Ecological Risk Assessment

for the Section 3 New Use Registration of 

Diphacinone (PC 067701)

For Use on Black-tailed Prairie Dogs

(Cynomys ludovicianus)

Prepared by:

N.E.Federoff, Wildlife Biologist

J. Lin, Environmental Engineer U. S. Environmental Protection Agency

Office of Pesticide Programs

Environmental Fate and Effects Division

Environmental Risk Branch II

1200 Pennsylvania Ave., NW

Mail Code 7507P

Washington, DC 20460

Reviewed by:

Kristina Garber, Senior Biologist

R. David Jones, Senior Fate Scientist

Branch Chief:

Brian Anderson

June 28, 2012
2
3

Table of Contents

1.0 Executive Summary................................................................................................ 7

1.1 Nature of Chemical Stressor................................................................................... 7

1.2 Exposure Characterization...................................................................................... 7

1.3 Effects Characterization.......................................................................................... 8

1.4 Potential Risks to Non-target Organisms................................................................ 8

1.5 Related Biological Opinions................................................................................. 10

1.6 Data Gaps.............................................................................................................. 11

2.0 Problem Formulation ............................................................................................ 12

2.1 Nature of Regulatory Action................................................................................. 12

2.2 Previous Risk Assessments................................................................................... 12

2.3 Stressor Source and Distribution .......................................................................... 13

2.3.1 Mechanism of Action............................................................................................ 13

2.3.2 Nature of the Chemical Stressor ........................................................................... 13

2.3.3 Environmental Transport Mechanisms................................................................. 17

2.4 Overview of Pesticide Usage ................................................................................ 17

2.4.1 Label for Proposed Registered Use....................................................................... 17

Figure 1. Historic Range of Black Tailed Prairie Dogs........................................................... 18

2.5 Receptors............................................................................................................... 19

2.6 Ecosystems Potentially at Risk ............................................................................. 20

2.7 Assessment Endpoints .......................................................................................... 20

2.8 Conceptual Model................................................................................................. 20

2.8.1 Risk Hypotheses.................................................................................................... 20

2.8.2 Conceptual Diagram ............................................................................................. 21

2.9 Analysis Plan ........................................................................................................ 23

2.9.1 Measures of Exposure........................................................................................... 23

2.9.2 Measures of Effect ................................................................................................ 24

2.9.3 Use of Probit Slope Response Relationship to Provide Information on the 

Endangered Species Levels of Concern................................................................................ 24

2.9.4 Data Gaps.............................................................................................................. 25

3.0 Exposure Assessment............................................................................................ 26

3.1 Label Application Rates and Intervals.................................................................. 26

3.2 Aquatic Exposure Assessment.............................................................................. 26
4

3.2.1 Aquatic EECs........................................................................................................ 26

3.3 Terrestrial Exposure Assessment.......................................................................... 26

3.3.1 Expected Diphacinone Ingestion through Bait Consumption............................... 26

3.3.2 Expected Diphacinone Ingestion through Consumption of Contaminated 

Carcasses28

4.0 Ecological Effects Characterization...................................................................... 29

4.1 Toxicity of Diphacinone to Aquatic Organisms................................................... 30

4.2 Toxicity of Diphacinone to Terrestrial Organisms............................................... 30

4.3 Birds and Mammals, Secondary Toxicity Tests................................................... 34

4.4 Incident Database Review..................................................................................... 36

5.0 Risk Characterization............................................................................................ 37

5.1 Risk Estimation: Integration of Exposure and Effects Data ................................. 38

5.1.1 Aquatic Risk Estimation ....................................................................................... 38

5.1.2 Bird and Mammal Risk Estimation....................................................................... 38

Risks from Direct Bait Consumption.................................................................................... 38

5.1.3 Secondary Risks from Consumption of Diphacinone-contaminated Carcasses... 40

5.1.4 Risk to Terrestrial Invertebrates............................................................................ 41

5.2 Risk Description.................................................................................................... 42

5.2.1 Risks to Aquatic Organisms.................................................................................. 42

5.2.2 Risks to Terrestrial Organisms.............................................................................. 42

5.2.3 Risks to Mammals directly consuming bait.......................................................... 43

5.2.4 Risks to carnivore and scavenger Mammals......................................................... 43

5.2.5 Risks to Reptiles ................................................................................................... 44

5.2.6 Risks to Terrestrial Invertebrates.......................................................................... 44

5.3 Threatened and Endangered Species Concern...................................................... 44

5.3.1 Action Area........................................................................................................... 44

5.3.2 Taxonomic Groups Potentially at Risk................................................................. 45

5.3.3 Probit Slope Response Analysis ........................................................................... 48

5.3.4 Indirect Effects Analysis....................................................................................... 49

5.3.5 Critical Habitat for Listed Species........................................................................ 49

6.0 Uncertainties......................................................................................................... 50

6.1 Fate and Transport Properties............................................................................... 50

6.2 Effects................................................................................................................... 50
5

7.0 Bibliography ......................................................................................................... 52
6

List of Appendices

Appendix A RQ Methods and LOC Definitions

Appendix B LOCATES Listing of Endangered Species
7

1.0 Executive Summary

1.1 Nature of Chemical Stressor

The proposed Section 3 registration is for the use of Kaput-D

®

Prairie Dog Bait on Black-tailed 

Prairie Dogs (BPD; Cynomus ludivicianus). This restricted use product is a bait that contains 

0.005% diphacinone (50 mg a.i./kg-bait) and must be hand applied on rangeland and adjacent 

non-crop areas at least 6” into active burrows. No bait is to be placed on or above ground level. 

The product label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, TX, MT and 

OK. 

Diphacinone is a first generation indanedione anticoagulant rodenticide and it is absorbed 

through the gut, inhibiting vitamin K. Indanediones are not only anticoagulants; they also 

uncouple oxidative phosphorylation (energy generation) in target animals (i.e., mammals). 

Diphacinone seems to have a different mode of action in reptiles. Mortality occurs but does not 

result from anticoagulation, and its mode of action in snakes is unknown. 

Anticoagulants are vitamin-K antagonists that disrupt normal blood-clotting mechanisms and 

induce capillary damage. Typically, death is delayed for four to ten or more days after a lethal 

dose is ingested, and animals may continue to feed and move about until shortly before death. 

Death results from hemorrhage, and exposed animals may exhibit behavior that may make them 

more susceptible to predation (Cox and Smith, 1992). This may result in secondary exposure to 

predatory animals. 

1.2 Exposure Characterization

The primary dissipation route of diphacinone is likely to be consumption of bait by target as well 

as non-target animals. Diphacinone is not expected to be transported to surface or ground water. 

This is based on its use pattern and formulation, low solubility (0.3 mg/L), and its range of Kd 

values of 3 to 5 mL/g, which suggest moderate mobility in soil. Because diphacinone is applied 

as a solid bait, spray drift is not expected. Consistent with a relatively low vapor pressure of 

approximately 1 x 10-8Torr and a Henry’s Law constant of approximately 2 x 10-10 atmm

3

/mole, volatilization is not expected to be a significant route of dissipation for diphacinone.

The estimated Kds of diphacinone are consistent with compounds, which leach to ground water. 

However, they are bounding estimates from column leaching studies, and it is very likely that the 

true values are much higher, closer to the regression estimates described in the fate assessment. 

The use pattern is such that the primary route of dissipation is likely consumption of treated bait. 

Because it is put into burrows, potential for movement by surface runoff is minimal. There is 

some small chance for leaching based on moderate mobiltiy, but the use pattern and low use

rates make contamination of groundwater unlikely. 

Available data suggest that diphacinone may degrade on soil via aerobic metabolism. The major 

degradate detected in the aerobic soil metabolism study, diphenylglycolic acid, comprised up to 

25% of the applied material in an aerobic soil metabolism study. This degradate appears to be 
8

persistent, but its mobility is unknown. Because diphacinone is formulated in pelleted bait or baitblock rodenticide products and primarily used in bait application or animal burrow treatment, it is 

probable that contact of this degradate with soil and water will be minimal. Exposures and 

resulting risks of non-target organisms to the degradate diphenylglycolic acid are, therefore, not 

quantified for this assessment.

For this assessment, it was assumed that terrestrial animals could be exposed to diphacinone 

through two different pathways. Animals may directly consume bait (primary consumption), or 

animals may consume contaminated carcasses either killed or scavenged by the consumer 

(secondary consumption). 

Based on diphacinone’s octanol to water partitioning ratio (Pow or Kow) of approximately

20,000 (log Pow = 4.3), there is potential for bioaccumulation. However, considering the use 

pattern as a bait, there is a little chance that bioaccumulation will occur in aquatic animals. 

Because Kaput-D is a bait and is confined to below ground terrestrial applications, exposure to 

the aquatic environment is expected to be limited. Screening level EECs confirmed that risk to 

aquatic organisms is below concern levels. 

1.3 Effects Characterization

Diphacinone exhibits high to very high acute and dietary toxicity to small mammals (LD50’s = 

0.2 to 57 mg ai/kg bw; LC50 = 2.08 ppm). It is moderately toxic to birds (LD50’s >400 to 3158 

mg ai/kg bw; LC50’s = 906 to >5000 ppm) on an acute toxicity basis. No chronic data were 

available for birds. Toxicity increases if diphacinone is ingested for several days rather than in a 

single dose. Toxicity data are also available for snakes, where LD50 values were 20.75-31.21 

mg/kg-bw.

Diphacinone is moderately toxic to freshwater fish (LC50 = 2.6 ppm) and invertebrates (LC50 = 

1.8 ppm) on an acute exposure basis. No data are available to evaluate chronic effects to 

freshwater fish or invertebrates. No data are available to evaluate acute or chronic effects to 

estuarine/marine fish or invertebrates. 

No toxicity data are available for aquatic or terrestrial plants. Given the mode of action of 

diphacinone, toxicity to plants is expected to be low.

1.4 Potential Risks to Non-target Organisms

Based on available screening-level information for the proposed use of diphacinone, there is a 

potential for mortality of non-listed and listed terrestrial mammals through consumption of bait. 

In addition, there is potential for mortality of secondary consumers through consumption of prey

that have eaten bait. Risks of mortality to secondary consumers were based on multiple lines of 

evidence including calculation of RQs (mammals and snakes) as well as incident reports of 

mortalities of mammals and birds exposed to diphacinone and secondary toxicity studies. When 
9

considering listed species, there is a potential concern for indirect effects upon the listed 

organisms due to decreased prey availability and habitat modification. Taxa-based risk 

conclusions for direct and indirect effects to listed species are listed in Table 1.1.

Table 1.1. Listed species risks associated with direct or indirect effects due to proposed 

diphacinone use on BPD

Listed Taxonomy Direct Effects Indirect Effects

Terrestrial and semi-aquatic plants – monocots No Yes

Terrestrial and semi-aquatic plants – dicots No Yes

Birds (surrogate for terrestrial-phase amphibians) Yes Yes

Mammals Yes Yes

Reptiles Yes Yes

Aquatic vascular plants No No

Aquatic non-vascular plantsa No No

Freshwater fish (surrogate for aquatic-phase amphibians) No No

Freshwater Invertebrates No No

Freshwater Benthic Invertebrates No No

Estuarine/Marine Fish No No

Estuarine/Marine Crustaceans No No

Estuarine/Marine Mollusks No No

a At the present time there are no Federally listed aquatic non-vascular plants. The taxonomic group is 

included here for the purposes of evaluating potential contributions to indirect effects to other taxonomy 

and as a record of exceedances should future listings of non-vascular aquatic plants warrant additional 

evaluation of Federal actions.

The LOCATES database (version 2.13) identifies those U.S. counties in which a given crop is 

grown for which there are co-located listed species. For this analysis, it was presumed that all 

federally-listed endangered or threatened terrestrial animals in the states of CO, KS, NE, NM, 

ND, SD, MT, OK, TX and WY may be directly or indirectly affected. With additional 

refinement by exploring more detailed use patterns and species biology (e.g., geographic 

location, specific feeding habits, time of year likely to utilize crop fields), some species listed 

may be determined to be not likely to be affected.

The lines of evidence supporting the conclusion of risk include:

 Incidents in the US reported in EIIS that were considered probable or highly probable for 

death due to diphacinone. Mortalities included species that were likely primary 

consumers of the bait and carnivorous species likely to have preyed upon the granivorous 

individuals. Reported incidents with known or potential impact on endangered species 

are:

o Take of an endangered species was reported. An adult kit fox was found dead in 

Bakersfield CA (weighing 5.75 lbs). Concentrations of diphacinone by

immunoassay were found in liver and blood at a concentration of 0.18 ppm 

(Incident #B000640-001). 

 Avian, mammalian and reptilian (snake) mortality through exposure to diphacinone was 

documented through low LD50 and LC50 values obtained in toxicity studies and/or the 

reported incident data. 
10

1.5 Related Biological Opinions

The Fish and Wildlife Service (FWS) recently completed a Biological Opinion on a registration 

action that allows use of another anticoagulant rodenticide (chlorophacinone; trade name of 

Rozol black-tailed prairie dog bait - hereafter referred to as Rozol) to control black tailed prairie 

dogs. Although the Agency’s endangered species risk assessment determined that 21 federally 

listed species may be affected, a number of conservation measures were developed with 

discussions with and agreement between the Fish and Wildlife Service, EPA, and the registrant 

to either reduce or preclude exposures to many of these species. The Agency’s effects 

determination for Rozol and resulting Biological Opinions are available on-line in the docket at 

regulations.gov and may be accessed using the following link: 

http://www.regulations.gov/#!docketDetail;dct=FR%252BPR%252BN%252BO%252BSR;rpp=

25;so=ASC;sb=docId;po=0;D=EPA-HQ-OPP-2011-0909

These conservation measures were part of the federal action and included label amendments to 

add specifications for a line-transect carcass search and reporting of dead/dying listed species 

and non-target animals in addition to prohibitions of Rozol use and timing restrictions via the 

Agency’s Endangered Species Protection Program (ESPP) Bulletins Live system available at: 

http://www.epa.gov/oppfead1/endanger/bulletins.htm. The Biological Opinion on Rozol 

(http://epa.gov/espp/2012/borozol-final.pdf) found that the conservation measures adequately

protected most of the endangered species, although FWS concluded that incidental take may still 

occur for the following three species: (1) black-footed ferret; (2) gray wolf; and (3) northern 

aplomado falcon. In order to minimize incidental take for these three species, FWS identified a 

number of additional reasonable and prudent measures (RPMs) including certified applicator 

training, development of website content to reduce the potential for secondary poisoning, 

requirements for the registrant to develop a stewardship training program and submit of Rozol 

distribution and production data, and additional bulletins for the northern aplomado falcon.

Additional details are included in the Biological Opinion available on the docket at the 

aformentioned link. 

The exposure and toxicity profiles are similar for chlorophacinone and diphacinone (see table 

below). In tests conducted in the same species, diphacinone is consistently less toxic to birds 

relative to chlorophacinone, and it is of similar toxicity to mammals (slight increases and 

decreases in median lethal doses/concentrations have been observed). Therefore, because the 

area of potential use is identical for Rozol and Kaput black-tailed prairie dog baits given that 

they are both assumed to be applied anywhere within the range of black tailed prairie dogs, the 

listed species that may be exposed are also identical. Also, given that the exposure and toxicity

profiles are similar, the conservation measures and RPMs identified for Rozol should also be 

protective of endangered species if implemented on Kaput black tailed prairie dog bait. In 

addition, labels for both Rozol and Kaput will stipulate that other anticoagulant rodenticides 

cannot be used if applying either of these products. For this reason, the Biological Opinion 

issued on Rozol is expected to be applicable to both Rozol and Kaput black tailed prairie dog 

bait, and is, therefore, expected to provide adequate protections for listed species for use of both 

rodenticides. 
11

Table 5.7. Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters

Comparative Parameter Chlorophacinone Diphacinone Comments

Exposure Concentration in Bait 0.005% 0.005% --

Food Consumption Not chemical dependent --

Time to mortality 4-13 days 3-9 days Time to mortality affects the 

amount of chemical that can 

be accumulated over time 

for target pests.

Liver Retention 35 days - mouse 3 days – Rat

5 days – Pig

>90 days - Cow

Lack of data in same species 

precludes definitive 

comparisons

Toxicity, Birds BWQ LD50s (mg/kg-bw) 260 1630 --

Other Avian LD50s (mg/kg-bw) None available Kestrel: 96.8

Mallard: 3200

--

Mallard LC50s (mg/kg-diet) 172 906 --

BWQ LC50 (mg/kg-diet) 56 >5000 --

Other Species (mg/kg-diet) 60 (Japanese Quail) Brown Tree Snake: 

20.75

--

Toxicity 

Mammals

Lab Rat – Gavage (mg/kg-bw) 6 1.9 --

Pine Vole – Gavage (mg/kg-bw) 14 58 --

Other Species – Gavage (mg/kgbw)1.9 (prairie dog) 0.2 (mongoose)

0.6 (Coyote)

0.9 to 15 (cats and 

dogs)

14 (meadow vole)

--

Lab Rat- Dietary (mg/kg-diet) 1 2.08 --

Green cells denote lower toxicity

1.6 Data Gaps

As cited in the RED (USEPA, 1998b), the only available environmental fate data from studies 

for diphacinone are 1) physicochemical properties, 2) hydrolysis, 3) aerobic soil metabolism, and 

4) aged column leaching. However, because of the specific results of studies and because the 

specific use and application rates for diphacinone, relative to established end-point toxicities, is 

low, EFED can satisfactorily evaluate/screen limits of potential environmental concentrations 

and ecological effects without additional environmental fate data.

Studies with a potential to add value to the risk assessment are:

 Field Testing for Terrestrial Wildlife (850.2500)

 Acute Passerine Study (850.2100) 

 Avian Reproduction Study (850.2300), two species
12

2.0 Problem Formulation

The purpose of problem formulation is to provide the foundation for the environmental fate and 

ecological risk assessment being conducted for the proposed new use of diphacinone. As such, it 

articulates the purpose and objectives of the risk assessment, evaluates the nature of the problem, 

and provides a plan for analyzing the data and characterizing the risk (USEPA, 1998a). 

2.1 Nature of Regulatory Action

The proposed Section 3 registration is for the use of Kaput-D

®

Prairie Dog Bait on Black-tailed 

Prairie Dogs (BPD; Cynomus ludivicianus). This restricted use product is bait that contains 

0.005% diphacinone (50 mg a.i./kg-bait) and must be hand applied on rangeland and adjacent 

non-crop areas at least 6” into active burrows. No bait is to be placed on or above ground level. 

The product label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, TX, MT and 

OK. Historic range of the BPD is depicted in Figure 1 below. 

2.2 Previous Risk Assessments

Since 1998, EPA has conducted a number of ecological risk assessments on diphacinone. These 

assessments have consistently identified potential risks to birds and mammals from primary and 

secondary exposure as the predominant risk concern for diphacinone. These assessments include 

the following:

 An assessment of the risks of rodenticide uses of diphacinone, along with seven other 

rodenticides, in the Registration Eligibility Decision (RED): Rodenticide Cluster that was 

published in 1998. This document included an ecological effects risk assessment that 

was based on environmental fate and ecotoxicological studies that had been submitted by

the registrants of diphacinone at that time. 

 An assessment of potential risks of nine rodenticides, including diphacinone, to birds and 

mammals was completed in 2004 (Erickson and Urban 2004). 

 Several Section 18 (emergency exemption) assessments involving diphacinone were 

completed in 2009. 

 An endangered species assessment was completed in 2011. It assessed risk from the use 

of diphacinone to the Federally Threatened Alameda Whipsnake (Masticophis lateralis 

euryxanthus) and the California Tiger Salamander (Ambystoma californiense), as well as 

risks to the Federally Endangered Salt Marsh Harvest Mouse (Reithrodontomys 

ravivertis), California Tiger Salamander (Ambystoma californiense) Sonoma County

Distinct Population Segment and Santa Barbara County Distinct Population Segment, and 

San Joaquin Kit Fox (Vulpes macrotis mutica). 

In addition, in March of 1993, a Biological Opinion (Effects of 16 Vertebrate Control Agents on 

Threatened and Endangered Species) was issued by the US Fish & Wildlife Service. The Service 

determined that risk was minimal to avian species from direct consumption of treated bait. 

However, secondary toxicity to raptors was possible from consumption of treated rodents. Risk 

to mammals was also indicated. Specific jeopardy was indicated for the Salt Marsh Harvest 

Mouse (SMHM) and the San Joaquin Kit Fox (SJKF).
2.3 Stressor Source and Distribution

2.3.1 Mechanism of Action 

Diphacinone is a first generation 

the gut. Anticoagulants are vitamin

mechanisms and induce capillary damage. Typically, death is delayed for four to ten or more 

days after a lethal dose is ingested, and animals may continue to feed and move about until 

shortly before death. Death results from hemorrha

that may make them more susceptible to predation (

indandiones also uncouple oxidative phosphorylation (energy generation) in mammals

Diphacinone seems to have a different mo

result from anticoagulation and its mode of action in snakes is unknown.

2.3.2 Nature of the Chemical Stressor

Diphacinone has moderate leaching potential. 

combined with a low solubility (0.3 mg/L) 

water. Consistent with a relatively low vapor pressure of approximately 1

Henry’s Law constant of approximately 2

be a significant route of dissipation for diphacinone.

mobile in soil; however, its chemical mobility is expected to be limited by the application 

method and formulation as a bait

a half-life value of approximately 30

a Kow of 20,000 indicating the potential for bioaccumulation in animal tissue. 

measures of bioaccumulation are not available. One primary dissipation pathway of diphacinone 

is expected to be consumption of bait by target and non

Table 2.1 lists the physical-chemical properties of 

environmental fate properties of 

detected in the submitted environmental fate and transport studies

Table 2.1. Physical-Chemical Properties of 

Property Value

Structure

Molecular 

Weight

340.48 g/mole

13

Stressor Source and Distribution

Mechanism of Action 

first generation indanedione anticoagulant rodenticide and is absorbed through 

Anticoagulants are vitamin-K antagonists that disrupt normal blood-clotting 

mechanisms and induce capillary damage. Typically, death is delayed for four to ten or more 

days after a lethal dose is ingested, and animals may continue to feed and move about until 

shortly before death. Death results from hemorrhage, and exposed animals may exhibit behavior 

that may make them more susceptible to predation (Cox and Smith, 1992). In addition, 

ndandiones also uncouple oxidative phosphorylation (energy generation) in mammals

Diphacinone seems to have a different mode of action in reptiles. Mortality occurs but does not 

result from anticoagulation and its mode of action in snakes is unknown.

Nature of the Chemical Stressor

iphacinone has moderate leaching potential. The range of possible Kd values is 3 to 

low solubility (0.3 mg/L) indicates that it is unlikely to contaminate ground 

Consistent with a relatively low vapor pressure of approximately 1 x 10-8

Henry’s Law constant of approximately 2 x 10-10 atm-m

3

/mole, volatilization is not expected to 

be a significant route of dissipation for diphacinone. Diphacinone is expected to be moderately 

; however, its chemical mobility is expected to be limited by the application 

method and formulation as a bait. Diphacinone is metabolized via aerobic soil metabolism

life value of approximately 30 days for one soil tested in a single study. Diphacinone has 

of 20,000 indicating the potential for bioaccumulation in animal tissue. However, direct 

s of bioaccumulation are not available. One primary dissipation pathway of diphacinone 

is expected to be consumption of bait by target and non-target animals.

chemical properties of diphacinone. Table 2.2 lists the 

environmental fate properties of diphacinone, along with the major and minor degradates 

detected in the submitted environmental fate and transport studies. 

hemical Properties of Diphacinone

Source Comment

--

Chemical Name: 

2-(diphenylacetyl)indan

-- --

is absorbed through 

clotting 

mechanisms and induce capillary damage. Typically, death is delayed for four to ten or more 

days after a lethal dose is ingested, and animals may continue to feed and move about until 

ge, and exposed animals may exhibit behavior 

In addition, 

ndandiones also uncouple oxidative phosphorylation (energy generation) in mammals.

de of action in reptiles. Mortality occurs but does not 

is 3 to 5 mL/g

that it is unlikely to contaminate ground 

8

Torr and a 

ization is not expected to 

Diphacinone is expected to be moderately 

; however, its chemical mobility is expected to be limited by the application 

aerobic soil metabolism with 

Diphacinone has 

However, direct 

s of bioaccumulation are not available. One primary dissipation pathway of diphacinone 

lists the other 

degradates 

Comment

(diphenylacetyl)indan-1,3-dione

--
14

Solubility 0.3 mg/L EXTOXNET http://extoxnet.orst.edu/pips/diphacin.htm

Vapor 

Pressure

1 x 10-8torr DP268619, 2000 --

Henry’s Law 2 x 10-10 atm-m

3

/mole DP268619, 2000 --

Kow 20,000 DP268619, 2000 --

Table 2.2. Summary of Diphacinone Environmental Fate Properties

Study

Value and 

unit

Major Degradate

Minor Degradates MRID # or 

Citation

Study Classification, 

Comment

Hydrolysis Stable at 7 

and 9. 44 

day half life 

at pH 5

-- DP268619, 2000 --

Photolysis in Water Stable -- DP268619, 2000 --

Photodegradation on Soil Stable -- Assumed to be stable

Aerobic Soil Degradation 30 days 

(half-life)

diphenylglycolic acid

(25%)

DP268619, 2000

30 days in single soil 

study 

Aerobic Aquatic 

Degradation

180 days 

(half-life)

-- 2X aerobic soil 

value

No study submitted, 

assumed value

Kd 3-5 -- DP268619, 2000 4 soils

Koc 300 -- DP268619, 2000 Supplemental column 

leaching study

Degradation/Metabolism

Laboratory aerobic soil metabolism data are available from a single sandy loam soil from North 

Dakota. The mean half-life of diphacinone in this soil was 30 days (average of 28 and 32 days) 

from studies done at two different radiolabeled positions. A major soil metabolite (> 10% of 

dose) was diphenylglycolic acid which peaked at a maximum of approximately 20-25% of the 

initial dose around one month after treatment. This metabolite then remained at a relatively 

constant concentration until the end of the 3.5 month study. Up to eight minor organic 

metabolites were separated at various times, but not identified; these generally reached relatively

constant concentrations ranging from a fraction of 1% to around 6% of the initial dose. (It is 

possible that some of these metabolites from separate labels may not be distinct compounds.) 

Volatile organics were negligible. Carbon dioxide was regularly and steadily produced from 

both radiolabels, and reached approximately 40% of the dose from both labels by the end of the 

study period. Recalcitrant soil residues also accounted for a large fraction of the dose from both 

labels, amounting to approximately 20-30% of the initial dose by study end. The evolved carbon 

dioxide and soil bound residuals are clearly indicative of ultimate biological deactivation of a 
15

large fraction of diphacinone and its transformation products. This study also indicated, 

however, that the major metabolite, diphenylglycolic acid, was somewhat resistant to further 

metabolism.

Diphacinone was stable against simple hydrolysis at pH 7 and 9. At pH 5, hydrolysis proceeded 

with an extrapolated half-life of approximately 44 days. Products of hydrolysis were not 

satisfactorily identified or quantified. However, since the apparent hydrolysis was appreciable 

only at low pH and aerobic metabolism provides a viable alternative transformation process for 

diphacinone, there is marginal value in additional information on hydrolytic degradates at low 

pH.

Mobility

In Air. Volatilization of diphacinone or degradates was not detected in the aerobic soil 

metabolism study. A relatively low vapor pressure of approximately 1 x 10-8torr and a Henry’s 

law constant for diphacinone of approximately 2 x 10-10 atm-m

3

/mole also indicates that 

volatilization should not be a significant route of dissipation.

In Soil. Because of numerous deficiencies in the aged column leaching study (including 

not using radiolabeling, irregular soil columns, no reporting of elution rate, inability to detect 

less than 2% of applied diphacinone in the eluate or soil columns, no estimation of sorption 

coefficients, no attempt to identify and quantify degradates), the RED and previous reviews 

simply gave qualitative statements to the effect that diphacinone was “relatively immobile” 

because it was not detected in the leachates of the four soils tested and moved significantly only 

in a loamy sand soil. However, if we were to take the column results at face value and accept the 

limit of six centimeter soil increment study resolution, we can approximate sorption coefficients 

by estimating column retardation factors and using nominal assumptions about soil bulk density

and porosity. For simplicity and convenience, we have assumed a nominal and uniform soil bulk 

density of 1.5 g/cm3and a porosity of 0.5 or 50%. Within error limits, choosing any reasonable 

specific values would not alter conclusions as explained in the following paragraph.

For three of the four soils (sandy loam, silt loam, sand), diphacinone was essentially confined to 

the top 6 cm of 30 cm columns (in one of these soils there was small migration into the 6 to 

12 cm interval). There is the possibility that, within the stated experimental error of 2%, as much 

as 2% of the applied could be in each of four remaining increments. However, for the purposes 

of approximation we non-conservatively assume no chemical in these increments. We also 

assume the chemical was centered at the half-way point of each 6 cm increment–for example, at 

3 cm for the top increment. This may or may not be a conservative assumption because of the 

6 cm limit of resolution. For the loamy sand soil column in which leaching occurred down to the 

18-24 cm increment, we calculated the center of mass of the incremental distribution of 

diphacinone and assumed that an undetected 2% reached the lowest 24-30 cm depth increment 

(which was essentially an inconsequential assumption). The resulting calculated sorption 

coefficient (Kd) for the three soils is roughly 5 mL/g with uncertain but “wide” error bars, if for 

no other reason than depth increment resolution. For the loamy sand soil, the corresponding 
16

calculated Kd is approximately 3 mL/g. Compounds with Kd values in this range are often 

classified as mobile. The loamy sand result should be a better approximation than that for the 

other soils because there were measurable concentrations at several depth increments. As stated 

previously, the registrant did not address the mobility of degradates in soil.

There is also an alternative approximation which can be made to estimate crudely the soil to 

water partitioning coefficient with the assumption that soil organic matter determines sorption. 

The estimation is based on an empirical correlation between the octanol to water partitioning 

ratio (Pow or Kow) and the soil organic matter to water sorption coefficient (Koc). The 

relationships1, assuming soil organic matter is typically about 58% carbon [i.e., organic 

carbon (oc) = organic matter (om)/1.72)], are 

log (Kom) = 0.904 log (Pow) - 0.779 (n = 12, r-squared = 0.989)

log (Koc) = 0.904 log (Pow) - 0.779 + log 1.72

Another empirical relationship given in a technical guidance document supported by the 

European Community (Directive 93/67/EEC) to calculate Koc from Pow is:

log Koc = 0.52 log (Pow) + 1.02

Log Pow for diphacinone is 4.27 (see comment on this value in Bioconcentration paragraph 

below). Thus, from the first relationship, log Koc = 3.32 and Koc is roughly 2100; from the 

second, log Koc = 3.24 and Koc is roughly 1700. These similar Koc values and the Kd values 

above place diphacinone in an intermediate range of mobility. 

Bioaccumulation

The registrant has not submitted a bioconcentration study at this time. However, judging from 

the diphacinone octanol to water partitioning ratio (Pow or Kow) of approximately 20,000 (log 

Pow = 4.3), there is potential for bioaccumulation in aquatic systems; however, there is limited 

potential for water contamination given the the use pattern to control black tailed prairie dogs. 

Also, diphacinone has an estimated octanol air partitioning coefficient of 12.5 (EPI Suite), which 

suggests that it may accumulate in terrestrial animals, which is also consistent with the secondary

exposure risks identified for diphacinone in the current and past assessments. 

 

1

Sawhney, B. L. and Brown, K., Editors. Reactions and Movement of Organic Chemicals in 

Soils. SSSA Special Publication Number 22. Soil Science Society of America, Inc. Madison, 

WI. 1989.
17

2.3.3 Environmental Transport Mechanisms

Potential transport mechanisms typically include pesticide surface water runoff, spray drift, and 

secondary drift of volatilized or soil-bound residues leading to deposition onto nearby or more 

distant ecosystems. However, because the uses of diphacinone are in bait application or animal 

burrow treatment, spray drift is presumed to be negligible. Because diphacinone bait may be 

used outdoors, some potential exist for residues of diphacinone to leach from the bait that is 

exposed to rainwater or runoff. Due to the low application rate, the low drift potential, and the 

formulation within a bait, the potential for contaminating surface water is believed to be 

insignificant. 

Another possible route of transport is within the bodies of animals which feed on the 

diphacinone bait. Because poisoned animals would not be killed immediately, they would travel 

some distance before dying, thereby exposing animals some distance away from the use site. 

This transport within animals is an important route of exposure for predatory birds and mammals 

since their diet includes small mammals, and thus vulnerable to secondary exposure from 

consuming poisoned rodents.

2.4 Overview of Pesticide Usage

2.4.1 Label for Proposed Registered Use

The proposed Section 3 label advises that KAPUT-D baits be hand applied on rangeland and 

adjacent non-crop areas at least 6” into active burrows. No bait is to be placed on or above 

ground level. The product label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, 

TX, MT and OK. The current black tailed prairie dog range is depicted in the following figure as 

presented in the Biological Assessment for Rozol Black Tailed Prairie Dog Bait (EPA, 2010). 
18

Figure 1. Historic Range of Black Tailed Prairie Dogs
19

2.5 Receptors

In order for a chemical to pose an ecological risk, it must reach ecological receptors in 

biologically significant concentrations. An exposure pathway is the means by which a 

contaminant moves in the environment from a source to an ecological receptor. For an 

ecological exposure pathway to be complete, it must have a source, a release mechanism, an 

environmental transport medium, a point of exposure for ecological receptors, and a feasible 

route of exposure. In addition, the potential mechanisms of transformation (i.e., which 

degradates may form in the environment, in which media, and how much) must be known, 

especially for a chemical whose metabolites/degradates are of greater toxicological concern. The 

assessment of ecological exposure pathways, therefore, includes an examination of the source 

and potential migration pathways for constituents, and the determination of potential exposure 

routes (e.g., ingestion, inhalation, and dermal absorption). 

Ecological receptors that may potentially be exposed to diphacinone include terrestrial wildlife 

(i.e., invertebrates, mammals, birds, and reptiles), terrestrial and semi-aquatic plants, aquatic 

plants, aquatic invertebrates and fish. In addition to terrestrial ecological receptors, aquatic 

receptors (e.g., freshwater and estuarine/marine fish and invertebrates, amphibians) may also be 

exposed to potential migration of pesticides from the site of application to various watersheds 

and other aquatic environments via runoff and drift.

Consistent with the process described in the Overview Document (USEPA, 2004a), this risk 

assessment uses a surrogate species approach in its evaluation of the proposed new uses of 

diphacinone. Data generated from surrogate test species, which are intended to be representative 

of broad taxonomic groups, are used to extrapolate to potential effects on a variety of species 

(receptors) included under these taxonomic groupings. 

Acute and chronic toxicity data from studies submitted by pesticide registrants are used to 

evaluate the potential direct effects of diphacinone to the aquatic and terrestrial receptors 

identified in this section, including toxicity data on the technical grade active ingredient, and 

when available, on formulated products. The evaluation of these data can also provide insight 

into the direct and indirect effects of diphacinone on biotic communities from loss of species that 

are sensitive to the chemical and from changes in structure and functional characteristics of the 

affected communities. 

The toxicity data used in this assessment are obtained from registrant-submitted guideline 

toxicity studies required for pesticide registration. These studies are typically performed on a 

number of organisms from several taxonomic groups, including birds, mammals, fish, and 

aquatic invertebrates (USEPA 2004a). The surrogate test species, assessment endpoints, and 

measures of effect derived from these studies used for this risk assessment are provided.

Additional information from other sources, such as public literature and incident reports, are 

used when relevant.
20

2.6 Ecosystems Potentially at Risk

The ecosystems at risk are often extensive in scope; therefore, it may not be possible to identify

specific ecosystems during the development of a baseline risk assessment. However, in general 

terms, terrestrial ecosystems potentially at risk could include the treated BTP habitat and areas 

immediately adjacent to the treated areas that may receive runoff. Areas adjacent to the treated 

field could include cultivated fields, fencerows and hedgerows, meadows, fallow fields or 

grasslands, woodlands, riparian habitats and other uncultivated areas. 

2.7 Assessment Endpoints

Assessment endpoints represent the actual environmental value that is to be protected, defined by 

an ecological entity (species, community, or other entity) and its attribute or characteristics 

(USEPA 2004a). For diphacinone, the ecological entities may include the following: birds, 

mammals, reptiles, amphibians, terrestrial plants and insects. The attributes for each of these 

entities may include growth, reproduction, and survival. 

Selection of the assessment endpoints is based on valued entities (i.e., ecological receptors), the 

ecosystems potentially at risk, the migration pathways of pesticides, and the routes by which 

ecological receptors are exposed to pesticide-related contamination. The selection of clearly 

defined assessment endpoints is important because they provide direction and boundaries in the 

risk assessment for addressing risk management issues of concern.

2.8 Conceptual Model

A conceptual model provides a written description and visual representation of the predicted 

relationships between diphacinone, potential routes of exposure, and the predicted effects for the 

assessment endpoint. A conceptual model consists of two major components: risk hypotheses 

and a conceptual diagram (USEPA, 1998a).

2.8.1 Risk Hypotheses

Based on the results of previous risk assessments for diphacinone, the following ecological risk 

hypotheses are being employed for this risk assessment:

 Terrestrial organisms are subject to adverse direct effects such as reduced survival, growth, 

and reproduction when exposed to diphacinone residues as a result of labeled use of the 

pesticide.

 Indirect effects, such as habitat, food web dynamics, perturbing forage or prey availability, 

and altering the extent and nature of nesting, will potentially occur if residue concentrations 

exceed levels of concern for acute or chronic exposure for terrestrial species. 

Based on previous assessments and the mode of action of diphacinone, the primary concern for 

this assessment is potential risks to non-target terrestrial animals either from direct consumption 

of bait (primary exposure) or from consumption of contaminated animals that have consumed 

treated bait (secondary exposure). 
21

2.8.2 Conceptual Diagram

The conceptual model assumes that KAPUT-D bait will be available to non-target organisms 

and, as toxic food bait, will adversely affect terrestrial species. Figure 2.3 illustrates the 

anticipated exposure pathways and transport routes to ecological receptors and identifies the 

predicted attribute changes from this exposure. The major sources of exposure of non-target 

terrestrial animals are expected to be ingestion of the bait and consumption of vertebrate body 

tissues that have eaten the food bait (USEPA 2004b). Exposure via these routes is expected 

primarily for birds and mammals, though it is likely that other terrestrial animals such as reptiles 

and terrestrial amphibians are at risk if they consume invertebrates or tissues of vertebrates that 

have eaten bait. 

Diphacinone is applied as a bait and little is expected to partition into drinking water source (e.g.

puddles) compared to that which is available for direct consumption on the bait itself; therefore,

this route of exposure was not assessed. Since diphacinone is not sprayed directly onto plants, 

and because so little is expected to leach from the bait and then be available for plant uptake, 

consumption of diphacinone on plants is also not being considered as a route of exposure. 

Dermal and inhalation routes of exposure are not expected to be important routes of exposure for 

a rodenticide bait such as KAPUT-D given the application method and formulation.
22

Figure 2.3. Conceptual Model Diagram of Diphacinone Exposure and Effects in Nontarget 

Species. Dotted lines indicate exposure pathways that have a low likelihood of contributing to 

ecological risk.

Stressor

Transport 

Pathways

Incidental Deposition in Water

Consumption by Target or NonTarget Terrestrial Vertebrates

Deposition on Ground

Exposure 

Media

Water Body/ Standing 

Water

Upland/Riparian Soil

Primary 

Exposure 

Route and 

Receptors

Ingestion of Water by

Terrestrial Vertebrates

Consumption and/or Uptake 

by Soil Macroinvertebrates

Exposed Intact Bait at 

Application Site

Secondary 

and Tertiary 

Exposure 

Routes and 

Receptors Consumption of Tissues and 

Eggs by Predators/Scavengers

Attribute 

Changes

Bioaccumulation and Food 

Chain Magnification

Reduced Reproduction and Survival of Terrestrial NonTarget Organisms

Diphacinone
23

2.9 Analysis Plan

In order to address the risk hypotheses, the potential for direct and indirect effects to the assessed 

species, prey items, and loss of burrows is estimated based on a taxon-level approach. In the 

following sections, the use, environmental fate, and ecological effects of diphacinone are 

characterized and integrated to assess the risks. This is accomplished using a RQ (ratio of 

exposure concentration to effects concentration) approach. Although risk is often defined as the 

likelihood and magnitude of adverse ecological effects, the RQ-based approach does not provide 

a quantitative estimate of likelihood and/or magnitude of an adverse effect. However, as 

outlined in the Overview Document (USEPA 2004a), the likelihood of effects to individual 

organisms from the use of diphacinone to control BPDs is estimated using the probit doseresponse slope and either the LOC (discussed below) or actual calculated RQ value. 

2.9.1 Measures of Exposure 

2.9.1.1 Estimating Aquatic Exposure

Because Kaput-D is a bait and is confined to below ground terrestrial applications, exposure to 

the aquatic environment should be very limited, thus no aquatic assessment was conducted 

because it was assumed that risk would be very low and below levels of concern. Concentrations 

from prior assessments were in pptr (ng/l). 

There are no aquatic monitoring data available for the use of diphacinone. For diphacinone 

usage as bait application or animal burrow treatment, it is possible to approximate a ‘high-end’ 

exposure scenario with two tier 1 models: GENEEC model for surface water exposure and 

SCIGROW model for ground water exposure. The peak surface water concentration (EEC) 

predicted by GENEEC is 483 ng/L. The peak ground water concentration (EEC) predicted by 

SCIGROW is 4.75 ng/L (concentrations taken from a previous assessment with equal or greater 

application rates). 

2.9.1.2 Estimating Primary Terrestrial Exposure

EFED’s exposure assessment for the rodenticides differs from that for most other pesticides. For

a rodenticide, the bait itself is the potential food item of concern. Thus, the amount of active 

ingredient in the formulated bait is used as an EEC. This information is used to estimate the 

amount of grain bait that birds and mammals of various sizes need to consume to obtain a dose 

expected to be lethal to 50% of the individuals in the population (i.e., LD50 dose). Estimates of 

food-ingestion rates (g dry matter per day) are determined from established allometric equations 

presented in the Wildlife Exposure Factors Handbook (USEPA 1993). The concentration of 

diphacinone in bait is also used to estimate initial dietary exposure (mg a.i. per kg in bait) which 

in turn is used to calculate avian and mammalian dietary RQs. 
24

2.9.1.3 Estimating Secondary Terrestrial Exposure

Secondary exposure analysis (from consumption of contaminated prey and/or scavenged 

carcasses) requires consideration of residues in the bodies of organisms that are commonly 

consumed by predators and scavengers. Details of the analysis are included in Section 3.

2.9.2 Measures of Effect

Data identified in Section 4 are used as measures of effect for direct and indirect effects. Data 

were obtained from registrant submitted studies or from literature studies identified by

ECOTOX. 

2.9.2.1 Integration of Exposure and Effects

Risk characterization is the integration of exposure and ecological effects characterization to 

determine the potential ecological risk from the use of diphacinone to control Black-tailed Prairie 

Dogs, and the likelihood of direct and indirect effects to the assessed species in terrestrial 

habitats. The exposure and toxicity effects data are integrated in order to evaluate the risks of 

adverse ecological effects on non-target species. The RQ method is used to compare exposure 

and measured toxicity values. EECs are divided by acute and chronic toxicity values. The 

resulting RQs are then compared to the Agency’s LOCs (USEPA 2004a). 

2.9.3 Use of Probit Slope Response Relationship to Provide Information on the 

Endangered Species Levels of Concern

As part of the risk characterization, an interpretation of acute RQs for listed species is discussed. 

This interpretation is presented in terms of the chance of an individual event (i.e., mortality or 

immobilization) should exposure at the EEC actually occur for a species with sensitivity to 

diphacinone on par with the acute toxicity endpoint selected for RQ calculation. To accomplish 

this interpretation, the Agency uses the slope of the dose response relationship available from the 

toxicity study used to establish the acute toxicity measures of effect for each taxonomic group 

that is relevant to this assessment. The individual effects probability associated with the acute 

RQ is based on the mean estimate of the slope and an assumption of a probit dose response 

relationship. In addition to a single effects probability estimate based on the mean, upper and 

lower estimates of the effects probability are also provided to account for variance in the slope, if 

available. 

Individual effect probabilities are calculated based on an Excel spreadsheet tool IECV1.1 

(Individual Effect Chance Model Version 1.1) developed by the U.S. EPA, OPP, Environmental 

Fate and Effects Division (June 22, 2004). The model allows for such calculations by entering 

the mean slope estimate (and the 95% confidence bounds of that estimate) as the slope parameter 

for the spreadsheet. In addition, the acute RQ is entered as the desired threshold. 
25

2.9.4 Data Gaps

Ecological

A data gap is identified for Field Testing for Terrestrial Wildlife (850.2500). 

The purpose of this study is to evaluate the availability of carcasses containing diphacinone 

residues to carnivores and scavengers. This study would also be useful for estimating 

diphacinone concentrations in prey. The purpose of this requested study is to provide 

quantitative data that are currently unavailable for KAPUT-D Prairie Dog Bait to assess the 

availability of carcasses. More specifically, the study should be designed to evaluate:

 the availability of poisoned/incapacitated prairie dogs and non-target animals to predators 

and scavengers,

 residue levels in dead and dying prairie dogs,

 the effect of carcass collection on mitigating exposure, and

 the carcass collection efficacy (per label directions).

This study has a potential to add considerable value to the ecological risk assessment. Because 

there is no Agency protocol for this study, a protocol for the carcass availability study should be 

submitted to EPA for approval prior to study initiation.

A data gap is identified for the Avian Reproduction Study (850.2300). 

Both the waterfowl and upland game bird reproduction studies have potential to add value to the 

ecological risk assessment. No acceptable data are available to assess possible reproductive 

effects to avian species from primary or secondary/tertiary exposure to diphacinone. Due to the 

increased use area and the fact that poisoned and moribund Prairie Dogs are easy prey and 

scavenge items for raptors and other birds, avian reproduction data are needed to adequately

quantify the risks this proposed use poses.

An additional data gap is identified for an Avian Acute Oral Toxicity Test (850.2100) conducted 

using a passerine species. Under 40 CFR Part 158, data are required for one passerine species 

and either one waterfowl species or one upland game bird species for terrestrial, aquatic, 

forestry, and residential outdoor uses. The current method of calculating a weight-adjusted LD50

using bobwhite quail or mallard duck data may over- or under-estimate risks to passerines 

because these birds may metabolize the chemical differently. Because of the documented high 

toxicity to birds through submitted studies and the reported incidents, information regarding 

relative toxicity to passerines is essential for further characterization of the avian risks of 

diphacinone. This study has a potential to add value to the ecological risk assessment. Because 

the 850.2100 guideline has not yet been finalized, protocols for the study of passerine species 

should be submitted to EPA for approval prior to study initiation.

Environmental Fate

As cited in the RED (USEPA, 1998b), the only available environmental fate data from studies 

for diphacinone are 1) physicochemical properties, 2) hydrolysis, 3) aerobic soil metabolism, and 

4) aged column leaching. However, because of the specific results of studies and because the 

specific use and application rates for diphacinone relative to established end-point toxicities is 
26

low, EFED can satisfactorily evaluate/screen limits of potential environmental concentrations 

and ecological effects without additional environmental fate data.

3.0 Exposure Assessment

3.1 Label Application Rates and Intervals

For this assessed use (i.e., control of BPD), diphacinone is formulated as a bait composed of 50 

mg a.i./kg-bait. The proposed Section 3 label submitted to EPA describes methods of application 

as detailed previously. It must be hand applied on rangeland and adjacent non-crop areas at least 

6” into active burrows.

3.2 Aquatic Exposure Assessment

Aquatic exposure from the use of diphacinone is expected to be negligible, and thus aquatic 

routes of exposure are not predicted to make significant contributions to total exposure. Also, 

indirect risk from potential effects on aquatic organisms was assumed to be negligible due to 

lack of direct effects to aquatic organisms. 

3.2.1 Aquatic EECs

There are no aquatic monitoring data available for the use of diphacinone. For diphacinone 

usage as bait application or animal burrow treatment, it is possible to approximate a ‘high-end’ 

exposure scenario with two tier 1 models: GENEEC model for surface water exposure and 

SCIGROW model for ground water exposure. The peak surface water concentration (EEC) 

predicted by GENEEC is 483 ng/L. The peak ground water concentration (EEC) predicted by

SCIGROW is 4.75 ng/L (concentrations taken from previous assessment). Thus, aquatic routes 

of exposure are not predicted to make significant contributions to total exposure

3.3 Terrestrial Exposure Assessment

For this assessment, it was assumed that terrestrial animals could be exposed in two different 

pathways. Animals may directly consume bait (primary consumption), or animals may consume 

contaminated carcasses either killed or scavenged by the consumer (secondary consumption). 

Both approaches and the expected exposure levels are detailed below. 

3.3.1 Expected Diphacinone Ingestion through Bait Consumption

Exposure through bait consumption will be calculated using two methodologies. For the first 

method, diphacinone exposure is calculated as mg a.i./kg-bwt, where kg-bwt is the kilograms of 

the consuming individual for three standard weight classes of passeriforms and rodents. 

Exposure (food dry weight consumption) estimates were derived using allometric equations from 

USEPA (1993). The allometric equations for passeriform birds and rodent mammals were used 

as these would best approximate those individuals with high potential for consuming grain and 

they would give the most conservative exposure estimates. Food dry weight was converted to 
27

wet weight assuming the bait contained 10% water, similar to the assumption that seeds for 

wildlife consumption contain 10% water (USEPA 1993). Formulas for calculation of dose 

estimates are provided in Table 3.1, and exposure estimates (on a dose basis) are provided in 

Table 3.2. RQs are generated by dividing these exposure estimates (mg a.i./kg-bwt) for a given 

weight class by the most conservative toxicity endpoint for the relevant taxa adjusted for the 

default body weights. Acute RQs using these exposure estimates were generated for birds and 

mammals (using LD50 data).

Table 3.1 Formulas for Calculation of diphacinone Intake based on Consumption of Bait.

Passeriform bird food intake (g, dry weight): FI (g dry-wt/day) = 0.398 * Wt(g)0.850

Rodent mammal food intake (g, dry weight): FI (g dry-wt/day) = 0.621 * Wt(g)0.564

Food intake (g, wet weight): FI (g wet-wt/day) = FI (g dry-wt/day) / 0.90

Diphacinone intake (mg a.i./kg-bwt/day) = FI (g wet-wt/day) * 50 mg a.i./kg-bait / Wt(g)

Where: Wt(g) = weight (in grams) of the bird or mammal consumer

Table 3.2 Expected Diphacinone Intake for Default Bird and Mammal Weights based on 

Consumption of Bait.

Weight (g) Food intake 

(g dry-wt/day)

Food intake 

(g wet-wt/day)

Dphacinone intake 

(mg a.i./kg-bwt/day)

Birds* 20 5.1 5.6 14.1

100 19.9 22.2 11.1

1000 141.2 156.9 7.8

Rodent 

Mammals

15 2.9 3.2 10.6

35 4.6 5.1 7.3

1000 30.6 34.0 1.7

*surrogate for terrestrial-phase amphibians

The second exposure method for primary consumption of bait results in a diet concentration of 

50 mg a.i./kg-bait (concentration of diphacinone as packaged in bait). RQs are calculated using 

this estimate of dietary concentration and the LC50 (mg a.i./kg-diet) available from dietary

toxicity studies. Acute RQs using these exposure estimates were generated for birds and 

mammals.
28

3.3.2Expected Diphacinone Ingestion through Consumption of 

Contaminated Carcasses

Mammals and birds have the potential to consume diphacinone bait. Therefore, if they consume 

bait and are taken as prey, then secondary exposure may result. The determination of 

diphacinone intake for individual secondary consumers of diphacinone poisoned animals or 

carcasses are calculated in a manner similar to the approach for individuals consuming bait

directly. Calculated and empirical residue data are used instead of bait concentration of 

diphacinone (Table 3.4). Diphacinone body burdens in studies were also determined in 

mammals after exposure to diphacinone bait. 

Measured residue data are available for the CA ground squirrel, which were used as the basis for 

exposure estimates for secondary exposure. It was assumed the concentrations were reported 

using wet weights of the mammals. The mean concentration was reported as 1.4 mg ai/kg bw 

with a maximum concentration of 3.4 mg ai/kg bw (Baroch 1994a, MRID# 43534601). Field 

collected data are subject to a number of uncertainties including, but not limited to:

 possible partial decomposition of bodies in field, 

 collection may miss carcasses with highest body burdens, and

 collection may miss individuals that were rapidly predated, died off site, or died 

underground.

An additional uncertainty with the use of these whole-body exposure estimates is that it may

underestimate exposure to animals that consume specific tissues, such as the liver, that may have 

more concentrated diphacinone levels relative to the whole-body estimates. 

Diphacinone exposure through carcass consumption is calculated as mg a.i./kg-bw, where kg-bw 

is the kilograms of the consuming individual for three weight classes of birds and mammals. For 

this analysis, bird weight classes of 50, 1000, and 2000 g individuals and mammal weight classes 

of 50, 1000, and 3000 g individuals were used to better represent the larger size of carnivores 

and scavengers relative to the full range of bird and mammal weights. Exposure (food dry weight 

consumption) estimates were derived using the generic bird and mammal allometric equations 

from (EPA 1993). Food dry weight was converted to wet weight assuming the consumed 

prey/carcass contained 68% water (USEPA 1993). Formulas for calculation of dose estimates are 

provided in Table 3.3, and exposure estimates (on a dose basis) are provided in Table 3.4. RQs 

are generated by dividing these exposure estimates (mg a.i./kg-bwt) for a given weight class by

the most conservative toxicity endpoint for the relevant taxa adjusted for the default body

weights. RQs using these exposure estimates were generated for acute bird and mammal (using 

LD50 data).
29

Table 3.3 Formulas for Calculation of Diphacinone Intake based on Consumption of Carcasses.

Bird food intake (g, dry weight): FI (g dry-wt/day) = 0.648 * Wt(g)0.651

Mammal food intake (g, dry weight): FI (g dry-wt/day) = 0.235 * Wt(g)0.822

Snake food intake (g dry weight): FI (g dry-wt/day) = FI = 0.621 W 0.564

Food intake (g, wet weight): FI (g wet-wt/day) = FI (g dry-wt/day) / 0.32

Diphacinone intake (mg a.i./kg-bwt/day) = FI (g wet-wt/day) * 3.4 mg a.i./kg-carcass / Wt(g)

Where: Wt(g) = weight (in grams) of the bird or mammal consumer

Table 3.4 Expected Diphacinone Intakes for Carnivorous Birds and Mammals based on 

Consumption of Contaminated Carcasses

Weight (g) Food intake 

(g dry-wt/day)

Food intake 

(g wet-wt/day)

Diphacinone intake 

(mg a.i./kg-bwt/day)1

Birds* 50 8.3 25.8 1.75

1000 58.2 181.7 0.62

2000 91.3 285.4 0.49

Mammals 50 5.9 18.3 1.24

1000 68.7 214.7 0.73

3000 169.5 529.8 0.60

Snake 400 N/A 1.36 3.40

*surrogate for terrestrial-phase amphibians

1

See Table 3.8 for derivation.

The maximum measured concentration of diphacinone in carcasses was 3.4 mg a.i./kg-carcass. 

This empirical measurement was also used to estimate exposures from consumption of 

contaminated carcasses. RQs are calculated using this estimate of dietary concentration and the 

LC50 (mg a.i./kg-diet) available from dietary toxicity studies. RQs using these exposure estimates 

were generated for acute bird and mammal (using LC50 data).

4.0 Ecological Effects Characterization

Toxicity endpoints are established based on data generated from guideline studies submitted by

the registrant, and from open literature studies that meet the criteria for inclusion into the 

ECOTOX database maintained by EPA/ORD. EFED policy is to use the effects endpoint from 

the most sensitive tested species for each taxa evaluated. In aquatic systems, taxa evaluated 

include aquatic plants, invertebrates, and fish. Freshwater fish serve as a surrogate for aquatic-
30

phase amphibians when amphibian data are unavailable or unacceptable for quantitative use. 

Where data are available, separate endpoints are used for freshwater and estuarine/marine 

organisms. In terrestrial systems, taxa evaluated include birds, mammals and honey bees. Bird 

endpoints are generally derived from guideline studies on bobwhite quail and/or mallard duck. 

Bird data are used as a surrogate for reptiles and terrestrial-phase amphibians when data for these 

taxa are unavailable or unacceptable for quantitative use. Mammal data are derived from 

guideline studies typically conducted on laboratory rats, mice, or rabbits.

In addition to registrant-submitted and open literature toxicity information, other sources of 

information, including use of the acute probit dose response relationship to establish the 

probability of an individual effect and reviews of ecological incident data, are considered to 

further refine the characterization of potential ecological effects associated with exposure to 

diphacinone. A summary of the available ecotoxicity information and the incident information 

for diphacinone are provided below.

4.1 Toxicity of Diphacinone to Aquatic Organisms

Table 4.1 summarizes the most sensitive aquatic toxicity endpoints, based on an evaluation of 

both the registrant-submitted studies and the open literature. Data on toxicity of diphacinone to 

aquatic plants are not available.

Table 4.1. Aquatic Toxicity Profile for Diphacinone

Assessment 

Endpoint

Acute/ 

Chronic

Species

TGAI % a.i.

Toxicity Value 

Used in Risk 

Assessment

Citation or 

MRID # 

(Author, 

Date)1

Classification

Freshwater 

fish 

(surrogate for 

aquatic-phase 

amphibians)

Acute Bluegill sunfish 

Lepomis 

macrochirus

TGAI 95.8%

Rainbow Trout

Oncorhynchus 

mykiss

TGAI 95.8%

96-hr LC50 = 7.5 

mg/L

96-hr LC50 = 2.6 

mg/L

MRID 

432495-01

(Machado 

1994)

MRID

432495-02

(Machado 

1994)

Acceptable (moderately toxic)

Acceptable (moderately toxic)

Freshwater 

invertebrates

Acute Daphnia

Daphnia manga

TGAI 98.7%

EC50 = 1.8 mg/L MRID 

422822-01 

(Putt 1992)

Acceptable (moderately toxic)

1-ECOTOX references are designated with an E followed by the ECOTOX reference number. 

Note: There were many other studies submitted but they were not listed here because they were classified as 

INVALID. 

4.2 Toxicity of Diphacinone to Terrestrial Organisms

Table 4.2 summarizes the most sensitive terrestrial toxicity endpoints, based on an evaluation of 

both the registrant-submitted studies and the open literature. Data on toxicity of diphacinone to 

terrestrial plants are not available.
31

Birds

Diphacinone is acutely moderately toxic to avian species. The most sensitive avian endpoint that 

corresponded to a standard test species (bobwhite quail or mallard duck) was an LD50 = 1630 mg 

a.i./kg-bwt from a bobwhite quail study (MRID 42245201). This endpoint is uncertain because 

the 95% confidence interval ranged from 0 to . Visual inspection of the data indicates that the 

LD50 is less than 2000 mg/kg but greater than 400 mg/kg. A second bobwhite quail study was 

available in the open literature (Rattner et al., 2010), which reported an LD50 of 2014 mg a.i./kgbwt. In addition, an LD50 = 3158 mg a.i./kg-bwt was determined for mallard ducks (MRID 

00128411). Also an LD50 of 96.8 mg a.i./kg-bwt was available for one raptor species (American 

kestrel, Falco sparverius) (Rattner et al. 2011). This study suggests that American kestrels may

be more sensitive than the standard test species (i.e., based on the available studies, 17 to 21 

times more sensitive than bobwhite quail and 33 times more sensitive than mallard ducks). 

Subacutely, diphacinone is considered moderately toxic (Mallard Duck LC50 = 906 ppm) to 

practically non-toxic (Bobwhite Quail LC50 = 5000 ppm).

Mammals

For mammals, the acute oral and dietary toxicity of diphacinone is classified as very highly 

toxic. This is expected, given the target of this rodenticide is mammals. The acute oral LD50 for 

males was calculated as 2.50 mg/kg, with 95% confidence limits of 1.82-3.44 mg/kg; for females 

it was 2.10 mg/kg, with 95% confidence limits of 1.55-2.86 mg/kg. The combined oral LD50 for 

both sexes was calculated as 2.31 mg/kg (95% confidence limits of 1.86 to 2.88 mg/kg). Dietary

LC50s ranged from 2.08 to 2.5 ppm. Symptoms occurred at all doses, and were not necessarily

associated with subsequent mortality. These included clear or colored nasal discharge, soft stool 

and/or diarrhea (possibly associated with the corn oil vehicle used), decreased motor activity and 

occasional drying of the corneal surface. Symptoms at higher dose levels included lacrimation, 

ataxia, cyanosis and bloody exudate from nose and eyes. Hemorrhage into the body cavities and 

of various organs was observed in animals which died. 

For diphacinone, the most sensitive laboratory rat data reported an LD50 = 1.9 mg a.i/kg-bwt

(MRID 05002272). Toxicity tests conducted on pine voles and meadow voles (Microtus 

pennsylvanicus) resulted in LD50s of 67.7 and 11.7 mg a.i/kg-bwt (Byers, 1978). LD50 values for 

dogs and cats ranged from 0.88 to 15 mg a.i/kg-bwt (LiphaTech, Inc, 1997). LD50s for the coyote 

(Canus latrans) and mongoose (Herpestes spp) were 0.6 and 0.18 mg a.i/kg-bwt, respectively

(Savarie et al. 1979 and MRID 405655-01). These data suggest that diphacinone may be more 

toxic to carnivores than those animals experiencing primary exposure to diphacinone. This 

increased sensitivity of secondary exposure animals relative to primary exposure animals was 

also observed in the avian acute oral toxicity studies. 

Reptiles

Brooks et al. (1998) conducted a study to determine the acute oral and dermal toxicity of 18 

chemicals, including diphacinone, to Brown Tree Snakes (Boiga irregularis). Acute exposure to 

diphacinone resulted in LD50s of 20.75 mg/kg (95% CI=10.1 to 44.9) with a slope of 4.2 
32

(ethanol used as a carrier) and 32.2 mg/kg (95% CI=18.8 to 55.9) with a slope of 5.0 (propylene 

glycol used as a carrier). Five snakes of either sex were used in each treatment group. All snakes 

were observed for 7 days after administration of Diphacinone. Diphacinone produced mortalities 

within 24 hrs of administration of doses of 10-40 mg/kg. At necropsy, there was no evidence of 

hemorrhaging. Diphacinone produced no mortalities from dermal application.

Table 4.2. Terrestrial Toxicity Profile for Diphacinone

Species Acute/ 

Chronic Species Toxicity Value

Citation

MRID/ECOTOX 

reference No.

Classification

Birds 

(surrogate for 

terrestrialphase 

amphibians

and reptiles)

Acute 

oral

LD50

Bobwhite Quail

Kestrel

>400<2000 

mg/kg (reported 

as >1630 

mg/kg)

96.8 mg/kg

422452-01

Rattner et al 2011

Supplemental

Supplemental

Acute 

Dietary 

LC501

Mallard Duck

Bobwhite Quail

906 ppm3

(LOAEC=1.6 ppm)

>5000 ppm2

424088-02

424088-01

Core

Core

Mammals Acute Rat 2.5 mg/kg M

2.1 mg/kg F

00060605 Core

Acute Rat 1.9 05002272 Supplemental

Acute 

Dietary 

LC50

Rat

Rat

2.08 ppm

2.5 ppm

TMN 075

TMN 051

Supplemental

Supplemental

Acute 

oral 

LD50

Coyote

Mongoose

0.6

0.2

Savarie et al. 19794

DWRC5 

405655-01

Supplemental

Supplemental

Reptile Acute 

oral 

LD506

Brown tree snake Ethanol carrier

20.75 mg/kg

Prop glycol 

carrier

32.2 mg/kg

Brooks et al. 1998 Supplemental

n/a: not applicable; ND = not determined; bw = body weight

1 test organisms (10/level; 6 test concentrations, 3 control groups) were observed an additional 20 days while on

untreated feed.

2 all mortality (10% at 5000 ppm, 30% at 1667 ppm, and 10% at 185 ppm) occurred within 18 days.

3 all mortality (20 of 60 birds dosed) occurred within 16 days.

4 reported by the Denver Wildlife Research Center (Savarie et al. 1979, ASTM STP 693, pp. 69-79)

5 reported by the Denver Wildlife Research Center in EUP application for mongoose control in Hawaii

6 no hemorrhaging was reported. All snakes died within 24hrs. Diphacinone had no dermal toxicity to brown tree 

snakes. 

Note: There were other studies submitted but they were not listed here because they were classified as INVALID. 
33

Elimination of Diphacinone

Available data on the retention times for diphacinone are provided in the table below. Yu et al. 

(1982) studied the metabolism and disposition of diphacinone in rats and mice. Rats given a 

single oral dose of radiolabeled diphacinone at either 0.18 or 0.4 mg a.i./kg, about 70% of the 

dose was eliminated in feces and 10% in urine within 8 days, whereas about 20% of the dose was 

retained in body tissues. Mice given a single dose of 0.6 mg a.i./kg eliminated most diphacinone 

within 4 days, and only 7% was retained in body tissues. In both rats and mice, most 

radioactivity (59 to 69%) was detected in the liver and the kidneys (9 to 12%). Radioactivity was 

also detected in the brain, heart, spleen, lungs, blood, muscle, fat, and gonads. Several major 

metabolites were identified, and parent diphacinone in excreta and liver accounted for only about 

20% of the dose. In another study, cattle that received a single injection of 1 mg a.i./kg had 

almost constant residue concentrations in liver and kidney at 30, 60, and 90 days after dosing 

(Bullard et al. 1979). 

Table 4.3. Retention of Diphacinone in Blood and Liver

Rodenticide Species Dose (mg 

a.i./kg) # doses Blood t1/2

a

(days)

Liver 

retentiona,b

(days)

Reference

(MRID)

Diphacinone Pig 12.5 3 or 5 NA 5.43 (t1/2) Fisher 2006

Diphacinone Rat 1.5 1 NA 3 (t1/2)

Fisher et al. 2003 

(48190801)

Diphacinone Cattle 1.0 1 NA > 90 Bullard et al. 1976

a

t1/2 for plasma and liver is the elimination half-life

b

liver retention is expressed as either the time period for which residues persist or as the elimination half-life

Diaz and Whitacre (1976) orally dosed rats with diphacinone (0.32 mg a.i./kg/day) for 1 or 2 

days. Rats dosed for 2 days were sacrificed 72 hours after the second dose and those dosed for 1 

day were sacrificed after 48 hours. In rats dosed for 2 days, about 45% of the total dose 

administered was excreted (86% in feces, 14% in urine) and 25% was retained in body tissues 72 

hours after the last dose. The remaining 30% of the dose was not recovered. The body tissues 

retaining the most diphacinone at 96 hours were the hide and tail, liver, intestine, blood, and the 

carcass. In rats dosed for 1 day and sacrificed after 48 hours, about 5% of the dose was excreted 

and 61% retained; the remainder was not recovered. 
34

Table 4.4. Percentage of Diphacinone Retained by Rats Dosed for 1 or 2 Days with 0.32 mg 

a.i./kg (adapted from Diaz and Whitacre 1976)

Organ

% of dose retained per groupa

48 h after 1 dose 72 h after 2 doses

Intestine 22.1 4.1

Liver 19.4 5.4

Hide and tail 10.9 6.5

Carcass 3.9 3.8

Blood 1.8 4.0

Muscle 0.8 0.4

Kidney 0.7 0.3

Testis not reported 0.8

Lung 0.5 0.2

Fat 0.2 0.4

Heart 0.1 0.2

Spleen 0.1 0.1

Brain < 0.1 < 0.1

a

because only 66-70% of the total dose was recovered, percentages in tissues are likely to be higher 

than the values tabulated

4.3 Birds and Mammals, Secondary Toxicity Tests

Available secondary poisoning studies involving diphacinone indicate that some mammals are 

susceptible to secondary poisoning from consuming diphacinone residue in animal tissue. The 

toxicant loads of the rats, mice, and nutria fed to raptors, mustelids, and dogs are not known but 

were sufficiently high to cause mortality in most species. The relationship between target loads 

in poisoned target species and the levels presented in coyote and sheep muscle was not 

established. Moreover, the amount of toxicant present in the coyote muscle was found to be 

considerably lower than that contained in the small intestine, liver, kidney, and heart tissues. The 

data are adequate to demonstrate that avian and mammalian predators may be killed from 

consuming target species poisoned with 0.01% a.i. bait and that mammals may be killed from 

consuming target animals exposed to either 0.01% or 0.005% bait. 
35

Table 4.5. Summary of Secondary Toxicity of Diphacinone to Mammals and Birds (from 

Erickson and Urban 2004)

Predator/

scavenger

(p/s)

Prey offered 

to p/s 

No. prey

offered daily

per p/s

No. days

p/s

exposed

No.

p/s

exposed

No.

p/s

dead

No. survivors

with signs of 

diphacinone 

toxicitya

Mink nutria fed 0.01% carrot 

bait for up to 10 days

ad lib. 5-18 3 3 no survivors

Mongoose rats fed 0.005% bait for 5 

days

1 1

3

5

6

7

8

10

1

1

2

1

1

1

1

0

1

2

1

1

1

1

nr

no survivors

no survivors

no survivors

no survivors

no survivors

no survivors

Ermine deer mice fed 0.01% bait 

for 10 days

2 5 2 1 nr

Striped skunk deer mice fed 0.01% bait 

for 10 days

2 5 5 0 nr

Deer mouse liver from diphacinonepoisoned owls1 g daily 7 4 1 3 (ct)

Rat meat containing 0.5 ppm 

ai 

ad lib. 6 8 4 nr

Dog 

(domestic)

nutria fed 0.01% carrot 

bait for up to 10 days

ad lib. 6-10 3 3 no survivors

a

eb = external bleeding; ih = internal hematoma; bl = bleeding (unspecified); ct = increased blood coagulation time; nr = not reported

Predator/

scavenger

(p/s)

Prey offered

to p/s 

No. prey

offered daily

per p/s

No. days

p/s

exposed

No.

p/s

exposed

No.

p/s

dead

No. survivors

with signs

of diphacinone toxicitya

Great horned 

owl

mice fed choice of 0.01% bait 

or untreated food for 10 days

2 5 3 2 1 (ct)

Saw-whet owl mice fed choice of 0.01% bait 

or untreated food for 10 days

2 5 1 1 no survivors

Barn owl rats fed choice of 0.005% bait 

or untreated food for 5 days

ad lib. 10 2 0 0

American 

crow

rats fed 0.005% bait until death 1

1-2

b

1

6

10

11

0

0

0

5 (eb/ct)

Golden eagle meat laced at 2.7 ppm ai 454 g 5

10

4

3

0

0

4 (eb/ct)

3 (eb/ct)c

a

eb = external bleeding; ih = internal hematoma; bl = bleeding (unspecified); ct = increased blood coagulation time; nr = not reported

b

offered 1 rat per crow for 5 days and 2 rats per crow on day 6

c

general weakness of all eagles was observed after 5 days
36

4.4 Incident Database Review

A review of the Ecological Incident Information System (EIIS, version 2.1), the ‘Aggregate 

Incident Reports’ (v. 1.0) database, and the Avian Monitoring Information System (AIMS) for

ecological incidents involving diphacinone was completed. The results of this review for 

terrestrial, plant, and aquatic incidents are discussed below. 

There were a total of 33 incidents listed in the database. Eleven (33.5%) were deemed “highly 

probable”, eight (24%) were deemed as “probable”, eleven (33.5%) were deemed “possible”, 

two (6%) were deemed “unlikely” and one (3%) was deemed “unrelated”. Descriptions of the 

incidents that are most likely associated with diphacinone are described below. These incidents 

confirm that exposure to and effects from diphacinone exposure can occur from both primary

consumers and secondary poisoning. 

Interpretation of incident data is difficult and may be confounded by a number of factors. The 

report of an incident can demonstrate that a complete exposure pathway resulting in an effect 

(i.e., mortality) has occurred. Only a small fraction of wildlife incidents are believed to be 

reported to the Agency. The lack of incidents or the presence of fewer incidents in and of itself 

does not necessarily suggest that fewer incidents are occurring or that one chemical is safer than 

another. However, incident data can corroborate results of deterministic risk assessments. 

There were a number of terrestrial incidents reported for diphacinone and involved both primary

and secondary toxicity. Eleven incidents were deemed “highly probable”. These included 

mortality to a mountain lion, a raccoon and a snowy owl through secondary poisoning, as well as 

nineteen gray squirrels, two white-tailed deer, a ground squirrel, a kit fox and a rat through bait 

consumption. Listed below are descriptions of “highly probable” incidents as well as those that 

resulted in measurable residues. Incidents located in EIIS are summarized below. 

Terrestrial Incidents

 Testing found diphacinone residue concentrations of 45 ppm and 55 ppm from blood and 

liver samples in a mountain lion and raccoon found dead in Northern CA respectively

(B000641-001). 

 Diphacinone was also found in a dead adult kit fox (found in Bakersfield CA and 

weighing 5.75 lbs) by immunoassay in liver and blood at a concentration of 0.18 ppm 

(B000640-001).

 A kangaroo rat was found in CA in a moribund state. An analysis of the liver detected 

diphacinone residues at 3.5 ppm (R000-02-001)

 A male deer fawn (found approximately 2 weeks post mortem) and an adult buck 

(weighing 202 lbs) was found dead on Fire Island NY and contained 0.2 ppm and 0.93 

ppm diphacinone in liver samples respectively (I004768-001 and I004769-001). 

 An adult male gray squirrel was found dead in Brookhaven NY and contained 2.0 ppm 

diphacinone (I005353-001). 
37

 A male snowy owl was found dead in Rhinebeck NY of likely secondary poisoning 

(I006306-01). Diphacinone was found in the liver but the concentration was not 

reported. 

 A number of gray squirrels were found dead in Richmond VA and pooled liver samples 

from two individuals (one weighed 490g and the other weighed 567g) contained 4.94 and 

3.4 ppm diphacinone respectively (I007488-004). 

 Although listed as a “probable” incident, a red-tailed hawk was found moribund (dying 

the next day) in Roslyn NY and contained 0.34 ppm diphacinone in a liver sample 

(I009270-001). 

 A gray squirrel was found dead in Albany NY of apparent blunt impact trauma (listed as 

a “possible” incident). However, the liver contained 1.02 ppm diphacinone (I011518-

001). 

 Eight squirrels were found dead in Conyers GA and 3.6 ppm diphacinone was found in 

the liver of one (410g) of the squirrels that was sampled (I019839-001). 

 A rat and a ground squirrel were found dead in Ventura CA and contained 4.12 and 1.99 

ppm diphacinone in liver samples respectively (I0I4886-001). 

 Two deer found dead in Suffolk NY contained 0.2 and 0.93 ppm diphacinone in liver 

samples respectively (I013810-007 and I013810-008, both incidents deemed as 

“probable”). 

 A radio-collared yearling female coyote was found dead in Ventura CA and contained 

diphacinone residues of 1.3 ppm in liver, 0.1 ppm in blood and 0.16 ppm in stomach 

contents but also contained 0.083 ppm Brodifacoum in the liver (I007107-014, I007165-

003). 

Plant Incidents

There were no incidents reported for diphacinone involving plants. 

Aquatic Incidents

There were no aquatic incidents reported for diphacinone. 

5.0 Risk Characterization

Risk characterization is the integration of exposure and effects characterization to determine the 

ecological risk from the use of diphacinone for BPD removal and the likelihood of effects on 

aquatic life, wildlife, and plants based on different pesticide-use scenarios. The risk

characterization provides estimation and description of the likelihood of adverse effects; 

articulates risk assessment assumptions, limitations, and uncertainties; and synthesizes an overall 

conclusion regarding the likelihood of adverse effects. In the risk estimation section, RQs are 

calculated using standard EFED procedures explained in the following section. In the risk 

description section, additional analyses may be conducted to help characterize the potential for 

risk.
38

5.1 Risk Estimation: Integration of Exposure and Effects Data

Results of the exposure and toxicity effects data are used to evaluate the likelihood of adverse 

ecological effects on non-target species. For the assessment of diphacinone risks, the risk 

quotient (RQ) method is used to compare exposure and measured toxicity values:

RQ = EEC / (Toxicity Endpoint)

The RQs are compared to the Agency’s levels of concern (LOCs). These LOCs are the Agency’s 

interpretive policy and are used to analyze potential risk to non-target organisms and the need to 

consider regulatory action. These criteria are used to indicate when a pesticide’s use as directed 

on the label has the potential to cause adverse effects on non-target organisms. 

5.1.1 Aquatic Risk Estimation

Based on the GENEEC estimated EECs, risk estimates for aquatic organisms are lower than 

concern levels. EECs in previous assessments were in pptr (ng/L), which is orders of magnitude 

lower than available toxicity reference values. Therefore, effects to aquatic organisms from the 

proposed diphacinone use are unlikely. 

5.1.2 Bird and Mammal Risk Estimation

Risks from Direct Bait Consumption

In the case of primary exposure, it is assumed the bait containing diphacinone is ingested by nontarget animals and evokes a toxic response. For toxic response elicited from gavage exposure 

route, exposure is measured as mg a.i./kg-bw. Toxicity is measured by the LD50 obtained from 

the single-gavage studies for birds and mammals. The LD50’s are adjusted for the weight of the 

assessed animals (birds: 20, 100, 1000 g; mammals: 15, 35, 1000 g). Avian and mammalian 

RQs based on acute single-dose gavage studies are provided in table 5.2. For birds, the LOCs 

were not exceeded. For mammals, the Listed Species, Restricted Use, and Acute LOCs were 

exceeded for all three weight classes.
39

Table 5.1 Formulas for Calculation of Weight-adjusted Avian and Mammalian 

Diphacinone LD50s.

Adjusted avian LD50: Adj LD LD

AW

TW

x

.

( )

50 50

1

















 where:

Adj. LD50 = adjusted LD50 (mg/kg-bw) 

LD50 = endpoint reported from bird study (mg/kg-bw)

TW = body weight of tested animal (178g bobwhite quail)

AW = body weight of assessed animal (20g, 100g, and 1000g)

x = Mineau scaling factor for birds; EFED default 1.15

Adjusted mammalian LD50: 

0.25

50 50 . 













AW

TW Adj LD LD where:

Adj. LD50 = adjusted LD50 (mg/kg-bw)

LD50 = endpoint reported from mammal study (mg/kg-bw)

TW = body weight of tested animal (350g)

AW = body weight of assessed animal (15g, 35g, 1000g)

Table 5.2 Bird and Mammal Acute RQs based on a Single-dose of Diphacinone through 

consumption of bait

Taxonomic 

Group

Weight (g) Diphacinone intake 

(mg a.i./kg-bwt/day)1

Adjusted LD50

(mg a.i./kg-bw)2

RQ3

Birds* 20 13 1163 0.01

100 10 1480 0.01

1000 7.1 2091 <0.01

Mammals 15 10 4.8 2.63***

35 6.6 3.9 2.24***

1000 1.5 1.7 1.20***

*surrogate for terrestrial-phase amphibians

1

See Table 3.5 and 3.6 for derivation.

2

See Table 5.2 for derivation.

3 Bolded (***) RQs exceed Listed Species, Restricted Use, and Acute Risk LOCs. 

For toxic response elicited from the dietary exposure route extended over several days, exposure 

is measured as mg a.i./kg-bait. Toxicity is measured by the LC50 obtained from the dietary

studies (5 days on treated diet) for birds and mammals. Avian and mammalian RQs based on 

dietary studies are provided in Table 5.3. For mammals, the Listed Species, Restricted Use, and 

Acute LOCs were exceeded (RQ = 24.04). 
40

Table 5.3 Bird and Mammal Acute RQs based on a 5-day Exposure to Diphacinone in the 

Diet (consumption of bait)

Taxonomic Group Diphacinone 

concentration in bait 

(mg a.i./kg-bait)

LC50

(mg a.i./kg-diet)

RQ1

Birds* 50 906 0.06

Mammals 50 2.08 24.04***

*surrogate for reptiles and terrestrial-phase amphibians

1 Bolded (***) RQs exceed Listed Species, Restricted Use, and Acute Risk LOCs.

5.1.3 Secondary Risks from Consumption of Diphacinone-contaminated Carcasses

To determine secondary exposure EECs, available literature concerning carcass residue 

concentrations were considered. For this assessment, a maximum carcass concentration found in 

a field study using diphacinone of 3.4 mg a.i./kg-carcass was used to estimate exposure to birds 

and mammals. Estimated diphacinone intake for secondary consumers (mg a.i./kg-bwt/day) was 

calculated in Table 3.4. As in the direct consumption risk estimation, the LD50’s are adjusted for 

the weight of the assessed animals (birds: 50, 1000, 2000 g; mammals: 50, 1000, 3000 g). These 

heavier weights are used to better represent carnivores and scavengers which tend to be larger 

individuals. For reptiles, an LD50 (20.75 mg/kg) was used to calculate the risk to a 400g snake 

eating a prey animal that has ingested diphacinone.

Carnivore/scavenger bird LOCs were not exceeded; however, RQs for carnivore and scavenger 

mammals exceeded Listed Species and Restricted Use LOCs while the snake RQ exceeded only 

the Listed Species LOC (Table 5.4).
41

Table 5.4 Carnivorous/Scavenger Bird and Mammal Acute RQs based on a Single-dose of 

Diphacinone by Gavage, consumption of contaminated carcasses

Taxonomic 

Group

Weight (g) Diphacinone intake 

(mg a.i./kg-bwt/day)1

Adjusted LD50 (mg 

a.i./kg-bwt)2

RQ3

Birds+ 50 1.75 87.2 0.02

1000 0.62 136.7 0.004

2000 0.48 151.7 0.003

Mammals 50 1.24 3.4 0.36**

1000 0.73 1.6 0.45**

3000 0.60 1.2 0.49**

Reptile (snake) 400 3.4 20.75++ 0.16*

+surrogate for terrestrial-phase amphibians

++ not adjusted

1

See Tables 3.8 and 3.9 for derivation.

2

See Table 5.2 for derivation with assessed body weights used in this table.

3 Bolded (*) RQs exceed Listed Species LOCs. 

Bolded (**) RQs exceed Listed Species & Restricted Use LOCs.

For toxic response elicited from the dietary exposure route extended over several days, exposure 

is measured as the concentration of contaminant in the carcass, 3.4 mg a.i./kg-carcass. Toxicity

is measured by the LC50 obtained from the dietary studies (5 days on treated diet) for birds and 

mammals. Avian and mammalian RQs based on dietary studies are provided in Table 5.5. 

Carnivore/scavenger bird LOCs were not exceeded; however, RQs for carnivore and scavenger 

mammals exceeded Listed Species, Restricted Use, and Acute LOCs.

Table 5.5 Bird and Mammal Acute RQs based on a 5-day Exposure to Diphacinone in the 

Diet, consumption of contaminated carcasses

Taxonomic Group Diphacinone 

concentration in 

carcass (mg a.i./kgcarcass)

LC50

(mg a.i./kg-diet)

RQ1

Birds* 3.4 906 0.004

Mammals 3.4 2.08 1.63***

*surrogate for reptiles and terrestrial-phase amphibians

1 Bolded (**) RQ exceed Listed Species & Restricted Use Risk LOCs. 

5.1.4 Risk to Terrestrial Invertebrates

Toxicity data were not available to assess risk to terrestrial invertebrates. 
42

5.2 Risk Description

The results of the screening-level risk assessment for the proposed uses of diphacinone for BPD

control suggest that levels of diphacinone in the environment, when compared with toxicity

values, may result in direct effects to listed and non-listed terrestrial animals. Although RQs only

exceeded LOCs for mammals, incident data and secondary exposure studies also suggest that 

birds may be affected as well. These direct effects may also result in indirect risk to non-target 

species.

5.2.1 Risks to Aquatic Organisms

Based on the screening level EECs calculated in previous assessments, risk estimates for aquatic 

organisms are lower than concern levels. Therefore, effects to aquatic organisms from the 

proposed diphacinone use are unlikely.

EFED presumed risks were below concern levels to aquatic plants given the expected low 

concentrations in water and the mode of action of diphacinone. 

5.2.2 Risks to Terrestrial Organisms

Risks to Birds directly consuming bait

Risk to birds (listed and non-listed species) are lower than concern levels from the proposed use 

of diphacinone. RQs for birds consuming a single-dose or doses over a 5 day period of 

diphacinone in bait do not exceed any LOCs. On an acute basis (single gavage or 5-day dietary

exposure), the calculated RQs ranged from <0.01 to 0.01, which do not exceed any LOCs.

Risks to Carnivore and Scavenger Birds 

Carnivorous and scavenger birds may be exposed to diphacinone through consumption of 

carcasses that were contaminated with diphacinone. Based on available ground squirrel carcass 

data, carcass diphacinone concentration was estimated at 3.4 mg a.i./kg-carcass. On an acute 

basis (single gavage or 5-day dietary exposure), the calculated RQs did not exceed any LOCs. 

The estimated concentrations were within a factor of 2 compared with the maximum carcass 

data. Also, using the kestrel endpoint (96.8 mg/kg) to calculate quotients increased risk

quotients slightly, but did not alter LOC exceedance conclusions. 

There is uncertainty associated with the RQs for carnivore and scavenger birds because the LD50

and LC50 used to calculate the RQs were based on granivorous species. These species prefer to 

eat a relatively consistent quantity of food on a daily basis. Carnivores and scavengers may

consume a large meal (an entire carcass) once every several days. Given this feeding strategy, 

these birds may get a larger single-dose exposure of diphacinone than is currently estimated in 

the risk quotient methodology. The kestrel data (Rattner et. al., 2011) indicate that diphacinone is 

21x more toxic to a carnivorous bird compared to the standard test species (quail).

Incidents were reported in the United States of carnivorous/scavenger birds that died that of 

diphacinone poisoning. The known species include a red-tailed hawk and a snowy owl. The 
43

presence of confirmed incidents of diphacinone poisoning under the currently registered labels 

that have similar or more restrictive application rates and methods indicates that exposure at 

sufficient levels to cause poisoning of these predator birds does occur. In addition, two secondary

exposure studies have been conducted that fed rats 0.005% diphacinone bait (American crows) 

or a choice of untreated or 0.005% diphacinone bait (barn owls) then fed the contaminated rats to 

birds. Neither of the studies resulted in mortality of birds; however, in the study where rats were 

not given a choice of treated or untreated food, approximately half of the birds (American crows) 

showed signs of treatment-related effects including increased incidents of external bleeding and 

increased coagulation time (see table 4.5). Together, the incident data and secondary exposure 

studies show that secondary exposure to birds can be sufficient to result in severe effects, 

including mortality. 

5.2.3 Risks to Mammals directly consuming bait

Mammals (listed and non-listed species) are at risk from this proposed use of diphacinone. 

Diphacinone presents primary exposure risk concerns for all mammal sizes assessed using all 

primary exposure assessment methods (Acute RQs were 1.20 to 2.63 and the acute dietary RQ 

was 24.04). 

Incidents were reported in the EPA database of non-carnivorous mammals that died of 

diphacinone poisoning. The known species include squirrels and deer. Omnivores such as the 

raccoon and San Joaquin kit fox were also cited in the reported incidents.

5.2.4 Risks to carnivore and scavenger Mammals

Carnivorous and scavenger mammals are exposed to diphacinone through consumption of 

carcasses that were contaminated with diphacinone. Based on available carcass data, carcass 

diphacinone concentration was estimated at 3.4 mg a.i./kg-carcass. This value represents the 

highest concentration of diphacinone found in any individual in all of the carcass residue studies. 

On an acute basis for the single gavage exposure, the calculated RQs (<0.01) did not exceed the 

Listed Species and Restricted Use LOCs. The RQ=1.6 for mammals based on the 5-day dietary

study does exceed Listed Species, Restricted Use, and Acute Risk LOCs. Therefore, mammals 

consuming contaminated carcasses over a several day period may expect mortality.

The LD50 and LC50 used to calculate the RQs were based on granivorous species. These species 

prefer to eat a relatively consistent quantity of food on a daily basis. Carnivores and scavengers 

may consume a large meal (an entire carcass) once every several days. Given this feeding 

strategy, these carnivorous mammals may get an even larger single-dose exposure of 

diphacinone than is currently estimated in the risk quotient methodology. 

Incidents were reported in the United States of carnivorous/scavenger mammals that died of 

diphacinone poisoning. The known species include San Joaquin kit fox, a mountain lion, a 

raccoon and a coyote. The presence of confirmed incidents of diphacinone poisoning under the 

currently registered labels that have similar or more restrictive application rates and methods 

indicates that exposure at sufficient levels to cause poisoning of these predator mammals does 

occur. The presence of confirmed incidents of diphacinone poisoning under the currently
44

registered labels that have similar application rates and methods corroborates the risk estimation 

process that identified risks to carnivorous/scavenger mammals. Although some uses have more 

or less restrictive use patterns. In addition, a secondary feeding study that fed 0.005% 

diphacinone bait to rats, then fed contaminated rats to mongoose also observed high rates of 

mortality (Table 4.5). 

5.2.5 Risks to Reptiles

For reptiles, an LD50 (20.75 mg/kg) was used to calculate the risk to a 400g snake eating a prey

animal that has ingested diphacinone. The snake RQ (0.16) exceeded only the Listed Species

LOC. Diphacinone seems to have a different mode of action in reptiles. Mortality occurs but 

does not result from anticoagulation and its mode of action in snakes is unknown. Diphacinone 

produced mortalities within 24 hrs of administration of doses of 10-40 mg/kg. At necropsy, 

there was no evidence of hemorrhaging. Diphacinone produced no mortalities from dermal 

application.

5.2.6 Risks to Terrestrial Invertebrates

No data were found to allow for an assessment of risk to terrestrial invertebrates.

5.2.6.1 Terrestrial and Semi-aquatic Plants 

RQs were not calculated for terrestrial plants as toxicity data were not available. EFED presumed 

direct risks were low to terrestrial plants given the mode of action of diphacinone and the lack of 

any reported incidents. Since diphacinone is not sprayed directly onto plants, and because so 

little is expected to leach from the bait and then be available for plant uptake, exposure is 

expected to be minimal. 

Terrestrial plants that rely on animals for pollination and seed dispersal do have the potential to 

experience indirect effects through reduction in the animals that perform those functions.

5.3 Threatened and Endangered Species Concern

5.3.1 Action Area

For listed species assessment purposes, the action area is considered to be the area affected 

directly or indirectly by the Federal action and not merely the immediate area involved in the 

action. At the initial screening-level, the risk assessment considers broadly described taxonomic 

groups and so conservatively assumes that listed species within those broad groups are colocated with the pesticide treatment area. This means that terrestrial plants and wildlife are 

assumed to be located on or adjacent to the treated site and aquatic organisms are assumed to be 

located in a surface water body adjacent to the treated site. The assessment also assumes that 

listed species are located within an assumed area, which has the relatively highest potential 

exposure to the pesticide, and that exposures are likely to decrease with distance from the 

treatment area. This risk assessment presents the proposed new use of diphacinone to remove 

BPD and establishes initial co-location of listed species with treatment areas. 
45

If the assumptions associated with the screening-level action area result in RQs that are below 

the listed species LOCs, a "no effect" determination conclusion is made with respect to listed 

species in that taxa, and no further refinement of the action area is necessary. Furthermore, RQs 

below the listed species LOCs for a given taxonomic group indicate no concern for indirect 

effects upon listed species that depend upon the taxonomic group covered by the RQ as a 

resource. However, in situations where the screening assumptions lead to RQs in excess of the 

listed species LOCs for a given taxonomic group, a potential for a "may affect" conclusion exists 

and may be associated with direct effects on listed species belonging to that taxonomic group or 

may extend to indirect effects upon listed species that depend upon that taxonomic group as a 

resource. In such cases, additional information on the biology of listed species, the locations of 

these species, and the locations of use sites could be considered to determine the extent to which 

screening assumptions regarding an action area apply to a particular listed organism. These 

subsequent refinement steps could consider how this information would impact the action area 

for a particular listed organism and may potentially include areas of exposure that are downwind 

and downstream of the pesticide use site.

5.3.2 Taxonomic Groups Potentially at Risk

Based on available screening-level information for the proposed use of diphacinone, there is a 

potential for direct effects to listed terrestrial animals. Consequently, there is a potential concern 

for indirect effects upon the listed organisms by, for example, perturbing habitat, forage or prey

availability. In conducting a screen for indirect effects, direct effect LOCs for each taxonomic 

group are used to make inferences concerning the potential for indirect effects upon listed 

species that rely upon non-endangered organisms in these taxonomic groups as resources critical 

to their life cycle. Terrestrial plants that rely on animals for pollination and seed dispersal do 

have the potential to experience indirect effects through reduction in the animals that perform 

those functions. 

Table 5.6. Listed species risks associated with direct or indirect effects due to proposed 

diphacinone use on the BPD

Listed Taxonomy Direct Effects Indirect Effects

Terrestrial and semi-aquatic plants – monocots No Yes

Terrestrial and semi-aquatic plants – dicots No Yes

Birds and terrestrial-phase amphibians Yes Yes

Mammals Yes Yes

Reptiles Yes Yes

Aquatic vascular plants No No

Aquatic non-vascular plantsa No No

Freshwater fish and aquatic-phase amphibians No No

Freshwater Invertebrates No No

Freshwater Benthic Invertebrates No No

Estuarine/Marine Fish No No

Estuarine/Marine Crustaceans No No

Estuarine/Marine Mollusks No No

a At the present time there are no Federally listed aquatic non-vascular plants. The taxonomic group is 

included here for the purposes of evaluating potential contributions to indirect effects to other taxonomy 

and as a record of exceedances should future listings of non-vascular aquatic plants warrant additional 

evaluation of Federal actions.
46

The LOCATES database (version 2.9.7) identifies those U.S. counties in which a given crop is 

grown for which there are co-located listed species. For this analysis, it was presumed that all 

federally-listed endangered or threatened terrestrial animals in the proposed States (i.e., CO, KS, 

NE, NM, ND, SD, WY, TX, MT and OK ) may be directly or indirectly affected. With additional 

refinement by exploring more detailed use patterns and species biology (e.g., geographic 

location, specific feeding habits, time of year likely to utilize crop fields), some species listed 

may be determined to be not likely to be affected.

The Fish and Wildlife Service (FWS) recently completed a Biological Opinion on a registration 

action that allows use of another anticoagulant rodenticide (chlorophacinone; trade name of 

Rozol black-tailed prairie dog bait - hereafter referred to as Rozol) to control black tailed prairie 

dogs. Although the Agency’s endangered species risk assessment determined that 21 federally 

listed species may be affected, a number of conservation measures were developed with 

discussions with and agreement between the Fish and Wildlife Service, EPA, and the registrant 

to either reduce or preclude exposures to many of these species. The Agency’s effects 

determination for Rozol and resulting Biological Opinions are available on-line in the docket at 

regulations.gov and may be accessed using the following link: 

http://www.regulations.gov/#!docketDetail;dct=FR%252BPR%252BN%252BO%252BSR;rpp=

25;so=ASC;sb=docId;po=0;D=EPA-HQ-OPP-2011-0909

These conservation measures were part of the federal action and included label amendments to 

add specifications for a line-transect carcass search and reporting of dead/dying listed species 

and non-target animals in addition to prohibitions of Rozol use and timing restrictions via the 

Agency’s Endangered Species Protection Program (ESPP) Bulletins Live system available at: 

http://www.epa.gov/oppfead1/endanger/bulletins.htm. The Biological Opinion on Rozol 

(http://epa.gov/espp/2012/borozol-final.pdf) found that the conservation measures adequately

protected most of the endangered species, although FWS concluded that incidental take may still 

occur for the following three species: (1) black-footed ferret; (2) gray wolf; and (3) northern 

aplomado falcon. In order to minimize incidental take for these three species, FWS identified a 

number of additional reasonable and prudent measures (RPMs) including certified applicator 

training, development of website content to reduce the potential for secondary poisoning, 

requirements for the registrant to develop a stewardship training program and submit of Rozol 

distribution and production data, and additional bulletins for the northern aplomado falcon.

Additional details are included in the Biological Opinion available on the docket at the 

aformentioned link. 

The exposure and toxicity profiles are similar for chlorophacinone and diphacinone (see table 

below). In tests conducted in the same species, diphacinone is consistently less toxic to birds 

relative to chlorophacinone, and it is of similar toxicity to mammals (slight increases and 

decreases in median lethal doses/concentrations have been observed). Therefore, because the 

area of potential use is identical for Rozol and Kaput black-tailed prairie dog baits given that 

they are both assumed to be applied anywhere within the range of black tailed prairie dogs, the 
47

listed species that may be exposed are also identical. Also, given that the exposure and toxicity

profiles are similar, the conservation measures and RPMs identified for Rozol should also be 

protective of endangered species if implemented on Kaput black tailed prairie dog bait. In 

addition, labels for both Rozol and Kaput will stipulate that other anticoagulant rodenticides 

cannot be used if applying either of these products. For this reason, the Biological Opinion 

issued on Rozol is expected to be applicable to both Rozol and Kaput black tailed prairie dog 

bait, and is, therefore, expected to provide adequate protections for listed species for use of both 

rodenticides. 

Table 5.7. Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters

Comparative Parameter Chlorophacinone Diphacinone Comments

Exposure Concentration in Bait 0.005% 0.005% --

Food Consumption Not chemical dependent --

Time to mortality 4-13 days 3-9 days Time to mortality affects the 

amount of chemical that can 

be accumulated over time 

for target pests.

Liver Retention 35 days - mouse 3 days – Rat

5 days – Pig

>90 days - Cow

Lack of data in same species 

precludes definitive 

comparisons

Toxicity, Birds BWQ LD50s (mg/kg-bw) 260 1630 --

Other Avian LD50s (mg/kg-bw) None available Kestrel: 96.8

Mallard: 3200

--

Mallard LC50s (mg/kg-diet) 172 906 --

BWQ LC50 (mg/kg-diet) 56 >5000 --

Other Species (mg/kg-diet) 60 (Japanese Quail) Brown Tree Snake: 

20.75

--

Toxicity 

Mammals

Lab Rat – Gavage (mg/kg-bw) 6 1.9 --

Pine Vole – Gavage (mg/kg-bw) 14 58 --

Other Species – Gavage (mg/kgbw)1.9 (prairie dog) 0.2 (mongoose)

0.6 (Coyote)

0.9 to 15 (cats and 

dogs)

14 (meadow vole)

--

Lab Rat- Dietary (mg/kg-diet) 1 2.08 --

Green cells denote lower toxicity

Black-Footed Ferret

Of special concern is the federally listed Black-Footed Ferret, which has an obligate relationship 

with prairie dogs for food and the use of their borrows for shelter. The Black-Footed Ferret may

consume poisoned prairie dogs or non-target animals that contain diphacinone residue in body 

tissues (secondary exposure) and decreases in prairie dogs may decrease the availability of 

suitable habitat for the ferret. Historically, where Prairie Dogs were found, so were BlackFooted Ferrets. A major cause for the decline in Black-Footed Ferrets is the reduction of the 

range of Prairie Dogs (USFWS 1988). This label targets a major food source of the Black 

Footed Ferret within much of its entire historic range, making recolonization and recovery 

problematic. Similar labels for chlorophacinone, a rodenticide of the same class, states, “Do not 
48

use this product within Prairie Dog towns in the range of the Black-Footed Ferret without first 

contacting U.S. Fish and Wildlife Service...” States/US Territories in which KAPUT-D is 

proposed for use and populations of Black-footed Ferrets are known to or are believed to occur

include Colorado, Kansas, Montana, Nebraska, New Mexico, North Dakota, South Dakota, and

Wyoming.

5.3.3 Probit Slope Response Analysis 

The Agency uses the probit dose-response relationship as a tool for providing additional 

information on the potential for acute direct effects to individual listed species and aquatic 

animals that may indirectly affect the listed species of concern (USEPA, 2004). As part of this 

evaluation, the acute RQ for listed species is presented in terms of the chance of an individual 

event (i.e., mortality or immobilization) should exposure at the EEC actually occur for a species 

with sensitivity to diphacinone on par with the acute toxicity endpoint selected for RQ 

calculation. To accomplish this interpretation, the Agency uses the slope of the dose-response 

relationship available from the toxicity study used to establish the acute toxicity measures of 

effect for each taxonomic group that is relevant to this assessment. The individual effects 

probability associated with the acute RQ is based on the mean estimate of the slope and an 

assumption of a probit dose-response relationship. In addition to a single effects probability 

estimate based on the mean, upper and lower estimates of the effects probability are also 

provided to account for variance in the slope, if available. Based on the available acute toxicity

for diphacinone, a summary of the probit dose-response analysis is provided below. If no dose 

response information is available to estimate a slope for this analysis, a default slope assumption 

of 4.5 (with lower and upper bounds of 2 to 9) (USEPA 1986) is used. 

Individual effect probabilities are calculated based on an Excel spreadsheet tool IECv1.1 

(Individual Effect Chance Model Version 1.1) developed by the U.S. EPA (OPP, Environmental 

Fate and Effects Division, June 22, 2004). The spreadsheet performs these calculations by 

entering the mean slope estimate (and the 95% confidence bounds of that estimate) as the slope 

parameter for the spreadsheet. The desired threshold for the probability of an individual effect is 

entered as the listed species LOC. In addition, the probability of an individual effect is also 

derived based on the calculated acute RQ. For these calculations, bird and mammal RQs from 

the primary exposure to bait (direct consumption) were used.
49

Summary of Diphacinone Probit Dose Response Analysis for Listed Species*

Taxa (study type) Acute Effect

Slope (95% C.I.)

Chance of Individual 

Effect at Listed Species 

LOC 1(95% C.I.)

Chance of Individual 

Effect at Derived Acute 

RQ (95% C.I.)

Snake oral dose

(max RQ = 1.08)

Mortality

Slope = 4.2 1 in 7.49E+04

Approximately 1 in 2 

assuming a range of slopes 

from 2 to 9.

Bird dietary

(max RQ = 0.06-0.11)

Mortality

Slope = 4.5

1 in 2.94E+05 <1 in 2.9E5

Mammal dietary fox

(max RQ = 6.1)

Mortality

Slope = 4.5

1 in 2.94E+05 Approximately 1 in 1 

assuming a range of slopes 

from 2 to 9.

Mammal dietary mouse

(max RQ = 21.7-43.5)

Mortality

Slope = 4.23 1 in 8.56E+04

Approximately 1 in 1 

assuming a range of slopes 

from 2 to 9.

1 Listed Species LOC = 0.10 for terrestrials, 0.05 for aquatic species

*Taken from the 2011 Endangered Species Assessment 

5.3.4 Indirect Effects Analysis

The Agency acknowledges that pesticides have the potential to exert indirect effects upon the 

listed organisms by, for example, perturbing forage or prey availability, altering the extent of 

nesting habitat, etc. In conducting a screen for indirect effects, direct effect LOCs for each 

taxonomic group are used to make inferences concerning the potential for indirect effects upon 

listed species that rely upon non-endangered organisms in these taxonomic groups as resources 

critical to their life cycle.

Based on direct risk to terrestrial animals, there may be potential indirect effects to terrestrial 

animals that depend on these organisms (including their surrogates) as a source of food or 

habitat. Reduction in bird, reptile and mammal populations will reduce the food base for any

obligate or opportunistic carnivores and scavengers. In addition, many of the potentially affected 

species (including the target species, BPD) provide unique habitats through the digging of 

burrows. Often these burrows are used concurrently with other species or other species may

utilize abandoned burrows. If the populations of these burrowing animals are significantly 

affected, a shift in the entire ecosystem structure in the targeted region may occur. 

Terrestrial plants that rely on animals for pollination and seed dispersal may have the potential to 

experience indirect effects through reduction in the animals that perform those functions.

5.3.5 Critical Habitat for Listed Species

In the evaluation of pesticide effects on designated critical habitat, consideration is given to the 

physical and biological features (primary constituent elements) of a critical habitat identified by

the U.S Fish and Wildlife and National Marine Fisheries Services as essential to the conservation 

of a listed species and which may require special management considerations or protection. The 

evaluation of impacts for a screening level pesticide risk assessment focuses on the biological 

features that are primary constituent elements and is accomplished using the screening-level 
50

taxonomic analysis (RQs) and listed species’ levels of concern (LOCs) that are used to evaluate 

direct and indirect effects to listed organisms.

The screening-level risk assessment for diphacinone has identified potential concerns for direct 

effects on listed terrestrial animals and indirect effect to those organisms dependent upon them 

for food and/or habitat. In light of the potential for indirect effects, the next step for EPA and the 

Service(s) is to identify which listed species and critical habitat are potentially implicated.

Analytically, the identification of such species and critical habitat can occur in either of two 

ways. First, the Services could determine whether the action area overlaps critical habitat or the 

occupied range of any listed species. If so, EPA would examine whether the pesticide’s potential 

impacts on non-endangered species would affect the listed species indirectly or directly affect a 

primary constituent element of the critical habitat. Alternatively, the Services could determine 

which listed species depend on biological resources, or have constituent elements that fall into 

the taxa that may be directly or indirectly impacted by a pesticide. Then EPA would determine 

whether or not use of the pesticide overlaps the critical habitat or the occupied range of those 

listed species. At present, the information reviewed by EPA is not sufficient to permit use of 

either analytical approach to make a definitive identification of species that are potentially 

impacted indirectly or critical habitats that are potentially impacted directly by the use of 

pesticides. EPA and the Service(s) are working together to conduct the necessary analysis.

This screening-level risk assessment for critical habitat provides a listing of potential biological 

features that, if they are primary constituent elements of one or more critical habitats, would be 

of potential concern. These correspond to the taxa identified above as being of potential concern 

for indirect effects and include birds, reptiles, terrestrial phase amphibians, mammals, terrestrial 

plants and aquatic organisms. This list should serve as an initial step in problem formulation for 

further assessment of critical habitat impacts outlined above, should additional work be 

necessary.

6.0 Uncertainties

6.1 Fate and Transport Properties

The most significant uncertainty regarding the fate and transport properties of diphacinone are

major metabolite, diphenylglycolic acid. Since the main exposure route is from consumption of 

the baits, this soil degradate is not expected to be digested. Therefore, the impact of this 

uncertainty will be minimal.

6.2 Effects

Uncertainty in the exposure assessment stems mainly from assumption made in the assessment 

related to the consumption of bait by various types of animals. Animals were assumed to 

consume an amount of bait equal to their predicted daily food ingestion rate. Ingestion of bait is 

most certain for omnivorous mammals because the bait is designed to be attractive to rodents and 

other mammals. However, small mammals could eat less bait than their average daily ingestion 
51

rate, either because they are also feeding on other food sources, or because they exhibit bait 

shyness. Alternatively, if other food is scarce and they find the bait to be very attractive, then 

they could exhibit gorging behavior, consuming bait in excess of their daily average daily intake 

rate. Incidents have shown that species other than small mammals have been killed due to 

diphacinone poisoning. Animals which feed predominantly on live prey, including snakes, may

not consume the bait. The use of allometric equations to estimate daily food intake rate 

introduces additional uncertainty. The food intake rate was estimated from the body weights of 

the animals using allometric equations. How well the generic allometric equations used predict 

the specific food intake rate of the assessed species is an uncertainty. For example, the 

relationship for the snake was based on an equation developed for insectivores, whereas snakes

can consume a wide variety of vertebrate prey in addition to terrestrial invertebrates. 

The assessment of secondary exposure involves additional uncertainties. A conservative 

assumption was made that the entire amount of active ingredient consumed by the prey is present 

in the prey animal when it is consumed by the carnivorous or scavenger animal. In reality, the 

amount of active ingredient in the prey may decrease between the time it consumes the bait by

the prey and the prey is consumed by the carnivorous or scavenger animal as the result of 

elimination and detoxification. Also, the amount of and rate of assimilation of diphacinone from 

the consumed prey into the secondary consumer is uncertain. Assimilation efficiency may be 

considerably less than the assumed 100%. Also, snakes and other reptiles typically digest large 

prey slowly over numerous days. Thus, the toxic residues in the ingested prey may be released 

and assimilated in some reptiles slowly over numerous days. In addition, it would seem that the 

mode of action that produces mortality in mammals is different than that in reptiles. Since 

diphacinone is an anticoagulant, mammals die through hemorrhaging. However, Brown Tree 

snakes dosed with diphacinone did not die of hemorrhage but by an unknown mechanism

(Brooks et.al. 1998).

The dose of diphacinone from secondary exposure is dependent on the size of the prey and its 

intake rate. The size of prey that a snake was predicted to be able to consume (100% of its BW) 

is uncertain. These equations may overestimate the maximum size prey which it may consume. 

Lastly, chronic risk is an uncertainty in part because no data is available but also because 

sublethal adverse effects to reproduction are not known. It is assumed that ingestion of enough 

diphacinone would induce mortality where reproduction would not be an issue. However, nonlethal effects to reproduction in these species are unknown. For example, foxes whelping pups 

do not hunt. Instead, the male fox will bring food to the female. In this case, the female may be 

killed if the prey brought by the male had ingested Diphacinone. In addition, since Diphacinone 

does not kill immediately, it is not known if Diphacinone may be excreted in mammalian milk, 

thus exposing whelping young. 

Overall, the reported incident data indicates pathways are complete and include species 

mortalities under the current assessment. Exposure assumptions are plausible and may result in 

mortality events.
52

7.0 Bibliography

Baroch, J. 1994a. Field efficacy of rodent bait diphacinone treated grains (0.01% SLN No. CA 

890022 and 0.005% SLN No. CA 890020) using spot-baiting applications to control the 

California ground squirrel (Spermophilus beecheyi). Unpublished report submitted to EPA by

the California Department of Food and Agriculture, Sacramento.

Brooks, J.E., P.J. Savarie and J.J. Johnston. 1998. The oral and dermal toxicity of selected 

chemicals to brown tree snakes (Boiga irregularis). Wildlife Research 25:427-435.

Bullard, R.W., R.D. Thompson, G. Holguin. 1976. Diphenadione residues in tissues of cattle. 

Journal of Agricultural and Food Chemistry. 24:261-263.

Byers, R. E. (1978). Performance of Rodenticides for the Control of Pine Voles in Orchards. J. 

Am. Soc. Hortic. Sci.103:65-69.

Cox , P. and R.H. Smith. 1992. Rodenticide ecotoxicology: pre-lethal effects of anticoagulants

on rat behavior. Proc. Vertebr. Pest Conf. 15:165-170.

Diaz, L.I. and D.M. Whitacre. 1976. Excretion and retention of 14C-diphacinone in rats.

Unpubl. report, submitted by Velsicol Chemical Corporation. 8 pp.

Fisher, P., C.O’Connor, G. Wright, and C.T. Eason. 2003. Persistence of four anticoagulant 

rodenticides in the liver of laboratory rats. Doc Science Internal Series 139. New Zealand 

Department of Conservation, Wellington, NZ. 19 pp. MRID# 48190801

Fisher, P. 2006. Persistence of residual diphacinone concentrations in pig tissues following 

sublethal exposure. DOC Research & Development Series 249. Department of Conservation, 

Wellington. 19p.

LiphaTech, Inc. 1997. The veterinarian’s guide to managing poisoning by anticoagulant 

rodenticides. Brochure available from Liphatech, Inc., 3600 West Elm Street, Milwaukee, WI. 

16 pp. Available at: (http://www.liphatech.com/vetguide.html)

Nagy, K.A. 1987. Field metabolic rate and food requirement scaling in mammals and birds. 

Ecol. Monogr. 57:111-128.

Rattner B.A., Horak K.E., Warner S.E. and J.J. Johnston. 2010. Acute toxicity of diphacinone in 

Northern bobwhite: Effects on survival and blood clotting. Ecotoxicol Environ Saf 73:1159–

1164.

Rattner B.A., Horak K.E., Warner S.E., Day D.D., Meteyer C.U., Volker S.F., Eisemann J.D.

and J.J. Johnston. 2011a. Acute toxicity, histopathology, and coagulopathy in American kestrels 

(Falco sparverius) following administration of the rodenticide diphacinone. Environ Toxicol 

Chem. 30:1213-1222.
53

Savarie, P.J. and R.K. LaVoie. 1979. Secondary toxicity hazards of the anticoagulant 

brodifacoum to American kestrels (Falco sparverius). Unpubl. report, Denver Wildlife Research 

Center. 24 pp. MRID# 80244, 00079312

Teeters W.R. 1981. Diphacinone technical: Toxicity to laboratory rat: Test No. 51 & 75 

(USEPA, PRD, Ag Research Center, Animal Biology Laboratory, unpublished report). 

U.S. Environmental Protection Agency. 1986. Hazard Evaluation Division Standard Evaluation 

Procedure: Ecological Risk Assessment. Office of Pesticide Programs. EPA-540/9-85-001

U.S. Environmental Protection Agency. 1993. Wildlife Exposure Factors Handbook. Office of 

Research and Development. December. EPA/600/R-93/187.

U.S. Environmental Protection Agency. 1998a. Guidelines for Ecological Risk Assessment. 

United States Environmental Protection Agency (USEPA). Risk Assessment Forum. Office of 

Research and Development. Available at 

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=12460 (Accessed June 19, 2009).

U.S. Environmental Protection Agency.1998b. Reregistration Eligibility Decision (RED): 

Rodenticide Cluster. July 1998. EPA 738-R-98-007.

U.S. Environmental Protection Agency. 2004a. Overview of the Ecological Risk Assessment 

Process in the Office of Pesticide Programs. US EPA Office of Pesticide Programs, 

Environmental Fate and Effects Division.

U.S. Environmental Protection Agency. 2004b. Potential Risks of Nine Rodenticides to Birds 

and Nontarget Mammals: a Comparative Approach. July 2004. US EPA Office of Pesticide 

Programs, Environmental Fate and Effects Division.

U.S. Environmental Protection Agency. 2009. Guidance for Selecting Input Parameters in 

Modeling the Environmental Fate and Transport of Pesticides; Version 2.1. Office of Pesticide 

Programs. Environmental Fate and Effects Division. October 22, 2009.

USFWS. 1988. Black-footed ferret recovery plan. U.S. Fish and Wildlife Service, Denver, CO 

154pp.

USFWS. 1993. U.S. Fish and Wildlife Service Biological Opinion. Effects of 16 Vertebrate 

Control Agents on Threatened and Endangered Species. U.S. Fish and Wildlife Service. 

Yu, C.C., Y.H. Atallah, and D.M. Whitacre. 1982. Metabolism and disposition of diphacinone

in rats and mice. Drug Metabolism and Disposition 10:645-648.
54

APPENDIX A

Risk Quotient Method and Levels of Concern

Risk characterization integrates the results of the exposure and ecotoxicity data to evaluate the 

likelihood of adverse ecological effects. The means of this integration is the quotient method. 

Risk quotients (RQs) are calculated by dividing exposure estimates (EECs) by acute and chronic 

ecotoxicity values. 

RQ = EXPOSURE/TOXICITY

RQs are then compared to OPP's levels of concern (LOCs). LOCs are used by OPP to determine 

potential risk to non-target organisms and identify the need to consider regulatory action to 

mitigate risk. The criteria indicate that a pesticide used as directed has the potential to cause 

adverse effects on non-target organisms. LOCs currently address the following risk presumption 

categories: 

 · acute risks - regulatory action may be warranted in addition to restricted use classification

 · acute restricted use - the potential for acute risk is high, but may be mitigated through 

restricted use classification

 · acute endangered species - endangered species may be adversely affected, and 

 · reproductive/chronic risk - the potential for chronic risk is high regulatory action may be 

warranted. 

EFED does not perform assessments for chronic risk to plants, acute or chronic risks to insects or 

chronic risk from granular/bait formulations to birds or mammals.

The ecotoxicity test values (measurement endpoints) used in the acute and reproductive/chronic 

RQs are derived from required studies. Examples of ecotoxicity values derived from short-term 

laboratory studies that assess acute effects are: 

 · LC50 (fish and birds)

 · LD50 (birds and mammals)

 · EC50 (aquatic plants and aquatic invertebrates), and 

 · EC25 (terrestrial plants - nonlisted species) 

 · NOAEC or EC05 (terrestrial plants - listed species)

Examples of toxicity test effect levels derived from the results of longer-term laboratory studies 

that assess reproductive/chronic effects are: 

 · LOAEC (birds, fish, and aquatic invertebrates)

 · NOAEC (birds, fish and aquatic invertebrates)

Other toxicity values may be used when justified. 
55

Risk presumptions and the corresponding RQs and LOCs, are tabulated below.

Risk presumptions for terrestrial animals based on risk quotients (RQ) and levels of 

concern (LOC)

Risk Presumption RQ LOC

Birds:

 Acute Risk EEC1/LC50 or LD50/ft2or LD50/day30.5

 Acute Restricted Use EEC/LC50 or LD50/ft2or LD50/day (or LD50 < 50 mg/kg) 0.2

 Acute Endangered

 Species

EEC/LC50 or LD50/ft2or LD50/day

0.1

 Chronic Risk EEC/NOAEC 1

Wild Mammals:

 Acute Risk EEC/LC50 or LD50/ft2or LD50/day 0.5

 Acute Restricted Use EEC/LC50 or LD50/ft2or LD50/day (or LD50 < 50 mg/kg) 0.2

 Acute Endangered

 Species

EEC/LC50 or LD50/ft2or LD50/day

0.1

 Chronic Risk EEC/NOAEC 1

1

 Estimated Environmental Concentration (EEC, ppm) on avian and mammalian food items

2

 mg/ft2

3

 mg of toxicant consumed/day

 LD50 * wt. of bird

 LD50 * wt. of bird 

Risk presumptions for aquatic animals based on risk quotients (RQ) and levels of concern 

(LOC)

Risk Presumption RQ LOC

 Acute Risk EEC1/LC50 or EC50 0.5

 Acute Restricted Use EEC/LC50 or EC50 0.1

 Acute Endangered Species EEC/LC50 or EC50 0.05

 Chronic Risk EEC/NOAEC 1

1

 EEC = (ppm or ppb) in water
56

Risk presumptions for plants based on risk quotients (RQ) and levels of concern (LOC).

Risk Presumption RQ LOC

Terrestrial and Semi-Aquatic Plants:

 Acute Risk EEC1/EC25 1

 Acute Endangered Species EEC/EC05 or NOAEC 1

Aquatic Plants:

 Acute Risk EEC2/EC50 1

 Acute Endangered Species EEC/EC05 or NOAEC 1

1

 EEC = lbs ai/A 

2

 EEC = (ppb/ppm) in water 
57

APPENDIX B

Listed Species Occurrence in KAPUT-D Use States for Selected Terrestrial Taxa: 

Mammal, Bird, Amphibian, Reptile by Use Site 

Species Occurrence in Selected States and Selected Taxa

No species were excluded

All Medium Types Reported

Mammal, Bird, Amphibian, Reptile

CO, KS, MT, NE, NM, ND, OK, SD, TX, WY

Colorado CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Owl, Mexican Spotted Strix occidentalis lucida Threatened Terrestrial Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No

Mouse, Preble's Meadow Jumping Zapus hudsonius preblei Threatened Terrestrial Yes

Wolf, Gray Canis lupus Endangered Terrestrial Yes

Kansas CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No

Montana CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No

Nebraska CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No
58

 New Mexico CH

Amphibian

Frog, Chiricahua Leopard Rana chiricahuensis Threatened Freshwater, Terrestrial No

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Falcon, Northern Aplomado Falco femoralis septentrionalis Endangered Terrestrial No

Owl, Mexican Spotted Strix occidentalis lucida Threatened Terrestrial Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial 

No

Jaguar Panthera onca Endangered Terrestrial No

Reptile

Rattlesnake, New Mexican Ridge-nosed Crotalus willardi obscurus Threatened Terrestrial Yes

North Dakota CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Mammal

Wolf, Gray Canis lupus Endangered Terrestrial Yes

Oklahoma CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

South Dakota CH

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No

Wolf, Gray Canis lupus Endangered Terrestrial Yes

Texas CH

Amphibian

Toad, Houston Bufo houstonensis Endangered Terrestrial, Freshwater Yes

Bird

Crane, Whooping Grus americana Endangered Terrestrial, Freshwater Yes

Falcon, Northern Aplomado Falco femoralis septentrionalis Endangered Terrestrial No

Owl, Mexican Spotted Strix occidentalis lucida Threatened Terrestrial Yes

Prairie-chicken, Attwater's Greater Tympanuchus cupido attwateri Endangered Terrestrial No

Warbler (=Wood), Golden-cheeked Dendroica chrysoparia Endangered Terrestrial No
59

Mammal

 

Jaguarundi, Gulf Coast Herpailurus (=Felis) yagouaroundi Endangered Terrestrial No

cacomitli

Jaguarundi, Sinaloan Herpailurus (=Felis) yagouaroundi Endangered Terrestrial No

tolteca

Ocelot Leopardus (=Felis) pardalis Endangered Terrestrial No

Reptile

Snake, Concho Water Nerodia paucimaculata Threatened Freshwater, Terrestrial Yes

Wyoming CH

Amphibian

Toad, Wyoming Bufo baxteri (=hemiophrys) Endangered Freshwater, Terrestrial No

Mammal

Ferret, Black-Footed Mustela nigripes Endangered Terrestrial No

Mouse, Preble's Meadow Jumping Zapus hudsonius preblei Threatened Terrestrial Yes