5c

Environ Geochem Health (2007) 29:45–50 DOI 10.1007/s10653-006-9060-2

ORIGINAL PAPER

Imidacloprid residues in fruits, vegetables and water samples from Palestine Ayman Daraghmeh Æ Amjad Shraim Æ Sameer Abulhaj Æ Ramzi Sansour Æ Jack C. Ng

Received: 4 June 2005 / Accepted: 22 August 2006 / Published online: 25 November 2006
Springer Science+Business Media B.V. 2006

Abstract The aim of this work was to report on imidacloprid [IUPAC name 1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] residues in some vegetables, fruits, and water samples collected from the West Bank, Palestine, in 1998 and 1999. Imidacloprid and its derivatives in the study samples were extracted by methanol/ water and oxidized into 6-chloronicotinic acid and subsequently derivatized into 6-chloronicotinic acid trimethylsilyl ester before being determined by GC/MS. Imidacloprid residues were detected in more than half of the analyzed samples. The

highest and lowest imidacloprid concentrations were found in eggplant (0.46 mg/kg) and green beans (0.08 mg/kg), respectively. An increase of 11–120% in imidacloprid concentration in the 1999 samples was observed when compared with those of 1998. This may suggest imidacloprid accumulation in the soil and/or increased use by local farmers. The imidacloprid residue concentrations in several crops were found to exceed the CODEX maximum residue limit.

A. Daraghmeh Central Public Health Laboratory, Ramallah West Bank, Palestine

Introduction

A. Shraim Chemistry Department, Faculty of Science, Taibah University, P. O. Box 344, Almadinah Almunawwarah, Kingdom of Saudi Arabia A. Shraim (&) Æ J. C. Ng National Research Centre for Environmental Toxicology, The University of Queensland, 39 Kessels Road, Coopers Plains, Brisbane, Queensland 4108, Australia e-mail: [email protected] S. Abulhaj Æ R. Sansour The Centre of Environmental Health, Berzeit University, Berzeit, West Bank, Palestine

Keywords Pesticides Æ 6-Chloronicotinic acid Æ 1-(6-Chloro-3-pyridylmethyl)-N-nitroimidazolidin2-ylideneamine Æ West Bank

Imidacloprid [IUPAC name 1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a chloronicotinyl insecticide first introduced by Bayer Agricultural Products (Leverkusen, Germany). It was commercially developed and marketed in the early to mid-1990s. Imidacloprid is a general-use pesticide used worldwide for crop-, fruit-, and vegetable pest control, termite control, and flea control in dogs and cats (Ishii et al., 1994; Cox, 2001). It is most commonly used on rice, cereal, maize, potatoes, vegetables, sugar beet, fruit, cotton, hops, and turf (Heijbroek & Huijbregts, 1995; Huijbregts, Gijssel, &

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Heijbroek, 1995; Ahmed, Kanan, Inanaga, Ma, & Sugimoto, 2001; Dikshit & Lal, 2002; Kuhar, Stivers-Young, Hoffmann, & Taylor, 2002). Imidacloprid is the active ingredient in a wide variety of products, such as Gaucho, Alcador, and Confidor. It is a relatively new type of insecticide, possessing potential activity and novel modes of action (Moriya et al., 1992; Moffat, 1993; MacDonald & Meyer, 1998). Imidacloprid degrades in soil and plants into a number of different derivatives, all of which contain the chloropyridine structure with the 6-chloronicotinic acid as a major metabolite (Ishii et al., 1994; de Erenchun, Gomez de Balugera, Goicolea, & Barrio, 1997). The halflife of the insecticide in soil is reported to range from 29–190 days (Sarkar, Roy, Kole, & Chowdhury, 2001). On the other hand, Cox (2001) reported that imidacloprid is persistent in soil, as the concentration of tested soil samples had not decreased 1 year after application. It is now accepted that successive treatments in soil can lead to its accumulation in soil and crops (Ishii et al., 1994; de Erenchun et al., 1997). Moreover, owing to its persistence (Cox, 2001; Sarkar et al., 2001), high solubility and mobility, imidacloprid has the potential to leach into the groundwater (Cox, 2001). According to Cox, Koskinen and Yen (1997), the solubility of imidacloprid in water was 500 mg/l, whereas the partition coefficient (Koc) for imidacloprid was found to be dependent on the initial solution concentration and type of soil [Koc 71–81 (initial concentration 250 mg/l), 415–531 (initial concentration 1.5 mg/l) and 802–1560 (initial concentration 0.05 mg/l)]. Imidacloprid is classified by the US Environmental Protection Agency (US EPA) as both a toxic class II and a class III agent, and must be labeled with the signal word ‘‘Warning’’ or ‘‘Caution’’. The acute and chronic reference doses for imidacloprid for all population subgroups [including infants ( < 1 year) and children (1–12 years)] as set by the US EPA (1999) are 0.42 mg/kg per day and 0.057 mg/kg per day, respectively. Imidacloprid was also reported to be very toxic to aquatic invertebrates, toxic to upland game birds, and of low toxicity to fish (Cox, 2001).

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In Palestine, imidacloprid (as Gaucho) has been used for agricultural purposes since the 1980s. The average use of commonly used agrochemicals, including imidacloprid, per growing season in Palestine was estimated to be 65.5 kg/ha for greenhouse tomatoes, 38.7 kg/ha for greenhouse cucumbers, and 74.6 kg/ha for open-field cucumbers. Owing to the local political situation, imidacloprid residues in Palestinian crops have never been determined before. Therefore, the objective of this study was to determine the residues of imidacloprid (parent compound) and its derivatives (after oxidation to 6-chloronicotinic acid) in some local crops as they reach the consumers. For this purpose, samples of various food crops were collected from the local markets in the West Bank, Palestine and analyzed by GC/MS. In order to check if imidacloprid residues have contaminated the water resources, we also collected and analyzed groundwater samples from an extensive area ofagricultural activity.

Experimental method Chemicals 6-Chloronicotinic acid (99%), sodium hydroxide (GR 97%), anhydrous sodium sulfate (99%), potassium permanganate (GR 99%), sodium bisulfite (ACS reagent grade), t-butylmethyl ether (99%), and Amberlite XAD-4 (20–60 mesh) were all obtained from Sigma–Aldrich, Germany. Acetonitrile and methanol (both HPLC grade) were obtained from Merck, Germany. Nmethyltrimethylsilyltrifluoroacetamide (MSTFA) was obtained from Alltech, USA. Analytical standard imidacloprid was a gift from Bayer, Germany. Nitrogen gas (99.999%) was obtained locally. All deionized water used was organic-free Milli-Q water (Millipore, USA). Equipment A gas chromatograph (GC 17A, Shimadzu, Japan) equipped with an auto-injector and a mass spectrometer detector (QP 5000 MSD) in the

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single ion-monitoring (SIM) mode was used. A DB-5 capillary column (5% phenylmethyl polysiloxane polymers), 30 m · 0.25 mm · 0.25 lm from J & W Scientific (Folsom CA, USA) was utilized. The carrier gas was helium at 100 kPa and flow rate of 1 ml/min. The injector temperature was 260C. The oven temperature was programmed as follows: initial temperature 100C, held for 1 min, ramped at 15C/min to 180C, then at 30C/min to 300C, and held for 3 min. The detector was switched on 3 min postinjection after the elution of the solvent at the beginning of the temperature program. The two ions used in the quantification of 6-chloronicotinic acid–trimethylsilyl ester were 214 as the target ion and 170 as the qualifying ion. Sample collection Eleven types of vegetables and fruits (apples, bananas, cauliflower, cucumber, eggplant, grapes, green beans, maize, peaches, potatoes, and watermelon) were purchased from the local markets of the West Bank, Palestine during 1998–1999. At the same time, 19 groundwater samples were collected from the extensive agricultural area around Jenin city. Extraction and analysis of imidacloprid Imidacloprid and its derivatives in the samples were extracted and oxidized to 6-chloronicotinic acid as per the method of Placke & Weber 1993. Briefly, approximately 1 kg of each vegetable or fruit sample was chopped into small pieces and made into a slurry. A representative portion of the slurry (50 g) was then taken and soaked in methanol:water (3:1 v:v, 300 ml) for 30 min. The mixture was then homogenized in a blender and filtered, and the filtrate volume was made up to 500 ml with methanol. A portion of this solution (100 ml) was concentrated to approximately 20 ml in a rotary evaporator at 60C and then transferred to a pre-conditioned column packed with XAD4 resin (for water samples, an aliquot of 250 ml was concentrated as above and transferred to the column). The column was flushed twice with methanol:water (3:1 v:v, 20 ml), then the

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retained compounds were eluted with methanol (100 ml) and concentrated to 1 ml in a vacuum rotary evaporator and gentle stream of nitrogen. Imidacloprid and other derivatives containing the 6-chloropicolyl moieties extracted in the 1 ml concentrate were oxidized to 6chloronicotinic acid as follows: the concentrate was diluted in water (100 ml) before we added the oxidizing solution [sodium hydroxide (32%, 5 ml) and potassium permanganate (5%, 50 ml—100 ml added to samples of hops)]. The mixture was then quickly refluxed with stirring for 5 min. Another 50 ml water was added. The flask was placed in an ice bath and allowed to cool to 15C under agitation for 10 min. Sulfuric acid (10%, 50 ml) was then added, followed by three additions of solid sodium bisulfite (approx 1 g each addition) under cooling and agitation. More sulfuric acid was added, when necessary, to maintain a pH value of £1. Subsequently, the solution was extracted with t-butylmethyl ether (3 · 50 ml). The organic phase was filtered through anhydrous sodium sulfate (30 g) and evaporated to dryness in a rotary evaporator. The extract was then dissolved in acetonitrile (2 ml). An aliquot (250 ll) of this solution was derivatized to 6-chloronicotinic acidtrimethylsilyl ester by mixing it vigorously with MSTFA (250 ll). An aliquot (1 ll) was then injected into the GC/MS under splitless mode. Standard solutions of 6-chloronicotinic acid were derivatized in the same way as above, and their aliquots were injected prior to those of the samples. The analytical method, as used in this work, was originally validated by Placke & Weber 1993. However, some of the quality control parameters were re-checked and found to be similar to the findings of Placke and Weber 1993; the method detection level (MDL) (calculated from three standard deviations of a blank treated by the same procedure as above) was found to be 0.03 mg/kg, and the spike recovery was better than 75%. The standard deviation of three repeated injections of the extract of cucumber (1998) was 0.04 mg/kg, whereas the standard deviation of three repeated analyses of eggplant (1998) was found to be 0.05 mg/kg.

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Results and discussion The chemical structures of imidacloprid, 6-chloronicotinic acid, and 6-chloronicotinic acid-trimethylsilyl ester are shown in Fig. 1. Under the above experimental conditions, 6-chloronicotinic acid-trimethyl silyl ester elutes at around 12 min. The concentrations of residues of imidacloprid in fruits, vegetables, and water samples are summarized in Table 1. The highest average concentration was found in the eggplant (0.41 mg/kg), followed by potatoes (0.40 mg/kg), peaches (0.36 mg/kg), and watermelons (0.32 mg/ kg). On the other hand, the lowest concentration was found in grapes (0.08 mg/kg), followed by green beans (0.10 mg/kg) and cucumber (0.11 mg/ kg). Although collected from an extensive area ofagricultural activity, the water samples were found to have imidacloprid concentrations below the MDL (0.03 mg/kg). According to a report by the US EPA 1997, imidacloprid residues were not detected in any wells, ponds, lakes, streams, etc. near sites where imidacloprid was applied. However, this situation may change, as other groundwater monitoring studies for imidacloprid residues are currently underway in several parts of the USA.

Fig. 1 Chemical structure of imidacloprid, 6-chloronicotinic acid, and 6-chloronicotinic acid trimethylsilylester

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Imidacloprid residues were found in 66% and 73% of the samples collected in 1998 and 1999, respectively. Of the 122 samples collected in the 2-year study, more than 90% were of domestic origin, whereas the rest were imported from Israel and/or were of unknown origin. Imidacloprid residues for most of the samples collected in 1999 were greater by 11–120% when compared with those of 1998. This increase may be explained by accumulation of imidacloprid in the soil and/or more quantities per hectare of the insecticide have been applied in 1999. However, larger scale and more in-depth research is needed before an accurate assessment of the situation of this compound can be made. Imidacloprid was reported to be persistent in soil (decay time for 50% of imidacloprid (DT50) is 2 years), with a high potential for carry-over and build-up of chemical residues (Sandler, 2001). A study on crops grown in soils treated with imidacloprid 1 year and 2 years before, but not during the year when the investigation was carried out, showed that the average concentration of imidacloprid in crops grown on soils 1 year before was 4.8 lg/kg compared with 8.6 lg/kg for crops grown on soils treated with imidacloprid for two consecutive years [Coordination des Apiculteurs de France (CAF, 2000)]. The CODEX 2005), Australian [Australian Pesticides and Veterinary Medicines Authority (APVMA, 2006)] and Canadian [Pest Management Regulatory Agency (PMRA, 2005)] maximum residue limits (MRLs) in most of the samples analyzed for this study are listed in Table 2. The highest imidacloprid residue concentration detected in either year in bananas was greater than the CODEX MRL (no MRL set for Australia and Canada); in eggplant it was greater than the CODEX and Canadian MRLs (but not more than the Australian MRL); in maize it was greater than the Australian and Canadian MRLs (no MRL in CODEX); in potatoes it was greater than the Canadian MRL (less than CODEX and Australian MRLs), and, in watermelon, it was greater than the CODEX and Australian MRLs (no MRL set for Canada). Little work has been published in the literature on the contamination of food crops by

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Table 1 Residues of imidacloprid in Palestinian crops and water samples collected in 1998 and 1999 Product

Apples Bananas Cauliflower Cucumber Eggplant Grapes Green beans Maize Peaches Potatoes Watermelon Water Total a

No. of samples analyzed

No. of samples with imidacloprid residue

Percentage of samples with imidacloprid residue

Imidacloprid residue (mg/kg)

1998

1999

1998

1999

1998

1999

Average

5 3 5 5 3 4 5 4 4 7 6 8 59

6 5 5 5 6 5 3 2 4 5 6 11 63

5 1 4 4 3 4 3 3 2 7 3 0 39

6 3 4 5 6 4 3 2 2 5 4 0 46

100 33 80 80 100 100 60 75 50 100 50 0 66

100 60 80 100 100 80 100 100 50 100 75 0 73

0.24 0.18 0.26 0.11b 0.41c 0.08 0.10 0.28 0.36 0.40 0.32 < DLd

a

1998

1999

0.23 0.16 0.30 0.13 0.36 0.05 0.12 0.27 0.32 0.38 0.33 < DLd

0.26 0.21 0.19 0.09 0.46 0.11 0.08 0.30 0.41 0.43 0.32 < DLd

Average value of imidacloprid residue (mg/kg) of all the samples collected during both years

b

Standard deviation of repeated injection is 0.04 mg/kg (n=3).

c

Standard deviation of repeated analysis is 0.05 mg/kg (n=3)

d

Below method detection level, which is 0.03 mg/kg

Table 2 The CODEX (2005), Australian (APVMA 2006), and Canadian (PMRA 2005) imidacloprid maximum residue limits (in milligrams per kilogram) in crops analyzed for this study Food Item

This study, maximum concentration

CODEX MRL

Australian MRL

Canadian MRL

Apple Banana Cauliflower Cucumber Eggplant Grapes Green beans Maize Peaches Potatoes Watermelon

0.26 0.21 0.30 0.13 0.46 0.11 0.12 0.30 0.41 0.43 0.33

0.5 0.05 0.5 1.0 0.2 1.0 2.0 – 0.5 0.5 0.2

0.3 _ 0.5 0.2 0.5 0.1 – 0.05 0.5 0.5 0.2

0.5 – 3.5 0.5 0.08 1.5 – 0.05 – 0.3 –

imidacloprid, probably because this insecticide is not included in the food monitoring programs of most countries (Cox, 2001). Fernandez-Alba, Valverde, Aguera, Contreras, & Chiron (1996) found residues of imidacloprid in the range of 0.01–0.3 mg/kg in all analyzed samples (45 samples of peppers, tomatoes, and cucumbers) tested one week after treatment. In a cropmonitoring program, where 200 samples of fruits and vegetables were analyzed for pesticide residues, including imidacloprid, the same

authors (Fernandez-Alba, Tejedor, Aguera, Contreras, & Garrido, 2000) found imidacloprid residues in 25–53% (average 21%) of the 200 samples analyzed. Only two tomato samples out of the total of 42 samples that contained imidacloprid residues were found to exceed the limit set by the Spanish government. The findings of this study are worrying. Therefore, it is highly recommended that a monitoring program for imidacloprid residues in food crops at the national level be initiated.

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50 Acknowledgments The authors thank both Dr. Aqel Abu Qaree and Dr. Aarif El-Mubarak for their technical support. The project was funded by NIEHS under the MERC program. EnTox is jointly funded by Queensland Health, Griffith University, Queensland University of Technology, and The University of Queensland, Australia.

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