Neonicotinoid Residues in Fruits and Vegetables: An Integrated

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Neonicotinoid Residues in Fruits and Vegetables: An Integrated Dietary Exposure Assessment Approach Chensheng Alex Lu, Chi-Hsuan Chang, Cynthia Palmer, Meirong Zhao, and Quan Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05596 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

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Environmental Science & Technology

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Neonicotinoid Residues in Fruits and Vegetables: An Integrated Dietary

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Exposure Assessment Approach

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Chensheng Lu 1, 2, Chi-Hsuan Chang 2, Cynthia Palmer 3, Meirong Zhao 1, Quan

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Zhang*1, 2

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1

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Zhejiang Province, College of Environment, Zhejiang University of Technology,

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Hangzhou, Zhejiang, 310032, People’s Republic of China

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2

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Boston, Massachusetts, 02215, USA

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Key Laboratory of Microbial Technology for Industrial Pollution Control of

Department of Environmental Health, Harvard T.H. Chan School of Public Health,

American Bird Conservancy, Washington DC, 20009, USA

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Corresponding Author:

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Quan Zhang

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18 Chaowang Road, Zhejiang University of Technology, Hangzhou, Zhejiang, China,

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310032.

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[email protected]

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1

ACS Paragon Plus Environment


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Table of contents

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Abstract

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Neonicotinoids have become the most widely used insecticides in the world

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since introduced in the mid-1990s, and yet the extent of human exposure and health

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impacts is not fully understood. In this study, the residues of seven neonicotinoids in

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fruit and vegetable samples collected from two cross-sectional studies, the U.S.

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Congressional Cafeteria study (USCS) and the Hangzhou China (HZC) study, were

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analyzed. We then employed a relative potency factor method to integrate all

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neonicotinoids in each food sample using the respective reference dose values as the

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basis for summation. The findings were compared with data published by the U.S.

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Department of Agriculture Pesticide Data Program (USDA/PDP). Imidacloprid and

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thiamethoxam were the most commonly detected neonicotinoids in fruits and

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vegetables with 68% and 51% detection in the HZC study, and 59% and 56%

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detection in the USCC study, respectively. The overall frequency of detection for

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neonicotinoids in the USDA/PDP samples was much lower than those reported here

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for the USCS or HZC studies, with imidacloprid the most frequently detected

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neonicotinoid at 7.3%. The high frequencies of neonicotinoid detection in fruits and

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vegetables in the USCC and HZC studies give us a snapshot of the ubiquity of

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neonicotinoid use in global agriculture and make it clear that neonicotinoids have

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become part of the dietary staple, with possible health implications for individuals as

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well.

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Introduction Neonicotinoids have quickly become the most widely used insecticides in the

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world since their introduction in the mid-1990s. They are commonly applied as insect

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controls in agricultural settings, particularly in seed treatments for crops, such as corn,

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soybeans, cereals, and oilseed rape. 1-4 It is estimated that more than 4 million pounds

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(about 1.8 million kilograms) of neonicotinoid active ingredients have been used on

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140 to 200 million acres (about 567 – 809 thousand square kilometers) of farmland

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annually in the United States, 5 accounting for more than 20 percent of the world

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insecticide market 6, and valued at approximately $1.4 billion to the U.S. economy. 7

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Because of their systemic properties, neonicotinoids can be absorbed by roots

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and then translocated to almost every tissue of the plants, including leaves, flowers,

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pollen, nectar, and crops grown by those plants once applied. 8, 9 Therefore, from the

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pest control perspective, neonicotinoids and other systemic pesticides have what

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sound like beneficial attributes. However, the adverse ecological, environmental, and

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public health implications of neonicotinoid residues in pollen, nectar, crops, and fruits

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can also be problematic, and should be examined. Recent studies have shown that

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approximately 73% of pollen and honey collected from bees and their hives were

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contaminated by at least one neonicotinoid, 10 a finding which may be related to the

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phenomenon of honey bee colony collapse disorder worldwide. 11 Besides potential

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exposure to non-target organisms like bees and birds, human beings could also be

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frequently exposed to neonicotinoids through ingesting neonicotinoid-contaminated

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foods and water, respectively. Sanchez-Bayo (2014) reported individual seeds of

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crops treated with neonicotinoids usually contained 1-17 mg/kg of neonicotinoids, and

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2-20% of neonicotinoids were taken up by the plants, with approximately 80-98% of

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neonicotinoids on the seeds releasing directly into the soil or groundwater. 12 These

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results suggest that neonicotinoids may be ubiquitous in the environment. Several

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studies have reported that neonicotinoids were detected in children’s urine in China

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and Japan. 13, 14 However, limited studies have identified the sources of neonicotinoids

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to which human beings could be exposed, regardless of the increasing trend of

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neonicotinoids use around the world.

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The extent of human health impacts resulting from neonicotinoid exposure is

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not yet fully understood because few exposure and toxicological data are available.

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Previous in vitro and in vivo studies have shown that the primary mode of action of

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neonicotinoids is to act on the nicotinic acetylcholine receptor (nAChRs) in the

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central nervous system, subsequently leading to neurobehavioral deficits and

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increased expression of glial fibrillary acidic protein in the motor cortex and

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hippocampus. 15, 16 U.S. EPA’s risk assessments note that in mammals, neonicotinoids

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are neurotoxic, and are also associated with liver, kidney, thyroid, testicular, and

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immune system effects. 17 Marfo et al. reported the association between urinary N-

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desmethyl-acetamiprid and the symptoms by a prevalence case-control study. 18

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Cimino et al. also confirmed sub-lethal effects of neonicotinoids on neurological

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impairments of human beings 19. Those findings on sub-lethal exposures are highly

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relevant to public health. Because of their systemic properties, neonicotinoids in foods

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cannot be easily removed by washing or peeling, so they could pose a dietary risk in

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individuals. Therefore, considering the ubiquity of neonicotinoids in the environment

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and foods, it is prudent to assess dietary intake of neonicotinoids at the population

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level so the potential health risks can be more closely examined. 20-22

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In this study, we report neonicotinoid residues measured in foods that are

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commonly consumed in cafeterias in the U.S. and by a group of Chinese elementary

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school children living in Hangzhou, China. We then compare the findings with data

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published by the U.S. Department of Agriculture Pesticide Data Program

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(USDA/PDP). The individual, as well as the total neonicotinoid residues in foods

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reported here, will be essential for facilitating future dietary exposure and health risk

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characterization and assessments.

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Materials and Methods

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Sample/Data collections

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vegetables collected in repeated sampling from two cross-sectional studies, the U.S.

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Congressional Cafeteria study (USCC) and the Hangzhou China (HZC) study. In the

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USCC study, we collected 36 and 28 samples from the U.S. Congressional cafeterias

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in winter (January) and spring (May) of 2015, respectively. Approximately half of the

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samples were purchased from the House Longworth Cafeteria and half from the

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Senate Dirksen Cafeteria. We set out to sample as many different food items as

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possible from both cafeterias to ensure a representative selection of foods that people

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often consume. Individual food items were placed in Ziplock bags, and then shipped

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in ice over-night from Washington DC to Boston MA for residue analysis in the lab at

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Harvard T.H. Chan School of Public Health (Chen et al. 2014).

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We analyzed neonicotinoid residues in fruits and

In the Hangzhou China Study (HZC), we collected fruit and vegetable samples

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from a group of 58 children ages 8-10 (26 male and 32 female) from an elementary

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school in Hangzhou, Zhejiang, China in 2015. We gave 24-hr dietary consumption

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questionnaires for 5 consecutive weekends to each child. We gathered information

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about food type, the amount of food, the time of consumption, and the sources of food

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purchased (supermarket, conventional market, etc.). We then bought food from the

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actual vendors, either in a traditional market or a supermarket, patronized by the

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parents in a market-basket approach, in order to better reflect neonicotinoid residues

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in the fruits and vegetables consumed. All the participants received approved

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informed consent prior to participating in the HZC study. All fruit and vegetable

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samples were processed and analyzed in the College of Environment at Zhejiang

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University of Technology in Hangzhou, following the same procedures (described

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below) as were used at the Harvard T.H. Chan School of Public Health. We also obtained neonicotinoid residue data for fruits and vegetables from

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USDA/ Pesticide Data Program (PDP) for 6 neonicotinoids (nitenpyram was not

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analyzed), measured in 22 fruits and 29 vegetable commodities from 2011 to 2014 for

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a total of 39,159 data points. USDA/PDP was initiated in 1991 to analyze pesticide

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residue in foods, and the data have been used primarily by the U.S. Environmental

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Protection Agency (EPA) for dietary exposure and risk assessments for the review of

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maximum residue limits (pesticide tolerances). USDA/PDP commodity samples were

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selected to reflect the highest U.S. consumption with an emphasis on foods consumed

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by infants and children. Commodities were collected at terminal markets and large

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chain store distribution centers from which food commodities are supplied to

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supermarkets and grocery stores that are typically available to the consumers.

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Commodities selected by USDA/PDP are cycled through approximately every five

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years.

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In the USCC study, we collected vegetable- and fruit samples for the purpose

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of detecting the residues of neonicotinoids. We selected this cafeteria to obtain

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samples because it is run by a food service company that publicly recognizes its

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corporate commitment to sustainability, as well as social and environmental

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responsibility. In the HZC study, were obtained the amount of fruits and vegetables

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consumed from the 24-hr dietary consumption questionnaires by children of ages 8-10

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from an elementary school in Hangzhou.

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We analyzed 64

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Analysis of neonicotinoid residues in fruits and vegetables

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fruit and vegetable samples collected by the USCC study for 7 neonicotinoids,

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including acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram,

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thiacloprid, and thiamethoxam, using a recently published analytical method by LC-

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MS/MS. 9 A total of 134 fruit and vegetable samples collected by the HZC study were

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also analyzed for the same 7 neonicotinoids, plus imidaclothiz a unique neonicotinoid

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only manufactured and used in China, using the same Chen et al. (2014) method with

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minor modifications to accommodate instrumental differences. Briefly, we used

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HPLC grade acetonitrile and formic acid from Merck (Rahway, NJ) and QuEChERS

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(containing 4 g of MgSO4, 1 g of NaCl, 500 mg of disodium citrate and 2 mL of SPE

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with a ceramic homogenizer containing of 25 mg of PSA, 7.5 mg of GCB, and 150

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mg of MgSO4) from Agilent (Shanghai, China) for sample extraction. Ten grams of

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fruit and vegetable samples were weighed, transferred to a 50 mL centrifuge tube, and

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then shaken for 30s. Ten mL of acetonitrile and 20 µL of internal standards solutions

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were added to the tube followed by one pack of QuEChERS salt and one ceramic

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homogenizer. The tubes were then shaken vigorously for 40s in the shaker and

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centrifuged for 4 min at 4000 g. Separation and detection of the neonicotinoids were

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achieved by the UPLC−MS/MS (Waters Corporation, Milford, MA) interfaced with a

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triple quadrupole mass spectrometer Xevo TQ-S (Waters Corporation) using an

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Acquity HSS T3 column (50 mm× 3.0 mm, 1.8µm, Waters Corporation). The system

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was run in isocratic mode with the mobile phase consisting of acetonitrile and Milli-Q

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water (95/5 V:V) acidified with 0.01% formic acid at a flow rate of 170 µl/min.

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Quality assurance (QA) and quality control (QC) samples using several commodity

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samples were also prepared and analyzed to ensure the accuracy and precision of

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analysis. The limits of detection (LOD) for individual neonicotinoids are listed in

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Tables 1-3.

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Data analysis

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fruits and vegetables across studies, we employed the relative potency factor (RPF)

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method, similar to a previously described method 10. We calculated the RPF based on

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the relative human toxicity of neonicotinoids compared to imidacloprid using

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reference dose (RfD – the maximum daily oral dose of a pesticide that the EPA deems

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acceptable) as shown in Equation (1). Then residues of imidacloprid, thiamethoxam,

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acetamiprid, clothianidin, thiacloprid, and dinotefuran measured in each fruit and

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vegetable sample were summed with respect to the RfD of imidacloprid, and

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expressed as IMIRFP, as shown in Equation (2). The reference dose for each

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neonicotinoid is listed in Tables 1-3. We did not include nitenpyram in the RPF

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calculation because no RfD is available for nitenpyram and there is no structurally

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similar neonicotinoid. For samples with neonicotinoid levels below the limit of

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detection (LOD), we applied half of the LOD in the RPF calculation.

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RPFi = RfDimidacloprid / RfDi (where i represents neonicotinoidi)……………Equation

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(1)

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IMIRPF (µg/kg) = Σi (neonicotinoidi *RPFi)

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= imidacloprid + thiamethoxam*9.5 + acetamiprid*0.8 + clothianidin*5.8 +

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thiacloprid*14.2 + dinotefuran*2.9 ……………………………………Equation (2)

In order to compare the residue levels of total neonicotinoids in

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Results Table 1 shows neonicotinoid residues measured in fruits and vegetables that

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we collected from the USCC study in January and May of 2015. We found 100%

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frequency of detection (FOD) for at least one neonicotinoid in all samples, except for

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grapes, sweet corn, and kale in which no neonicotinoids were detected. Samples

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containing either multiple residues or relatively high levels of neonicotinoids included

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cantaloupe, honeydew melon, sweet peppers, tomatoes (including cherry tomatoes),

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and zucchini. One apple and one squash sample contained very high levels of

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acetamiprid (50.8 µg/kg) and thiamethoxam (43.1µg/kg), respectively. We found

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approximately 79% and 65% of fruits and vegetables contained more than one

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neonicotinoid, respectively, and about 62% and 38% of fruits and vegetables

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contained more than 3 neonicotinoids, respectively. Thiamethoxam was the most

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frequently detected neonicotinoid with 59% detection in fruits and vegetables

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collected from these two congressional cafeterias, followed by imidacloprid (56%

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detection). Thiamethoxam’s RfD is 9.5 times smaller than the RfD for imidacloprid

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indicating a higher relative toxicity of thiamethoxam in human beings.

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Table 2 shows neonicotinoid residues measured in fruits and vegetables that

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we collected from a conventional market and a supermarket in the HZC study in 2015.

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Similar to the results of the USCC study, most of the samples contained at least one of

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the seven neonicotinoids that we analyzed, and only asparagus, taro roots, and spinach

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had frequency less than 100%. Samples containing noteworthy average neonicotinoid

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levels included bamboo shoots (59.3 µg/kg of dinotefuran), carrots (45.8 µg/kg of

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acetamiprid), celery (28.9 µg/kg of thiamethoxam), Chinese greens (110 µg/kg of

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acetamiprid), eggplant (28.6 µg/kg of imidacloprid), ginger (151 µg/kg of

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imidacloprid), green onion (37 µg/kg of imidacloprid), tomatoes (15 µg/kg of

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acetamiprid and 21.6 µg/kg of thiamethoxam), apples (28.9 µg/kg of dinotefuran, 45.4

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µg/kg of imidacloprid, and 25.9 µg/kg of thiamethoxam), bananas (35.9 µg/kg of

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thiamethoxam), mango (191 µg/kg of imidacloprid), oranges (20.2 µg/kg of

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acetamiprid), pineapple (50.3 µg/kg of imidacloprid), and strawberries (46.4 µg/kg of

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acetamiprid). We were able to detect all seven neonicotinoids in cucumbers, 6

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neonicotinoids in cabbages and eggplant, and 5 neonicotinoids in carrots, Chinese

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greens, kidney beans, tomatoes, and apples. We found approximately 57% and 63%

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of fruits and vegetables contained more than one neonicotinoid, respectively, and 30%

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and 42% of fruit and vegetable samples contained 3 or more neonicotinoids,

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respectively. Imidacloprid was the most commonly detected neonicotinoid with 68%

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detection in fruits and vegetables collected from HZC, followed by thiamethoxam

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(51% detection).

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Table S1 shows neonicotinoid residues measured in foods by USDA/PDP

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from 2011 to 2014. The frequency for neonicotinoids in the food samples was lower

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than those reported by the USCS or HZC studies. This is most likely due to the fact

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that much higher limits of detection (LODs) for the 6 neonicotinoids were used in

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analyzing food samples collected by the USDA/PDP, as shown in Table S1.

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Imidacloprid was the most frequently detected neonicotinoid, but with only 7.3%

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frequency in fruits and vegetables reported by USDA/PDP, followed by acetamiprid

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(5.1% frequency). Neonicotinoids were detected most frequently in cherries, with

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frequency of 94%, followed by apples (59%) and strawberries (47%). Minimal or no

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neonicotinoids were detected in avocado, carrots, soybean, mushrooms, bananas,

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papaya, soy-based infant formula, and a variety of vegetable-based baby foods. The

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average concentrations of the 6 neonicotinoids in fruit and vegetable samples from

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USDA/PDP were noticeably lower than those reported in our USCC and HZC studies,

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mostly because a large portion of USDA/PDP samples fell below the LODs.

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Regardless, several samples contained neonicotinoid levels that are worth highlighting

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here. USDA/PDP found all 6 neonicotinoids in 3.4% of 744 onion samples with the

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average concentrations of 10.4, 23.3, 1.5, 11.7, 0.4, and 20.4 µg/kg of acetamiprid,

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clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam, respectively.

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Five neonicotinoids were found together in 47% of 927 sweet bell pepper samples

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with the average concentrations of 2.9, 18.7, 10.2, 10.7, and 10 µg/kg of acetamiprid,

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clothianidin, dinotefuran, imidacloprid, and thiamethoxam, respectively. Among 396

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samples of spinach analyzed, approximately 5% of them were found to contain at

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least one neonicotinoid with average concentrations of 8.1, 20.1, and 20.1 µg/kg of

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acetamiprid, clothianidin, and imidacloprid, respectively.

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In order to quantify the total dietary intake from a mixture of neonicotinoids in

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a food sample and compare the total neonicotinoid levels across different studies

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conducted in different regions/countries, we utilized the relative potency factor (RPF)

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method as shown in Equation (2) to integrate all neonicotinoid residues found in each

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food sample using the respective reference dose values as the basis for summation.

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The RPF method normalizes each neonicotinoid to imidacloprid, an index

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neonicotinoid, based on the reference dose (RfD, mg/kg bw/day) reported by U.S.

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EPA (2010). Imidacloprid was chosen because it is the most widely used

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neonicotinoid worldwide, and because it has been better studied in regard to its

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toxicity than other neonicotinoids. 23 The value of IMIRPF (µg/kg) represents the total

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amount of all neonicotinoids, normalized to imidacloprid, in a food sample. Table 3

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and Table 4 show the average IMIRPF for fruits and vegetables that were analyzed in

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each of the 3 studies, ranking from the highest to the lowest IMIRPF concentrations. In

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the USCC study, honeydew melon had the highest IMIRPF, containing 54.9 µg/kg of

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imidacloprid-equivalent total neonicotinoids, whereas apples and cherries contained

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the highest IMIRPF of 384.1 and 401.8 µg/kg of imidacloprid-equivalent total

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neonicotinoids as measured in the HZC and USDA/PDP, respectively. Those three

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fruit samples not only contained the highest levels of IMIRPF, but also had the highest

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frequency ranging from 94 to 100%. For vegetables, squash, asparagus, and spinach

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had the highest IMIRPF of 427, 321, and 569 µg/kg in the USCC, HZC, and

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USDA/PDP, respectively. However, neonicotinoids were detected in only 5% of the

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total 396 samples of spinach collected by the USDA/PDP. For purposes of comparison, Figures 1 and 2 show the distributions of IMIRPF

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in the box plots for the same seven fruit and vegetable samples that were collected and

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analyzed by those three studies. Although the IMIRPF values for apples, cantaloupe,

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and strawberries were consistently lower in the USCC study than those measured in

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the HZC and USDA/PDP (Figure 1), only IMIRPF for apples and cantaloupe are

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significantly different (ANOVA, p

Neonicotinoid Residues in Fruits and Vegetables: An Integrated

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