National Wildlife Research Center

FY 2019

Analytical and Formulation Chemistry Services

Contact Information:

David Goldade Chemist NWRC Headquarters 4101 LaPorte Avenue Fort Collins, Colorado 80521

Phone: (970) 266-6080 E-mail: david.a.goldade@usda.govWeb site: www.aphis.usda.gov/wildlifedamage/nwrc/

Groups Affected:

Agricultural producersConsumers of agricultural productsIndustry groupsState wildlife and natural resourcemanagersU.S. citizens

Major Cooperators:

APHIS Veterinary ServicesCalifornia Department of Food andAgricultureColorado State UniversityIsland ConservationLandcare Research (New Zealand)LiphatechArkion Life SciencesMonell Chemical Senses CenterU.S. Department of DefenseU.S. Department of the InteriorOffice of Insular AffairsU.S. Fish and Wildlife Service

National Wildlife Research Center Scientists Use Chemistry to Resolve Wildlife Damage

Wildlife Services' (WS) National Wildlife Research Center (NWRC) is the only Federal research facility devoted exclusively to resolving conflicts between people and wildlife through the development of effective, selective, and socially responsible methods, tools, and techniques.

NWRC's analytical chemistry and formulations laboratories support all NWRC scientists and WS operational programs, as well as other Federal and State agencies that need help with chemical screening, analysis, formulation, and modeling. This work supports a variety of research topics, ranging from wildlife contraceptives and wildlife disease to invasive species eradication and wildlife repellents. NWRC's chemistry and formulations staff are experienced in a variety of scientific disciplines, including pharmacology, environmental fate, chemical synthesis, toxicology, analytical chemistry, computer modeling, and formulations process development. Experts design methods to identify, analyze, and develop new drugs, repellents, toxicants, and other chemically-based wildlife damage management tools. They also enhance the delivery of compounds to target species through microencapsulation and by developing food products, baits, and lures that are stable in various environments and situations. Such methods enhance NWRC's research efforts and support product registration with the U.S. Environmental Protection Agency (EPA) and U.S. Food and Drug Administration (FDA).

Applying Science and Expertise to Wildlife Challenges

Oral Wildlife Contraceptive for Black-tailed Prairie Dogs. Black-tailed prairie dogs (Cynomus ludovicianus) exist in prairies throughout the Great Plains, from northern Mexico to southern Canada. In some areas, their burrowing, grazing, and damage to ornamental plants can lead to conflicts with people. In many urban areas, where prairie dog densities can be up to 5 times higher than in rural environments, wildlife managers prefer using non-lethal methods to reduce conflicts. DiazaCon (20,25-diazacholesterol) is a cholesterol-inhibiting compound shown to reduce fertility in gray squirrels. To determine if it would be effective in reducing reproduction in black-tailed prairie dogs, NWRC researchers fed DiazaCon-coated rolled oats to free-ranging prairie dogs at a natural area in Fort Collins, CO. For comparison, prairie dogs on an adjacent site were fed nontreated bait. Researchers observed a 96-percent reduction in the number of pups per DiazaCon-treated adult prairie dog, compared to animals in the nontreated site. Blood samples taken 11 months after treatment showed lower levels of cholesterol in treated animals. However, the levels were not high enough to prevent successful breeding in subsequent years. DiazaCon may offer an alternative tool to help manage urban prairie dog populations in locations where lethal control is undesirable.

Biomarkers for Use with Mongoose Oral Rabies Vaccine. The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus in Puerto Rico and comprises over 70 percent of animal rabies cases reported annually. Oral rabies vaccination (ORV) is the primary strategy used to control rabies in wildlife reservoirs, but currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and optimal bait types for mongooses has been done in Puerto Rico with promising results. To help evaluate ORV strategies targeting free-ranging mongooses in Puerto Rico, NWRC researchers tested the effectiveness of two biomarkers (ethyl-iophenoxic acid and methyliophenoxic acid) incorporated into placebo ORV baits to estimate bait uptake by captive mongooses. A biomarker is a measurable substance in an animal that can indicate that it has at least partially eaten a bait. Researchers fed biomarker-treated baits to mongooses and collected blood samples from mongooses prior to treatment, one day post-treatment, and then weekly up to 8 weeks post-treatment. Results showed mongooses that ate greater than or equal to 25 percent of the marked baits had robust short and long-term (4 to 8 weeks) levels of iophenoxic acid biomarker in their blood, which will be useful in evaluating future ORV programs for mongooses on Puerto Rico. Similar NWRC studies have looked at the effectiveness of iophenoxic acid biomarkers for use in white-tailed deer and raccoons.

Using Pentosidine as a Biomarker for Aging Turtles. Pentosidine is a naturally occurring compound in an animal's skin. Differing concentrations of pentosidine have been useful for estimating the age of bird and mammal species, but no study has examined its usefulness for estimating the ages of longer-lived species, such as turtles. Such a tool could be useful in understanding age distributions for species of conservation concern. NWRC researchers evaluated pentosidine concentrations in the skin of 117

Wildlife Services Protecting People ♦ Protecting Agriculture ♦ Protecting Wildlife

The U.S. Department of Agriculture is an equal opportunity employment provider and employer

117 female yellow mud turtles of known age in western Nebraska. Although pentosidine levels were low, they correlated positively to age. Results were too variable to allow for precise age estimates. Further studies using pentosidine to estimate the age of both long-lived cold-blooded and warm-blooded animals are needed to determine its usefulness.

Flushing Invasive Snakes from Cargo Using Heat. The brown treesnake is an invasive species on Guam, and its accidental export to other Pacific Islands in outbound cargo is a concern. Current attempts to avoid such exports rely mainly on snake detector dogs to inspect outbound cargo and vehicles. In past work, NWRC researchers showed that thermal fumigation could cause snakes to exit cargo, but the method does not work well with tightly packed cargo. More recently, researchers have shown that radiative heating at temperatures of 111-118° Fahrenheit (44-48° Celsius) can cause snakes to exit cargo; however, passive solar heating of cargo does not reach high enough temperatures. An active radiant-heating system has promise as a reliable way to treat tightly packed cargo. Treatment times are short enough that the method would not interrupt normal cargo-handling procedures.

DRC-1339 Residue Levels in Bird Tissues. DRC-1339 (also known as 3-chloro-4-methylaniline hydrochloride) is a slow-acting bird toxicant registered for controlling blackbirds, European starlings, pigeons, collared doves, gulls, magpies, crows, and ravens that damage agricultural crops, property or prey upon federally-listed threatened and endangered species. Time to death for birds that eat DRC-1339 coated bait is 1 to 3 days. Public concerns exist regarding non-target species exposure to DRC-1339 through animals eating dead or dying DRC-1339 treated birds. To improve upon methods of detecting and measuring DRC-1339 residues in bird tissues, NWRC chemists combined a new gas chromatography-tandem mass spectrometry method with improved extraction techniques. To evaluate the new method, 37 red-winged blackbirds were randomly assigned to groups and exposed to one of three doses of purified DRC-1339. After 3 days, birds were humanely euthanized and tissue samples were collected for analysis. The limits of detection (DL) for previously reported methods for detecting DRC-1339 residues were between 12 and 25 ng/g for breast, gastrointestinal tract, liver and kidney tissues. The new method showed DL between 2.6 and 5.9 ng/g for these same tissues. This increase in precision allows for greater confidence in secondary hazard studies and risk assessments with the use of DRC-1339.

Rodenticide Use in Marijuana Fields Threaten Wildlife. California is the largest producer of marijuana in the United States, with a mix of illegal and legal fields grown for medical and recreational purposes. One environmental side effect of marijuana production is the extensive use of anticoagulant rodenticides (AR) to prevent rodent damage to marijuana plants. NWRC and partners conducting owl surveys in California found a dead northern spotted owl in an area near seven active marijuana growing operations (MGO). NWRC chemists tested liver and blood samples from the bird for 11 anticoagulant rodenticides and 1 neurotoxicant rodenticide metabolite using a novel method developed for quantifying parts per billion levels of rodenticides in small tissue samples (< 100 mg). Samples were extracted using a small-scale dispersive solid-phase extraction procedure and quantified using an ultra-performance liquid chromatograph with tandem mass spectrometer detector. Results showed the bird likely died from brodifacoum AR exposure. The researchers note

• WS researchers observed a 96% reduction in thenumber of pups per adult black-tailed prairie dog treatedwith the oral contraceptive DiazaCon.

• WS research demonstrated the feasibility of including aiophenoxic acid biomarker in oral rabies vaccination baits forinvasive mongooses.

• WS researchers evaluated the usefulness of pentosidineconcentrations as a biomarker for estimating age inyellow mud turtles. Although pentosidine levels were low,they correlated positively to age.

• WS chemists used high temperature radiative heating toflush invasive brown treesnakes from tightly packedshipping cargo containers.

• WS chemists developed new methods for quantifyingDRC-1339 and rodenticide residues in targeted bird species.

the exposure may have been caused by the owl feeding on AR-contaminated prey. The proliferation of MGOs and their use of ARs in forested California landscapes may serve as an added stressor to this threatened species and other wildlife.

Selected Publications:

Berentsen, A.R., R.T. Sugihara, C.G. Payne, I. Leinbach, S.F. Volker, A. Vos, S. Ortmann, and A.T. Gilbert. 2019. Analysis of iophenoxic acid analogues in small Indian mongoose (Herpestes auropunctatus) sera for use as an oral rabies vaccination biological marker. JoVe 147:e59373. doi: 10.3791/59373

Franklin, A.B., P.C. Carlson, A. Rex, J.T. Rockweit, D.Garza, E. Culhane, S.F. Volker, R.J. Dusek, V.I. Shearn-Bochsler, M.W. Gabriel, and K.E. Horak. 2018. Grass is not always greener: rodenticide exposure of a threatened species near marijuana growing operations. BMC Research Notes 11:94. doi: 10.1186/s13104-018-3206-z

Goldade, D.A., K.R. Kim, J.C. Carlson, and S.F. Volker. 2019. Determination of residue levels of the avicide 3-chloro-4-methylaniline hydrochloride in red-winged blackbirds (Agelaius phoeniceus) by gas chromatography-tandem mass spectrometry. Journal of Chromatography B 1104:141-147. doi: 10.1016/j.jchromb.2018.11.009

Iverson, J.B., R.S. Stahl, C. Furcolow, and F. Kraus. 2017. An evaluation of the use of pentosidine as a biomarker for ageing turtles.Conservation Physiology 5(1):cow076. doi: 10.1093/conphys/cow076

Kraus, F., R. Stahl, and W. Pitt. 2017. An assessment of radiative heating as a thermal treatment for invasive snakes in cargo. International Journal of Pest Management 2:1–12. doi: 10.1080/09670874.2017.1293309

Yoder, C.A., R.E. Mauldin, J.P. Gionfriddo, K.A. Crane, D.A. Goldade, and R.M. Engeman. 2017. DiazaCon reduces black-tailed prairie dog reproduction in Colorado. Wildlife Research 43:655–661. doi: 10.1071/WR15210

Major Research Accomplishments: