research advances in organophosphorus pesticide

© by PSP Volume 25 – No.1/ 2016,

Fresenius Environmental Bulletin

RESEARCH ADVANCES IN ORGANOPHOSPHORUS PESTICIDE DEGRADATION: A REVIEW Xiyan Ji, Qiang Wang, Wudi Zhang, Fang Yin Yunnan Normal University, Kunming 650500, P. R. China

ABTRACT The technologies related to the microbial biodegradation of organophosphorus pesticide biodegradation are currently being heavily researched. This article reviews various types of microbial degradation of organophosphorus pesticide, degradation mechanism and degradation genes and discusses the recent development trend of microbial degradation of organophosphorus pesticide. This paper also introduces the current research status of biodegradation technologies around the world including China. This paper primarily presents the research results of organophosphorus pesticide biodegradation. Lastly, the authors exhibited some suggestions for further researches. KEYWORDS: organophosphorus pesticide (OPs); biodegradation; microbial degradation; microbial species; degrading mechanism; degradation genes

INTRODUCTION Pesticide is termed as chemical that kills or slows down the growth of undesirable organisms. Today, organophosphorus pesticide (OPs) is the most commonly used category of pesticide used in agriculture and horticulture practices, with as many as 150 kinds of organophosphorus commodity pesticides available in the world [1]. In China, about 30 pesticides including insecticide, herbicide and bactericide have been commonly used. Pesticides are biologically active compounds designed to interfere with metabolic processes and environmental safety. And many researchers pay attention to the field of OPs degradation. Fodale’s result shows that 47 strains were isolated from Sicilian soils under different management systems [2].Wang’s result suggests that MTM ( mesoporous TiO2 membrane) TiO2 membrane has good potential for treatment of aqueous organophosphorus pesticides [3]. Kotonia used gas chromatography-mass spectrometry

(GC-MS) to determinate 14 insecticides and metabolites in grapes and peaches [4]. Generally, OPs contain two primary varieties, phosphate, and O-ethyl-S-propyl phosphorothiolatex, as seen in Fig1. Among the OPs that most widely produced and used in China, R1 or R2 represents methoxyl (CH3O) or ethoxy (C2H5O). X is for oxygen atom (O) or sulphur atom (S). Z is for alkoxy, phenoxyl or other complicated substituent group. It can form a variety of compounds depending on which different substituent group is present. According to their structures, organophosphorus pesticides can be divided into phosphate, phosphonic acid ester, and phosphorous amides with corresponding glucosinolates derivatives. Among the toxic pesticides, organophosphorus pesticide is the most common. Organophosphorus pesticide toxicity studies had shown that organophosphorus pesticides have alkylation function which can poison animals and human body. They can be absorbed by inhalation, ingestion, and skin penetration. Thousands of humans including children are affected by OPs toxicity across the world annually. For instance, children exposed to OPs are more likely to suffer from attention deficit hyperactivity disorder (ADHD) (Samuels and Obare, 2011). Importantly, frequent use of OPs in agricultural practices and their presence as residues in fruits, vegetables, livestock, poultry products and municipal aquifers are considered vectors for the exposure. As such, OPs degradation has been a major concern in environmental chemistry and is currently being studied worldwide. This has lead to major advancements in OPs degradation technologies. The definition of OPs degradation is that OPs chemical structure changes under some factors correlated with environment, where the OPs molecules are degrade into smaller molecules such as CO2, PO43- and H2O. The degradation phenomenon is divided into 3 steps [5]: ①Initial degradation: OPs parent structure breaks down, leading the change in characteristics; ②Secondary degradation: The products of



R1 and R2 represent methoxyl (CH3O) and ethoxy (C2H5O), respectively. X is for oxygen atom (O) or sulphur atom (S). Z is for alkoxy, phenoxyl or other complicated substituent group.

FIGURE 1 Organophosphorus pesticide general structure formula and several typical organophosphorus pesticide chemical structures degradation don’t pollute the environment; ③ Final degradation: The substrate (organophosphorus pesticide) fully transformed into inorganic substances such as CO2, PO43- and H2O. Although, there are a lot of studies on organophosphorus pesticide degradation very few reports touch on microbial degradation. We seek to fill this void and will primarily discuss biodegradation methods and microbial degradation. Biodegradation Method. Although traditional physical and chemical degradation treatments are effective against OPs, has good effect, the cost is too high and easy to cause secondary pollution, so it only can be used as an assessment method. In contrast, bioremediation technology has been developed to remove and control the organic matter pollution, and has recently become the most popular method. The basic purpose of this technology is to use a variety of methods to strengthen the microbial community and accelerate the degradation of organic pollutants [5] in the environment . Moreover, microbiological degradation is considered as an effective and environment friendly method to eliminate OPs pollution. Since the 1940’s research has confirmed the microbial degradation of pesticide played an important role, and has attempted to isolate of pesticide degrading bacteria and the degradation pathway [6,7].

High Efficient Bacteria Separation. Longterm mass production and extensive use of organophosphorus pesticide in China has lead to serious water and soil pollution as well as, lead to excessive pesticide residues on agricultural products, sparking food safety problems. Excessive OPs residue has become a major inhibitor to the expansion of China’s agricultural product exports. Therefore, it is necessary to establish an effective method to control environmental pollution and remove agricultural farming pesticides residues. With the strong metabolic diversity, microorganisms have many advantages in the degradation [8] process of pesticides . At present, the separated microorganisms include bacteria, fungi and actinomycete. Bacteria played an important role in the degradation process because of its various biochemical adaptability and good ability of inducing mutants. In recent years, the in-depth study on biodegradation pathway has provided the possibility for bioremediation. Any microorganisms have been isolated and identified, including Agrobacterium Escherichia coli, Flavobacterium sp., Bacillus stearothemophilus, Pseudomonas, strain B-1, strain SC, F. sp. Strain ATCC27551, Alteromonas species, Arthrobacter, Nocardiasp., Corynebacterium glutamicum, Burkholderia sp. Strain NF100, Bacillus cereus J5, WBC-3, cyanobacterium Anabaena



sp. Strain PCC7120, Ochrobacterum sp. B2. Some studies have reported the effects of microorganism on biodegradation of pesticides. For instance, Zhang et al. [8] investigated degradation of methyl parathion in soil and Chinese chive by strain DLL-1at concentrations of 7.5, 15, 22.5, 75 kg(a.i.)ihm-2. From their result, usage of methyl parathion at 7.5, 15 and 22.5 kg(a.i.)ihm-2 resulted in the average amount of residue of 0.663, 1.270 and 1.901mgnt -1, respectively in Chinese chive. The natural degradation rate was 98.94% ， 96.44%，and 96.04% corresponding to the 3 levels of usage. At the concentration of 75 kg·hm-2 of degradation bacterium, the amount of pesticide residue in Chinese chive and its soil were 0.269 and 0.099 mg·kg-1 respectively which were decreased by 78.82% and 98.68% compared with the control. Thus, the amount of pesticide residue could be significantly decreased through the application of high effective degrading microbial agents. However, usage of degradation bacterium more than 75kg·hm-2 did not increase the degradation rate further. In bacterial isolation study, Jia et al. [9] have found that a strain Pm-6 of methamidophos-degrading bacterium was successfully isolated from the pesticidescontaining soil in a pesticide factory. The strain was identified as Acinetobacter sp. This bacterium was stable when store for 30 days at 4℃ with slight change in capability. Likewise, Meiqin Yi [10] isolated three strains of Fungi (Hw 23, Hw 26 and Hw 27) using liquid enrichment culture from active sludge at Huayang Pesticide Factory for degradation of methyl parathion. They were able to use methyl parathion as the carbon and energy for growth. Daiet al [11] and Qiu et al [12] argued convincingly that a triazophos degrading bacterium designated as mp-4 wereisolated from soils that have long been subjected to organophosphate pollution. Strain mp-4 was identified as Ochrobactrum.sp based on its biochemical-physiological characteristic and the 16 SrDNA homologue sequence analysis. Strain mp-4 can grow with triazophos as its sole carbon source and degrade it at a rate of 98.3％. The optimal growth temperature and pH for mp-4 are 30℃ and 6.6, respectively. At the temperature of 27-32℃ and pH of 7.5-8.8, Mp-4 can degrade triazophos well. Thus, many research showed principal mechanism for microorganism adaption to organophosphorus pesticides contained environment and efficiency of microorganism for pesticides degradation or removal. Different studies showed that some organophosphorus pesticide

can have a variety of degrading bacteria present at the same time, while one kind of bacteria can degrade kinds of OPs. This reflects the diversity of microorganisms and microbial functional diversity. With the continuous development of biotechnology, people have made a lot of beneficial explorations on OPs degradation finding that mesophilic microorganisms exert a great pole course in the OPs degradation in the natural environment. The use bioremediation technology is effective, economical and environmental friendly technology to [13, 14] accelerate the degradation of OPs . However, most of the studies on OPs degradation recommend moderated temperature and high concentration conditions, so mostly the microorganisms are usually isolated on room temperature. In high altitude area or cold region, the temperature is mostly below the normal temperature and degradation efficiency will be affected, while also requiring a low concentration in the water [15]. All of these disadvantages constitute a barrier to microbial growth. As a consequence, He Li [16, 17] found that the bacterial strain, which was isolated from the polluted soil in north-east china, was capable of degrading various organophosphorus pesticides effectively. Strain SA-8 was initially identified as Plwsimonas sp. according to its biochemical characters. The optimal temperature and pH value for strain SA-8 growth were 20℃ and 7.0 respectively. The biodegradation rate of methyl-parathion can reach 93% after 24h cultivation in suitable conditions. When the temperature is 10℃, its degradation rate is 66.2%, but only 27.3% degradation rate at 35 ℃. The experiment demonstrated that strain SA-8 is a good psychrotroph. Microbial Degrading Mechanism of OPs. Organophosphorus pesticides usually contain P-O and S- P bonds. In addition, some OPs (e.g. methamidophos) also contain P-N bond. The degradation of OPs by bacteria is generally carried out through the hydrolases and transfer enzymes function, acting on P-Oalkyl, P-O-aryl, O=P-NH2, =C-NH2. The study on the OPs degradation pathway found that microorganisms can break the P-O bond or P-S bond and some depurated organophosphorus pesticide hydrolase (OPH) have significantly difference in substrate specificity, sensitivity to the chemicals and molecular weight. Initial metabolism of OPs is hydrolytic reaction, and its initial products are O, O-dimethyl dithiophosphoric acid and p-nitrophenol [18,19]. M. B. Angelica also confirmed that the P-S bonds of organophosphorus pesticides can be



cut off by chlorinated pesticides. Some researchers for instance, Donna & Ulrica [20] and, Thomas and Macaskie [21] studies showed that there is a certain correlation between the microbial phosphate solubilizing capacity and the pH of culture medium, however they also found that decrease in pH value is not the necessary conditions of the phosphate solubilizing. Further research on this area could be interesting and important. According to Liu et al. [22] and, Liang et al. [23] degradation process caused by intracellular enzyme can be divided into 3 steps: firstly, as is a kind of dynamic balance, i.e. OPs compounds absorb on cell membrane; secondly, OPs compounds that absorb on cell membrane go into cell under the constant biomass condition and the penetration rate determines the amount of compounds inside cell; thirdly, as a enzymatic reaction in the cell which is a quick process and also not a limiting step. Pseudomonas stutzeri JHY01 was capable of degrading Chlorpyrifos, a typical organophosphorus pesticide [24, 25]. The ultrasonic cell-break method was used to extract the degrading enzyme. Lan et al [24] or [25] Jeon et al conducted a study on the chlorpyrifos degradation through extracellular enzyme, intracellular enzyme and cell fragment. Their experimental results showed the degradation rates of chlorpyrifos by extracellular enzyme, intracellular enzyme and cell fragment were 8.41%, 79.85% and 77.14%, respectively, indicating that Chlorpyrifosdegrading enzymes were typical intracellular enzymes. Later, orthogonal experiments were designed by them to optimize the extraction conditions of the degrading enzymes. The degrading activity of the crude enzymes was maximal (with the degradation rate 84.47%) at the following ultrasonic conditions: the ratio of cells biomass to extraction buffer volume 1:4 (g/ml), ultrasonic treatment for 4 seconds, totally 30 times at interval of 4 seconds (UAS, Sonics-VCX500, 500W, 25KHz). According [26] to Wang et al , an organophosphorus hydrolase from strain Aspergillus niger J6 was isolated and purified by means of cell

disruption, ammonium sulfate precipitation and Sephadex G-75 chromatography. In addition, enzymatic properties were also studied. The results showed that the degradation enzyme was located on the cell membrane, with its molecular weight being 66 000 by SDS-PAGE. The optimal reactive temperature and pH of the degradation enzyme was 70 ℃ and pH 7.0 respectively. Some metal ions (Fe3+,Cu2+,Li+,Zn2+,Mg2+) and chemical reagents (NaN3, EDTA, SDS and PSMF) showed synergistic effect on the degradation enzyme activity, while Mn2+ displayed slight inhibition, and Ca2+ had strong inhibition [26]. Organophosphorus Pesticides Degradation Microorganisms. In recent years, many researchers had screened various kinds of bacteria (fungi, actinomycetes, algae and other microbial strains) which have the ability to degrade the organic phosphorus pesticide through the enrichment, isolation and screening technology from the natural soil. These microorganisms can be classified into Shah Ray Ties, Pseudomonas, Burkholderia spp., Roseomonas, Ochrobactrum, Klebsiella, Bacillus E6, Plesiomonas sp. M6, fecal production alkali bacillus, sp. Diaphorobacter, Klebsiella sp., and Holder Burke (sp. Burkholderia). Table 1 summarizes the microorganism and types of organic phosphorus which can degrade by corresponding bacteria. Xu Gangming [27,28] reported that strains from three pesticide factory sewage treatment pool of activated sludge, which degraded Chlorpyrifos effectively. These strains are bacterial and accessory bacteria (Paracoccus sp. DM/TRP), serratia sp. TCR, and Trichosporon sp.TCF. Based on the expressed mpd gene sequence and opd gene, he designed several pair gene primers. Gene amplification in different conditions was made using general PCR and Touch-Down PCR (TD-PCR) by taking total bacterial NDA as a template. These strains can be completely mineralized and degrade chlorpyrifos, the same with intermediate product trichloropyrindinol.

TABLE 1 Organophosphorus pesticide degrading microorganism OPs. Isocarbophos Triazophos Chlorpyrifos Parathion-methyl Fenitrothion

Microorganism Bacillus latersprorus, Serratia, Pseudomonas Roseomonas, Ochrobactrumanthropi, Alcaligenesfaecalis, Klebsiellaaerogenes Pseudomonas, Diaphorobacter sp. Pseudomonas, Diaphorobacter sp. Burkholderia cenocepacia, Diaphorobacter sp.



PROBLEMS AND OUTLOOK Substantial progress has been accomplished in the development of technologies for OP pesticides degradation in recent years. However, at present, the separation of strains and microbial degradation are limited to the lab-scale, and still remains to be applied in the field practice. This could be mainly due to need for degradation conditions for the effective use of microbial degradation of organophosphorus pesticides that are hard to achieve outside of the lab. Temperature, pH, concentration of organophosphorus pesticide and microbial essential nutrients will change a relatively large range, under natural conditions. It can’t ensure the effect of the microbial breeding, even may inhibit its growth. Therefore, further future study of microbial degradation of organophosphorus pesticide should emphatically focus on the following several aspects: ①Explore the degradation strains with growing conditions which are similar to the natural environment; cultivation, development and utilization of high efficient bacteria for organophosphorus pesticide, establish high efficient degradation bacterium seed (gene) bank. ②Extract the related genes and remove into a common species, such as ecoli, etc. for degradation enzyme gene cloning and expression, constructing engineering bacteria, improve the ability of degradation, prepare degradation enzyme, on the basis of identification of degradation enzyme gene. ③Field practice of biological pesticide which can help to reduce the use of heavy toxicity pesticides; ④Promotion of more applied research on the microbial method that dealing with degradation of contaminants practically in real field to the practice. ACKNOWLEDGEMENTS This work was supported by Research Fund for the Doctoral Program of Higher Education (20135303110001), National Natural Science Foundation of China (51366015), Applied Basic Research Programs of Yunnan Province (2014FA030), and Science and Technology Innovation Platform Construction Plan of Yunnan Province (2013DH041). REFERENCES [1] S. G. Dai, Environmental Chemistry [M], Beijing, Higher Education Press (1997). [2] R.Fodale, C.De Pasquale, L.Lo Piccolo, et al, (2007) Isolation of organophosphorus-

degrading bacteria from agricultural mediterranean soils, Fresenius Environmental Bulletin,19 (10B), 2396-2403. [3] P.Wang, D.S.Bi, Y.Chen, (2015) Ultraviolet irradiation of a mesoporous TiO2 membrane for removing organophosphorus pesticides from water, Fresenius Environmental Bulletin,24 (5A), 1779-1785. [4] C.A. Kotonia, K.S.Liapis, V.N.Ziogas, Determination of residues of 14 insecticides and metabolites in grapes and peaches by gas chromatography mass spectrometry, Fresenius Environmental Bulletin, 16 (3), 223226. [5] Z. G. Jin, T. Zhang, H. L. Zhu, Pollutant Biodegradation [M], Shanghai, East China University of Science and Technology Press (1997). [6] D. S. Tara, H. Irene, J. L. Michael, (2000) Enrichment of an endosulfan-degrading mixed bacterial culture, Applied and Environ Microbiol,66 (7), 2822–2828. [7] Z. Liu, S. P. Li, (2003) Mutation breeding of methyl parathion degrading strain DLL-1 (Pseudomonas sp.), ActaPedologicaSinica, 40 (2), 293-298. [8] R. F. Zhang, J. D. Jiang, Z. L. Cui. (2004) Degradation of methyl parathion in soil and Chinese chive by strain DLL-1, Chinese journal of applied ecology, 15 (2), 295-298. [9] R. Jia, Z. R. Hong, Q. Li, Y. Wang, (1997) Isolation of methamidophos-degrading bacterium and studies on its properties, Journal of Anhui University Natural Science Edition, 21 (1), 98-101. [10] M. Q. Yi, K. Y. Wang, X. Y. Jiang, Y. J. Wang, (2000) Studies on the isolation and characterization of fungi for degradation of methyl parathion, Chinese Journal of Pesticide Science, 2 (4),40-43. [11] Q. H. Dai, R. F. Zhang, J. D. Jiang, L. F. Gu, S. P. Liu, (2005) Isolation, identification and characterization of triazophosdegrading bacterium mp4, ActaPedologicaSinica, 42 (1), 111-115. [12] S. L Qiu, Z. L. Cui, B. Fan, Q. H. Dai, S. P. Li, (2004) Construction of GFP-Labelled pseudomonas putida DLL-1, a methylparathion-degrading bacterium, Chinese Journal of Applied and Environmental Biology, 10 (6), 778-781. [13] A. Bonnechere, V Hanot, C Bragard, et al. (2013) Effect of household and industrial processing on the levels of pesticide residues and degradation products in melons, Food Additives and Contaminants Part A, Chemistry, Analysis, Control, Exposure and Risk Assessment, 29 (7), 1058-1066. [14] D. Sun, J. Qiu, Y. Wu. (2012)



Enantioselective degradation of indoxacarb in cabbage and soil under field conditions, Chirality, 24, 628-633. [15] F. Dong, B. Chankvetadze (2013). Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil, Environment Science and Technology, 47, 3386-3394. [16] H. Li, W.Q. Liang, X. Y. Wu, Y. H. Liu, (2010) Research on biodegradation of organophosphorus insecticide by a novel psychrotrophic Bacterium SA-8, ActaScientiarum Nature AliumUniversitatisSunyatseni, 43 (3), 131-132. [17] S. Dayananda, K. Syed, M. Mike (2003). Transposon-like organization of the plasmidborne organophosphate degradation gen cluster found in Flavobacterium sp., Appl Environ Microbiol, , 69, 2533-2539. [18] F. Oppong, G. Sagar, (1992) Degradation of triasulfuron in soil under laboratory conditions, Weed Research, 32, 167-173. [19] N. Sethunathan, T. Yoshida. (1973) A flavobacterium sp. that de-grades diazinon and parathion. Can J Microbial, 19, 873 -875. [20] C. Donna, S. Ulrica, (2001) Cocomposting of cattle manure and hydrocarbon contaminated flare pit soil, Compost Science and Utilization, 9( 4), 322335. [21] R. A. Thomas,L. E. Macaskie. (1998) The effect of growth conditions on thebiodegradation of acid mine drainage waters by a naturally-occurringmixed microbial culture. Appl. Microbial.Biotechnology, 49, 202-209. [22] Y.H. Liu, Y. C. Chung, Y. Xiong. (2001) Purification and characterization of a dimethoatedegrading enzyme of Aspergillus niger ZHY256， isolated from sewage, Appl. Environ Microbial,67, 3746- 3749. [23] W.Q.Liang, Wang Z. Y., Li H. (2005) Purification and char-acterizationof a novel pyrethroid hydrolase from aspergil-lusniger ZD11,Journal of Agricultural and Food, Chemistry,53, 7415- 7420. [24] Y. H. Lan, M. Xie, F. L. Chen, F. H. Wang, S. Y. Zhou. (2008) Localization and

extraction conditions of chlorpyrifosdegrading enzyme from Pseudomonas stutzeri, Chinese Journal of Biological Control,24 (4), 349-353. [25] Y. H. Jeon, S. P. Chang, I. G. Hwang. (2003) Involvement of growth-promoting rhizobacteriumPaenibacilluspolymyxa in root rot of stored Korean Ginseng,Journal of Microbiology and Biotechnology, 13 (6), 881891. [26] J. W. Wang, J. Zhen, B. E. Xie, Y. Y. Liu, G. J. Li.(2012) Purification of a organophosphorus pesticidedegrading enzyme from As-pergillusniger J6 and its properties, Jiangsu J. of Agr ． Sci., 28(3), 549-554. [27] G.M. Xu. (2007) Isolation and Characterization of Organophosphorus Degrading Microbes and Their Mineralizing Mechanism [D], Shandong Agricultural University. [28] Tova A. Samuels and Sherine O. Obare (2011). Advances in Analytical Methods for Organophosphorus Pesticide Detection, Pesticides in the Modern World - Trends in Pesticides Analysis, Dr. Margarita Stoytcheva (Ed.),ISBN:978-953307-437-5, InTech, Availablefrom: http://www.intechopen.com/books/pesticidesin-themodern-world-trends-in-pesticidesanalysis/advances-in-analytical-methods-fororganophosphorus-pesticidedetection.

Received: Accepted: CORRESPONDING AUTHOR Wudi Zhang, Energy and Environmental Science School, Yunnan Normal University No.768, Jingming St. Kunming City, Yunnan Prov., China, 650500 Tel: +86 13508714255 Email: [email protected]