Imidacloprid in Melon Guttation Fluid: a Potential Mode of Exposure for Pest and Beneﬁcial Organisms

ECOTOXICOLOGY Imidacloprid in Melon Guttation Fluid: A Potential Mode of Exposure for Pest and Beneﬁcial Organisms

1 ERIC J. HOFFMANN AND STEVEN J. CASTLE

USDA Arid Lands Agricultural Research Center, 21881 N. Cardon Lane, Maricopa, AZ 85238

J. Econ. Entomol. 105(1): 67Ð71 (2012); DOI: http://dx.doi.org/10.1603/EC11251 ABSTRACT ELISA techniques were used to detect imidacloprid in guttation ßuid of young cantaloupe plants in Arizona. Imidacloprid was detected at up to 4.1 ␮g/ml (ppm) in a coincidental guttation collection 3 d after a top label rate soil application and at 37 ␮g/ml one d after a separate top label rate soil application study. These imidacloprid titers exceed reported median oral toxicities for several insect species by factors of 10 or more. Pesticides in guttation ßuid are a relatively unexplored route of exposure for both pest and beneÞcial insects, and could represent an important risk for both of these groups in guttation-prone environments.

KEY WORDS guttation, imidacloprid, ELISA, melon

Soil-applied plant-systemic insecticides are versatile was treated with a drip application of Admire Pro (551 compounds that have the beneÞt of focusing exposure g/L, BayerAG, Research Triangle Park, NC) at a top to target organisms. However, a recent report on toxic label rate of 767.3 ml/ha on 18 August 2010. Canta- levels of neonicotinoids in the guttation ßuid of maize loupe main stems had grown to approximately the seedlings after seed treatment demonstrates a novel sixth node stage at the time of treatment and had exposure risk for water-seeking honey bees and non- begun to bloom. The application was made using an target organisms in general (Girolami et al. 2009). in-line injector (D14MZ2, Dosatron, Clearwater, FL) Guttation is the expulsion of xylem water through set at 1:100 vol:vol and attached to drip tubing that leaf hydathodes, most commonly occurring in scenar- drew up the Admire Pro solution at a rate of 2.5 ios of high root pressure and low transpiration capacity liters/100 m/min. This is double the label application (reviewed in Ivanoff 1963). The collection of this ßuid rate on an area-basis, but equivalent on a plant-basis on leaf surfaces can be confused with dew condensa- because of a doubling of plant density using our halved tion, but guttation originates completely from the row spacing (203 cm is standard for melons). Total plant vascular system. water volume used was Ϸ13,000 liters, which included Residues and associated risks of pesticides in gut- prewetting of the ground, treatment injection, line tation ßuid are a proposed area of assessment for the clearing, and watering in. The second Þeld remained International Commission for Plant Bee Relationships untreated (1,200 m separation) but was furrow-irri- Bee Protection Group (Thompson 2010). This paper gated on the same schedule and to a similar soil mois- presents the Þrst data on guttation ßuid from canta- ture level as the treated Þeld. loupes that were treated with imidacloprid as a soil On the morning of 22 August 2010, it was coinci- application, as well as a contextual overview of imi- dentally observed that melon plants in both the dacloprid ingestion toxicities to various insects. treated and untreated Þelds had guttation ßuid at the leaf margins. Because of the typically low humidity and high temperatures, guttation is rarely observed Materials and Methods during summer months in central Arizona. However, Opportunistic Observation. Two cantaloupe (Cu- an evening monsoon storm event (2 cm rainfall) on 21 cumis melo cultivar ÔSol RealÕ) Þelds were planted on August served to increase both soil moisture and hu- 23 July 2010 for a study on the impact of soil-applied midity in the Þelds, both are necessary for guttation imidacloprid on honey bees (Apis mellifera L.). Plant- (Thompson 2010). Guttation samples were collected ings were at The University of Arizona Maricopa Ag- from Þve treated and two control plants using a mi- ricultural Center in Maricopa, AZ. Seeds were planted cropipette and stored in centrifuge vials at Ϫ80ЊC until at 28 cm plant spacing and 101.5 cm row width. Plants analysis. Guttation droplets had evaporated from were watered via furrow irrigation every 7Ð10 d. Fields leaves by 0800 hours. were 0.2 Ha conÞgured in 12 rows of 167 m. One Þeld Targeted Guttation Study. Because of the surprising Þndings of the imidacloprid titers in the August gut- 1 Corresponding author, e-mail: [email protected]. tation samples, a small plot of cantaloupe was planted 68 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 1

Fig. 1. Histograms of imidacloprid recoveries from melon guttation ßuid (A and B) and estimated imidacloprid leaf deposition (C and D) 1 and 5 d after a 21 Oct soil treatment at 21.8 and 14.7 mg imidacloprid per plant. on 15 September 2010 with the speciÞc goal of mon- tiÞcation threshold of 4 ng/ml (ppb) imidacloprid. itoring plants for guttation events that might occur Samples exceeding the linearity range were serially under cooler fall conditions. After germination, plants diluted by 20-fold until they fell within the linear were watered regularly, and an additional fertilizer standard range. Estimates of imidacloprid quantities application (Miracle-Gro 24Ð8-16, Scotts, Marrysville, deposited on leaves were made by multiplying the OH) was made to encourage guttation and promote collected volume for each sample by the analyzed cantaloupe growth. On 21 October, individual plants concentration. were treated with drenches of imidacloprid. Plants were treated individually at either 14.7 or 21.8 mg (AI) Results imidacloprid in 118 ml water. These application rates correspond to commercially used label rates of 282 and Opportunistic Observation. Imidacloprid was de- 422 g/ha. Row irrigation was applied immediately af- tected in the treated melon Þelds at mean Ϯ SEM rate ter treatment to disperse the application into the root of 2.155 Ϯ 0.547 ␮g/ml (ppm). The Þve samples col- zones of plants. lected from different cantaloupe plants ranged from Plants were observed daily at sunrise for any gut- 1.073 to 4.115 ␮g/ml. There was no detection of imi- tation events. Two events were noted, on 22 October dacloprid from untreated melons (n ϭ 2). and 26 October. All guttation drops from a single leaf Targeted Guttation Study. Imidacloprid was de- were collected using a micropipette into a 1.5 ml tected above the 4 ng/ml threshold at both 1 and 5 d centrifuge vial and volumes were estimated to the after treatment (Fig. 1A and B) for both application nearest 10 ␮l. For 22 October, 11 and 15 plants were rates. The maximum concentration detected was 37.35 sampled for the 14.7 and 21.8 mg doses, respectively, ␮g/ml. There was a single detection of 72 ng/ml out with Þve samples from untreated plants. For 26 Oc- of six untreated samples, but this is likely because of tober, 8 and 18 plants were sampled for the 14.7 and experimental error (see Discussion). Collected gut- 21.8 mg doses, respectively. Plants sampled on 22 Oc- tation volumes ranged from 10 to 380 ␮l per leaf and tober were not resampled. estimated imidacloprid deposition ranged from 3 to Chemical Residue Analysis. Imidacloprid levels 2,267 ng per leaf (Fig. 1C and D). were determined using a competitive ELISA kit (EP006, Envirologix, Portland, ME) that has a linear Discussion range of 0.2Ð6.0 ng/ml (ppb). Samples were initially screened at 20-fold dilutions to avoid possible matrix The ELISA technique used here is a useful tool for effects (Byrne et al. 2005), yielding a minimum quan- assessment of imidacloprid presence in guttation ßuid. February 2012 HOFFMANN AND CASTLE:IMIDACLOPRID IN MELON GUTTATION 69

Table 1. Examples of oral imidacloprid toxicities to selected pest and beneﬁcial insects

Insect Method Response Rate Citation

Acyrthosiphon pisun ad libitum Spiked ArtiÞcial LC50 48 h: 140 ppb Sadeghi et al. 2009 Diet 72 h: 30 ppb Myzus persicae ad libitum Spiked ArtiÞcial LC50 48 h: 73 ppb Nauen and Elbert 1997 Diet Bombus terrestris Oral dose LD50 24 h: 40 ng/bee Marletto et al. 2003 48 h: 20 ng/bee Bombus impatiens ad libitum Spiked pollen Reduced foraging rate 30 ng/g pollen Morandin et al. 2005 ad libitum Spiked pollen Reduced worker lifespan, worker 19.2 ␮g/g pollen Gradish et al. 2010 oviposition inhibition Apis mellifera Oral dose LD50 3.7Ð40.9 ng/bee Schmuck et al. 2001 Oral dose Medium term memory impact 12 ng/bee Decourtye et al. 2004 ad libitum Spiked syrup at Foraging impacts 46 ppb Ramirez-Romero et al. 2005 feeder 50 ppb Yang et al. 2008 Orius laevigatus ad libitum Spiked water Fifth instar LC50 1.1 ppm Delbeke et al. 1997 Adult LC50 2.1 ppm Podisus maculiventris ad libitum Spiked water Fifth instar LC50 4.15 ppm De Cock et al. 1996 Adult LC50 0.46 ppm

The intraplant variability in imidacloprid titers was and hydroxy forms of imidacloprid as well as the rel- over 100-fold, but this type of variability has been atively inactive urea, guanidine (desnitro) and N- observed in xylem ßuid after soil applied applications nitroso metabolites (Lagalante and Greenbacker to citrus (Castle et al. 2005) and across leaf ages in 2007). Plant metabolism of imidacloprid is variable, sugar beets (Westwood et al. 1998). but the insecticidal components represent a signiÞ- The competitive ELISA technique used by the com- cant proportion of the metabolite proÞle (Laurent and mercial imidacloprid test kits has been validated in Rathahao 2003, Sur and Stork 2003). The concentra- both water and plant-derived matrices. Because of tions reported here may be an overestimate of the nonspeciÞc binding of proteins in the original sample, imidacloprid parent compound, but may actually be dilutions are necessary to eliminate false positives and an informative test as it reports the contributions of inßated imidacloprid titers. Necessary dilutions vary multiple toxic imidacloprid metabolites. Such a total by matrix, but 20-fold dilutions have been found suf- report, in conjunction with time-series behavioral Þcient to avoid interferences in grapevine xylem sap studies can give a perspective on the contribution of (Byrne et al. 2005a). Byrne et al. (2005b) compared active and inactive metabolites (Byrne et al. 2005). the standard curve of imidacloprid-spiked water with The imidacloprid titers found in melon guttation those of 20-fold and 50-fold dilutions of spiked leaf (maximum of 37 ␮g/ml) are in line with those recently homogenates and found no signiÞcant difference. Wa- reported in corn seedling guttation. In Þeld and green- tanabe et al. (2007) found that 20-fold dilutions were house studies with corn germinated from imidaclo- sufÞcient to eliminate matrix effects for apple and prid-coated seeds, imidacloprid titers in seedling gut- grape juices and a 50-fold dilution was recommended tation ranged from 17 to 346 ␮g/ml at application rates for orange juice. The ELISA technique is extremely of 0.5Ð1.25 mg/seed (Gaucho, Bayer CropScience, robust when compared with HPLC reference meth- Research Triangle Park, NC) (Girolami et al. 2010, ods, with Ն99% correlations between the two in Tapparo et al. 2011). spiked sample pairings across various juice and agri- Imidacloprid has both ingestion and contact activ- cultural homogenates (Watanabe et al. 2004a,b, 2007) ity, and guttation drops could serve as a water source and over 94% in more complex leaf homogenates (Xu for both pest and nontarget organisms. Most published et al. 2006, Fischer et al. 2009). The xylem-derived bioassays report on direct topical application results or cantaloupe guttation ßuid has very little protein, and mixed exposure modes (leaf dip or Þeld residual as- was not found to present any matrix-based deviations says) that are not easily compared with these guttation when multiple dilutions were applied to the same data. However, there are some pest and nontarget sample. Similarly, we had no detections of imidaclo- examples of high-resolution ingestion data derived prid in the 20-fold dilutions of control samples, where from methods that assess per-insect dosing or re- matrix-induced titer inßation would be most likely. sponses to known pesticide concentrations (Table 1).

Our single control detection was not because of matrix Oral LC50 rates for the pea aphid Acyrthosiphon pisun effects; the sample was replicate-tested at 20- and (Harris) and the green peach aphid Myzus persicae 50-fold dilutions and both gave the same adjusted (Sulzer) were Ͻ200 ppb (ng/ml) imidacloprid, and concentration. The imidacloprid detection may be the the bumblebee Bombus terrestris (L.) had a 24 h oral result of treatment drift from an adjacent treated plant LD50 rate of 40 ng/bee (Nauen and Elbert 1997, Mar- after watering, misapplication, or mishandling of the letto et al. 2003, Sadeghi et al. 2009). Similar per-insect sample. LC50 rates were seen in the honey bee Apis mellifera In addition to imidacloprid, the ELISA test kit used (L.) (Schmuck et al. 2001). For context, the A. mel- here is mildly cross-reactive to the insecticidal oleÞn lifera honey stomach (also used for water-carrying) 70 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 1

has a volume of around 50 ␮l; 10 ␮lof10ppm(␮g/ml) Thripidae), using a combined bioassay and ELISA ap- imidacloprid represents a 100 ng load of active ingre- proach. Pest. Manag. Sci. 61: 754Ð758. dient. Sublethal individual and hive-level effects have Castle, S. J., F. J. Byrne, J. L. Bi, and N. C. Toscano. 2005. been assessed using several methods. Medium-term Spatial and temporal distribution of imidacloprid and memory was impacted at doses of 12 ng/bee (De- thiamethoxam in citrus and impact on Homalodisca co- courtye et al. 2004). Colony-level foraging impacts agulata populations. Pest. Manag. Sci. 61: 75Ð84. Curtis, L. C. 1943. Deleterious effects of guttated ßuids on from exposure to treated sugar syrup were not noted foliage. Am. J. Bot. 30: 778Ð781. at Þve ppb (Faucon et al. 2005) but have been seen at Curtis, L. C. 1944. The inßuence of guttation ßuid on pes- higher rates (Ramirez-Romero et al. 2005, Yang et al. ticides. Phytopathology 34: 196Ð205. 2008) (Table 1). Guttation ßuid is generally low in De Cock, A., P. De Clercq, L. Tirry, and D. Degheele. 1996. sugar content, and thus not highly attractive for for- Toxicity of diafenthiuron and imidacloprid to the pred- aging honey bees. However, water collecting in honey atory bug Podisus maculiventris (Heteroptera: Pentato- bees is intensive in arid regions and it is unclear to midae). Environ. Entomol. 25: 476Ð480. what degree ephemeral and rare sources such as gut- Decourtye, A., C. Armengaud, M. Renou, J. Devillers, S. tation would be used over more permanent water Cluzeau, M. Gauthier, and M. H. Pham-Delegue. 2004. supplies. Imidacloprid impairs memory and brain metabolism in the honeybee (Apis mellifera L.). Pestic. Biochem. The reviewed data of imidacloprid oral toxicities Physiol. 78: 83Ð92. suggest that imidacloprid concentrations below 20 Delbeke, F., P. Vercruysse, L. Tirry, P. De Clercq, and D. ␮ ppm ( g/ml) are toxic to a broad range of insects. For Degheele. 1997. 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