# United States Department of the Interior

FISH AND WILDLIFE SERVICE
Ecological Services 420 South Garfield Avenue, Suite 400

Pierre, South Dakota 57501

- 5408



### August 22, 2011

MEMORANDUM

SUBJECT: Review and Management Recommendations for study titled “ Retention time of chlorophacinone in the tissues of black

- tailed prairie dogs e x posed to chlorophacinone bait ”

FROM: Matt Schwa rz, Environmental Contaminants Specialist, South Dakota Ecological Services Field Office , and Joy Gober, Fish and Wildlife Biologist, Natural Resource Program Center, Colorado

THRU: Scott Larson, Field Office Supervisor, South Dakota Ecological Services
F ield Office

TO: Kevin Johnson, Regional EC Coordinator, Region 6 Office

Attached is the final report and management recommendations for the Off

- Refuge Environmental Contaminants Investigation titled “ Retention time of chlorophacinone in the tissues of b lack

- tailed prairie dogs exposed to chlorophacinone .” The main component of the investigation included a laboratory study performed by the U.S. Department of Agriculture’s National Wildlife Research Center (Center) in Fort Collins, Colorado. The South Da kota Ecological Services Field Office has reviewed the study report submitted by the Center (Witmer, 2011) and has prepared comments and management recommendations in light of the study findings.

Witmer (2011) provides valuable information on prairie do g chlorophacinone excretion, residue concentrations, and observed effects from toxicity; however, the limited prairie dog exposure to chlorophacinone bait in the current study underestimates what would occur during a field application. For reasons specifi ed in our enclosed review, the amount of bait (53 grams each) and time of exposure (1

- 2 days) for the lab study are much lower than what would be expected for a field application.

Concentrations of chlorophacinone in prairie dog tissues from the current study exceeded those previously reported for common v oles ( Vidal et al., 2009 ) but were lower than those previously reported for prairie dogs carcasses retrieved 11 days after a field application ( Primus, 2007 )

. Study results also indicate that chloropha cinone is metabolized over time so that death may occur after the parent compound is nearly gone. Therefore, “trace amounts” of chlorophacinone , as

### 2

reported previously in raptor diagnostic necropsy examination reports, s hould be considered as acceptable e vidence that rodenticide exposure may have attributed to untimely death

. Furthermore the behavioral incapacitation and lethargy effects of chlorophacinone may result in raptors being more susceptible to being shot or hit by vehicles or succumbing to injur ies that would typically be considered non

- life threatening (e.g., territorial infighting or capture of prey).

The primary weakness of the current study is that it underestimates the amount of bait available, and the length of time bait is available, compared to field applications. Although three prairie dogs were offered ad libitum Rozol for two days (T2 group), valid conclusions cannot inferred from the T2 group due to small sample size, the limited 2 days of ad libitum availability and variability in the amount of bait consumed.

F urther field evaluations are needed to evaluate concentrations of chlorophacinone in prairie dogs exposed during actual field applications to supplement less robust assessments that are currently available

. However, resul ts of the current study combined with knowledge from other studies and field observations indicate that chlorophacinone residues in prairie dogs are elevated enough to cause concern for secondary poisoning of non

- target avian and mammalian species. Therefo re, we recommend that future assessments focus more on the effects of chlorophacinone toxicity to non

- targets that consume poisoned prairie dogs. More studies are needed to determine how sub

- lethal exposure may result in decreased non

- target reproduction and survival.

References

See enclos ureReview of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### U.S. FISH AND WILDLIFE SERVICE

DIVISION OF ENVIRONMENTAL QUALITY

REGION 6



## R ETENTION TIME OF CHL OROPHACINONE IN THE TISSUES OF BLACK



## - TAILED

PRAIRIE DOGS EXPOSED TO CHLOROPHA CINONE BAIT

Final Report

Region 6
DEC ID : 20096000 3
FFS: 6F56 Congressional District : SD00

Prepared August, 2011, by:
Matthew S. Schwarz 1 Joy Gober 2

1 U.S. Fish and Wildlife Service Division of Environmental Quality
South Dakota Field Office Pierre, South Dakota 57501

2 US Fish & Wildlife Service Natural Resource Program Center 1201 Oakridge Drive, Suite 320 Fort Collins, CO 80525Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### Reviewer’s Comments

The current study (Witmer, 2011) was funded by the U.S. Fish and Wildlife Service through the Environmental Contaminants Program as an Environmental Contaminants Investigatio n. The objectives of this investigation were:

1) To determine chlorophacinone tissue residues in prairie dogs at incremental time periods post

- exposure from a limited feeding

.

2) To determine potential exposure of predators from consuming prairie dogs
killed by chlorophacinone at incremental time periods post

- exposure.

The current study provides data on Objective 1 : however, the limited exposure of bait to prairie dogs (i.e., short feeding duration and limited amount of bait) is not reflective of actual prairi e dog consumption rates when chlorophacinone bait is used in the field as a
rodenticide. T herefore, while inferences can be made towards Objective 2

, they are limited because the current study was not designed to replicate how prairie dogs forage on poiso ned bait in the field. We recognize that to examine the issue of secondary poisoning of predators and scavengers from consumption of poisoned prairie dogs, it was necessary to start with a controlled exposure experiment that could provide levels of
chloro phacinone in various prairie dog tissues at specific time intervals post exposure. However in doing so, study O bjective 2 was not fully satisfied because the limited feeding duration in the current study underestimates exposure that may occur from
repeat ed small daily doses consumed during an actual field application of chlorophacinone to control prairie dogs. Prairie dogs that feed on chlorophacinone bait over several days following a field application would likely have higher tissue residues
than those in the current study. Secondary exposure risk to prairie dog predators would also be expected to occur for a longer duration and at higher uptake levels than the current study results suggest.

Chlorophacinone bait availability is expected to be greater a fter a field application to control prairie dogs than in the current study. In the current study, prairie dogs in group T1 were provided bait for two consecutive days (53 grams total) and excess bait was removed. Prairie dogs were then either euthanized at predetermined time intervals or maintained on a clean diet for up to 25 days. For field applications, the Rozol label recommends a dose of 53 grams per active burrow and there are on average 3.9 active
burrow

- entrances per black

- tailed prairie dog (Big gins et al., 1993). Inactive burrows may also be mistakenly baited resulting in further bait availability. Furthermore, there is no effort to remove excess bait or provide clean food during field applications and thus prairie dogs may continue to consume bait after they have accumulated a lethal dose. AsReview of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### noted on page 35 of the report, it has been observed that “animals continue to feed on the baits for several days, then become lethargic and eventually stop feeding." Lee and Hygnstrom (2007) reported f inding prairie dogs carcasses 10

- 25 days after field applications.

Observations during the current study also indicate that prairie dogs were exposed to less bait than what would typically occur during a field application. For example, only 3 of 36 po isoned prairie dogs died and only 9 showed signs of being poisoned

. The current study also reported that only 56 percent of T1 animals showed evidence of hemorrhaging. Although first generation indandiones (i.e., chlorophacinone and diphacinone) can caus e mortality without showing hemorrhaging in mammals, the low number of dead and morbid prairie dogs in the current study indicates a lower dose was received than reported elsewhere for field applications. For example, Lee and Hygnstrom (2007) reported

pra irie dog population reductions between 85

– 96 percent following field applications of chlorophacinone.

Previous reports (Lee and Hygnstrom, 2007; Primus, 2007) that include chlorophacinone residues in prairie dog tissues after a field application also s uggest that prairie dogs in the current study consumed less bait than what would likely occur under a field application. The current study reported a mean concentration of chlorophacinone in liver of 0.82 ± 0.70 micrograms per gram (μg/g) in prairie dogs euthanized 11 days after bait (0.005% chlorophacinone) was first presented. In comparison, eight prairie dog carcasses collected 10 to 25 days after a field application of similar bait had a mean concentration of chlorophacinone in liver of 2

. 19 ± 1.8 0 μg /g (Lee and Hygnstrom, 2007; Primus, 2007). E ight additional prairie dog carcasses collected from other non

- experimental field sites (dates of application unknown) reported a mean concentration of chlorophacinone in
liver of 5. 86 ± 1.88 (Primus, 2007). R esidues in live rodents are expected to be greater than in their carcasses, especially for relatively short

- lived, chronic rodenticides such as diphacinone and chlorophacinone as the rodent may continue to consume bait above a
lethal dose and close to the onset of morbidity . The longer the lag time (between exposure and death), the more time is available for the target rodent to continue consuming bait. Therefore, chlorophacinone residues in live prairie dogs could be substantially higher than those in carcasses , but further study is needed to predict residue levels in live prairie dogs that have undergone multiple feedings of poisoned bait.

Repeated small daily doses of anticoagulants are also known to result in higher toxicity than a single acute dose (Godfrey et al., 1981; Jackson and Ashton, 1992). For example, the median lethal dose (LD50) from a single exposure of chlorophacinone to Nor way rats ( Rattus norvegicus ) is 20.5 milligrams per kilograms (mg/kg) whereas a 5

- day daily dose LD50 is twenty times lower at 0.95 mg/kg (Jackson and Ashton, 1992). We expect thatReview of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### the difference in toxicity between a single acute dose and repeated daily doses would also apply to non

- target species that feed on prairie dogs. Additionally, the extended period of
1 to 4 weeks between application of poisoned bait and observed death s of prairie dog s ( Vyas, 2010, Lee and Hygnstrom, 2007 ) extends the time durin g which prairie dogs are available to non

- target predatory animals.

Results from the current study indicate that tissue concentrations of chlorophacinone were highly variable among individuals necropsied on the same day (not evident in Figures 3 and 4 but note standard deviations in Table 1). Silberhorn and others (2003) also reported large variations in residues in individual ground squirrel carcasses even for those squirrels that died on the same day. All the prairie dogs in the current study were

expos ed at the same time so the variability may be due to individual differences in the amount of bait consumed, size, and metabolism and excretion of chlorophacinone. Due to variability in individual residue loads, it is recommended that future assessments
in clude at least 10 individuals collected on the same day post application.

Toxicity to chlorophacinone among individuals also appeared to be highly variable and not necessarily related to the amount of bait consumed. Observational data on prairie dog

re sponse after consuming the maximum amount of bait (53 grams) ranged from appearing normal to severe incapacitation and death. Animal health logs indicate that the three prairie dogs found dead had appeared normal during prior observation checks and only o ne showed signs of external bleeding. Variability in the susceptibility of target species to chlorophacinone toxicity may also apply to non

- target species and should be
further evaluated.

Although chlorophacinone residues in the current study likely under estimate exposure from field applications, dead and euthanized prairie dogs still had residue levels that were high enough to warrant concern for secondary exposure to non

- target birds or mammals.

Non

- target species that feed on prairie dogs would be expo sed to chlorophacinone in
whole

- body and liver tissues. In the current study, prairie dogs had concentrations of chlorophacinone in whole

- body and liver that ranged from 0.053

- 1.78 μg/g and 0.061

- 8.407 μg/g, respectively. Liver chlorophacinone concen trations exceeded those previously reported for common voles (0.082

- 3.800 μg/g) but secondary exposure risk to vole predators was not evaluated (Vidal et al., 2009). Whole

- body concentrations in prairie dogs from the current study were also greater than those estimated in laboratory rats (0.18

– 0.81 μg/g) that resulted in the death of 11 of 20 domestic ferrets when fed upon for five consecutive days (Ahmed et al., 1996 as cited by Erickson and Urban, 2004).Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### Observations from the current study indicate that chlorophacinone is metabolized over time so that death can occur after the parent compound is nearly gone. Prairie dogs that lived the longest but eventually were euthanized based on condition had liver chlorophacinone levels similar to "trace amoun ts" reported for wildlife mortality incident investigations. For example, two prairie dogs (KQ

- 18 and KQ

- 26) ate 36 and 53 grams of bait during the first two days, respectively. They were then euthanized due to their condition on days 22 and 26 and had l iver concentrations of chlorophacinone of 0.265 and 0.187 μg/g, respectively. These concentrations are similar to those previously reported as "trace" amounts of chlorophacinone (e.g., 0.25 μg/g) in wild raptors opportunistically found. For example, a ba ld eagle found near a chlorophacinone poisoned prairie dog town in Nebraska had a liver concentration of 0.3 μg/g chlorophacinone and forensic necropsy results indicated that the eagle died from chlorophacinone ingestion (USFWS, 2007). Chlorophacinone was also detected at 0.18 μg/g in a red

- tailed hawk from New York State (Stone et al., 2003). Other raptors, for which chlorophacinone exposure may have contributed to death, include a ferruginous hawk and great

- horned owl, both collected from Kansas with “t race amounts” of 0.25 μg/g chlorophacinone (USFWS, 2009).

The current study indicates that lethargy can persist in poisoned prairie dogs for several days before they die or need to be euthanized based on morbidity. Despite only a single
exposure to rozo l bait, many of the prairie dogs suffered from delayed incapacitation. For example, prairie dog KQ26 was lethargic for 16 days starting 10 days after exposure and was euthanized on Day 26 due to poor condition. Incapacitation in these animals
occurred de spite receiving a clean maintenance diet post exposure to rozol. Prolong ed lethargy would likely result in increased susceptibility to predation and these same prairie dogs that are more easily captured by predators may present the highest risk of seconda ry exposure if they continue to eat chlorophacinone after receiving a lethal dose and thus accumulate higher tissue residues.

The current study included a T2 group of three prairie dogs that were provided chlorophacinone bait ad libitum for two days. V alid conclusions cannot be made from this T2 group. The small sample size of this group (n = 3) and high variability in both the amount of bait consumed per individual (i.e., range of 7.0

- 54.6) and tissue concentrations (see Table 2B) preclude statistic al analysis. The prairie dog that consumed only 7.0 g of bait had the lowest tissue concentrations of chlorophacinone in the T2 group and ingested much less bait than any other prairie dog in either treatment group (the next lowest was 27.5 g of bait cons umed), leading to the question of whether some other factor was affecting this test animal. Furthermore, the time period of two days for ad libitum exposure is less than what would be expected in a field application.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### We do not agree with the current stu dy conclusions that “the highest risk of secondary exposure to chlorophacinone residues by non

- target animals consuming prairie dogs exposed to the bait would occur within a few days after bait application and would drop quickly thereafter.” As specified above, the current study is not representative of prairie dog exposure to chlorophacinone from a typical field application and prairie dogs that continue to consume bait after they have accumulated a lethal dose may have the highest chlorophacinone tissue residues. This would result in risk of secondary exposure to non

target animals over a more extended time period.

The current study also suggests that “because birds are less susceptible to chlorophacinone poisoning than mammals, secondary risks are pro bably higher for predatory or scavenging mammals (coyotes) than for predatory birds” and based this conclusion on a review by Primus and others (2001). The risk assessment by Primus and others (2001) did not evaluate sub

- lethal effects leading to indirect mortality, which is our greatest concern regarding avian consumption of chlorophacinone poisoned prairie dogs.
Sub

- lethal effects have been documented in raptors exposed to anticoagulants and can occur despite low tissue residue concentrations. For exam ple, American kestrels ( Falco sparverius ) administered diphacinone and with liver residues just above the diphacinone
method detection limit s of 0.263 and 0.280 μg/g diphacinone had histological evidence of hemorrhage in lung and liver (Rattner et al., 201 1a). Golden eagles ( Aquila chrysaetos ) fed muscle from diphacinone

- treated sheep exhibited extreme weakness, hemorrhages, and ataxia (Savarie et al., 1979). These studies indicate that raptors are susceptible to indandione’s multiple modes of action which include both the blocking of prothrombin formation and the uncoupling of oxidative phosphorylation (Van Den Berg and Nauta,
197 5 ). Ample evidence exists to indicate that avian predators and scavengers are susceptible to secondary toxicity risks and additional study is needed to further evaluate
the issue.

Management Recommendations

More data that are representative of field conditions are needed to adequately evaluate Objectives 1 and 2. We recommend a more robust assessment of chlorophacinone
resi dues in prairie dogs that mimics operational application exposures of chlorophacinone bait. The assessment is needed to determine residues in prairie dogs that receive repeated small doses of chlorophacinone and should include at least 10 individuals that are euthanized as soon as they exhibit signs of lethargy or morbidity.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### Studies indicate that avian lethality tests required to register first generation indandione rodenticides can result in toxicity values that ultimately underestimate risk and that ne w test requirements are needed. Standardized tests for avian lethality that are required by the U.S. Environmental Protection Agency (USEPA) to support pesticide registration include the single

- dose acute oral toxicity test and the five

- day sub

- acute diet ary toxicity test that are used to derive an LD50 and median lethal concentration (LC50), respectively (USEPA, 2007). First generation indandione rodenticides have a mode of action that results in cumulative effects over several days of feeding

, thus the required single

- dose acute oral toxicity test tends to result in large LD50s values that ultimately underestimate risk ( Ashton et al., 1986; Jackson and Ashton, 1992)

. The standardized five

- day sub

acute dietary toxicity test includes multiple exposures ov er several days but has little value as a quantitative descriptor of lethal toxicity and is more of a measure of vulnerability to a contaminated diet, with results that can be highly dependent on a species willingness to eat the bait and ability to cope wi th reduced nutriment (Hill, 1993; Mineau et al., 1994; Hoffman, 2003). Studies that do not follow required methodologies for registration but provide supplemental information, such as the previously mentioned
five

- day sub

- acute oral toxicity tests (Godfre y et al., 1981; Jackson and Ashton, 1992), indicate that a repeated low dose oral sub

- acute toxicity test for anticoagulant rodenticides can result in a more toxic LD50 than a single

- dose acute oral test. Likewise, a dietary toxicity test that measured th e diphacinone

- treated diet consumed daily by
Eastern screech

- owls ( Megascops asio ) found that repeated low

- dosage exposure over seven days increased diphacinone toxicity by more than an order of magnitude compared to an acute oral toxicity test (Rattner et al., 2011b; N. Vyas pers comm.). These studies indicate a need to change current required avian oral and dietary lethality tests for first generation indandione rodenticides to include multiple day low

- dose exposures that measures individual daily dosage . Factors associated with extrapolating laboratory derived risk quotients to the field can further underestimate risk (Matz et al, 1998; Vyas et al., 2006), and this may be especially true when considering the sub

- lethal effects from first generation inda ndione rodenticides. Th us , methods for the lethality tests should also be expanded to include observational periods for sub

- lethal effects and protocols that include gross pathology and histopathological examination of tissues to evaluate internal
hemorrh aging. The USEPA has the responsibility and authority under the Federal Insecticide, Fungicide, and Rodenticide Act to determine the potential of a pesticide to cause adverse effects and require further testing when needed (USEPA, 2007). We
recommend tha t USEPA develop new standardized testing requirements for first generation indandione rodenticides and require additional long term field studies to allow for a more adequate determination of whether continued registration approval is
warranted for use of f irst generation indandione rodenticides to control prairie dogs.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### Active surveillance is needed to further examine the extent of non

- target mortalities from the use of anticoagulant rodenticides to control prairie dogs. Lee and Hygnstrom (2007)
included searchers for non

- target carcasses on and immediately around baited plots while performing field assessments designed to assess the efficacy of chlorophacinone and did not report any indications that avian non

- targets were adversely affected from feeding on poisoned prairie dogs. However, recovery of poisoned raptors from baited prairie dog downs is expected to be highly unlikely given the chronic nature of chlorophacinone that allows wide ranging birds to move away from the site of application.

Additio nal assessments of secondary risks to avian species from exposure to chlorophacinone are needed and should consider interspecific differences in exposure and susceptibility. Although there is a paucity of sub

- lethal threshold effects data following
repeat ed exposure for birds of prey to chlorophacinone; a few studies of diphacinone toxicity to raptors (Savarie et al., 1979; Mendenhall and Pank, 1980; Rattner et al., 2011a) indicate that they may be especially sensitive to anticoagulants. Acute diphacinone toxicity tests indicate that American kestrels are over 20 times more sensitive than Northern bobwhite ( Colinus virginianus ), and over 30 times more sensitive than mallards ( Anas platyrhynchos ), two test species required by USEPA for pesticide registratio n (Rattner et al., 2010 and 2011a). Furthermore , golden eagles appear to be

even more sensitive to diphacinone than kestrels (Savarie et al., 1979; Rattner et al., 2011a). Mendenhall and Pank (1980) observed differences in diphacinone toxicity between gr eat

- horned owls and barn owls and suggested that explanations for such a discrepancy may include interspecific differences in susceptibility or differences in prey species that result in dissimilar exposure. These studies indicate that future assessments on the effects of chlorophacinone on avian species that consume prairie dogs should include multiple species. Ferruginous hawks may be especially susceptible to anticoagulant use on prairie dogs as they are a primary predator of prairie dogs and have
been frequently reported near prairie dog towns poisoned with anticoagulants. In 2010, Audubon of Kansas reported finding the remains of 17 dead hawks in 2009 following anticoagulant use to control prairie dogs in the area. Unfortunately, these carcasses wer e not recovered for necropsy or chemical analysis.

Based on sub

- lethal effects to non

- target species as reported from laboratory studies as well as reported mortalities and concerns based on opportunistic recoveries (Littrell, 1990; Ruder et al., 2008), there is clearly a need for field studies that evaluate anticoagulant exposure and effects to the many species that may consume poisoned prairie dogs. Littrell (1990) ranked exposure to diphacinone/chlorophacinone second only to strychnine as the most haz ardous vertebrate pesticide to non

- targets based on his 10 years of experience in reviewing vertebrate pesticides. Ruder and others (2008)Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### reported three mortality events involving several species, including wild turkeys ( Meleagris gallopavo ), a raccoon ( Procyon lotor ), and an American badger ( Taxidea

taxus ) after a chlorophacinone application to control black

- tailed prairie dogs in Kansas. The authors concluded that their opportunistic findings of non

- target mortalities likely
underestimate actual non

- ta rget losses and warrant further investigation. This conclusion seems justified as a four year survey of possible anticoagulant poisonings of wildlife in France that was based on a wildlife disease surveillance network yielded 59 confirmed diagnoses for br omadiolone and 41 for chlorophacinone (Berny et al., 1997 as cited by Stone et al., 1999). A similar surveillance network is needed to evaluate non

- targets after anticoagulant use to control prairie dogs in the United States, especially given all of the

a vian predators that key in on and consume prairie dogs including golden eagles, northern goshawks, northern harriers, peregrine falcons, prairie falcons, Cooper’s hawks, ferruginous hawks and red

- tailed hawks. A few laboratory studies indicate that some of these species survive after being fed anticoagulant poisoned rodents, at least until time of necropsy (Savarie et al., 1979; Mendenhall and Pank, 1980; Radvanyi et al., 1988). However, the sub

- lethal effects described in these studies (e.g., fatigue, w ing

- dropping, and lung, heart and liver hematomas) are likely to result in decreased survival or reproduction and need to be evaluated under field conditions.

Chlorophacinone is slow acting and non

- target species are often highly mobile, thus tracking i ndividual birds via radio or satellite telemetry is needed to evaluate secondary toxicity to avian predators that consume chlorophacinone poisoned prairie dogs. Avian carcasses are also quickly scavenged in the wild (Vyas, 1999) and tracking may aid in

re covering intact carcasses for necropsy and residue analyses. Tracking studies also have the added benefit of allowing for evaluation in the field where animals also are exposed to other stressors and can also help assess potential sub

- lethal effects that can include
decrease d survival and reproduction.

Conclusions

The current study provided some information on chlorophacinone residue concentrations over time and observed effects from toxicity; however, the limited exposure of chlorophacinone in the current study underestimates what would occur during a field application. Chlorophacinone loss from metabolism and excretion indicates that prairie dogs that continue to consume bait over several days will likely have higher chlorophacinone residues than prairie dogs that are found dead or euthanized after several days of being too sick to eat. Field observations and results from previous studies indicate that field applications would likely result in higher prairie dog residue burdensReview of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

10 

### than indicated in t he current study and risk of secondary exposure to prairie dogs predators could last for weeks after application. Predators that consume prairie dogs would also have an increased risk to secondary toxicity from repeated small doses above
what is inferred from a single reference dose.

Study results indicate that injury from exposure to chlorophacinone may not be related to concentrations of chlorophacinone in whole

- body or liver tissues as measured at the time of death. Chlorophacinone is ingested, resul ts in internal and sometimes external hemorrhaging and is then metabolized and excreted. Internal bleeding in non

- targets from repeated exposure to chlorophacinone likely result s in sub

- lethal effects that contribute to increased mortality without resulti ng in high concentrations in tissue. Thus even low concentrations of chlorophacinone detected in poisoned carcasses may be indicative of cause of death, either directly or indirectly, and should be considered with other biological evidence in determining whether harmful exposure to anticoagulants
occurred.

This study was an acute dietary toxicity test that provided some useful information, as previously noted. However, chlorophacinone lethality increases with multiple low

- dose feedings and prairie dogs exposed to chlorophacinone bait under field conditions can live
for several weeks before death occur s . Therefore, further study is needed to adequately evaluate Objectives 1 and 2. We recommend a more robust assessment of chlorophacinone res idues in prairie dogs that are exposed to repeated low doses of chlorophacinone and are immediately euthanized after they exhibit signs of morbidity. Previous studies indicate that raptors are likely more sensitive to the first generation
indandione roden ticides than traditional avian test species and further evaluation of threshold effects from repeated daily doses are needed. Lastly, we recommend that future field assessments incorporate tracking techniques to evaluate decreased survival and reproductio n for multiple avian species.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

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### References

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Ashton AD, Jackson WB, Peters H. 1986. Comparative evaluation of LD50 values for various anticoagulant rodenticides. Tropical Pest Management 32:187

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Berny PJ, Buronfosse T, Buronfosse F, Lamarque F, Lorgue G. 1997. Field evidence of secondary poisoning of foxes ( Vulpes vulpes )and buzzards ( Buteo buteo ) by bromadiolone, a four

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Rattner BA, Horak KE, Warner SE, Day DD, Johnston JJ. 2010. Comparati ve toxicity of diphacinone to northern bobwhite ( Colinus virginianus ) and American kestrels ( Falco sparverius ). Proceedings, 24th Vertebrate Pest Conference, Sacramento, CA, USA, February 22

- 25, 2010, pp 145

- 152.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

13 

### Rattner BA, Horak KE, Warner SE, Day DD, Meteyer CU, Volker SV, Eisemann JD, Johnston JJ. 2011a. Acute toxicity, histopathology, and coagulopathy in American kestrels ( Falco sparverius ) following administration of the rodenticide diphacinone. Environmental Toxicology and Chemistry 30(5):1213

- 1222.

Rattner BA, Horak KE, Eisenreich KM, Lazarus RS, Eisemann JD, Johnston JJ. 2011b. Comparative toxicity and risk evaluation of the anticoagulant rodenticide diphacinone in various species of birds. Society of Environmental Toxicology and
C hemistry Europe 21st Annual Meeting. Abstract 311 ET10B

- 2. May 15

- 19, 2011, Milan, Italy.

Ruder MG, Poppenga RH, Bryan II JA, Bain M, Pitman J, Keel MK. 2008. Intoxication of nontarget wildlife with rodenticides in Northwestern Kansas . Journal of
Wildli fe Diseases 47(1):212

- 216.

Savarie PJ, Hayes DJ, McBride RT, Roberts JD. 1979. Efficacy and safety of diphacinone as a predacide. In Kenaga EE, (ed), Avian and Mammalian Wildlife Toxicology, pp 69

- 79. STP 693 American Society for Testing Materials,
Phila delphia, PA.

Silberhorn EM, Schnabel DL, Salmon TP. 2003. Ecological risk assessment for grain

based field

- use anticoagulant rodenticides registered by the California Department of Food and Agriculture for special local needs. California Department of F ood and Agriculture, Sacramento, CA. 109 pp.

Stone WB, Okoniewski JC, Stedelin JR. 1999. Poisoning of wildlife with anticoagulant
rodenticides in New York. J ournal of Wildlife Diseases 35:187

- 93

.
Stone WB, Okoniewski JC, Stedelin JR. 2003. Anticoagula nt rodenticides and raptors: recent findings from New York, 1998

- 2001. Bulletin of Environmental Contamination and Toxicology 70:34

– 40.

U.S. Environmental Protection Agency (USEPA. 2007. Pesticides; data requirements for conventional chemicals. Federal Register 72(207): 60934

- 60988. http://edocket.access.gpo.gov/2007/pd f/E7

- 20826.pdf

U.S. Fish and Wildlife Service (USFWS). 2007. Veterinary medical examination report.
Lab Case # 07

- 000015. National Fish and Wildlife Forensics Laboratory, Ashland, Oregon. 6 pp.Review of Witmer (2011) USFWS Project: FFS# 6F56 Final Report

14 

### U.S. Fish and Wildlife Service (USFWS). 2009. Veterinar y medical examination report.
Lab Case # 09

- 000049. National Fish and Wildlife Forensics Laboratory, Ashland, Oregon. 11 pp.

Van Den Berg G, Nauta WT. 1975. Effects of anti

- inflammatory 2

- aryl

- 1,3

- indandiones on oxidative phosphorylation in rat liver mitochondria. Biochemical
Phar ina macology 24(7):815

- 821.

Vidal D, Alzaga V, Luque

- Larena J, Mateo R, Arroyo L, Vinuela J. 2009. Possible interaction between a rodenticide treatment and a pathogen in common vole ( Microtus arvalis ) during a population pea k. Science of the Total Environment
408:267

- 271.

Vyas NB. 1999. Factors influencing estimation of pesticide

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

- 191.

Vyas NB, Spann JW, Hulse CS, Borges SL, Bennett RS, Torrez M, Williams B I, Leffel
R. 2006. Field evaluation of an avian risk assessment model. Environmental Toxicology and Chemistry 25: 1762

- 71

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Vyas NB. 2010. Annual Report 2010 : Characterization of a vian h azards f ollowing

c hlorophacinone (Rozol®) u se for p rairie d og c ontrol

. U

. S

. Geological Survey,
Patuxent Wildlife Research Center . 39 pp.

Witmer G. 2011. Retention time of chlorophacinone in the tissues of black

- tailed prairie dogs exposed to chlorophacinone bait. Final Report: QA

- 1682.
USDA/APHIS/WS National Wil dlife Research Center, Fort Collins, CO. 59 pp.FINAL REPORT Study ID: QA

- 1682

Page 1 of 59 

### VOLUME

STUDY TITLE

Retention time of chlorophacinone in the tissues of black

- tailed prairie dogs exposed to chlorophacinone bait

DATA REQUIREMENT(S):

None

AUTHORS

Gary Witmer, Ph.D., Study Director

STUDY COMPLETION DATE

March 2 3 , 201 1

PERFORMING LABORATORY

† 

### National Wildlife Research Center USDA/APHIS/WS 4101 LaPorte Avenue Fort Collins, Colorado 80521

- 2154

LABORATORY PROJECT ID:

QA

- 1682

CITATION

Witmer , Gary. 2011. Retention time of chlorophacinone in the tissues of b lack

- tailed prairie dogs exposed to chlorophacinone bait . Final Report: QA

- 1 682

. USDA/APHIS/WS National Wildlife Research Center, Fort Collins, CO. 5 9 p p

.FINAL REPORT Study ID: QA

- 1682

Page 2 of 59FINAL REPORT Study ID: QA

- 1682

Page 3 of 59FINAL REPORT Study ID: QA

- 1682

Page 4 of 59FINAL REPORT Study ID: QA

- 1682

Page 5 of 59 

### TABLE OF CONTENTS

COVER PAGE

... .. ....................................................................................... . .. .......................... 1

STATEMENT OF DATA CO NFIDENTIALITY CLAIMS

................... ERROR! BOOKMARK NOT DEFINED.

GOOD LABORATORY PRAC TICE COMPLIANCE STAT EMENT

................................ ................................ 3

QUALITY ASSURANCE ST ATEMENT

................................ ................ ERROR! BOOKMARK NOT DEFINED.

TABLE OF CONTENTS

................................ ................................ ................................ ................................ ........... 5

EXECUTIVE SUM MARY

................................ ................................ ................................ ................................ ........ 6

INTRODUCTION

................................ ................................ ................................ ................................ ...................... 6

STUDY OBJECTIVE...............................................................................................................7

MATERIALS

................................ ................................ ................................ ................................ .............................. 7

T EST M ATERIAL

................................ ................................ ................................ ................................ ........................ 7
T EST O RGANISM

................................ ................................ ................................ ................................ ........................ 7

METHODS

................................ ................................ ................................ ................................ ................................ .. 8

T EST C ONDITIONS

................................ ................................ ................................ ................................ ..................... 8 O BSERVATIONS

................................ ................................ ................................ ................................ ......................... 9 S TATISTICAL A NALYSIS

................................ ................................ ................................ ................................ ............ 9

RESULTS

................................ ................................ ................................ ................................ ................................ .... 9

CONCLUSIONS AND DISC USSION

................................ ................................ ................................ .................... 11

ARCHIVE

................................ ................................ ................................ ................................ ................................ . 11

KEY PERSONNEL

................................ ................................ ................................ ................................ .................. 12

LITERATURE CITED

................................ ................................ ................................ ................................ ............ 12

TABLES

................................ ................................ ................................ ................................ ................................ .... 1 4

FIGURES

................................ ................................ ................................ ................................ ................................ .. 18

APPENDICES

................................ ................................ ................................ ................................ ........................... 2 2

A PPENDIX I

- S TUDY P ROTOCOL

................................ ................................ ................................ .............................. 22
A PPENDIX II

- P ROTOCOL A MENDMENTS /D EVIATIONS

................................ ................................ ............................ 3 9
A PPENDIX III

- ACP A NALYTICAL S ERVICES R EPORT (L IVER AND WHOLE BODY RESIDUES )

................................ .. 4 3
A PPENDIX IV

– NWRC BAIT ANALYSIS A ND CERTIFICATE PROVI DED BY THE MANUFACTU RER

................................ 5 7FINAL REPORT Study ID: QA

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### EXECUTIVE SUMMARY
Rozol prairie dog bait (0.005% chlorophacinone) was fed to male and female adult/sub a dult black

- tailed prairie dogs ( Cynomys ludovicianus ) over a 2

- day period. The residue levels of chlorophacinone in prairie dogs were determined over a 2 7 day period. Most prairie dogs ( n = 36 ; T1 group) were allowed to eat up to 53 g (EPA label application rate) while a group of 3 prairie dogs (T2 group) were allow ed to eat bait ad libitum for a 2

- day period. All remaining animals (n

= 11 ) served as the control group. The T1 group consumed an average of 48.5 g (SD = 7.2 g) of bait, while the T 2 group consumed an average of 33.0 g (SD = 24.1 g) of bait. Highest re sidue levels were found on Day 3 after the bait was first offered: an average of 5. 499 μg/g (SD = 2.0 34 μg/g) in livers and 1. 281 μg/g (SD = 0. 369 μg/g) in whole bodies. Levels quickly declined after Day 3 and a half life of about 5

- 6 days in livers and w hole bodies was interpolated from the graphed results. No chlorophacinone was found in the control animals (all values below the Method Limit of Detection). Residue levels were not significantly different in males and females nor in animals that died ver sus those euthanized in the same time period.

No control animals died during the course of the study. The 3 T2 animals were euthanized 3 days after bait was offered and no conclusive signs of anticoagulant poisoning (hemorrhaging) were observed from this limited time of exposure . Of the 36 T1 animals, 3 died of anticoagulant poisoning and 9 were considered to be moribund or had bleeding injuries and were euthanized. The remaining 24 animals in the T1 group appeared healthy when they were euthanized as p er the schedule. The first clinical symptoms of anticoagulant poisoning (lethargy ) were observed on Day 5

, while main symptoms ( external bleeding or blood in feces) began to be observed in Day 8 after the bait was first offered. Twenty of the 36 (56%) T 1 animals showed evidence of hemorrhaging (external and/or internal) when necropsied.

INTRODUCTION

Black

- tailed prairie dogs ( Cynomys ludovicianus ) are one of five species of prairie dogs found in North America. Black

- tailed prairie dog population sizes are sometimes controlled because of the conflicts that arise with humans (e.g., property damage, consumption of range forage meant for livestock, threat of plague to humans and companion animals) and citizen attitudes about prairie dogs and their manageme nt vary widely (Zinn and Andelt 1999)

. Management of prairie dogs in the past has included poisoning, fumigants, barriers, and relocation (Franklin and Garrett 1989, Robinette et al. 1995, Andelt and Hopper 1998). Anticoagulants are commonly used to

cont rol rodent populations , but have not been registered for use with prairie dogs until recent years (Witmer and Fagerstone 2003)

. Fisher and Timm (1987) demonstrated in a cage trial that chlorophacinone was an effective rodenticide for prairie dogs, but als o demonstrated the potential for secondary hazards to carnivores (using domestic ferrets) from consumption of poisoned prairie dogs. Lee et al. (2005) demonstrated the field efficacy of a chlorophacinone bait when placed in prairie dog burrows. Unlike zi nc phosphide, the traditional toxicant for prairie dogs, anticoagulants persist in tissue (Eason et al. 2010) . Symptoms of chlorophacinone exposure typically take several days after ingestion to manifest, and it may take 7

- 20 days for mortality to occur a fter a single gavage dose (Yoder 2007 ). Chlorophacinone (Rozol ® 

### ) was approved under a Special Local Need or 24(c) registration for use on prairie dogs in several states. Because prairie dog colonies are utilized by various mammalian and avian predators, theFINAL REPORT Study ID: QA

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Page 7 of 59 

### US Fish and Wildlife Service was concerned about the potential poisoning of these animals. The concern seems well

- founded because, for example, Fournier

- Chambrillon et al. (2004) and Albert et al. (2010), found chlorophacinone residues in the livers o f mustelids and owls, respectively. To allow that assessment, managers need information on the levels of chlorophacinone levels that can occur in prairie dog tissues after feeding on rodenticide baits. This study is designed to provide the requested data set of the sponsor

, USFWS . It will also be submitted to the US EPA to assist in making registration decisions on this anticoagulant rodenticide.

STUDY OBJECTIVE

The specific objective of this study was to determine the chlorophacinone residue levels in prairie dog livers and whole bodies at various time intervals after the animals have consumed chlorophacinone rodenticide baits. We hypothesize d that residue levels w ould peak at some point and then decline over time.

MATERIALS

Test Material

Name: Rozol for Prairie Dog s EPA Reg. No. : 7173

- 286 CAS number : CAS #3691

- 35

- 8 (chlorophacinone) Lot/Batch No.: 28709A Source: LiphaTech, Inc., Milwaukee WI Description : Coated grain rodenticide food bait Purity: 0.005% active ingredient Active Ingredien t : chlorophacinone Stability of Compound Under Test Conditions: Listed as stable on MSDS

Storage Conditions of Test Chemicals: T est material was maintained in a p lastic, sealed, black container (original container) in a fume hood at room temperature ( maintained at about 21 o 

### C )

.

Test O rganism

Species : Black

- tailed prairie dog ( Cynomys ludovicianus)

Age at study initiation: all > 9 months (all adults or subadults)

Weight at study initiation: Ave. = 813.2 g (range : 590

- 1 ,060 g)

Source: Wild capture at Buckley Air Force Base, Aurora COFINAL REPORT Study ID: QA

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Page 8 of 59 

### METHODS

The protocol for this study was prepared according to NWRC standards and procedures and approved on 11/2 3 /2009 (note: study initiation is considered as the date of Study Director signature, 11/20/2009) . It was assigned NWRC Study Number QA

- 1682 (Appendix I). Details of the methods of the approved protocol are presented in Appendix I ; a mendments and deviations to the protocol are provided in Appendix II.

Test Co nditions

Quarantine Period: Approximately 2 weeks

.

Conditions : Animals were dusted with an insecticide (Drione) powder while in their capture cages in the field. When brought to NWRC, a nimals were held individually in raccoon

- sized cage traps (25 cm wid e, 81 cm deep, 30 cm height) in an outdoor building under ambient conditions for the approximately 2

- week quarantine period. They were then brought into a climate controlled animal room where conditions were maintained at about 5.6 o 

### C, a relative humidity of 25

- 30 %, and a 12 hrs on:12 hrs off light cycle. Room conditions were monitored by daily checking the room condition panel in the antechamber room and recording the temperature

. Additionally, a H OBO data logger in the actual animal room was maintained and checked periodically to assure that the room’s settings were actually occurring as programmed. Animals were allowed 3 days to acclimate indoors in their new cages before rodenticide bait was added.

Feeding: Animals were fed a maintenance diet of gras s hay, a slice of apple, and a slice of carrot each day.

Health: A health log was maintained for each animal and each animal was checked twice daily (morning and afternoon) beginning with the afternoon check on 1/27/10 . The Study Director and Attending V eterinarian were consulted when any abnormalities were observed and animals were euthanized if deemed appropriate.

Pen size and construction materials : Stainless steel rabbit rack cages ( 48 cm wide, 61 cm deep, 41 cm height) were used to house the animals during the study with one animal per cage.

Test duration: 2 7 days from the day the Rozol Prairie Dog Bait was first offered (January 28, 2010 = Day 1)

.
Test Material Application : All maintenance food was removed from treatment animal cages in late af ternoon the day (1/27/10) before bait was offered. At 8 am, 1/28/10 (henceforth called Day 1), rodenticide bait was offered to each animal in a ceramic bowl for 2 days with no alternative food available . At 8 am on 1/30/10, all remaining rodenticide bait was removed. T he animals were then put back on the maintenance diet. Bait was weighed before being offered and when removed so that the amount consumed could be determined. Animals were randomly assigned to one of 3 groups: T1 (received 53 g of bait fo r a 2

- day period, T2 (received ad limitum bait for a 2

- day period), and a control group maintained o n the maintenance diet throughout the study.FINAL REPORT Study ID: QA

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Page 9 of 59 

### Chemical analysis : The Study Director received a certificate of analysis of the Rozol bait at the time of rec eipt from the manusfacturer . Additionally, the Analytical Chemistry Unit of NWRC analyzed the bait using NWRC Analytical Method 163A. Liver residue levels were determined with NWRC Analytical Method 1 4 3A; whole body residue levels were determined with NW RC Analytical Method 1 42 A.

Observations

Parameters recorded : initial and final body weight, bait consumption, animal condition (twice daily), mortality , necropsy results (external/internal hemorrhaging) . Animals were observed twice daily. Any animal appearing to be moribund (substantial lethar g y, unresponsive to probing, and/or substantial bleeding) was euthanized for purposes of humaneness after consultation with the Study Director and/or Attending Veterinarian. Otherwise, animals were euthanized a ccording to a predetermined schedule. A group of 4 randomly selected animals was euthanized according to a predetermined schedule; that is, on days 3, 5, 7, 9, 11, 14, 18, 2 7 with the bait presentation day being D ay 1. Animals were euthanized by anesthet izing with isoflourane gas and then exposing to carbon dioxide. Animals were then necropsied and prepared for residue analysis with signs of external and internal hemorrhaging noted. Samples taken and frozen for chemical analysis were livers, whole bodie s (less pelt, head, paws, tail), and rodenticide bait samples (freezer temperature maintained at about

- 10.6 o 

### C )

. Animals found dead in their cages were processed in the same way.

Statistical Analysis

Statistic al tests: Software program “Statistix 9” (Analytical Software, Tallahassee FL) was used to perform ANOVA and t tests to determine the significance of differences in the variables food

co n sumption, body weight, and residue levels

. A P value of < 0.05 was considered to indicate a significant diffe rence. Non

- linear regression was performed on the average residue levels to generate the decay curves, associated regression coefficients, and regression equations.

Randomization method : The r andom numbers table from the book, Tables for Statisticians by Arkin and Colton (1963) was used to assign animals to treatment groups and to select animals for euthanasia

.

RESULTS
The chlorophacinone concentration as determined by the NWRC Analytical Chemistry Unit was 0.005% (Appendix I V ). The concentration of ac tive ingredient (chlorophacinone) in the Rozol prairie dog bait used in this study was also determined by the manufacturer (LiphaTech, Inc. ) to be 44.86 mg/kg or 0.0045% ( Appendix IV ).

The levels of chlorophacinone residues in black

- tailed prairie dogs were determined over a 2 7 day period (Table s 1 and 2 ) . Rozol prairie dog bait (0.005% chlorophacinone) was fed to male and female adult/sub a dult prairie dogs over a 2

- day period with no other food present duringFINAL REPORT Study ID: QA

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Page 10 of 59 

### those 2 days (Day 1 and 2) . Most prairie d ogs ( n= 36; T1 group) were allowed to eat up to 53 .0 g (EPA label application rate) while a group of 3 prairie dogs (T2 group) were allowed to eat bait ad libitum for the 2

- day period. An additional 11 prairie dogs served as a control group. Table 2 provi des the data set for all animals. The T1 group consumed an average of 48.5 g (SD = 7. 3 g) of bait, while the T 2 group consumed an average of 33.0 g (SD = 24.1 g) of bait (Table 2)

. The difference in food consumption between the two groups was not signifi cant (t = 0.82, P = 0.4 565 ). Table 1 provides a summary of the residue levels data set for T1 animals with values averaged by days after bait first presented. Highest residue levels in T1 animals were found on Day 3 after the bait was offered: an average of 5. 499 μg/g (SD = 2.0 3 4 μg/g) in livers and 1. 28 1 μg/g (SD = 0. 3 69 μg/g) in whole bodies. [Note: μg/g = ppm.] Residue levels declined significantly over time in livers (F = 20.88, P = 0.0000) and in whole bodies (F = 25.67, P = 0.0000). Levels quickl y declined after Day 3 (Tables 1 and 2; Figure 1) and the levels on Day 7 averaged 1

. 0 69 μg/g (SD = 0.4 09 μg/g) in livers and 0

. 25 1 μg/g (SD = 0. 12 4 μg/g) in whole bodies. These levels are significantly lower than the levels on Day 3 in livers ( t = 4.27, P = 0.0053 ) and in whole bod ies ( t = 5.34, P = 0.0018). Non

- linear regression fit a curvilinear line very well to the decline in liver residues (pseudo R 2 

### = 0.82) and to whole body residues (pseudo
R 2 

### = 0.94; Figure 1). A half life of about 5

- 6 days in li vers and whole bodies can be interpolated from the graphed results (Figure 1 , Figure 2 )

. The rate of decline in residue levels slowed after Day 7, suggesting a biphasic degradation curve (Figure 2) which is common of other anticoagulants such as diphacino ne (J. Eisemann, pers. comm.). Levels of residues were not significantly different in the livers (t = 1. 3 4, P = 0.2 371 ) of T1 Day 3 animals versus T2 animals. Levels of residues were significantly higher (t = 3.13, P = 0.02 59 ) in whole bodies of T1 Day 3 animals versus T2 animals, but were not significantly different (t = 1.22, P = 0.2 756 ) between T1 Day 5 animals and T2 animals (Table 2, Figures 3 and 4 )

. No chlorophacinone was found in the control animals (all values below the Method Limit of Detection ).

We compared the residue levels between T1 males and females that had been euthanized on Day 3 and Day 5 (4 males; 4 females). The highest residue levels occurred in animals euthanized on those two days. There were no significant differences in residu e levels in the livers (t = 0.07, P = 0.9448) of males (mean = 4.4150 μg/g, SD = 2.8166) versus females ( m ean = 4.5200 μg/g, SD = 0.7386) or in whole bodies (t = 0.13, P = 0.8996) of males (mean = 0.9675 μg/g, SD = 0.6091) versus females (mean = 1.0100 μg/ g, SD = 0.2149). We also compared residue levels in animals that were found dead (n = 3) versus levels in animals that were euthanized in that same time period (n = 11). There were no significant differences in residue levels in the livers (t =

- 1.23, P = 0.2408) of animals that were found dead (mean = 0.4300 μg/g, SD = 0.3579 ) versus those euthanized (mean = 0

. 8391 μg/g, SD = 0. 5341 ) or in whole bodies (t =

- 0. 77 , P = 0. 4564 ) of animals found dead (mean = 0. 1700 μg/g, SD = 0. 1908 ) versus those euthanized (mean = 0

. 2690 μg/g, SD = 0. 1957 ).

No control animals died during the course of the study. The 3 T2 animals were euthanized 3 days after bait was offered and no conclusive signs of anticoagulant poisoning (hemorrhaging) were observed from this limited t ime of exposure. Of the 36 T1 animals, 3 died of anticoagulant poisoning and 9 were considered to be moribund and were euthanized for purposes of humaneness

. The average days to death (or moribund state resulting in euthanasia) was 1 5

. 3 days ( n = 12, ran ge = 9

- 2 6

, SD = 5. 5 ). This is similar to the days to death reported by Yoder (2007) in her LD50 determination study: most deaths in 9

- 14 days with a smaller peak in deathsFINAL REPORT Study ID: QA

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Page 11 of 59 

### in 17

- 20 days. The remaining 24 animals in the T1 group appeared healthy when they were euthanized as per the schedule. The first clinical symptoms of anticoagulant poisoning were

o bserved on Day 5 (lethargy) and especially on Day 8 ( external bleeding or blood in feces) a fter the bait was first offered. Twenty of the 36 (56%) T1 anim als showed evidence of hemorrhaging (external and/or internal) when necropsied.

All animals, including those of the control group, lost a significant amount of weight over the course of the study (for control animals : t =

- 6.48, P = 0.000 1 ; for T1 animals : t =

- 10.16, P = 0.0000 ). This may be attributed to the fact that a relatively low nutrition maintenance diet was provided (grass hay, apple, carrot) which was done to avoid confounding the anticoagulant effects by providing a diet relatively high in vit amin K (the antidote to anticoagulant poisoning). This would have occurred if the standard rodent chow pellets were provided to study animals. An additional factor that may have played a role in weight loss was that the study was conducted in winter (alb eit indoors) when the wild

- caught animals would normally be less active, would have only low nutrition foods available, and would be losing weight.

CONCLUSIONS AND DISCUSSION

Chlorophacinone levels quickly peaked in prairie dogs after being fed Rozol prai rie dog bait

. H ighest levels were obtained from animals euthanized on the third day after being offered the bait. Levels quickly declined thereafter and were significantly lower by Day 7. Chlorophacinone residues in our liver samples (maximum average on Day 3 of 5. 499 μg/g) were higher than the 2008 data reported by the Colorado Division of Wildlife (L. Baeten, unpubl. data; received from Francie Pusateri) for prairie dogs recovered dead after a field application of Rozol prairie dog bait (average = 1.34 μg/g, SD = 1.21) perhaps because of the relatively rapid metabolism and excretion of chlorophacinone residues after consumption of the bait and/or a late collection date of carcasses after death in the field study (see review by Primus et al. 2001). Pri mus et al (2001) reported varying levels of residues, depending on the rodent species. Vidal et al. (2009) reported somewhat lower levels of chlorophacinone residues ( 0.082

- 3. 800 μg/g ) in the livers of voles ( Microtus arvalis ) t han our maximum average lev els in prairie dogs. In their risk assessment, they suggested that the risks to av i an scavengers are minimal to negligible while there may be higher risks to some mammalian scavengers.

Our results also demonstrated that p rairie dogs allowed to feed ad lib itum on the bait did not consume more bait nor did they have higher residue levels than those offered only 53 g of bait. The overall study results suggest that the highest risk of secondary exposure to chlorophacinone residues by non

- target animals consum ing prairie dogs exposed to the bait would occur within a few days after bait application and would drop quickly thereafter. Additionally, it has been suggested that because birds are less susceptible to chlorophacinone poisoning than mammals the secondar y risks are probably higher for predatory or scavenging mammals (coyotes) than for predatory birds (barn owls, American kestrels; see review by Primus et al. 2001).

ARCHIVE
All raw data, documentation, records, protocols, specimens, correspondence and o therFINAL REPORT Study ID: QA

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Page 12 of 59 

### documents relating to interpretation and evaluation of data, and final reports generated as a result of this study are retained in the archives of the National Wildlife Research Center at Fort Collins, Colorado.

KEY PERSONNEL
Key personnel involved i n the study include the following staff of the NWRC : Name Title Duties related to study Gary Witmer Supervisory Research Wildlife Biologist Study Director, major participant in all aspects of study
Nathan Snow Biological Science Technician Major particip ant in all aspects of study Rachael Piergross Biological Science Technician Major participant in all aspects of study
David Goldade Supervisory Chemist Residue analysis
Doreen Griffin QA

- QC Specialist Sample log

- in, archiving
Chris topher Campton Biol. Sci. Lab Tec h n ician Tissue preparation
Dustin Keller C O State Univ. Work

- Study Student Tissue preparation

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E A SON


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### , B. D ASSE


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### . 1995. Effe ct of group size on survival of relocated prairie dogs. Journal of Wildlife Management 59:867

- 874.

W ITMER


### , G., AND 

### K. F AGERSTONE 

### . 2003. The use of toxicants in black

- tailed prairie dog management: an overview. Proceedings of the Wildlife Damage Manage ment Conference 10:359

- 369.

V IDAL


### , D., V. A LZAGA


### , J. L UQUE



### - L ARENA


### , R. M ATEO


### , L. A RROYO


### , AND 

### J. V INUELA



### . 2009. Possible interaction between a rodenticide treatment and a pathogen in common vole ( Microtus arvalis ) during a population peak. Science of the Total Environment 408:267

- 271.

Y ODER 

### , C. 2007. Acute oral toxicity (LD50) of chlorophacinone in black

- tailed prairie dogs ( Cynomys ludovicianus ). Unpublished report, QA

- 1446. USDA National Wildlife Reesarch Center, Fort Collins, Colorado. 86 pp.

Z I NN


### , H. C., AND 

### W. F. A NDELT



### . 1999. Attitudes of Fort Collins, Colorado, residents toward prairie dogs. Wildlife Society Bulletin 27:1098

- 1106

.FINAL REPORT Study ID: QA

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### Table 1. Average liver and whole body c hlorophacinone residue levels of T1 sacrificed prairie dogs by days after b ait first presented. T1 prairie dogs were presented with 53 g of Rozol for Prairie Dog s on Day 1 (January 28, 2010).

T1 Groups Sacrificed (No. Animals in Group a 

### ) Days After Bait First Presented Ave. Liver Residues, μg/g (S.D.) Ave. Whole Bod y Residues, μg/g (S.D.)

1 (4) 3 5.499 (2.034) 1.281 (0.369) 2 (4) 5 3.4 31 (1.22 3 ) 0.691 (0.22 5 ) 3 (4) 7 1.069 ( 0.40 9 ) 0.251 (0.12 4 ) 4 ( 4) 9 1.10 1 (0.310) 0.435 (0.0 70 )
5 (4) 11 0.82 1 (0.69 8 ) 0.22 4 (0.191)
6 (6) 14 0.470 (0.38 9 ) 0.106 (0.130)
7 (5) 1 8 0.216 (0.137) 0.053 (0.000)
8 (5) 2 7 0.217 (0.14 6 ) 0.07 2 (0.028)

a 

### Groups with more than 4 animals resulted from animals dying or having to be euthanized for humaneness purposes within a few day s of a scheduled euthanasia day

.FINAL REPORT Study ID: QA

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. Data set for a ll animals by a nimal number, sex, weights, bait consumption, fate and date, and residue levels and treatment group

.

(A) Animals offered 53 g of Rozol for Prairie Dog s (T1 group) on January 28, 2010.

Prairie Dog No. Sex (F/M) Assigned

Treatment Initial Weig ht

(g) End Weight

(g) Difference in Weights (g) Bait
Offered

(g) Bait Remaining (g) Bait Consumed (g) Euthanized or died Date of fate Liver
Residue (μ g/g) Whole Body
Residue (μ g/g)

KQ

- 02 Male T1 710.0 665.0

- 45.0 53.0 0.0 53.0 Euthanized 1/30/ 2010 3.66 0 1.085

KQ

- 04 Female T1 925.0 910.0

- 15.0 53.0 0.0 53.0 Euthanized 1/30/2010 4.905 0.935

KQ

- 28 Female T1 655.0 605.0

- 50.0 53.0 7.5 45.5 Euthanized 1/30/2010 5.025 1.325

KQ

- 41 Male T1 810.0 745.0

- 65.0 53.0 0.2 52.8 Euthanized 1/30/2010 8.407 1 .78

KQ

- 17 Male T1 935.0 895.0

- 40.0 53.0 0.0 53.0 Euthanized 2/1/2010 3.803 0.51 8

KQ

- 27 Female T1 675.0 610.0

- 65.0 53.0 0.4 52.6 Euthanized 2/1/2010 4.71 0 0.89

KQ

- 32 Female T1 630.0 565.0

- 65.0 53.0 7.1 45.9 Euthanized 2/1/2010 3.425 0.881

KQ

- 48 Male T1 740.0 700.0

- 40.0 53.0 0.9 52.1 Euthanized 2/1/2010 1.785 0.47 7

KQ

- 15 Female T1 895.0 835.0

- 60.0 53.0 21.0 32.0 Euthanized 2/3/2010 0.79 4 0.09 6

KQ

- 29 Female T1 715.0 665.0

- 50.0 53.0 25.5 27.5 Euthanized 2/3/2010 1.675 0.309

KQ

- 34 Male T1 915.0 825. 0

- 90.0 53.0 0.9 52.1 Euthanized 2/3/2010 0.945 0.218

KQ

- 37 Male T1 730.0 635.0

- 95.0 53.0 0.6 52.4 Euthanized 2/3/2010 0.864 0.382

KQ

- 20 Male T1 915.0 815.0

- 100.0 53.0 0.1 52.9 Euthanized due to condition 2/5/2010 1.537 0.37 7

KQ

- 21 Female T1 840.0 755 .0

- 85.0 53.0 15.3 37.7 Euthanized 2/5/2010 0.937 0.53 2

KQ

- 40 Female T1 825.0 770.0

- 55.0 53.0 0.4 52.6 Euthanized 2/5/2010 1.096 0.44

KQ

- 50 Male T1 1045.0 960.0

- 85.0 53.0 0.8 52.2 Died 2/5/2010 0.83 4 0.393

KQ

- 08 Female T1 670.0 500.0

- 170.0 53.0 18.6 34.4 Euthanized 2/7/2010 0.87 7 0.3 30

KQ

- 13 Male T1 800.0 705.0

- 95.0 53.0 0.0 53.0 Euthanized 2/7/2010 0.502 0.053

KQ

- 24 Male T1 1060.0 900.0

- 160.0 53.0 0.0 53.0 Died 2/7/2010 0.1 4 1 0.07 3

KQ

- 35 Female T1 805.0 660.0

- 145.0 53.0 14.3 38.7 Euthanized due to condition 2/7/2010 1.765 0.439

KQ

- 12 Male T1 765.0 700.0

- 65.0 53.0 0.0 53.0 Died 2/9/2010 0.321 0.053

KQ

- 01 Female T1 590.0 530.0

- 60.0 53.0 0.1 52.9 Euthanized due to condition 2/10/2010 0.0 90 0.053

KQ

- 30 Male T1 870.0 760.0

- 110.0 53.0 0.1 52.9 E uthanized 2/10/2010 1.19 0 0.053

KQ

- 33 Female T1 675.0 540.0

- 135.0 53.0 0.1 52.9 Euthanized due to condition 2/10/2010 0.57 6 0.372

KQ

- 45 Male T1 820.0 745.0

- 75.0 53.0 1.8 51.2 Euthanized 2/10/2010 0.235 0.053

KQ

- 19 Male T1 775.0 635.0

- 140.0 53.0 0.0 5 3.0 Euthanized due to condition 2/11/2010 0.413 0.053

KQ

- 03 Male T1 765.0 720.0

- 45.0 53.0 0.1 52.9 Euthanized 2/14/2010 0.127 0.053

KQ

- 39 Female T1 855.0 840.0

- 15.0 53.0 4.0 49.0 Euthanized 2/14/2010 0.14 5 0.053FINAL REPORT Study ID: QA

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- 42 Male T1 895.0 860.0

- 35.0 53.0 0. 2 52.8 Euthanized 2/14/2010 0.13 1 0.053

KQ

- 44 Female T1 895.0 805.0

- 90.0 53.0 11.4 41.6 Euthanized 2/14/2010 0.22 9 0.053

KQ

- 49 Female T1 810.0 730.0

- 80.0 53.0 13.0 40.0 Euthanized due to condition 2/15/2010 0.45 1 0.053

KQ

- 46 Male T1 985.0 695.0

- 290.0 53.0 0.8 52.2 Euthanized due to condition 2/17/2010 0.44 2 0.116

KQ

- 06 Male T1 760.0 660.0

- 100.0 53.0 0.0 53.0 Euthanized 2/18/2010 0.132 0.053

KQ

- 18 Female T1 855.0 660.0

- 195.0 53.0 16.6 36.4 Euthanized due to condition 2/18/2010 0.265 0.053

KQ

- 26 Fe male T1 875.0 505.0

- 370.0 53.0 0.0 53.0 Euthanized due to condition 2/22/2010 0.187 0.084

KQ

- 47 Female T1 790.0 730.0

- 60.0 53.0 0.2 52.8 Euthanized 2/23/2010 0.061 0.053

Average 813.2 717.6

- 95.6 53.0 4.5 48.5 1.463 0.355

SD 11 1. 3 117.5 7 1

. 9 0.00 7. 3 7. 3

(B) Animals offered ad libitum Rozol for Prairie Dog s (T2 group) : bait was presented on January 28, 2010.

Prairie

Dog
No. Sex (F/M) Assigned

Treatment Initial Weight

(g) End Weight

(g) Differe nce in Weights

(g) Bait
Offered

(g) Bait Remaining (g) Bait
Consumed (g) Euthanized
or died Date of
fate L iver
Residue (μg/g) Whole Body
Residue (μg/g)

KQ

- 07 Male T2 880.0 810.0

- 70.0 150.0 143.0 7.0 Euthanized 1/31/2010 0.14 6 0.053

KQ

- 25 Female T2 925.0 875.0

- 50.0 150.0 95.4 54.6 Euthanized 1/31/2010 3.02 0.648

KQ

- 31 Female T2 765.0 730.0

- 35.0 150.0 1 12.5 37.5 Euthanized 1/31/2010 5.923 0.609

Average 856.7 805.0

- 51.7 150.0 117.0 33.0 3.0 30 0.437

SD 82.5 72.6 17.6 0.0 24.1 24.1 2.88 9 0.33 3

(C) Animals in c ontrol (C) group (fed only maintenance diet). All resi due levels below the Minimum Limit of Detection.

Prairie

Dog
No. Sex (F/M) Assigned

Treatment Initial Weight

(g) End Weight

(g) Difference
in Weights

(g) Bait
Offered

(g) Bait Remaining (g) Bait
Consumed (g) Euthanized
or died Date of
fate Liver
Residue (μg/g) Whole Body
Residue (μg/g)

KQ

- 05 Male C 795.0 775.0

- 20.0 0.0 0.0 0.0 Euthanized 1/30/2010 < 0.061 < 0.053

KQ

- 43 Female C 950.0 915.0

- 35.0 0.0 0.0 0.0 Euthanized 1/30/2010 < 0.061 < 0.053

KQ

- 10 Male C 900.0 760.0

- 140.0 0.0 0.0 0.0 Euthani zed 2/7/2010 < 0.061 < 0.053FINAL REPORT Study ID: QA

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- 11 Female C 805.0 725.0

- 80.0 0.0 0.0 0.0 Euthanized 2/7/2010 < 0.061 < 0.053

KQ

- 14 Female C 825.0 765.0

- 60.0 0.0 0.0 0.0 Euthanized 2/18/2010 < 0.061 < 0.053

KQ

- 16 Male C 860.0 715.0

- 145.0 0.0 0.0 0.0 Euthanized 2/18/2010 < 0 .061 < 0.053

KQ

- 09 Male C 965.0 850.0

- 115.0 0.0 0.0 0.0 Euthanized 2/23/2010 < 0.061 < 0.053

KQ

- 22 Male C 645.0 580.0

- 65.0 0.0 0.0 0.0 Euthanized 2/23/2010 < 0.061 < 0.053

KQ

- 23 Female C 840.0 690.0

- 150.0 0.0 0.0 0.0 Euthanized 2/23/2010 < 0.061 < 0.053

KQ

- 36 Female C 885.0 760.0

- 125.0 0.0 0.0 0.0 Euthanized 2/23/2010 < 0.061 < 0.053

KQ

- 38 Female C 745.0 685.0

- 60.0 0.0 0.0 0.0 Euthanized 2/23/2010 < 0.061 < 0.053

Average 837.7 747.3

- 90.5 0.0 0.0 0.0

SD 91. 8 87.6 46. 3 0.00 0.00 0.00FINAL REPORT Study ID: QA

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### F igure 1

. Non

- linear regression of a verage chlorophacinone residue levels in prairie dog livers ( top) and whole bodies ( bottom ) by date of euthanasia or death . Animals were offered Rozol for Prairie Dog s on January 28

, 2010 ( Day 1)

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### Figure 2. Average chlorophacinone residue levels in prairie dog livers (upper line) and whole bodies (lower line) by date of euthanasia or death. Animals were offered Rozol for Prairie Dog s on January 28, 2010 (Day 1).FINAL REPORT Study ID: QA

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### Figure 3

. Averag e chlorophacinone residue levels in livers of control animals

, over time in animals presented with 53 g of bait, and in animals allowed to feed ad libitum for 2 days. Animals were offered Rozol for Prairie Dog s on January 28, 2010 (Day 1). All control an imal values were below the Method Limit of Detection (MLOD). For liver samples the MLOD = 0.061 μg/g.FINAL REPORT Study ID: QA

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### Figure 4

. Average chlorophacinone residue levels in whole bodies of control animals, over time in animals presented with 53 g of bait, and in animals allowed to feed ad libitum for 2 days. Animals were offered Rozol for Prairie Dog s on January 28, 2010 (Day 1). All control animal values were below the Method Limit of Detection (MLOD). For whole body samples the MLOD = 0. 053 μg/g.

0 0.2 0.4 0.6 0.8

1 1.2 1.4

Control Day 3 Day 5 Day 7 Day 9 Day 11 Day 14 Day 18 Day 23 Ad lib Ave. μg/gFINAL REPORT Study ID: QA

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### APPENDICES

Appendix I

- Study Protocol

Appendix II

- Protocol Amendments/Deviations

Appendix III

- ACP Analytical Services Report (Liver and Whole Body Residue Levels)

Appendix IV

– NWRC Bait Analysis and Certif icate Provided by the ManufacturerFINAL REPORT Study ID: QA

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- Study ProtocolFINAL REPORT Study ID: QA

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### Appendix II

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### Appendix II , cont. Amendments to, and deviations from, the approved stud y protocol. The deviations to the approved protocol (as identified in the quality assurance inspection reports and described in the two amendments attached to this appendix and in the analytical chemistry report (Appendix III) were:
1. Animals were not weighe d and sexed in the field; instead, they were weighed and sexed when brought into the animal research building after quarantine. This allowed us to determine the weight and sex more accurately and closer to the start of the study.

2. A few (5 females and 1 ma le) of the 50 prairie dogs used in the study were below the minimum weight cut

- off levels of 600 g for females and 700 g for males. However, because all animals were captured in January, all were considered to be adults or subadults approaching adult size

. 3. A random numbers table was used instead of a statistical software program to assign animals to treatment group. Memo

- to

- File on this change was put in the study records.

4. The study completion date and date of archiving was extended because of a delay

exp erienced in getting the final analytical chemistry report on residue levels.
5. The analytical chemistry method used in the study was slightly modified (as detailed in the report in Appendix III) when some difficulties were encountered in achieving
consistent residue levels from tissue samples.FINAL REPORT Study ID: QA

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### Appendix III

- ACP Analytical Services Report (Liver and Whole Body Residue Levels)FINAL REPORT Study ID: QA

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### Appendix IV

– NWRC Bait Analysis and Certificate Provided by the ManufacturerFINAL REPORT Study ID: QA

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