized as below:

 

In a prediction done using theKOCWIN Program(v2.00) of Estimation Programs Interface (EPI Suite, 2017), the soil adsorption coefficient i.e Koc value of Pyrazine-2-carboxylic acid was estimated to be 4.192 L/kg (log Koc=0.6225) by means of MCI method (at 25 deg C).

 

The Soil Adsorption Coefficient i.e Koc value of test substance Pyrazine-2-carboxylic acid was estimated using Adsorption Coefficient module (v12.1.0.50374) program as Koc 2.76, 3.89, 3.95, 3.20, 1.09 and 1 at pH 0, 1, 2, 3, 4 and at pH range 5-14 respectively (log Koc ranges from 0.0 ± 1.0 to 0.6 ± 1.0)(ACD (Advanced Chemistry Development)/I-Lab predictive module, 2017).

 

In another prediction done by using ChemSpider Database (2017), the Soil Adsorption Coefficient i.e Koc value of test substance Pyrazine-2-carboxylic acid (CAS no. 98 -97 -5) was estimated. The adsorption coefficient (Koc) value of substance Pyrazine-2-carboxylic acid was estimated to be 1 (Log Koc = 0) at pH 5.5 and 7.4, respectively.

 

Additional soil adsorption coefficient i.e Koc value of test chemical Pyrazine-2-carboxylic acid (CAS No. 98 -97 -5)was estimated using the SciFinder database (American Chemical Society (ACS), 2017). The soil adsorption coefficient i.e Koc value of Pyrazine-2-carboxylic acid was estimated to be 10.1, 10.2, 8.28, 2.82 and 1 at pH 1, 2, 3, 4 and 5-10, respectively (log Koc = 0 to 1) (at 25 deg C).

 

On the basis of above overall results for target chemical Pyrazine-2 -carboxylic acid (from EPI suite, ACD labs,ChemSpider and SciFinder database, 2017), it can be concluded that the Koc value of test substance Pyrazine-2-carboxylic acid ranges from 1 –10.2 indicating that the test chemical Pyrazine-2-carboxylic acid has a negligible sorption to soil and sediment and therefore have rapid migration potential to ground water.