Why is Mosquito Control Important?

According to the CDC, some mosquitoes can carry and spread viruses like West Nile, dengue, Zika and parasites like malaria. Other mosquitoes bother people and are considered “nuisance” mosquitoes. Understanding the risks and preventing mosquitoes from breeding are the best and most practical means of keeping you and your family safe from mosquito-borne threats.

Q: What is the City of Mansfield doing to address the problem of West Nile Virus? A: The City of Mansfield has developed a comprehensive plan aimed at reducing the risk of illness due to West Nile Virus. The main goal of this plan is decreasing the number of adult mosquitoes by eliminating mosquito-breeding sites wherever possible. In areas where the elimination of mosquito breeding grounds is not possible, larvicides may be applied. The City also partners with the Tarrant County Health Department to perform mosquito surveillance activities during times of the year when mosquito populations are high. The City’s mosquito control responses are based on the likelihood of threats to human health from WNV or other mosquito-borne illness.

Q: What is the most effective means of mosquito control? A: According the Center for Disease Control and Prevention, source reduction, or elimination of larval habitat, is often the most effective and economical method of providing long-term mosquito control. Source reduction can include activities such as the proper disposal of used tires, cleaning rain gutters, emptying and refilling bird baths on a weekly basis, and any activity which reduces small pools of water. The City provides educational information about mosquito borne illnesses and prevention methods through the City’s website and social media sites. Additionally, public health education material is available at various locations throughout the City where residents frequent.

Q: Will the City Ever Spray for Mosquitoes? A: Adulticide (targeted ground spraying) isn’t the most effective first line of defense against mosquitoes. However, if the problem does progress and there is a need for steps beyond surveillance and larvicide, the City of Mansfield is prepared to move forward with targeted ground spraying. A positive case for a mosquito borne illness may prompt targeted ground spraying in the affected area.

Q: Is Spraying Safe? A: Effect on human health is one of the primary factors considered in regulation of pesticides. The City of Mansfield uses a water-based permethrin product for mosquito control, which has been determined by the EPA not to pose an unreasonable risk to human health. Furthermore, no specific risks to animals or the environment are expected when applied as directed and no special precautions are required.

Q: How Can I Learn More? A: The EPA, CDC and Tarrant County Health all have excellent information. For specific scholarly articles please see attached materials. Research

A Human-Health Risk Assessment for West Nile Virus and Insecticides Used in Mosquito Management Robert K.D. Peterson, Paula A. Macedo, and Ryan S. Davis Agricultural and Biological Risk Assessment, Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana, USA

approach extending from deterministic models West Nile virus (WNV) has been a major public health concern in North America since 1999, when (tier 1) based on conservative assumptions to the first outbreak in the Western Hemisphere occurred in New York City. As a result of this ongoing probabilistic models (tier 4) using refined disease outbreak, management of mosquitoes that vector WNV throughout the United States and assumptions [Society for Environmental Canada has necessitated using insecticides in areas where they traditionally have not been used or Toxicology and Chemistry (SETAC) 1994]. In have been used less frequently. This has resulted in concerns by the public about the risks from risk assessment, conservative assumptions in insecticide use. The objective of this study was to use reasonable worst-case risk assessment method- lower-tier assessments represent overestimates ologies to evaluate human-health risks for WNV and the insecticides most commonly used to control of effect and exposure; therefore, the resulting adult mosquitoes. We evaluated documented health effects from WNV infection and determined quantitative risk values typically are conserva- potential population risks based on reported frequencies. We determined potential acute (1-day) and tive and err on the side of safety. subchronic (90-day) multiroute residential exposures from each insecticide for several human sub- Unfortunately, few, if any, science-based groups during a WNV disease outbreak scenario. We then compared potential insecticide exposures considerations of the risks of insecticide use to toxicologic and regulatory effect levels. Risk quotients (RQs, the ratio of exposure to toxicologic versus the risks from vectorborne diseases have effect) were < 1.0 for all subgroups. Acute RQs ranged from 0.0004 to 0.4726, and subchronic RQs been examined. An understanding of the ranged from 0.00014 to 0.2074. Results from our risk assessment and the current weight of scien- human-health risks for both vectorborne dis- tific evidence indicate that human-health risks from residential exposure to mosquito insecticides are eases and associated vector controls would aid low and are not likely to exceed levels of concern. Further, our results indicate that, based on human- greatly in decision making by all stakeholders. health criteria, the risks from WNV exceed the risks from exposure to mosquito insecticides. Therefore, the objective of this study was to Key words: comparative risk assessment, mosquito control, organophosphates, pesticide exposure, use risk assessment methodologies to evaluate pyrethroids, risk analysis, vectorborne disease. Environ Health Perspect 114:366–372 (2006). human-health risks from WNV and from the doi:10.1289/ehp.8667 available via http://dx.doi.org/ [Online 28 October 2005] insecticides used to control adult mosquitoes. Materials and Methods West Nile virus (WNV) has become a major 1.5 million people were infected with the Problem formulation. Although WNV has public health concern in North America since virus in 2003. important effects on horses and birds, our 1999, when the first outbreak in the Western As a result of this ongoing disease out- assessment of health risks from WNV focused Hemisphere occurred in New York City, break, management of mosquitoes that vector only on humans. Currently, effect and expo- causing 62 cases of human encephalitis and WNV throughout the United States and sure factors for WNV are poorly understood 7 deaths [Centers for Disease Control and Canada has necessitated using insecticides in (Loeb et al. 2005), making quantitative mod- Prevention (CDC) 1999]. The initial out- areas where they traditionally have not been eling of risk difficult. Therefore, we evaluated break in New York City is thought to have used or have been used less frequently. This documented health effects from WNV infec- affected 2.6% of the population (Hubalek practice has raised concerns by the public tion and determined potential population 2001). In 2000, WNV spread to three states, about risks from insecticide use. In a survey by risks based on reported frequencies. Because with 21 human cases of WNV infection and Hinten (2000), 54% of 880 people surveyed of the relatively recent emergence of WNV in 2 deaths. In 2001, 66 human cases and were either equally afraid of WNV and pesti- North America, information on prevalence of 9 deaths were reported in 10 states, before cides or were more afraid of the insecticides. various effects of the disease should be WNV spread westward, affecting all but Since 1999, numerous concerns have been regarded as tentative. 6 states in 2002 and causing the largest raised by the public regarding the safety of Our tier-1 quantitative assessment of arboviral encephalitis epidemic in U.S. history using insecticides to control mosquitoes human-health risks associated with insecticides (Huhn et al. 2003). A total of 4,156 human (Cohen 2003; Fehr-Snyder 2004; Fitz 2003). cases were documented, with 284 deaths Some of those concerned have even suggested Address correspondence to R.K.D. Peterson, reported (CDC 2003), and numbers contin- that the health risks from the insecticides Department of Land Resources and Environmental ued to grow in 2003, when 46 states reported exceed those of WNV (Cohen 2003; Ziem Sciences, Montana State University, Bozeman, MT 9,862 human cases with 264 deaths (CDC 2005). These concerns by the public are not 59717 USA. Telephone: (406) 994-7927. Fax: (406) 2004a). In 2004, 2,539 human cases and exclusive to the WNV issue, but reflect long- 994-3933. E-mail: [email protected] 100 deaths were reported in 41 states (Hayes standing perceptions of risk from pesticides We thank the authors of the New York City et al. 2005). Since the first appearance of (Peterson and Higley 1993; Slovic 1987). Environmental Impact Statement, whose assessment made it possible for us not to have to reinvent the WNV in the United States in 1999, the CDC Risk assessment is a formalized basis for the wheel. has reported 16,706 documented human cases objective evaluation of risk in which assump- This study was funded by a grant from the U.S. and 666 deaths (CDC 2004b; Hayes et al. tions and uncertainties are clearly considered Armed Forces Pest Management Board’s Deployed 2005); however, large numbers of human and presented [National Research Council War Fighter Protection Research Program and by the infections may not be detected because of sig- (NRC) 1983, 1996]. Human-health and eco- Montana Agricultural Experiment Station, Montana nificant underreporting of milder cases of logic risk can be described in quantitative terms State University. The authors declare they have no competing West Nile fever (Hubalek 2001; Huhn et al. as a function of effect (also termed “hazard” or financial interests. 2003). Given the infection rate observed for “toxicity”) and exposure (NRC 1983). Risk Received 20 September 2005; accepted 28 October previous years, Peleman (2004) estimated that assessment typically uses a tiered modeling 2005.

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used in mosquito control focused on acute and mean body weight for females between 13 and dividing the most sensitive toxic effect [typically subchronic residential exposures after truck- 54 years of age (U.S. EPA 1996). Children 5–6 the no observed adverse effect level (NOAEL)] mounted ultra-low-volume (ULV) spraying of and 10–12 years of age were assumed to weigh by a series of uncertainty factors (typically a fac- mosquito adulticides. The dissemination of 21.1 and 40.9 kg, respectively. Infants tor of 100 to account for intraspecies and inter- mosquito adulticides by ULV application gen- (0.5–1.5 years of age) and toddlers (2–3 years species uncertainty) (Table 1). erates fine aerosol droplets that remain aloft of age) were assumed to weigh 9.4 and Environmental concentrations and fate of and target flying mosquitoes [U.S. Environ- 14.3 kg, respectively. All weights for children insecticides. We used the AERMOD, version mental Protection Agency (EPA) 2002b]. were derived from mean body weight values 1.0 tier 1 air dispersion model (U.S. EPA Acute exposures were defined as single-day for male and female children within their 1999) to predict the 7.6 m (25 ft) and 91.4 m exposures immediately after a spray event. respective age groups (U.S. EPA 1996). (300 ft) air concentrations (micrograms per Subchronic exposures were defined as expo- Hazard identification. We conducted cubic meter) of each insecticide within 1- and sures per day over a 90-day seasonal multispray human-health risk assessments for six insecti- 6-hr time ranges after ULV application by a event. A total of 10 spray events were assumed cide active ingredients (permethrin, pyrethrins, truck-mounted sprayer. Estimates of environ- to occur on days 1, 4, 14, 17, 27, 30, 40, 43, resmethrin, phenothrin, malathion, and naled) mental concentrations are presented only for 53, and 56. This design represents a reasonable and one synergist (piperonyl butoxide). truck-mounted ULV applications because our worst-case mosquito insecticide seasonal appli- Malathion and naled are in the organophos- modeling suggested that delivery of ULV cation scenario, including during a human epi- phate class of insecticides, and permethrin, applications by aircraft resulted in substan- demic of WNV [Karpati et al. 2004; New pyrethrins, resmethrin, and phenothrin are in tially less aerial and surface deposition (and York City Department of Health (NYCDOH) the pyrethroid class. The synergist, piperonyl therefore less human exposure and risk). This 2001]. Chronic exposures (> 6 months) to butoxide, is present in many formulations with was also observed by the NYCDOH (2001). mosquito adulticides are unlikely. Additionally, pyrethroids. All compounds are currently regis- We used the following conservative extrapolation of subchronic exposures to tered by the U.S. EPA for adult mosquito assumptions: a) each chemical had a 24-hr chronic exposure time frames would result in management in the United States. half-life in air except for naled, which was lower risks than with subchronic risks (NYC- Toxicity end points. Toxicity and dose– given a 18-hr half-life; b) the insecticides were DOH 2001). Therefore, chronic risks were not response information for each compound were applied at the maximum application rate as assessed in this study. reviewed for acute and subchronic exposure stated on each label; c) all of the insecticides Exposures to several population subgroups durations. Toxicity end points in this assess- were susceptible to the same weather condi- were estimated to account for potential age- ment were chosen based on U.S. EPA regula- tions using standardized weather data from related differences in exposure. Groups tory end points.We used inhalation, dermal, Albany, New York, in 1988; d) all spray events included adult males, adult females, infants and ingestion toxicity end points for each occurred at 2100 hr; and e) each spray release (0.5–1.5 years of age), and children (2–3, 5–6, respective exposure route and duration. was at 1.5 m. The chemical properties, appli- and 10–12 years of age). Adult males were Ingestion reference doses (RfDs) were used as cation rates, and predicted environmental con- assumed to weigh 71.8 kg, which represents the toxicity end points (acceptable daily expo- centrations for each active ingredient are listed the mean body weight for all males > 18 years sures) and were compared with total estimated in Table 2. of age, and adult reproductive females were exposures (total body burden). Acute and Receptors were established within the assumed to weigh 60 kg, which represents the subchronic ingestion RfDs were calculated by model on a Cartesian grid at five intervals of

Table 1. Toxicologic effects and regulatory end points for the active ingredients. Acute Subchronic Compound End point Study and toxicologic effects End point Study and toxicologic effects Malathion NOAEL = 50 mg/kg/daya Based on reduction in maternal bw gain in NOAEL = 2.4 mg/kg/daya Based on inhibition of blood enzyme activity at RfD = 0.5 mg/kg/day a study with pregnant rabbitsa RfD = 0.024 mg/kg/day 50 ppm malathion in the diet in a 24-month UF = 100 UF = 100 study in ratsa Naled NOAEL = 1.0 mg/kg/dayb Based on inhibition of blood and brain NOAEL = 1.0 mg/kg/dayb Based on inhibition of blood and brain enzymes RfD = 0.01 mg/kg/day enzymes in a 28-day study in ratsb RfD = 0.01 mg/kg/day in a 28-day study in ratsb UF = 100 UF = 100 Permethrin NOAEL = 25 mg/kg/dayc Acute neurotoxicity study in rats NOAEL = 25 mg/kg/dayc Acute neurotoxicity study in rats RfD = 0.25 mg/kg/day LOEL = 75 mg/kg based on observations RfD = 0.25 mg/kg/day LOEL = 75 mg/kg based on observations of UF = 100 of clinical signs such as aggression, UF = 100 clinical signs such as aggression, abnormal/decreased movement, and abnormal/decreased movement, and increased body temperaturec increased body temperaturec Resmethrin NOEL = 10 mg/kg/dayd Based on liver weight increases in a NOEL = 10 mg/kg/dayd Based on liver weight increases in a 6-month RfD = 0.1 mg/kg/day 6-month study in dogsd RfD = 0.1 mg/kg/day study in dogsd UF = 100 UF = 100 Phenothrin NOEL = 70 mg/kg/daye 13-week study in rats NOEL = 70 mg/kg/daye 13-week study in rats RfD = 0.7 mg/kg/day LOEL = 216 mg/kg-day based on increases in RfD = 0.7 mg/kg/day LOEL = 216 mg/kg-day based on increases in UF = 100 liver weights and decreases in cholesterol UF = 100 liver weights and decreases in cholesterol in both male and female ratse in both male and female ratse Pyrethrins NOAEL = 20 mg/kg/dayf Acute neurotoxicity study in rats NOAEL = 4.37 mg/kg/dayf Rat chronic toxicity study RfD = 0.07 mg/kg/day LOAEL = 63 mg/kg/day based on tremors RfD = 0.044 mg/kg/day LOAEL = 42.9 mg/kg/day based on increased UF = 300 in femalesf UF = 100 incidence of thyroid follicular cell hyperplasia in males.f Piperonyl NOAEL = 630 mg/kg/dayg Developmental toxicity study in rats NOAEL = 89 mg/kg/dayg Two generation reproduction study in rats butoxide RfD = 6.3 mg/kg/day LOAEL = 1,065 mg/kg/day based on decreases RfD = 0.89 mg/kg/day LOAEL = 469 mg/kg/day based on decrease in g g UF = 100 in maternal bw gain UF = 100 bw gain of F1 and F2 pups at postnatal day 2 Abbreviations: bw, body weight; LOAEL, lowest observed adverse effect level. LOEL, lowest observed effect level; NOEL, no observed effect level; UF, uncertainty factor used to determine the RfD. aU.S. EPA 2000c. bU.S. EPA 2002a. cU.S. EPA 2005c. dU.S. EPA 2000a. eU.S. EPA 2000b. fU.S. EPA 2005b. gU.S. EPA 2005a.

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25 m at 7.6 m and 91.4 m from the edge of through day 90 using the following multiple Acute inhalation exposure. Acute inhala- the spray emission area. The receptors were at a degradation model: tion exposures were estimated as height of 1.5 m. Each receptor estimated the 1- PE = (EEC × RR ×D × CF ) ÷ bw, [2] and 6-hr average air concentrations for each [1] insecticide. An average was then taken of the estimates from the six receptors at 7.6 m that where PE is potential exposure [milligrams per were not at the edges of the spray zone. The where D is the sum of the deposition over one kilogram body weight (bw)], EEC is the 6-hr following data were obtained using this net- spray, P is the peak deposition after a spray average estimated environmental concentration work of receptors: the 1-hr average concentra- event, r1 is the rate of decay calculated by using of an active ingredient in the air 1.5 m high at tion at 7.6 m, the 6-hr average at 7.6 m, and the aerobic soil half-life of each active ingredi- 7.6 m from the spray source (micrograms per the peak value at 91.4 m. ent, r2 is the rate of decay calculated by using cubic meter), RR is the respiratory rate under We used the screening Industrial Source the soil photolysis half-life of each active ingre- moderate activity (cubic meters per hour), D is Complex Short-Term (ISCST3) model (U.S. dient, t is the time in hours, and j is the spray the duration of exposure (hours), CF is the EPA 1995) to estimate particle deposition day. The average daily exposure was then deter- conversion factor to account for the conversion (milligrams per square meter) at 7.6 m and mined by dividing the deposition sum by 90. of units from micrograms per cubic meter to 91.4 m from the spray area at a 1-hr average. The same deposition and degradation milligrams per cubic meter, and bw is body The following assumptions were made in model was used to characterize deposition and weight (kilograms). addition to those from AERMOD: a) all of persistence on garden produce by using a RRs were assumed to be 1.6 m3/hr for the insecticides were susceptible to the same Kenaga nomogram to estimate the deposition adults and 1.2 m3/hr for children, including weather conditions using standardized weather (milligrams per kilogram dry weight) of each infants. These rates are indicative of moderate data from Salem, Massachusetts; b) the ULV insecticide on respective plant parts. Because physical activity (U.S. EPA 1996). The dura- particle size applications had 3% of the emit- the nomogram represents a linear relationship tion of exposure was 6 hr. Therefore, the ted particles greater than the allowable particle between application rate and maximum assumption was that the person was outside and size as stated on the label; and c) the particles residues, it can be used to estimate the maxi- 7.6 m from the spray truck when it passed him were assigned a density in accordance with the mum residues on plant surfaces for a given or her. Moreover, the person remained outside, specific gravity of each insecticide. application rate (Fletcher et al. 1994). For this 7.6 m from the emission, for the following A Cartesian grid was used for ISCST3 that analysis, maximum application rates were used 6 hr, respiring as if under moderate physical was similar to that used in AERMOD. for each insecticide, and each estimated con- activity during the entire time. Body weight for Receptors were added at 15.24-m intervals centration was then applied to the model the different age groups is discussed above. between 7.6 m and 91.4 m from the spray above, using the surface photolysis half-life to Acute dermal exposure from spray deposi- source to obtain a more accurate estimate of estimate the rate of degradation. tion. Acute dermal exposures from deposition the average deposition within 91.4 m of the Acute exposure. We assumed that multi- of spray drift on skin were estimated as source. The receptors were also at the same route exposures immediately after a single-spray PE = (TDE × AB) ÷ bw, [3] height of 1.5 m. All of the same methods were event were limited to 24 hr. Routes of insecti- used to calculate the average deposition at cide exposure included inhalation, dermal con- 7.6 m and 91.4 m. The middle receptors were tact with spray, hand-to-mouth ingestion by where PE is potential exposure (milligrams per included to calculate an average deposition infants and toddlers from spray deposition on kilogram bw), TDE is total dermal exposure within 91.4 m. The following data were hands, and ingestion of garden produce. We (milligrams), AB is dermal absorption rate, obtained from this information: deposition at also assumed that residents did nothing to limit and bw is body weight (kilograms). There is 7.6 m, deposition at 91.4 m, and the average their exposure to the spray. In its assessment of no publicly available information on dermal deposition within 91.4 m of the spray source. acute and subchronic exposures from several deposition immediately after truck-mounted For estimating subchronic exposures, we mosquito adulticides, the NYCDOH (2001) ULV sprays. Therefore, we used the U.S. EPA used the estimated deposition values within concluded that exposures from potable water Pesticide Handler Exposure Database (PHED; 91.4 m for each insecticide in an exponential and swimming were negligible. Using environ- U.S. EPA 1998) as a conservative surrogate. decay model to characterize their persistence mental fate models, we also concluded that the The PHED contains pesticide-handler scenar- on surfaces such as soil within a spray program chemical properties of the insecticides result in ios derived from field studies and exposure that included 10 sprays on days 1, 4, 14, 17, negligible concentrations in water. Therefore, estimates based on physical factors such as 27, 30, 40, 43, 53, and 56. Insecticide con- we did not include these exposures in our application rate, hectares treated per day, type centrations for each spray event were followed assessment. of clothing worn, methods of application, and

Table 2. Application rates, chemical properties, and predicted environmental concentrations of active ingredients. Active ingredient Property Piperonyl butoxide Phenothrin Permethrin Resmethrin Malathion Naled Pyrethrins Application rate (kg ai/ha) 0.0392 0.004 0.0078 0.0078 0.0639 0.0224 0.009 Density (g/mL) 0.898a 0.898a 0.8657b 0.87c 1.23d 1.67e 0.81f Surface photolysis half-life (days) NAg 6c 23h 0.14i 6.5i 2.4i 0.5j Soil aerobic half-life (days) 14i 7i 37k 30h 1h 1h 1j Acute air concentration (µg/m3)l 7.39 0.81 1.55 1.61 9.76 1.68 1.7 1-Day acute produce concentration (mg/kg dry wt) 0.525 0.054 0.105 0.105 0.855 0.3 0.12 90-Day mean surface concentration (mg/m2)m 15.42 0.43 4.14 0.22 2.18 0.65 0.54 90-Day mean produce concentration (mg/kg dry wt) 2.88 0.055 0.096 0.012 0.73 0.13 0.21 Abbreviations: ai/ha, active ingredient per hectare; NA, not available; wt, weight. aClarke Mosquito Control Products (1999b). bClarke Mosquito Control Products (1999a). cBayer Environmental Science (2004). dGriffin (2001). eAMVAC (2003). fMcLaughlin Gormley King Co. (2004). gSurface and produce concentrations determined from soil aerobic half-life only. hU.S. Department of Agriculture (USDA 2005). iNYCDOH (2001). jFood and Agricultural Organization (2000). kU.S. EPA (2005c). l6-Hr mean concentration at 7.6 m from spray source. m90-Day mean surface concentration within 91.4 m of the spray source.

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formulation type. We used the PHED sce- deposited on tomatoes is discussed above. We PE = (EEC × SEF × SA × DR × FA × D) nario in which a flagger (person marking the assumed that the resident did not wash the ÷ bw, [6] location for pesticide application while the tomatoes after picking. The residue concentra- application is occurring) was exposed to a liq- tion also did not change with processing of the where PE is potential exposure (milligrams per uid application. We assumed that the person tomatoes. The amount of insecticide ingested kilogram bw per day), EEC is the 90-day aver- was not wearing clothing and that the expo- was estimated as the product of the residue con- age environmental concentration of the active sure was 10 times greater than the flagger sce- centration and the quantity of food consumed. ingredient deposited on soil or turf within nario. We believe this scenario conservatively Tomato consumption patterns were deter- 91.4 m from the spray source (milligrams per estimated residential dermal exposure for two mined using the Dietary Exposure Evaluation square meter), SEF is saliva extraction factor, reasons: a) we added a 10-fold increase in Model [(DEEM)-Food Commodity Intake SA is surface area for three fingers (square exposure, and b) the U.S. EPA has not consid- Database (FCID) version 2.04; Exponent, meters), DR is dislodgeable residue, FA is fre- ered acute dermal contact from ULV applica- Washington, DC]. The model determines quency of activity (events per hour), D is tions for pyrethrins, piperonyl butoxide, and dietary consumption for the U.S. population exposure duration (hours), and bw is body permethrin because it was believed to be negli- and several subgroups by using individual weight (kilograms). Assumptions for estimat- gible (U.S. EPA 2005a, 2005b, 2005c). The food consumption records collected by the ing subchronic environmental concentrations values for percent dermal absorption were U.S. Department of Agriculture (USDA) are discussed above. The saliva extraction fac- 0.22% for pyrethrins (U.S. EPA 2005b), 2% Continuing Surveys for Food Intake by tor was assumed to be 50% (U.S. EPA 2005a, for piperonyl butoxide (U.S. EPA 2005a), Individuals for 1994–1998. Translation fac- 2005c), and the palmar surface area for three 10% for malathion and resmethrin (U.S. EPA tors used to convert foods-as-eaten to com- fingers was assumed to be 20 cm2 (U.S. EPA 2000a, 2000c), 15% for permethrin (U.S. modities are based on a U.S. EPA/USDA 2005c). Dislodgeable insecticide residue from EPA 2005c), 70% for phenothrin (U.S. EPA FCID recipe set. For this assessment, we soil or turf grass was assumed to be 20% (U.S. 2000b), and 100% for naled (U.S. EPA determined the acute food consumption pat- EPA 1997). The frequency of hand-to-mouth 2002a). terns by subgroup using the 95th percentile activity in children was assumed to be Acute hand-to-mouth exposure from 1-day consumption values for tomatoes, 20.5 events/hr and is based on the maximum spray deposition on hands. Acute hand-to- tomato baby food, tomato paste, tomato paste frequency observed (Freeman et al. 2005). mouth exposures were estimated for only two baby food, tomato puree, tomato puree baby The duration of exposure was assumed to be subgroups (toddlers and infants), because food, dried tomato, dried tomato baby food, 4 hr/day. Therefore, the toddler or infant was young children are more likely than adults to and tomato juice. Therefore, the respective assumed to be engaging in hand-to-mouth be exposed to pesticides as a result of hand-to- individuals in these subgroups ate all of these activities outside each day for 4 hr over 90 days. mouth contact (Cohen Hubal et al. 2000). tomato food products within 1 day of applica- Subchronic ingestion of garden produce. Exposures were calculated as tion at the 95th percentile of U.S. national Our assumptions for subchronic ingestion of consumption. garden produce were the same as for acute PE = [(THD ÷ HSA) × AHS × SEF] ÷ bw, [4] Subchronic exposure. We assumed multi- ingestion of produce, with the following differ- route exposures per day over 90 days after ences: a) the insecticide was deposited onto where PE is potential exposure (milligrams per multispray events. Routes of insecticide expo- both tomatoes and head- and leaf-lettuce, b) all kilogram bw), THD is total hand dermal expo- sure included inhalation, dermal contact with tomato and lettuce consumption by the resi- sure (milligrams), HSA is adult hand surface spray, ingestion of garden produce, hand-to- dents over the 90 days was from the garden, area (square meters), AHS is adjusted hand sur- mouth ingestion by infants and toddlers from and c) tomato and lettuce consumption pat- face area for each subgroup (square meters), spray deposition on hands, hand-to-mouth terns were determined using chronic food con- SEF is saliva extraction factor, and bw is body ingestion by infants and toddlers from deposi- sumption patterns (3-day average). weight (kilograms). Total hand dermal expo- tion on surfaces, dermal contact with soil and Subchronic dermal contact with soil and sure was determined using the PHED database other surfaces, and soil ingestion. other surfaces. Exposures from contact with and the assumptions discussed above. The Subchronic inhalation, dermal, and soil, turf, and other outdoor surfaces were hand surface area of toddlers (2–3 years of age) hand-to-mouth exposures. Exposures for each calculated as was assumed to be 0.035 m2, which represents exposure type were estimated as PE = (EEC × SA ×SS ×AB × DR × CF) the 50th percentile total surface area values for PE = (PE × SE) ÷ D, [5] males and females in the 2–3 and 3–4 year age acute, type ÷ bw, [7] groups, multiplied by the mean percentage of the total body represented by hands for males where PE is the potential exposure (milligrams where PE is potential exposure (milligrams per and females of that age (U.S. EPA 1996). The per kilogram bw per day), PEacute, type is the kilogram bw per day), EEC is the 90-day aver- hand surface area for infants was assumed to be acute exposure type (e.g., acute inhalation) age environmental concentration of the active 0.007 m2 and was also calculated as a percent from each spray event (milligrams per kilo- ingredient deposited on soil or turf within of total body surface area for infants (U.S. EPA gram bw), SE is the number of spray events, 91.4 m from the spray source (milligrams per 1996). We calculated the total body surface and D is the duration of exposure (days). We square meter), SA is body surface area in con- area of infants using the formula by Current assumed that the insecticides were sprayed on tact with surface (square centimeters), SS is (1998). We assumed that, on the day of appli- days 1, 4, 14, 17, 27, 30, 40, 43, 53, and 56 weight of soil adhered to skin (milligrams per cation, 50% of the insecticide deposited on the (10 spray events per season) in any given area. square centimeter), AB is dermal absorption hand was available through saliva extraction The exposure duration was 90 days. rate, DR is dislodgeable residue, CF is the con- (U.S. EPA 2005a, 2005c). Subchronic hand-to-mouth exposure version factor to account for square meters to Acute ingestion of garden produce. We from deposition on surfaces. Subchronic square centimeters, and bw is body weight assumed that the insecticide settled onto a hand-to-mouth exposures were estimated for (kilograms). The body surface area in contact tomato garden and that the resident picked, only two subgroups (toddlers and infants) with the surface was assumed to be the sum of processed, and ate tomatoes the next day. The based on the rationale discussed above. surface areas for face (head ÷ 2), hands, arms, estimated maximum insecticide residue Exposures were calculated as legs, and feet (U.S. EPA 1996). Therefore, we

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assumed residents were minimally clothed fever, and approximately 2.6% of the popula- 2001). The risk increases 43 times for persons while outside. Contact with surfaces was asso- tion in outbreak areas in New York were ≥ 80 years of age (Sampathkumar 2003). ciated with certain human activities. The activ- infected during the epidemic of 1999. Loeb Few data exist regarding long-term mor- ities were assumed to be gardening for adults et al. (2005) reported a 3.1% outbreak infec- bidity after WNV infection. Substantial mor- (0.55 mg soil/cm2 skin) and soccer for chil- tion rate in Oakville, Ontario, Canada, in bidity may follow hospitalization for WNV dren, including infants (0.164 mg soil/cm2 2002. Unfortunately, the seroprevalence of infection (Petersen et al. 2003) and is observed skin) (U.S. EPA 1996). We assumed that these WNV antibodies across larger time and geo- in patients with WN fever (Watson et al. activities occurred each day over the 90 days. graphic scales has not been determined. 2004). Encephalitis cases seem to have more The assumptions for dermal absorption rate Overall, 20% of infected persons develop mild variable outcomes than meningitis cases, which and dislodgeable residues are discussed above. febrile illness (Mostashari et al. 2001), and tend to recover well (Granwehr et al. 2004). A Subchronic soil ingestion. Exposures from 0.67% develop neurologic disease (Fratkin poor prognosis and very limited recovery have incidental ingestion of soil were calculated as et al. 2004). A total of 0.43% develop been observed in acute flaccid paralysis cases encephalitis, and 0.24% develop meningitis (Saad et al. 2005; Sejvar et al. 2003a, 2003b). PE = [(EEC ÷ SW) × SI ] ÷ bw, [8] (Asnis et al. 2001; Brilla et al. 2004; Emig and Although patients with WN fever tend to Apple 2003; Klee et al. 2004; Sejvar et al. recover well, median recovery time was 60 days where PE is potential exposure (milligrams per 2003a; Weiss et al. 2001). for patients in Illinois in 2002 (Watson et al. kilogram bw per day), EEC is the 90-day aver- Case-fatality rates in the United States 2004). The disease also has a significant effect age environmental concentration of the active ranged from 4 to 18% among hospitalized on the lifestyle of patients with WN fever. Of ingredient deposited on soil or turf within patients (Brilla et al. 2004; Emig and Apple 98 respondents with WN fever, 57 (58%) 91.4 m from the spray source (milligrams per 2003; Nash et al. 2001b; Pepperell et al. 2003; missed work/school, 82 (84%) had household square meter), SW is soil weight (milligrams Sejvar et al 2003a; Weiss et al. 2001) and from activities limited, 47 (49%) had difficulty per cubic meter), SI is soil ingestion (mil- 2.7 to 14% among cases reported to the CDC walking, and 89 (91%) had outside-of-home ligrams per day), and bw is body weight (kilo- (CDC 2004b). activities limited (Watson et al. 2004). grams). Because the insecticide would only be No difference in distribution of WNV In a long-term follow-up study on 42 WN surface-deposited on soil, we assumed that the infection among age groups and between sexes encephalitis survivors 1 year after illness onset, concentration (milligrams per square meter) is apparent (Nash et al. 2001a, 2001b; Tyler only 37% presented full physical, functional, would be the same for a cubic meter of soil. 2001), but for unknown reasons, males seem and cognitive recoveries, and there was a sub- Soil weight was assumed to be 3.86 kg/m3 to be at higher risk for WN neuroinvasive ill- stantially higher prevalence of impairment based on reported densities for Scotts lawn soil ness (O’Leary et al. 2004; Petersen and Marfin compared with baseline (Nash et al. 2001a). (The Scotts Company, Marysville, OH). Soil 2002). Children infected with WNV usually Similarly, only 2 of 8 patients in a study in ingestion rates were assumed to be 100 mg/day show no symptoms or have only a mild fever New York presented full recovery after 1 year; for children and 50 mg/day for adults (U.S. (Hayes and O’Leary 2004). The incidence of 3 patients had neurologic sequelae, and EPA 1996). We assumed that all soil ingestion encephalitis and death increases with age (Nash 1 patient had minimal impairment after each day was from soil containing residues of et al. 2001a, 2001b; O’Leary et al. 2004; Tsai 18 months (Asnis et al. 2001). the active ingredients. et al. 1998; Weinberger et al. 2001). Weiss Acute risks from insecticides. Table 3 shows Risk characterization. Human-health risks et al. (2001) reported that persons ≥ 50 years the calculated RQs for each active ingredient in in this study were assessed by integrating toxic- of age were more likely to present meningo- terms of total acute PE. Exposures and risks ity and exposure. We assessed risks using the encephalitis and had increased mortality rates; also were determined for each exposure route. risk quotient (RQ) method. For each popula- other reports show that the incidence of neuro- Potential acute inhalation exposures of the six tion subgroup, an RQ was calculated by divid- logic symptoms and death may increase 10- to human subgroups to the adulticides ranged ing the PE by the appropriate toxicity end 20-fold among persons ≥ 50 years of age (Nash from 0.00011 to 0.0075 mg/kg bw, and the point (e.g., the RfD). Therefore, the RQ is the et al. 2001a; Sampathkumar 2003; Tyler environmental concentrations were lower than ratio of exposure to effect. RQs < 1 are typi- cally below regulatory levels of concern. Table 3. Acute RQs for the active ingredients for each subgroup.a Exposures by similar route of exposure and Piperonyl duration (e.g., subchronic dermal contact with Subgroup Malathion Naled Permethrin Resmethrin Phenothrin Pyrethrins butoxide spray and surfaces) were compared with the appropriate RfD (e.g., subchronic dermal Adult males 0.0076 0.1496 0.0020 0.0052 0.0004 0.0081 0.0004 Adult females 0.0079 0.1576 0.0021 0.0055 0.0004 0.0085 0.0004 RfD). Multiroute exposures (dermal + inges- Children (10–12 years) 0.0105 0.2123 0.0029 0.0072 0.0006 0.0113 0.0006 tion + inhalation) were compared with the Children (5–6 years) 0.0177 0.3631 0.0049 0.0123 0.0010 0.0190 0.0009 ingestion RfD. The ingestion RfD provided a Toddlers (2–3 years) 0.0225 0.4726 0.0063 0.0159 0.0013 0.0245 0.0012 conservative toxicity end point because it typi- Infants (0.5–1.5 years) 0.0188 0.4495 0.0058 0.0147 0.0012 0.0218 0.0010 cally was based on the most sensitive NOAEL. aRQ = total acute PE ÷ RfD. Therefore, it represented the largest dose in which no adverse effects on human health Table 4. Subchronic RQs for the adulticides for each subgroup.a would occur during the relevant exposure Piperonyl duration. Subgroup Malathion Naled Permethrin Resmethrin Phenothrin Pyrethrins butoxide Results Adult males 0.0360 0.0259 0.0007 0.0004 0.0001 0.0056 0.0032 Adult females 0.0363 0.0269 0.0007 0.0004 0.0001 0.0056 0.0032 West Nile virus risks. According to a sero- Children (10–12 years) 0.0470 0.0290 0.0008 0.0005 0.0001 0.0074 0.0043 epidemiologic survey conducted by Mostashari Children (5–6 years) 0.0676 0.0447 0.0012 0.0009 0.0002 0.0104 0.0059 et al. (2001), for every diagnosed case of West Toddlers (2–3 years) 0.1815 0.1294 0.0204 0.0037 0.0009 0.0270 0.0262 Nile (WN) meningoencephalitis, there were Infants (0.5–1.5 years) 0.2074 0.1661 0.0301 0.0054 0.0013 0.0292 0.0325 approximately 30 additional people with WN aRQ = total subchronic PE ÷ RfD.

370 VOLUME 114 | NUMBER 3 | March 2006 • Environmental Health Perspectives West Nile virus and mosquito insecticide risks

the inhalation reference concentrations for all (2005) assessed human exposure to ULV- expressing neurologic disease. Depending on active ingredients evaluated. Potential acute applied naled, permethrin, and phenothrin in the insecticide, her acute exposure would be dermal exposures to the adulticides ranged Mississippi, North Carolina, and Virginia as 0.0415–15.76% of the RfD (0.0004–0.1576% from 0.0000001 to 0.0011 mg/kg bw, with a result of emergency large-scale mosquito of the NOAEL). Consequently, her acute risks RQs ranging from 0.0000005 to 0.0113. For abatement. Using biomonitoring of urine, from the insecticide would be lower than her acute exposure due to ingestion (hand-to- they did not observe an increase in insecti- acute risks from WNV. Subchronic insecticide mouth exposure from spray deposition on cide metabolite concentrations among risks would also be negligible (Table 4), hands and ingestion of produce), total PEs exposed residents. Karpati et al. (2004) and whereas subchronic and chronic WNV risks ranged from 0.0001 to 0.0061 mg/kg bw, with O’Sullivan et al. (2005) did not observe (disease sequelae) would be greater. Therefore, RQs ranging from 0.00014 to 0.2142. Total increases in hospital emergency department once exposed to the insecticide (based on the acute RQs ranged from 0.0004 to 0.4726. visits for asthma after wide-scale spraying of tier 1 exposure assumptions from this study), Subchronic risks from insecticides. Table 4 residential neighborhoods. the risk of any adverse health effects to the shows the calculated RQs for each active Uncertainties. Despite the conservatism of adult female would be negligible. ingredient in terms of total subchronic PE. our risk assessment, uncertainties were Results from our risk assessment and the Potential subchronic inhalation exposures of revealed. Many of the uncertainties associated current weight of scientific evidence (Currier the six subgroups to the adulticides ranged with residential exposure estimates are dis- et al. 2005; Karpati et al. 2004; NYCDOH from 0.000012 to 0.00083 mg/kg bw. For cussed above. The principal uncertainty was 2001; O’Sullivan et al. 2005; U.S. EPA 2000c, subchronic dermal exposures to the adulticides for environmental concentrations of the active 2005a, 2005b, 2005c) indicate that human- (dermal and contact with soil), total PEs ingredients. Data for actual aerial concentra- health risks from residential exposure to mos- ranged from 0.00000006 to 0.00015 mg/kg, tions and surface deposition of active ingredi- quito adulticides are very low and are not likely with RQs ranging from 0.0000001 to 0.0015. ents need to be generated to more accurately to exceed levels of concern. Further, by virtu- Potential subchronic exposures due to inges- characterize risks. Because of the nature of ally any current human-health measure, the tion (ingestion of produce and soil, hand-to- ULV application methods, it is likely that con- risks from infection by WNV exceed the risks mouth activity after contact with surfaces, centrations of active ingredients are much from exposure to mosquito insecticides. and hand-to-mouth activity after contact lower than those predicted using the AER- Therefore, perceptions that human-health risks with spray drift) ranged from 0.00001 to MOD and ISCST3 tier 1 models. Toxicologic from the insecticides used to control adult 0.0283 mg/kg bw, with RQs ranging from uncertainties include mammalian toxicities to mosquitoes are greater than the risks from 0.00007 to 0.1709. Total subchronic RQs combinations of piperonyl butoxide and adul- WNV currently cannot be supported by cur- ranged from 0.00014 to 0.2074. ticides and to inert ingredients in the formu- rent scientific evidence. Our results, and the None of the subgroups had RQs ≥ 1.0 lated products. The addition of piperonyl results from other studies, should be used by (i.e., PEs did not equal or exceed the RfDs) for butoxide to the adulticides increases the mos- the U.S. EPA, public health officials, and the any of the active ingredients evaluated. The quito toxicity of the pyrethroids approxi- general public to make better-informed deci- lowest acute RQs were to phenothrin and mately 10-fold, but mammalian toxicity is not sions about risk–risk tradeoffs. piperonyl butoxide for adults and the highest likely to be proportionally increased (Knowles acute RQ was to naled for toddlers (Table 3). 1991). Even if mammalian toxicity were CORRECTION The lowest and highest subchronic RQs were increased 10-fold to the pyrethroids, RQs to phenothrin for adults and malathion for would still be well below levels of concern. In the original manuscript published infants, respectively (Table 4). Human exposures to solvents and other inert online, the acute air concentration for naled ingredients are likely to be low, resulting in in Table 2 and the RQ ranges for acute Discussion low risks (NYCDOH 2001). Future research inhalation exposures and acute subchronic Conservatism. Based on the exposure and toxi- should be directed toward reducing toxicity dermal exposures were incorrect. These city assumptions above, we believe our and exposure uncertainties associated with have been corrected here. assumptions were sufficiently conservative and mosquito adulticides. In addition, future most likely overestimated risk. For example, assessments should address ecologic risks. assuming an acute RR of 0.8 m3/hr for 2 hr Comparing risks. Although it is difficult to REFERENCES and no dermal or ingestion exposures [which compare the risks directly, several conclusions were the U.S. EPA assumptions for mosquito can be drawn by considering both human AMVAC. 2003. Trumpet EC Insecticide. 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372 VOLUME 114 | NUMBER 3 | March 2006 • Environmental Health Perspectives APICULTURE AND SOCIAL INSECTS Minimizing the Impact of the Mosquito Adulticide Naled on Honey Bees, Apis mellifera (Hymenoptera: Apidae): Aerial Ultra-Low-Volume Application Using a High-Pressure Nozzle System

1 2 2 HE ZHONG, MARK LATHAM, STEVE PAYNE, AND CATE BROCK

John A. Mulrennan, Sr., Public Health Entomology Research and Education Center, College of Engineering Sciences, Technology and Agriculture, Florida A&M University, 4000 Frankford Ave., Panama City, FL 32405

J. Econ. Entomol. 97(1): 1Ð7 (2004) ABSTRACT The impact of the mosquito adulticide naled on honey bees, Apis mellifera L., was evaluated by exposingtest beehives to nighttimeaerial ultra-low-volume (ULV) applications usinga high-pressure nozzle system. The tests were conducted during routine mosquito control missions at Manatee County, Florida, in summer 2000. Two treatment sites were sprayed a total of four times over a 10-wk period. Honey bees, which clustered outside of the hive entrances, were subjected to naled exposure during these mosquito control sprays. The highest average naled ground deposition was 2,688 ␮g/m2 at the Port Manatee site, which resulted in statistically signiÞcant bee mortality (118) compared with the controls. At the Terra Ceia Road site, an intermediate level of naled deposition was found (1,435 ␮g/m2). For this spray mission, the range of dead bees per hive at Terra Ceia was 2 to 9 before sprayingand 5 to 36 after naled application. Means of all other naled grounddepositions were Ͻ850 ␮g/m2. We concluded that substantial bee mortality (Ͼ100 dead bees) resulted when naled residue levels were Ͼ2,000 ␮g/m2 and honey bees were clustered outside of the hive entrances during mosquito adulticide applications. Compared with the ßat-fan nozzle systems currently used by most of FloridaÕs mosquito control programs, the high-pressure nozzle system used in this experiment substantially reduced environmental insecticide contamination and lead to decreased bee mortality. Statistical analysis also showed that average honey yield at the end of the season was not signiÞcantly reduced for those hives that were exposed to the insecticide.

KEY WORDS aerial ULV, Apis mellifera, honey bees, honey yield, naled

FLORIDA IS CURRENTLY the fourth largest honey pro- athion deposition and bee mortality. Bee colonies ex- ducer in the United States. Hodges et al. (2001) re- posed to ultra-low-volume (ULV) malathion applied ported that FloridaÕs apicultural industry, with an es- at 210 ml/ha weighed signiÞcantly less for up to 28 d timated 258,000 honey bee, Apis mellifera L., colonies, compared with control colonies (Pankiw and Jay produced 25.58 million pounds of honey in 1999. In 1992b). Hill et al. (1971) reported substantial bee addition, honey bees pollinate many important fruit losses, but rapid reestablishment of the colonies when and vegetable crops, and the economic impact of bees hives were not covered duringaerial ULV application to FloridaÕs agriculture is invaluable. Insecticide poi- of malathion. In another study, Caron (1979) reported soningof honey bees is one of the most serious prob- consistent losses to bee colonies duringdaylightap- lems faced by honey producers (Tew 1998). The neg- plication of ground-based ULV malathion, but night- ative impact of insecticide application on honey bees time application had no measurable effect on the bee is well documented (Shaw and Armstrong1966; Levin colonies. Subsequent research by Hester et al. (2001) et al. 1968; Colburn and Langford 1970; Hill et al. 1971; also demonstrated that proper ground ULV applica- Atkins 1972; Caron 1979; Atkins et al. 1981; Gary and tion of malathion at night had no signiÞcant impact on Mussen 1984; Pankiw and Jay 1992a, b). Atkins et al. honey bees. (1981) compared the relative toxicity of four insecti- The insecticide naled (1,2-dibromo-2,2-dichloro- cides against caged honey bees and ranked them from ethyl dimethyl phosphate) is one of the most effective most to less toxic as follows: bendiocarb, chlorpyrifos, and widely used mosquito adulticides currently reg- malathion, and pyrethrins. Pankiw and Jay (1992a) istered for aerial ULV application in Florida. Colburn demonstrated a signiÞcant correlation between mal- and Langford (1970) observed naled to be more toxic to honey bees than malathion. Some Florida bee- 1 E-mail: [email protected]. keepers have reported bee kills that presumably re- 2 Manatee County Mosquito Control District, P.O. Box 386, Pal- sulted from operational aerial application of naled for metto, FL 34220. control of mosquitoes (personal communication with

0022-0493/04/0001Ð0007$04.00/0 ᭧ 2004 Entomological Society of America 2JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 1

Gary Ranker, beekeeper). In addition, they suspected these beehives duringthe entire experimental period. that reduction of honey yield was associated with the Sixteen hives, arranged in clusters of four, were placed abnormal number of dead bees. Previous work by at each of the three study sites. The hives in these Caron (1979) found that caged bees could be killed up four-member clusters abutted each other on two sides to 60 m from the path of ground ULV application of forminga “square” with cardinal orientation of the naled. Shaw and Armstrong(1966) found that naled hive entrances. At the treatment sites (sites 1 and 2), was toxic to bees clustered outside of the hives, but it the hives were divided equally and positioned within was nonhazardous to bees inside their hives. Rind- two different topographic areas. At site 1, eight hives ßeisch (1983) reported that reduction of bee mortality were placed in an open area (referred to as the open from aerial application of naled could be achieved by Þeld area), and eight were located in a forested area. light water misting and proper hive placement. At site 2, eight of the hives were situated in an open Aerosol deposits of naled have been shown to cause area, and the remaininghives were placed at the edge adverse impacts on honey bees through direct contact of a wooded area (referred to as the near forest area). (Zhong et al. 2003b). After foraging, honey bees ag- At the control site (site 3), all 16 hives were placed in gregating in front of entrances to hives are more likely the same open area because this site would not be to be exposed to mosquito control insecticides, which exposed to naled spray. could possibly weaken the bee colonies and reduce A modiÞed dead bee collector (Gary 1960, Hester et honey production and pollination activities. In our al. 2001) was positioned in front of each beehive to previous research, we observed increased bee mor- monitor for bee mortality. The collector was made tality when naled ground deposits were Ͼ3,500 ␮g/m2 from wire mesh screen (0.3-cm mesh) fashioned in the (Zhonget al. 2003b). This observation was based on shape of an open box measuring40.6 by 38.1 by 25.4 cm nighttime aerial ULV application of naled using a con- (length by width by height). Every 24 h, the number ventional ßat-fan nozzle system. We surmised that the of dead bees in each trap was counted and recorded, heavy deposits around hives were probably from large and the traps were emptied. The eight hives in each naled droplets (50Ð60 ␮m volume median diameter treatment area were used as replicates for this study. [VMD]) generated by the ßat-fan spray system. We Dead bees were to be counted 12 h before and 12 h hypothesized that decreasingthe aerosol droplet size after naled treatment. Duringthe initial spray mission, would reduce naled ground deposits and would, however, the collectors were not emptied before thereby, lead to a reduction in bee mortality. To test treatment, and no prespray mortality counts were this hypothesis, an experimental high-pressure hy- made. Natural mortality rates for honey bees were draulic spray system replaced the ßat-fan nozzle sys- estimated from the dead bee counts at both the control tem previously used for the aerial ULV application. hives and at the prespray hives. In addition to bee The high-pressure system is capable of generating counts, all hives were observed for bee behavior after droplets with a 20Ð30-␮m VMD (Zhonget al. 2003a). naled application, around mid-morningwhen the bees Research has already shown that the smaller droplets were actively crawlingon the hive surfaces. Any ab- generated by a high-pressure system increase mos- normal behavior was recorded. The beekeeper also quito control efÞcacy by enhancingimpingement opened several of the hives (by smokingthe hives) to onto adult mosquitoes (Dukes et al. 2001). Our cur- inspect the frames inside the beehives and to check for rent research was designed to evaluate the impact of dead bees. At the end of the study season, the honey a high-pressure spray system on insecticide deposi- produced by each hive was extracted from the combs, tion, honey bee mortality, and seasonal honey yield weighed, and recorded. Two hives at site 1 (one in when used for aerial ULV application of naled for each typographic area) did not produce enough mosquito control. honey to be extracted and weighed. Aerial ULV Application. Aerial ULV applications were conducted duringroutine mosquito control mis- Materials and Methods sions usinga high-pressurenozzle system in summer Research Site. This study was conducted in Manatee 2000. DIBROM Concentrate (ADAPCO, Inc., San- County, Florida, in the city of Palmetto. Two treat- ford, FL), which contains 87.4% naled as the active ment sites (sites 1 and 2) and one control site (site 3 ingredient, was applied. The two treatment sites were without naled exposure) were chosen. Site 1 was po- sprayed a total of four times over a 10-wk period by the sitioned near Terra Ceia Road and had global posi- Manatee County Mosquito Control District. Spraying tioningsystem (GPS) coordinates of 82.57419 W and began Ϸ2 h after sunset usingan MBB BO105 CBS4 27.57822 N. Site 2 was located at Port Manatee, Ϸ6.04 N2LA helicopter equipped with a high-pressure noz- km north northeast of site 1 and had GPS coordinates zle system. The high-pressure nozzle system was an of 82.54491 W and 27.62531 N. Site 3 was located at experimental model conÞgured by district personnel. Manatee Fruit, Ϸ15.85 km south southwest of site 1 The nozzles were positioned on the lateral boom and had GPS coordinates of 82.61992 W and 27.44194 pointingtoward the rear of the helicopter. The system N. was operated at 17,237 KPa (2,500 psi) and delivered Honey Bee Bioassay. Forty-eight hives were used to DIBROM Concentrate at a rate of 49.25 ml/ha evaluate the impact of naled exposure on honey bees. (0.667oz/acre). The insecticide was applied in multi- The beehives were obtained from, and maintained by, ple swaths, with a ßight line separation of 322 m. The a local beekeeper. No acaricides were used on any of helicopter was ßown at 177 km/h (110 miles/h), at an February 2004 ZHONG ET AL.: MINIMIZING THE IMPACT OF NALED SPRAY ON HONEY BEES 3 altitude of 30Ð46 m. The aircraft was equipped with a standard used for the calibration was obtained from Star 5000 GPS (ARNAV Systems, Inc., Puyallup, WA) AMVAC Chemical Corporation (Los Angeles, CA) to conÞrm the accuracy of ßight lines. Weather in- and was certiÞed as 99.8% pure naled. The analytical- formation, includingwind speed and direction, tem- grade naled was dissolved in a small volume of hexane perature, and relative humidity, was collected at ap- to make a stock standard solution with a concentration plication altitude by usinga Kitoon system (ADAPCO, of 1 mg/ml. This naled stock solution was subse- Inc.). The Kitoon system was positioned in an open quently diluted to prepare calibration solutions. In the area between the two treatment sites. laboratory, continuous calibration at a 2-␮g/ml level Naled Residue Sample Collection. Two Þlter papers was conducted every 10 samples usinga recovery (24 cm, Whatman, Maidstone, United Kingdom), criterion of 100 Ϯ 10%. Laboratory control spikes and placed side by side on a styrofoam board (40 by 80 by Þeld spikes were used, respectively, to check for ex- 2 cm) covered with aluminum foil, were used to col- traction efÞciency and Þeld degradation of naled. The lect naled residue. One styrofoam board was placed recovery criterion for the laboratory control spike horizontally on the ground at each treatment area and samples was 100 Ϯ 20%. Field blanks, laboratory at the control site in the immediate vicinity of the test blanks, and instrument blanks were used to check for hives. The residue samples were collected 2 h after possible contamination. treatment to allow sufÞcient time for the naled spray Statistical Analyses. A randomized complete block droplets to settle. Each Þlter paper sample was re- design was used for this study. Data analyses were moved from the styrofoam board, folded usingtwo blocked for each spray mission (spray date) so that pairs of clean forceps, and placed into a 40-ml Pyrex meteorological variations could be kept to a minimum. screw-top culture tube (CorningGlassworks, Corn- All naled residue data and bee mortality data were ing, NY). The tube was immediately Þlled with 30 ml analyzed by analysis of variance (ANOVA) usingSTA- of hexane to prevent further degradation of the naled TISTICA software (StatSoft, Inc. 2001), with a signif- residue duringsample storageand transport. Residue icance level of P Ͻ 0.05 chosen for all statistical tests. recovery in the Þeld samples was monitored by spiking Statistically different means were separated usingthe 50 ␮l of a naled standard (1 mg/ml in hexane) onto Student-Newman-Keuls test (Sokal and Rohlf 1981). clean Þlter papers at the start of the aerial application. For each of the four spray missions, naled residue data These naled Þeld spikes, which served as quality con- were analyzed by comparingthe amount of residue trol samples, were placed in the Þeld at an upwind found at each treatment area with that at both the location and subjected to the same environmental control site and at the other treatment areas. The conditions as the residue samples. Field spike samples naled residue data were transformed using( x ϩ 1)1/2 were the Þrst to be put out and the last to be collected before analysis, though only nontransformed means from the Þeld. All samples were stored in a refrigerator and standard errors are presented in the tables. For the at 4ЊC until shipment. The samples were transported bee mortality data, multiple comparisons of means in a cooler with icepacks to the chemistry laboratory were made to evaluate the impact of naled exposure at the Public Health Entomology Research and Edu- on honey bees. The statistical analyses planned for cation Center (Panama City, FL) for naled residue each spray mission included comparisons of the num- analysis. ber of dead bees per hive: 1) at pre- and posttreatment Naled Residue Analysis. Within 36 h of arrivingat intervals, and 2) at exposed (treated) and unexposed the laboratory, all samples were analyzed for naled (control) hives. For the initial spray mission (24 July residue by usinga method developed by Zhongand 2000), however, the analyses were restricted to com- Latham (2001). A Varian 3400 gas chromatograph con- parisons amongthe posttreatment control and test Þgured with a thermionic-speciÞc detector and an areas because no prespray dead bee counts were 8200 autosampler (Varian Analytical Instruments, made. Walnut Creek, CA) was used for the analysis. Data At the end of the study season, honey yield from the were collected on a Dell Computer (Dell Computer hives at each treatment area was compared with the Corporation, One Dell Way, Round Rock, TX) yield from the hives at the control site to further assess equipped with data-handlingsoftware Star Chroma- if the colonies were negatively impacted by naled tography Workstation, version 4.51 (Varian Analytical exposure. Finally, overall mortality of the exposed bee Instruments). A DB-5 capillary column (30 m by groups was pooled and compared with that of the 0.25-mm inner diameter, 0.1-␮m Þlm thickness) unexposed bees (prespray and control groups). Sim- bonded with fused silica was used (J&W ScientiÞc, ilarly, honey yield at all exposed hives was pooled and Folsom, CA). The injector was operated at 250ЊCin compared with that at all unexposed hives. Two hives splitless mode, and the detector was maintained at at site 1 were excluded from data analyses because of 300ЊC. The column oven temperature was held for 0.5 insufÞcient honey production. min at 120ЊC, ramped at 25ЊC/min to 260ЊC, and held for 1 min. Retention time of naled was 3.65 min. An Results injection volume of 1 ␮l was used for all standards and samples. Table 1 provides weather information, as well as the A Þve-point calibration curve (with R2 Ն 0.995), number of ßight passes conducted and the liters of coveringa naled concentration rangefrom 0.1 to 10 naled applied usingthe high-pressurenozzle system ␮g/ml, was generated for the analysis. The analytical duringeach of the spray missions (24 July, 26 July, 25 4JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 1

Table 1. Number of flight passes, amount of naled applied, and associated weather information during each of the acrial ULV mosquito spray missions conducted using a high-pressure nozzle system

No. of Amt. of naled Wind Wind speed Relative Spray date Temp. (ЊC) passes applied (liters)a direction (kph) humidity (%) 24 July 2000 26 165.4 E-SE 3.2Ð6.4 22.2 100 26 July 2000 44 347.4 E 4.8 23.8 95 25 Sept. 2000 41 397.0 W-NW 8.1Ð12.9 25.5 88 28 Sept. 2000 26 330.8 E-NE 1.6Ð4.8 28.3 85

a The amount of naled reported here was applied to a substantial geographic area and not limited to the treatment sites during these mosquito spray missions. Naled dosage rate, however, was similar for all treatments. Naled was applied as DIBROM Concentrate, which contains 87.4% naled as the active ingredient.

September, and 28 September 2000). Naled was ap- to justify any corrections to the Þeld residue data (no plied to a substantial geographic area and not limited adjustments were made to sample analysis results). In to the treatment sites duringthese mosquito control addition, naled recovery was Ͼ90% for all laboratory spray missions. The naled dosage rate, however, was control spikes, indicatingthe highextraction efÞ- similar for all treatments. ciency of the analytical protocol. Throughout the Quality control data showed that naled residue re- chemical analysis, the recovery of naled from the con- covery was adequate. The average recovery of naled tinuingcalibration veriÞcation samples was consis- Þeld spikes was 61 and 76%, respectively, for the Þrst tently 100 Ϯ 10%. All blank samples were free of naled and second spray missions. For the last two spray contamination as well. missions, only Ϸ43% of the naled residue was recov- Duringthis study, natural bee mortality for the ered. A number of factors, includinghigherÞeld tem- control hives ranged from 0 to 33 dead bees per day peratures and accuracy of spikingvolume, may have (data from July through November 2000). Bee mor- contributed to this reduction in naled recovery. Also, talities associated with each of the four mosquito con- because the residue samples were in the Þeld for a trol spray missions are summarized in Table 2, along shorter length of time than the Þeld spike samples, less with the amount of naled residue found. degradation of naled was expected. Therefore, the After the Þrst mosquito spray mission (24 July lower spike recovery alone was not deemed sufÞcient 2000), the level of naled deposition at the two treat-

Table 2. Average (mean ؎ SE) naled residue (␮g/m2) found around the test hives and the associated honey bee mortality following aerial ULV mosquito spray missions conducted using a high-pressure nozzle system

Naled residue Dead bees/hive Spray Date Location Mean Ϯ SEb No. of samples Mean Ϯ SEa No. of beehives Prespray Postspray 24 July 2000 Site 1 (open Þeld) 2 726.8 Ϯ 93.2cd 8 N/Ac 126.0 Ϯ 51.9ABd Site 1 (in forest) 2 297.5 Ϯ 4.2b 8 N/Ac 62.6 Ϯ 8.0Ad Site 2 (open Þeld) 2 767.0 Ϯ 2.2d 8 N/Ac 279.0 Ϯ 41.0Bd Site 2 (near forest) 2 652.8 Ϯ 1.3c 8 N/Ac 200.4 Ϯ 24.2Bd Control (open Þeld) 2 0a 16 N/Ac 93.9 Ϯ 21.1ABd 26 July 2000 Site 1 (open Þeld) 2 442.0 Ϯ 0.7b 8 12.9 Ϯ 3.2A 2.6 Ϯ 1.1A Site 1 (in forest) 2 834.4 Ϯ 9.6c 8 49.5 Ϯ 29.9B 3.6 Ϯ 0.9A Site 2 (open Þeld) 2 2,688.4 Ϯ 63.4e 8 5.4 Ϯ 1.4A 117.8 Ϯ 7.0C Site 2 (near forest) 2 2,334.1 Ϯ 152.8d 8 8.3 Ϯ 2.6A 106.1 Ϯ 23.5C Control (open Þeld) 2 0a 16 7.3 Ϯ 2.2A 3.9 Ϯ 1.0A 25 Sept. 2000 Site 1 (open Þeld) 2 524.7 Ϯ 1.4b 8 7.3 Ϯ 2.7A 10.0 Ϯ 1.7A Site 1 (in forest) 2 623.7 Ϯ 36.7c 8 9.6 Ϯ 2.0A 14.9 Ϯ 6.4A Site 2 (open Þeld) 2 741.2 Ϯ 18.1d 8 9.1 Ϯ 2.3A 53.4 Ϯ 10.5C Site 2 (near forest) 2 766.8 Ϯ 19.0d 8 6.4 Ϯ 2.2A 30.0 Ϯ 6.3B Control (open Þeld) 2 0a 16 7.5 Ϯ 1.7A 6.3 Ϯ 0.8A 28 Sept. 2000 Site 1 (open Þeld) 2 1,434.9 Ϯ 2.3e 8 5.1 Ϯ 1.3AB 20.3 Ϯ 5.8C Site 1 (in forest) 2 731.8 Ϯ 3.0d 8 9.0 Ϯ 3.2AB 7.8 Ϯ 2.0AB Site 2 (open Þeld) 2 601.0 Ϯ 30.1c 8 8.8 Ϯ 2.0AB 14.9 Ϯ 3.0BC Site 2 (near forest) 2 464.6 Ϯ 3.8b 8 6.5 Ϯ 1.3AB 5.1 Ϯ 1.6A Control (open Þeld) 2 0a 16 3.2 Ϯ 0.6A 2.7 Ϯ 0.9A

a For each spray mission, means of naled residue were compared and are signiÞcantly different (P Ͻ 0.05) when followed by unlike lower case letter(s). Analyses were performed on transformed data [(x ϩ 1)1/2], but means presented are from nontransformed data. b For each spray mission, means of dead bees counts were compared across all pre- and postspray counts (all row and column entries) and are signiÞcantly different (P Ͻ 0.05) when followed by unlike capital letter(s). c Duringthe initial spray mission, the dead bee collectors were not emptied before treatment and no prespray counts were taken. d Bee mortality counts were taken 12 h after treatment but included Ͼ15-d accumulations rather than the intended 24-h counts. Therefore, testingof the statistical signiÞcanceof bee mortality for this mission was restricted to comparisons amongthe posttreatment control and test areas. February 2004 ZHONG ET AL.: MINIMIZING THE IMPACT OF NALED SPRAY ON HONEY BEES 5 ment sites ranged from Ϸ300 to 800 ␮g/m2. Statistical Table 3. Comparison of means and SE of honey yield per hive analysis of bee mortality for this mission was restricted at end of 2000 season after four aerial ULV mosquito spray missions with naled using a high-pressure nozzle system to comparisons amongthe posttreatment control and Ͼ test areas using 15-d accumulations, which may No. of Honey yield/hive Location P Valueb present limitations for the use of these data. The hives Mean Ϯ SE (kg)a ANOVA did reveal a signiÞcant difference in bee mor- Site 1 (open Þeld) 7c 33.3 Ϯ 7.8a 0.166 tality for this posttreatment group (F ϭ 7.38; df ϭ 4, Site 1 (in forest) 7c 44.6 Ϯ 7.7a 0.795 43; P Ͻ 0.001). Bee mortality at the site 1 forest area Site 2 (open Þeld) 8 48.4 Ϯ 6.3a 0.893 Ϯ was signiÞcantly less than that at both the open Þeld Site 2 (near forest) 8 49.2 1.5a 0.727 Control (open Þeld) 16 52.1 Ϯ 4.3a and near forest treatment areas for site 2, but it was not signiÞcantly different than that at the site 1 open Þeld a Means followed by the same letter are not signiÞcantly different area. This reduction in the number of dead bees may (P Ͼ 0.05) with the StudentÐ NewmanÐKeuls mean separation test reßect that bee mortality could be partially inßuenced (Sokal and Rohlf 1981). b Reported value represents comparison between the average by site variations in addition to insecticide levels. honey yield per hive at the treatment area and at the control site. For the second mosquito spray mission (26 July c Two hives at site 1 (one in each typographic area) did not produce 2000), the level of naled deposition at the two treat- enough honey to be extracted and weighed and, therefore, were ment sites ranged from Ϸ440 to 2,750 ␮g/m2. For these excluded from data analyses. naled residue levels, a comparison of all pre- and posttreatment dead bee counts showed that there was bees at these test hives only ranged from 2 to 14 before a signiÞcant difference in one or more of the means as sprayingand 5 to 36 after naled application. ϭ ϭ determined by ANOVA analysis (F 16.81; df 9, 86; Finally, honey yield at the end of the 2000 season Ͻ P 0.001). The number of dead bees at site 2 after was not signiÞcantly different among the treatment naled treatment was signiÞcantly greater than the and control sites (Table 3). Furthermore, honey yield counts recorded before spraying, at site 1, and at the comparisons based on pooled data showed no signif- control site. The highest levels of naled residue were icant difference in yield between the exposed and the found on this spray date at site 2 and ranged from unexposed hive groups (Table 4). Bee mortality, 2 Ϸ2,170 to 2,750 ␮g/m . At site 1, the range of naled though, was signiÞcantly higher when the data for the deposition was less, Ϸ440Ð850 ␮g/m2, and bee mor- bees exposed to naled were combined and then com- tality was not signiÞcantly different at either of the pared with the pooled data for the unexposed bees. treatment areas compared with that at the control site. The site 1 forest area did have signiÞcantly greater prespray bee mortality (average of 50 dead bees) than Discussion all of the other prespray locations. We found 256 dead Several factors contributed to the variation in bee bees at one of the eight hives in this treatment area; but mortality that we observed amongtest hives. First, the reason for this sizeable 1-d mortality count re- duringour study a wide rangeof natural bee mortality mains unknown. This high number may have been was found for the control hives: 0 to 33 dead bees per caused by noninsecticide factors that introduce con- day. This is important in the overall bee mortality siderable variation into natural bee mortality. assessment because our data sometimes showed that After the third spray mission (25 September 2000), the amount of naled deposited in all four treatment Ϸ Table 4. Comparison of means and SE for bee mortality and areas was fairly similar, ranging from 520 to 785 honey yield per hive between exposed and unexposed hives after 2 ␮g/m . For these naled residue levels, a comparison of four aerial ULV mosquito spray missions with naled using a high- all pre- and postspray dead bee counts showed that pressure nozzle system there was a signiÞcant difference in the means as Sample determined by ANOVA (F ϭ 12.76; df ϭ 9, 86; P Ͻ Treatment Mean Ϯ SEa P Value No. 0.001). Even though there were fewer dead bees at site Ϯ Ͻ 2 after naled treatment compared with the previous Mortality of exposed bee 96 32.2 4.56b 0.001 groupsb mission (26 July), the number of dead bees was still Mortality of unexposed bee 192 8.3 Ϯ 1.40a signiÞcantly greater than the numbers recorded be- groupsb fore spraying, at site 1, and at the control site. At site Honey yield per hive by 30 44.2 Ϯ 3.15A 0.140 c 1, there was no signiÞcant difference in bee mortality exposed hives (kg) Honey yield per hive by 16 52.1 Ϯ 4.35A before or after spraying, or at the control site. unexposed hives (kg) For the Þnal spray mission (28 September 2000), the amount of naled deposited in all four treatment areas a Means were compared and are signiÞcantly different (P Ͻ 0.05) varied, ranging from Ϸ460 ␮g/m2 to an intermediate when followed by unlike letter(s) with the StudentÐNewmanÐKeuls ␮ 2 mean separation test (Sokal and Rohlf 1981). level of 1, 440 g/m . ANOVA analysis of all the dead b Bee mortality data from the initial spray mission (24 July 2000) bee counts showed a signiÞcant difference in bee were excluded from the overall bee mortality analysis because these mortality for this spray mission (F ϭ 5.96; df ϭ 9, 86; data were based on Ͼ15-d accumulation of dead bees, rather than the P Ͻ 0.001). Although signiÞcant bee mortality was scheduled 24-h accumulation. c Two hives at site 1 (one in each typographic area) did not produce found at the open Þeld treatment areas after spraying enough honey to be extracted and weighed and, therefore, were compared with the control site, the number of dead excluded from data analyses. 6JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 1 postspray dead bee counts of 15 to 30 were statistically deposition with the conventional spray system varied signiÞcant. An even wider range in natural bee mor- from 558 to 6,922 ␮g/m2 (Zhonget al. 2003a), whereas tality (0Ð100 dead bees per day) was presented by with this studyÕs high-pressure nozzle system, naled Tew (1998) as an extension guideline for beekeepers. ground deposits were reduced, ranging from 293 to He also provided other bee ranges to assist beekeepers 2,752 ␮g/m2. Because of unexpectedly heavy naled in assessinginsecticide impacts to hives as follows: low deposition (Ͼ2,000 ␮g/m2) on 26 July 2000, the num- kill (200Ð400 dead bees per day), moderate kill (500Ð ber of dead bees at site 2 was somewhat Ͼ100 per hive. 900 dead bees per day), and high kill (Ͼ1,000 dead The reason for the heavy deposition with the high- bees per day). pressure nozzle system needs to be further investi- Second, we observed substantial variations in the gated. numbers of honey bees clustered outside the hives at On the day after aerial ULV application (Ϸ12 h night throughout the summer. Bees are not actively posttreatment), we did not observe any abnormal bee pollinatingafter twilightor early in the morningbe- behavior or additional mortality when inspectingthe fore dawn when aerial ULV mosquito adulticide treat- frames inside the beehives. This observation agreed ment is typically conducted. However, warm night- with those of Shaw and Armstrong(1966) that naled time temperatures and high humidity may induce bee was highly toxic to bees outside of the hives upon aggregation at the hive entrances. When honey bees direct contact but was not hazardous to bees inside are clustered in front of the hives, they are more likely their hives. Also rapid degradation of naled in a Þeld to be exposed to mosquito control insecticides and, environment (Tietze et al. 1996, Zhongand Latham therefore, have an increased risk of beingkilled. At- 2001) should not promote long-term adverse impacts kins et al. (1981) indicated that precautions should be to the bee colony. taken to make sure that bees are not clustered in front Honey yield at the end of the 2000 harvest season of the hive entrances at the time of insecticide appli- was evaluated to assess the Þnal effect of naled expo- cation. sure to the beehives. Two hives at site 1 (one in each The most important factor contributingto bee kills typographic area) did not produce enough honey to was the amount of naled that deposited around the be evaluated and were excluded from data analysis at hives during nighttime mosquito spraying. Although the end of this study. The weaken hives were evident we observed variation in bee mortality amongthe test before naled testingbegan,and accordingto the bee- hives, generally as the naled ground deposition in- keeper, the lack of honey production was most likely creased so did the number of dead bees observed due to poor performance by the queen bees. Although duringeach of the spray missions. We found signiÞ- honey yield was slightly less at hives exposed to naled cant bee mortality associated with naled ground dep- compared with that at control hives, the reduction in osition Ͼ2,000 ␮g/m2. The dead bees, based on our honey yield was not statistically signiÞcant. Also, in Þeld observations, were those clustered outside of the contrast to our previous study (Zhonget al. 2003a), hive entrances duringmosquito adulticide applica- honey yield was not signiÞcantly reduced when data tion. After beingexposed to lethal doses of naled, bees for exposed hives were pooled and compared with that fell into the dead bee collectors in front of the hive for unexposed hives. entrances. Either decreasingnaled grounddeposition In conclusion, a high-pressure nozzle system is able or minimizingthe size of the clusters of bees in front to generate smaller droplets than a conventional ßat- of the hives can achieve a reduction in honey bee fan nozzle system. These smaller droplets (Ͻ30 ␮m mortality. VMD) have been shown to elicit better and more Other factors possibly contributingto variations in efÞcient adult mosquito control than those from ßat bee mortality are hive orientation toward the prevail- fans (Dukes et al. 2001). At the same time, the smaller ingwind, wind direction and speed, and protective droplets are less likely to be deposited on the ground vegetation around the hives at the time of insecticide and, therefore, contribute to less bee mortality even application. Further study is necessary to clarify the though large bee clusters may be exposed to the naled impact of these factors on insecticide deposition and spray cloud. This is a great improvement over the bee mortality. Some observations were notable with ßat-fan spray system. However, to ensure that the the high-pressure system. In general, the highest droplet size is appropriate (Ͻ30 ␮m VMD), the spray postspray bee mortality counts occurred at an open equipment must be calibrated regularly and ade- Þeld treatment area. And, at the “in forest” treatment quately maintained. Because our results showed a pos- area, postspray dead bee counts never differed signif- itive correlation between increased naled deposition icantly from the control site counts. and bee mortality, we also recommend that mosquito Our Þndings not only show that a high-pressure control programs periodically monitor ground depo- nozzle system substantially reduces naled contamina- sition of insecticides associated with ULV applications tion compared with the conventional ßat-fan nozzle duringtheir routine operations. systems currently used by most of FloridaÕs mosquito control programs, it also leads to decreased bee mor- tality. The dead bee count per hive decreased from a Acknowledgments maximum of 302 for the ßat-fan nozzle system (Zhong We acknowledge the assistance of Gary Ranker in setting et al. 2003a) to a maximum of 132 for the high-pressure up and maintainingthe beehives used duringthis Þeld study. nozzle system. In our previous study, naled ground We thank James Cilek for critical reviews and comments on February 2004 ZHONG ET AL.: MINIMIZING THE IMPACT OF NALED SPRAY ON HONEY BEES 7 the draft manuscript. We also thank W. Preston, J. T. Reeder, ture industry. University of Florida, Institute of Food and J. Nanney, the landowners, for permission to use prop- and Agricultural Sciences, Florida Cooperative Exten- erty as experimental sites, and AMVAC Chemical Corpora- sion Publication No. FE273. http://edis.ifas.uß.edu/ tion for the gift of naled analytical standard. Finally, we are BODY_FE273. deeply indebted to employees at Manatee County Mosquito Levin, M. D., W. B. Forsyth, G. L. Fairbrother, and F. B. Control District for invaluable support duringthis study. This Skinner. 1968. Impact of colonies of honey bees of ultra- research was partially supported by the Florida Department low-volume (undiluted) malathion applied for control of of Agriculture and Consumer Services (grant 4930). grasshoppers. J. Econ. Entomol. 61: 58Ð62. Pankiw, T., and S. C. Jay. 1992a. Aerially applied ultra-low- volume malathion effects on caged honey bees (Hyme- References Cited noptera: Apidae), caged mosquitoes (Diptera: Culici- Atkins, E. L. 1972. Rice Þeld mosquito control studies with dae), and malathion residues. J. Econ. Entomol. 85: 687Ð low volume Dursban sprays in Colusa County, California. 691. V. Effects upon honey bees. Mosq. News 32: 538Ð541. Pankiw, T., and S. C. Jay. 1992b. Aerially applied ultra-low- Atkins, E. L., D. J. Womeldorf, and L. A. Lewis. 1981. Com- volume malathion effects on colonies of honey bees (Hy- parative effects of aerosols of bendiocarb, chlorpyrifos, menoptera: Apidae). J. Econ. Entomol. 85: 692Ð699. malathion, and pyrethrins upon caged honey bees and Rindfleisch, J. 1983. Aerial application of Dibrom-14: honey mosquitoes, pp. 76Ð81. In Proceedings and Papers, 49th bee mortality in relation to hive placement and protec- Annual Conference of California Mosquito and Vector tion by mistingwith water. Am. Bee J. 123: 121Ð122. Control Association, Inc., 26Ð28 April 1981, Redding, CA. Shaw, F. R., and R. L. Armstrong. 1966. Preliminary obser- California Mosquito and Vector Control Association, Inc., vation on the effects of a naled fogon honey bees. Am. Sacramento, CA. Bee J. 106: 248. Caron, D. M. 1979. Effects of some ULV mosquito abate- Sokal, R. R., and F. J. Rohlf. 1981. Biometry, 2nd ed. Free- ment insecticides on honey bees. J. Econ. Entomol. 72: man, San Francisco, CA. 148Ð151. StatSoft, Inc. 2001. STATISTICA (data analysis software Colburn, R. B., and G. S. Langford. 1970. Field evaluation of system), version 6. www.statsoft.com some mosquito adulticides with observations on toxicity Tew, J. E. 1998. Protectinghoney bees from pesticides. Al- to honey bees and houseßies. Mosq. News. 30: 518Ð522. abama A&M University and Auburn University, Alabama Dukes, J., H. Zhong, M. Greer, P. Hester, and D. Hogan. Cooperative Extension Publication No. anr-1088. http:// 2001. Downwind movement and deposit of insecticides www.aces.edu/pubs/anr/anr-1088/anr-1088.htm applied by Þxed wingaircraft for adult mosquito control. Tietze, N. S., K. R. Shaffer, and P. G. Hester. 1996. Half-life WingBeats 12: 18Ð25. of naled under three test scenarios. J. Am. Mosq. Control Gary, N. E. 1960. A trap to quantitatively recover dead and Assoc. 12: 251Ð254. abnormal honey bees from the hive. J. Econ. Entomol. 33: Zhong, H., and M. Latham. 2001. Development and valida- 782Ð785. tion of a rapid analytical method to quantify naled resi- Gary, N. E., and E. C. Mussen. 1984. Impact of Mediterra- due. J. Am. Mosq. Control Assoc. 17: 225Ð230. nean fruit ßy malathion bait spray on honey bees. Envi- Zhong, H., J. C. Dukes, M. Greer, P. G. Hester, M. Shirley, ron. Entomol. 13: 711Ð717. and B. Anderson. 2003a. Ground deposition impact of Hester, P. G., K. R. Shaffer, N. S. Tietze, H. Zhong, and N. L. aerially applied fenthion on Þddler crabs, Uca pugilator. Griggs Jr. 2001. EfÞcacy of ground-applied ultra-low- J. Am. Mosq. Control Assoc. 19: 47Ð52. volume malathion on honey bee survival and productivity Zhong, H., M. Latham, P. G. Hester, R. L. Frommer, and C. in open and forest areas. J. Am. Mosq. Control Assoc. 17: Brock. 2003b. Impact of naled on honey bee Apis mel- 2Ð7. lifera L. survival and productivity: aerial ULV application Hill, E. F., D. A. Eliason, and J. W. Kilpatrick. 1971. Effects usinga ßat-fan nozzle system. Arch. Environ. Contam. of ultra-low volume application of malathion in Hale Toxicol. 45: 216Ð220. County, Texas. J. Med. Entomol. 8: 173Ð179. Hodges, A., D. Mulkey, E. Philippakos, G. Fairchild, and M. Received for publication 7 March 2003; accepted 3 September Sanford. 2001. Economic impact of the Florida apicul- 2003. Journal of the American Mosquito Control Association, 26(1):57-66, 2010 187806 Copyright © 2010 by The American Mosquito Control Association, Inc.

EVALUATION OF EFFICACY AND HUMAN HEALTH RISK OF AERIAL ULTRA-LOW VOLUME APPLICATIONS OF PYRETHRINS AND PIPERONYL BUTOXIDE FOR ADULT MOSQUITO MANAGEMENT IN RESPONSE TO WEST NILE VIRUS ACTIVITY IN SACRAMENTO COUNTY, CALIFORNIA

PAULA A. MACEDO,! JEROME J. SCHLEIER 111,2 MARCIA REED,! KARA KELLEY,! GARY W. GOODMAN,! DAVID A. BROWN! AND ROBERT K. D. PETERSON2

ABSTRACT. The Sacramento and Yolo Mosquito and Vector Control District (SYMVCD, also referred to as "the District") conducts surveillance and management of mosquitoes in Sacramento and Yolo counties in California. Following an increase in numbers and West Nile virus (WNV) infection rates of Culex tarsalis and Culex pipiens, the District decided on July 26, 2007, to conduct aerial applications ofEvergreen® EC 60-6 (60% pyrethrins: 6% piperonyl butoxide) over approximately 215 km2 in the north area of Sacramento County on the nights of July 30, July 31, and August 1, 2007. At the same time, the District received notification of the first human WNV case in the area. To evaluate the efficacy of the applications in decreasing mosquito abundance and infection rates, we conducted pre- and post-trapping inside and outside the spray zone and assessed human health risks from exposure to the insecticide applications. Results showed a significant decrease in abundance of both Cx. tarsalis and Cx. pipiens, and in the minimum infection rate of ex. tarsalis. Human-health risks from exposure to the insecticide were below thresholds set by the US Environmental Protection Agency.

KEY WORDS West Nile virus, aerial spraying, insecticide, mosquito control, risk assessment

INTRODUCTION intensive larviciding and public education efforts, the District intervened by aerially applying a West Nile virus (WNV, family Flaviridae, formulation ofpyrethrins and piperonyl butoxide genus Flavivirus) was first detected in the United (PBO) over an urban/suburban area in Sacra­ States in 1999 in New York City, and reached mento County (Elnaiem et al. 2008), which most California in the summer of 2003 (Reisen et al. likely interrupted the WNV transmission cycle 2004). In 2004, WNV amplified to epidemic levels (Carney et al. 2008). and dispersed to all 58 counties in the state, and Although traditionally used in response to was associated with low-level transmission to epidemics and as part of a sustainable public humans and horses in Sacramento and Yolo counties that year (Armijos et al. 2005, Hom et al. health program (Rose 2001), the application of 2005). In 2005, there was a severe outbreak in pesticides often generates public concerns and Sacramento County, with 177 human cases and controversy about the safety of these chemicals to 40 equine cases (Elnaiem et al. 2006). people and the environment as well as the efficacy of such practice (Thier 2001, Roche 2002, Hodge The Sacramento and Yolo Mosquito and Vector Control District (SYMVCD, also referred and O'Connell 2005). A human health risk to as "the District") conducts routine surveillance assessment conducted by Peterson et al. (2006) and management of mosquito populations in for truck-mounted ultra-low volume (ULV) Sacramento and Yolo counties. The District applications of adulticides commonly used in monitors weekly mosquito abundance and West mosquito management programs determined Nile, western equine encephalitis (WEE), and St. risks to be below levels established by the US Louis encephalitis (SLE) viral infection. The Environmental Protection Agency (USEPA), District follows the California Mosquito-Borne which agrees with the current scientific weight Virus Surveillance and Response Plan (Kramer of evidence (NYCDOH 2001, Karpati et al. 2004, 2005) and its own Mosquito and Mosquito-Borne Currier et al. 2005, O'Sullivan et al. 2005). The Disease Management Plan (SYMVCD 2005), and results from Peterson et al. (2006) indicated that applies the principles of integrated pest manage­ potential health risks from WNV exceed risks ment (IPM) in its program. When WNV reached from exposure to these pesticides when used at epidemic levels in 2005 despite SYMVCD's label rates to control adult mosquitoes. Their study used extremely conservative assumptions and estimated exposure after truck-mounted I Sacramento-Yolo Mosquito and Vector Control District, 8631 Bond Road, Elk Grove, CA 95757. ULV applications as a worse-case scenario, used 2 Department of Land Resources and Environmental application rates greater than the ones used by Sciences, Montana State University, 334 Leon Johnson SYMVCD, and therefore likely overestimated the Hall, Bozeman, MT 59717. exposure that would be seen for the application

57 58 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, No.1 by the District. Schleier et al. (2009b) evaluated Mosquito-Borne Virus Surveillance and Re­ probabilistically the deterministic risk estimates sponse Plan (Kramer 2005) and its Mosquito presented by Peterson et al. (2006) and found and Mosquito-Borne Disease Management Plan them to be very conservative. Davis et al. (2007) (SYMVCD 2005), the District made the decision evaluated ecological risks posed by adult mos­ on July 26,2007, to aerially apply pyrethrins and quito management programs and concluded that PBO (Evergreen EC-60-6®) over the area of risks to nontarget organisms from pesticides concern. On the same day that this decision was applied for adult mosquito management are low made, the District received notification ofthe first and not likely to exceed regulatory levels of human case in the area. The insecticide applica­ concern. tions took place on the nights of July 30 and 31, Although vector control strategies and their and August 1, 2007. The objectives of this study effectiveness have generated concern in past were to 1) evaluate the efficacy of the aerial years, there have been few published studies applications in reducing adult mosquito popula­ addressing the efficacy of these operations in tions and infection rates of the two main species reducing mosquito populations, infection rates, implicated in WNV transmission in Sacramento and virus transmission. Sacramento County County and 2) assess human health risks for the experienced a WNV epidemic for the first time aerial application of adulticide conducted in in 2005. Although the evaluation of the aerial Sacramento County in 2007 in response to the adulticiding conducted during that year suggested WNVactivity. interruption of transmission (Carney et al. 2008) and reduction of vector abundance (Elnaiem et al. 2008), some uncertainties were identified by MATERIALS AND METHODS SYMVCD staff to be addressed in future Aerial applications and study area evaluations, particularly the absence of fixed 2 locations for trapping mosquitoes before and The spray zone was a 215 km area in northern after the applications, the small number of Sacramento County, located in the Central Valley mosquito pools collected from those areas, and of California (Fig. 1). The insecticide Evergreen other confounding factors such as the effect of EC 60-6 (60% PBO and 6% pyrethins; McLaugh­ wind shadow at some of the locations. lin Gormley King, Golden Valley, MN) was In 2006, WNV reached epidemic levels in the applied by a fixed-wing Piper Aztec and a Cessna cities of Davis and Woodland in Yolo County, 402 aircraft (VDCI/ADAPCO Vector Control and the SYMVCD, with the collaboration of the Services, Greenville, MS) for three consecutive Center for Vectorborne Diseases at the University nights on July 30 and 31, and August 1,2007. The of California-Davis, monitored abundance and application rate was 2.8 g/ha (0.0025 lb/ac) of infection rates and conducted aerial applications pyrethins and 28 g/ha (0.025 lb/ac) of PBO. The of pyrethrins and PBO on the nights of August 8 release altitude was 91 m (300 ft), the wind speed and 9, 2006 (Macedo et al. 2007a, Nielsen et al. ranged from 3.7 km/h to 18.5 km/h, and the 2007). Analysis of data from 2005 and 2006 temperature at the time ofthe applications ranged showed that the aerial applications could have from 34°C to 36°C. Application start times ranged been more successful in interrupting virus trans­ from 7:34 p.m. to 7:55 p.m. and application end mission to people if they had been conducted times ranged from 9:20 p.m. to 9:51 p.m. 1 wk or 2 wk before, when mosquito abundance and infection rates were higher (Macedo et al. Mosquito abundance 2008). The first indication of active WNV transmis­ To evaluate the effect of the aerial applications sion in the District in 2007 was the detection of on mosquito abundance, the District used en­ WNV in a dead American crow on May 31. The cephalitis virus surveillance traps (EVS) baited first positive mosquito pool was obtained on July with dry ice, herein referenced to as CO2 traps 4, 2007, in a pool of Culex pipiens L. West Nile (Rohe and Fall 1979), and gravid-female traps virus continued to amplify during the month of (Cummings 1992) to collect mosquitoes inside July, and mosquito abundance and maximum and outside of the aerial spray zone for 3 days likelihood estimates of minimum infection rates before and 3 days after the application events. continued to be monitored. The District identi­ Trap collections were brought to the laboratory, fied an area of approximately 215 km2 in the where mosquitoes were anesthetized with trieth­ north part of Sacramento County as higher risk ylamine, identified to species and counted, and and intensified all the aspects of its IPM program then frozen at - 80°C for later virus testing. Three in an attempt to reduce mosquito populations. CO2 traps and one gravid trap were placed at On the week of July 24,2007, infection rates had each of the 12 fixed sites in the aerial spray zone reached 10.85 and 7.87 per 1,000 mosquitoes for and six fixed sites in the untreated control zone Culex tarsalis Coquillett and Cx. pipiens, respec­ (Fig. 1). Counts of females per trap-night were tively. Following the guidelines of the California transformed by In(y + 1) and expressed as MARCH 2010 MOSQUITO CONTROL EFFICACY AND HUMAN HEALTH RISK 59

A N II Aerial Treatment Area W+E • Untreated Control Trap Sites S .A Treatment Area Trap Sites

Fig. 1. Map of California showing (A) the location of Sacramento and Yolo counties, (B) the 2007 spray zone in north Sacramento, and (C) the locations of trapping sites used in the spray zone and untreated control area during 3 days before and 3 days after the aerial applications of pyrethrins and piperonyl butoxide in 2007. geometric means (Reisen and Lothrop 1999), and et al. (1971), which accounts for reductions or analyzed by two-way analysis of variance increases in the untreated areas. (ANOVA) and paired t-tests (SAS, version 9.1, SAS Institute, Cary, NC). Percentage of reduc­ Sentinel cages tion of ex. tarsalis and Cx. pipiens abundance was estimated by the formula described by Mulla Sentinel mosquitoes were exposed within dis­ et al. (1971). posable bioassay cylindrical cardboard cages, 15 cm diam and 4.5 cm deep, with 14 X 18-mesh polyester screens on both vertical circular surfac­ West Nile virus infection rates es (modified from Townzen and Natvig 1973), Mosquitoes from trapping conducted in the 18 with a hole in the cardboard for cotton pads fixed sites 3 days before and 3 days after the aerial moistened with 10% sugar water. One cage applications, as well as from other traps set inside containing approximately 25 wild-caught adult the spray zone during the week before and after Cx. tarsalis and another containing 25 wild­ the adulticide applications were collected and caught Cx. pipiens were placed at each of the 12 brought to the laboratory where they were then sites within the aerial spray zone and at the six anesthetized with triethylamine, identified to control sites during each application. Cages were species, pooled in groups of one to 50 females, placed vertically at a I-m height with a screened frozen at -80°C, and tested for arboviral RNA surface positioned to face the prevailing wind (WN, SLE, and WEE viruses) by multiplex real­ direction. Mosquito mortality was evaluated at time reverse transcriptase-polymerase chain re­ the time of the placement of the cages (before the action (Shi et al. 2001), using WNV primers insecticide application), and at 1, 2, and 12 h after published previously (Lanciotti et al. 2000). each application. Results were expressed as Infection rates were calculated for the week percentage of mortality. before and the week after the aerial application events using bias-corrected maximum likelihood Human health risk assessment estimation (Biggerstaff 2006). In addition, per­ centage of reduction of minimum infection rates Human health risk assessments had been w"as estimated by the formula described by Mulla previously conducted for truck-mounted applica- 60 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, NO.1 tions of pyrethrins and PBO using greater values that could occur. This was performed by application rates than the ones used by using 20,000 iterations with the assumptions SYMVCD for adult mosquito control, and for outlined below and in Table 1. Respiratory rate, a different application schedule (Peterson et al. BW, percentage of surface area of two hands, air 2006). We modified the risk assessments by concentrations, and spray deposition were trun­ Peterson et al. (2006) and Schleier et al. (2009b) cated at zero because it is not possible for these to more accurately represent the application type, quantities to have negative values. rate, and schedule used by SYMVCD in 2007. In Environmental concentrations: We used the addition to those modifications to the previous environmental concentration data from Schleier risk assessments, we incorporated more recent et al. (2008) at ground level using the same deposition data from Schleier et al. (2008). We application rates listed above (see Schleier et al. estimated the human health risk from exposure to 2008 for details of the applications). To model 3 days of aerial applications of pyrethrins and the deposition of pyrethrins and PBO onto PBO at the rates specified above. To account for surfaces, we created distributions using concen­ age-related differences, exposures were estimated trations measured 1 hand 12 h after applica­ for adult males and adult females (18-65 years of tion because the concentrations at these times age), youth (10-12 years of age), children (5-6 were not significantly different (Schleier et al. years of age), toddlers (2-3 years of age), and 2008). Distributions for deposition onto surfac­ infants (0.5-1.5 years of age). es were chosen based on the Anderson-Darling Toxicity and dose-response relationships: Dose­ goodness-of-fit test, which for non-normalized response information for each compound was data weights the differences between two reviewed and endpoints were chosen based on distributions at their tails (Pettitt 1977, Oracle acute and subchronic exposures. The toxicity 2007). The distribution fit of the environmental thresholds used in this assessment were ingestion concentration data for PBO was log-normal reference doses (RID) established by the USEPA. with a mean 0.01 }.1g/cm2 and a standard Ingestion RIDs were based on the no-observed­ deviation of 0.01. To model air concentrations adverse-effect level (NOAEL) with a 100-fold of PBO we assumed the same amount that safety factor for intra- and interspecies extrapo­ deposited on the ground would be available in lation uncertainties. The acute oral RID for 1 m 3 of air. We used the same distribution for pyrethrins and PBO are 0.07 and 6.3 mg/kg body air concentrations as we did for ground weight (BW)/day, respectively (USEPA 2006a, deposition. Schleier et al. (2008) did not detect 2006b). any pyrethrins during their study; therefore we Risk characterization: Total acute exposure to modeled deposition and air concentrations each active ingredient for each group was using the same assumptions as for PBO, scaling estimated by summing inhalation, dermal, hand­ the distributions based on the application rate. to-mouth, turf-dislodgeable, and ingestion expo­ Pyrethrins were applied at an application rate sure routes which are outlined below. The risk 10% of that for PBO. quotient (RQ) was calculated by dividing the Acute exposure: We assumed that acute multi­ total potential exposure for each group and route exposures immediately after a single-spray chemical by its respective ingestion toxic endpoint value (RID). The multi-route exposure was event were limited to 24 h. Routes of insecticide compared to the ingestion RID because it exposure to each group were inhalation, dermal, provided a conservative endpoint, which is based and dietary and non-dietary ingestion. Assump­ on the most sensitive NOA-EL. Estimated RQs tions of body weight, respiration rate, and were compared to a RQ level of concern (LOC), frequency of hand-to-mouth activity are present­ which is set by the USEPA or other regulatory ed in Table 1. agencies to determine if regulatory action is Because the data from Schleier et al. (2008) needed. The RQ LOC used in the assessment demonstrated that the inhalation exposure is was 1.0. An RQ > 1.0 means that the estimated most likely limited to 1 h, we assumed that each exposure was greater than the relevant RID. group would be outside when the aerial spray Probabilistic analysis: Monte Carlo simulation began and that the duration of the exposure was (Crystal Ball® 7.3; Decisioneering, Denver, CO) 1 h. Instead of using modeled environmental was used to generate the exposures and RQs. concentrations we incorporated the deposition Probabilities of occurrence of RQ values were rates of Schleier et al. (2008). The exposure determined by incorporating sampling from the modeling assumptions for dermal, hand-to­ statistical distribution of each input variable used mouth, and turf-dislodgeable residues follow the to calculate the RQs. Each of the input variables assumptions of Schleier et al. (2009b), except was sampled so that its distribution shape was actual environmental concentrations were used reproduced. Then, the variability for each input instead of modeled environmental concentra­ was propagated into the output of the model so tions. The modifications to inhalation and that the model output reflected the probability of ingestion exposure are outlined below. MARCH 2010 MOSQUITO CONTROL EFFICACY AND HUMAN HEALTH RISK 61

Table 1. Assumptions for body weight, respiratory rate, and frequency of hand-to-mouth activity for each group assessed. Input Variables Group Parameter! Values Units Distribution Source Body weight Adult males2 Mean 78.65 kg Log-normal (truncated) Portier et al. (2007) SD 13.23 Adult females3 Mean 65.47 kg SD 13.77 Youth4 Mean 36.16 kg SD 7.12 Children5 Mean 19.67 kg SD 2.81 Toddlers6 Mean 13.27 kg SD 1.62 Infants7 Mean 9.1 kg SD 1.24 Respiratory rate Adult males Mean 17.53 m 3/day Log-normal (truncated) Brochu et al. (2006) SD 2.8 Adult females Mean 13.78 SD 2.1 Youth Mean 11.3 SD 2.14 Children Mean 7.74 SD 1.04 Toddlers Mean 5.03 SD 0.94 Infants Mean 3.72 SD 0.81 Hand-ta-mouth Toddlers Location 5.3 events/h Weibull (truncated) Xue et al. (2007) frequency Scale 3.41 Shape 0.56 Infants Location 14.5 events/h Scale 15.98 Shape 1.39

I SD = standard deviation. 2 18-65 years of age. 3 18-65 years of age. 4 10-12 years of age. s 5-6 years of age. 6 2-3 years of age. 70.5-1.5 years of age.

Inhalation exposure was estimated by is the actual environmental concentration of 2 insecticide that settles onto surfaces (J.lg/cm ), PE1nhaiation = (AEC x RR x D)/BW (1) 2 2 CF is the conversion from f.lg/cm to mg/m , SAT where PElnhalation is potential exposure from is the surface area of tomatoes consumed as 2 inhalation (mg/kg BW), AEC is actual environ­ estimated by Eifert et al. (2006) (m ), and BW is 3 mental air concentrations (f.lg/m ), RR is respira­ body weight (kg). The average amount plus the tory rate for each group (m3/h), D is duration of standard error of tomatoes consumed per day by exposure, and BW is body weight (kg) for each adult males and females, youth, children, tod­ group (Table 1). dlers, and infants is 0.804, 0.804, 0.874, 1.19, 1.77, For acute ingestion exposure from tomatoes and 1.21 g/kg BW, respectively (USEPA 1997). that were exposed to the pesticide, we assumed that all foods containing tomatoes eaten per day were consumed from tomatoes grown in a home RESULTS garden without being washed. In addition, we assumed there would be no degradation in the Two-way ANOVA showed significant differ­ preparation process. Acute ingestion was esti­ ences in Cx. tarsalis and Cx. pipiens abundance in mated by the CO2 -baited traps before and after the applications (F = 14.59; df 1, 16; P = 0.0015; PElngestion = [(AEC x CF) x SAT]/BW (2) and F = 8.49; df 1, 13; P = 0.0121 respectively). where PEIngestion is potential exposure from There was no interaction between time and consuming exposed produce (mg/kg BW), AEC treatment, so a paired t-test was used to compare 62 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, NO.1

Table 2. Mean female mosquitoes per trap night and standard deviation before and after the insecticide applications inside (spray zone) and outside (control) of the spray area. l Spray zone Control Species, trap type Before After Before After

Culex pipiens, CO2 traps 4.94 (4.70) a 2.46 (2.24) b 10.33 (13.59) a 8.48 (13.68) a Culex pipiens, gravid traps 14.79 (12.27) a 9.79 (9.75) a 12.33 (12.50) a 14.47 (9.65) a Culex tarsalis, CO2 traps 8.75 (7.44) a 3.00 (1.61) b 17.78 (19.08) a 14.28 (20.00) a

I Means within a column in spray zone or control followed by the same letter were not significantly different (P > 0.05). abundance before and after in the spray zone and mortality at the untreated control area (F control areas separately. There was a significant 142.91; P < 0.0001 and F= 185.34; P < 0.0001 reduction in abundance of host-seeking Cx. for Cx. pipiens and Cx. tarsalis, respectively). tarsalis and Cx. pipiens inside of the spray zone The human health risk assessment from and not in the untreated control areas (Table 2). exposure to three aerial ULV applications of There was no significant difference in the number pyrethrins and PBO at rates used by the of Cx. pipiens females captured by the gravid SYMVCD indicated that total acute exposure traps before and after the aerial adulticide for pyrethrins at the 95th percentile of exposure applications in the spray zone or in the control ranged from 0.000004 to 0.0003 mg/kg BW/day areas. Percentage of reduction calculated by for the groups assessed (Table 5). Risk quotients Mulla's formula was estimated to be 57.33% for for pyrethrins at the 95th percentile ranged from Cx. tarsalis and 40.81% for Cx. pipiens. 0.00003 to 0.002 for all groups (Table 6). Total Maximum likelihood estimates of WNV infec­ acute exposure for PBO at the 95th percentile tion rates for Cx. pipiens and Cx. tarsalis before ranged from 0.00008 to 0.003 mg/kg BW/day for and after the application events are shown in the groups assessed (Table 5). Risk quotients for Table 3. Infection rates for Cx. tarsalis decreased PBO at the 95th percentile ranged from 0.00001 significantly in the spray zone after the aerial to 0.0005 for all groups (Table 6). No chemical or adulticide applications, but not for Cx. pipiens. In group exceeded the RQ LOC. Toddlers and contrast, infection rates for both species increased infants were the highest-risk groups whereas in the untreated control area after the applica­ adult males were the lowest-risk group assessed tions. Percentage of reduction of the minimum in this study (Table 6). infection rates calculated by Mulla's formula was Our results showed that median inhalation estimated to be 77.41% for Cx. tarsalis and exposure contributed <0.01 % to the overall 21.56% for Cx. pipiens. exposure of all groups. Median dermal exposure Sentinel cage bioassay data showed that contributed about 53% to the overall exposure of average mortality 1 h after application was 400/0 adult males and females, youth, and children; (range 0% to 91 %) for Cx. pipiens and 51 0/0 however, the median dermal exposure only (range 00/0 to 94%) for Cx. tarsalis. Results for contributed 180/0 to the overall exposure of mortality at 1,2, and 12 h are shown in Table 4. toddlers and infants. Median exposure from We observed a high variability among sentinel hand-to-mouth exposure from insecticide settling cage mortality, suggesting that the insecticide onto their hand contributed about 17% to the application did not reach all sites. Nonetheless, overall exposure of toddlers and infants. Median mortality of mosquitoes in the sentinel cages in exposure from hand-to-mouth turf-dislodgeable the spray zone was significantly different than residue contributed about 16% to the overall

Table 3. Maximum likelihood estimates of WNV infection rates for mosquito pools collected in the spray zone and untreated control area before and after the application events. No. No. No. positive % positive Area Time Species MLEl (95% CI) females pools pools pools Spray Before Culex pipiens 7.87 (5.02-11.86) 2,968 188 21 11.17 zone Culex tarsalis 10.85 (6.54-17.09) 1,605 118 16 13.56 After Culex pipiens 7.51 (2.82-16.65) 705 69 5 7.25 Culex tarsalis 3.42 (0.20-16.54) 292 50 1 2.00 Control Before Culex pipiens 5.31 (1.76-12.70) 781 45 4 8.89 Culex tarsalis 4.53 (1.51-10.81) 910 37 4 10.81 After Culex pipiens 6.46 (2.84-12.92) 1,205 68 7 10.29 Culex tarsalis 6.32 (2.38-14.05) 855 47 5 10.64

1 MLE, bias-corrected maximum likelihood estimate of infection rate in 1,000 mosquitoes (Biggerstaff 2006); CI, confidence interval. MARCH 2010 MOSQUITO CONTROL EFFICACY AND HUMAN HEALTH RISK 63

Table 4. Culex pipiens and Culex tarsalis average percentage of mortality (standard deviation) in bioassay cages. Spray zone Control Time (hr) Cx. pipiens Cx. tarsalis Cx. pipiens Cx. tarsalis 1 40.07 (25.83)1 51.41 (29.03)2 2.94 (12.13) 8.51 (23.95) 2 56.47 (26.3)1 72.27 (29.43)2 6.96 (18.28) 9.14 (24.8) 12 68.48 (28.48)1 86.56 (21.74)2 7.55 (18.2) 9.14 (24.8)

I Significantly different than Cx. pipiens control (P < 0.05). 2 Significantly different than Cx. tarsalis control (P < 0.05). exposure of toddlers and infants. Mean ingestion abundance between the two areas even in the exposure contributed about 43% to the overall presence ofground treatments at the control sites. exposure of all groups. Another variable out of our control is that mosquito abundance tends to vary markedly among trap sites. Moreover, routine vector DISCUSSION control strategies continued to be conducted by Although evaluation of efficacy is an essential SYMVCD following its IPM program, and component of assessing pesticide applications source reduction and larvicide applications were (Carney et al. 2008) and vector abundance is an performed before, during, and after the aerial important measure of efficacy of control strate­ adulticide applications throughout all areas of gies (Nielsen et al. 2007), it remains a difficult Sacramento and Yolo counties. task for mosquito control districts because there Our analysis indicates that the aerial applica­ are many variables that cannot be controlled. In tions were made at a time when Cx. pipiens 2007, when the decision was made to conduct populations in Sacramento County were already aerial applications of pyrethrins and PBO to declining, but Cx. tarsalis populations were manage adult populations of mosquitoes in the increasing. Analysis of the data and population north Sacramento area, the District selected areas trends indicate that most Cx. pipiens collected at outside of the aerial spray zone to be the the time of the aerial adulticide applications were untreated control sites, and as such, they should gravid females, suggesting that the population of not have received any insecticide application. But these mosquitoes was composed of older, blood­ as a vector control agency, once one of these fed females, presenting a different behavior than control sites shows either high abundance of the host-seeking mosquitoes. That may explain mosquitoes or positive mosquito pools, it is the why there was a greater reduction in host-seeking District's responsibility to respond to those Cx. tarsalis than Cx. pipiens. Nonetheless, there surveillance parameters and follow its manage­ was still a significant reduction in host-seeking ment plan. Therefore, although the control sites Cx. pipiens abundance in the spray zone. were not sprayed aerially, some did receive Although not statistically significant, a reduction ground treatments with trucks and backpack of 33.8% in Cx. pipiens captured in gravid traps foggers in response to high trap counts, positive was also observed in the spray zone. That is dead birds, and positive mosquito pools. Al­ important because, at the same time, Cx. pipiens though this may be a confounding factor when captured in gravid traps in the untreated control comparing abundance of mosquitoes and infec­ area increased 17.40/0. tion rates inside and outside of the aerial spray Sentinel mosquitoes were used to evaluate the zone, our results showed significant differences in deposition of the pesticide into the target areas.

Table 5. Acute total potential exposure means at 50th and 95th percentile confidence intervals for each group and chemical assessed. Chemical PEl Adult males2 Adult females3 Youth4 Children5 Toddlers'6 Infants7 Pyrethrins 50th 0.000001 0.000001 0.000002 0.000003 0.00001 0.00002 95th 0.000005 0.000005 0.000008 0.00001 0.00005 0.00009 Piperonyl 50th 0.00003 0.00003 0.00004 0.00006 0.0001 0.0003 butoxide 95th 0.0001 0.0001 0.0002 0.0002 0.0006 0.001

I PE, potential exposure. Total acute exposure to each active ingredient for each group was estimated by adding together inhalation, dermal, hand-to-mouth, turf-dislodgeable, and ingestion exposure routes (mglkg body weight/day). 2 18-65 years of age. 3 18-65 years of age. 4 10-12 years of age. 5 5-6 years of age. 6 2-3 years of age. 7 0.5-1.5 years of age. 64 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 26, No.1

Table 6. Acute risk quotient means at 50th and 95th percentile confidence intervals for each group and chemical assessed. Chemical RQl Adult males2 Adult females3 Youth4 Children5 Toddlers6 Infants7 Pyrethrins 50th 0.000008 0.00001 0.00001 0.00002 0.00008 0.0002 95th 0.00003 0.00004 0.00006 0.0001 0.0004 0.0007 Piperonyl 50th 0.000004 0.000005 0.000006 0.000009 0.00002 0.00004 butoxide 95th 0.00002 0.00002 0.00003 0.00004 0.0001 0.0002

1 Risk quotient. 2 18-65 years of age. 3 18-65 years of age. 4 10-12 years of age. s 5-6 years of age. 6 2-3 years of age. 7 0.5-1.5 years of age.

Deposition may be markedly altered by local tion rates for Cx. pipiens in the spray zone, rates meteorological conditions that are affected by the for this species were also higher in the control presence of heavy vegetation (Barber et al. 2007, areas after the application events, and more Elnaiem et al. 2008), which also filters out the positive mosquito pools were found in the control pesticide (Taylor and Schoof 1971). To penetrate area after the adulticide applications than before. the canopy, wind direction must be perpendicular Therefore, our data indicate that the aerial DLV to the spray line and wind speeds must move the treatments conducted by the SYMVCD in 2007 pesticide through the canopy (Barber et al. 2007). may have reduced the risk of WNV transmission Wind direction and speed are usually measured at to humans by effectively reducing the population the application point, but may be different from of infected adult mosquitoes at the target area. conditions at vegetated locations. Mortality The probabilistic risk assessment suggests that results from our bioassay cages varied signifi­ human risk from exposure to the insecticide cantly, with different locations presenting very applications was below regulatory levels of low mortality at different application events. concern, so the benefits likely exceeded risks. Environmental conditions may have been respon­ The current weight of evidence from biomonitor­ sible for the reduced spray movement through ing, epidemiology, risk assessments, and reduc­ some of these target zones in different days. tion in disease incidence rates after ULV appli­ The probabilistic risk assessment showed that cations (Kutz and Strassman 1977; Karpati et al. RQs for a single truck-mounted application are 2004; Currier et al. 2005; O'Sullivan et al. 2005; about 10-fold greater than those estimated for Carr et al. 2006; Peterson et al. 2006; Macedo et three applications of aerial ULV. Although the al. 2007b; Duprey et al. 2008; Schleier et al. rates used for aerial applications may be greater 2009a, 2009b) demonstrate that the benefit of than for truck-mounted, deposition on the reducing WNV incidence rates outweigh public ground is lower after aerial ULV (Lothrop et al. health risks from insecticide applications to 2007, Schleier and Peterson 2009, Schleier et al. manage adult mosquitoes. 2009b). These results support the findings of previous risk assessments and regulatory docu­ ACKNOWLEDGMENTS ments that the risks from aerial ULV are lower than those of truck-mounted ULV (NYCDOH The authors thank the County of Sacramento 2005, Peterson et al. 2006). Our results are Department of Health and Human Services supported by biomonitoring studies that showed Public Health Laboratory for providing Biosafety no increase in urinary metabolites after aerial Laboratory-3 containment space; Leslie Shama ULV applications of naled (Kutz and Strassman for assistance in an earlier risk assessment; 1977, Duprey et al. 2008). Our assessment Rhonda Laffey for assistance with mapping; determined that exposures after three aerial and Vicki Kramer, William K. Reisen, and Mark ULV applications are 0.001 % of the acute RID, Novak for critically reviewing the manuscript. and are below regulatory LaCs. We are grateful for the support from the Center The main objective of the aerial adulticide for Vectorborne Diseases (University of Califor­ applications conducted by SYMVCD in 2007 was nia, Davis, CA). to decrease the number of infected and infective adult mosquitoes in the target area. Infection rates were significantly lower for Cx. tarsalis in REFERENCES CITED the spray zone after the aerial adulticide applica­ Armijos V, Wright S, Reisen W, Kelley K, Yamamoto tions, but not in the control areas. Even though S, Brown D. 2005. West Nile virus in Sacramento and we did not observe a significant decrease in the Yolo counties, 2004. Proc Pap Mosq Vector Control maximum likelihood estimate of minimum infec- Assoc Calif 73:24--27. MARCH 2010 MOSQUITO CONTROL EFFICACY AND HUMAN HEALTH RISK 65

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Ary Farajollahi1,2*, Sean P. Healy1,3, Isik Unlu1,2, Randy Gaugler1, Dina M. Fonseca1 1 Department of Entomology, Center for Vector Biology, Rutgers University, New Brunswick, New Jersey, United States of America, 2 Mercer County Mosquito Control, West Trenton, New Jersey, United States of America, 3 Monmouth County Mosquito Extermination Commission, Eatontown, New Jersey, United States of America

Abstract Aedes albopictus, the Asian tiger mosquito, continues expanding its geographic range and involvement in mosquito-borne diseases such as chikungunya and dengue. Vector control programs rarely attempt to suppress this diurnal species with an ultra-low volume (ULV) adulticide because for maximum efficacy applications are conducted at night. During 2009–2011 we performed experimental nighttime applications of a novel adulticide (DUETH) against field populations of Ae. albopictus within an urban site composed of approximately 1,000 parcels (home and yard) in northeastern USA. Dual applications at mid label rate of the adulticide spaced one or two days apart accomplished significantly higher control (85.065.4% average reduction) than single full rate applications (73.065.4%). Our results demonstrate that nighttime ULV adulticiding is effective in reducing Ae. albopictus abundance and highlight its potential for use as part of integrated pest management programs and during disease epidemics when reducing human illness is of paramount importance.

Citation: Farajollahi A, Healy SP, Unlu I, Gaugler R, Fonseca DM (2012) Effectiveness of Ultra-Low Volume Nighttime Applications of an Adulticide against Diurnal Aedes albopictus, a Critical Vector of Dengue and Chikungunya Viruses. PLoS ONE 7(11): e49181. doi:10.1371/journal.pone.0049181 Editor: Clive Shiff, Johns Hopkins University, United States of America Received September 5, 2012; Accepted October 9, 2012; Published November 8, 2012 Copyright: ß 2012 Farajollahi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a cooperative Agreement between USDA and Rutgers University (USDA-ARS-58-6615-8-105) entitled ‘‘Area-wide Pest Management Program for the Asian Tiger Mosquito in New Jersey.’’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction Most federal and state guidelines for protecting the public during outbreaks of mosquito-borne diseases recommend adulti- Chikungunya fever is an emerging tropical mosquito-borne cides from aircraft and truck-mounted equipment as the most disease caused by the chikungunya virus (CHIKV, genus effective method of reducing transmission risk to humans [12]. Alphavirus, family Togaviridae) that has become widespread in the These adulticide interventions are generally applied as ultra-low Indian Ocean region, resulting in millions of disease cases and volume (ULV) cold aerosol sprays during night-time campaigns over 250 deaths [1]. While the acute febrile phase of the disease is when a thermal inversion has occurred to keep the insecticide from usually resolved in a few days, the associated joint pain may persist dispersing upwards and light winds aid in the spread of the indefinitely; further causing health and economic impact [2]. insecticide droplets [13]. Because prior ULV applications have not Although historically not an important vector of CHIKV, the been efficacious or long lasting in controlling diurnally active Asian tiger mosquito, Aedes albopictus (Skuse) has recently emerged urban mosquitoes, such as Aedes aegypti (L.) [14,15] and Ae. albopictus as the principal driver of epidemics of chikungunya [3] after [16], they have been declared ineffective in reducing dengue a single amino acid mutation in the envelope protein of CHIKV transmission [17]. One reason for failure of control may be the increased its vector competence [4,5]. nocturnal resting behavior of day-biting mosquitoes in natural and Due to the widespread and increasing distribution of Ae. artificial places that are sheltered from the insecticide plume [18]. albopictus in temperate regions of North America and Europe The ineffectiveness of nighttime ULV applications against diurnal [6,7,8] and the escalating diagnoses of cases in travelers returning mosquitoes has become the conventional wisdom within the from endemic or epidemic areas [9,10] the risk of local CHIKV modern vector control community in the USA and many transmission in these continents is no longer conjectural, as mosquito abatement programs simply do not attempt to adulticide revealed by an epidemic comprising over 200 autochthonous cases against Ae. albopictus (D. Ninivaggi, personal communication). in Italy during 2007 [11] as well as sporadic autochthonous cases Since the public health implications of an Ae. albopictus-driven in France [3]. Due to the absence of a vaccine for CHIKV, arboviral epidemic are great, vector control officials must be mosquito control, particularly the reduction of biting populations adequately prepared to intervene with efficacious application of the primary vector Ae. albopictus, is the only effective means of strategies and products. A critical need exists for novel methods of reducing chikungunya fever cases during an epidemic.

PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49181 Nighttime ULV Efficay against Aedes albopictus insecticide application or new formulations to achieve successful The unit was fitted with a SmartFlow (Clarke Mosquito Control, control. Roselle, IL, USA) system used in tandem with radar ground speed DUETTM Dual-action Adulticide (ClarkeH, Roselle, IL, USA) is of the vehicle to ensure appropriate flow of insecticide and a new commercially available adulticide for mosquito control that accurate reporting and tracking of amount of chemical used along causes a benign agitation [a non-biting excitation of mosquitoes] with distance and area sprayed (Mention of trade names or potentially flushing mosquitoes from resting places and increasing commercial products are solely for the purposes of providing contact with airborne droplets that are more likely to impinge on specific information and do not imply endorsement by the authors flying adults [19]. DUET adulticide combines the pyrethroids or other involved parties). The sprayer was mounted in the back of sumithrin (5%, 44.94 g/L Active Ingredient) and prallethrin (1%, a flatbed truck at a height of 1.8 m, and the spray boom was 8.99 g/L AI) with the synergist piperonyl butoxide (5%, 44.94 g/ angled 45.5u pointing backwards. The vehicle was driven at an L AI). Prallethrin is reported to induce an excitatory response at average speed of 16.1 km/h. Droplet size measurements were sublethal concentrations and may drive mosquitoes from a resting obtained for the Cougar ULV machine prior to operational state and expose them to lethal doses of airborne sumithrin and applications using a DC-III portable droplet measurement system piperonyl butoxide [19]. This adulticide may have advantages (KLD Laboratories, Huntington Station, NY, USA). For vector against not only resting gravid or engorged mosquitoes but also spraying a droplet size range of 5 to 25 mm is most efficient, against diurnal mosquitoes such as Ae. albopictus which may be because this size is most likely to impinge on a mosquito and inactive during routine nighttime ULV applications by mosquito deliver a toxic dose [22]. abatement programs. Droplet measurements for mosquito control are often provided The objective of this study was to evaluate the area-wide as a mass median diameter or a volume median diameter (VMD). efficacy of nighttime (01:00–06:00) ground-applied ULV adulti- The VMD is also routinely provided as DV0.5, a term used to cide applications of DUET against Ae. albopictus within an urban represent a statistic where 50% of the spray volume or mass is residential community; we compared the abundance of Ae. contained in droplets smaller than this value. Most often, values albopictus populations within treated and untreated areas of Mercer for a DV0.1 and a DV0.9 are also provided, to describe 10% and County, New Jersey during 2009–2011. Our ultimate goal was to 90% of the cloud volume, respectively. Droplet size and develop a successful ULV adulticide application strategy to be distribution are two of the most important factors affecting the used in an integrated pest management (IPM) program for success of an ULV application [23]. Additionally, adulticide labels, suppression of Ae. albopictus, both for nuisance reduction and to which are interpreted as federal law, require that given equipment address imminent future outbreaks of chikungunya and dengue adhere explicitly to required VMD values. We conducted two fever. readings using the DC-III during our calibration of the Cougar ULV sprayer and acquired a DV0.1 value of 2.9 mm, a VMD Materials and Methods (DV0.5) value of 15.2 mm, and a DV0.9 value of 30.8 mm. A total of 4,015 drops were counted, with only 6 droplets above 32 mmin Study Area size, and none above 48 mm. During 2009, a highly urbanized residential field site was chosen The pesticide label for DUET requires ground-based spray in Mercer County, New Jersey, USA (40u 139 N, 74u 449 W) as equipment to be adjusted to deliver aerosolized droplets within part of an area-wide management of the Asian tiger mosquito a VMD of 8 to 30 mm (DV0.5,30 mm) and a DV0.9 value of less [20]. The field site (Treatment Site) is located within the City of than 50 mm. For all field trials, DUET was applied at a flow rate Trenton (population , 83,000, area 21.1 km2) and consists of of 136.04 ml/min. Applications during 2009 were conducted at 48.4 ha, including 1,251 parcels (Figure 1). Parcels correspond to maximum allowable label rate for a ground ULV spray (86.2 g/ a structure or house with surrounding yard, and are most often ha). This full label rate results in 0.81 g/ha AI of prallethrin, built as adjoining row homes or duplexes, indicative of the type of 4.04 g/ha AI of sumithrin, and 4.04 g/ha AI of piperonyl housing in this area. Almost all adjoining parcels contain butoxide. Subsequent applications during 2010–2011 were a sheltered alcove area between two homes, where vegetation conducted at recommended mid label rate (42.7 g/ha), resulting and trash proliferate, affording mosquitoes a shaded and humid in 0.40 g/ha AI of prallethrin, 2.02 g/ha AI of sumithrin, and area for a resting place. Additionally, socioeconomic conditions 2.02 g/ha AI of piperonyl butoxide. Only single adulticide within the field site have led to a large number of abandoned applications were conducted during 2009, however, in order to homes that have been boarded shut by the City of Trenton, but increase efficacy by compensating for gaps in coverage and often house transient humans and large amounts of trash [20]. missed targets, we conducted dual applications of the adulticide Lack of ownership and responsibility for hygiene has increased spaced one or two days apart during 2010 (twice) and 2011 mosquito populations within these parcels. Our field site consists of (once). Our intention was to control adult populations with the roughly 26 residential blocks, each containing a residential street first ULV application, wait one or two days, and conduct on all four sides, and divided between parallel parcels by a drivable another adulticide application to control any newly emerged alley. During ULV adulticide applications, streets and alleys are adults or mosquitoes that may have been missed with the initial both driven to maximize dispersal of insecticide. A second field site application. (40u 129 N, 74u 439 W), similar in both socioeconomic conditions Truck-mounted adulticide applications were conducted at night and Ae. albopictus levels [20], was chosen as an untreated control using a single vehicle to drive the entire treatment site. Routes (Control Site), where no active interventions were performed were designed to follow all available roads and alleys to provide against Ae. albopictus. This site consisted of 62.4 ha, including 1,064 maximum coverage. Each application took about 2 hours to parcels and was solely used to sample adult mosquito populations complete and was conducted between 01:30–06:30, depending on using the same protocol used in the treatment site [21]. the date of the application.

Ultra-low Volume Adulticide Application Adult Mosquito Surveillance and Analysis A CougarH (Clarke Mosquito Control, Roselle, IL, USA) cold Mosquitoes were sampled in our treatment site and control site aerosol ULV generator was used during all adulticide applications. on a weekly basis during 2009–2011 utilizing a network of

PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49181 Nighttime ULV Efficay against Aedes albopictus

PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49181 Nighttime ULV Efficay against Aedes albopictus

Figure 1. Map of ULV adulticide treatment site in Mercer County, New Jersey, USA, 2009–2011. Inset of Mercer County in the top left displays locations of treatment and no intervention sites, and detailed map below displays locations of BGS traps, parcels, and roads/alleys within only the treatment site. A typical block within this highly urbanized location is about 90 m wide and 150 m long, with each block divided by a drivable alley behind each parcel. All roadways and alleys were driven during an adulticide application. doi:10.1371/journal.pone.0049181.g001

BioGents SentinelTM (BGS) traps (Biogents AG, Regensburg, 10 m heights for thermal inversion observation. A Vantage Pro2 Germany). Specific details of surveillance protocols are outlined (Davis Instruments, Hayward, CA, USA) portable weather station elsewhere [21]; but briefly, locations were chosen by overlaying was set up within the treatment site 2 hours prior to application a grid of specific distance intervals. We used a 175–200 m distance and maintained until 2 hours post application. Additional between BGS traps for each site. Locations were determined using meteorological data was obtained from a permanent weather the Fishnet tool within ArcGIS Desktop 9.2 (Environmental station located at Trenton-Mercer Airport, situated 7.5 km from Systems Research Institute, Redlands, CA, USA). These distances the application site. were based on current knowledge of Ae. albopictus flight range [24] and the available resources within each site. Two hundred meter Results sampling resulted in 12 traps within the treatment site and 15 traps within the control site during 2009–2010, while 175 meter The experiments were performed during the 2009, 2010, and sampling resulted in 16 traps within the treatment site and 24 2011 active seasons for Ae. albopictus. Adulticide applications were traps within the control site during 2011. Sampling was performed conducted in unison with an intensive surveillance program and with BGS traps deployed weekly for 24 hours and deployed in were one of the components of an IPM strategy being developed backyards (near vegetation or shade) of each parcel selected. Each for control of Ae. albopictus. We conducted our first application of week, traps were placed in the same location within the backyards. DUET at full label rate and then proceeded to evaluate mid label Permissions to place BGS traps within each parcel were acquired rate applications in different combinations (Table 1). Although at the beginning of each season from individual property owners. most applications of adulticide were initiated when the mean The BGS trap was used with a solid BG-lure (Biogents AG, number of adults (male+female) captured in BGS traps were above Regensburg, Germany) containing ammonia, lactic acid and fatty 5, on one occasion we started with lower numbers (4.161.4) acids, components known to be particularly attractive to Ae. because we were testing the effect of adulticiding on populations of albopictus [25]. Ae. albopictus at the end of the season. Although evaluating the Mosquitoes recovered from traps were placed in containers and efficacy of control measures may be more difficult when adult transported to the laboratory on dry ice for identification and numbers are already low, this test yielded control levels similar to pooling. We calculated the mean number of Ae. albopictus adults those at other mid label rate single applications (Table 1). As (male+female) collected during each sampling session in BGS traps a result, the removal of this treatment from the analysis does not within each site. Adulticide applications were performed when affect the overall results (data not shown). The number of post- treatment adults was measured for 24 hrs starting the afternoon of environmentally, logistically, and operationally feasible within the the day (night) when treatment occurred. For duplicate treatments, treatment site when a threshold mean of $5 Ae. albopictus the post-treatment counts were made after the second treatment (male+female) adults were detected in our weekly BGS surveil- only. In all cases post-treatment values were lower than 5 lance. This number was chosen because 3 bites have been reported (2.360.7). The absence of significant wind was a constant (Table 1) as a common nuisance threshold driving residents indoors [26], as well as high humidity and air temperatures at night in the mid and an average of 5 bites/day by Ae. albopictus in Italy has been 20 C range, which are characteristic of urban areas in mid recorded as intolerable [27]. Percent control after ULV applica- u Atlantic states during the summer months [30]. tion of adulticides was calculated by using an algebraic variation of We found that single ULV adulticide applications at the full Henderson’s method [28] using the formula: percent con- label rate of 86.2 gm/ha resulted in a percent reduction of trol = 100–[(T/U)100], where T is the post application mean 72.765.4% (SE), which is significantly higher [p = 0.04] than divided by the pre application mean in the treatment site and U is single ULV applications at the mid label rate of 42.7 gm/ha the post application mean divided by the pre application mean in (54.064.7%). However, dual applications at mid label rate were the control (no intervention) site. Although additional integrated significantly more effective (p = 0.003) than single applications at pest management intervention efforts such as education, source full rate and resulted in an average percent reduction of reduction, and application of larvicides were being conducted 85.065.4%. Dual applications at the full label rate could not be within our treatment site as part of a larger project [21], none conducted without exceeding label guidelines. Overall the two would have an immediate effect on adult populations. Thus, our variables, application rate (full versus mid) and application type analyses concentrated on the overnight percent reduction of adult (single versus dual), explained 75% of the variance in percent populations. We used ANOVA (JMP 8, SAS Institute, Cary, NC, control (R2 = 0.75). USA) to examine the efficacy of a single ULV application versus a dual application, and full label rate versus mid label rate. Percentages were arcsin transformed prior to analysis [29]. No Discussion specific permits were required for the collection of adult Evaluating the efficacy of aerosol sprays for adult mosquito mosquitoes or the described field studies, which were developed control is critical to assessing their suitability, especially during with homeowners assent by professional county mosquito control epidemics when fast reduction in populations of biting females is personnel. These studies did not involve endangered or protected paramount. Over three years and multiple nighttime adulticide species. applications, we observed an overall significant average percent reduction in adult populations of day-biting Ae. albopictus mosqui- Meteorological Data Collection toes as measured using BGS trap surveillance. Our results provide During each application, meteorological data was recorded for direct evidence that nighttime applications of an ULV adulticide wind speed, direction, humidity, and temperature at 1 m and are effective in reducing Ae. albopictus abundance.

PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49181 LSOE|wwpooeog5Nvme 02|Vlm su 1|e49181 | 11 Issue | 7 Volume | 2012 November 5 www.plosone.org | ONE PLOS Table 1. Summary of adulticide applications and BGS trap results during 2009–2011, Mercer County, New Jersey.

Relative Application Application Application Humidity Wind Speed Year Date Time (am) Rate (gm/ha) Temp (uC) (RH %) (km/h) Treatment Site Control Site Percent Control* Pre-treatment Post-treatment Pre-treatment Post-treatment Mean (6SE) Mean (6SE) Mean (6SE) Mean (6SE)

2009 05-Aug-09 02:45 to 05:00 86.2 22.2 94% ,1.6{ 8.362.0 2.060.7 27.165.3 16.562.8 61% 19-Aug-09 03:15 to 06:00 86.2 22.8 93% 5.6 14.364.0 4.160.9 18.164.7 21.465.0 74% 16-Sep-09 03:00 to 05:00 86.2 19.2 76% 10.5 7.862.1 1.160.4 19.763.9 16.363.8 83% 2010 29-Jul-10 & 04:00 to 05:30 & 42.7 25.3 & 18.9 85% & 65% 8.1 & 9.9 10.862.9 2.660.7 9.161.7 13.963.6 82% 30-Jul-10{ 04:00 to 06:00 18-Aug-10 & 05:00 to 06:30 & 42.7 22.2 & 21.1 69% & 73% ,1.6 & ,1.6 10.562.3 0.960.3 14.262.8 11.461.4 90% 20-Aug-10{ 05:30 to 07:00 02-Sep-10 05:00 to 06:00 42.7 22.8 73% ,1.6 5.761.2 3.161.0 12.162.3 11.561.7 43% 2011 4-Aug-11 & 01:45 to 03:25 & 42.7 20.6 & 22.2 87% & 95% 5.6 & ,1.6 6.661.2 1.660.6 10.361.6 14.961.8 83% 5-Aug-11{ 03:30 to 05:00 14-Sep-11 02:30 to 05:00 42.7 21.1 87% 3.2 15.562.8 3.261.3 26.365.0 17.363.4 68% 27-Sep-11 02:30 to 04:00 42.7 21.7 93% ,1.6 4.161.4 2.560.7 12.762.8 16.464.0 54%

*Percent control following Henderson’s equation: 100–[(T/U)*100]. {Wind speeds of ,1.6 km/h indicate that wind was negligible during ULV application. {Denotes a tandem application of adulticide. doi:10.1371/journal.pone.0049181.t001 ihtm L fia against Efficay ULV Nighttime ee albopictus Aedes Nighttime ULV Efficay against Aedes albopictus

Our measures of adult population reductions were derived from following ULV adulticide applications have shown that only 8% BGS traps, a relatively new sampling device for capturing of female mosquitoes dissected post-treatment were parous, as container-inhabiting Aedes mosquitoes. The BGS trap has been compared with parity rates of 30% in the pre-treatment area and proven as an effective alternative to other collection devices and 40% in an untreated area [37]. The reduction in parous females, traps such as backpack aspirators, gravid traps, variations of which are most likely to be infected, makes ULV adulticiding carbon dioxide-baited traps, and the Fay-Price trap [25,31,32] for a very important component of a comprehensive intervention obtaining estimates of field abundance of Ae. albopictus, and program geared towards protection of public health from approximates human landing rate estimates [32,33]. By targeting mosquito-borne diseases. Careful examination of the 2007 out- adult mosquitoes, BGS traps provide an actual estimate of the break of chikungunya fever in Italy, the first large outbreak in biting populations, and hold an immediate advantage over other a temperate climate region, indicates that a larger epidemic was sampling and population assessment methods (e.g. Breteau, thwarted by timely control interventions [39]. Although it is still container, house indices or pupae per person) which are relatively debated what level of reduction in adult populations is necessary more labor intensive and plagued with levels of assumptions, and sufficient to prevent disease outbreaks, transmission models imprecision, and unpredictability [34]. BGS traps provide an developed for Ae. aegypti and dengue suggest that the degree of opportunity for improved adult entomological surveillance and suppression required to eliminate summertime spread of the have been used successfully as a rapid response tool for detection disease may be lower than 83% in some cases but closer to 90% in of Ae. albopictus [35] and to gauge efficacy of various control others [34,40]. The reduction in Ae. albopictus abundance we measures targeted against this species [21]. Furthermore, we achieved through nighttime adulticiding (85%) would likely result utilized not only before/after numbers, but also comparisons in a decrease in the number of infective bites received by the between treated and untreated sites to determine the immediate human population and would consequently impact the trans- percent reduction effects of adulticide applications on populations mission of an arbovirus such as dengue or CHIKV. of Ae. albopictus in temperate North America. In conclusion we provide evidence that a nighttime ULV Significantly, we found a greater effect on adult Ae. albopictus application of a synthetic pyrethroid is efficacious in reducing the populations through utilization of dual or repeated applications of abundance of Ae. albopictus in an urban environment and that dual adulticide at mid label rate. Previous studies have indicated that applications using mid label rates, spaced one or two days apart, two adulticide treatments using dieldrin (a chlorinated hydrocar- provide levels of reduction in the adult populations of Ae. albopictus bon similar to DDT which is now banned in most of the world) as in the upper range of which is necessary for interruption of a thermal fog during the day and spaced a week apart, increased arboviral transmission. The large and growing populations of Ae. and prolonged control of Ae. albopictus for up to eight weeks [36]. albopictus in several northeastern urban centers such as Washington Adulticide interventions by aircraft during the day against Ae. aegypti using malathion applied twice 4 days apart have also shown (DC), Philadelphia, Trenton, and New York City [6,8] make upwards of 90% control for over 10 days post application [37]. We a large autochthonous outbreak of an arbovirus such as CHIKV conducted dual ULV applications of adulticide at mid label rate or dengue a clear and present danger. We recommend that resulting in an average reduction of 85% in Ae. albopictus. nighttime applications of ULV adulticides in areas with large Furthermore, although previous studies have indicated that ULV populations of Ae. albopictus be considered as part of an integrated adulticides need to be applied at maximum rate [13,38], we found mosquito management approach for public health protection. that even mid label rate applications of the insecticide had a significant effect on Ae. albopictus. Our field applications were Acknowledgments conducted in a highly urbanized area in which we were able to We thank T. Crepeau, C. Borow, N. Indelicato, M. Milewski, R. drive both roadways and alleys to further enhance penetration of Oppenheimer, and S. Copeland for technical support, and J. McNelly and product and contact with mosquitoes. This finding has promising G. Lizarraga for logistical support during some applications. potential for vector control programs that are often under scrutiny about pesticide costs and also usage/exposure from the general Author Contributions public and must face increasing regulations and adulticide amount limits from local/federal government. Conceived and designed the experiments: AF SPH IU RG DMF. The rationale for adulticiding during epizootics or epidemics of Performed the experiments: AF SPH IU. Analyzed the data: AF DMF. Contributed reagents/materials/analysis tools: AF SPH RG DMF. Wrote arboviruses is to reduce the number of infected mosquitoes and the paper: AF RG DMF. thus interrupt pathogen transmission. Studies of Ae. aegypti

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COMMUNICATION SHORT

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insec-

rates

depending d-phenotrhin ml/ha, (10-100 ticides aircraft deltamethrin and the beneficial from

to terres-

on

formulation; Barber the of 2007) Latham and form trial in insects.

droplet

efficient information effects aerosol available Little the of using

size is

range

spray, an an on vec-

(Zhong applications

(Dvo. bees and label control al. Dvo. <60/xm 100/xm, 2003,

tor < et

per

as rec- on

5 9

flying ommendations), 2007), adult al. involves literature and Boyce 2004; mosquitoes. To

target et most to

caged applications efficacy, bees immediate the the

the of the

maximize insecti- to

spray exposure

occur

(Colburn during during crepuscular crepuscular hours cidal cloud when nocturnal hours and

some or

pathogen-transmitting Langford and Womeldorf al. 1970, 1974, 1979, major mosqui- Caron nuisance

et

2007). while beneficial Pankiw and These field al. Jay Boyce 1992,

active, organisms,

nontarget et toes

are

bioassays biology butterflies, (Connelly

bees and take and do of such the the

into

not not account

as are

bees, during night 2009). Carlson hives which their the

return to

Zhong Pesguard Aqua-K-Othrine (Seeley investigated

(2004) 1996). and al. the

S102

two et water-

are

unsynergized applications based, organophos- effects formulations of deltamethrin and of aerial of the

ULV

d-phenothrin, respectively, phate beehives; available however, for Naled the effects (acute mosquito

on

sublethal) adulticiding pyrethroid application (type Both and of aerial deltamethin Europe. II

ULV in

py- on

rethroid) d-phenothrin pyrethroid) investigated.

and beehives have been (type I

not

are

adulticiding honey applications (LCsos beneficial bees such

aerial

using ULV toxic insects

to two

as wa-

high pyrethroids mortality bee for deltamethrin ter-based d- caused and and 0.067/xg 0.05

mosquito

per

phenothrin, respectively, during experimental Pesticide trials National Informa- field conducted the in rice

(Chaskopoulou of northern al. Greece

ecosystem et

The

2011). of the received

majority

treatment

areas

Entomology, Bldg. Florida,

of of Department

University 970 Nat-

by

and uniform the cloud

accurate

coverage spray

as

Gainesville ural

32611-0620,

Drive, Area FL.

by high droplet evidenced (Dvo. densities the

35-40

Laboratory, Biological Control Tsimiski European

43, USDA-ARS

5

caged

/zm) and ob- uniform mortalities mosquito Thessaloniki, 54623,

Greece.

Corresponding [email protected]. author, e-mail:

served within the this

manuscript In sites. treatment

Apieulture-Sericulture, Laboratory

Agriculture,

of of School Ar-

1) immediate the

effects

of

toxic

report: acute

we or

Thessaloniki, Thessaloniki, of istotle

University

Greece.

applications

aforementioned the of aerial the

two

mos- 2nd Control County Mosquito Ave., West, District, Manatee 2317

adulticides beneficial

terrestrial

quito insects, in- Palmetto, FL, 34221. on

Entomological of Society 0022-2585/14/0720-0724504.00/0 (C) 2014 America

d-phenothrin .,000 ha Area

deltamethrin .,000 ha Area

hive location Bee

clud0g

bees, honey

the sublethal effects 2)

(Fig. and of the 1). The site

sites

at treatment sepm-ate

were a

products beehives. performance

agricultural the of

by

ba (A I,(XX) in size 4

on

open

areas

and by kin). The control 9.5 km B 3.21 -3.5 sites

assigned

for botb

the similar

treatment

to

years were

Methods Materi,Ms

mad

located from and km the

-3-5 sites treatment

areas.

Application Technologies. Insecticides and Three terrestrial beneficial

Nontarget Insects. Two

commercially

(]hrysopel-la

used: lacewing insecticides registered available hu'vae ULV

in

sects

green were

adulticiding ground Chrysopidae),

Europe for adult Stepbens (Neuropterm mosquito

cmae

were

mealybug for destroyers acdal Aqua-K-Othne (2% Cylptolaemus the Mul-

nmntrouzierl treatme.ts: wt:wt

deRametbfin; (Coleoptera: Environmental Coccinellidae), Bayer Science, domesticated and Lyon,

sant

d-phenothtn; (10% boney Pesguard France) .u.ll.ra Apidae). bees and (Hymeuoptera: S102 Apis L.

:wt

Osaka, Japan). Chemical Ltd., the Sumitomo Over Co.

prod- We conducted biological tdals

cmtrol with each 2

(Koppert, Berkel

were yr, spray

company

en

reeommedations of llealth Rodcnrijs, Netherlands}. based the

World The Tbe

larvae uct

on

were re-

applications

for

Organization (WHO) bottles ceived first second and of and

ULV insec- instars in

cs were

adtflticiding for 1997) ticides (WHO mosquito using

registered

g/ha label

(I.0 Chrysperla Epbestia of larvae other

fed 15C. kuh-

countries in

rtes

tot at

were

[AI[) (Lepidoptera: Aqua-K-Otbrine PyraIidae) lbr ingredient apbids. and Zeller and

7.5 niella active

eggs

JAIl aphids g/ha Pesguard

significantly for SI02). The of food

som'ee

re-

applications degree Inseccide conducted

tbe cannibadism duced the using o{

rearing

in

were a

helicopter

{5C model; Dougl McDonnell turbine fourth the third- aad experiments, For instar tainers.

Information

Helicopters, application Mesa, AZ).

on

technologies (nozzle navigational systems) and

type

(altitude, flight

successfully and conditions,

weather adults and (Nasreen

pupate

parameters

et

emerge

;

widths, provided offsets) and Approximately elsewhere al, 200,5). swath before trial. h the spraying 24

m'e

(Chkopoulou

}. al. the larvae moved incubator from

the the 201

et to

were

Experimented

expmSmental laboratory acclimatize for 23C them field During Sites. 28, two to at

expo-

purchased chosen: whe all Adult

A), from (Area site C. sites

monh-ouzieri

treatment

were

a sure.

were

biological insecticide

conducted, and Greek eontroI (BioInsoeta, control Tbes-

treatments

company

were

place.

where salonlkl, adults received flay took Greece). site, The of the During treatments

were

beehives added the because tbc experiments immediately, and and used experiment in

were

were

separately exposed

bees be tbe had each insec-

to to

ticide, additional

(Langstrotb. their blves. Fifteen by by included (-ea hives site 41 24

51 treatment

an

was

applied B)

insecticide that allow for each be would

obtained from era) Aristotle Tbes- University at to were

Vol. JOURNAL 51, 722 ENTOMOLOGY

MEDICAL 3 OF no.

experimental sa]oniki delivered the

and

sites Mortality ofnontarget exposed Table to organisms the aerial 1.

to were

Aqua-K-Othrine, Pesguard adulticide with and (five Thes- hives five hives and five

in B, A, Area Area treatments in in

saloniki,

Greece

the wk area) hives the control before 2 experiments in

hives bee acclimatization. allow for The

to

were

mortality

%

placed of

the the

(mean SEM) sites in treatment center to

___

Application Replicates

ensure

Treatment

(ml/ha) (N) (Fig. the of insecticidal cloud the

majority rate

Cnjptolaemus

Chmjsoperla to

exposure

adults larvae located that 1). studies shown hives well have Previous

insecticide treated within losses

50.0 AKO 1.9 2.5 5 sustain

1.1a 1.0

_ _

area an more

75.0 PESG 5 3.3 1.7 1.2 0.8a ___ _+ (Anderson situated Atkins elsewhere and than hives

Control

5 1.0"

1.0 0.0 3.3

1.6a

__+ ___

experimental

left the 1968). The hives for in sites

were

when the the trials

entire

Aqua-K-Othrine; Pesguard AKO,

PESG,

S102.

summer season were con-

delivery,

(2.5 too).

ducted each hive by Upon within followed the column

letter "Means

not

a are same con-

significantly different

[SAS (P Student-Newman-Keuls 0.05,

In- tained four brood frames and of

12,000 minimum

a

2003]

).

Inc.

stitute

provided bees bees adult and that the with

syrup

was

could food their within in

case sources access new

(beginning

insufficient. of summer) before and the after

environment

treat-

were

Sampling (end

compared of summer), for the Bioassay. all

8-14

Assessment stations:

ments

sam- were

deployed preassigned piing and control locations stations

at treatment

were areas.

sampling and each within

5 site, stations treatment

placed sampling

the control All

in stations

were area.

Results

deployed and Each handled similar

in

were manner. a

holding Mortality. sampling contained devices for each All species Insect station Acute

insect

nontarget

exposed bees, products which had low for the the mortalities when species

insect nontarget

except to

very

(Table mortality their hives. tested ). and

"2.5 Mean 1 C. in

were carnae

was

sampling mortality had Beneficial and Every and 1.7%

C.

insects: 1.9

station

montrouzieri

one mean

was

Plastics, exposed (American Pesguard, Aqua-K-Othrine 237-ml Chatta- when and 3.3% container

to

open

respectively. and Control TN) mortalities low remained and containing C. 10 montrouzieri, two

nooga,

significantly

different each than the 5 containing C. containers

prevent

to not treatment

were carnae

overcrowding. resulting cannibalism mortality from mortalities. unusual There of adult To

prevent

was no

daily mortality Vaseline bees of and film the from thin exceeded

escaping,

insects

average

never a was

applied along edge shiny bees (Fig. the of

the The dead hive 2). bees 10

No containers.

per

upper were

placed observed the with the field hive. containers insects in

in

any were

application before the commenced and beehives Beehive Performance. The the --15

min in two

spray

performed remained similarly there until after the

beehives the

spraying 30 min in

treatment to

areas was

completed weight insecticidal cloud the increased (25-30%), the untreated and

entire

in to

area, ensure

passed Approximately through population (14-18%), intended adult bees and brood (15- the 60

size

area.

significant

all returned There 19%). the adult bee

postspraying,

containers rain increase

in

to

were was no

laboratory. populations (Table

however, all 2); three at

areas

significant monitored The beehives for dead there of brood for the

increase in

treat-

presence

were was a

before,

bees and the and the h There control postspraying, morning 12

Area B

sig-

at ment

area. was a

wk). weekly (for

beehives weight All intervals of the nificant beehives the of total the 10 increase

in in

were

with bees. weight collect fitted the dead all

attributed three The

In

increase in is traps entrance to not

areas.

mortality, only bee population addition bee adult brood the of and brood adult and but

increase acute to to

weight

hive, populations, production. honey recorded also and of each

to

were

shiny (bees weekly. The bees fallen of with

presence

exposure) setae--indicative of sublethal insecticide

Discussion

by recorded observation of careful each frame for

was

looking droplet adult bees. high of unusual aerial the The trials resulted den-

ULV

in

presence

Analysis. analyses plots drops All

(400-4,000 Statistical statistical within

sities

treatment

per- per our were

package slides) high (SAS formed the software with and Institute SAS

mosquito

centimeter

square

on con-

analyzed replicates sentinel-caged NC). both trol levels and Cary, wild Inc., Five

mosquitoes in

per

were

(Chaskopoulou analysis populations insecticide. of (ANOVA)

2011). al. One-way However,

variance et

we

significant performed

mortality observed effect determine the of the of

insec-

insects nontarget to

no was

Chrysrperla exposed ticide and the adulticides When

mosquito percentage treatments. treatment to

on

Cryptolaemus droplet mortality.

delivered the similar

appropriate Data size, insect arcsine-

in

to

were are

square-root-transformed presented here, before used the there research low ANOVA.

Means

in

is

were one

separated ground deposition pesticides (P Student-Newman-Keuls of and 0.05; measurable using

no

(Dukes [SAS 2003] ). effect al. Institute

Inc. negative

species

nontarget to

et

through paired performed 2004). biolog- determine release the of Crop A protection

t-test to

any

was

development

Chrysoperla insecticide Cryptol- the beehives of effect the ical and such control

agents,

on as

[SAS 2003]). bee (Tzanakakis (P Adult and 0.05;

Institute important Inc. practice Greece

is in

aemus, an

populations, weight study provides 1980). brood the of beehives evidence and the that aerial Our ULV

May 2014 MosQurro CHASKOPOtrLOU

NOaOT Ec'rs

723 ADUI,T1CIDIN': AatJ 'r .: oF

t6

14

12

,

(PESG) Area A []

(AKO) IIIArea B

I0 (CONTROL) C mArea :

mortality daily bee (+SD) sampled Fig. (dates before the Averge (dates boxes) and the after 2. morning morning

in

boxes) without application. each spray

adultieiding,

applied properly, foraging flight has slow when mosquito afftct

the bee and

the perception

no

significant

biological effects foraging these control of bees

and (Desneux 2007) al.

time in agents.

et

on

being highly honey susceptible productivity, bees Despite

both beehive

whereas cbemicals several to

re-

products tested low

bee mortalities,

exceed- duce bee hatch brood production (Erickson

not

very egg

or

dily,

bees hive

stady, observed ing beehives Erickson the and 1983). 20 the experi-

In in

in per

were

our

daily

mortality mental beehives. Normal bee

performed treated well and increased adult bee

varies

areas

(Delabie population, al. brood, from weight, (Gary and 1985), 20 25 tbat and 30 suggesting 80 et to

to

insec-

deposits (.lobasen and bees hive 1984),

biologically significant ticide

Mussen effect had -<100

per

o on

1977). higher foraging Pesticides losses bees

when bees

within the

to

treatment

cause

areas.

applit during day and Waller but late (Byrne 1990), mosqulto-borne of pr(walence With the increasing

pathogenic

diseases bees when the of the evening sprang such European majority community,

in not

are

foraging beehives (Seeley and sheltered

epidemic 156) the Nile

North West Virus in

in recent

are

minimal ofbees results

(Hellenic the insecticides. deaths with hnman and in Greece '26]

34

to exposure

cases

pollen Insecticide residues

[HCDC] fbr and and Control Centre Disease Prevention

water,

nectar

any

on or

pyrethroids, the hive and could

d-phenothrin such 2010), kill and delta- contaminate

stress source,

or

as

example, pathion bees. methrin, the widely sublethal doses of used

will be For control mosqui- to more

Vol. JOURNAL 51, 724

MEDICAL ENTOMOLOGY 3 OF no.

increasingly

glasshouse It important appropriate field is and Pestic. Sci. experiments. toes. 16: to oratory,

use

efficacy methods that 409-415. against mosquitoes maximize

Delpuech.

and The sub- N., Desneux, J. Decourtye, 2007. A. while environmental Aerial

minimizing impact.

ULV

pesticides arthropods. effects

lethal of beneficial

study adulticiding products this with the evaluated

on in

Entomol.

52:

Annu. Rev. 81-106.

be sublethal effects done and with

acute

can no on

Zhong, Dukes, andJ.A.S.

Hogan, J.,

Hester, Greer,

P. H. M. D.

beneficial adult meth- This control

mosquito insects.

Barber.

of nozzle

A comparison 2004.

systems

two

spray

odology environmentally sound,

is sensitive

even over aerially apply the ultra-low-volume adulticide used

to

honey bees, long

late

containing

environments

fenthion. Control

Mosq. J. as as Assoc. 20: 27-35. Am.

technologies applications

evening using

Erickson, and bees

Erickson. and J., Honey H.

E. B. proper 1983. spray

navigational (nozzles systems) and used.

pesticides and

J.

Facts Bee Am. 2.

past. are common sense.

123:797-814.

of Mediterra- and E., Impact Gary, C. Mussen. N. E.

1984.

Acknowledgments

fly honey fruit malathion bait bees.

Envi-

spray nean

on

Entomol. 13:711-717.

ron.

Applications, Development thank the ASNF Air We

(HCDC) Hellenic and for Disease Prevention Con- Centre

Thessaloniki,

of of and the Local Agency Communities

Union

(http://www. trol. Nile Greece. West

2010. virus in

Municipalities

of and Thessaloniki for their County

support

keelpn elpno.gr ke id990 2010

o

funding. thank Greek Rural and the of Devel- Ministry We

report_total_20100917.pdf) (accessed 2010). October

20

of Health and Food and the and Social Ministry

opment

Johansen, pollinators. Pesticides and

Annu. Rev. C.A. 1977.

experimental Solidarity for the thank We issuing permits.

Entomol.

22: 177-192.

Chemical for the and Bayer CropScience Sumitomo

test

Latham,

Barber.

control Flor- and J. Mosquito M.,

2007. in

Agrolab products. for thank of evaluation also

We S.A.

ex-

adulticiding. focus Outlooks aerial ida with

Pest

a on

agricultural perimental procedures, and the of the residents

Manage. 18:178-183.

hospitality Thessaloniki for

of their and for the region

support

Mustafa,

Ashfaq. Mortality and of Nasreen,

A., G.

M. 2005.

Deployed Fighter

thank

the Program

We War experiments.

(Stephens) Chrysoperla Chrysopi- (Neuroptera:

carnea

(DWFP) their for

support.

dae) laboratory

after insecticides; stud-

to

exposure

some

South Stud. Pac. 26: 1-6. ies.

(NPIC) Information National Pesticide Center. Pes- 2010.

Cited

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Niemela, malathion and and Kimsey, F. Reisen. M. C. K. K. W. mosquitoes, noptera:

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on

by

of the Control: The defined of the naled adulticide the Minimizing mosquito impact

mosquito

mission

state

as

regulators, honay controllers, mellifera bees, Apidae): and environmental (Hymenoptera: Apis

managers.

on

Entomology application high-pressure Laboratory, Beach, Florida Medical

aerial nozzle ULV using Vero

sys-

a

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of 23(3):335-339, Journal the Mosquito Association, Control American 2007

Copyright by Mosquito Association, American The Control 2007 Inc. (C)

MOSQUITO NONTARGET EFFECTS ADULTICIDE OF THE PYRETHRIN

APPLIED AERIALLY OUTBREAK A AN DURING WEST VIRUS NILE IN

CALIFORNIA URBAN ENVIRONMENT

BOYCE, LAWLER, McCAULEY, SCHULTZ, WALTER SHARON M. SHANNON P. JENNIFER M. J. 2'3 2'3

NIEMELA, KIMSEY, MICHAEL LYNN S. CARRIE NIELSEN K. WILLIAM REISEN F. K. AND

August pyrethrin synergized piperonyl 2006, ABSTRACT. (EverGreen (R) insecticide butoxide with In

a

Crop McLaughlin Gormley King Valley, Protection MN) 60-6, Company, sprayed EC Golden in ultra-

was

city Mosquito by Davis, CA, low the volumes the Sacramento-Yolo and District of Vector Control

to over

mosquitoes transmitting impact Concurrently, Nile control evaluated the insecticide virus. of the West

on we

arthropods comparing mortality 1) arthropods, by groups.of of control and sentinel and nontarget

treatment

measuring diversity 2) arthropods placed and abundance the of dead found and control

tarps

treatment

on

ground. including dragonflies spraying species the found effect of sentinel We

nontarget

on no on

(Sympetrum spiders (Argiope aurantia), (Colias eurytheme), honeybees corruptum), (Apis butterflies and

mellifera). higher significantly diversity arthropods numbers found and of In

nontarget contrast,

were on

placed ground species sprayed unsprayed in small-bodied of the dead All

tarps

nontarget

versus areas. were

arthropods

opposed large-bodied mortality sentinels the affected. of sentinel that The

to

not as were

placed mosquitoes ground ranged the sites the sentinels from and 0% 100%.

nontarget

at tarps to same as

mosquitoes spraying pyrethrins ground Dead conclude aerial found the with that We had

tarps.

not on were

impact arthropods placed

large-bodied impact the in measurable

the

but did have

spray no zone, on a on

organisms. wide

small-bodied of range a

mortality, pyrethrins adulticide, Ultra-low KEY WORDS volume nontarget

they wildlife wider because toxic INTRODUCTION

to to

are a

variety species bacterial larvi- of than

nontarget

family (WNV; Flaviviridae, Nile virus The West

typically Logomasini (Their 2004) 2001, cides and

Flavivirus) of invasion North has

America

genus

applied

Adulticides broad

be

areas. may are over

seriously impacted

equine, human, and wildlife

applied suburban in urban

target

to

or areas

(Komar 2003), health 2003, al. it Marra and

et

mosquitoes applied they people, be

may near or

frequency increased has

the of ultra-low volume

mosquito popula-

reduce adult wetlands

to

near

(ULV)

applications mitigate

adulticide

to

ongo-

tions their

Larvicides used

most at

source. are

ing pending

Although outbreaks disease. of the

or

effectively mosquito early in the before

season

currently adulticides considered in low

use are

mosquitoes large numbers adult of

present,

are or

theory risk health, effects for

vertebrate in

on

during surface of the when the

extent summer

mortality their could of

nontarget

cause use

limited. adulticides is In

contrast, most

water

are

potentially arthropods,

wildlife

and affect in-

likely equine

used after be human

to

or cases

directly reducing by (Jensen

invertebrate

et

prey

and when is immediate need there

to

occur an

1999). Conversely, al. activities control

vector

interrupt

virus transmission.

(especially birds) reducing by benefit wildlife

may

approached epidemic Transmission of WNV

transmission within WNV wildlife and

among

during CA, County, in levels Yolo the of

summer

populations.

Recognizing spraying that aerial adulti- of

2006.

applied Adulticides in if California

the

are

community cide imminent urban of the

was over

(Emergency risk is elevated Level

2

to

response

August advantage 2006,

took Davis the in of

we

Planning

Conditions) (Epidemic Level 3

or

adulticiding

opportunity the effects of evaluate

to

Conditions)

(California Department Health of

arthropod species. specific

Our

nontarget

on 2006).

Services Adulticides that be used in

may

objectives mortality 1)

between to compare were organophosphates (malathion, California include

arthro- of sentinel control and treatment

groups pyrethrins haled) (permethrin, resmethrin,

and

pods, diversity

2) and and the

to

sumithrin). measure

Adulticides

threat

greater

.may pose a

arthropods abundance dead found of treat-

on

placed ground. control the and tarps

ment

on University California, Center, Wildlife Health of

Avenue, Davis, Shields One 95616. CA

Entomology, Department University of Califor- of

METHODS MATERIALS AND

nia, Avenue, Davis, Shields

One 95616. CA

application Study Population Biology, University 3Center insecticide for and of

area

California,

Davis, Avenue, One Shields CA 95616.

city

of Davis is located the Central The in

Diseases, University for Vectorborne Center

of

just California, Valley of of Sacramento California, west Davis, at Avenue, Shields CA One 95616. 335

MOSQUITO 23, AMERICAN 336 JOURNAL CONTROL ASSOCIATION

VOL. No. OF THE

protective 3833'14"N, cool, in 12144'17"W elevation of housed containers in dark

at

an a

population placed 60,000 and the The of about the field within h lives in in of 2 16

start

area m.

application. setting They 23,000 aerial insecticide with of the households urban in

an were an

prior greenbelts, just parks extensive network removed the with from field the sunrise of and

to

producing morning, examined,

of abundant recorded mosaic and live

tree next open cover a

as or

city canopy-covered is surrounded dead. The and

areas.

irrigated by Dragonflies farmland. separately housed in mesh

were

placed agricul- Coq. enclosures. Culex abundant is in the Three individuals tarsalis net at

were

Davis, sites, each tural around whereas of and is pipiens 6 10 Cx. L. 3 7

treatment at areas

non- or

species control found within habitat. Davis sites. the urban enclosures Both Two

treatment

at

are

(Goddard reducing vandalized, dragonfly sample 2002). of al. sites WNV competent

vectors et were

spiders garden infection from Yellow

size elevated in 30 28. WNV In

rates to to response were

individually mosquitoes, hoop-style in these identical marked increase the housed in

vector cages

a

by public, reported mosquito number of dead corvids those used sentinels. the for Two

to

during placed July and individuals the of late human each of and 10

onset at treatment

were cases

Mosquito August sites, 2006, and each Sacramento-Yolo the of non-Davis 6 control 3 7 at or

Alfalfa District aerial housed sites. butterflies in of and Control conducted

Vector groups

were

spraying virus transmission. Aerial in decrease enclosures each of mesh sites. 9

at net

treatment to

just (9 application place August 3) Controls consisted of after butterflies of

took 27

sunset

groups on

synergized pyrethrin insecticide, placed single with and The site and with 8 9. covered

at

treatment

a

plastic bags. piperonyl Honeybees Crop pairs (EverGreen (R) butoxide Protection housed in in

were

hoop-style King placed McLaughlin Gormley Company, 60-6,

EC each Two

at

cages cages. were

Valley, applied sites, MN) of and

Golden lb/acre site 12 0.0025 each

treatment at at cage was was

(maximum mosquito control) plastic bag label covered with for control. in

to rate a serve as a

city by ultra-low volumes entire twin- the

over a

flying approximately engine above aircraft 95

m

Diversity

and abundance

ground while winds krrdh. 10 <

were

diversity measured abundance the and of We

arthropods placed dead muslin found

tarps

on on

arthropods Sentinel

application ground before and after aerial the of

pyrethrin. hoop-style placed Twenty sentinel

Target 2-m Two species: tarps

cages

were

overnight containing Natvig (Townzen 1973), the each established sites of 10 and 21-

at treatment

spraying placed August tarsalis, field-collected adult when canceled due 2 35

Cx.

to on was were

conditions, breezy again perpendicular exposed August wind and the in and when 8

to on

spraying previously sampling August settings each of occurred. The sheltered 21 2

at

was

mosquito samples control used Davis established surveillance locations in and

group, as a

spraying

following August Davis, collected sites established well control 10 8

at

as were on as

Davis Twenty used the outside of the Davis Sentinels

treatment

group. spray as

zone. were

placed placed ust prior evening identical also outside the field in insecticide of the tarps

to were

(non-Davis application control) appli- Davis

retrieved within after and h 2

at spray zone

site) (7 placed plastic night locations sites Cages and cation. in individual the 6 2

at at

were

spraying. bags plugs toweling located The of with wetted the and

tarps

wet cotton at same were

arthropods Mortality posi- the sentinel and sites recorded with

10%

at water. sugar as were was

boundary again tioned time the between the of collection and about after and h 8

at

canopy

exposed placed ground Tarps the insecticide

exposure.

areas. were on

just prior of within removed and dragonflies h 2 (Syrnpe- Nontarget Sentinel species:

sunset

to

Any arthropod sunrise examined. dead

yellow and garden 1861) Hagen, and

corruptum trum

was

retained and identified later order spiders and (Argiope 1833) Lucas,

aurantia to

as

were

family; arthropods

live found spraying August

8,

when occurred evaluated tarps

were on on

released. eurytheme (Colias alfalfa butterflies Boisdu- and

mellifera honeybees 1852) (Apis 1758) val, L., and

spraying evaluated when occurred

on were

RESULTS

August Twelve selected sites .9.

treatment were

mosquito arthropods previously from established of sentinel the The survival 21

treat- at

provide geographic surveillance sites broad and control sites is shown in Table There 1.

to ment

Corresponding dragonflies spiders, city. control

the sites and of survival of 100% and

coverage was

approximately chi-square analysis significant established indicated km of the dif- 3

west were no

sites). (non-Davis 0.055)or (P in Davis ference survival butterflies

of control

spray zone

spiders, Dragonflies, honeybees unsprayed sprayed butterflies, 0.125) (P and bees in and

were

surviving captured proposed arthropods in All the within the control

spray areas. were area

spraying. period They flight (dragonflies, capable prior butterflies, aerial of normal 24-h to were

SEvrEMBER 2007 337 NONTARGET EFFECTS PYRETHRINS OF

placed in

found the

tarps

treatment toes

were on

arthropods Survival Table sentinel of 1.

treatment at

control sites.

applications pyrethrin sites. Aerial of and control or

piperonyl synergized butoxide with occurred

August Davis, 8-9, 2006, CA. in

on DISCUSSION

Common Control Treatment

light study this shed conducted We

to

on

Species

survival survival % %

name

impacts

whether there detectable

not

were or on

Dragonfly Sympetrum O0 O0

arthropods synergized pyrethrin when

nontarget

tum

corrup

aerially applied piperonyl butoxide

with

to

was

garden

Yellow Argiope O0 O0

community control urban WNV trans-

to

an

spider

aurantia

spraying

found mission. effect of We

no

on

Alfalfa Colias 85 100

relatively arthropods, large'sized sentinel includ-

butterfly

theme

y

eur

butterflies, ing dragonflies, spiders, honey- and

Honeybee mellifera Apis 92 75

significantly (Table 1). (P bees Mosquito In

Culex tarsalis contrast, O0

53 <

higher diversity 0.001) and of numbers non-target

arthropods killed in the

treatment versus were

(Table 2). the dead All of

control

groups

non-

(spiders) honeybees) their

movement

arthropods, species small-bodied upon or

target

were as

mortality release back into the environment. The

large-bodied opposed sentinels that the

to

were

mosquitoes of within the

sentinel

spraying

aerial conclude that affected. We spray

zone was

not

exposed (10-100%), variable

locations but

impact large-bodied arthropods

had the

among

no on

significantly

(F 13.4, 0.002)

1, 18, df

P

placed did the have in but

was

spray zone, measur-

a

placed exposed

in

than in greater

impact

small-bodied wide

able of

cages cages

range on a

vegetative (Table 2). under There

organisms. canopy was

no

mortality mosquitoes the

sentinel

at placed

sentinel and the

We among nontarget

cages

our

(Table 1). sites control

edge ground of the locations the tarps at at

same

study, the results the sentinel of In

contrast to both the

next to

canopy open areas, so were

significantly diversity higher (P 0.001) and exposed similar of adulticide. dead levels The <

to

arthropods th numbers of found

organisms nontarget either fell from found

tarps

were

on

dead in sites the the overhanging dropped

midair,

branches, from tarps treatment

on versus

or

(Table 2). and non-Davis Davis sites control All and crawled the died. The ratio tarp greater

onto

species

the dead of small-bodied body

surface of for the small-bodied nontarget

to were area mass

arthropods belonging explain arthropods partially they families in why the >25 had

to

may

following Coleoptera, Blatodea, mortality large-bodied higher orders: Collem- sentinels. than the

Hemiptera, Diptera, bola, Hymenoptera, Pscop- mortality mosquitoes placed The sentinel of

at

Thysanoptera, Acari, ranged study and Araneae. There sites from the

tera,

nontarget

same our as

mortality relationship mosquitoes between and dead found

0-100%

apparent

was no

were no on

mosquitoes mortality survivorship expected, of and sentinel of of the the As

nontarget

tarps.

species sampling significantly mosquitoes (P mosqui- sites.

dead sentinel No < among was

free-flying moving Mortality mosquitoes arthropods Table and sentinel 2. of and nontarget

treatment at

or

pyrethrins application Davis, August sites before 2006, control Twenty and after aerial CA. 8, of in 2-m

on

days prior placed overnight (Davis night spraying (2/site) control) locations Davis of in and 10 the 6 tarps

at

to

were

spraying (Davis exposed boundary placed treatment). between The the and relative

tarps

to at canopy were areas

mosquitoes. by compared the sentinel numbers Mean t-tests. were

mosquito Sentinel of of families of of orders of No. No. No.

mortality organisms arthropods arthropods dead % dead dead

Canopy Exposed Site Control Control Control Treatment Treatment Treatment

A19 26.1 6 96.3 5 4

B13 14.3 96.2 3 3 7

C10 16.7 22.7 44 2 2

12.5 13 D7 58.3 2 5 7

E4 15.4 69.0 19 3 3 2 2 5

F5 18.2 65.2 13 3 4 3 7 4

G6 100.0 0 9 151 5

Hll 8.0 9.7 6 37 3

I14 27.3 32.0 8 10 20

J12 13.0 15.4 8 24 7

Mean 56.5 15.2 1.9 4.4 0.7 5.9 0.7 32.8

value <0.005 <0.001 <0.001 <0.001 P

MOSQUITO 23, 338 AMERICAN CONTROL ASSOCIATION No. JOURNAL VOL.

THE OF

vegetative higher 0.005) tives orders families from of in 10 and >25 under than

canopy

arthropods. species exposed (Table 2). diurnal These nocturnal and

The

canopy cover areas

plant predators, likely provides by included feeders and shrubs and created

trees scavengers,

normally variety in protected partially microhabitats that of

environment that

may occur a

or a

ranging overhanging arthropods from the soil completely surface with shield from

to

contact

vegetation. Although diversity applied the of aerially 1996, (Mount affected insecticide the al.

et

high, species the overall Lothrop 2002). numbers of

al. control

From

et vetztor any was a

given

quite exception perspective, low. The difficult

this that is it

taxon

to one were means

Argentine

Formicidae), (Hymenoptera: high mosquito achieve extensive beneath kill

ants

rates was

organisms which the ecological abundant dead perspective,

From

most

were canopy

cover. an

why know detected. this do nonnative We proportion the this that substantial

of not

means a

species sampling. of abundant in populations

arthropod in under and

ant nontarget was so our

contrasting study

sentinel results The the of shielded from Al-

exposure. canopy

cover are

importance ground illustrate the

and of the population- study though

did

tarps

not

our measure

using impacts approaches different detect impacts, speculate impacts

level that to nontarget

on

we

species. findings view

We heavily

vegetated nontarget Davis in like will be

not our as

areas

meaningful, strongly

but

other organism significant

unless has

encourage

we

very

an a re-

investigators

impacts these studies. The only spatial distribution, repeat in stricted to

occurs

(vertebrate invertebrate) of and wildlife WNV exposed grassland (i.e., areas), habitats and is

on

isolation will in from control susceptible. endangered highly Threatened and not vector

occur

carefully activities,

additional, and controlled important species particularly would be focus

a

fully studies needed understand the

to future research that for address attempts

to more are

long-term

impacts

short- and nontarget impacts aerially applied

insecticides of on popu-

on

species.

persistence. lation numbers

or

design experimental the believe of the We

study sentinel robust and would have

was

ACKNOWLEDGMENTS large impacts they The detected had occurred.

application

adulticides after done of ULV

was

Veronica thank Sarah and Armi- Wheeler We

night-flying maximize of

to exposure sunset

jos staging mosquito assistance for with the

pesticide mosquitoes with minimize and

contact

providing Mogel sentinels, honey- and for Karl

diurnally species. the With active nontarget

hoop by bees. Sentinel donated

the

garden spiders, exception yellow all the of cages of were

Mosquito Sacramento-Yolo and Control Vector

species sentinels diurnal

used

nontarget

we were

District, Gary

and Brown, David thank

night. active However, that should be we

not at

some

Goodman, Wright Reed, Marcia

and Stan the

depending level of where

exposure may occur on

Laboratory entire staff of District for their the

night, organisms spend attempted the and

to

we

collaborating help Funding project. with this in

placing by sentinel mimic natural

cages exposure

by University provided

of the California

Dragonflies edge. butterflies the and was

at canopy

Wildlife Health Davis Center.

night, placement perch vegetation of their in

at so

vegetative (rather edge the of

at canopy cages

provided inside) deep than maximum natural

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exposed spin spiders in their

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were response

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[accessed 2007],

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