Biodegradation of the organophosphorus insecticide diazinon by Serratia sp. and Pseudomonas sp. and their use in bioremediation of contaminated soil

International Biodeterioration & Biodegradation 132 (2018) 59–65 Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod Biodegradation of organophosphorus insecticides with PeS bonds by two Sphingobium sp. strains T Jae-Hyung Ahn, Shin-Ae Lee, Soo-Jin Kim, Jaehong You, Byeong-Hak Han, Hang-Yeon Weon, Se-Weon Lee∗ Agricultural Microbiology Division, National Institute of Agricultural Sciences, Rural Development Administration (RDA), Wanju-gun, Jeollabuk-do, 55365, Republic of Korea A R T I C LE I N FO A B S T R A C T Keywords: Organophosphorus Cadusafos Biodegradation Soil Bacteria Although cadusafos, an aliphatic organophosphorus (OP) insecticide, is not approved by the European commission, it is used in several countries and sometimes detected as a residue in soils and agricultural products. In this study, two cadusafos-degrading Sphingobium sp. strains, K22212 and Cam5-1, were isolated and characterized for use as detoxifying agents of the insecticide. Both strains degraded 100 mg L−1 of cadusafos in mineral medium within 12 h through a common metabolite, which was supposed to be dimerized thiophosphates based on its molecular weight. Degradation of cadusafos increased cell growth for Cam5-1 but not for K22212. K2212 and Cam5-1 degraded cadusafos in soil (15 mg kg−1 dry soil) within 5 and 2 days, respectively. Both strains also degraded ethoprophos, phenthoate and phorate but not chlorpyrifos and diazinon, indicating that they are specialized for degradation of OP insecticides with at least one single bond connecting phosphorus and sulfur atoms (PeS bond). For both strains, the degradation rate was the largest for ethoprophos, followed by cadusafos, phenthoate, and phorate. Our results indicate that these bacterial strains are effective degraders of OP insecticides with PeS bonds, and in particular, Cam5-1 is more promising for removal of the OP insecticides in soils. 1. Introduction At present organophosphorus (OP) insecticides are the largest class of insecticides, occupying nearly 44% shares of the global insecticide market in 2016 (Marketsandmarkets, 2017), with more than 100 OP insecticides commercialized. Due to their widespread use, OP insecticides are sometimes detected in agricultural products (Bai et al., 2006; Kang et al., 2015). In most cases they were reported to be below the maximum residue level (MRL), the maximum concentration of a pesticide residue permitted in or on food commodities and animal feed (GEMS/Food, 1997), but considering the possibility that long-term low-level exposure of some OP insecticides are involved in developmental toxicity (Costa et al., 2013), endocrine disruption (Aguilar-Garduño et al., 2013), affective disorders (Stallones and Beseler, 2016), cancer (Lerro et al., 2015), and hypospadias (Michalakis et al., 2014), continuous monitoring of OP insecticide levels in food and humans is necessary. In addition, OP insecticides have been detected in diverse environments (Abdel-Halim et al., 2006; Fadaei et al., 2012; Kawahara et al., 2005; Kumari et al., 2008) and several studies have showed the toxic effects of OP ∗ Corresponding author. E-mail address: leeseweon@korea.kr (S.-W. Lee). https://doi.org/10.1016/j.ibiod.2018.05.006 Received 22 January 2018; Received in revised form 11 May 2018; Accepted 11 May 2018 0964-8305/ © 2018 Elsevier Ltd. All rights reserved. insecticides on ecosystems (Díaz-Resendiz et al., 2015; John and Shaike, 2015). Microbial degradation of OP insecticides has been extensively studied as an insecticide-detoxifying tool. The release of the X group (Fig. S1a) by hydrolysis of the single bond between phosphorus and oxygen (PeO bond) or phosphorus and sulfur (PeS bond) has been considered the most significant step of detoxification (Havens and Rase, 1991; Lai et al., 1995; Singh and Walker, 2006). Many bacterial and fungal strains were reported to degrade various OP compounds, and their hydrolysis enzymes were identified and characterized (Iyer et al., 2013; Singh and Walker, 2006; Theriot and Grunden, 2011). The known bacterial organophosphorus hydrolases have no or much lower activity on PeS bonds than on PeO bonds in OP insecticides (Alvarenga et al., 2015; Horne et al., 2002; Iyer et al., 2013; Lai et al., 1995) while two fungal enzymes were shown to effectively cleave PeS bonds (Liu et al., 2001, 2004). Among OP insecticides, cadusafos is an aliphatic organophosphorus insecticide with two PeS bonds (Fig. S1b) that targets soil-born insects and nematodes. The World Health Organization has classified cadusafos as a Class Ib (highly hazardous) toxin based on the LD50 for the rat International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. treatment was performed in duplicate. (World Health Organization, 2010) and it was also reported to be ecotoxic towards earthworms and soil microbial communities (Fouché et al., 2017). By the European commission, cadusafos has not been approved as a plant protection product because it did not meet the safety requirements (https://ec.europa.eu/food/plant/pesticides_en); however, it is still being used by several other countries, including South Korea. Although cadusafos is reported to have a relatively short soil half-life of 38 days (PPDB, https://sitem.herts.ac.uk/aeru/ppdb/ en/), it is sometimes detected in agricultural products and soil samples in South Korea (government investigation); thus, efficient and safe methods to effectively degrade the insecticide are required. In this study, we isolated two bacterial strains that rapidly degrade cadusafos from agricultural soils and characterized their physiology and degradation abilities to potentially use them as cadusafos-detoxifying agents. 2.3. Physiological and biochemical characterization In addition to R2A agar, growth of cadusafos-degrading strains on trypticase soy agar (TSA; Difco), nutrient agar (NA; Difco), and LuriaBertani (LB) agar (Difco) was tested. Growth in the presence of sodium chloride (0–3.0% at intervals of 0.5%) and at various temperatures (8–50 °C at intervals of 5–7 °C) was investigated using R2A broth medium. The pH for optimal growth was tested in R2A broth with a pH range of 3.0–12.0 in increments of 1.0 unit. In all growth experiments, the absorbance at 600 nm of the culture medium in a 96-well plate was measured using a microplate reader (SpectraMax 340; Molecular Devices) to determine growth. Other biochemical characteristics were determined using the API 20NE system (bioMérieux) according to manufacturer's instructions. 2. Materials and methods 2.4. Identification of metabolic intermediates 2.1. Isolation and identification of cadusafos-degrading microorganisms To identify metabolic intermediates of cadusafos degradation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) and gas chromatography-mass spectrometry (GC-MS) with Purge & Trap were performed. A mineral medium containing 100 mg L−1 of a technical product of cadusafos was inoculated with strain Cam5-1, incubated at 28 °C with shaking for 18 h and filtered through a syringe filter with 0.2 μm Supor® membrane (Pall) for the analyses. LC-HRMS analysis was performed on an ACQUITY UPLC System coupled with a SYNAPT G2-Si Mass Spectrometer with an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, Waters). A mixture of acetonitrile and water (70:30, v/v) was used as the mobile phase at a flow rate of 0.5 mL min−1. The injection volume was 5 μL. The mass range was 50–1200 Da with a resolution of 20,000. Cone voltage was 30 V and trap collision energy was 6 V. The automated Purge & Trap Sampler JTD-505III (Japan Analytical Industry) was used under the following conditions: desorption temperature, 280 °C; desorption time, 30 min; desorption gas flow rate, 50 mL min−1; cold-trap for sample trapping, −40 °C; for pyrolysis, 280 °C; transfer-line temperature, 280 °C; needle heater, 280 °C; coldtrap heater, 200 °C; head press, 86 MPa; column flow, 1.0 mL min−1; split ratio, 1/100. GC-MS analysis was performed using a GC-MS QP 2010 plus (Shimadzu) under the following conditions: DB-624 column (30 m × 0.251 mm × 1.40 mm, Agilent Technologies); 30–600 mass scan; 0–35 min oven temperature program (40 °C for 3 min hold, 10 mL min−1 up to 260 °C, 5 min hold); ion source, 200 °C; transfer line, 250 °C; EM voltage, 20 eV. Identification of chromatographic peaks was performed based on NIST and WILEY libraries. Forty-one agricultural soil samples were collected throughout South Korea in 2016 and information of the sampling sites is indicated in Table S1. Soils were sampled from around the crop roots, transferred to a laboratory in an ice box, and preserved at 4 °C until use. Two enrichment procedures for isolating cadusafos-degrading bacteria were used; for enrichment procedure 1, 1 g of soil sample was transferred to 5 mL mineral medium containing 100 mg L−1 of a technical product of cadusafos (92.2%), which was kindly donated by NongHyup Chemical, and incubated in a shaking incubator (150 rpm) at 28 °C. Composition of the mineral medium is indicated in Table S2. When a complete degradation of cadusafos was observed, 10% of the culture medium was transferred to fresh medium and the same procedure was repeated two more times. Finally, some of the culture medium was spread on R2A agar medium and incubated at 28 °C. Bacterial strains with distinct colony morphologies were pure-cultured using R2A agar and examined for degradation of cadusafos. In enrichment procedure 2, 20 g of each soil sample were mixed together and a portion of 400 g was sampled from the mixture, to which 10 mL acetone containing 10 mg of a technical product of cadusafos was added (the final cadusafos concentration was 25 mg kg−1 soil). Treated soil was dried at room temperature for 3 h to evaporate acetone completely and then incubated in a glass cylinder (diameter, 11 cm; height, 11 cm) at room temperature. The soil mixture was mixed with a spatula every day and the concentration of cadusafos was monitored periodically. When a complete degradation of cadusafos was observed, enrichment procedure 1 was performed. Isolated bacterial strains were identified by sequencing 16S rRNA genes and their phylogenetic relationships with nearest type strains were inferred by constructing a maximum-likelihood tree using the EzBioCloud (Yoon et al., 2017) and the MEGA program (Tamura et al., 2013). 2.5. Degradation of cadusafos in soil Upland soil was collected from the experimental upland field of the National Institute of Agricultural Sciences, Wanju, Korea (35°49′29.9″N, 127°02′41.1″E), dried in a greenhouse for two days, and sifted through a 2-mm pore sieve. The soil sample (300 g) was treated with 4.5 mg of a technical product of cadusafos dissolved in 10 mL of acetone (15 mg cadusafos kg−1 dry soil) and dried for 3 h to completely evaporate acetone. After treated soil was transferred to a glass cylinder (diameter, 11 cm; height, 11 cm), 10 mL of 0.85% NaCl solution containing 3.2 × 108 CFU of strain K22212 or 2.4 × 108 CFU of strain Cam5-1 (corresponding to 3.2 × 106 CFU of K22212 and 8.1 × 105 CFU of Cam5-1 g−1 dry soil) and 30 mL of distilled water were added. Control soil was treated with 10 mL of 0.85% NaCl solution and 30 mL of distilled water. All cylinders were covered with aluminum foil and incubated at 28 °C. Soil pH (1:10) was 6.4 and water content, which was determined by drying at 105 °C for 4 h, ranged between 16 and 17%. Each treatment (control, K22212 treatment, or Cam5-1 treatment) was performed in triplicate and temporal variation of cadusafos 2.2. Degradation of cadusafos in a mineral medium Cadusafos-degrading bacteria were cultured on R2A agar medium at 28 °C for 3 days and then the colonies were suspended in a 0.85% NaCl solution until the turbidity reached 1.65 McFarland units, determined by a DEN-1B densitometer (Biosan). The suspension (0.5 mL) was used to inoculate 5 mL mineral medium containing 100 mg L−1 of a technical product of cadusafos. Test tubes containing inoculated media were incubated in a shaking incubator (150 rpm) at 28 °C, and cadusafos concentration and cell density were estimated periodically using highperformance liquid chromatography (HPLC) and a DEN-1B densitometer, respectively. At the start and end of the experiments, viable cells were counted by the dilution plate count technique using R2A agar medium. A non-inoculated medium was used as a control and each 60 International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. Fig. 1. Degradation of cadusafos by K22212 and Cam5-1 in mineral media containing 100 mg L−1 of cadusafos. (a) Temporal variation of cadusafos concentration; (b) Temporal variation of medium turbidity. Temporal variations of cadusafos concentration, area of the HPLC chromatographic peak at the retention time of 7.5 min, and medium turbidity for (c) K22212 and (d) Cam5-1. The unit of the HPLC peak area is arbitrary. Values are presented as means ± SD, n = 2. soil samples, 10 g of wet soil was mixed with 15 mL acetonitrile, and shaken at 150 rpm for 30 min. The supernatant was filtered through a 0.2-μm PVDF filter and HPLC analysis was performed as described above. concentration was monitored. 2.6. Degradation of other OP insecticides Cadusafos-degrading strains were used to inoculate 5 mL mineral media containing 25 mg L−1 of cadusafos, chlorpyrifos, diazinon, ethoprophos, phenthoate or phorate (all chemicals purchased from Sigma-Aldrich) and incubated as described above except for the turbidity of 0.85% NaCl solution inoculated with the bacterial strains, which was 5 McFarland units instead of 1.65. After a 7-day incubation, concentrations of the OP insecticides were estimated. For insecticides degraded by the bacterial strains, additional experiments were performed to calculate degradation rate constants and half-lives, in which cadusafos-degrading strains were inoculated into 3 mL mineral media containing 100 mg L−1 of the insecticides and incubated as described above. 2.8. Deposition of 16S rRNA gene sequences and bacterial strains The 16S rRNA gene sequences of K22212 and Cam5-1 were deposited in the NCBI GenBank under accession numbers MF582324 and MF582325, respectively. The bacterial strains were also deposited in the Korean Agricultural Culture Collection (KACC) under KACC numbers 19414 for K22212 and 19413 for Cam5-1. 3. Results and discussion 3.1. Isolation and characterization of cadusafos-degrading microorganisms From each enrichment procedure, one bacterial strain showing cadusafos degradation was isolated; strain K22212 was isolated by enrichment procedure 1 and strain Cam5-1 by enrichment procedure 2. The 16S rRNA gene sequences of the two bacterial strains shared high similarity with each other (98.5% identical) and were found affiliated with the genus Sphingobium (Fig. S2), thereby identifying them as Sphingobium sp. K22212 and Sphingobium sp. Cam5-1. This is not surprising considering Spingobium sp. strains were known to degrade various xenobiotic compounds (Guo et al., 2009; Kiran Kumar Reddy et al., 2014; Yao et al., 2015; Zheng et al., 2011). Both K22212 and Cam5-1 showed the fastest growth on R2A agar medium and also showed good growth on TSA and NA media but did not grow on LB agar medium. K22212 was found to grow between 8 2.7. HPLC analysis OP insecticide concentrations were determined using a Shimadzu SCL-10Avp HPLC System equipped with a Shimadzu SPD-10Avp photodiode array (PDA) detector (Shimadzu). The liquid sample (0.2 mL) was mixed with 0.8 mL of acetonitrile and filtered through a 0.2-μm PVDF filter (Pall) for HPLC analysis. HPLC analysis was performed using an YMC-Triart C18 column (S-3 μm/12 nm, 150 × 4.6 mm I.D.) at a constant column temperature of 40 °C. A mixture of acetonitrile and water (70:30, v/v) was used as the mobile phase at a flow rate of 0.7 mL min−1. The injection volume was 20 μL. Cadusafos, chlorpyrifos, ethoprophos, diazinon, phenthoate and phorate were analyzed by the PDA detector at 198, 198, 193, 193, 194 and 193 nm, respectively. For 61 International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. 3.4. Degradation of cadusafos in soil and 45 °C (optimum 28 °C), pH 5.0–10.0 (optimum 6.0), and in the presence of 0.0–0.5% NaCl (optimum 0.0%), while for Cam5-1 growth was observed between 15 and 45 °C (optimum 35 °C), pH 5.0–11.0 (optimum 6.0), and in the presence of 0.0–1.0% NaCl (optimum 0.0%). Other biochemical characteristics are indicated in Table S3. Inoculation with K22212 (3.2 × 106 CFU g−1 dry soil) and Cam5-1 (8.1 × 105 CFU g−1 dry soil) of upland soil contaminated with cadusafos (15 mg kg−1 dry soil) resulted in a complete degradation of cadusafos within 5 and 2 days, respectively, compared with more than 32 days in control soil (Fig. 3). This indicates that these bacterial strains can greatly increase the removal rate of cadusafos in upland soils. To our knowledge, three bacterial strains capable of degrading cadusafos were pure-cultured in previous studies, Flavobacterium sp. CadI, Sphingomonas sp. CadII, and Pseudomonas sp. PC1 (Abo-Amer, 2012; Karpouzas et al., 2005). Considering that CadII (4.3 × 108 CFU g−1 dry soil) degraded 80% of 10 mg cadusafos kg−1 soil within 6 days (Karpouzas et al., 2005) and PC1 (2.1 × 106 CFU g−1 dry soil) completely degraded 10 mg cadusafos kg−1 soil within 4 days (Abo-Amer, 2012), Cam5-1 appears to be more efficient in cadusafos removal from soil than both of these strains. The ability to utilize cadusafos as a carbon source is significant, as this will allow cadusafos-degrading activity to continue in nutrient-poor soils and will increase the survival rate of microorganisms with this ability. The soil enrichment procedure used for isolation of Cam51—where all 41 agricultural soil samples were mixed together and incubated with cadusafos—was a relatively straightforward way of isolating the most efficient among many cadusafos-degrading microorganisms in the soil samples. If safety is proven, we believe that Cam51 can be used for detoxification of OP insecticides on agricultural products. 3.2. Degradation of cadusafos in a mineral medium In mineral medium, 100 mg L−1 of cadusafos was completely degraded within 7 h by K22212, within 12 h by Cam5-1, and remained unchanged in the non-inoculated medium for 73 h (Fig. 1a). Cell density, estimated by turbidity, slightly decreased for K22212 and increased for Cam5-1 after 73 h of incubation (Fig. 1b). This was supported by the dilution plate count technique results (Fig. S3), in which viable cells after a 73-h incubation period decreased by 45% for K22212 and increased to more than two folds for Cam5-1. In HPLC analysis, the disappearance of cadusafos peak at retention time of 10.7 min coincided with the appearance of a new peak at retention time of 7.5 min for both strains (Fig. S4). This new peak also gradually disappeared for both strains. When the area of the peak eluted at 7.5 min was plotted with cadusafos concentration and medium turbidity, it was found that the compound corresponding to this peak reached its maximum when cadusafos was completely consumed, and then it was also completely consumed for 73 h of incubation with K22212 (Fig. 1c) and 49 h with Cam5-1 (Fig. 1d). Therefore this compound appears to be an intermediate of cadusafos metabolism. While the turbidity of the K22212-inoculated medium did not increase during the consumption of cadusafos and the putative intermediate (Fig. 1c), the turbidity of the Cam5-1-inoculated medium initially decreased and then remained unchanged during the consumption of cadusafos, and increased during the consumption of the putative intermediate (Fig. 1d). Therefore, it appears that Cam5-1 can utilize the intermediate as a carbon source unlike K22212. 3.5. Degradation of other OP insecticides Among the OP insecticides tested, cadusafos, ethoprophos, phenthoate and phorate were degraded in mineral media by more than 50%, while the concentrations of chlorpyrifos and diazinon remained unchanged for 7 days of incubation. This result suggests that the two bacterial strains are specialized for the degradation of OP insecticides with PeS bonds, since the degraded compounds have at least one PeS bond while chlorpyrifos and diazinon have only PeO bonds (Fig. S1). To compare degradation rates among the OP insecticides, the two bacterial strains were inoculated into a mineral medium containing 100 mg L−1 of cadusafos, ethoprophos, phenthoate, or phorate and then incubated with monitoring of concentrations of the OP insecticides. For both strains, the degradation rate was the largest for ethoprophos, followed by cadusafos, phenthoate, and phorate (Table 1 and Fig. S6). For the preceding three OP insecticides, the degradation rate seems to be correlated with the size of the functional group attached to the sulfur atom; the functional group is the largest in phenthoate, followed by cadusafos, and then ethoprophos (Fig. S1). For phorate, another sulfur atom besides that in the PeS bond is supposed to impede the degradation further (Fig. S1e). The degradation of both cadusafos and ethoprophos by other bacterial strains was reported in a previous study and attributed to the structural similarity between the two OP insecticides (Karpouzas et al., 2005); these strains could not utilize OP insecticides with only PeO or PeN bonds, indicating that they are also specialized for the degradation of OP insecticides with PeS bonds. 3.3. Identification of metabolic intermediates To identify the metabolic intermediate of cadusafos degradation, LC-HSMS analysis was performed. We presumed that the peak eluted at 7.5 min in HPLC analysis (Fig. S5a) corresponded to the peak eluted at 0.72 min in UPLC analysis (Fig. S5b), thus the mass spectrum of which was determined. In this analysis, it appeared that a peak at 282.0993 m/z was the dominant (Fig. 2a). The molecular mass 282.0993 was higher than that of cadusafos (270.4), suggesting that the intermediate may be generated by the assemblage of smaller intermediates. In GC-MS analysis, cadusafos peak obtained in non-inoculated medium completely disappeared after the inoculation of Cam 5-1 and 18-h incubation, while 2-butanone, 2-butanol, and sec-butyl disulfide peaks newly appeared (Fig. 2b and c). Since 2-butanethiol was detected both before and after inoculation with Cam5-1 and sec-butyl disulfide can form spontaneously from two molecules of 2-butanethiol (Chebbi et al., 2015), it is unclear if these two compounds are metabolic intermediates. Considering the above results, we hypothesized that Cam5-1 hydrolyzes both PeS bond and SeC bond in cadusafos, resulting in the formation of 2-butanethiol which is dimerized into sec-butyl disulfide, and 2-butanol which is further oxidized to 2-butanone, and a metabolite with a thiol group(s) which is dimerized into a disulfide or thioether such as shown in Fig. 2a. Molecular mass of the combined products are 282, corresponding to that of the intermediate of cadusafos degradation. To our knowledge, such a degradation pathway of OP insecticides has not been reported to date (Singh, 2008; Singh and Walker, 2006). Identification of the complete degradation pathway of cadusafos by Cam5-1 is future research topics of interest. 4. Conclusions Two cadusafos-degrading bacterial strains were isolated from agricultural soils and affiliated with the genus Sphingobium. Degradation of cadusafos increased growth of strain Cam5-1 but not strain K22212. Both strains can also degrade ethoprophos, phenthoate and phorate but not chlorpyrifos and diazinon, indicating that they are specialized for OP insecticides with PeS bonds. Cam5-1 degraded cadusafos in soil with faster rate than K22212, thus it is supposed that Cam5-1 is an efficient agent degrading OP insecticides with PeS bonds. 62 International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. (caption on next page) 63 International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. Fig. 2. Analysis of metabolic intermediates of cadusafos degradation by Cam5-1. (a) LC-HRMS spectrum of the peak eluted at 0.72 min in Fig. S5b and expected chemical structures of the intermediate. GC chromatograms of volatile components of the mineral medium containing 100 mg L−1 of cadusafos (b) before inoculation with Cam5-1 and (c) after inoculation and 18-hr incubation. The selected peaks were identified based on their mass spectra and NIST/WILEY libraries; only those with similarity indices > 94% were given except for cadusafos. Aguilar-Garduño, C., Lacasaña, M., Blanco-Muñoz, J., Rodríguez-Barranco, M., Hernández, A.F., Bassol, S., González-Alzaga, B., Cebrián, M.E., 2013. Changes in male hormone profile after occupational organophosphate exposure. Longitud. Study. Toxicol. 307, 55–65. Alvarenga, N., Birolli, W.G., Porto, A.L.M., 2015. Biodegradation of organophosphate and pyrethroid pesticides by microorganims. In: Lichtfouse, E., Schwarzbauer, J., Robert, D. (Eds.), Pollutants in Buildings, Water and Living Organisms. Springer International Publishing, Cham, pp. 85–121. Bai, Y., Zhou, L., Wang, J., 2006. Organophosphorus pesticide residues in market foods in Shaanxi area, China. Food Chem. 98, 240–242. Chebbi, A., Mhiri, N., Rezgui, F., Ammar, N., Maalej, A., Sayadi, S., Chamkha, M., 2015. Biodegradation of malodorous thiols by a Brevibacillus sp. strain isolated from a Tunisian phosphate factory. FEMS Microbiol. Lett. 362 fnv097-fnv097. Costa, L.G., Giordano, G., Cole, T.B., Marsillach, J., Furlong, C.E., 2013. Paraoxonase 1 (PON1) as a genetic determinant of susceptibility to organophosphate toxicity. Toxicology 307, 115–122. Díaz-Resendiz, K.J.G., Toledo-Ibarra, G.A., Girón-Pérez, M.I., 2015. Modulation of immune response by organophosphorus pesticides: fishes as a potential model in immunotoxicology. J. Immunol. Res. 2015, 10. Fadaei, A., Dehghani, M.H., Nasseri, S., Mahvi, A.H., Rastkari, N., Shayeghi, M., 2012. Organophosphorous pesticides in surface water of Iran. Bull. Environ. Contam. Toxicol. 88, 867–869. Fouché, T.C., Claassens, S., Maboeta, M.S., 2017. Ecotoxicological assessment of chemical fumigants utilising an earthworm (Eisenia andrei) bioassay and soil microbial communities. Water, Air, Soil Pollut. 228, 154. GEMS/Food, 1997. Guidelines for Predicting Dietary Intake of Pesticide Residues (Revised). World Health Organization, Switzerland. Guo, P., Wang, B., Hang, B., Li, L., Ali, S.W., He, J., Li, S., 2009. Pyrethroid-degrading Sphingobium sp. JZ-2 and the purification and characterization of a novel pyrethroid hydrolase. Int. Biodeterior. Biodegrad. 63, 1107–1112. Havens, P.L., Rase, H.F., 1991. Detoxification of organophosphorus pesticide solutions. In: Tedder, D.W., Pohland, F.G. (Eds.), Emerging Technologies in Hazardous Waste Management II. American Chemical Society, Washington, DC. Horne, I., Sutherland, T.D., Harcourt, R.L., Russell, R.J., Oakeshott, J.G., 2002. Identification of an opd (organophosphate degradation) gene in an Agrobacterium Isolate. Appl. Environ. Microbiol. 68, 3371–3376. Iyer, R., Iken, B., Damania, A., 2013. A comparison of organophosphate degradation genes and bioremediation applications. Environ. Microbiol. Rep. 5, 787–798. John, E.M., Shaike, J.M., 2015. Chlorpyrifos: pollution and remediation. Environ. Chem. Lett. 13, 269–291. Kang, N., Kim, S., Kang, Y., Kim, D., Jang, J., Won, S., Hyun, J., Kim, D., Jung, I., Rhee, G., Shin, Y., Joung, D., Kim, S., Park, J., Kwon, K., Y, J., 2015. Monitoring and exposure assessment of pesticide residues in domestic agricultural products. Korean J. Pestic. Sci. 19, 32–40. Karpouzas, D.G., Fotopoulou, A., Menkissoglu-Spiroudi, U., Singh, B.K., 2005. Non-specific biodegradation of the organophosphorus pesticides, cadusafos and ethoprophos, by two bacterial isolates. FEMS Microbiol. Ecol. 53, 369–378. Kawahara, J., Horikoshi, R., Yamaguchi, T., Kumagai, K., Yanagisawa, Y., 2005. Air pollution and young children's inhalation exposure to organophosphorus pesticide in an agricultural community in Japan. Environ. Int. 31, 1123–1132. Kiran Kumar Reddy, G., Nancharaiah, Y.V., Venugopalan, V.P., 2014. Biodegradation of dibutyl phosphite by Sphingobium sp. AMGD5 isolated from aerobic granular biomass. Int. Biodeterior. Biodegrad. 91, 60–65. Kumari, B., Madan, V.K., Kathpal, T.S., 2008. Status of insecticide contamination of soil and water in Haryana, India. Environ. Monit. Assess. 136, 239–244. Lai, K., Stolowich, N.J., Wild, J.R., 1995. Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase. Arch. Biochem. Biophys. 318, 59–64. Lerro, C.C., Koutros, S., Andreotti, G., Friesen, M.C., Alavanja, M.C., Blair, A., Hoppin, J.A., Sandler, D.P., Lubin, J.H., Ma, X., Zhang, Y., Beane Freeman, L.E., 2015. Organophosphate insecticide use and cancer incidence among spouses of pesticide applicators in the Agricultural Health Study. Occup. Environ. Med. 72, 736. Liu, Y.-H., Chung, Y.-C., Xiong, Y., 2001. Purification and characterization of a dimethoate-degrading enzyme of Aspergillus Niger ZHY256, isolated from sewage. Appl. Environ. Microbiol. 67, 3746–3749. Liu, Y.-H., Liu, Y., Chen, Z.-S., Lian, J., Huang, X., Chung, Y.-C., 2004. Purification and characterization of a novel organophosphorus pesticide hydrolase from Penicillium lilacinum BP303. Enzym. Microb. Technol. 34, 297–303. Marketsandmarkets, 2017. Inseticides Market by Type (Pyrethroids, Organophosphorus, Carbamates, Organochlorine, Botanicals), Crop Type, Mode of Application, Formulation (WP, EC, SC, EW, ME, GR), and Region - Global Forecast to 2022. Michalakis, M., Tzatzarakis, M.N., Kovatsi, L., Alegakis, A.K., Tsakalof, A.K., Heretis, I., Tsatsakis, A., 2014. Hypospadias in offspring is associated with chronic exposure of parents to organophosphate and organochlorine pesticides. Toxicol. Lett. 230, 139–145. Singh, B.K., 2008. Organophosphorus-degrading bacteria: ecology and industrial applications. Nat. Rev. Microbiol. 7, 156. Singh, B.K., Walker, A., 2006. Microbial degradation of organophosphorus compounds. FEMS Microbiol. Rev. 30, 428–471. Fig. 3. Degradation of cadusafos in upland soil by K22212 and Cam5-1. Control soil was not inoculated with the bacterial strains. Values are presented as means ± SD, n = 3. Table 1 Monod kinetic parameters of the degradation of OP insecticides by K22212 and Cam5-1. OP insecticide Strain Rate constant (h−1) t1/2 (h) r2 Ethoprophos K22212 Cam5-1 1.432 2.058 0.48 0.34 0.8987 0.9890 Cadusafos K22212 Cam5-1 0.466 0.350 1.49 1.98 0.9795 0.8436 Phenthoate K22212 Cam5-1 0.0175 0.0156 39.61 44.43 0.9403 0.9508 Phorate K22212 Cam5-1 0.0083 0.0099 83.51 70.01 0.9994 0.9729 Declarations of interest None. Acknowledgments This work was supported by the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea [project no. PJ010949]. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.ibiod.2018.05.006. References Abdel-Halim, K.Y., Salama, A.K., El-khateeb, E.N., Bakry, N.M., 2006. Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses. Chemosphere 63, 1491–1498. Abo-Amer, A.E., 2012. Characterization of a strain of Pseudomonas putida isolated from agricultural soil that degrades cadusafos (an organophosphorus pesticide). World J. Microbiol. Biotechnol. 28, 805–814. 64 International Biodeterioration & Biodegradation 132 (2018) 59–65 J.-H. Ahn et al. Yao, L., Jia, X., Zhao, J., Cao, Q., Xie, X., Yu, L., He, J., Tao, Q., 2015. Degradation of the herbicide dicamba by two sphingomonads via different O-demethylation mechanisms. Int. Biodeterior. Biodegrad. 104, 324–332. Yoon, S.-H., Ha, S.-M., Kwon, S., Lim, J., Kim, Y., Seo, H., Chun, J., 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617. Zheng, G., Selvam, A., Wong, J.W.C., 2011. Rapid degradation of lindane (γ-hexachlorocyclohexane) at low temperature by Sphingobium strains. Int. Biodeterior. Biodegrad. 65, 612–618. Stallones, L., Beseler, C.L., 2016. Assessing the connection between organophosphate pesticide poisoning and mental health: a comparison of neuropsychological symptoms from clinical observations, animal models and epidemiological studies. Cortex 74, 405–416. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Theriot, C.M., Grunden, A.M., 2011. Hydrolysis of organophosphorus compounds by microbial enzymes. Appl. Microbiol. Biotechnol. 89, 35–43. World Health Organization, 2010. The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 2009. Germany, Stuttgart. 65