Pesticides and Wildlife - ACS Publications - American Chemical Society

Chapter 2

Bioaccumulation of Pesticides in Bats from Missouri 1

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Angela Schmidt , Virgil Brack, Jr. , Russ Rommé , Karen Tyrell , and Alan Gehrt

Downloaded by NORTH CAROLINA STATE UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: November 1, 2000 | doi: 10.1021/bk-2001-0771.ch002

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ΒΗΕ Environmental, Inc., 11733 Chesterdale Road, Cincinnati, O H 45246 B H E Environmental, Inc., 7039 Maynardville Highway, Suite 7, Knoxville, T N 37918 Kansas City District, U.S. Army Corps of Engineers, Kansas City, M O 64106 3

Bats may be exposed to pesticides through direct exposure (e.g., ingestion of contaminated food or water, inhalation of aerosols, dermal absorption) and through indirect exposure (e.g., ingestion of contaminated prey by adults and contaminated milk by young). This study assesses the potential for bioaccumulation of pesticides by federally endangered Indiana bats (Myotis sodalis) and gray bats (M. grisescens). Both species occur on Fort Leonard Wood, Missouri where the pesticides chlorpyrifos and malathion have been used since the mid-1980's. This study began in 1997 and will continue until 2003. Surrogate species for the endangered bats were captured during spring and fall from Fort Leonard Wood and nearby Mark Twain National Forest. Insects were collected to determine pesticide concentration of the bats' food. Bat and insect samples also were screened for organochlorine and organophosphorous pesticides. Surrogate bats and insects collected in 1997 and 1998 had detectable amounts of DDE, heptachlor epoxide, and dieldrin.

Introduction Two federally endangered species, the Indiana bat {Myotis sodalis) and gray bat ( M grisescens) occur on Fort Leonard Wood (FLW), a 63,000 acre U.S. Army facility in Missouri. As required by the Endangered Species Act, the National Environmental Policy Act (ΝΕΡΑ), and applicable Army regulations, FLW assesses 8

© 2001 American Chemical Society In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


9 effects of actions on the Installation that have the potential to affect endangered species. This study is being conducted to evaluate the effects of pesticides used at FLW on Indiana bats and gray bats. After reviewing results of a Biological Assessment completed to evaluate effects of pesticide use and other activities at FLW, the U.S. Fish and Wildlife Service (FWS) issued a Biological Opinion (BO) and Take Statement containing Reasonable and Prudent Measures (RPMs), designed to monitor, reduce, or eliminate harmful effects to endangered bats from certain activities and training. One RPM required FLW to collect and analyze surrogate bats that reside on the installation and have a similar potential for exposure to pesticides as Indiana bats and gray bats. FLW is conducting other biomonitoring studies to assess secondary effects from pesticides and other training materials to Indiana bat and gray bat insect prey populations and habitat quality for the bats and prey. These studies are not addressed here. The Installation uses the pesticides chlorpyrifos (0,0-diethyl 0-3,5,6 trichloro-2pyridyl phosphorothioate or Dursban) and malathion (S-{ 1,2-bis (ethoxy carbonyl) ethyl} Ο,Ο-dimethyl phosphorodithioate). Chlorpyrifos is used to control webworms, ticks, and chiggers; malathion is used to control mosquitoes. Both chlorpyrifos and malathion are organophosphorous pesticides and latent cholinesterase inhibitors. Fort Leonard Wood has minimized use of these pesticides, however, they have been applied in large areas and in locations at or near where Indiana bats and gray bats occur. Chlorpyrifos is generally applied by a handsprayer and malathion is applied as a fog from a pressurized aerosol generator. The Indiana bat hibernates in caves (hibernacula) on FLW that provide suitable conditions for efficient hibernation. Indiana bats occur in significant numbers on FLW between August and April. However a few Indiana bats have been captured on FLW during the summer. Prior to hibernation, swarming (autumn active foraging period) begins in August and lasts several weeks. They roost in trees during the spring, summer, and fall. Typically, maternity roosts of up to 100 adult females are formed under slabs of exfoliating bark; no such roosts have been found on FLW. Indiana bats may form nursery roosts a few miles from hibernacula, or travel hundreds of miles to summer habitat. In autumn and spring, these bats may frequent hibernacula but roost in trees and forage outside the caves. The Indiana bat appears to be a selective opportunist, foraging around tree crowns in both riparian and non-riparian habitat (/, 2, 3). Their diet varies by location, often containing insects with a terrestrial-based life cycle as well as insects that are aquatic-based (3, 4, 5). Terrestrial-based insect prey include moths, leafhoppers, and beetles. Aquatic-based prey include mayflies, stone flies, and caddis flies (/, 2, 6, 7), and comprise a large portion of the bats' diet. Gray bats hibernate in caves in winter and form maternity roosts in caves during summer. No gray bats are known to hibernate on FLW. There are several gray bat maternity roosts on FLW. This species frequently forages over streams and other water bodies, and a large portion of the diet consists of insects with an aquatic larval stage, such as mayflies, stone flies, and caddis flies (/, 2, 6, 7).

In Pesticides and Wildlife; Johnston, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Purpose This study is being conducted to determine if Indiana bats or gray bats bioaccumulate chlorpyrifos or malathion used on FLW. The primary objective of this multi-year study is to assess whole-body burdens of the pesticides in species of bats (surrogates) with diets and habitat preferences similar to those of Indiana bats and gray bats. Additionally, insect samples are being evaluated to characterize the presence of pesticides in prey. Bioaccumulation of pesticides in bats is affected by how and when the exposure to the pesticide occurs. Pesticide contamination/exposure has been suspected as a contributing factor in population declines of Indiana bats and gray bats, but only limited data exist upon which to base this premise. An Exposure Pathway Analysis was completed to determine when Indiana bats and gray bats occur on FLW, what activities (e.g., forage, hibernate, swarm) they perform while on the installation, and other relevant life history information (e.g., body size). The Exposure Pathway Analysis indicated both species may inhale, ingest, or dermally absorb pesticides. To assess the bioaccumulation of chlorpyrifos and malathion in bat tissue and insects, sample locations were selected on FLW (exposure sites) and on Mark Twain National Forest (reference sites located approximately 75 miles from FLW). A comparison of pesticide residues in bats and insects at reference sites to those in bats and insects inhabiting FLW will help establish the source of pesticides detected in samples. In addition to concerns for chlorpyrifos and malathion, FLW is assessing bioaccumulation of other pesticides in bats and insects. Surrogate bats and insect samples were screened for organochlorine and other organophosphorous pesticides used in Missouri during the past 30 years.

Methods

Surrogate Species Selection Fifteen species of bats (Order Chiroptera) are known from Missouri (8). Four species having similar habitat and diet preferences to Indiana bats and gray bats were selected as surrogate species. The most appropriate surrogate species that occur on FLW are the eastern pipistrelle bat (Pipistrellus subflavus), red bat {Lasiurus borealis), and little brown bat (Myotis lucifugus). Eastern pipistrelles and red bats, as well as big brown bats {Eptesicus fuscus), and little brown bats are insectivorous, and as established from extensive mist net surveys on FLW, occur in the same areas as Indiana bats and gray bats. Little brown bats and big brown bats are relatively uncommon on FLW and subsequently only a few were included in this study. The



11 eastern pipistrelle and red bats are the most common species collected on the installation. The eastern pipistrelle and red bat have similar lifespans (7 to 10 years), similar body weights (5 to 12 grams), and similar low fecundity rates (1 to 2 offspring per year) as Indiana bats and gray bats. The eastern pipistrelle hibernates in caves during winter and forms maternity roosts in trees during summer. The species isfrequentlycaptured in riparian habitat. The eastern pipistrelle often forages over or near streams (2), and aquatic-based insects may be common in the diet (6). The red bat roosts in woodlands during summer. Although the species may be migratory, the wintering location of a majority of the population is unknown. Red bats forage over trees and in open areas (1,2) and fly in corridors along or over streams. The diet consists largely of terrestrial-based insects (6, 9).

Field Collection Techniques Eleven exposure sites were selected on FLW. Two of the sites are located in areas where malathion and chlorpyrifos are used regularly (during spring, summer, and fall); Cantonment Area and golf course. Both malathion and chlorpyrifos have been applied on at least 20 acres in these two areas, four times a year. The other nine exposure areas selected for this study have variable pesticide use. Pesticides are applied only during military operations. All exposure sites are within 20 miles (approximately) of each other and occur throughout the entire installation. FLW applies the pesticides on an as-needed basis and follows the installation's Pest Management Plan. When possible, exposure sites were sampled immediately after application of malathion or chlorpyrifos. Surrogate bats were collected with mist nets between late April and early June, and between August and September in 1997 and 1998. The mist nets used in this study were made of monofilament nylon with a mesh size of approximately 3.8 cm, and varied in length and height to cover the bats' flight corridor at each site. The nets were placed across roads or streams likely to be used by bats as travel corridors or for foraging. Specific mist net sites were selected to maximize canopy closure above the nets whenever possible. One or two nets were deployed at 11 exposure and 3 reference sites for at least one calendar night in 1997 and 1998. Biologists raised nets at dusk and monitored them approximately every 20 minutes until sufficient surrogate bats were captured for analysis, or until 0400 hours. A minimum of two surrogate bats were collected from each exposure site and at least five surrogate bats from each reference site. The species, sex, maturity, and reproductive condition of captured bats were identified. Body weight and right forearm length of each captured bat were measured. The height at which the bat was captured and time of capture were recorded. Indiana bats and gray bats were banded with a numbered, colored, celluloid, split-ring forearm band, and released.



12 Insects were collected during fall using black (ultraviolet) and white (fluorescent) light traps. Insects were sampled in fall to ensure adequate representation of insects available for consumption by Indiana bats and gray bats (all insects were adults). Additionally, based on surrogate bats using the installation and their feeding habits, it is believed their diets may differ slightly from that of gray bats. In order to adequately represent gray bats' dietary exposure to pesticides, we collected insects during the time when gray bats would have increased energy demands. Traps consisted of a light source surrounded by Plexiglas® dividers over a funnel with a mesh bag to retain insects. Traps were suspended from trees at heights of at least 2-m above ground to capture flying insects. One black light trap and one white light trap were deployed in separate trees at dusk (approximately between 1900-2130 hours) at exposure and reference sites. Insects captured in black light traps and white light traps were composited into a single sample per site. Insect samples were weighed and information including date, sample location, collection time, sample number, and sample weight was recorded.

Laboratory Techniques A 5-g to 10-g sample of composited insects or an entire bat was weighed into a Teflon extraction jar and spiked with the appropriate surrogate internal standards (these contain organochlorine and phosphorous pesticides). Samples were combined with methylene chloride (100-mL of 75%/25%) and approximately 50 g of sodium sulfate and macerated with a Tekmar Tissumizer equipped with a stainless steel probe and centrifuged at 2000 RPM. An additional 5-g aliquot of tissue was removed for percent moisture determination. The extract from the Teflon extraction jar was decanted into an Erlenmeyer flask. After each maceration (total of two 50 mL solvent additions) the centrifuged solvent extracts were combined in the flask. An additional extraction (the third) was performed using shaking techniques, the sample centrifuged a third time, and the extracts combined with the others. A 10-mL aliquot of the combined extracts was removed from the Erlenmeyer flask and air-dried for lipid weight determination (methylene chloride extractables). The remainder of the extract was dried over sodium sulfate, processed through a gravity fed alumina cleanup column (20 g, 2% deactivated), and concentrated to 900 in a KudernaDanish apparatus under nitrogen. The concentrated extract was further cleaned using size-exclusion High Performance Liquid Chromatography (HPLC). This procedure removed common contaminants which could interfere with instrumental analysis, including lipid and elemental sulfur. The post-HPLC extract was concentrated to approximately 500 under nitrogen and the recovery internal standards added to quantify extraction efficiency. The extract was solvent-exchanged with hexane for chlorinated hydrocarbon analysis by GC/ECD (gas chromatography/ electron capture detector). Sample extracts were analyzed for pesticides by using a GC equipped with electron capture detectors (ECD) following procedures defined in and modified from EPA's SW846 Method #8081. A five-level calibration was analyzed before any



13 sample extracts, with the lowest level just above the detection limit, and the range of the calibration encompassing the expected concentration range of the samples. The instruments were equipped with two ECD detectors and two 60-m capillary columns of different polarities (DB-5 and DB-1701). The 60-m DB-5 column, electronic pressure controlled inlet, hydrogen carrier gas, and optimized long temperature program, provide an extra measure of accuracy above the standard 30-m column (i.e., reduction in coelution, more stable baseline) to provide high quality GC/ECD data in a single data set. A portion of the samples was also injected into a Hewlett Packard 5890 gas chromatograph second with a nitrogen phosphorous detector (NPD). The NPD detector was used to detect the presence of nitrogen or phosphorous containing compounds (e.g., organophosphorous pesticides). Confirmation analysis was performed using the dual column GC and ECD.

Results

Exposure Pathway Analyses An Exposure Pathway Analysis was completed for Indiana bats and gray bats on FLW to determine potential direct and indirect exposure pathways and routes (Figure 1). This study addresses only primary (direct) exposure pathways. Review of the life histories of Indiana bats and gray bats indicated individuals of both species may be exposed to pesticides through inhalation, ingestion, and dermal absorption on FLW (Figure 2).

Sample Locations and Bat Capture Results Four or more bats were collectedfromeach of 11 exposure sites during 1997 and 1998. Reference sites were selected in Mark Twain National Forest where we believe chlorpyrifos, malathion, or other pesticides have not been used. Eighteen bats were collected during both 1997 and 1998 from each of three reference sites. One hundred-twenty five bats were collected in spring and fall of 1997; 38 were analyzed for pesticides. Some bats were released due to their status (e.g., state-listed) and many of the bats collected were analyzed to assess the presence of polycyclic aromatic hydrocarbons (PAHs), metals, PAH metabolites, and Cytochrome P450 activity. Eighty percent of those analyzed were eastern pipistrelles (η = 15) or red bats (n = 20). Other species sampled and analyzed for pesticides included big brown bat (n = 2), and little brown bat (n = 1). Approximately half of the bats collected for laboratory analysis were female. Bat capture results in 1998 were similar to those of 1997. One hundred-twenty nine bats were collected and 36 were analyzed for pesticides. Eighty percent of those


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Direct Exposure

Indirect Exposure

Primary Effects

Secondary Effects


Inhalation while roosting while foraging while hibernating Ingestion from prey from fur while grooming from lactation Dermal absorption from roost from air from other bats

Prey

Pesticide

Figure 1. Pesticide Exposure Pathwaysfor Indiana bats and gray bats on Fort Leonard Wood

Figure 2 Points and routes of pesticide exposurefor Indiana bats and gray bats on Fort Leonard Wood


15 analyzed were eastern pipistrelles (η = 11) or red bats (n = 24). The only other species collected and analyzed for pesticides was big brown bat (n = 1). Insect samples were collected from each of 11 exposure sites and each of 3 reference sites in both 1997 and 1998. Each of the 28 samples were analyzed for pesticides.


Pesticide Analysis and Screening Neither chlorpyrifos or malathion were detected (method detection limit of 0.00^g/g) in any bat or insect sample collected on FLW or at reference sites during 1997 or 1998. Every bat sample (whole body sample) (38 bats from 1997 and 36 from 1998) contained detectable concentrations of DDE and heptachlor epoxide. Dieldrin was detected in 22 of the 36 bats tested in 1998. Because the sample size is small, the usefulness of statistical analysis is limited. An preliminary review of pesticide residue content in bats yielded no identifiable or correlative relationships with sex, species, body size, or body weight (e.g., larger and heavier bats did not have the greatest concentration of pesticide in their tissues). A thorough statistical analysis of the data will be conducted at the end of the study (year 2003). The mean concentration of DDE in bats collected in 1997 and 1998 was 0.652 μg/g (sd ± 1.314) and ranged from < 0.001 to 8.9 μg/g (Table 1). The mean concentration of heptachlor epoxide in bats collected in 1997 and 1998 was 0.020 μg/g (sd ± 0.063) and values ranged from < 0.001 to 4.8 μg/g (Table 1). Dieldrin was only detected in 22 bat samples in 1998. The mean concentration was 0.060 μg/g (sd ± 0.102), and concentrations ranged from < 0.001 to 0.38 μg/g (Table 1). Fourteen of the 28 insect samples contained low levels of DDE, with the mean ranging from < 0.001 to 0.005μg/g; 8 insect samples had detectable concentrations of heptachlor epoxide, with the mean ranging from < 0.001 to 0.005 μg/g.

Table 1. Mean concentration, standard deviation, and range of heptachlor epoxide and DDE in bat samples in spring and fall of 1997 and 1998, and dieldrin in spring and fall of 1998 at 11 exposure sites on Fort Leonard Wood and three reference sites in Missouri. Range * SD Pesticide Sample Mean Concentration* Size O.001 to 4.8 0.063 Heptachlor epoxide 74 0.020 DDE

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0.652

1.314

Pesticides and Wildlife - ACS Publications - American Chemical Society

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