1 

## Annual Report 2010 Characterization of Avian Hazards Following Chlorophacinone (Rozol®) Use for Prairie Dog Control

Nimish B. Vyas, US Geological Survey, Patuxent Wildlife Research Center

Submitted to US Fish and Wildlife Service Region 6 Kevin Kritz, Kimb erly Dickerson , Joy Gober, Scott Larson , Matt S chwarz2 Abstract : Black

- tailed prairie dogs (BTPD) , Cynomys ludovicianus , are a keystone species of the prairie. However m any in the agricultural community consider them to be pests and BTPDs are subjected to large

- scale private and governmental eradication campaigns. The US Environmental Agency's recent registration of the rodenticide Rozol® ( active ingredient chlorophacinone) for BTPD control has raised c oncern s because of its potential primary and secondary poisoning hazard s to birds. I conducted a preliminary study to better understand avian exposure and effects following operational Rozol® applications to control BTPDs. My findings show that birds can be lethally exposed to Rozol®. Background and Justification : Black

- tailed prairie dog ecology : Black

- tailed prairie dogs, (BTPD) Cynomys ludovicianus , currently occupy more than 970,000 ha of habitat in 10 western states. BTPDs are highly social, diurnal fossorial rodents and live in colonies that are divided into coteries. Colonies vary in size and may range from a fraction of a hectare to hundreds of hectares

. Ecologically, BTPDs are a keystone species because they influence the prairie ecosystem’s structure, function , and composition

. Unlike other species of prairie dogs, BTPDs remain active above ground throughout the year , making them a valuable food source for wintering raptors ( Plum pton and Anders e n 199 7 ; Weber 2004 ; Kotliar et al

. 2006 ). Black

- tailed prairie dog control : Many in the agricultural community contend that BTPDs damage crops, compete with livestock for forage, and their burrows drain fields of water and create a hazard t o livestock and farm equipmen t ( Derner et al. 2006; Lamb et al.3 2006 ). Consequently BTPDs are considered a nuisance and are subjected to large

- scale eradication s , often times promoted and conducted by county, state, and federal agencies (Zinn et al. 1999) . Intentional BTPD eradication commonly involves placing poisons either in or outside the burrows ( Forrest and Luchsinger 2006; Hoogland 2006)

. Until recently, z inc phosphide, aluminum phosphide, and carbon ( for use in ignitable gas

c artridges ) had been the only active ingredients to have received full registration ( Section
3 registration) for BTPD control under the Federal Insecticide, Fungicide, and Rodenticide Act by the US Environmental Protection Agency (EPA )

. However, in the
pas t few years, six states (Colorado, Kansas, Nebraska, Oklahoma, Texas, and Wyoming) were granted the authority by EPA, under the Federal Insecticide, Fungicide, and Rodenticide Act Section 24c, to issue S pecial L ocal Needs registration s for the rodenticide Rozol® (0.005 % active ingredient chlorophacinone, 2

- [(p

- chlorophenyl) phenylacetyl)] 1,3

- indandione) to control BTPDs ( see for example, http://www.epa.gov/opprd001/24c/; http://www.colorado.gov/cs/Satellite/Agriculture

Main/CDAG/1214812857241). Avian laboratory acute oral toxicity tests show technical grade chlorophacinone to be Moderately Toxic, however, on a subacute dietary basis chlorophacinone is categorized as Highly Toxic (http://www.epa.gov/oppsrrd1/REDs/2100red.pdf). Furthermore, EPA identi fied the risk of secondary poisoning from Rozol® as being greater than the risks from other rodenticides (Erikson and Urban 2004). In 2009, EPA granted full registration (Section
3 ; EPA registration number 7173

- 286 ) to chlor o phacinone for BTPD control an d thereby expand ed Rozol® 's availability to 10 states : Colorado, Kansas, Montana, Nebraska, New Mexico, North Dakota, Oklahoma, South Dakota , Texas, and Wyoming ( a ttachment 1 )

.4 BTPDs occupy an estimated 971,000 h a in the US and Rozol® is becoming the rode nticide of cho ice for controlling BTPD s ( http://www.fws.gov/mountain

prairie/species/mammals/btprairiedog/ ; Mr. Fred Raish, supervisor, Yuma County Pest Control District [YCPCD] , Colorado, pers onal comm unication )

. In 1993, at EPA’s request, the US Fish and Wildlife Service (FWS) completed a Biological Opinion on 16 vertebrate control agents including chlorophacinone. However chlorophacinone was not registered for BTPD control at that time and therefore, its risk
to non - target species at BTPD colonies was not reviewed . EPA subsequently noted in their 1998 Reregistration Eligibility Decision that additional consultation with FWS may be necessary if new uses are proposed. However, EPA allowed the new use of Rozol® fo r BTPD control without consultation with FWS despite documented non

- target mortalities and letters stating ecological concern to EPA from state wildlife agencies, FWS, and non

- governmental organizations. In consideration of the expanded use of Rozol® for BTPD control, Rozol®'s mode of action, wildlife incident reports, and the Rozol® label 's requirements for preventing take of non

- target wildlife, research is warranted to better quantify the hazards of chlorophacinone t o avian species. Little is known ab out the hazards of chlorophacinone

- contaminated BTPDs to free

- ranging non

target species. However, a few serendipitous non

- target mammalian and avian carcass recoveries raise the possibility of the adverse effects of Rozol® on a larger scale (Scott
Larson , Joy Gober, Kimberly Dickerson, FWS Region 6, personal communication ; Stone et al. 2003). The paucity of incident data does not imply a lack of hazard, but highlights insufficient monitoring (Vyas 1999). I nformation on the effects of operational5 applic ations of Rozol® to avian species is much needed by FWS Region 6 since it includes seven of the 10 states where Rozol® is now registered for BTPD control. Objectives and Methods : This study is a three

- year project. The current annual report summarizes the observations, methods, and findings from three field visits that were conducted as part of a preliminary study during year 1 (2010) of the project

. The overall objective of this project is t o determine if Rozol® applications for BTP D control result in adverse effects to birds. In 2010 , data w ere collected with respect to the following s p ecific objectives listed in the Study Plan that was submitted to the FWS : 1. Determine the effects of chlorophacinone on the above

- ground activity of BTPDs. 2. Document the above

- ground BTPD morbidity and mortality. 3. Measure chlorophacinone residue concentrations in BTPDs.
4. Measure BTPD s’ susceptibility to predation. 5. Determine avian exposure and effects of chlorophacinone. Field Visit 1 : This visit was conducted in early January 2010. The purpose of this visit was to observe and learn about BTPDs and the birds associated with BTPD colonies in winter. I observed BTPDs and birds at two protected BTPD colonies at The Nature Conservancy's Fox Ranch , Kirk, Yuma County, Colorado and I was privileged to be invited by Mr. Fred Raish, supervisor, YCP C D , to visit three fields that had been treated with Rozol® for BTPD control in the past month. The three Rozol

- treate d fields were in proximity to each other and together they formed an almost contiguous area of approximately 65.5 ha

or 162 acres ( Mr. Fred Raish, supervisor, YCP C D , personal communication). Because of6 the high density of BTPD burrows, Rozol ® application to this area occurred over approximately 3 weeks ( Mr. Fred Raish, supervisor, YCP C D, personal communication)

. Observations. I observed BTPDs and birds at two BTPD colonies at Fox Ranch over
two days. BTPDs were active above ground at both colonies. L arge flocks of horned larks ( Eremophila alpestris ) were the only avian species seen feeding at the colon ies

. Two golden eagles ( Aquila chrysaetos ) and one red

- tailed hawk ( Buteo jamaicensis ) were seen near but not at the colonies. By contrast, an hour at the Rozol ® treated fields revealed six or seven ferruginous hawks ( Buteo regalis ) in the BTPD colon ies (figure 1)

. Large flocks of horned larks were also observed feeding in the Rozol ®

- treated fields. Walking through the fields revealed that Rozol® was visible in many BTPD burrow
entrances (please see below section Field Visit 2: Results: Rozol® availability to wildlife and figure 5 for details). Rozol ® is a green colored winter wheat based anticoagulant indandione rodenticide product ( figure 2) . EPA r equires rodenticides, other than those used exclusively at agricultural sites, to be formulated with an indicator dye in order to help identify a human's or a household pet's exposure to the rodenticide ( EPA 1998 ). For Rozol ® , the indicator dye color is g reen and it can be used to determine exposure in BTPD and birds. I also frequently encountered bird droppings in burrow entrances

. Based on the dropping size and shape and their abundance, I believe the droppings to be of horned larks. The majority of the droppings were grey

- white in color (typical dropping coloration) but occasionally, green droppings were observed in the burrow
entrances (figure 3)

. I did not see horned lark droppings in burrow entrances at the BTPD colon ies at Fox Ranch. A l arger g reen - colored dropping that , based on its size, shape, and texture and the common occurrence of the species in Yuma County, I believe7 the dropping to be from a ring

- necked phe a sant ( Phasianus colchicus ) , w as discovered in one of the treated field s . Additio nally, grey

- white droppings were seen on several BTPD entrance mounds and similar but green droppings were observed on some entrance mounds. Based on the droppings' size and consistency, I believe these droppings to be from a raptors such as the ferrugino us hawks in the fields (figure 3)

. No BTPDs were observed at the Rozol ®

- treated fields due to the weather (BTPDs are not active above ground when it is overcast and very windy ; Mr. Fred Raish, supervisor, YCPCD, personal communication). Field Visit 2 : The actual preliminary field study was conducted during the second field visit (March

- April, 2010)

. Study Site
Wo rk was conducted in a private pasture located along County Road CC, approximately 1.3 km (0.8 miles) south of Vernon, Yuma County, Colorado ( a ttachment
2 ). The BT PD colony on the pasture was scheduled to be poisoned with Rozol ® during the 2009

- 2010 application season by the YCPCD as part of their on

- going BT PD control program in Yuma County, Colorado. This site was identified as a potential st udy site and the landowner's permission for access onto the pasture was secured by the generous assistance of Mr. Fred Raish, supervisor, YCPCD . The BT PD colony in the pasture was divided into two locations . T he main colony occupied approximately 17 .3 ha (42.8 acres) of the pasture. The secondary part of the colony was composed of only a few BT PD burrows and was located approximately 0.6 km (0.4 miles) away from the primary colony.8 I focused the research on the primary colony. Hereafter the term ' stu dy site ' refers to the primary and secondary colonies and the term ' colony ' refers to the primary colony.
Methods Application : Rozol® was applied to the study site according to the label by certified pesticide applicators of the YCPCD

. Based on YCPCD 's calculations, approximately 231 kg ( 510 lbs ) of Rozol ® w ere applied, treating 4080 holes. Th e southern half of the colony was treated on March 15, 2010 and the northern half of the colony and the secondary colony were treated on March 16, 2010. Within the colony , the area used by
BTPDs contained low heavily grazed vegetation with patches of bare ground and was encircled by taller grasses and herbaceous vegetation

. A barbed wire fence formed the perimeter along t h ree sides of the colony ( figure 4 ). Since it took two days to complete the Rozol ® application ( March 15 and 16, 2010, ) , it can take at least a week for mortality to occur after exposure, and BTPDs in the southern part of the northern half of the colony could easily venture into the southern half of the colony and be come exposed to Rozol ® after the first application day , hereafter the two application days are combined as d ay 0 (day of application) . S ubsequent days of the study will be identified with respect to day 0. In general, I walked east

- west transects through the colony to search for evidence that may potentially create an exposure scenario to birds and to document the effects of Rozol ®

. Each search lasted 2

- 4 hrs and searches were conducted on 14 days over the 29 day post application period (table 1). The transects were about 3m (10 ft) apart in short vegetation but were closer in taller, dens er vegetation. Since the objective was to find evidence of poisoning and not to estimate the BT PD population , transect widths and rate9 of walking the transects were not restricted by rigid methodology. In general, transects were walked in late afternoon following t he Rozol ® label

. Information on the application, wildlife occurrence and behavior, signs of Rozol ® exposure to wildlife, and ad verse effects to wildlife were documented and appropriate samples were collected. Observations and sample collection s were conducted f or 14 days during the 29 day post

application study period. Observations : I recorded avian and non

- target mammal use of the colony and noted evidence of Rozo l ® exposure in BTPDs and other wildlife in the colony. Signs of
expo sure included mortality, morbi dity, discolored droppings, scavenging, possible blood stained soil, and a general change in the number of BTPDs and oth er wildlife following Rozol ® application. I also noted if Rozol ® was observed outside the burrow. In addition to making observations in the late afternoon, when my daily schedule allowed, I drove past the study site in late morning and early afternoon to observe avian predators and scavengers at the colony and BTPD activity . These observations were made while driving the car slowly along County Road CC in order to not frighten BTPDs in the pasture ; BTPDs ran into thei r burrows if I stopped the car. Sample c ollection and h andling : Rozol ® was collected from the applicator on day 0 for chlorophacinone analysis. Additionally, Rozol ® that was still visible either on the ground surface or in the burrow entrances was collected on day 27 post

- application for chlor ophacinone analysis

. These samples were collected by either a trowel or a long handled scooper. The collection was sifted through several different sized mesh to separate the Rozol ® from the soil and litter

. Dead and moribund BTPDs and non

- target wildli fe were collected for chlorophacinone residue analysis and for histopatholog y10 examination

. I also used l ive animal box traps, baited with oats, Cheerios®, peanut butter, and commercially available timothy hay (green hay), coated with molasses, peanut oil, and vegetable oil , either singly or in various combinations over the 29 day post application period but did not trap any BTPDs. Dark colored BTPD droppings were collect ed to dete rmine the occurrence of blood since chlorophacinone is an anticoagulant rodenticide

. Presence of blood will be determined with a phenophthalein presumptive blood test kit at the Patuxent Wildlife Research Center. All samples were stored in a freezer before shipping them to Patuxent Wildlife Research Center with the exception of a western meadowlark ( Sturnella neglecta ) which was placed in a refrigerator overnight and dissected the next day to harvest tissues for histopatholog y examination. Ten tissue samples from the skeletal muscle , heart, lung, kidney, spleen, brain, liver, and intestine were collected and placed in 10% neutral buffered formalin following protocol provided by Dr. Carol Meteyer, wildlife pathologist, USGS National Wildlife Health Center, Madison, Wisconsin. The tiss ues were shipped to Patuxent Wildlife Research Center, prepared into slides by American Histo labs Inc. , Gaithersburg, Maryland, and sent to Dr. Meteyer for examination.
Results Rozol® availability to wildlife : The angle of the BTPD burrow entrance in r elation to the tunnel appears to influence the availability of Rozol® from the surface (figure 5)

. After application, Rozol® was visible from the surface in burrows with shallow

- angle d
entrance s whereas no Rozol® could be seen from the surface in steep

- angled burrow
entrances

. Beginning day 2 post

- application , an increasing number of shallow

- angled burrows had Rozol® exposed into their entrances and on to the ground surface outside11 the ir entrance s by BTPDs performing burrow maintenance (figure 6 )

. By da y 27 post

application , fewer burrows displayed Rozol ® in their entrances and on the ground surface outside

. In late April, some of the wheat seeds from the Rozol ® bait had started germinating in burrow entrances. Sprouting seeds may be attractive to wild life. Avian species observed in the BTPD colony : In winter, BTPDs are a valuable food source for raptors and colonies of dead and dying BTPD s can attract flocks of raptors as documented in Field Visit 1 (Plumpton and Andersen 1997; Weber 2004; Kotliar et al.
2006 ; Mr. Carl Stogsdill, Stogsdill Ranch, personal communication ). Field V isit 2 was conducted after the wintering raptor flocks had begun migrating north and no raptor flocks were seen at the colony. However, f our species of r aptor s ( turkey vult ure, Cathartes aura , northern harrier, Circus cyaneus , r ed

- tailed hawk, and burrowing owl, Athene cunicularia ) were observed at the colony . A pair of burrowing owls, established territory in the colony and eventually nested in a Rozol® treated BTPD burrow

. Additionally, a pair of great horned owls ( Bubo virginianus ) were detected on a tree close to the colony at dusk

. Several resident and transient passerine species were observed using the colony for feeding and nesting. Western meadowlarks were seen in the taller vegetation in the colony during the early study period but later began foraging in the
BTPD

- grazed section of the colony

. Several pairs of horned larks established territories in the BTPD

- grazed part of the colony and often perched on BTPD moun ds. A Say's phoebe ( Sayornis saya ) was observed for few days using BTPD mounds as platform s for darting after insects. Large flocks of chestnut collard longspurs ( Calcarius ornatus ) and horned larks used the colony as a migratory stopover site . Several unidentified sparrows mainly perched on the fence along the southern boundary of the colony and flew into the12 colony to feed. Other birds using the BTPD colony included m ourning doves ( Zenaida

macroura ) and r ing

- necked pheasants that foraged in the colony and a pair of killdeer ( Charadrius vociferus ) that est ablished their territory in the grazed portion of the colony. Signs of Rozol® exposure and adverse effects : Carcass searches over 11 days recovered two intact t hirteen

- lined g round s quirrel s ( Spermophilus tridecemlineatus ), nine intact BTPDs, seven scavenged BTPDs and one intact western meadowlark ( table 1; figures 7,
8 , 9 )

. Several BTPD remains (for example, f eet , hide , head ) were also found but were not collected. All carcasses were found a bove ground. All intact BTPD carcasses were within 3 m of their burrow entrances. The first mortality, a thirteen

- lined ground squirrel was found on day 5 post

- application on BTPD mound located close to taller vegetation. The second thirteen

- lined squir rel was also collected from the taller vegetation in the colony on day 13 post

- application

. The first BTPD poisonings were obse rved on day 12 post

- application and included two intact carcasses, two scavenged car casses, and 2 moribund BTPDs (figure10). Ma ny of the dead and moribund BTPDs had brownish

- red coloration on their underside and under their tails (figure 11 ). A red

- tailed hawk and a northern harrier were observed scavenging on BTPDs on day 12 post

- application and turkey vultures were seen feeding on BTPD carcasses on days 23 and 24 post

- application (figure 1 2 )

. Most moribund BTPDs were seen close to their burrow entrances . The death of one moribund BTPD was documented on video. The BTPD was on its side with its legs moving in a cycling motion and displayed heavy breathing before dying. Signs of poisoning were observed up to the last day of the study, day 29 post

- applica tion

. A total of eight moribund BTPDs were observed. On day 29 post application, o ne moribund13 BTPD was captured in a net but died shortly afterwards (included in the nine intact
BTPDS collected above)

. One intact male western meadowlark carcass was found in the grazed section of the colony ( f i g u r e 9 )

. No overt signs of trauma were apparent (e.g. wounds or loose feathers) and no signs of external bleeding were observed. The meadowlark was dissect ed to harvest tissues for histopat hology examination and Rozol® residue analysis. Upon opening the carcass, the bird appeared to be healthy but the pectoral muscles displayed gross hemorrhag ing ( f i g u r e 1 3 )

. The gizzard and intestine were full ; t he gizzard contained insects parts and the intesti ne had a dark greenish liquid. The histopathology report documented abundant hemorrhage associated with the skeletal muscle and one focus of hemorrhage in the brain (Dr. Carol Meteyer, wildlife pathologist, USGS National Wildlife Health Center, Madison, W isconsin, personal communication). A green bird dropping , I believe it to be from a ring

- necked pheasant based on the dropping's size and texture and the presence of ring

- necked pheasants in the colony , was found on day 1 post

- application

. Another green bird dropping was encountered under the barbed wire fence on day 24 post

- application

. I believe this dropping to be from a western meadowlark based on its size and texture and the fact that meadowlarks were practically the only species that perched on that segment of the fence during Field Visit 2

. A third green dropping of an unknown avian species (probably a passerine) was also found in the colony (figure 1 4 )

. Other avian droppings appeared normal. Green and dark bl ack colored BTPD droppings were observed beginning day 2 post

- application and continued at least until day 29 post

- application . The black droppings were mostly seen14 stuck to the inside wall of burrows near the entrances or on the burrow mounds at the
entr ances (figure 1 5 )

. Two BTPD burrow mound s were stained and the staining went down into the tunnel entrance , possibly by blood (figure 1 6 )

. The BTPD population declined over the study period . Moribund BTPDs were still observed above ground on day 29 post

- application

. No ground squirrels were seen in the colony by day 29 post

- application

. Field Visit 3 : Transects as above were walked in June 2010 to locate affected animals and document breeding birds in the BTPD colony. Birds seen at the colony include: a pair of burrowing owls nest ing in a Rozol® treated BTPD burrow, western kingbirds ( Tyrannus verticalis ), horned larks, l ark buntings ( Calamospiza melanocorys ) , w estern meadowlarks, l ark sparrows ( Chondestes grammacus ) , and mourning doves. One nest with nestlings of a ground nesting passerine species was found and one egg , possibly of killdeer, was encountered. The surviving BTPDs appeared active and alert and had pups but n o ground squirrels were observed. Soil from a mound with possible blood was

co llected, along with burrowing owl castings and pellets and BTPD droppings to test for the presence of blood as described above

. Discussion : Implications of the findings Though the work described above was conducted as a preliminary study, the results document avian exposure and effects following operational application of Rozol ® for BTPD control. Rozol ® is a green colored product winter wheat based anticoagulant indandione rodenticide product

. Discolored (green) bird droppings impl y that birds have15 been exposed to Rozol ®

. Green raptor droppings were found in Rozol ® treated colonies during the field visit in January. The dead and dying BTPDs h ad attracted ferruginous hawks who in turn were exposed to Rozol ® via secondary poisoning

. A concentrated

a nd debilitated (easy) food source is highly attractive and becomes a magnet for scavengers ( see Vyas 1999 for review ; see Vyas et al. 2003 for review ; Chesser 1979)

. No raptors were seen at BTPD colonies on Fox Ranch but a flock of ferruginous hawks had congregated at the Rozol ® treated colony that was less than 32 km (20 miles ) away. The day I visited the Rozol ® treated colonies in January 2010, no BTPDs were seen above ground because of an overcast sky and strong winds. However, ferruginous hawks were seen soaring low over the BTPD colony, perched on the fields' perimeter barbed wire fence, and perched on the ground. These are typical hunting behaviors for ferruginous hawks on BTPD colonies ( Wakeley 1978 )

. These observation and reports from FWS reports show that ferruginous hawks are attracted to Rozol ® treated colonies because of the availabili ty of dead and moribund prey. The fact that these birds were present at a colony without above

- ground BTPD activity on the day I visite d and that they had not moved to an other location indicates that the hawks have preyed on BTPDs at this location in the recent past and expect to encounter BTPDs in the near future

. It is likely that the green stained raptor droppings are evidence of seco ndary poisoning to the hawks. My observations of a red

- tailed hawk, a northern harrier and two turkey vultures feeding on BTPDs at the study colony during the second field visit also demonstrate the importance of a con centrated food source to birds. Typi cally, BTPDs either sounded their alarm calls and ran to their burrows by the time I was approximately 15 m (50 ft) from them. However, the moribund BTPDs allowed me to approach them within 3 m16 before slowly raising themselves onto their legs and moving to wards their burrow. My being able to get so close to the moribund BTPD s and netting one of them implies that they are easy prey (Hunt et al. 1992)

. An unexpected finding is the exposure to songbird species. Traditional concerns about the risks of avian poisoning from Rozol ® have focused on raptors becoming exposed via secondary poisoning (Erikson and Urban 2004)

. However, my observations also show that other groups of birds are also exposed by i ngesting Rozol ® bait

. H orned lark droppings were found in BTPD burrow entrances of Rozol ®

- treated colonies but not in burrow entrances of the BTPD colony at Fox Ranch. Horned larks are birds of open country (Beason 1995) and in general , they would not be expected to go into an enclosed area such as a burrow but t he fact that their droppings were in burrow entrances in the Rozol ® treated colonies implies that these birds were attracted into the burrow entrances by Rozol ®

. The ir actual exposure to Rozol ® i s supported by the presence of green droppings found in the entrances

. The western meadowlark mortality during Field Visit 2 was also surprising

. While his liver has not yet been analyzed to confirm for Rozol ® residue, the gross pectoral muscle hemorrhaging and the hemorrhage in the brain, lack of evidence of trauma, injuries, and illness, and a gastrointestinal track with food, suggest anticoagulant poisoning. Furthermore, western meadowlarks primarily utiliz ed the barbed wire fence along the west boundary of colony for perching and singing and they were also the primary species which used the west fence. Birds flew from the fence into the grazed and ungrazed sections of the colony to feed. Based on these ob servations and the size and texture of the dropping found under the west fence, I speculate that the green dropping was from a meadowlark. The green dropping was discovered during the carcass17 search the day before recovering the dead meadowlark. The w est ern meadowlark s' diet consists of grain during winter and early spring, and insects in lat e spring and summer ( Davis and Lanyon , 2008 ). The upper gastrointestinal track of the meadowlark found during the study was replete with insect parts yet there is ev idence of rodenticide
exposure. This suggests that meadowlarks are attracted to Rozol®. Adverse effects to BTPDs and non

- target wildlife following operational application of Rozol ® were first documented in this study on day 5 post

- application and continu ed at least until my last day at the colony during Field Visit 2 (day 29 post

application ). The Rozol ® label states that the treatment period is "October 1 and March 15 of the following year, or before spring green

- up of prairie grasses, whichever occurs later, when animals will most readily take the grain bait" ( attachment 1 )

. The reason Rozol ® is to be applied before green

- up is to ensure that BTPDs feed on Rozol ® instead of the vegetation. In the current study, Rozol ® was applied on March 15 and March 16, 2010 and green

- up was e xpected later in the week (Mr. Fred Raish, supervisor, YCPCD, personal communication ; figure 1 7 )

. Despite competition from fresh vegetation over the course of Field Visit 2 , adverse effects to BTPDs and non

- target wildlife were observed in this study. Documentation of adverse effects to birds depends on how readily the effect can be detected. An observer is more likely to detect an adverse effect when discovering a mortality event than when co ming across a sublethally poisoned bird with no overt signs of stress. Additionally, the definitive nature of mortality requires no speculation about the consequences of the effect to the birds. Therefore mortality is the most commonly reported adverse e ffect for birds.18 A major obstacle to gathering evidence of avian poisonings following Rozol ® applications is that dead birds are difficult to find in the field. On a landscape level, avian mortality may be overlooked because of the vastness of the areas that are treated with Rozol ® and the spacio

- temporal mosaic of the applications. The size of the area s involved renders it impossible to monitor all locations subjected to Rozol ® applications

. The situation is further complicated by the fact that Rozol ® use occurs primarily on private properties, making the sites inaccessible for regular monitoring. On the local scale, a mortality even can also go undetected. The initial discovery of a carcass and the outcome of a subsequent search are moderated by the scavenger and predator populations, animal behavior, carcass morphology, habitat, vegetative structure, searcher experience , and search intensity. Rozol ® 's mode of action also is a critical variable for successful recovery of a carcass because 1) Rozol ® is not an acute toxin so bird mortalities would not be confined to treated locations, 2) Rozol ® is an anticoagulant that requires multiple feedings and death can occur 1

- 3 weeks after application, 3) birds are mobile and can die away from the treated locat ion. An avian carcass found away from the treated site may be discounted as resulting from Rozol ® use (see Vyas 1999 for review)

. Many mortalities that are discovered are not reported because of the public’s ignorance that the wildlife mortality should be reported and to whom it should be reported, fear of prosecution, camaraderie, procrastination, and apathy. When a mortality event is reported to the correct authorities, an investigation to determine the cause of death is not always immediately possibl e by the authorities due to distance, weather, terrain, private property issues, and other on

- going activities . Furthermore, because of19 limited resources, only one of two searchers may be able to respond and yet be required to search large areas, thereby limiting search intensity. An increasing interval between the onset of mortality and recovery of evidence reduces the chances of salvaging carcasses that would be suitable for laboratory analysis to determine the cau se of death because of scavenging , deco mposition, and c hemical degradation (see Vyas 1999 for review)

. Mortality reports confirm the hazard s from the operational use of Rozol ® to birds, however the magnitude of the effects is not known. In general, t he percent of

carcasses that are detected, reported, and confirmed for the cause of death is small but it provides invaluable information on the hazards to birds. On the landscape level, these

carcasses strongly imply that operational Rozol ® applications result in avian mortalities and on a local level, the evidence state that the actual number of avian losses per mortality event typically exceeds the number of carcasses recovered. I believe, in the current study , the recovery of only a single avian carcass to be an artifact primarily of breaks in the days I conducted carcass searches during the 29 days post

- application o f the study. In light of the obstacles in finding carcasses, it would not be unreasonable to assume that more birds may have died from Rozol ® application in the study colony.
Re lating the findings to t he pesticide label The pesticide label is considered the law for directing the pesticide's use. The findings reported here raise concerns regarding the adequacy of t he Rozol® label t o ensure safety to birds ( a ttachment 1 )

. 1. Th e label states: "Apply...bait at least 6 inches down active prairie dog burrows. Do not apply bait on or above ground level. Make sure no bait is left on the20 soil surf ace at the time of application. Applicator must retrieve and dispose of any bait that is spilled above ground or placed less than 6 inches down the burrow entrance. " First, t he angle of the BTPD burrow entrance in relation to the tunnel appears to influence the availability of Rozol® from the surface. During Field Visits 1 and 2, I notice d that, though applied 6" down the tunnel, Rozol® was visible from the surface in burrows with shallow

- angled entrance s . This may be an artifact of the application equipment used to place Rozol® in the burrow. The bend in the tunnel may inhibit the application equipment from reaching deeper into the burrow (figure 5). C onsequently birds are able to reach the Rozol®

. Second, as part of their burrow maintenance, BTPDs kick the Rozol ® out of burrows with shallow

- angled entrance s onto the ground su rface. The fact that Rozol ® was placed on a shallow entrance ramp may allow the BTPDs to kick it out while maintaining their burrows (figure 5)

. Rozol ® was seen on the ground surface beside the burrow entrance s and in the burrow entrance s during Field V isits 1, and 2. Rozol ® on the ground surface as a result of continuous burrow maintenance and not misapplication, was observed b eginning day 2 post

- application through day 29 post

- application during Field Visit 2. The label's requirement that Rozol ® be p laced in the burrow to minimize surface availability is undermined by BTPD behavior. 2. The label states: " The applicator must return to the [application] site within 5 to 10 days after bait application to collect and properly dispose of any bait or dead or dying prairie dogs that may have come to the surface. The applicator must also return to the site 14 to 21 days after bait application to collect and properly dispose of any additional bait or dead or dying prairie dogs that may have come to the surface

. Bury carcasses on site21 in holes dug at least 18 inches deep or in inactive burrows

... to avoid non

- target animal scavenging. Burial includes covering and packing the hole or burrow with soil". First, the label requires the applicator to return to the application site and collect and dispose bait or dead or dying prairie dogs that may have come to the surface in order to minimize secondary poisoning of scavengers. The label, however, does not specify
h ow many times the applicator must return to the site within 5 to 10 days and during 14 to 21 days post

- application . In the current study, dead and moribund BTPDs were documented on each day that a carcass search was conducted starting on day 5 post

applic ation . Therefore if the applicator does not visit the application site daily for at least one month post

- application and remove carcasses and the exposed Rozol ® , any protection provided to birds by the Rozol ® label is considerably diminished

. Second, t he label does not specify how the search for spilled and surfaced Rozol ® and dead and moribund BTPDs must be conducted in order to ensure that the label requirements are satisfied. Currently, the label requirements could be fulfilled by scanning the area wit h binoculars from the field perimeter or by conducting the search while driving a vehicle through the colony . With either method or with similar methods, the applicator will not be able to satisfy the intent of the label requirement (Vyas 1999). To satis fy the label, searchers need to walk at a moderate pace in a systematic method to ensure coverage of the entire area. Third, t he label does not instruct the applicator as to what must be done with dead or moribund non

- target wildlife at the treated site. These poisoned animals are also a haz ard to predators and scavengers. Additionally, the label provides no information for reporting non - target mortalities to state and federal wildlife authorities.22 Fourth, the label recommends disposing of the carcasses by burying them in holes and in inactive burrows. However, scavengers such as the North American badger s ( Taxidea taxus ) have a highly developed sense of smell and are powerful diggers and would be expected to reach the BTPD carcass. Additionally, BTPD burrows may have more than one entrance so plugg ing one entrance will not exclude fossorial scavengers from reaching buried BTPD carcasses.
3. The label states: " Collect and bury carcasses in late afternoon near sundown, to reduce the potential of scavenging animals finding carcasses. " First, I observed raptors feeding on BTPDs in late morning, and in early and late afternoon. The above label statement may lessen the availability of poisoned prey for nocturnal animals, but it does not necessarily reduce the chances of secondary poisoning for diurnal avian predators and scavengers. Second, because mortality from Rozol ® may occur over several weeks, BTPDs who have been exposed to Rozol ® and are still active above ground, remain a source for second ary poisoning to diurnal avian predators. While the label tries to reduce hazard to

non - target wildlife from dead and dying BTPDs and from Rozol ® that has surfaced, the label does not address reduction of hazard created by contaminated yet active BTPDs

. T he current label requirements for Rozol® regarding BTPD carcass re moval by the applicator are laborious but still fall short of mitigating for the risks from Rozol® to federally protect avian species under the auspices of the Migratory Bird Treaty Act. At a recent meeting between the Director of the Division of Registration of the Office of Pesticide Programs of the EPA and Rozol® applicators in North Dakota , it was brought to light by the applicators that the carcass removal requirements as stated on th e current23 label are, in addition to being laborious, unrealistic and therefore label non

- compliance is commonplace (Mr. Scott Larson, FWS Region 6, personal communication)

. I am aware of similar label non

- compliance in two other states. D ead and dying animals can be quickly removed by predators and scavengers (Vyas 1999; Vyas et al. 2003). If the label requirements for returning to the application site for removing BTPD carcasses are interpreted by applicators as requiring only one visit within 5 to 10 days and during 14 to 21 days post

- application , the interval between application and carcass collection will be too great. Consequently, many BTPD and other carcasses will be missed during searches, thereby reducing the perception of the hazard but not t he actual poisonings

. Since the label requirements for carcass removal address only BTPDs, applicators returning to the application site would confine their search to the treated site . Since it may take an animal 1

- 3 weeks to die following a Rozol® appli cation, a highly mobile

non - target species such as birds can die away from the BTPD colony and thereby be missed in any effort to document Rozol® 's hazards to birds. The EPA finalizes and approves the pesticide label that will be placed on the product. T he function of a pesticide label is not just to provide information on application rates, times, and methods but also to provide this information to minimize environmental harm. Based on the findings of this study thus far, it would not be unreasonable to state that the current Rozol® l abel is inadequate in provid ing proper safety to federally protected birds. Acknowledgements I thank the US FWS Region 6 for their support in conducting this study especially Kevin Kritz, Kimb erly Dickerson , Joy Gober, Scott Larson , Matt Schwarz , and Larry Gamble . I24 also thank Fred Raish and the staff of the Yuma County Pest Control District for their assistance. I am grateful to William Burnidge and Chris Pague of The Nature Conservancy for their assistance. I express my gratitude to Dr. Carol Meteyer, USGS, for helping me with the tissue collection protocol and conducting the histopathology examination of tissues , and Josh Melby, Colorado Division of Wildlife, for guidance and for providing BTPD carcasses. I also sincerely acknowledge Nathan Andrews, Laura Quakenbush of the Colorado Division of Agriculture, the staff of the PWRC, especially John B. French, Barnett Rattner, Gary Heinz, and Kinard Boo ne for their help and guidance.25 Literature Cited : B eason RC . 1995. Horned Lark ( Eremophila alpestris )

. The Birds of North America Online (A. Poole, Ed.). Ithaca: Cornell Lab of Ornithology; Retrieved from the Birds of North America Online: h ttp://bna.birds.cornell.edu/bna/species/195doi:10.2173/bna.195 Chesser, RK. 1979. Opportunistic feeding on man

- killed prey by ferruginous hawks

. The Wilson Bulletin

. 91 : 330

- 331

. Davis SK and Lanyon WE. 2008. Western Meadowlark ( Sturnella neglecta )

. The Birds of North America Online (A. Poole, Ed.). Ithaca: Cornell La b of Ornithology; Retrieved from the Birds of North America Online: http://bna.birds.cornell.edu/bna/species/104doi:10.2173/bna.104 Derner JD, Detling JK, Antolin MF. 2006. Are livestock weight gains affected by black

tailed prairie dogs? Front iers in Ecol ogy and the Environment 4: 459

- 464. EPA. 1998. Rodenticide Cluster. Reregistration Eligibility Decision

. Environmental Protection Agency. Washington, DC. Available at: http://www.epa.gov/oppsrrd1/REDs/2100red.pdf Erikson W and Urban D
. 2004. Potential risks of nine rodenticides to birds and nontarget mammals: a comparative approach , United States Environmental Protection Agency. h ttp ://www.regulations.gov/search/Regs/home.html#documentDetail?R=0900006 4800b7862 Forrest SE and Luchsinger JC. 2006. Past and current chemical control of prairie dogs. in Saving North America's Western Grasslands , Hoogland J. Ed. Island Press, Washington, DC, p p. 115

- 128
H oogland JL

. 2006. Why have so many prairie dogs disappeared? in Saving North America's Western Grassl ands, Hoogland J. Ed. Island Press, Washington, DC, pp. 89

- 93

. Hunt KA, Bird DM , Mineau P , and Shutt L. 1992. Selective predation of organophosphorus

- exposed prey by American kestrels. Anim al Behavio u r. 43:971

- 976. Kotliar NB, Miller MJ , Reading RP , Clark TW . 2006. The prairie dog as a keystone species. in Saving North America's Western Grasslands, Hoogland J. Ed. Island Press, Washington, DC, p p 53

- 64. Lamb BL, Reading RP, Andelt WF. 2006 Attitudes and perceptions about prairie dogs. in Saving N orth America's Western Grasslands, Hoogland J. Ed. Island Press, Washington, DC, p p 108

- 114.26 Plumpton DL and Andersen DE. 1997. Habitat use and time budgeting by wintering ferruginous hawks. The Condor. 99:888

- 893. Vyas NB. 1999. Factors influencing t he estimation of pesticide

- related wildlife mortality. Journal of Toxicology and Industrial Health. 15:186

- 191. Vyas NB, Spann JW , Albers E , and Patterson D

. 2003. Pesticide

- laced predator baits: considerations for prosecution and sentencing. Environmen tal Lawyer. 9: 589

- 608. Wakeley JS. 1978. Hunting methods and factors affecting their use by ferruginous hawks. Condor

. 80:327

- 333. Weber DA. 2004. W inter raptor use of prairie dog towns in Denver, Colorado vicinity. in Proceedings of the 4th International Symposium on Urban Wildlife C onservation Symposium. Shaw WW, Harris LK, Vandruff, LE

. Ed s. May 1

- 5, 1999. Tuscon, Arizona. pp. 195

- 199. Zinn H and Andelt W . 1999. Attitudes of Fort Collins, Colorado r esidents t oward p rairie d ogs. Wildlife Society Bulletin. 27:1098

- 1106.27 Table 1. Timeline of observations and sample collection following Rozol® application. Day 0

- Rozol® application.

- Rozol® collected. Day 1

- Green colored gamebird dropping found. Day 2

- Rozol® being kicked out of burrows.

- Green colored BTPD droppings found. Day 3

- No searches conducted due to weather. Day 4

- Nothing remarkable observed. Day 5

- Green and black colored BTPD droppings.

- 1 dead intact thirteen

- lined ground squirrel collected. Day 6

- Day 11

- No searches conducted due to weather. Day 12

- 1 red

- tailed hawk and 1 northern harrier seen feeding on dead BTPDs.

- 2 intact dead BTPDs collected.

- 2 scavenged BTPDs collected.

- 2 moribund BTPDs observed. Day 13

- 1 red

- tailed hawk and 1 northern harrier overhead, hazed by my presence.

- 1 intact dead thirteen

- lined ground squirrel collected.

- 2 intact dead BTPDs collected.

- 1 scavenged BTPD collected.

- 1 moribund BTPD observed.

- Possible blood stains found at burrow entrance.

- Several BTPD carcass fragments observed. Day 14

- Day 22

- No searches conducted: trap acclimation period. Day 23

- 1 turkey vulture observed scavenging on a BTPD.

- No searches conducted: trap setting. Day 24

- Green colored meadowl ark dropping found under west fence.

- 2 turkey vultures observed scavenging on BTPD.

- 2 intact dead BTPDs collected.

- 2 scavenged BTPDs collected.

- 1 moribund BTPD observed.
Day 25

- 1 intact dead western meadowlark collected.

- 1 scavenged BTPD collected. Day 26

- 1intact dead BTPD collected.

- 1 scavenged BTPD collected.

- 1 moribund BTPD observed.

- Green colored passerine dropping observed. Day 27

- 1intact dead BTPD collected.

- Rozol® collected.

- BTPD droppings collected.

- Soil from burro w entrance with possible blood stains collected. Day 28

- No searches conducted due to weather. Day 29

- 1 moribund BTPD collected.

- 2 moribund BTPDs observed.28 Attachment 1. Rozol® label.29 Attachment 2. Preliminary study site location.30 Figure 1

. One of the f er rugi nous hawk s over a Rozol ® treated BTPD colony during Field Visit 1. No BTPDs were above ground because of the weather.

Figure 2 . Rozol® .31 (a) (b) (c) Figure 3. Bird droppings at a Rozol®

- treated colony: (a) normal colored horned lark droppings in burrow entrance, (b) few green colored horned lark droppings along with normal horned lark droppings in bu rrow entrance

. Rozol® is visible from the surface in images (a) and (b). ( c) green colored raptor dropping.

Figure 4. Study colony. Grazed by BTPD (foreground) and un

- grazed habitats.32 ( a) (b)

(c) Figure 5. Possible influence of BTPD burrow entrances in relation to their tunnels, Rozol® placement and Rozol® availability to birds: (a) burrow with steep

- angled en trance (b) burrow with a shallow

- angled entrance. Green arrows and shape depict Rozol® placement and Rozol® location after BTPDs kick it out of the burrows. (c) the e quipment used during Rozol® application at the study colony include d a rigid pole with a n attached cup.33 Figure 6. Examples of Rozol® in burrow entrances and above ground.34 Figure 7

. Thirteen

- lined ground squirrels (top two photos) and some of the BTPD mortalities recovered intact. All intact BTPD carcasses were found near burrows.35 Figure 8. Some of the scavenged BTPDs found during carcass searches. The first photo shows a BTPD that was snagged on the fence as northern harrier , who was feeding on the BTPD at the study colony, f lew off with the BTPD but the prey's weight forced her to fly low and the BTPD did not clear the fence.36 Figure 9. Western meadowlark carcass detected in the study colony.

Figure 10. One of the moribund BTPDs from the study colony.

Figure 11. Many of the dead and moribund BTPDs had brownish

- red coloration on their underside and un der their tails . Similar discoloration in Figure 10.37 Figure 12. A turkey vultures scavenging a BTPD on day 23 post

- application.

Figure 13.Western meadowlark recovered from the study colony displaying hemorrhaging on the pectoral muslces.38 Figure 14. Two of the green (Rozol®

- exposure) avian droppings found in the study colony.

Figure 15

. Dark black BTPD dropping s were seen starting day 5 post

- application and continued to be found at least until day 29 post

application.39 Figure 16. BTPD mound stained possibly by blood.

Figure 17. View of the vegetation in the study colony on day 0. Green

- up expected later in the week.