Organophosphorus Pesticides Analysis

The organophosphorus (OP) pesticides are synthetic esters, amides, or thiol derivatives of the phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids (Corbett et al., 1984; Eto, 1974; Gupta, 2006; Hassall, 1982; Quin, 2000). The structural diversity of this family of compounds is reflected in their physicochemical and biological properties: vapour pressure, solubility in water, chemical stability and toxicity (Corbett et al., 1984; Hassall, 1982; WHO, 1986), which determine their specific application (Hassal, 1982). Compared to the mostly banned in U.S. and Europe organochlorine pesticides, the OP ones are less persistent in the environment, are not subject of bioaccumulation and biomagnification, and do not release toxic break down products (Krieger, 2001). These features justify their application in the agricultural and veterinary practices of the modern world. In 1999 the OP insecticides represented ≈37% of the pesticides in use at a global scale (Table 1) and 72% of the insecticides used in U.S. (Kiely et al., 2004). The top ten OP insecticides active ingredients include malathion, chlorpyrifos, terbufos, diazinon, methyl-parathion, phorate, acephate, phosmet, azinphos-methyl, and dimethoate (Kiely et al., 2004).


Introduction
The organophosphorus (OP) pesticides are synthetic esters, amides, or thiol derivatives of the phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids (Corbett et al., 1984;Eto, 1974;Gupta, 2006;Hassall, 1982;Quin, 2000).The structural diversity of this family of compounds is reflected in their physicochemical and biological properties: vapour pressure, solubility in water, chemical stability and toxicity (Corbett et al., 1984;Hassall, 1982;WHO, 1986), which determine their specific application (Hassal, 1982).Compared to the mostly banned in U.S. and Europe organochlorine pesticides, the OP ones are less persistent in the environment, are not subject of bioaccumulation and biomagnification, and do not release toxic break down products (Krieger, 2001).These features justify their application in the agricultural and veterinary practices of the modern world.In 1999 the OP insecticides represented ≈37% of the pesticides in use at a global scale (Table 1) and 72% of the insecticides used in U.S. (Kiely et al., 2004).The top ten OP insecticides active ingredients include malathion, chlorpyrifos, terbufos, diazinon, methyl-parathion, phorate, acephate, phosmet, azinphos-methyl, and dimethoate (Kiely et al., 2004).
Table 1.Summary of the annual OP insecticides consumption (metric tons of active ingredients) and of the annual total pesticides consumption (metric tons of active ingredients), according to the Statistics Division of the Food and Agriculture Organization of the United Nations (FAOSTAT, 1990(FAOSTAT, -2007)).
Nevertheless, because of the high acute toxicity of the OP pesticides (Eddleston et al., 2008;Gupta, 2006;Roberts & Aaron, 2007), as well as because of the registered chronic effects (Gupta, 2006), the OP pesticides residues limits in food, drinking water and environmental samples are subject of regulation and control.The European Council Directive 98/83/EC on the quality of water intended for human consumption (Council Directive 98/83/EC, 1998) sets the limit value of the individual pesticides in drinking water at 0.1 µg L -1 and that of the total pesticides at 0.5 µg L -1 .According to the U.S. Environmental Protection Agency Office of Ground Water and Drinking Water (OGWDW), the health advisory levels for some OP pesticides in drinking water are: diazinon 3 µg L -1 , parathion-methyl 2 µg L -1 , disulfoton 1 µg L-1, fenamiphos 2 µg L-1, etc., the following 22 OP pesticides being on the U. S. National Pesticide Survey List: diazinon, dichlorfos, dicrotophos, dimethoate, diphenamiphos, sulfone, disulfoton, disulfoton sulfone, disulfoton sulfoxide, fenamiphos sulfone, fenamiphos sulfoxide, fenitrothion, methyl  EEC, 1990) set the maximum residue limits (MRLs) for pesticides in food commodities.Other organisations involved in establishing the pesticide residues levels are the World Health Organization (WHO), the Food and Agricultural Organization of the United Nations (FAO), the Codex Alimentarius Commission, and the U.S. Environmental Protection Agency (EPA).Currently, EPA is reassessing pesticide residue limits in food to ensure that they met the safety standard established by the Food Quality Protection Act of 1996 (FQPA, 1996).Some relevant data on MRLs are presented in Table 2.

Pesticide
MRLs (varie according to the product, mg kg -1 ) Pesticide MRLs (varie according to the product, mg kg The MRL of a number of OP pesticides is set up at or about the limit of their determination by the currently available analytical methods.Thus, in this work are reviewed the developed during the last years (2005)(2006)(2007)(2008)(2009)(2010)(2011) procedures for OP pesticides analysis, including sample pretreatment and determination in the context of the implementation of modern reliable, high sensitive, selective, rapid, cost-effective and environmental friendly analytical techniques for OP pesticides residues quantification.This overview comments on their advantages and limitations.
The tendencies in the application of gas chromatography and liquid chromatography to pesticides residues determination in environmental samples and food, together with sample preparation procedures are revised by several authors (Le Doux, 2011;Pareja et al., 2010;Sharma et al., 2010;Yusà et al., 2009).The trends in liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry development are highlighted in the published in 2007, 2009, and 2010reviews (Kuster et al., 2009;;Malik et al., 2010;Petrovic et al., 2010;Soler & Picó, 2007).
Relevant data collected for the period 2010-2011, revealing the analytical performances of some selected chromatographic methods are summarized in Table 3. Table 3. Analytical performances of some chromatographic methods applied to OP pesticides analysis, namely LOD.

Electrochemical methods for OP pesticides analysis
The electrochemical activity of the OP pesticides containing nitro-phenyl groups: parathionmethyl, parathion, paraoxon, fenitrothion, etc. makes possible their direct electrochemical determination.The mechanism of the redox process, selecting parathion-methyl as a model, is the following: The electrochemical method of choice applied to OP pesticides analysis, considering the period 2005-2011, is square-wave voltammetry (Li and al., 2011;Parham and Rahbar, 2010;Sbaï et al., 2007;Tan et al., 2010;Wang and Li, 2008).It is recognized as a high sensitive regarding the detection of organic molecules.Current efforts are directed toward sensitivity and selectivity improvement by chemical modification of the electrode surface.The reported techniques include: nano-ZrO 2 modification of a carbon paste electrode (Parham and Rahbar, 2010), fabrication of ZrO 2 /Au nano-composite films on alumina substrates (Wang and Li, 2008), electrodeposition of molecularly imprinted porous silicate (Tan et al., 2010) or of gold-sodium dodecylbenzene sulfonate nanoparticles onto a glassy carbon electrode (Li and al., 2011a), tetrasulfonated phtalocyanine electrodeposition combined with Nafion coating of carbon fibres (Sbaï et al., 2007), etc. ZrO 2 nanoparticles in particular, used as a selective sorbents for solid-phase extraction, demonstrate excellent performance in OP pesticides detection, because of their affinity toward the phosphate group on OP pesticide molecule (Parham and Rahbar, 2010;Wang and Li, 2008).
The developed electrochemical methods are applied for OP pesticides determination in pears (Li and al., 2011;Tan et al., 2010), and in water samples (Parham and Rahbar, 2010).Some relevant data revealing the sensitivity of the determinations are presented in Table 4. Table 4. Analytical performances of square wave voltammetry, applied to OP pesticides analysis, namely LOD.
The analysis of the reported studies confirms that the electrochemical methods have the advantage to be rapid, sensitive, selective, and accurate.In addition, they use affordable, portable, and miniaturized instrumentation.These characteristics make them appropriate for the "in field" determination of the low persistent in the environment OP pesticides.

Immunochemical methods for OP pesticides analysis
Only few immunochemical methods for OP pesticides analysis were reported during the surveyed period 2005-2011.Gui and al. (2006) synthesize two haptens of the OP insecticide triazophos and develop an enzyme-linked immunosorbent assay based on monoclonal antibody, demonstrating high affinity and specificity to triazophos.Guo and al. (2009) investigate two formats of gold-labeled antibody lateral-flow strips for the simultaneous detection of triazophos and of the carbamate pesticide carbofuran.The application of the immunogold labeling technique in immunoassays is reviewed by Lai et al. (2010).Garcés-García and al. (2006) Gui and al., 2006 Table 5. Analytical performances of some immunochemical methods, applied to OP pesticides analysis, namely LOD.

Biosensors based methods for OP pesticides analysis
The overview of the publications covering the period 2005-2011 shows that almost all of the described biosensors for OP pesticides analysis are electrochemical ones.The OP pesticides determination is based on the quantification of the acetylcholinesterase inhibition, they provoke.The enzyme activity is determined by electrochemically monitoring the thiocholine formed upon enzymatic hydrolysis of acetylthiocholine.Three alternative routes are explored as response-generating electrochemical reactions: i. Direct electrochemical oxidation of thiocholine at 0.80 V/Ag, AgCl (Ivanov et al., 2010;Marinov et al., 2010;Ovalle et al., 2009): 2(CH 3 ) 3 N + (CH 2 ) 2 SH → (CH 3 ) 3 N + (CH 2 ) 2 S-S(CH 2 ) 2 N + (CH 3 ) 3 +2H + +2e − It is important to note that potential lowering and hence interferences elimination could be achieved by using nanostructured materials for electrode modification.Nanoparticles reduce the working potential by catalysing the electrochemical thiocholine oxidation (Du, 2007).ii.Mediated thiocholine oxidation at lower electrode potential (0.1÷0.45 V/Ag, AgCl), using cobalt phtalocyanine (Alonso et al., 2010;Law & Higson, 2005), tetracyanoquinodimetane (Hildebrandt et al., 2008) or hexacyanoferrate (III) (Ovalle et al., 2009) as electron mediators in a heterogeneous or in a homogeneous phase: Chemisorption of thiocholine at -0.7 V/Ag, AgCl and electrochemical desorption in KOH, giving a measurable reductive peak current (Du et al., 2008).The majority of the publications report the application of screen-printed electrodes (Alonso et al., 2010;Dondoi et al., 2006;Hildebrandt et al., 2008, Law & Higson, 2005) as transducers.Recent developments in the field of screen-printed electrodes and their related applications are comprehensively reviewed by Renedo et al. (2007).Disposable screen-printed electrodes are considered as an alternative to the traditional electrodes for "in situ" analysis.As immobilization matrices in electrochemical acetylcholinesterase-based sensors for OP pesticides determination were preferentially used various nanomaterials: nanostructured polymer membranes with integrated multiwall carbon nanotubes (Ivanov et al., 2010) or gold nanoparticles (Marinov et al., 2010), multiwall carbon nanotubes-chitosan composite (Du et al., 2007), etc., to achieve sensitivity increase and sensor stability improvement.Recent trends and challenges in developing nanomaterials-based biosensors for OP pesticides detection are discussed by a number of authors (Balasubramanian & Burghard, 2006;Eftekhari, 2008;Gorton, 2005;Guo & Wang, 2007;Kerman et al., 2008;Kumar, 2007;Liu et al., 2008, Luo et al., 2006;Merkoçi & Alegret, 2005;Merkoçi, 2009;Pumera et al., 2007, Wang & Lin 2009).The alternative route leading to biosensors sensitivity, selectivity and stability increase involves the incorporation in the biosensing platform of biorecognition elements with tailor designed properties.Genetically modified enzymes are extensively used in inhibition based biosensors for OP pesticides determination (Bucur et al., 2005;Marques et al., 2005;Valdés-Ramírez et al., 2008), allowing attaining LOD as low as 10 -17 M (Sotiropoulou et al., 2005).Current research efforts are reviewed by Campás et al. (2009).Another important issue associated with electrochemical biosensors development is that concerning chemometrics.It was demonstrated that artificial neural networks implementation could resolve mixtures of pesticides (Alonso et al., 2010).Exhaustive reviews on enzyme inhibition-based biosensors, including inhibition determination in organic phase are provided by Amine et al. (2006) and López et al. (2006).The application of various enzymes: acetylcholinesterase, acid phosphatase, alkaline phosphatise, organophosphorus hydrolase, and tyrosinase for the quantification of OP pesticides in the environment is extensively revised by Van Dyk et al. (2011).Another group of electrochemical biosensors for OP pesticides analysis is that of the microbial sensors.Such sensors, based on Clark dissolved oxygen electrode modified with recombinant p-nitrophenol degrading/oxidizing bacteria endowed with OPH activity were reported by Lei et al. (2005Lei et al. ( , 2006)).The surface-displayed OPH catalyzes the hydrolysis of OP pesticides with nitrophenyl substituent to release products, metabolized by the bacteria while consuming oxygen.The oxygen consumption is measured and correlated to the OP concentration.A microbial biosensor for direct determination of nitrophenyl-substituted organophosphate nerve agents using genetically engineered Moraxella sp. has been proposed by Mulchandani et al. (2006).However, the reached LOD is over the OP concentration in environmental samples and higher than that for acylcholinesterases inhibition-based sensors, immunoassays, and gas, liquid and thin layer chromatography (Mulchandani et al., 2006).Recently, an electrochemical hybrid biosensor for OP pesticides trace level concentrations determination was developed and characterized (Stoytcheva et al., 2009).It integrates a hybrid biorecognition element consisting of immobilized Arthrobacter globiformis and free acetylcholinesterase (ACh) with a Clark type oxygen probe transducer.The bacteria convert the ACh-generated choline to betaine with oxygen consumption measured as a Clark probe current change.This change, representing the sensor response, correlates to the concentration of the OP pesticides inhibiting the Ach catalyzed acetylcholine hydrolysis to choline.Current progress in microbial electrochemical and optical biosensors are reported by Su and al. (2011).
Finally, the few works commenting on optic-and immuno-biosensors development and application to OP pesticides analysis have to be pointed out, too (Llorent-Martínez et al., 2011;Mauriz et al., 2006aMauriz et al., , 2006b;;Suri et al., 2009).Special attention should be paid to the overview of Jiang and al. (2008), presenting the various transduction systems used in immunosensors: electrochemical, optical, piezoelectric, and nanomechanic, and the immobilization protocols.Some relevant data are presented in Table 6.Table 6.Analytical performances of some biosensors-based methods, applied to OP pesticides analysis, namely LOD.

Chemometrics applied to OP pesticides analysis
Current progress on the application of chemometrics to evaluate the occurrence of organic pollutants, including OP pesticides in the environment are reviewed by Mas et al. (2010).
The interpretation of the results of the analytical determination of these substances, applying chemometric approaches is among the addressed issues.
On the improvement of the electroanalytical techniques in particular with the aid of chemometrics (partial least squares, artificial neural networks, and multiple curve resolution methods) comment Ni and Kokot (2008).Some of the suggested methods are successfully applied to OP pesticides analysis.
The strategies for the enhancement of the spectroscopic photochemistry by chemometrics are discussed in the overview presented by Liu et al. (2009).The chemometric methods revealed their efficacy for the simultaneous and selective enzymatic spectrophotometric determination of carbamate (carbofuran, carbaryl) and OP pesticides (chlorpyrifos, dichlorfos, phoxim) (Ni and al., 2007;Rhouati and al., 2010), and for the simultaneous determination of OP pesticides residues: dipterex, dichlorvos and omethoate in vegerable samples by continuous-flow chemiluminescence without any previous separation (Li et al., 2007).

Conclusion
The continuous lowering of the maximum residue limits of the OP pesticides in food and in the environment calls for the development of sensitive methods for their determination.Such high effective techniques, well suited for testing complex matrices, are the chromatographic ones.Nevertheless, the review of the advances in OP pesticides analysis during the period 2005-2011 demonstrated that despite of the efforts to reduce solvent consumption and to simplify sample pretreatment, the chromatographic determinations remain expensive and time consuming.In addition, they require experienced personnel and sophisticated laboratory equipment, inappropriate for "in field" application.Therefore, a number of alternative electrochemical and biosensors-based techniques were suggested.
Because of the inexpensive instrumentation, the simple operation procedure without or with a minimum sample pretreatment, and the high sensitivity, they gain more and more importance in OP pesticides analyses, as an excellent complement to the classical analytical techniques, for "in situ" and "on line" determinations.

Table 2
. Maximum residue limits (MRLs) of pesticides in or on food and feed of plant and animal origin, set by the European Council (EC) regulation No 396/2005 (Reg.EC No 396/2005), and MRLs in food set by the Codex Alimentarius Commission (Codex pesticides residues in food online database, 1996)

2.1 Chromatographic methods for OP pesticides analysis
point out the development of plate immunoassays for routine determination of residues: diazinon, fenthion, malathion, and chlorpyrifos in extra virgin olive oil.The achieved by the mentioned methods LOD is shown in Table5.