Biopesticide residues in water

chapter six

Biopesticide residues in water Edmond Sanganyado, Basil Munjanja, and Vincent T. Nyakubaya Contents 6.1 Introduction.............................................................................................. 93 6.2 Physicochemical properties.................................................................... 94 6.2.1 Sources of biopesticides in water............................................... 96 6.2.2 Fate of biopesticides in water..................................................... 97 6.2.3 Toxicity of biopesticides.............................................................. 99 6.3 Biochemical pesticides............................................................................ 99 6.3.1 Sample preparation...................................................................... 99 6.3.1.1 Liquid–liquid extraction............................................ 102 6.3.1.2 Hollow fiber liquid phase microextraction............. 102 6.3.1.3 Ultrasonic assisted extraction................................... 103 6.3.1.4 Solid phase extraction................................................ 103 6.3.2 Separation and detection.......................................................... 103 6.3.2.1 Liquid chromatography............................................. 104 6.3.2.2 Gas chromatography.................................................. 108 6.3.2.3 Immunoassay.............................................................. 108 6.3.2.4 Capillary electrophoresis........................................... 109 6.4 Microbial pesticides and plant-incorporated protectants................ 109 6.4.1 Culture and phenotypic identification methods................... 109 6.4.2 Nucleic acid detection methods................................................110 6.5 Knowledge gaps..................................................................................... 112 6.6 Conclusion.............................................................................................. 112 References......................................................................................................... 112

6.1 Introduction Since the publication of Silent Spring by Rachel Carson in 1962, the public has been made conscious of environmental and human health risks of exposure to organic pollutants, especially pesticides. Conventional pesticides are very toxic, less specific, have low efficacy, and high persistence, hence the need to develop new pesticides which are less toxic, have highly 93

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specific, high efficacy, and less persistence. Synthetic pesticides are costly, challenging, and take long to develop. However, development of biopesticides is relatively cheap and quick; hence, biopesticides have increasingly become a choice alternative for conventional pesticides. By definition, biopesticides are a group of pesticides derived from natural products such as plants, bacteria, fungi, and animals and are classified as microbial, biochemical or plant incorporated pesticides (Tanwar et al., 2012). The amount and type of biopesticides found in the aquatic environment continues to increase as their production and use continues to grow. In the last few decades, production of biopesticides has increased considerably and is projected to continue in the next half decade. Despite the reluctance of end users in exploiting biopesticides for pest management, around 1200 biopesticides are commercially available, and only 270 are registered in EU and the United States (Chandler et al., 2011). About 50%, 30%, and 12% of all biopesticides are used in horticulture, animal husbandry, and crop production, respectively (Glare et al., 2012). The global market of biopesticides is expected to increase from US$ 1.2 billion in 2008 to US$ 3.3 billion in 2014 (Glare et al., 2012). Due to the current limited usage, few biopesticides have been detected by determining their residues in environmental matrices, such as soil and water. For example, azoxystrobin, a fungicide commonly used in agriculture in European nations and United States has been detected in surface and groundwater. Battaglin et al. (2011) detected in azoxystrobin in 45% of 103 samples collected from US streams. The mean of the detections was 0.163 µg/L and the maximum concentration was 1.13 µg/L. The goal of this chapter is to help researchers study biopesticide residues in aquatic environment by:

a. Discussing the toxicity, sources, and fate of biopesticides in aquatic systems b. Comparing sample preparation and clean-up techniques in current use and c. Considering methods of separation and detection, principally chromatographic techniques

6.2 Physicochemical properties The application of biopesticides in plant protection often results in loading of their residues in the aquatic environment. Studies on biopesticide residues in water should permit the occurrence, fate and transport of the organic pollutants to be correlated to their physical and chemical properties, and spatial and temporal trends in use. Depending on their physicochemical properties in water matrices, biopesticides can dissolve freely

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in water, sorb to suspended particles, or might bioaccumulate which may lead to adverse effects in nontarget organisms (Rodrigues et al., 2013). Oftentimes, the behavior of organic pollutants in the aquatic environment can be predicted by their physical and chemical properties. For example, highly polar compounds are usually found in aquatic environment as freely dissolved solutes whereas less polar pollutants will be either sorbed on suspended matter or sediment. Volatile, compounds will readily partition into the atmosphere (Figure 6.1). Physicochemical properties also influence the choice of the analytical technique used in the separation and detection of biopesticide residues in water samples. Basically, an analytical process involves sampling, sample pretreatment and clean-up, separation, detection, and quantification (Figure 6.2). The structural complexity of biopesticides varies remarkably from simple molecules such as 5-decenol to large proteins such as Cry1Ab (University of Hertfordshire, 2013). Such structural diversity implies biopesticide residue analysis can be done using a variety of analytical techniques ranging from simple immunoassays using ELISA kit, to elaborate methods such as chromatography and electrophoresis. Chromatographic methods are mainly used in the analysis of biochemical pesticides; nonchromatographic methods such as immunoassay are often used in the analysis of microbial pesticides and plant-incorporated protectants.

Figure 6.1 Sources of biopesticides in aquatic environment.

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Cry5ba

5-decenol

Azadirachtin

112

OH

Domain II

O

HO O

H O HO

O H

H O

Domain I

H3CO

OCH3 OH O O O

O O O

Domain III

H3C

170 698 173

Figure 6.2 Chemical structures of selected biopesticides chemical structures of selected biopesticides. (Reprinted with permission from Hui, F, U Scheib, Y Hu, R J Sommer, R V Aroian, and P Ghosh. 2012. Structure and Glycolipid Binding Properties of the Nematicidal Protein Cry5B. Biochemistry 51 (49), 9911–21. Copyright (2012), American Chemical Society.)

6.2.1 Sources of biopesticides in water Biopesticides naturally occur in the environment or enter through anthropogenic activities, mainly agriculture. Examples of naturally occurring biopesticides include microbials such as Coniothyrium minitans, Bacillus thuringiensis, Sclerotinia minor, and Chondrosterum purpureum, and biochemicals such as rotenone, nicotine, and pyrethrums (Szewczyk et al., 2006; Chandler et al., 2011; Glare et al., 2012; Ntalli and Caboni, 2012). However, with increase in production and use of biopesticides, anthropogenic activities are quickly becoming a major source of biopesticides in the aquatic environment (Chandler et al., 2011). After application in pest control, biopesticides are either metabolized by target organisms or are washed off from soil or plant surface and then transported to rivers, lakes, or dams by surface run-off. Surface run-off and leaching in agriculture fields are the main routes of entry of biopesticides into the aquatic environment (Jørgensen et al., 2012; Rodrigues et al., 2013). The aquatic environment is the sink of most organic pollutants. Contamination of

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water by biopesticides may pose human health risk and adverse ecotoxicologic effects to nontarget aquatic species (Plimmer, 1999; Raizada et al., 2001; Winkaler et al., 2007; Chandler et al., 2011). Therefore there is a need to determine biopesticide residues in water. This chapter describes the methods used to extract biopesticides in water and how they are detected. Several field studies have been carried out to determine the transport of biopesticides from crop fields into streams (Douville et al., 2005, 2007, 2009; Deb et al., 2009). Jørgensen et al. (2012) investigated the leaching of azoxystrobin and its transformation product in crop fields. They found azoxystrobin residue in drainage water ranging from 0.03 to 1.40 µg/L. However, azoxystrobin strongly adsorb to sand and was not detected in surface water sampled from fields with sandy soil (Jørgensen et al., 2012).

6.2.2 Fate of biopesticides in water The concentrations of the biopesticides found in rivers, lakes, and other water bodies depend on various factors such as the total amount used, half-life of the biopesticide, and partition coefficient. The partition coefficient of the biopesticide determines the distribution of the biopesticides in environmental compartments such as soil and water. Moreover, another important factor is volatilization; highly volatile biopesticides readily enter the atmosphere. However, the distribution of biopesticides is often affected by their physicochemical properties (Table 6.1). Highly soluble biopesticides such as nicotine and spinosad readily partition into water, whereas less soluble abamectin and pyrethrin I do not readily enter water bodies. Understanding the fate of biopesticides in the aquatic environment is very important in their environmental risk assessment. When biopesticides enter the aquatic environment they are dissipated by processes such as photolysis, biodegradation, volatilization, sorption, and hydrolysis (Cleveland et al., 2002; Thompson et al., 2004; Sanderson et al., 2007; Prihoda and Coats, 2008; Schleier et al., 2008; Prasse et al., 2009). B. thuringiensis is widely used as either a microbial pesticide or plant-incorporated protectant in pest control. Douville et al. (2005) showed B. thuringiensis Cry1Ab endotoxin had a half-life of 4 and 9 days in water and soil, respectively. Cleveland et al. (2002) investigated the abiotic and biotic degradation processes of spinosyns A and D. The researchers made four important observations; spinosads were dissipated in water via photolysis, biodegradation, partitioning, and abiotic hydrolysis, in order of importance. Similar studies have been conducted on other biopesticides such as azadirachtin (Thompson et al., 2002), azoxystrobin (Rodrigues et al., 2013) and ivermectin (Sanderson et al., 2007; Prasse et al., 2009). However, further studies need to be carried out on the fate of biopesticides in the aquatic environment. Even though there is widespread global growth in the production of biopesticides, there are few studies on biopesticide residues in water.

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1,000,000 1,000 0.96 15.0 235

21 24

10

1.21

1.17 −1.22 5.9 4.16 4.0

5.0 5.0

–

4.4

Log P

9.0 × 10−04 1.72 × 10−20 7.83 × 10−02 1.13 × 10−08 1.89 × 10−07

– 1.7 × 10−04

–

2.70 × 10−03

Henry’s Law constant at 25°C (Pa m3 mol−1)

2 18 2 2 14

300

–

2.7

30

Half-life in soil (aerobic) (days)

Source: Data from University of Hertfordshire. 2013. The Bio-Pesticides Database (BPDB) developed by the Agriculture and Environment Research Unit. http://sitem.herts.ac.uk/aeru/bpdb/index.htm.

Nicotine Oxytetracycline Pyrethrin I Rotenone Spinosad

Fungicide, bactericide Insecticide, acaricide, veterinary treatment, bactericide Insecticide Bactericide, antimicrobial, veterinary treatment Insecticide, acaricide, veterinary treatment Insecticide, acaricide, veterinary treatment Insecticide

Insecticide, acaricide, nematicide, veterinary treatment Insecticide, bactericide

Abamectin

Bacillus thuringiensis Berliner subsp. israelensis Emamectin B1a Emamectin benzoate

Use

Biopesticide

Solubility in water at 20°C, (mg L−1)

Table 6.1 Use and Physicochemical Properties of Biopesticides

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Pesticide residue analysis in water is often done to establish their occurrence and fate in the aquatic environment for risk assessment or determining the efficacy of the pesticide. Most of the studies on the fate of biopesticides in the aquatic environment sort to establish their persistence after application to crops for efficacy and not risk assessment.

6.2.3 Toxicity of biopesticides Since biopesticides are produced from natural products, they are often assumed to be environmentally friendly and harmless. Yet, the purpose of biopesticides is to avert, kill, or mitigate specific organisms through a specific mechanism of toxicity that could be pursued in no-target organisms. Various studies have been conducted on the toxicity and ecotoxicity of biopesticides such as azadirachtin (Boeke et al., 2004; Thompson et al., 2004; Senthil Nathan et al., 2006, 2007; Morgan, 2009), spinosads (Sparks et al., 2001; Cisneros et al., 2002; Yano et al., 2002; Galvan et al., 2005; Biondi et al., 2012) and avermectins (McCracken, 1993; Chung et al., 1999; Shipp et al., 2000; Pitterna et al., 2009; Castanha Zanoli et al., 2012). Even though the ecotoxicity of biopesticides is comparatively lower than that of synthetic pesticides, the occurrence of biopesticides in aquatic environment is important in the determination of their environmental risk assessment.

6.3 Biochemical pesticides Generally, there four types of biochemical pesticides currently used in pest control; namely enzymes, semiochemicals, natural plant regulators, and hormones. Instrumental analysis of biochemical pesticides currently focus on their characterization, isolation, and detection for insecticidal activity studies, and biopesticide residue analysis in crops. Very few studies have been conducted on their occurrence in aquatic environment. However, current instrumental analysis involves immunoassay and high-pressure liquid chromatography (HPLC) and capillary electrophoresis (CE).

6.3.1 Sample preparation In biopesticide residue analysis, the method for extracting biopesticides in water is often determined by the type, chemical structure, or physicochemical properties of the biopesticide and the type of the detection and quantitation instrument to be employed. Sample preparation in biopesticide residue analysis aims to isolate the compound from water and concentrate it with negligible matrix interferences. This is difficult because biopesticide residues are often found in the aquatic environment at trace

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Biopesticides handbook Sampling process

Homogenization

Sample preparation

Liquid–liquid extraction

Microextraction

Microwave assisted extraction

Ultrasound extraction

Solid phase extraction

Accelerated solvent extraction

Molecular imprinted solid phase extraction

Solid phase microextraction

Molecular imprinted solid phase microextraction

Detection Gas chromatography

Liquid chromatography

Nonchromatographic methods

Analysis

Figure 6.3 Steps in biopesticide residue analysis in water.

levels. Thus, it is usually the bottleneck in the analytical process. Steps in sample preparation include sampling process, homogenization, extraction, and clean-up (Figure 6.3). The first step in sample analysis is sample pretreatment. After sample pretreatment the subsample undergoes extraction and clean-up. The analytes are extracted from water samples using liquid–liquid extraction (LLE), ultrasonic-assisted extraction (UAE), solid phase extraction (SPE), solid phase microextraction (SPME) and molecular imprinted polymers (MIP). Table 6.2 discusses the advantages and disadvantages of the techniques commonly used in biopesticide residue analysis. Sample

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LME

LLE

SPME

Biopesticides are retained selectively on a sorbent and then partitioned into a solvent Analyte is extracted by equilibrium partitioning of the biopesticides between the water sample and a sorbent Biopesticide have different partition coefficients in different solvents Biopesticides are extracted with the aid of a microporous membrane either in an aqueous– organic–aqueous or aqueous–organic system

SPE

Principle of operation

Ultrasonic vibrations aid release of analytes in the water matrix

UAE

Method

Selectivity can be tuned through adjusting solvent chemistry Uses less solvent, and no additional clean-up step is usually required Can be automated

Flexible, easy to do, and can be applied to diverse compounds

Many samples can be extracted simultaneously Can be used to extract thermolabile biopesticides Relatively cheap, fast, and easily automated Low solvent use Very flexible in use Reduces costs and amount of solvent used No additional clean-up step might be required

Advantages

Several different solvents are often required Requires use of additional equipment, including vacuum pump and manifold Additional steps might be required for extraction of a broad spectrum of biopesticides Optimizing the extraction technique is often challenging Uses lots of solvent Time consuming, relatively expensive, and difficult to automate Extraction optimization can be challenging The flux often declines and results in loss of selectivity

Requires an extra step for isolating the biopesticide

Disadvantages

Table 6.2 Advantages and Disadvantages of Sample Extraction Techniques Commonly Used in Biopesticide Residue Analysis

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pretreatment is often carried out to obtain a subsample which is both homogenous and representative to the sample. However, water samples are basically homogenous in nature and do not usually require a separate homogenization step. Sonication (Sanderson et al., 2007) or mechanical shaking can be used for enhancing the homogeneity of the water samples. The most commonly used sample pretreatment procedure in biopesticide residue analysis is filtration which is used to remove suspended particles that cause interferences during analysis.

6.3.1.1 Liquid–liquid extraction Liquid–liquid extraction is also called solvent extraction and is an example of a classical technique used in extraction of biopesticide residue in aqueous liquid samples (Table 6.2). Water samples are mixed with an organic solvent in a separation funnel where the analyte partition differentially between the immiscible liquids. Various factors affect the extraction efficiency of LLE, and these include the solubility of the biopesticide in the solvent and the number of times extraction will be repeated. The choice of the solvent is very important in biopesticide residue analysis because it can affect the recoveries of the extraction technique. Generally, nonpolar compounds are extracted using cyclohexane, acetone, n-hexane or dichloromethane (DCM) and polar compounds using methanol (MeOH), ethyl acetate (EtOAc), and acetonitrile (ACN). However, recoveries are usually poor for polar biopesticides and they can be improved by either using a set of solvents or adjusting the pH. The pH of the solvent can be changed in a way that can prevent the biopesticides from ionizing thus improving their quantitative recovery. Spinosyns A and D have pKa of 8.10 and 7.87, respectively, and are highly soluble in water. In order to successfully extract them from water, methanol and dichloromethane are used as extraction solvents, and sodium hydroxide is added to change the pH to ≥12 (West, 1997; Cleveland et al., 2002).

6.3.1.2 Hollow fiber liquid phase microextraction The disadvantage of SLME and MMLE is that they require a peristaltic pump for the flow system. To avoid this problem, hollow fiber liquid phase microextraction (HF-LPME) was developed where the acceptor phase was placed inside a porous hollow fiber usually made of polypropylene (Martín-Esteban, 2013). Like SLME and MMLE, HF-LPME has two configurations, two-phase mode where the organic phase is on the wall and inside the hollow fiber and three-phase mode where the acceptor phase is aqueous or organic (Ghambarian et al., 2012). However, unlike LLE, SPE, SLME, MMLE, and other exhaustive extraction methods, HF-LPME is an equilibrium extraction technique, wherein the analyte distributes between the donor and acceptor phase until equilibrium is attained. In order to speed up equilibration of the system, a sonic bath

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and a magnetic stirrer are often used. The partition coefficient of the analyte is then used to determine the concentration of the biopesticide in the water sample. Raich-Montiu et al. (2008) extracted avermectins in water using threephase HF-LPME. The water sample was the donor aqueous phase and the acceptor aqueous phase was the HPLC mobile phase with 10 mM ammonium bicarbonate at pH 10. Park et al. (2013) developed a method for extracting avermectins in surface water using two-phase HF-LPME. They used dihexyl ether as the extracting solvent followed by methanol to desorb the analytes from the polypropylene membrane. Recovery ranged from 80.1% to 93.7% and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.15 and 0.5 µg/L, respectively (Park et al., 2013).

6.3.1.3 Ultrasonic assisted extraction Ultrasonic-assisted extraction uses ultrasound to aid in the isolation of the analytes from the water matrix (Table 6.2). It is important to note that this technique is at times used as a complementary step in biopesticide residue analysis to assist a tentative extraction or clean-up technique (West, 1997; Belmonte Vega et al., 2005; Mazzotti et al., 2009). Ultrasonics-assisted extraction usually involves adding a solvent to the sample and speeding up extraction by placing the mixture in a sonic bath.

6.3.1.4 Solid phase extraction Solid phase extraction (SPE) is the most commonly used extraction technique in biopesticide residue analysis. In solid phase extraction, the analyte is sorbed on a sorbent such as C18, Florisil, alumina, activated carbon, or hydrophilic lipophilic balance (HLB) copolymer, and then isolated by desorption using an eluting solvent. There are three steps involved in SPE: conditioning the cartridge with solvents, loading the sample, and eluting the analyte fractions. However, sometimes, a washing step is included after loading to remove any potential matrix interferents. Generally, no additional clean-up step is required after SPE. Rosen and Zang (2007) extracted cevadine, cevine, cevacine, sabadine, veratridine, ryanodine, and dehydroryanodine in aqueous sample using Envi-18 cartridge. After SPE, the analytes were derivatized by methylation using diazomethane. In some studies, azadirachtin (Thompson et al., 2002, 2004) and rotenone (Mazzotti et al., 2009) were extracted in water using C18 cartridge and the recoveries were both greater than 95%. It is important to note that C18 usually extracts polar compounds poorly. Spinosad was extracted in agricultural water samples with pH adjusted to 3.5 using an HLB cartridge (Belmonte Vega et al., 2005). The recovery was about 70% and the LOD and LOQ were 0.025 and 0.050 µg/L, respectively. In a similar study, azoxystrobin was also extracted in water samples using HLB cartridge (Reilly et al., 2012).

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6.3.2 Separation and detection Instrumental analysis to separate and detect the biopesticides in water is the last step in the analytical process. However, they are very few studies on biopesticide residues analysis in aqueous samples. Biopesticide residue analysis is often used in occurrence and fate studies; thus, the analytical instruments used in analysis of biopesticides in water should be highly selective and sensitive. The LOD and LOQ of the analytical technique should preferably fit the intended purpose. Various factors influence the choice of an instrumental technique for biopesticide residue analysis in water. Examples of such factors include boiling point, thermal stability, solubility in particular organic or aqueous solvents, molecular structure, and electron ionization and fragmentation. Compounds with high boiling point and poor thermal stability cannot be analyzed by gas chromatography without derivatization. For a compound to be analyzed by liquid chromatography (LC) it should be soluble in the solvents used as mobile phase and also interact with the stationary phase in the LC column. Various detectors can be used in quantitation of biopesticides, and the choice of detector usually depends on the availability of certain functional groups in their chemical structure.

6.3.2.1 Liquid chromatography Liquid chromatography is the most widely used technique in biopesticide residue analysis in water because most of biochemical pesticides are not amenable to GC analysis. Highly polar, less volatile, or thermolabile biopesticides cannot be analyzed with GC without derivatization. Unfortunately, derivatization is time consuming, laborious, and prone to inaccuracies and poor reproducibility. Therefore, LC is usually the most viable analytical technique for biochemical pesticide residue analysis in water. Furthermore, LC offers another advantage of skipping the sample extraction step through direct aqueous injection. Various studies have employed LC to analyze biochemical pesticides like azoxystrobin (Jørgensen et al., 2012; Reilly et al., 2012; Rodrigues et al., 2013), azadirachtins (Thompson et al., 2002, 2004), spinosads (West 1997; Cleveland et al., 2002), and avermectins (Krogh et al., 2008; Thompson et al., 2009; Park et al., 2013) in water. Generally, the recoveries and LOD obtained in these studies ranged from 38% to 97% and 0.0025 to 1.0 µg/L (Table 6.3). Generally, in liquid chromatography separation is achieved because of the differential interaction of the analyte with the stationary phase in the analytical column. Another important factor that influences separation of the analyte is its affinity for the mobile phase. Consequently, to improve the sensitivity and selectivity of the LC, optimization of the mobile phase is often done through adjusting pH, using organic modifiers, changing mobile phase flow rate, or altering temperature. Separation in LC is often

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Technique

Clean-up/ derivatization Detection

SPME

LLE

Abamectin, SPE doramectin, ivermectin, emamectin benzoate, eprinomectin, moxidectin and selamectin

Bacillus thuringiensis Cry1Ab endotoxin Azoxystrobin

Conditioned with n-heptane, acetone, MeOH, and H2O Eluted with MeOH

–

Centrifugation at 150 mM NaCl, 5 mM 12,000g for 30 min KH2PO4, 1 mM NaHCO3, 0.2% Tween at 4°C 20 and 1% albumin SPME polyacrilate fiber; – direct injection

LC–MS/MS

GC–MS

ELISA

Restek RTX-1 MS 0.02 µg/L 66–98 column (30 m × 0.25 mm, i.d. × 0.25 µm film thickness) Zorbax Eclipse 2.5– 38–67 XDB-C8 column; 14 µg/L gradient elution with acetonitrile and 10 mM ammonia at pH 4.0 using formic acid

–

–

YMC AQ-301 ODS, 20 ppb 10 cm × 4.6 mm i.d., 1:1:1 (v/v/v) ACN/MeOH/2% (w/v) aqueous ammonium acetate; 0.8 mL/min – –

Rec. (%) –

LOD –

–

Conditions

Analytical method

Immunomagnetic Enzyme-conjugated Reactant chromogen, RaPID beads based spinosad and magnetic 3,3′,5,5′-tetramethyl photometric extraction beads coated with benzidine analyzer antibodies – HPLC-UV Spinosyns A–D LLE Dilute with NaOH; elute with MeOH and methylene chloride

Spinosad

Biopesticide

Extraction solvent/ sorbent

Sample preparation

Table 6.3 Determination of Biopesticide Residues in Water

continued

Krogh et al. (2008)

Filho et al. (2010)

Douville et al. (2005)

Cleveland et al. (2002)

Cleveland et al. (2002)

Reference


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Technique

Immunobased extraction

SPE

Hollow-fiberassisted liquid phase microextraction

Hollow fiber-supported liquid membrane

Biopesticide

Spinosyn A

Rotenone

Abamectin, ivermectin, moxidectin, and doramectin

Ivermectin

Polypropylene hollow fiber; desorption with 10 mM ammonium bicarbonate

Accurel polypropylene membrane; desorption with MeOH

C18 cartridge; Conditioned with ACN and H2O. Eluted with ACN

Donor was fluorescein and quencher tetramethylrhodamine

Extraction solvent/ sorbent

Sample preparation

–

LC–MS/MS

LC–MS/MS

LC/MS with ACPI

–

–

Fluorescent excitation transfer immunoassay

Detection 0.01 ppb

LOD

0.20 µg/L C8 4.6 mm × 250 mm column; gradient elution with mobile phase A: ACN; MeOH and ammonium formate buffer 10 mM in H2O (43:34:23, w/w/w). Mobile phase B: ACN

Reference

97

–

RaichMontiu et al. (2008)

Park et al. (2013)

Mazzotti et al. (2009)

96–120 Lee et al. (1999)

Rec. (%)

C18 (100 × 2.1 mm, 0.15 µg/L 80.1– 93.7 3 µm) column; gradient elution with ACN and 10 mM ammonium acetate in H2O

Pursuit C18 column 0.04 (5.0 cm × 2.0 mm); µg/kg gradient elution with ACN and H2O containing formic acid

–

Conditions

Analytical method

–

Clean-up/ derivatization

Table 6.3 (continued) Determination of Biopesticide Residues in Water


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SPE

SPE

LLE

Ivermectin

Azadirachtins A and B

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Spinosyns A and D

Extraction using MeOH and dichloromethane

C-18 SPE cartridge, eluted with ACN.

–

–

HLB 6 cc cartridges, Derivatized by conditioned with adding MeOH and H2O, eluted N-methylimidazole with methylene solution followed by chloride trifluoroacetic anhydride solution 1.0 µg/L

HPLC-UV

ODS-AQ (5-µm 3.0 µg/L particle size, 120 Å, 150 × 4.6 mm i.d.); isocratic elution with ACN, MeOH, and aqueous ammonium acetate in acetonitrile

HPLC with Phenomenex 1.0 µg/L photo diode Prodigy 5 µm ODS array detector column (150 × 4.1 mm i.d.), isocratic elution with ACN and H2O (20:80)

Sigma Discovery C18 Column (15 cm × 4.6 nm, 5 µm); gradient elution with ACN and H2O.

Sanderson et al. (2007)

Thompson et al. (2002, 2004)

West (1997)

–

>95

>70


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carried out in normal phase, reverse phase, polar organic mode, or polar ionic mode. However, in biopesticide residue analysis reversed phase is the most commonly used mode. After the analytes are separated they are then detected and quantified using detectors such as diode array detector (DAD), ultraviolet–visible wavelength detector (UV), mass spectrometry detector (MSD), and tandem mass spectrometer (MS/MS).

6.3.2.2 Gas chromatography

Gas chromatography separates and detects analytes based on their difference in volatilities. It is a highly sensitive and effective technique. Basically, in GC, the analyte is volatilized on the injector and enters the analytical column where there is gas–liquid interaction between the analyte and the stationary phase. Separation is achieved through differential partitioning of the gaseous analyte with the stationary phase. The separated analyte is then detected and quantified using a detector such as flame ionization detector, electron capture detector, nitrogen–phosphorus detector, or mass spectrometer. The selectivity of GC analysis is also affected by the stationary phase in the analytical column. There are various types of columns available commercially for pesticide residue analysis. Most of the stationary phases are made of 100% polydimethylsiloxane, 95% dimethyl-5% diphenylpolysiloxane, and 14% cyanopropyl-phenyl-86% dimethylpolysiloxane in increasing order of polarity. Gas chromatography coupled to either MS or MS/MS is commonly used in the analysis of biochemical pesticides. Several studies have determined the occurrence of azoxystrobin in water using GC–MS. Filho et al. (2010) developed a method of analyzing pyraclostrobin and azoxystrobin together with several synthetic pesticides using GC–MS with direct injection SPME. The recoveries ranged from 68% to 97% and the LOD was 0.02 µg/L. In a separate study, Reilly et al. (2012) found azoxystrobin in surface water and groundwater ranging from 9.0 to 59 ng/L using GC–MS. However, most biopesticides commonly used in crop production and animal husbandry have highly complex chemical structures and large molecular weights. Such chemical structures entail the commonly used biopesticides have high boiling points and cannot be analyzed by gas chromatography. As a result, in biopesticide residue analysis, LC and immunoassay techniques are the predominant methods of instrumental analysis used.

6.3.2.3 Immunoassay Chromatographic analysis methods are widely used in the determination of biopesticides in water (Cleveland et al., 2002; Krogh et al., 2008; RaichMontiu et al., 2008). However, chromatographic analysis is expensive and requires laborious sample preparation steps. Immunoassay (IA) is an

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alternative and complimentary method that does not require the strenuous extraction step. Furthermore, IA is very rapid and highly sensitive. Few studies have been conducted to determine biopesticides in water using IA. Cleveland et al. (2002) determined Spinosad in water using RaPID assay immunoassay (IA) kit. The technique did not distinguish specific spinosyns and their related metabolites. LC was employed in order to determine the individual spinosyns. However, Lee et al. (1999) developed an IA that could differentiate individual spinosyns in water: a fluorescent excitation transfer immunoassay. The recoveries ranged from 96% to 120% and the LOD was 0.01 ppb.

6.3.2.4 Capillary electrophoresis Capillary electrophoresis (CE) is a flexible method where separation of target species is based on their difference in electrophoretic mobility inside a capillary. There are different types of CE and the most widely used is capillary zone electrophoresis in which the analytes are separated in a buffer solution. Other CE techniques include micellar electrokinetic capillary chromatography, microemulsion electrokinetic capillary chromatography, and capillary electrochromatography. After separation in the capillary, analytes are detected using detectors such as UV, DAD, MS, and MS/MS. Despite its wide potential, according to the authors’ knowledge, CE has not been used in biopesticide residue analysis in water samples.

6.4 Microbial pesticides and plant-incorporated protectants A microbial pesticide is a microbial species such as virus, eukaryotic, or prokaryotic microorganism that can destroy, prevent, or alleviate pests or can be used as a plant growth regulator. Microbial pesticides can be determined in the water samples using immunoassay, polymerase chain reaction (PCR), or culture and metabolic techniques (Plimmer, 1999). Plant-incorporated protectants are biopesticides produced by genetic modification plants. B. thuringiensis Cry1Ab gene encodes for an insecticidal protein and has been incorporated in corn so that corn can produce the insecticide on their own without the bacteria. Various genes have also been incorporated in different crops for pest control. However, after harvesting, the detritus from the crop decomposes and may release the biopesticides into the environment where it can be transferred into aquatic microorganisms. Biopesticide residue analysis in water generally involves two steps. The first step determines the selectivity of the technique and involves isolating, labeling, and amplifying the target microorganism (Noble and Weisberg, 2005). After differentiating, the target microorganism is then

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detected or quantified using an appropriate detector, and this step is responsible for the sensitivity of the method.

6.4.1 Culture and phenotypic identification methods Traditional methods are often used in the determination of microorganisms in water and these include culture and metabolic techniques. Basically, the culture method consists of incubating the agar after inoculation with the water sample. The bacteria are then isolated, inspected, and identified with an appropriate technique. Douville et al. (2005) cultured B. thuringiensis subsp. kurstaki strain Pab4 in a minimal salts media and then identified the microbial pesticide Cry1Ab protein. Metabolic method is a phenotypic identification method which involve analyzing the spent media or the metabolic products of the microorganism using either chromatographic or electrophoretic technique (Plimmer, 1999). Additional techniques include whole-cell, surface recognition analysis where the microbial pesticide is labeled in order to detect cell-surface lipids and proteins (Noble and Weisberg, 2005). Surface proteins can be detected using immunoassay techniques and m olecule-specific probes. The most commonly used immunoassay technique in biopesticide analysis is the enzyme-linked-immunosorbent assay (ELISA). Douville et al. (2005) determined the residues of Cry1Ab endotoxin in surface water using ELISA, and the maximum concentration they obtained was 0.2 ppb. However, these traditional methods have several disadvantages because they are tedious, labor intensive, and time consuming. In addition, the technique has low sensitivity, and some microbial pesticides cannot be cultured in the growth media.

6.4.2 Nucleic acid detection methods A specific sequence of nucleic acids in microbial pesticides and plant incorporated protectants can be identified using genetic techniques. These nucleic acid methods use a genetic probe to identify the specific sequence. The use of a gene probe offers several advantages. Genetic techniques can analyze unculturable microorganisms; there is no need to use a marker, the whole genome can be tracked. Gene probes also offer the versatility of analyzing the gene even if it has been transferred to another organism and this was exploited by Douville et al. (2009) when they determined the occurrence of Bt Cry1 and transgenic Cry1Ab genes in mussels. Examples of genetic techniques include polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), and quantitative PCR (Q-PCR (Noble and Weisberg, 2005). Quantitative PCR is the most widely used genetic technique in microbial pesticide residue analysis (Douville et al., 2007, 2009). In qPCR, the nucleic target of

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the biopesticide is amplified, detected, and quantified simultaneously by observing fluorescent labeled PCR products (Botes et al., 2013). The qPCR techniques can be classified according to how measurement is done. When the measurement is done after the polymerase chain reaction has terminated, then the method is called end-point qPCR. However, the commonly used technique involves taking measurements during the elongation phase and is termed real-time PCR. Douville et al. (2007, 2009) determined the occurrence of the Cry1Ab gene in surface water using real-time qPCR with HSP70 as a forward primer (5′-GAT GCC TTC TCC CTA GTG TTG A-3′) and Cry1Ab as reverse primer (5′-GGA TGC ACT CGT TGA TGT TTG-3′).

6.5 Knowledge gaps There are very few studies on the occurrence and fate of biopesticides in water. This is because biopesticides have only recently gained prominence and are not yet used in considerably large quantities. By design, most of the biopesticides are considered inherently environmentally friendly and thus their possible human health and environmental risk are ignored. However, biopesticides can cause adverse effects to nontarget aquatic organism when they enter the aquatic system. There is great need, therefore, to study the transport, fate, and occurrence of biopesticides in the aquatic environment. Such studies will provide knowledge on the persistence, dissipation, and levels of biopesticides in water systems. In order to close the gap on the exposure of aquatic organism to biochemical pesticides, modern sample preparations methods need to be exploited. Current methods of biopesticide residue extraction use only SPE and LLE. There is need to use other microextraction methods besides HF-LPME such as liquid–liquid microextraction, molecular imprinted polymer extraction, solid phase microextraction, and single drop cloud extraction (Table 6.4). These methods offer various advantages including low cost, less solvent consumption, and higher selectivity.

6.6 Conclusion Since few studies have been carried out to date on biopesticides residues in water, more work needs to be done on their transport, fate, and occurrence in water. The global market of biopesticides is projected to increase in the coming years. However, with the growing use, their occurrence in aquatic ecosystems could probably increase in the future. Therefore, more studies need to be carried out on method development and the occurrence of biopesticides residues in water. The data may then be used in carrying out risk assessment programs globally.

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Matrix

Surface and groundwater Surface and groundwater Stream Surface and groundwater Surface water

Spain USA USA Brazil Spain

Country LC–MS GC/IT-MS GC–MS GC–MS LC–MS

Analytical method 0.037 ± 0.023 0.009 – 0.13 0.54

Minimum 0.493 ± 0.636 0.598 1.13 0.19 1.20

Maximum

Concentration (µg/L) Reference Herrero-Hernández et al. (2013) Q1 Reilly et al. (2012) Battaglin et al. (2011) Filho et al. (2010) Belmonte Vega et al. (2005)

Source: Adapted from Rodrigues, E T, I Lopes, and M Â Pardal. 2013. Environment International 53 (March): 18–28. doi:10.1016/j.envint.2012.12.005. http://www.ncbi.nlm.nih.gov/pubmed/23314040.

Spinosad

Azoxystrobin

Biopesticide residue

Table 6.4 Occurrence of Biopesticides in Water


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Cat#: K20781

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