New analytical method for chlorpyrifos determination in biobeds constructed in Brazil: Development and validation

Highlights

• A new method for chlorpyrifos determination in biobeds has been developed.

• The proposed method is simple and fast, allowing analysis in very complex matrices.

• Biomixture degrades chlorpyrifos pesticide in southern Brazil climatic conditions.

Abstract

A quick and efficient method was optimized and validated to determine chlorpyrifos in biobeds using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Chlorpyrifos was extracted from the matrix with 30 mL of a mixture of acetone, phosphoric acid and water 98:1:1 (v/v/v). After homogenization, centrifugation and filtration, 125 µL of the extract was evaporated and reconstituted in 5 mL of methanol acidified with 0.1% acetic acid. Validation was performed by studying analytical curve linearity (r²), estimated instrument and method limits of detection and limits of quantification (LOD_i, LOD_m, LOQ_i and LOQ_m, respectively), accuracy, precision (expressed as relative standard deviation, RSD), and matrix effect. Accuracy and precision were determined from the amount of pesticide recovered from biobed blank samples (i.e. without pesticide residue) spiked with chlorpyrifos at three different concentrations (2, 10 and 50 mg kg⁻¹), with seven replicates at each concentration. For all three concentrations studied, the average recovery values obtained were between 96 and 115% with RSD values lower than 20%. The validated LOQ obtained was 2 mg kg⁻¹ (from recovery studies) and the matrix effect observed was lower than ±20%, which demonstrated that there was neither considerable suppression nor enhancement of the analyte signal. The biobed system efficiently degraded chlorpyrifos in both 1) simulation of accidental spillage and 2) application of diluted pesticide solution. In the latter case, all the values obtained at the final sampling time (14 months) were below the validated LOQ_m.

Introduction

Pesticides play an important role in modern agriculture and food production [1]. However, the extensive use of pesticides is a serious public health problem in developing countries, especially those with economies based on agribusiness, as Brazilian case. Since 2008, Brazil has been considered the world's largest consumer of pesticides [2], due to the country’s vast agricultural area in addition to its tropical and subtropical conditions, which favor the occurrence of pests and diseases during all over the year, due the lack of insect diapause period or stopping the growth of pathogens.

During repetitive application of concentrated pesticide at a given location, contamination spots can readily occur. This increases the risk that pesticide residue concentrations exceed the limit that it is considered safe in the environment [3], [4], [5]. For example, when filling and rinsing sprayer equipment at the same place, year after year, high pesticide residue concentrations have been detected [6], [7].

A biobed is a in situ biological reactor system, developed in Sweden during the 1990′s and nowadays implemented in several European countries, that adsorbs and degrades pesticides in order to minimize environmental contamination [8], [9], [10]. Biobed is a simple and low cost construction, designed to retain and degrade pesticides from spills on farms [6]. Due to its easy installation and use, it has also become an important environmental safety tool for Latin American countries [11].

The original Swedish biobed, whose substrate was replicated for this experiment, is an unlined system consisting of three layers: clay, biomixture and grass. Clay is used as an impermeable layer to decrease the water flow downward and to increase the pesticide retention time. Biomixture should have a good absorption capacity and a high microbial activity and it comprises straw, soil and peat in the ratio of 50:25:25 (w/w/w) [8]. Grass layer increases the efficiency of the reactor, retaining part of the pesticides, controlling the leaching of those with high mobility and regulating the system humidity [8], [12]. The purpose of straw is to stimulate growth of white rot fungi (Phanerochaete chrysosporium) that degrade lignin through the production of ligninolytic enzymes, mainly phenoloxidase enzymes that have a high specificity and are thereby able to degrade a wide range of pesticides residues [10], [13], [14]. Soil provides sorption and should contain an appropriate amount of humus and clay to promote microbial activity [8], [15]. Peat contributes to the sorption capacity, moisture control and abiotic pesticide degradation. It also decreases the biomixture’s pH, favoring microbial activity [16], [8].

Since the biomixture matrix used in the reactor is highly complex and it is usually based on materials found locally, it is necessary to have analysis methods suitable for each environment. In addition, the effectiveness of the pesticide degradation process in biobeds requires highly sensitive and validated analytical methods [11].

Chlorpyrifos is an organophosphate insecticide [17]. It was introduced in the United States in 1965 by The Dow Chemical Company and it is known by several trade names, including Lorsban®. Lorsban® is one of the most used products for insect control worldwide [18], [19]. Since its introduction into Brazil in 1972, Lorsban® has shown to be an important pest control agent, used for 36 plagues in 13 different crops [20], [21]. This pesticide acts as an inhibitor of enzymes (e.g., cholinesterase) causing cholinergic syndrome and thus neurotoxic effect [22].

It is known that due to biological degradation occurring in the substrate of the biobed, chlorpyrifos will present some by-products (3,5,6-trichloro-2-pyridinol (TCP), 3,5,6-trichloro-2-methoxypyridine (TMP), O-ethyl O-(3,5,6-trichloro-2-pyridinol) (CYPO), which may have an inhibitory effect on the microbial activity inside the reactor [23], [24]. From these, 3,5,6-trichloro-2-pyridinol (TCP) is considered one of the most important due to its antimicrobial characteristics, given the risk of affecting microorganisms populations that act on the biodegradation process in the system. However, there are no evidences that such metabolites were able to impede the efficiency of the system, even when applying chlorpyrifos alone or in combination to other pesticides [25], [26], [27], [28].

The analytical methods for chlorpyrifos determination in biobeds are based on extraction with organic solvents. Most methods require>30 mL of solvent, which generates large amounts of residue [29], [30], [31], [32]. Other methods use less volume of organic solvents but are time-consuming [33], [25].

The aim of this study was to optimize and validate an efficient and rapid analytical method to determine chlorpyrifos in a biobed system developed in southern Brazil [1], [14]. The degradation of chlorpyrifos was studied in biobeds involving two situations: 1) simulating contamination by accidental pesticide spillage and 2) contamination with a diluted solution coming from the washing of agricultural machines used to apply pesticides. Those biobeds systems used in this study were assembled at the Embrapa Grape & Wine. Method optimization and validation, as well as biobeds sample analysis were performed at the Center of Research and Analysis of Residues and Contaminants (CEPARC) at the Federal University of Santa Maria (UFSM) - Brazil.