Residues of Thiamethoxam and Chlorantraniliprole in Rice Grain

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RESIDUES OF THIAMETHOXAM AND CHLORANTRANILIPROLE IN RICE GRAIN Gustavo Mack Telo, Scott Allen Senseman, Enio Marchesan, Edinalvo Rabaioli Camargo, Trevor Jones, and Garry McCauley J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5042504 • Publication Date (Web): 27 Jan 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry 1

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RESIDUES OF THIAMETHOXAM AND CHLORANTRANILIPROLE

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IN RICE GRAIN

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Gustavo Mack Teló†*, Scott Allen Senseman‡, Enio Marchesan†, Edinalvo Rabaioli

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Camargo£, Trevor Jones , Garry McCauley¥

§

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†

Research Group in Irrigated Rice, Department of Plant Science, Universidade Federal de

8

Santa Maria (UFSM), ZIP CODE 97105-900, Santa Maria, RS, Brazil.

9

‡

Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA

10

£

Department of Crop Protection, Universidade Federal de Pelotas (UFPel), Pelotas, RS,

11

Brazil. §

Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, USA.

12 13

¥

14

TX, USA.

Texas A&M AgriLife Research, David Wintermann Rice Research Center, Eagle Lake,

15 16 17 18 19 20 21 22 23 24 25 26

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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RESIDUES OF THIAMETHOXAM AND CHLORANTRANILIPROLE

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IN RICE GRAIN

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ABSTRACT: Thiamethoxam and chlorantraniliprole insecticides have been important tools

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for controlling pests in rice. However, food safety issues related to pesticide residues are

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important when dealing with to consider with a food crop such as rice. Therefore, the

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objective of this study was to analyze thiamethoxam and chlorantraniliprole residues in rice

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hull, bran and polished rice grains. The study was conducted during the 2012 cropping

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season at the Texas A&M Agrilife Research, David R. Wintermann Rice Research Station

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near Eagle Lake, TX. Rice was planted on May 5th, 2012, using the cultivar “Presidio”.

37

Pesticide applications were performed at 5, 15, 25 and 35 days after flowering (DAF) using

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1 and 2x of the recommended rate of 30 g a.i. ha-1 for thiamethoxam and 30 g a.i. ha-1 for

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chlorantraniliprole. Sequentially, two treatments using the insecticides at recommended

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rate were applied at 5 and 25 DAF and at 5, 25 and 35 DAF. Insecticide residues were

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analyzed in different sample fractions: rice hull, bran and polished rice grains. The

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samples were subjected to extraction using an accelerated solvent extraction (ASE)

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technique. Sample aliquots were analyzed using ultra high performance liquid

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chromatography tandem mass spectrometry (UHPLC-MS/MS), with limit of quantification

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(LOQ) of 5x10-5 mg kg-1. Residues of thiamethoxam and chlorantraniliprole were detected

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in rice hull, bran and polished rice grains, and the quantified values were greater in hull

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and in rice bran.

48 49

KEYWORDS: Oryza sativa L, accelerated solvent extraction, rice hull, rice bran, polished

50

rice grains, accelerated solvent extraction.

51 52


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INTRODUCTION

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Rice (Oryza sativa L.) is one of the main components of a basic daily diet

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throughout the world, the third most popular grain worldwide and a major food crop for

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more than 60% of the world’s population1, and its consumption has increased over recent

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decades2. Thus, the quest for high grain yield and quality through management practices

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and technology is a challenge for scientists and researchers.

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The use of insecticides in irrigated rice has intensified in recent years due to greater

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insect pressure at economically important levels, thus, efficiency in controlling insecticide

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use in irrigated rice is a pressing issue. Lepidoptera3,4 and heterotera5,6,7 are common

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insects in irrigated rice crops, and their attack can occur both in the vegetative and

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reproductive phase of the rice plants. In many cases, occurrence of pests, which cause

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crop injury, has forced farmers to apply pesticides near harvest. Therefore, the use of

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insecticides is characterized as an essential management practice to ensure adequate

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agricultural yield, maintenance of the crops yield potential8 as well as quality of the rice

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grain9,10.

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The use of insecticides from the neonicotinoid and anthranilicdiamide chemical

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groups are recommended for use on irrigated rice crops11,12,13, providing higher efficiency

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in the control of insects due to its wide spectrum control.

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The insecticide thiamethoxam {3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-

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oxadiazinan-4-ylidene(nitro)amine} represents the fastest growing class of insecticides

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called neonicotinoids14. Neonicotinoid insecticides acts selectively on the central nervous

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system of insects with minimal effects on beneficial insects and with low toxicity toward

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mammals without causing either teratogenic or mutagenic effects15, which are active

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against many sucking and biting insects, including aphides, whiteflies and some

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lepidoptera species16.

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The thiametoxam is a versatile insecticide, which may be applied to the areal parts



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of plants or to seeds with relatively low rate. It belongs to the systemic insecticide class to

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its high solubility in water and low partition coefficient (Kow)17, being characterized as a

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polar compound which tends to ascend via xylem18, the insecticides thiamethoxam are

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highly efficient in controlling insects, and there are no records of cross resistance with

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other classes of insecticides19. The use of neonicotinoid insecticides at various crop stages

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and during postharvest storage plays an important role in food security and quality

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preservation.

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Chlorantraniliprole

{3-bromo-N-[4-chloro-2-methyl-6-[(methyl-amino)carbonyl]

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phenyl]-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide}

is

a

new

insecticide

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pertaining to the

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ryanodine receptors (RyR) in insect muscles resulting in an uncontrolled release of

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calcium from internal stores in the sarcoplasmic reticulum21, causing impaired regulation of

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muscle contraction leading to feeding cessation, lethargy, paralysis, and eventual death of

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target organisms22. Its use provides good lepidoptera control23,24, it is a systemic

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insecticide which tends to be translocated long distances ascendingly3 in the plant and is

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recommended for and widely used in irrigated rice crops by foliar application11,12. The

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insecticide moves within the foliar tissue where it is protected from leaching while

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remaining available to chewing insects in both top and bottom surfaces of the leaves. This

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trans foliar activity associated to its insecticide potential, resistance and photo degradation

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are the bases for efficient protection in the tomato plant. It is noteworthy that this

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insecticide presents low toxicity to non-target organisms such as mammals, birds, fish and

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others25,26, a good options for pest control with low environmental impact. The

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anthranilicdiamide class has no cross resistance with other insecticide chemical groups27,

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and is an efficient alternative in the insecticide rotation28.

anthranilicdiamide class20. Chlorantraniliprole selectively binds to

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In rice crops the increasing application of pesticides directly on the organ of the

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plant that is used for consumption and associated with the fact that rice does not receive


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intensive industrial processing, the analysis of residues of insecticides on the grain

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becomes essential, as a way to ensure food safety, due to the fact that insecticide

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residues can persist until the harvest stage, resulting in the contamination of the rice

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grain29,30,31.

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However, pesticides may also affect human life due to pesticide build-up throughout

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the food chain may lead to an ecologic phenomenon called bio magnification which is the

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bioaccumulation of a pesticide through an ecological food chain by transfer of residues

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from the diet into body tissues. The tissue concentration increases at each trophic level in

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the food web when there is efficient uptake and slow elimination32. However knowledge

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pertaining to the long-term effects of pesticides in human health are still very restricted33,

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but indicate that its effects may be associated with several health problems34,35,36,37.

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Literature presents several studies related to pesticide residues in food, however, there is

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little information on the use of insecticides in rice fields and its persistence in the grains. It

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is important to analyze pesticide residues not only on the rice grain itself but also on rice

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sub-products such as rice bran which can be used in whole rice38 and animal feed39,40.

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Overall, the results of these few studies show variation in the presence of pesticide

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residues in rice grain, likely due to environmental factors that can interact and interfere

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with the persistence of pesticides. Literature presents several hypotheses regarding what

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may influence the persistence of pesticides in food, and many such reasons are not fully

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understood due to the characteristics of the active ingredients41,42, the moment of

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application43, solar radiation, temperature44, and the persistence of pesticides in

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plant45,46,47.

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Thus, studies analyzing pesticide residues in rice grain are pertinent and provide a

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better understanding of if and how technologies used in commercial crops compromise

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grain quality. Another important issue may be related to the rate of pesticides applied to

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the area parts of plants, however, reports on the use of the recommended rate and double



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the recommended rate for the insecticide thiamethoxam have not been able to detect

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residues in polished rice grains46. However it is important to analyze pesticide residues in

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rice as a whole because in a study with the insecticide thiamethoxam, the highest residue

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concentration was detected on the rice (62% of total detected), followed by rice grain (38%

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of total detected) no residues were detected on the rice grain48.

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Regarding, chlorantraniliprole in rice, residues were detected in polished rice grains

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varying with the applied rate (30 and 60 g a.i. ha-1). The addition of a double rate of

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chlorantraniliprole presented the highest values of residues detected in polished rice

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grains, which was 85% higher than the recommended rate49.

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The need for information regarding the persistence of pesticides in irrigated rice is

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relevant because of the nutritional and toxicological issues associated to rice

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consumption48. Therefore, it is necessary to study pesticide residues in rice grains to

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provide basic information for the use of thiamethoxam and chlorantraniliprole in pest

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management strategies for rice and grain quality.

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The objective of this study was to analyze residues of the insecticides

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thiamethoxam e chlorantraniliprole in rice hull, bran and polished rice grains when applied

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on the areal part of the rice plants.

148 149 150 151

MATERIALS AND METHODS The study was conducted in two stages; the first stage was conducted in the field and the second in the laboratory.

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Experimental procedures in the field

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The first phase of the experiment was conducted during the 2012 cropping season

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at Texas A&M Agrilife Research, David R. Wintermann Rice Research Station near Eagle

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Lake, TX. The rice was seeded in May 5th, 2012, using the cultivar “Presidio”, with a


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seeding rate of 80 kg ha-1. Each plot had seven rows spaced at 0.19 m from each other

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and measuring 4 m long. The management practices were conducted according to the

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technical recommendations for rice cultivation in Texas50.

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Pesticide applications were performed at 5, 15, 25 and 35 days after flowering

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(DAF) using 1 and 2x of the recommended rate, that is, 30 g a.i. ha-1 of thiamethoxam and

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30 g a.i. ha-1 of chlorantraniliprole (Table 1). Besides, two treatments using the insecticides

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recommended rate were applied sequentially at 5 and 25 DAF plus at 5, 25 and 35 DAF.

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Insecticide residues were analyzed in different sample fractions: rice hull, bran and

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polished rice grains.

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The applications were made with a CO2 pressurized backpack sprayer calibrated to

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dispense 205 L ha-1 of spraying volume. The system was attached to a spray boom with

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three nozzles operating at 276 kPa. The experiment was conducted using a randomized

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complete block design with four replications.

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For quantification of residues in grains, rice plants were harvested when the

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average moisture content in the grains reached 20%, based on Texas recommendations50.

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Rice harvest was performed 40 days after flowering from an area totaling 3.8 m2 (4.0 x

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0.95 m) in each plot. After grain harvest, a homogeneous sample of 2 kg was separated

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for each treatment. Subsequently, samples were cleaned and dried with forced air

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ventilation at 35±2 C until reaching average moisture content of 13%. The samples were

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then stored at -20 C, during the thirty-day period until extracted for pesticides residues.

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Insecticide residues were analyzed using different fractions of rice samples including

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1) rice hull, 2) rice bran and, 3) polished rice grains. These fractions were obtained by the

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processing the samples in a rice milling equipment (Zaccaria®, model PAZ-1-DTA,

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Limeira-SP-Brazil).

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Insert Table 1



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Determination of the pesticide residues

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The second stage the study was conducted in the Department of Soil and Crop

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Sciences at Texas A&M University. Prior to chromatographic analysis, samples were

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extracted using an accelerated solvent extraction - Dionex ASE 200 system (Dionex Co.,

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Sunnyville, CA, USA), and afterwards the samples were analyzed using ultra high

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performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).

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Sample preparation for ASE

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The sample preparation proceeded with 2 g of the matrix (rice hull, bran or polished

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rice grains), 3 g Chem tube hydromatrix (Agilent Technologies) and 2 g of Florisil (Acros

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Organics). Sample preparation was carried out in a 50-mL beaker and the contents were

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mixed with a spatula to obtain a free-flowing powder. The cell size used was a 22-mL

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stainless steel cell, using two circular cellulose filters (size 19.8 mm in diameter) at the low

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end of the cell. Fortification was done by adding appropriate volumes of solution to the

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samples where they were equilibrated in the extraction in cells at room temperature for 10

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min.

200 201

Optimization of ASE conditions

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Optimal extraction conditions demonstrating the highest chemical recoveries

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included were extraction temperatures of 75 C for rice hull and 100 C for rice bran and

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polished grains; pressure 10.3 MPa; solvent-acetonitrile (100%) determined from a

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previous experiment (unpublished data). The duration of static phase was 5 min, following

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1 min of pre-heating and 5 min of equilibration. The solvent was collected in 60-mL vials

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with Teflon septa. Subsequently, each extraction cell containing the same sample went

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through another identical extraction cycle and the solvent was collected in the same vial.


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Optimization of evaporation conditions

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The total extracted volume of 60 mL was transferred into 15-mL volumetric tubes 10

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mL at a time for sample evaporation using a TurboVap® LV Evaporator (Zymark Center,

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Hopkinton, MA). Samples were evaporated at 45 C to dryness using a gentle stream of

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nitrogen. Pesticides were re-suspended in 2 mL of acetonitrile and shaking in vortex mixer

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(Fisher Vortex Genie2®, model G-560 – Fisher Scientific, Bohemia, N.Y.) The samples

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were stored at 4 C during the two-hour period to complete the preparation of all samples

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and begin analyzed by chromatograph.

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Chromatography conditions

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Analysis were performed using an ultra high performance liquid chromatography

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tandem mass spectrometry (UHPLC-MS/MS), model Acquity® TQD (Waters Corporation,

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Milford, MA). Chromatographic separation was done with a ethylene bridged hybrid (BEH)

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C18 column (50 x 2.1 mm; 1.7 µm) with controlled temperature of 30 C and a 5-µL injection

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volume. Mobile phase was pumped from two solvent reservoirs consisting of 0.05% of

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formic acid in water (A) and acetonitrile (B) in an isocratic run (50 A: 50B) with a flow rate

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of 0.4 mL min-1, and the estimated void time for this method is 0.3 min. The mass

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spectrometer was operated using electrospray ionization (ESI) in positive mode, capillary

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voltage at 2.2 kV, source and dissolution temperature were 150 C and 400 C, dissolution

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and cone gas flow of 800 L h-1 and 50 L h-1. Nitrogen (AOC, Bryan, TX) was used as

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dissolution and cone gas, and argon (AOC, Bryan, TX) as collision gas. The pesticides

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analyzed were confirmed using selected reaction monitoring (SRM) mode in which

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ionization of the parent and one product were analyzed and monitored for each compound

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studied (Table 2).

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Extraction efficiency and statistical design

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The efficiency of the extraction method was determined by sample fortification and

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analysis of quality control samples. Untreated rice samples were fortified to a

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concentration of 0.75 mg kg1. The samples were then processed according to the

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extraction procedure previously described and analyzed six repetitions each sample.

240 241

Limit of detection and limit of quantification

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The quantification was based on a seven-point calibration curve (0.0005, 0.001,

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0.002, 0.01, 0.1, 0.5 and 1.0 mg kg1). A matrix effect was obtained by comparing each

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response generated from calibration standards, which were prepared in a pure acetonitrile

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and in matrix extracts. ME (%) was calculated as follow: (B/A) × 100, where B is a

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response of matrix-matched calibration standard, and A is a slope of non-matrix-matched

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calibration standard. Matrix effect values between -20 and +20% are considered

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acceptable51.

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Limit of detection (LOD) and limit of quantification (LOQ) were calculated according

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to the peak-to-peak noise method52. The baseline of the unfortified blank was magnified to

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obtain the instrument noise response and converted into a concentration estimate from the

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known concentration of the fortified extract. The limit of detection (LOD) was estimated by

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multiplying the response of the method noise level by approximately three and then

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converting the total response into an estimated concentration. Limit of quantification (LOQ)

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was estimated by multiplying the response of method noise level by approximately ten and

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then converting the total response into concentration.

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Statistical analysis

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The experimental design was a completely randomized factorial arrangement with

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eight replications in laboratory. Data were subjected to analysis of variance and means


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were compared using Scott-Knott test (p 50%) matrix effects, it is necessary to use certain calibration methods to

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overcome the influence of matrix. For hull, grains and bran extracts, thiamethoxam

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presented high suppression (-94, -34 and -88%, respectively). Moreover, for the same

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extracts chlorantraniliprole presented high suppression for hull (-75%) and bran (-59%).

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Otherwise, for grains the matrix effect for this pesticide was positive (31%). These results

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prove the importance to calculate the matrix effect.

(suppression

or

enhancement of

20-50%)

or

strong (suppression

or

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The recovery study was conducted for hull, bran and polished rice grain. Average

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recovery ranged from 89.6 to 109.7% with a relative standard deviation

Residues of Thiamethoxam and Chlorantraniliprole in Rice Grain

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