# Ecological Risk **Assessment**

**for the Section 3 New Use Registration of Di** **phacinone (**  **PC 06770**  **1 )**  **For Use on**  **Black -**  **tailed Prairie Dogs (**  **Cynomys ludovicianus )** 

**Prepared by:** N.E.Federoff

, Wildlife Biologist J. L in, Environmental Engineer **U. S. Environmental Protection Agency Office of Pesticide Programs**  **Environmental Fate and Effects Division Environmental Risk Branch I**  **I 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 2**  **8 ,**  **201 2** 23 **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 Formul ation
................................ ................................ ............................ 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 Ecosy stems Potentiall y at Risk
................................ ................................ ............. 20 2.7 Assessment Endpoints
................................ ................................ .......................... 20 2.8 Conceptual Model
................................ ................................ ................................ . 20 2.8.1 Risk H ypotheses
................................ ................................ ................................ .... 20 2.8.2 Conceptual Diagram
................................ ................................ ............................. 2 1 2.9 Analy sis 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 Ass essment ................................ ................................ ............................ 26 3.1 Label Application Rates and Intervals
................................ ................................ .. 26 3.2 Aquatic Exposure Assessment
................................ ................................ .............. 264

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 Carcasses 28 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 In cident Datab ase 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 Analy sis
................................ ................................ ........... 48 5.3.4 Indirect Effects Anal ys is
................................ ................................ ....................... 49 5.3.5 Critical Habitat for Listed Species
................................ ................................ ........ 49 6.0 Uncertainties
................................ ................................ ................................ ......... 50 6.1 Fate and Transport Properties
................................ ................................ ............... 50 6.2 Effects
................................ ................................ ................................ ................... 505

7.0 Bibliograph y ................................ ................................ ................................ ......... 526 

### List of Appendices **Appendix A**  **RQ Methods and LOC Definitions Appen**  **dix 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 b ait that contains 0.0 05% 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 p roduct label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, TX, MT and OK. Diphacinone is a first generation i ndanedione anticoagulant rodenticide and it is absorbed through the gut, inhibiting vitamin K. Indan e diones are not onl y 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 bloo d
- clotting mechanisms and induce capillary

damage. Ty

pically , death is delayed for four to ten or more day s 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 relativel y low vapor pressure of approximately

1 x 10 - 8 Torr and a 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. The estimated Kd s of diphacinone are consistent with compounds , which leach to ground water. However, they

are bounding estimates from column leaching studies , and it is very likel y 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, diphen yl gl ycolic acid , comprised up to 25 % of the a

pplied material in an aerobic soil metabolism study
. This degradate appears to be8

persistent, but its mobility is unknown. Because diphacinone is formulated in pelleted bait or bait
block rodenticide products and primaril y use d 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 diphenylg lycolic acid are , therefore , not quantified f

or this assessment. Fo r this assessment, it was assumed that terrestrial animals could be exposed to diphacinone through two different pathway s. Animals may directly consume bait (primary

consumption), or animals may

consume contaminated carcasses either killed or scavenged b y 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 li ttle 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 (LD 50 ’s = 0.2 to 57 mg ai/kg bw; LC 50 = 2.0 8 ppm) . It is moderate ly toxic to birds (LD 50 ’s > 400 to 3158 mg ai/kg bw; LC 50 ’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 day s rather than in a single dose. Toxicity

data are also available for sn akes, where LD50 values were 20.75
- 31.21 mg/kg
- bw. Diphacinone is moderately toxic to freshwater fish ( LC 50 = 2. 6 ppm ) and invertebrates ( LC 50 = 1.8 ppm ) on an acute exposure basis. No data are available to evaluate chronic effects to freshwater fish or i nvertebrates. No data are available to evaluate acute or chronic effects to estuarine/marine fish or invertebrates. No toxicity

d ata are available for aquatic or terrestrial plants. G iven 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 w ere based on multiple lines of evidence including calculation of RQs (mammals and snakes) a s well as incident reports of mortalities of mammals and birds exposed to diphacinone and secondary toxicity studies
. When9

considering listed species, there is a potential concern for indirect effects upon the listed organisms due to decreased prey availab ility

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

– mo nocots 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 plants a 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 anal ys is, 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 directl y or indirectly affected
. With additional refinement b y exploring more detai led 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 o f risk include:  Incidents in the US reported in EI IS that were considered probable or highly probable for death due to diphacinone. Mortalities included species that were likel y 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 (wei ghing 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) m ortality through exposure to diphacinone was documented through l ow LD 50 and L C 50 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 federall y listed species may

be affected , a number of conservation measures were developed with discussions with and agreement between the F ish 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 Biolo gical 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=docI d;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/dyinglisted species and no n - target animals in addition to prohibitions of Rozol use and timing restrictions via the Agency ’s Endangered Species Protection Program (ESPP) Bulletins L ive sy stem 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 endangere d 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 progra m 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 pro files are similar for chlorophacinone and diphacinone ( s ee t able 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 de creases 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 anywh ere within the range of bla ck 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 appl yi ng either of these products. For this reason, the Biological O pinion issued on R ozol is expected to be applicable to both Rozol and Kaput black tailed prairie dog bait, and is, therefore, expected to provide adequate protections for l isted species for use of both rodenticides.11

Table 5.7. Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters **Comparative Param eter**  **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
- mo use 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: 9 6 . 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: 2 0.75 -- Toxicity Mammals Lab Rat

– Gavage (mg/kg
- bw) 6 1.9 --

Pine Vole

– Gavage (mg/kg
- bw) 14 58 -- Other Species

– Gavage (mg/kg
bw) 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) phy sicochemical properties, 2) hydroly sis, 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/sc reen 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 W ildlife (850.2500)  Acute Passerine Study (850. 2100 )  Avian Reproduction Study (850.2300), two species12



### 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 di phacinone. 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 (US EPA, 1998 a). 2.1 **Nature of Regulatory Action** The proposed Se ction 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 b ait that contains 0.0 05% diphacinone (50 mg a.i./kg
- bait) and must be hand applied on rangeland and a djacent 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 bel ow. 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 d iphacinone, 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 potenti al 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 assessme nt was completed in 2011. Itassessed risk from the use of diphacinone to the Federall y Threatened Alameda Whipsnake ( Masticophis lateralis euryxanthus ) and the California Tiger Salamander ( Ambystoma californiense ), as well as risks to the Federall y Endan gered 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 muti ca )
. 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 ( SMH M ) 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. Ty

pically , death is delayed for four to ten or more day s 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 ( i ndandiones also uncouple oxidative phosphory lation (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** D iphacinone has moderate leaching potential. combined with a low solubility (0.3 mg/L) water. Consistent with a relativel y 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. measure s 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**  **- C**  **hemical Properties of Property**  **Value** Structure

Molecular Weight 340.48 g/mole 13

**Stressor Source and Distribution Mechanism of Action**  first generation i ndanedione anticoagulant rodenticide and is absorbed through Anticoagulants are vitamin
- K antagonists that disrupt n ormal blood
- clotting mechanisms and induce capillary damage. Ty

pically , death is delayed for four to ten or more day s after a lethal dose is ingested, and animals may continue to feed and move about until shortly before death. Death results from hemorrha ge, and exposed animals may exhibit behavior that may make them more susceptible to predation ( Cox and Smith, 1992 ). In addition, ndandiones also uncouple oxidative phosphory lation (energy

generation) in mammals Diphacinone seems to have a different mo de 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. T he range of possible K d values is 3 to

low solubility

(0.3 mg/L) indicates that it is unlikely to contaminate ground Consistent with a relativel y low vapor pressure of approximately 1 x 10 - 8 Henry ’s Law constant of approximately 2 x 10 - 10 atm - m 3 /mole, volatil ization 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
. D ipha cinone is metabolized via aerobic soil metabolism life value of approximately 30 day s 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. Ty

pically , death is delayed for four to ten or more day s 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 phosphory lation (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 - 8 torr 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 D**  **iphacinone Environmental Fate Properties**  **Study Value and**  **unit Major Degradate** 

**Minor Degradates MRID # or**  **Citation Study Classification,** 

**Comment** Hydroly sis 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 3 0 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 day s (average of 28 and 32 days) from studies done at two different radiolabeled positions. A major soil metabolite ( > 10% of dose) was diphen yl gl yco lic 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 relativel y constant concentration until the end of the 3.5 month study . Up to eight minor organic metaboli tes 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 regularl y 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 clearl y indicative of ultimate biological deactivation of a15

large fraction of diphacinone and its transformation products. This study also indicated, however, that the major metabolite, diphenylg lycolic acid, was somewhat resistant to further metabolism. Diphacinone was stable against simple h ydrol ys is at pH 7 and 9. At pH 5 , hydrol ys is proceeded with an extrapolated half
- life of approximately

44 day s. Products of hydroly sis were not satisfactoril y identified or quantified. However, since the apparent hydrolysis was appreciable only at low pH and aerobic metabol ism provides a viable alternative transformation process for diphacinone, there is marginal value in additional information on hydroly tic 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 - 8 torr and a Henry ’s law constant for diphacinone of approximately 2 x 10 - 10 atm - m 3 /mole also indi cates 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 t o 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 diphacin one was “relatively immobile” because it was not detected in the leachates of the four soils tested and moved significantly onl y 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 b ulk density of 1.5 g/cm 3 and 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), diphacinon e 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 th e top increment. This may or may not be a conservative assumption because of the 6 cm limit of resolution. For the loam y 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/gwith uncertain but “wide” error bars, if for no other reason than depth increment resolution. For the loamy sand soil, the corresponding16

calculated Kd is approximately 3 mL/g. Compounds with Kd values in this range are often classified as mobile. The loamy sand r esult 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 alter

native approximation which can be made to estimate crudel y 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 partit ioning ratio (Pow or Kow) and the soil organic matter to water sorption coefficient (Koc). The relationships 1 , assuming soil organic matter is typically about 58% carbon [i.e., organic carbon (oc) = organic matter (om)/1.72)], are

log (Kom) = 0.904 l og (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 Lo g 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 rough ly 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 sy stems; 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 (EPISuite), 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** P otential transport mechanisms ty pically include pesticide surface water runoff, spray drift , and secondary drift of volatilized or soil
- bound residues leading to deposition onto nearb y or more distant ecosy stems. 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 diphacinon e 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 dyi ng, thereb y 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. Histo**  **ric 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 biologicall y 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, especiall y for a chemical whose metabolites/degradates are of greater toxicological concern. The as sessment of ecological exposure pathway s, therefore, includes an examination of the source and potential migration pathway s for constituents, and the determination of potential exposure routes ( e.g., ingestion, inhalation, and dermal absorption). Ecolog ical receptors that may potentiall y 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 v ia runoff and drift. Consistent with the process described in the Overview Document ( US EPA, 2004 a), this risk assessment uses a surrogate species approach in its evaluation of the proposed new uses of diphacinone. Data generated from surrogate test speci es, 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 p esticide 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 th ese 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 ty pically performed on a number of organisms from several taxonomic groups, including birds, mammals, fish, and aquatic invertebrates ( US EPA 2004 a). The surrogate test species, assessment endpoints, and measures of effect derived from these studies used for this risk assessment are provided. Additional informati on from other sources, such as public literature and incident reports, are used when relevant.20

2.6 **Ecosystems Potentially at Risk** The ecos ys tems at risk are often extensive in scope ; therefore, it may not be possible to identify specific ecos ys tems during the development of a baseline risk assessment. However, in general terms, terrestrial ecos ys tems 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 i s to be protected, defined b y an ecological entity

(species, community , or other entity) and its attribute or characteristics (US EPA 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 ecos ys tems potentially

at risk, the mi gration pathway s of pesticides, and the routes b y which ecological receptors are exposed to pesticide
- related contamination. The selection of clearl y defined assessment endpoints is important because they provide direction and boundaries in the risk assess ment 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 hy pothes e s and a conceptual diagram (US EPA, 1998 a). 2.8.1 **Risk Hypothes e**  **s** Based on the results of previous risk assessments for diphacinone, the following ecological risk hy potheses are being employed for this risk assessment:

 Terrestrial o rganisms 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. B ased 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 b ait will be available to non
- target organisms and, as toxic food bait, will adversel y affect terrestrial species. **Figure 2.**  **3** illustrates the anticipated exposure pathway s 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 in gestion of the bait and consumption of vertebrate body tissues that have eaten the food bait (USEPA 2004 b ). Exposure via these routes is expected primarily for birds and mammals, though it is likely that other terrestrial animals such as reptiles and terre strial amphibians are at risk if they consume invertebrates or tissues of vertebrates that have eaten bait. Dip hacinone 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 wa s 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, consumpt ion 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 formulati on .22

Figure 2.3. Conceptual Model Diagram of Diphacinone Exposure and Effects in Nontarget Species. Dotted lines indicate exposure pathway s that have a low likelihood of c ontributing to ecological risk. **Stressor Transport**  **Pathways** Incidental Deposition in Water

Consumption by Target or Non Target Terrestrial Vertebrates Deposition on Ground **Exposure Media**  Water Body/ Standing Water Upland/Riparian Soil

**Primary Exposure**  **Route and Receptors**  In gestion 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 Ti ssues and Eggs by Predators/Scavengers
**Attribute Changes**  Bioaccumulation and Food Chain Magnification

Reduced Reproduction and Survival of Terrestrial Non
Target Organisms Diphacinone23

2.9 **Analysis Plan** In order to address the risk hy pothes e s, 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 se ctions, the use, environmental fate, and ecological effects of di phacinone 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 d efined 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 2004 a) , the like lihood of effects to individual organisms from the use of diphacinone to control BPD s is estimated using the probit dose
response 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 approximat e a ‘high
- end’ exposure scenario with two tier 1 models: GENEEC model for surface water exposure and SCI GROW model for ground water exposure. The peak surface water concentration (EEC) predicted b y GENEEC i s 483 n g/ L. The peak ground water concentration (EEC) predicted b y SCI GROW i s 4.75 n g/ 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., LD 50 dose). Estimates of food
- ingestion rates (g dry matter per day) are determined from established allometric equations presented in the Wildlife Exposure Factors Handbook (US EPA 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 Secon**  **dary Terrestrial**  **Exposure** Secondary

exposure analy sis (from consumption of contaminated prey and/or scavenged carcasses ) requires consideration of residues in the bodies of organisms that are commonl y consum ed b y predators and scavengers
. Details of the anal ys is 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 In**  **tegration 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 exp osure 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 2004 a ). 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 expos ure 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 o f 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 s ingle 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 b y 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 o f 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 W ildlife (850.2500). The purpose of this study is to evaluate the availability of carcasses containing diphacinone residues to carnivores and scavengers. T his study would also be useful for estimating diphacinone concentrations in prey
. T he purpose of this requested study is to provide quantitative data that are currentl y unavailable for KAPUT - D Prairie Dog Bait to a ss ess the availability

of carcasses. More specificall y, 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 e cological 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 Dog s are eas y prey and scavenge items for raptors and other birds, avian reproduction data are needed to adequately quantify the risks this proposed u se 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 LD 50 using bobwhite quail or mallard duck data may over - or under
- estimate risks to passerines because these birds may

metab olize the chemical differentl y. 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 dip hacinone. 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. **E nvironmental**  **F ate**  As cited in the RED (USEPA, 1998b), the only available environmental fate data from studies for diphacinone are 1) phy sicochemical properties, 2) hydroly sis, 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 is26

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**  F or this assessed use ( i.e ., control of BPD ) , dip hacinone 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** A quatic 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 SCI GROW model for ground water exposure. The peak surface water concentration (EEC) predicted b y GENEEC i s 483 n g/ L. The peak ground water concentration (EEC) predicted by SCI GROW i s 4.75 n g/ L (concentrations taken from p revious assessment) . Thus , aquatic routes of exposure are n ot 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 pathway s. Animals may

directly consume bait (primary

consumption), or animals may consume contaminated carcasses either killed or scavenged by the consumer (seco ndary consumption). Both approaches and the expected exposure levels are detailed below. 3.3.1 **Expected Di**  **phacinone Ingestion through Bait Consumption** Exposure through bait consumption will be calculated using two methodologies. For the first method, diphacinon e 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 US EPA ( 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 we ight was converted to27

wet weight assuming the bait contained 10% water, similar to the assumption that seeds for wildlife consumption contain 10% water (US EPA 1993). Formulas for calculation of dose estimates are provided in **Table 3. 1**  , and exposure estimat es (on a dose basis) are provided in **Table 3. 2**  . RQs are generated b y dividing these exposure estimates (mg a.i./kg
- bwt) for a given weight class b y the most conservative toxicity endpoint for the relevant taxa adjusted for the default body weights. Acute RQs using these exposure estimates were generated for bird s and mammal s (using LD 50 data)
. **Table 3. 1**  **Formulas for Calculation of di**  **phacinone 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 Di phacinone intake (mg a.i./kg
- bwt/day) = FI (g wet
- wt/day) * 50 mg a.i./kg
-bai t / Wt(g) Where: Wt(g) = weight (in grams) of the bird or mammal consumer

**Table 3. 2**  **Expected Di**  **phacinone 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)**  **D phacinone intake**  **(m g a.i./kg**  **- bwt/day)**  B irds* 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 LC 50 (mg a .i./kg
- di et) available from dietary toxicity studies. Acute RQs using these exposure estimates were generated for bird s and mammal s.28 3.3.2 **Expected Di phacinone Ingestion through Consumption of**  **Contaminated Carcasses** M ammals 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 s econdary consumers of diphacinone poisoned animals or carcasses are calculated in a manner similar to the approach for individuals consuming bait directly
. Calculated and e mpirical 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 d ata 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 co ncentration was reported as 1.4 mg ai/kg bw with a maximum concentration of 3.4 mg ai/kg bw ( Baroch 1 994a , 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 thes e 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 carc ass 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 analy sis, 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 mamm al allometric equations from (EPA 1993). Food dry weight was converted to wet weight assuming the consumed prey /carcass contained 68% water (US EPA 1993). Formulas for calculation of dose estimates are provided in **Table 3. 3**  , and exposure estimates (on a dos e basis) are provided in **Table 3. 4**  . RQs are generated b y 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 LD 50 data)
.29

**Table 3. 3**  **Formulas for Calculation of Di**  **phacinone 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 Di phacinone 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 Di**  **phacinone 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) Di**  **phacinone intake (m**  **g 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 carcasse s . RQs are calculated using this estimate of dietary concentration and the LC 50 (mg a.i./kg
- diet) available from dietary toxicity studies. RQs using these exposure estimates were generated for acute bird and mammal (using LC 50 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 E COTOX 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 s ys tems, taxa evaluated include aquatic plants, invertebrates, and fish. F reshwater f ish 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 sy stems, taxa evaluate d include birds , mammals and honey bees . Bird endpoints are generall y 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 ty

pically conducted on laboratory rats, mice, or rabbits. In addition to registrant
- submitted and open literature toxicity information, other sources of informat ion, 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 Di**  **phacinone 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 lit erature . Data on toxicity of diphacinone to aquatic plants are not available. **Table 4.1. Aquatic Toxicity Profile for**  **Diphacinone Assessment**  **Endpoint A**  **cute/**

**Chronic Species** 

**TGAI % a**  **. i**  **. Toxicity Value** 

**Used in Risk Assessment**  **Citation or**

**MRID # (Author**  **, Date)**  **1 Classification** 
Freshwater fish (surrogate for aquatic
- phase am phibians) Ac ute Bluegill sunfish Lepomis macrochirus TGAI 95.8 % Rainbow Trout Oncorhynchus mykiss TGAI 95.8 % 96 - hr LC 50 = 7.5

mg/L
96 - hr LC 50 = 2.6 mg/L MRID

432495 - 01 (Machado 1994) MRID 432495 - 02 (Machado 1994) Acceptable (moderately toxic)

Acceptable (moderately toxic)
Freshwater invertebrates Ac ute Daphnia Daphnia manga TGAI 98.7 % E C 50 = 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 Di**  **phacinone to Terrestrial Organisms** Table 4.2 summarizes the most sensitive terrestrial toxicity endpoints, based on an ev aluation of both the registrant
- submitted studies and the open lit erature . Data on toxicity of diphacinone to terrestrial plants are not available.31

**Birds** Diphacinone is acutely moderatel y toxic to avian species. T he most sensitive avian endpoint that corresponded to a standard test species (bobwhite quail or mallard duck) was an LD 50 = 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 LD 50 of 2014 mg a
. i
. /kg
bwt. In additi on, an LD 50 = 3158 mg a . i
. /kg
- bwt was determined for mallard ducks (MRID 00 128411 ). Also an LD 50 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 kestr els 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 moderatel y toxic (Mallard Duck L C50 = 906 ppm) to practicall y non - toxic (Bobwhite Quail L C50 = 5000 ppm). **Mammals** For mammals, the a cute oral and dietary toxicity of diphacinone is classified as very highl y toxic. This is expected, given the target of this rodenticide is mammals. The acute oral LD 50 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 LD 5 0 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. Sy mptoms occurred at all doses, and were not necessarily associated with subsequent mortality . These included clear or colored nasal discharge, soft stool and/or diarrhea (possibl y associated with the corn oil vehicle used), decreased motor activity and occasional drying of the corneal surface. S ymptoms 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 LD 50 = 1.9 mg a.i/kg
- bwt (MRID 05002272) . Toxicity tests conduct ed on pine voles and meadow voles ( Microtus pennsylvanicus ) resulted in LD 50 s of 67.7 and 11.7 mg a.i/kg
- bwt ( Byers, 1978). LD 50 values for dogs and cats ranged from 0.88 to 15 mg a.i/kg
- bwt (L iphaTech, Inc, 1997). LD 50 s 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 toxic ity studies. **Reptiles** Brooks et al
. (1998) conducted a study t o determine the acute oral and dermal toxicity o f 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 4.232

(ethanol used as a carrier) and 32.2 mg/kg (95% CI=18. 8 to 55.9) with a slope of 5.0 (prop yl ene gl ycol used as a carrier). Five snakes of either sex were used in each treatment group. All snakes were observed for 7 day s after administration of Diphacinone. Diphacinone produced mortalities within 24 hrs of administration of doses of 10 - 40 mg/kg. At necrops y, 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.**  **C lassification**  Birds (surrogate for terrestrial
phase am phibians and reptiles ) A cute

oral LD50 Bobwhite Quail

Kestr e l >400<2000

mg/kg (reported as > 1630 mg/kg) 9 6 . 8 mg/kg 422452 - 01

Rattner et al 2011 Supplemental

Supplemental Ac ute Dietary LC50 1 Mallard Duck

Bobwhite Quail 906 ppm3

( LOAEC=1.6 ppm) >5000 ppm2 424088 - 02

424088 - 01 Core

Core Mammals A cute Rat 2.5 mg/kg M 2.1 mg/kg F 00060605 Core Ac ute Rat 1.9 05002272 Supplemental Ac ute Dietary LC50 Rat

Rat 2.08 ppm

2.5 ppm TMN 075

TMN 051 Supplemental

Supplemental
Ac ute oral LD50 Coyote

Mongoose 0.6

0.2 Savarie et al . 1979 4

DWRC 5 405655 - 01 Supplemental

Supplemental Reptile Ac ute

oral LD50 6 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 mga.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 day s, and onl y 7% was retained in body tissues. In both rats and mice, most radioactivity

(59 to 69%) was detected in the liver and the kidney s (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 onl y 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 9 0 day s after dosing (Bullard et al. 1979). **Table 4.3. Retention of Diphacinone in Blood and Liver**  **Rodenticide Species**  **Dose (m g**  **a.i./kg) # doses**  **Blood t 1/2**  **a (days)**  **Liver**

**retention a,b**  **(days) Reference** 

**(M RID)**  Diphacinone Pig 12.5 3 or 5 NA 5.43 (t 1/2 ) Fisher 2006 Diphacinone Rat 1.5 1 NA 3 (t 1/2 ) Fisher et al. 2003 (48190801) Diphacinone Cattle 1.0 1 NA > 90 Bullard et al. 1976 a t 1/2 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) orall y dosed rats with diphacinone (0.32 mg a.i./kg/day) for 1 or 2 day s. Rats dosed for 2 day s were sacrificed 72 hours after the second dose and those dosed for 1 day were sacrificed after 48 hours. In rats dosed for 2 day s, 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 dos e 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 group**  **a 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 s econdary

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 sufficientl y high to cause mo rtality 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 co yote muscle was found to be considerabl y l ower 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 kil led 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 diphacinone
poisoned owls 1 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 Gr eat 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 Am erican 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 days36

4.4 **Incident Database Re view**  A review of the Ecological Incident Information Sy stem (EIIS, version 2.1), the ‘Aggregate In cident Reports’ (v. 1.0) database, and the Avian Monitoring Information Sy stem (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 “highl y probable”, eight ( 24 %) were deemed as “probable”, eleven ( 33. 5 %) wer e deemed “possible”, two ( 6 %) w ere 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 diphac inone 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 resultin g in an effect (i.e., mortality) has occurred. O nl y 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 incide nts are occurring or that one chemical is safer than another. However, incident data can corroborate results of deterministic risk assessment s. There were a number of terrestrial incidents reported for diphacinone and involved both primary and secondary

toxicity . Eleven incidents were deemed “highl y 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. L isted below are descriptions of “highl y probable” incidents as well as those that resulted in measurable residues. In cidents 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) b y 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 analy sis of the liver detected diphacinone residues at 3.5 ppm ( R000 - 02 - 001)  A mal e 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 (I 004768 - 001 and I004769
- 001).  An adult male gray

squ irrel 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 likel y 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 respectivel y (I007488
- 004).  Although listed as a “probable” in cident, a red
- tailed hawk was found moribund (dying the next day) in Rosly

n 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 squirre l were found dead in Ventura CA and contained 4.12 and 1.99 ppm diphacinone in liver samples respectively (I 0I4886 - 001).  Two deer found dead in Suffolk NY contained 0.2 and 0.93 ppm diphacinone in liver samples respectively

(I 013810 - 007 and I013810
- 008, both incidents deemed as “probable”).  A radio
- collared ye arling female co yot e 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, I 007165 - 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 i s 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 sy nthesizes 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 anal ys es 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 anal yz e potential risk to non
- target organisms and the need to consider regulatory

action. These criteria are used to indicate wh en 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 low er than concern levels . EECs in previ ous 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 unlikel y. 5.1.2 **Bird and Mammal Risk Estimation**

**Risks from Direct Bai t Consumption**  In the case of primary

exposure, it is assumed the bait containing diphacinone is ingested b y non target animals and evokes a toxic response. For toxic response elicited from gavage exposure route, exposure is measured as mg a.i./kg
- bw. Toxi city is measured by

the LD 50 obtained from the single
- gavage studies for birds and mammals. The LD 50 ’s are adjusted for the weight of the assessed animals (birds: 20, 100, 1000 g; mammals: 15, 35, 1 0 00 g). Avian and mammalian RQs based on acute single
- dos e gavage studies are provided in table 5.2 . For birds, the LOCs were not exceeded. For mammals, the L isted Species, Restricted Use, and Acute LOCs were exceede d for all three weight classes.39

**Table 5. 1**  **Formulas for Calculation of Weight -**  **adjusted Avian and Mammalian**  **Di phacinone LD**  **50 s.**  Adjusted avian LD 50 : 

### Adj LD LD AW TW x



### . ( ) 50 50 1 

###         whe re: Adj. LD 50 = adjusted LD 50 (mg/kg
- bw) LD 50 = endpoint reported from bird study (mg/kg
- bw) TW = body weight of tested animal ( 178g bobwhite quail ) AW = body

w eight of assessed animal (20g, 100g, and 1000g) x = Mineau scaling factor for birds; EFED default 1.15 Adjusted mammalian LD 50 :   25 . 0 50 50 . 

      AW TW LD LD Adj where: Adj. LD 50 = adjusted LD 50 (mg/kg
- bw) LD 50 = endpoint reported from mammal study (mg/kg
- bw) TW = body weight of tested animal ( 350 g) AW = body

w eight 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) Di**  **phacinone intake (m**  **g a.i./kg**  **- bwt/day)**  **1 Adjusted LD**  **50 (m**  **g a.i./kg -**  **bw) 2**  **RQ 3**  Birds* 20 13 1163 0.0 1 100 10 1480 0.0 1 1000 7. 1 2091 < 0.0 1 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 day s, exposure is measured as mg a.i./kg
- bait. Toxicity is measured by

the LC 50 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 L isted 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)**  **Taxonom i**  **c Group Di**  **phacinone concentration in bait**  **(m g a.i./kg**  **- bait)**  **LC 50**  **(m g a.i./k** **g -**  **diet) RQ**  **1**
Birds * 50 906 0.0 6 Mammals 50 2.08 **2 4**  **.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**  **Di p**  **hacinone -**  **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 LD 50 ’s are adjusted for the weight of the assessed animals (birds: 50, 1000, 2000 g; mammals: 50, 1000, 3 000 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 ri sk to a 400g snake eating a prey

animal that has ingested diphacinone. Carnivore/scavenger bird L OCs were not exceeded; however, RQs for carnivore and scavenger mammals exceeded Listed Species and Restricted Use LOCs while the snake RQ exceeded onl y the L isted 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) Di**  **phacinone intake (m**  **g a.i./kg -**  **bwt/day) 1**  **Adjusted LD 50**  **(m g**  **a.i./kg -**  **bwt) 2**  **RQ 3**  Birds + 50 1.75 87.2 0.0 2

1000 0.62 136.7 0.0 04 2000 0.48 151.7 0.00 3 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 am phibians ++ 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 day s, exposure is measured as the concentration of contaminant in the carcass, 3.4 mga.i./kg
- carcass. Toxicity is measured b y the LC 50 obtained from the dietary studies (5 days on tr eated diet) for birds and mammals. Avian and mammalian RQs based on dietary studies are provided in **Table 5. 5**  . Carnivore/scavenger bird L OCs were not exceeded; however, RQs for carnivore and scavenger mammals exceeded Listed Species, Restricted Use, and A cute 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 (m g a.i./kg**  **** **carcass) LC**  **50**

**(m g a.i./kg**  **- diet)**  **RQ 1** 

Birds* 3.4 906 0.004 Mammals 3.4 2. 0 8 **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 dip hacinone 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 low er than concern levels
. Therefore, effects to aquatic organisms from the proposed diphacinone use are unlikel y. 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 b irds (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 0.0 1 , which do not exceed an y LOCs .

**Risks to Carnivore and Scavenger Birds** Carnivorous and scavenger birds may be exposed to diphacinone through consumpt ion of carcasses that were contaminated with diphacinone . Based on available ground squir r el carcass data, carcass diphacinone concentration was estimated at 3.4 mga.i./kg
- carcass. On an acute basis (single gavage or 5
- day dietary exposure), the calculate d 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 (9 6 . 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 LD 50 and L C 50 used to calculate the RQs were based on granivorous species. These species prefer to eat a relativel y 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 currentl y estimated in the risk quotient methodology
. T he k estrel data (Rattner et. al., 2011) indicate that diphacinone is 21x more toxic to a carnivorous bird compared to the standard test species (quail)
. In cidents 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. The43

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 secon dary 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. D iphacinone present s 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)
. In cidents were reported in the EPA database of non - carnivorous mammals that died of diphacinone poisoning. The known species include squirrels and deer
. O mnivore s such as the raccoon and San Joaquin kit fox were also cited i n 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 c oncentration was estimated at 3.4 mga.i./kg
- carcass. This value represents the highest concentration of diphacinone found in an y individual in all of the carcass residue studies. On an acute basis for the single gavage exposure , the calculated RQs ( < 0. 0 1 ) 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 L isted Species, Restricted Use, and Acute Risk LOCs. Therefore, mammals consuming contaminated carcasses over a several day period may

expect mortality
. The LD 50 and L C 50 used to calculate the RQs were based on granivorous species. These species prefer to eat a relativel y consistent quantity of food on a daily

basis. Carnivores and scavengers may consume a large meal (an entire c arcass) once every several day s. 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
. In cidents 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 co yote. The presence of confirmed incidents of diphacinone poisoning under the currentl y 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 currently44

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 fee ding 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 p rey animal that has ingested diphacinone. T he snake RQ (0.16) exceeded only the Listed Species LOC . Diphacinone seems to have a different mode of action in reptiles. M ortality 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 necrops y, there was no evidence of hemorrhaging. D iphacinone 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 directl y onto plants, and because so little is expected to leach from the bait and then be available for plant uptake, exposure is exp ected 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 indirectl y b y the Federal action and not merel y the immediate area involved in the action. At the initial screening
- level, the risk assessment consid ers broadl y described taxonomic groups and so conservatively assumes that listed species within those broad groups are co
located 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 relativel y 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 a rea 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 ext end 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 effe cts 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 f or each taxonomic group are used to make inferences concerning the potential for indirect effects upon listed species that rel y upon non - endangered organisms in these taxonomic groups as resources critical to their life cycle. Terrestrial plants that rel y 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

– mo nocots 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 plants a No No Freshwater fish and a quatic
- 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 anal ys is, it was presumed that all federally
- listed endangered or threatened terrestrial animals in the proposed S tates (i.e., CO, KS, NE, NM, ND, SD, WY, TX, MT and OK ) may be directl y or indirectly affected. With additional refinement b y 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 determi ned 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 federall y listed species may

be affected , a number of conservation measures were developed with discu ssions 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=docI d;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/dyinglisted 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 L ive sy stem 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 ade quately 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 t o 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 ( s ee t able below ). In tests conducted in the same species, diphacinone is consistently less toxic to birds relative to chlorophacinone, and it is of similar toxic ity 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 anywh ere within the range of black tailed prairie dogs, the47

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 appl yi n g either of these products. For this reason, the Biological O pinion 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 l isted species for use of both rodenticides. Table 5.7. Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters **Comparative Param eter**  **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
- mo use 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: 9 6.8 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: 2 0.75 -- Toxicity Mammals Lab Rat

– Gavage (mg/kg
- bw) 6 1.9 --

Pine Vole

– Gavage (mg/kg
- bw) 14 58 -- Other Species

– Gavage (mg/kg
bw) 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 -**  **F ooted 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 p rairie d ogs 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
. Historicall y, where Prairie Dog s were found, so were Black
Footed Ferrets. A major cause for the decline in Black
- Footed Ferret s is the reduction of the rang e of Prairie Dog s (USFWS 1988). T his 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 clas s, states , “Do not48

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 population s of Black
- footed Ferret s are known to or are believed to occur include Colorado , Kansas , Montana , Nebraska , New Mexico, North Dakota , South Dakota , and W yo ming
. 5.3.3 **Probit Slope Response Analysis**  The Agency

uses the probit dose
- response relationship as a tool for providing additional i nformation 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 inter pretation, 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 probabili ty

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 probabili ty 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 anal ys is is provided below . If no dose response information is available to estimate a s lope for this anal ys is, 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 o n an Excel spreadsheet tool IECv 1.1 (Individual Effect Chance Model Version 1.1) developed by the U.S. EPA ( OPP, Environme ntal Fate and Effects Division, June 22, 2004). The spreadsheet performs these calculations b y entering the mean slope estimate (and the 95% confidence bounds of that estimate) as the slope parameter for the sprea dsheet. 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 c alculated 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 *T aken 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 b y, 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 rel y 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 carn ivores 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 utili ze abandoned burrows. If the populations of these burrowing animals are significantly affected, a shift in the entire ecos ys tem structure in the targeted region may occur. Terrestrial plants that rely on animals for pollination and seed dispersal may hav e 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 phy sical 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 consid erations 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
- level50

taxonomic analy sis (RQs) and listed sp ecies’ 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 i ndirect 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. Analy tically , the identification of such species and critical habitat can occur in either of two way s. First, the Services could determine whether the action area overlaps critical habitat or the occupied range of an y listed species. If so, EPA would examine whether the pesticide’s potential impacts on non
- endangered species would affect the listed species indirectly or directl y affect a primary constituent element of the critical habitat. Alternativel y, 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 indirectl y impacted by a pesticide. Then EPA would determine whether or not use of the pesticide overlaps the critical habitat or the occupied range of tho se listed species. At present, the information reviewed by EPA is not sufficient to permit use of either anal yt ical approach to make a definitive identification of species that are potentiall y impacted indirectl y or critical habitats that are potentially impacted directl y by the use of pesticides. EPA and the Service(s) are working together to conduct the necessary analy sis. 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 Propert ies**  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 ty

pes of animals. Animals were assumed to consum e 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 ingestion51

rate, either because they

are also feeding on other food sources, or because they exhibit bait shy ness. Alternativel y, if other food is scarce and they find the bait to be very attractive, then they could exhibit gorging behav ior, consuming bait in excess of their daily average dail y 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 con sume 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 equa tions 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 insectivore s, 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 pre y 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 considerabl y less than the as sumed 100%. Also, snakes and other reptiles ty picall y digest large prey slowly over numerous day s. Thus, the toxic residues in the ingested prey may be released and assimilated in some reptiles slowly over numerous day s. In addition, it would seem that the mode of action that produces mortality in mammals is different th an that in reptiles . Since diphacinone is an anticoagulant, mammals die through hemor rh ag ing
. However, Brown T ree snakes dosed with diphacinone did not die of hemo r r h age but b y an unkno wn 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. T hese equations may overestimate the maximum size prey which it may

consume. Lastl y, 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, non
lethal effects to reproduction in these species are unknown. For example, f oxes whelping pups do not hunt. Instead, the male fox will bring food to the female. In th is 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 pathway s 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% SL N No. CA 890022 and 0.005% SLN No. CA 890020) using spot
- baiting applications to control the California ground squirrel (Spermophilus beecheyi). Unpubl ished 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 14 C - 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, Wellingto n. 19p. LiphaTech, Inc. 1997. The veterinarian’s guide to managing poisoning b y 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. Jo hnston. 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 coagulopath y in American kestrels ( Falco sparverius ) following administration of the rodenticide diphacinone. Environ Toxicol Chem
. 30:1213 - 1222.53

Savarie, P.J. and R.K. L aVoie. 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 L aboratory , unpublished report). U.S. Environmental Protection Agency . 1986. Hazard Evaluation Division Standard Evaluation Procedure: Ecological Risk Assessment. Office of Pesticide Progr ams. 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 Assessme nt. 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 .19 98b. Reregistration Eligibility Decision (RED): Rodenticide Cluster. July 1998. EPA 738 - R - 98 - 007. U.S. Environmental Protection Agency . 2004 a . 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 Ef fects 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 an d 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 b y 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 currentl y 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 speci es may be adversel y 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 laborat ory

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, f

ish and aquatic invertebrates) Other toxicity

values may

be used when justified.55

Risk presumptions and the corresponding RQs and L OCs, are tabulated below. **Risk presu mptions for terrestrial animals**  **based on risk quotients (RQ) and levels of concern (LOC)**  **Risk Presumption RQ**  **LOC Birds:**  Acute Risk EEC 1 /L C 50 or LD 50 /ft2 or LD 50 /day3 0.5 Acute Restricted Use EEC/L C 50 or LD 50 /ft2 or LD 50 /day (or LD 50 < 50 mg/kg) 0.2 Acute Endangered Species EEC/L C 50 or LD 50 /ft2 or LD 50 /day 0.1 Chronic Risk EEC/NOAEC 1 **Wild Mammals:** Acute Risk EEC/L C 50 or LD 50 /ft2 or LD 50 /day 0.5 Acute Restricted Use EEC/L C 50 or LD 50 /ft2 or LD 50 /day (or LD 50 < 50 mg/kg) 0.2 Acute Endangered Species EEC/L C 50 or LD 50 /ft2 or LD 50 /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 LD 50 * wt. of bird LD 50 * wt. of bird **Risk presumptions for aquatic animals based on risk quotients (RQ) and levels of concern (LOC)**  **Risk Presumption RQ**  **LOC** Acute Risk EEC 1 /L C 50 or EC 50 0.5 Acute Restricted Use EEC/L C 50 or EC 50 0.1 Acute Endangered Species EEC/L C 50 or EC 50 0.05 Chronic Risk EEC/NOAEC 1 1 EEC = (ppm or ppb) in water56

**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 EEC 1 /EC 25 1 Acute Endangered Species EEC/EC 05 or NOAEC 1 **Aquatic Plants:** Acute Risk EEC 2 /EC 50 1 Acute Endangered Species EEC/EC 05 or NOAEC 1 1 EEC = lbs ai/A 2 EEC = (ppb/ppm) in water57

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 T yp es Reported Mammal, Bird, Amphibian, Reptile CO, KS, MT, N E, NM, ND, OK, SD, TX, WY **Colorado** CH Bird Crane, W hooping 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 Wo lf, Gray Canis lupus Endangered Terrestrial Yes **Kansas** CH Bird Crane, W hooping Grus americana Endangered Terrestrial, Freshwater Yes Mammal Ferret, Black - Footed Mustela nigripes Endangered Terrestrial No **Montana** CH Bird Crane, W hooping Grus americana Endangered Terrestrial, Freshwater Yes Mammal Ferret, Black - Footed Mustela nigripes Endangered Terrestrial No **Nebraska** CH Bird Crane, W hooping Grus americana Endangered Terrestrial, Freshwater Yes Mammal Ferret, Black - Footed Mustela nigripes Endangered Terrestrial No58

**New Mexico** CH Amphibian Frog, Chiricahua Leopard Rana chiricahuensis Threatened Freshwater, Terrestrial No Bird Crane, W hooping 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, W hooping Grus americana Endangered Terrestrial, Freshwater Yes Mammal Wo lf, Gray Canis lupus Endangered Terrestrial Yes **Oklahoma** CH Bird Crane, W hooping Grus americana Endangered Terrestrial, Freshwater Yes **South Dakota** CH Bird Crane, W hooping Grus americana Endangered Terrestrial, Freshwater Yes Mammal Ferret, Black - Footed Mustela nigripes Endangered Terrestrial No Wo lf, Gray Canis lupus Endangered Terrestrial Yes **Texas** CH Amphibian Toad, Houston Bufo houstonensis Endangered Terrestrial, Freshwater Yes Bird Crane, W hooping 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 Wa rbler (=W ood), Golden - cheeked Dendroica chrysoparia Endangered Terrestrial No59

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 W ater Nerodia paucimaculata Threatened Freshwater, Terrestrial Yes **Wyoming** CH Amphibian Toad, W yom ing 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