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 exposed 

to chlorophacinone bait”

FROM: Matt Schwarz, 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 

Field 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 black-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 Dakota 

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 dog 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 specified 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 voles (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 chlorophacinone 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, should be considered as 

acceptable evidence 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 injuries 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.

Further 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, results 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. 

Therefore, 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 enclosure
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U.S. FISH AND WILDLIFE SERVICE

DIVISION OF ENVIRONMENTAL QUALITY

REGION 6

RETENTION TIME OF CHLOROPHACINONE IN THE TISSUES OF BLACK-TAILED 

PRAIRIE DOGS EXPOSED TO CHLOROPHACINONE BAIT

Final Report

Region 6

DEC ID : 200960003

FFS: 6F56

Congressional District: SD00

Prepared August, 2011, by:

Matthew S. Schwarz1

Joy Gober2

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 80525
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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 

Investigation. 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 prairie dog consumption rates when chlorophacinone bait is used in the field as a 

rodenticide. Therefore, 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 poisoned 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 

chlorophacinone in various prairie dog tissues at specific time intervals post exposure. 

However in doing so, study Objective 2 was not fully satisfied because the limited 

feeding duration in the current study underestimates exposure that may occur from 

repeated 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 after 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 (Biggins 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. As 
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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 finding 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 poisoned 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 cause 

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 

prairie 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 suggest 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 ± 1.80 μg/g (Lee and Hygnstrom, 2007; Primus, 

2007). Eight 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). Residues 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 Norway 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 that
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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 deaths of prairie dogs

(Vyas, 2010, Lee and Hygnstrom, 2007) extends the time during 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 

exposed 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 

include 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 

response 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 one 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 underestimate 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 exposed 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 concentrations 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). 
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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 amounts" 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 liver 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 bald 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 “trace 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 rozol 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 despite receiving a clean maintenance diet post exposure to rozol. Prolonged 

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 secondary 

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. Valid 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 statistical 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 consumed), 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. 
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We do not agree with the current study 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 nontarget 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 probably 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 example, American kestrels (Falco 

sparverius) administered diphacinone and with liver residues just above the diphacinone 

method detection limits of 0.263 and 0.280 μg/g diphacinone had histological evidence of 

hemorrhage in lung and liver (Rattner et al., 2011a). 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, 

1975). 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 

residues 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. 
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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 new 

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 dietary 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 subacute dietary toxicity test includes multiple exposures over 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 with 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 (Godfrey 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 the 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 indandione rodenticides. Thus, 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 

hemorrhaging. 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 that 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 first generation indandione rodenticides to control prairie dogs. 
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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. 

Additional 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 

repeated 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 

registration (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 great-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 were 

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 hazardous vertebrate pesticide to non-targets based on his 

10 years of experience in reviewing vertebrate pesticides. Ruder and others (2008) 
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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-target 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 bromadiolone 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 

avian 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, wing-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 individual 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 

recovering 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 

decreased 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 burdens 
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than indicated in the 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, results in internal and sometimes external 

hemorrhaging and is then metabolized and excreted. Internal bleeding in non-targets 

from repeated exposure to chlorophacinone likely results in sub-lethal effects that 

contribute to increased mortality without resulting 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 occurs. Therefore, further study is needed to adequately 

evaluate Objectives 1 and 2. We recommend a more robust assessment of 

chlorophacinone residues 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 rodenticides 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 reproduction for multiple avian species. 
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dogs exposed to chlorophacinone bait. Final Report: QA-1682. 

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

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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 23, 2011

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 black-tailed prairie 

dogs exposed to chlorophacinone bait. Final Report: QA-1682. USDA/APHIS/WS National 

Wildlife Research Center, Fort Collins, CO. 59 pp. 
FINAL REPORT Study ID: QA-1682

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TABLE OF CONTENTS

COVER PAGE.........................................................................................................................1

STATEMENT OF DATA CONFIDENTIALITY CLAIMS...................ERROR! BOOKMARK NOT DEFINED.

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ................................................................ 3

QUALITY ASSURANCE STATEMENT ................................................ERROR! BOOKMARK NOT DEFINED.

TABLE OF CONTENTS ........................................................................................................................................... 5

EXECUTIVE SUMMARY ........................................................................................................................................ 6

INTRODUCTION ...................................................................................................................................................... 6

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

MATERIALS.............................................................................................................................................................. 7

TEST MATERIAL ........................................................................................................................................................ 7

TEST ORGANISM........................................................................................................................................................ 7

METHODS.................................................................................................................................................................. 8

TEST CONDITIONS ..................................................................................................................................................... 8

OBSERVATIONS ......................................................................................................................................................... 9

STATISTICAL ANALYSIS ............................................................................................................................................ 9

RESULTS.................................................................................................................................................................... 9

CONCLUSIONS AND DISCUSSION .................................................................................................................... 11

ARCHIVE ................................................................................................................................................................. 11

KEY PERSONNEL.................................................................................................................................................. 12

LITERATURE CITED ............................................................................................................................................ 12

TABLES .................................................................................................................................................................... 14

FIGURES .................................................................................................................................................................. 18

APPENDICES........................................................................................................................................................... 22

APPENDIX I - STUDY PROTOCOL.............................................................................................................................. 22

APPENDIX II - PROTOCOL AMENDMENTS/DEVIATIONS............................................................................................ 39

APPENDIX III - ACP ANALYTICAL SERVICES REPORT (LIVER AND WHOLE BODY RESIDUES).................................. 43

APPENDIX IV – NWRC BAIT ANALYSIS AND CERTIFICATE PROVIDED BY THE MANUFACTURER ................................ 57
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EXECUTIVE SUMMARY

Rozol prairie dog bait (0.005% chlorophacinone) was fed to male and female adult/subadult 

black-tailed prairie dogs (Cynomys ludovicianus) over a 2-day period. The residue levels of 

chlorophacinone in prairie dogs were determined over a 27 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 allowed 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 T2 group consumed an average of 33.0 g (SD = 24.1 g) of bait. Highest residue 

levels were found on Day 3 after the bait was first offered: an average of 5.499 μg/g (SD = 2.034

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 whole 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 versus 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 per 

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%) T1 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 management 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 

control 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 also 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 zinc 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 after 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, the 
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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 of 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 hypothesized that residue levels would peak at some 

point and then decline over time. 

MATERIALS

Test Material 

Name: Rozol for Prairie Dogs

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 Ingredient: chlorophacinone

Stability of Compound 

Under Test Conditions: Listed as stable on MSDS

Storage Conditions of 

Test Chemicals: Test material was maintained in a plastic, sealed, black container

 (original container) in a fume hood at room temperature (maintained at 

about 21oC).

Test Organism

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 CO
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METHODS

The protocol for this study was prepared according to NWRC standards and procedures and 

approved on 11/23/2009 (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; amendments and 

deviations to the protocol are provided in Appendix II.

Test Conditions

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, animals were held individually in raccoon-sized 

cage traps (25 cm wide, 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 oC, 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 HOBO 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 grass 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 Veterinarian 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: 27 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 afternoon 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. The 

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 for a 2-day period, T2 (received ad 

limitum bait for a 2-day period), and a control group maintained on the maintenance diet 

throughout the study.
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Chemical analysis: The Study Director received a certificate of analysis of the Rozol bait at the 

time of receipt 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 143A; whole body residue levels were determined with NWRC 

Analytical Method 142A.

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 lethargy, 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 according 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, 27 with 

the bait presentation day being Day 1. Animals were euthanized by anesthetizing 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 bodies (less pelt, head, paws, tail), and 

rodenticide bait samples (freezer temperature maintained at about -10.6 oC). Animals found dead 

in their cages were processed in the same way.

Statistical Analysis

Statistical 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 

consumption, body weight, and residue levels. A P value of < 0.05 was considered to indicate a 

significant difference. Non-linear regression was performed on the average residue levels to 

generate the decay curves, associated regression coefficients, and regression equations.

Randomization method: The random 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 IV). The concentration of active 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 27

day period (Tables 1 and 2). Rozol prairie dog bait (0.005% chlorophacinone) was fed to male 

and female adult/subadult prairie dogs over a 2-day period with no other food present during 
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those 2 days (Day 1 and 2). Most prairie dogs (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 

provides the data set for all animals. The T1 group consumed an average of 48.5 g (SD = 7.3 g) 

of bait, while the T2 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 significant (t = 0.82, P = 

0.4565). 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 = 2.034 μg/g) in livers and 1.281

μg/g (SD = 0.369 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 quickly declined after Day 3 (Tables 1 and 2; Figure 1) and the levels on Day 7

averaged 1.069 μg/g (SD = 0.409 μg/g) in livers and 0.251 μg/g (SD = 0.124μg/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 bodies (t = 5.34, P = 0.0018). Non-linear regression fit a curvilinear line 

very well to the decline in liver residues (pseudo R2 = 0.82) and to whole body residues (pseudo 

R2 = 0.94; Figure 1). A half life of about 5-6 days in livers 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 diphacinone (J. Eisemann, pers. comm.). Levels of residues were not 

significantly different in the livers (t = 1.34, P = 0.2371) of T1 Day 3 animals versus T2 animals. 

Levels of residues were significantly higher (t = 3.13, P = 0.0259) in whole bodies of T1 Day 3

animals versus T2 animals, but were not significantly different (t = 1.22, P = 0.2756) 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 residue levels in the livers (t = 0.07, P = 

0.9448) of males (mean = 4.4150 μg/g, SD = 2.8166) versus females (mean = 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 time 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 15.3

days (n = 12, range = 9-26, 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 deaths 
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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

observed on Day 5 (lethargy) and especially on Day 8 (external bleeding or blood in feces) after 

the bait was first offered. Twenty of the 36 (56%) T1 animals 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 = 0.0001; 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 vitamin 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 (albeit 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 prairie dog bait. 

Highest 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). 

Primus 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) than our maximum average levels in prairie dogs. In their 

risk assessment, they suggested that the risks to avian scavengers are minimal to negligible while 

there may be higher risks to some mammalian scavengers.

Our results also demonstrated that prairie dogs allowed to feed ad libitum 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 consuming 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 secondary 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 other 
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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 in 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 participant 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

Christopher Campton Biol. Sci. Lab Technician Tissue preparation

Dustin Keller CO State Univ. Work-Study Student Tissue preparation

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Contamination and toxicology 58:451-459.

ANDELT, W. F., AND S. N. HOPPER. 1998. Managing prairie dogs. Colorado State University 

Cooperative Extension Bulletin Number 6.506, Fort Collins, Colorado.

EASON, C., R. HENDERSON, S. HIX, D. MACMORRAN, A. MILLER, E. MURPHY, J. ROSS, AND S.

OGILVIE. 2010. Alternatives to brodifacoum and 1080 for possum control and rodent control—

how and why? New Zealand Journal of Zoology 37:175-183.

FISHER, D. D., AND R. M. TIMM. 1987. Laboratory trial of chlorophacinone as a prairie dog 

toxicant. Proceedings of the Great Plains Wildlife Damage Control Workshop 8:67-69.

FOURNIER-CHAMBRILLON, C., P. BERNY, O. COIFFIER, P. BARBEDIENNE, B. DASSE, G. DELAS, H.

GALINEAU, A. MAZET, P. POUZENC, R. ROSOUX, AND P. FOURNIER. 2004. Evidence of secondary 

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40:688-695.

FRANKLIN, W. L., AND M. G. GARRETT. 1989. Nonlethal control of prairie dog colony expansion 

with visual barriers. Wildlife Society Bulletin 17:426-430.

LEE, C., P. GIPSON, AND J. WILSON. 2005. In-burrow application of Rozol to manage black-
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tailed prairie dogs. Proceedings of the Wildlife Damage Management Conference 11:349-353.

PRIMUS, T., J. EISEMANN, G. MATSCHKE, C. RAMEY, AND J. JOHNSTON. 2001. Chlorophacinone 

residues in rangeland rodents: as assessment of the potential risk of secondary toxicity to 

scavengers. Pp. 164-180 In: J. Johnston, ed. Pesticides and Wildlife. American Chemical 

Society Symposium Series 177. American Chemical Society, Washington, D.C.

ROBINETTE, K. W., W. F. ANDELT, AND K. P. BURNHAM. 1995. Effect of group size on survival 

of relocated prairie dogs. Journal of Wildlife Management 59:867-874.

WITMER, G., AND K. FAGERSTONE. 2003. The use of toxicants in black-tailed prairie dog 

management: an overview. Proceedings of the Wildlife Damage Management Conference 

10:359-369.

VIDAL, D., V. ALZAGA, J. LUQUE-LARENA, R. MATEO, L. ARROYO, AND J. VINUELA. 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.

YODER, 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.

ZINN, H. C., AND W. F. ANDELT. 1999. Attitudes of Fort Collins, Colorado, residents toward 

prairie dogs. Wildlife Society Bulletin 27:1098-1106. 
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Table 1. Average liver and whole body chlorophacinone residue levels of T1 sacrificed prairie 

dogs by days after bait first presented. T1 prairie dogs were presented with 53 g of Rozol for 

Prairie Dogs on Day 1 (January 28, 2010).

T1 Groups Sacrificed

(No. Animals in 

Groupa)

Days After Bait First 

Presented

Ave. Liver Residues, 

μg/g (S.D.)

Ave. Whole Body 

Residues, μg/g (S.D.)

1 (4) 3 5.499 (2.034) 1.281 (0.369)

2 (4) 5 3.431 (1.223) 0.691 (0.225)

3 (4) 7 1.069 (0.409) 0.251 (0.124)

4 (4) 9 1.101 (0.310) 0.435 (0.070)

5 (4) 11 0.821 (0.698) 0.224 (0.191)

6 (6) 14 0.470 (0.389) 0.106 (0.130)

7 (5) 18 0.216 (0.137) 0.053 (0.000)

8 (5) 27 0.217 (0.146) 0.072 (0.028)

a Groups with more than 4 animals resulted from animals dying or having to be euthanized for 

humaneness purposes within a few days of a scheduled euthanasia day.
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Table 2. Data set for all animals by animal number, sex, weights, bait consumption, fate and date, and residue levels and treatment group.

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

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-02 Male T1 710.0 665.0 -45.0 53.0 0.0 53.0 Euthanized 1/30/2010 3.660 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 0.518

KQ-27 Female T1 675.0 610.0 -65.0 53.0 0.4 52.6 Euthanized 2/1/2010 4.710 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 0.477

KQ-15 Female T1 895.0 835.0 -60.0 53.0 21.0 32.0 Euthanized 2/3/2010 0.794 0.096

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 0.377

KQ-21 Female T1 840.0 755.0 -85.0 53.0 15.3 37.7 Euthanized 2/5/2010 0.937 0.532

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 0.834 0.393

KQ-08 Female T1 670.0 500.0 -170.0 53.0 18.6 34.4 Euthanized 2/7/2010 0.877 0.330

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 0.141 0.073

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 0.090 0.053

KQ-30 Male T1 870.0 760.0 -110.0 53.0 0.1 52.9 Euthanized 2/10/2010 1.190 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 0.576 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 53.0 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 0.145 0.053
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KQ-42 Male T1 895.0 860.0 -35.0 53.0 0.2 52.8 Euthanized 2/14/2010 0.131 0.053

KQ-44 Female T1 895.0 805.0 -90.0 53.0 11.4 41.6 Euthanized 2/14/2010 0.229 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 0.451 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 0.442 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 Female 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 111.3 117.5 71.9 0.00 7.3 7.3

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

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-07 Male T2 880.0 810.0 -70.0 150.0 143.0 7.0 Euthanized 1/31/2010 0.146 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 -35.0 150.0 112.5 37.5 Euthanized 1/31/2010 5.923 0.609

Average 856.7 805.0 -51.7 150.0 117.0 33.0 3.030 0.437

SD 82.5 72.6 17.6 0.0 24.1 24.1 2.889 0.333

(C) Animals in control (C) group (fed only maintenance diet). All residue 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 Euthanized 2/7/2010 <0.061 <0.053
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KQ-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 0.00
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Figure 1. Non-linear regression of average chlorophacinone residue levels in prairie dog livers 

(top) and whole bodies (bottom) by date of euthanasia or death. Animals were offered Rozol for 

Prairie Dogs 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 Dogs

on January 28, 2010 (Day 1).
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Figure 3. Average 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 Dogs on January 28, 2010 (Day 1). All control animal 

values were below the Method Limit of Detection (MLOD). For liver samples the MLOD = 

0.061 μg/g.
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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 Dogs 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/g
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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 Certificate Provided by the Manufacturer
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Appendix I - Study Protocol
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Appendix II - Protocol Amendments/Deviations 
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Appendix II, cont. Amendments to, and deviations from, the approved study 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 weighed 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 male) 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 

experienced 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.
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Appendix III - ACP Analytical Services Report (Liver and Whole Body Residue Levels)
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Appendix IV – NWRC Bait Analysis and Certificate Provided by the Manufacturer
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