Document ID: EPA-HQ-OPP-2012-0739-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-09-10T04:00Z

Ecological Risk Assessment 
                  for the Section 3 New Use Registration of 
                            Diphacinone (PC 067701)
                     For Use on Black-tailed Prairie Dogs
                            (Cynomys ludovicianus)
                                       
                                       
                                       
	
                                       
                                       
                                       
                                       
Prepared by:
N.E.Federoff, Wildlife Biologist
J. Lin, Environmental Engineer
U. S. Environmental Protection Agency
Office of Pesticide Programs
Environmental Fate and Effects Division
Environmental Risk Branch II
1200 Pennsylvania Ave., NW
Mail Code 7507P
Washington, DC 20460
Reviewed by:
Kristina Garber, Senior Biologist
R. David Jones, Senior Fate Scientist

Branch Chief:
Brian Anderson

                                       
                                       
                                June 28, 2012 

                               Table of Contents

1.0	Executive Summary	6
1.1	Nature of Chemical Stressor	6
1.2	Exposure Characterization	6
1.3	Effects Characterization	7
1.4	Potential Risks to Non-target Organisms	7
1.5	Related Biological Opinions	9
1.6	Data Gaps	10
2.0	Problem Formulation	11
2.1	Nature of Regulatory Action	11
2.2	Previous Risk Assessments	11
2.3	Stressor Source and Distribution	12
2.3.1	Mechanism of Action	12
2.3.2	Nature of the Chemical Stressor	12
2.3.3	Environmental Transport Mechanisms	16
2.4	Overview of Pesticide Usage	16
2.4.1	Label for Proposed Registered Use	16
Figure 1.  Historic Range of Black Tailed Prairie Dogs	17
2.5	Receptors	18
2.6	Ecosystems Potentially at Risk	19
2.7	Assessment Endpoints	19
2.8	Conceptual Model	19
2.8.1	Risk Hypotheses	19
2.8.2	Conceptual Diagram	20
2.9	Analysis Plan	22
2.9.1	Measures of Exposure	22
2.9.2	Measures of Effect	23
2.9.3	Use of Probit Slope Response Relationship to Provide Information on the Endangered Species Levels of Concern	23
2.9.4	Data Gaps	24
3.0	Exposure Assessment	25
3.1	Label Application Rates and Intervals	25
3.2	Aquatic Exposure Assessment	25
3.2.1	Aquatic EECs	25
3.3	Terrestrial Exposure Assessment	25
3.3.1	Expected Diphacinone Ingestion through Bait Consumption	25
3.3.2	Expected Diphacinone Ingestion through Consumption of Contaminated Carcasses	27
4.0	Ecological Effects Characterization	28
4.1	Toxicity of Diphacinone to Aquatic Organisms	29
4.2	Toxicity of Diphacinone to Terrestrial Organisms	29
4.3	Birds and Mammals, Secondary Toxicity Tests	33
4.4	Incident Database Review	35
5.0	Risk Characterization	36
5.1	Risk Estimation: Integration of Exposure and Effects Data	37
5.1.1	Aquatic Risk Estimation	37
5.1.2	Bird and Mammal Risk Estimation	37
Risks from Direct Bait Consumption	37
5.1.3	Secondary Risks from Consumption of Diphacinone-contaminated Carcasses	39
5.1.4	Risk to Terrestrial Invertebrates	40
5.2	Risk Description	41
5.2.1	Risks to Aquatic Organisms	41
5.2.2	Risks to Terrestrial Organisms	41
5.2.3	Risks to Mammals directly consuming bait	42
5.2.4	Risks to carnivore and scavenger Mammals	42
5.2.5	Risks to Reptiles	43
5.2.6	Risks to Terrestrial Invertebrates	43
5.3	Threatened and Endangered Species Concern	43
5.3.1	Action Area	43
5.3.2	Taxonomic Groups Potentially at Risk	44
5.3.3	Probit Slope Response Analysis	47
5.3.4	Indirect Effects Analysis	48
5.3.5	Critical Habitat for Listed Species	48
6.0	Uncertainties	49
6.1	Fate and Transport Properties	49
6.2	Effects	49
7.0	Bibliography	51

                              List of Appendices

Appendix A		RQ Methods and LOC Definitions
Appendix B		LOCATES Listing of Endangered Species
Executive Summary
Nature of Chemical Stressor
The proposed Section 3 registration is for the use of Kaput-D[(R)] Prairie Dog Bait on Black-tailed Prairie Dogs (BPD; Cynomus ludivicianus). This restricted use product is a bait that contains 0.005% diphacinone (50 mg a.i./kg-bait) and must be hand applied on rangeland and adjacent non-crop areas at least 6" into active burrows.  No bait is to be placed on or above ground level. The product label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, TX, MT and OK.  

Diphacinone is a first generation indanedione anticoagulant rodenticide and it is absorbed through the gut, inhibiting vitamin K.  Indanediones are not only anticoagulants; they also uncouple oxidative phosphorylation (energy generation) in target animals (i.e., mammals).  Diphacinone seems to have a different mode of action in reptiles.  Mortality occurs but does not result from anticoagulation, and its mode of action in snakes is unknown. 

Anticoagulants are vitamin-K antagonists that disrupt normal blood-clotting mechanisms and induce capillary damage.  Typically, death is delayed for four to ten or more days after a lethal dose is ingested, and animals may continue to feed and move about until shortly before death.  Death results from hemorrhage, and exposed animals may exhibit behavior that may make them more susceptible to predation (Cox and Smith, 1992).  This may result in secondary exposure to predatory animals. 
Exposure Characterization
The primary dissipation route of diphacinone is likely to be consumption of bait by target as well as non-target animals.  Diphacinone is not expected to be transported to surface or ground water. This is based on its use pattern and formulation, low solubility (0.3 mg/L), and its range of Kd values of 3 to 5 mL/g, which suggest moderate mobility in soil.  Because diphacinone is applied as a solid bait, spray drift is not expected. Consistent with a relatively low vapor pressure of approximately 1 x 10[-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 Kds of diphacinone are consistent with compounds, which leach to ground water. However, they are bounding estimates from column leaching studies, and it is very likely that the true values are much higher, closer to the regression estimates described in the fate assessment. The use pattern is such that the primary route of dissipation is likely consumption of treated bait. Because it is put into burrows, potential for movement by surface runoff is minimal.  There is some small chance for leaching based on moderate mobiltiy, but the use pattern and low use rates make contamination of groundwater unlikely.  

Available data suggest that diphacinone may degrade on soil via aerobic metabolism. The major degradate detected in the aerobic soil metabolism study, diphenylglycolic acid, comprised up to 25% of the applied material in an aerobic soil metabolism study.  This degradate appears to be persistent, but its mobility is unknown.  Because diphacinone is formulated in pelleted bait or  bait-block rodenticide products and primarily used in bait application or animal burrow treatment, it is probable that contact of this degradate with soil and water will be minimal.   Exposures and resulting risks of non-target organisms to the degradate diphenylglycolic acid are, therefore, not quantified for this assessment.

For this assessment, it was assumed that terrestrial animals could be exposed to diphacinone through two different pathways. Animals may directly consume bait (primary consumption), or animals may consume contaminated carcasses either killed or scavenged by the consumer (secondary consumption).  

Based on diphacinone's octanol to water partitioning ratio (Pow or Kow) of approximately 20,000 (log Pow = 4.3), there is potential for bioaccumulation.  However, considering the use pattern as a bait, there is a little chance that bioaccumulation will occur in aquatic animals.  
 
Because Kaput-D is a bait and is confined to below ground terrestrial applications, exposure to the aquatic environment is expected to be limited.  Screening level EECs confirmed that risk to aquatic organisms is below concern levels. 

Effects Characterization
Diphacinone exhibits high to very high acute and dietary toxicity to small mammals (LD50's = 0.2 to 57 mg ai/kg bw; LC50 = 2.08 ppm). It is moderately toxic to birds (LD50's >400 to 3158 mg ai/kg bw; LC50's = 906 to >5000 ppm) on an acute toxicity basis.  No chronic data were available for birds. Toxicity increases if diphacinone is ingested for several days rather than in a single dose.  Toxicity data are also available for snakes, where LD50 values were 20.75-31.21 mg/kg-bw.

Diphacinone is moderately toxic to freshwater fish (LC50 = 2.6 ppm) and invertebrates (LC50 = 1.8 ppm) on an acute exposure basis. No data are available to evaluate chronic effects to freshwater fish or invertebrates. No data are available to evaluate acute or chronic effects to estuarine/marine fish or invertebrates.  

No toxicity data are available for aquatic or terrestrial plants. Given the mode of action of diphacinone, toxicity to plants is expected to be low. 
Potential Risks to Non-target Organisms
Based on available screening-level information for the proposed use of diphacinone, there is a potential for mortality of non-listed and listed terrestrial mammals through consumption of bait.  In addition, there is potential for mortality of secondary consumers through consumption of prey that have eaten bait. Risks of mortality to secondary consumers were based on multiple lines of evidence including calculation of RQs (mammals and snakes) as well as incident reports of mortalities of mammals and birds exposed to diphacinone and secondary toxicity studies. When considering listed species, there is a potential concern for indirect effects upon the listed organisms due to decreased prey availability and habitat modification. Taxa-based risk conclusions for direct and indirect effects to listed species are listed in Table 1.1.

Table 1.1. Listed species risks associated with direct or indirect effects due to proposed diphacinone use on BPD
                                Listed Taxonomy
                                Direct Effects
                               Indirect Effects
Terrestrial and semi-aquatic plants  -  monocots
                                      No
                                      Yes
Terrestrial and semi-aquatic plants  -  dicots
                                      No
                                      Yes
Birds (surrogate for terrestrial-phase amphibians)
                                      Yes
                                      Yes
Mammals
                                      Yes
                                      Yes
Reptiles
                                      Yes
                                      Yes
Aquatic vascular plants
                                      No
                                      No
Aquatic non-vascular 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 analysis, it was presumed that all federally-listed endangered or threatened terrestrial animals in the states of CO, KS, NE, NM, ND, SD, MT, OK, TX and WY may be directly or indirectly affected.  With additional refinement by exploring more detailed use patterns and species biology (e.g., geographic location, specific feeding habits, time of year likely to utilize crop fields), some species listed may be determined to be not likely to be affected.

The lines of evidence supporting the conclusion of risk include:
   * Incidents in the US reported in EIIS that were considered probable or highly probable for death due to diphacinone. Mortalities included species that were likely primary consumers of the bait and carnivorous species likely to have preyed upon the granivorous individuals.  Reported incidents with known or potential impact on endangered species are:
         o Take of an endangered species was reported. An adult kit fox was found dead in Bakersfield CA (weighing 5.75 lbs). Concentrations of diphacinone by immunoassay were found in liver and blood at a concentration of 0.18 ppm (Incident #B000640-001).   
   * Avian, mammalian and reptilian (snake) mortality through exposure to diphacinone was documented through low LD50 and LC50 values obtained in toxicity studies and/or the reported incident data. 

Related Biological Opinions
The Fish and Wildlife Service (FWS) recently completed a Biological Opinion on a registration action that allows use of another anticoagulant rodenticide (chlorophacinone; trade name of Rozol black-tailed prairie dog bait -  hereafter referred to as Rozol) to control black tailed prairie dogs.  Although the Agency's endangered species risk assessment determined that 21 federally listed species may be affected, a number of conservation measures were developed with discussions with and agreement between the Fish and Wildlife Service, EPA, and the registrant to either reduce or preclude exposures to many of these species.  The Agency's effects determination for Rozol and resulting Biological Opinions are available on-line in the docket at regulations.gov and may be accessed using the following link:  

http://www.regulations.gov/#!docketDetail;dct=FR%252BPR%252BN%252BO%252BSR;rpp=25;so=ASC;sb=docId;po=0;D=EPA-HQ-OPP-2011-0909 

These conservation measures were part of the federal action and included label amendments to add specifications for a line-transect carcass search and reporting of dead/dying listed species and non-target animals in addition to prohibitions of Rozol use and timing restrictions via the Agency's Endangered Species Protection Program (ESPP) Bulletins Live system available at:  http://www.epa.gov/oppfead1/endanger/bulletins.htm.  The Biological Opinion on Rozol (http://epa.gov/espp/2012/borozol-final.pdf) found that the conservation measures adequately protected most of the endangered species, although FWS concluded that incidental take may still occur for the following three species:  (1) black-footed ferret; (2) gray wolf; and (3) northern aplomado falcon.  In order to minimize incidental take for these three species, FWS identified a number of additional reasonable and prudent measures (RPMs) including certified applicator training, development of website content to reduce the potential for secondary poisoning, requirements for the registrant to develop a stewardship training program and submit of Rozol distribution and production data, and additional bulletins for the northern aplomado falcon. Additional details are included in the Biological Opinion available on the docket at the aformentioned link.  

The exposure and toxicity profiles are similar for chlorophacinone and diphacinone (see table below).  In tests conducted in the same species, diphacinone is consistently less toxic to birds relative to chlorophacinone, and it is of similar toxicity to mammals (slight increases and decreases in median lethal doses/concentrations have been observed).  Therefore, because the area of potential use is identical for Rozol and Kaput black-tailed prairie dog baits given that they are both assumed to be applied anywhere within the range of black tailed prairie dogs, the listed species that may be exposed are also identical.  Also, given that the exposure and toxicity profiles are similar, the conservation measures and RPMs identified for Rozol should also be protective of endangered species if implemented on Kaput black tailed prairie dog bait.  In addition, labels for both Rozol and Kaput will stipulate that other anticoagulant rodenticides cannot be used if applying either of these products.  For this reason, the Biological Opinion issued on Rozol is expected to be applicable to both Rozol and Kaput black tailed prairie dog bait, and is, therefore, expected to provide adequate protections for listed species for use of both rodenticides.  

Table 5.7.  Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters
Comparative Parameter
Chlorophacinone
Diphacinone
Comments
Exposure
Concentration in  Bait
0.005%
0.005%
                                      --

Food Consumption
Not chemical dependent
                                      --

Time to mortality
4-13 days
3-9 days
Time to mortality affects the amount of chemical that can be accumulated over time for target pests.

Liver Retention
35 days - mouse
3 days  -  Rat
5 days  -  Pig
>90 days - Cow
Lack of data in same species precludes definitive comparisons
Toxicity, Birds
BWQ LD50s (mg/kg-bw)
260
1630
                                      --

Other Avian LD50s (mg/kg-bw)
None available
Kestrel: 96.8
Mallard: 3200
                                      --

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

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

Other Species (mg/kg-diet)
60 (Japanese Quail)
Brown Tree Snake: 20.75 
                                      --
Toxicity Mammals
Lab Rat  -  Gavage (mg/kg-bw)
6
1.9
                                      --

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

Other Species  -  Gavage (mg/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
      
Data Gaps
As cited in the RED (USEPA, 1998b), the only available environmental fate data from studies for diphacinone are 1) physicochemical properties, 2) hydrolysis, 3) aerobic soil metabolism, and 4) aged column leaching.  However, because of the specific results of studies and because the specific use and application rates for diphacinone, relative to established end-point toxicities, is low, EFED can satisfactorily evaluate/screen limits of potential environmental concentrations and ecological effects without additional environmental fate data.

Studies with a potential to add value to the risk assessment are:
   * Field Testing for Terrestrial Wildlife (850.2500)
   * Acute Passerine Study (850.2100) 
   * Avian Reproduction Study (850.2300), two species
      
      
Problem Formulation
The purpose of problem formulation is to provide the foundation for the environmental fate and ecological risk assessment being conducted for the proposed new use of diphacinone.  As such, it articulates the purpose and objectives of the risk assessment, evaluates the nature of the problem, and provides a plan for analyzing the data and characterizing the risk (USEPA, 1998a).  
Nature of Regulatory Action
The proposed Section 3 registration is for the use of Kaput-D[(R)] Prairie Dog Bait on Black-tailed Prairie Dogs (BPD; Cynomus ludivicianus). This restricted use product is bait that contains 0.005% diphacinone (50 mg a.i./kg-bait) and must be hand applied on rangeland and adjacent non-crop areas at least 6" into active burrows.  No bait is to be placed on or above ground level. The product label restricts use to the 10 States of CO, KS, NE, NM, ND, SD, WY, TX, MT and OK.  Historic range of the BPD is depicted in Figure 1 below.  
Previous Risk Assessments
Since 1998, EPA has conducted a number of ecological risk assessments on diphacinone. These assessments have consistently identified potential risks to birds and mammals from primary and secondary exposure as the predominant risk concern for diphacinone. These assessments include the following:
   * An assessment of the risks of rodenticide uses of diphacinone, along with seven other rodenticides, in the Registration Eligibility Decision (RED): Rodenticide Cluster that was published in 1998.  This document included an ecological effects risk assessment that was based on environmental fate and ecotoxicological studies that had been submitted by the registrants of diphacinone at that time.  
   *  An assessment of potential risks of nine rodenticides, including diphacinone, to birds and mammals was completed in 2004 (Erickson and Urban 2004).  
   * Several Section 18 (emergency exemption) assessments involving diphacinone were completed in 2009. 
   * An endangered species assessment was completed in 2011.  It assessed risk from the use of diphacinone to the Federally Threatened Alameda Whipsnake (Masticophis lateralis euryxanthus) and the California Tiger Salamander (Ambystoma californiense), as well as risks to the Federally Endangered Salt Marsh Harvest Mouse (Reithrodontomys ravivertis), California Tiger Salamander (Ambystoma californiense) Sonoma County Distinct Population Segment and Santa Barbara County Distinct Population Segment, and San Joaquin Kit Fox (Vulpes macrotis mutica).  
           
In addition, in March of 1993, a Biological Opinion (Effects of 16 Vertebrate Control Agents on Threatened and Endangered Species) was issued by the US Fish & Wildlife Service. The Service determined that risk was minimal to avian species from direct consumption of treated bait.  However, secondary toxicity to raptors was possible from consumption of treated rodents.  Risk to mammals was also indicated.  Specific jeopardy was indicated for the Salt Marsh Harvest Mouse (SMHM) and the San Joaquin Kit Fox (SJKF).
Stressor Source and Distribution
Mechanism of Action 
Diphacinone is a first generation indanedione anticoagulant rodenticide and is absorbed through the gut.    Anticoagulants are vitamin-K antagonists that disrupt normal blood-clotting mechanisms and induce capillary damage.  Typically, death is delayed for four to ten or more days after a lethal dose is ingested, and animals may continue to feed and move about until shortly before death.  Death results from hemorrhage, and exposed animals may exhibit behavior that may make them more susceptible to predation (Cox and Smith, 1992).  In addition, indandiones also uncouple oxidative phosphorylation (energy generation) in mammals.

Diphacinone seems to have a different mode of action in reptiles.  Mortality occurs but does not result from anticoagulation and its mode of action in snakes is unknown.
Nature of the Chemical Stressor
Diphacinone has moderate leaching potential.  The range of possible Kd values is 3 to 5 mL/g combined with a low solubility (0.3 mg/L) indicates that it is unlikely to contaminate ground water.  Consistent with a relatively 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.  Diphacinone is expected to be moderately mobile in soil; however, its chemical mobility is expected to be limited by the application method and formulation as a bait.  Diphacinone is metabolized via aerobic soil metabolism with a half-life value of approximately 30 days for one soil tested in a single study.  Diphacinone has a Kow of 20,000 indicating the potential for bioaccumulation in animal tissue. However, direct measures of bioaccumulation are not available.  One primary dissipation pathway of diphacinone is expected to be consumption of bait by target and non-target animals.

Table 2.1 lists the physical-chemical properties of diphacinone.  Table 2.2 lists the other environmental fate properties of diphacinone, along with the major and minor degradates detected in the submitted environmental fate and transport studies.  
Table 2.1.  Physical-Chemical Properties of Diphacinone
                                   Property
                                     Value
                                    Source
                                    Comment
Structure
                                       
                                      --
Chemical Name: 
2-(diphenylacetyl)indan-1,3-dione
Molecular Weight
340.48 g/mole
                                      --
                                      --
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 Diphacinone Environmental Fate Properties

Study

Value and unit

                                Major Degradate
                               Minor Degradates
                                       
                              MRID # or Citation
                                       
                         Study Classification, Comment
Hydrolysis
Stable at 7 and 9.  44 day half life at pH 5
                                      --
DP268619, 2000
                                      --
Photolysis in Water
Stable
                                      --
DP268619, 2000
                                      --
Photodegradation on Soil
Stable
                                      --

Assumed to be stable
Aerobic Soil Degradation
30 days (half-life)

diphenylglycolic acid (25%)
DP268619, 2000
30 days in single soil study 
Aerobic Aquatic Degradation
180 days (half-life)
                                      --
2X aerobic soil value
No study submitted, assumed value
Kd
3-5
                                      --
DP268619, 2000
4 soils
Koc
300
                                      --
DP268619, 2000
Supplemental column leaching study

Degradation/Metabolism

Laboratory aerobic soil metabolism data are available from a single sandy loam soil from North Dakota.   The mean half-life of diphacinone in this soil was 30 days (average of 28 and 32 days) from studies done at two different radiolabeled positions.  A major soil metabolite (> 10% of dose) was diphenylglycolic acid which peaked at a maximum of approximately 20-25% of the initial dose around one month after treatment.  This metabolite then remained at a relatively constant concentration until the end of the 3.5 month study.  Up to eight minor organic metabolites were separated at various times, but not identified; these generally reached relatively constant concentrations ranging from a fraction of 1% to around 6% of the initial dose.  (It is possible that some of these metabolites from separate labels may not be distinct compounds.)  Volatile organics were negligible.  Carbon dioxide was regularly and steadily produced from both radiolabels, and reached approximately 40% of the dose from both labels by the end of the study period.  Recalcitrant soil residues also accounted for a large fraction of the dose from both labels, amounting to approximately 20-30% of the initial dose by study end.  The evolved carbon dioxide and soil bound residuals are clearly indicative of ultimate biological deactivation of a large fraction of diphacinone and its transformation products.  This study also indicated, however, that the major metabolite, diphenylglycolic acid, was somewhat resistant to further metabolism.
					
Diphacinone was stable against simple hydrolysis at pH 7 and 9.  At pH 5, hydrolysis proceeded with an extrapolated half-life of approximately 44 days.  Products of hydrolysis were not satisfactorily identified or quantified.  However, since the apparent hydrolysis was appreciable only at low pH and aerobic metabolism provides a viable alternative transformation process for diphacinone, there is marginal value in additional information on hydrolytic degradates at low pH.

Mobility

	In Air.  Volatilization of diphacinone or degradates was not detected in the aerobic soil metabolism study.  A relatively low vapor pressure of approximately 1 x 10[-8] torr and a Henry's law constant for diphacinone of approximately 2 x 10[-10] atm-m[3]/mole also indicates that volatilization should not be a significant route of dissipation.

	In Soil.  Because of numerous deficiencies in the aged column leaching study (including not using radiolabeling, irregular soil columns, no reporting of elution rate, inability to detect less than 2% of applied diphacinone in the eluate or soil columns, no estimation of sorption coefficients, no attempt to identify and quantify degradates), the RED and previous reviews simply gave qualitative statements to the effect that diphacinone was "relatively immobile" because it was not detected in the leachates of the four soils tested and moved significantly only in a loamy sand soil.  However, if we were to take the column results at face value and accept the limit of six centimeter soil increment study resolution, we can approximate sorption coefficients by estimating column retardation factors and using nominal assumptions about soil bulk density and porosity.  For simplicity and convenience, we have assumed a nominal and uniform soil bulk density of 1.5 g/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), diphacinone was essentially confined to the top 6 cm of 30 cm columns (in one of these soils there was small migration into the 6 to 12 cm interval).  There is the possibility that, within the stated experimental error of 2%, as much as 2% of the applied could be in each of four remaining increments.  However, for the purposes of approximation we non-conservatively assume no chemical in these increments.  We also assume the chemical was centered at the half-way point of each 6 cm increment - for example, at 3 cm for the top increment.  This may or may not be a conservative assumption because of the 6 cm limit of resolution.  For the loamy sand soil column in which leaching occurred down to the 18-24 cm increment, we calculated the center of mass of the incremental distribution of diphacinone and assumed that an undetected 2% reached the lowest 24-30 cm depth increment (which was essentially an inconsequential assumption).  The resulting calculated sorption coefficient (Kd) for the three soils is roughly 5 mL/g with uncertain but "wide" error bars, if for no other reason than depth increment resolution.  For the loamy sand soil, the corresponding calculated Kd is approximately 3 mL/g.  Compounds with Kd values in this range are often classified as mobile.  The loamy sand result should be a better approximation than that for the other soils because there were measurable concentrations at several depth increments.  As stated previously, the registrant did not address the mobility of degradates in soil.

There is also an alternative approximation which can be made to estimate crudely the soil to water partitioning coefficient with the assumption that soil organic matter determines sorption.  The estimation is based on an empirical correlation between the octanol to water partitioning ratio (Pow or Kow) and the soil organic matter to water sorption coefficient (Koc).  The relationships1, assuming soil organic matter is typically about 58% carbon [i.e., organic carbon (oc) = organic matter (om)/1.72)], are  

		log (Kom) = 0.904 log (Pow) - 0.779 (n = 12, r-squared = 0.989)
		log (Koc)  = 0.904 log (Pow) - 0.779 + log 1.72

Another empirical relationship given in a technical guidance document supported by the European Community (Directive 93/67/EEC) to calculate Koc from Pow is:

		log  Koc = 0.52 log (Pow) + 1.02

Log Pow for diphacinone is 4.27 (see comment on this value in Bioconcentration paragraph below).  Thus, from the first relationship, log Koc = 3.32 and Koc is roughly 2100; from the second, log Koc = 3.24 and Koc is roughly 1700.  These similar Koc values and the Kd values above place diphacinone in an intermediate range of mobility.  

Bioaccumulation

The registrant has not submitted a bioconcentration study at this time.  However, judging from the diphacinone octanol to water partitioning ratio (Pow or Kow) of approximately 20,000 (log Pow = 4.3), there is potential for bioaccumulation in aquatic systems; however, there is limited potential for water contamination given the the use pattern to control black tailed prairie dogs.  Also, diphacinone has an estimated octanol air partitioning coefficient of 12.5 (EPI Suite), which suggests that it may accumulate in terrestrial animals, which is also consistent with the secondary exposure risks identified for diphacinone in the current and past assessments.  

Environmental Transport Mechanisms
Potential transport mechanisms typically include pesticide surface water runoff, spray drift, and secondary drift of volatilized or soil-bound residues leading to deposition onto nearby or more distant ecosystems.  However, because the uses of diphacinone are in bait application or animal burrow treatment, spray drift is presumed to be negligible. Because diphacinone bait may be used outdoors, some potential exist for residues of diphacinone to leach from the bait that is exposed to rainwater or runoff.  Due to the low application rate, the low drift potential, and the formulation within a bait, the potential for contaminating surface water is believed to be insignificant. 

Another possible route of transport is within the bodies of animals which feed on the diphacinone bait.  Because poisoned animals would not be killed immediately, they would travel some distance before dying, thereby exposing animals some distance away from the use site. This transport within animals is an important route of exposure for predatory birds and mammals since their diet includes small mammals, and thus vulnerable to secondary exposure from consuming poisoned rodents.
Overview of Pesticide Usage
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).  

            Figure 1.  Historic Range of Black Tailed Prairie Dogs

Receptors
In order for a chemical to pose an ecological risk, it must reach ecological receptors in biologically significant concentrations.  An exposure pathway is the means by which a contaminant moves in the environment from a source to an ecological receptor.  For an ecological exposure pathway to be complete, it must have a source, a release mechanism, an environmental transport medium, a point of exposure for ecological receptors, and a feasible route of exposure. In addition, the potential mechanisms of transformation (i.e., which degradates may form in the environment, in which media, and how much) must be known, especially for a chemical whose metabolites/degradates are of greater toxicological concern. The assessment of ecological exposure pathways, therefore, includes an examination of the source and potential migration pathways for constituents, and the determination of potential exposure routes (e.g., ingestion, inhalation, and dermal absorption).  
 
 Ecological receptors that may potentially be exposed to diphacinone include terrestrial wildlife (i.e., invertebrates, mammals, birds, and reptiles), terrestrial and semi-aquatic plants, aquatic plants, aquatic invertebrates and fish.  In addition to terrestrial ecological receptors, aquatic receptors (e.g., freshwater and estuarine/marine fish and invertebrates, amphibians) may also be exposed to potential migration of pesticides from the site of application to various watersheds and other aquatic environments via runoff and drift.
 
Consistent with the process described in the Overview Document (USEPA, 2004a), this risk assessment uses a surrogate species approach in its evaluation of the proposed new uses of diphacinone.  Data generated from surrogate test species, which are intended to be representative of broad taxonomic groups, are used to extrapolate to potential effects on a variety of species (receptors) included under these taxonomic groupings.  

Acute and chronic toxicity data from studies submitted by pesticide registrants are used to evaluate the potential direct effects of diphacinone to the aquatic and terrestrial receptors identified in this section, including toxicity data on the technical grade active ingredient, and when available, on formulated products.  The evaluation of these data can also provide insight into the direct and indirect effects of diphacinone on biotic communities from loss of species that are sensitive to the chemical and from changes in structure and functional characteristics of the affected communities.  

The toxicity data used in this assessment are obtained from registrant-submitted guideline toxicity studies required for pesticide registration.  These studies are typically performed on a number of organisms from several taxonomic groups, including birds, mammals, fish, and aquatic invertebrates (USEPA 2004a).  The surrogate test species, assessment endpoints, and measures of effect derived from these studies used for this risk assessment are provided. Additional information from other sources, such as public literature and incident reports, are used when relevant.
Ecosystems Potentially at Risk
The ecosystems at risk are often extensive in scope; therefore, it may not be possible to identify specific ecosystems during the development of a baseline risk assessment.  However, in general terms, terrestrial ecosystems potentially at risk could include the treated BTP habitat and areas immediately adjacent to the treated areas that may receive runoff.  Areas adjacent to the treated field could include cultivated fields, fencerows and hedgerows, meadows, fallow fields or grasslands, woodlands, riparian habitats and other uncultivated areas.  
   Assessment Endpoints
Assessment endpoints represent the actual environmental value that is to be protected, defined by an ecological entity (species, community, or other entity) and its attribute or characteristics (USEPA 2004a).  For diphacinone, the ecological entities may include the following:  birds, mammals, reptiles, amphibians, terrestrial plants and insects. The attributes for each of these entities may include growth, reproduction, and survival.  
 
Selection of the assessment endpoints is based on valued entities (i.e., ecological receptors), the ecosystems potentially at risk, the migration pathways of pesticides, and the routes by which ecological receptors are exposed to pesticide-related contamination. The selection of clearly defined assessment endpoints is important because they provide direction and boundaries in the risk assessment for addressing risk management issues of concern.
   Conceptual Model
A conceptual model provides a written description and visual representation of the predicted relationships between diphacinone, potential routes of exposure, and the predicted effects for the assessment endpoint. A conceptual model consists of two major components: risk hypotheses and a conceptual diagram (USEPA, 1998a).
            Risk Hypotheses
Based on the results of previous risk assessments for diphacinone, the following ecological risk hypotheses are being employed for this risk assessment:

* Terrestrial organisms are subject to adverse direct effects such as reduced survival, growth, and reproduction when exposed to diphacinone residues as a result of labeled use of the pesticide.
* Indirect effects, such as habitat, food web dynamics, perturbing forage or prey availability, and altering the extent and nature of nesting, will potentially occur if residue concentrations exceed levels of concern for acute or chronic exposure for terrestrial species. 
   
Based on previous assessments and the mode of action of diphacinone, the primary concern for this assessment is potential risks to non-target terrestrial animals either from direct consumption of bait (primary exposure) or from consumption of contaminated animals that have consumed treated bait (secondary exposure).  
            Conceptual Diagram
The conceptual model assumes that KAPUT-D bait will be available to non-target organisms and, as toxic food bait, will adversely affect terrestrial species.  Figure 2.3 illustrates the anticipated exposure pathways and transport routes to ecological receptors and identifies the predicted attribute changes from this exposure.  The major sources of exposure of non-target terrestrial animals are expected to be ingestion of the bait and consumption of vertebrate body tissues that have eaten the food bait (USEPA 2004b). Exposure via these routes is expected primarily for birds and mammals, though it is likely that other terrestrial animals such as reptiles and terrestrial amphibians are at risk if they consume invertebrates or tissues of vertebrates that have eaten bait. 

Diphacinone is applied as a bait and little is expected to partition into drinking water source (e.g. puddles) compared to that which is available for direct consumption on the bait itself; therefore, this route of exposure was not assessed. Since diphacinone is not sprayed directly onto plants, and because so little is expected to leach from the bait and then be available for plant uptake, consumption of diphacinone on plants is also not being considered as a route of exposure. Dermal and inhalation routes of exposure are not expected to be important routes of exposure for a rodenticide bait such as KAPUT-D given the application method and formulation.

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
                 Ingestion of Water by Terrestrial Vertebrates
Consumption and/or Uptake by Soil Macroinvertebrates
                    Exposed Intact Bait at Application Site
Secondary and Tertiary Exposure Routes and Receptors
            Consumption of Tissues and Eggs by Predators/Scavengers
Attribute Changes
                 Bioaccumulation and Food Chain Magnification
     Reduced Reproduction and Survival of Terrestrial Non-Target Organisms
                                  Diphacinone

Figure 2.3. Conceptual Model Diagram of Diphacinone Exposure and Effects in Nontarget Species. Dotted lines indicate exposure pathways that have a low likelihood of contributing to ecological risk.
Analysis Plan
In order to address the risk hypotheses, the potential for direct and indirect effects to the assessed species, prey items, and loss of burrows is estimated based on a taxon-level approach.  In the following sections, the use, environmental fate, and ecological effects of diphacinone are characterized and integrated to assess the risks.  This is accomplished using a RQ (ratio of exposure concentration to effects concentration) approach.  Although risk is often defined as the likelihood and magnitude of adverse ecological effects, the RQ-based approach does not provide a quantitative estimate of likelihood and/or magnitude of an adverse effect.  However, as outlined in the Overview Document (USEPA 2004a), the likelihood of effects to individual organisms from the use of diphacinone to control BPDs is estimated using the probit dose-response slope and either the LOC (discussed below) or actual calculated RQ value.  

            Measures of Exposure 

                 Estimating Aquatic Exposure

Because Kaput-D is a bait and is confined to below ground terrestrial applications, exposure to the aquatic environment should be very limited, thus no aquatic assessment was conducted because it was assumed that risk would be very low and below levels of concern.  Concentrations from prior assessments were in pptr (ng/l).  

There are no aquatic monitoring data available for the use of diphacinone.  For diphacinone usage as bait application or animal burrow treatment, it is possible to approximate a `high-end' exposure scenario with two tier 1 models: GENEEC model for surface water exposure and SCIGROW model for ground water exposure.  The peak surface water concentration (EEC) predicted by GENEEC is 483 ng/L.  The peak ground water concentration (EEC) predicted by SCIGROW is 4.75 ng/L (concentrations taken from a previous assessment with equal or greater application rates).  

                 Estimating Primary Terrestrial Exposure

EFED's exposure assessment for the rodenticides differs from that for most other pesticides.  For a rodenticide, the bait itself is the potential food item of concern. Thus, the amount of active ingredient in the formulated bait is used as an EEC.  This information is used to estimate the amount of grain bait that birds and mammals of various sizes need to consume to obtain a dose expected to be lethal to 50% of the individuals in the population (i.e., LD50 dose). Estimates of food-ingestion rates (g dry matter per day) are determined from established allometric equations presented in the Wildlife Exposure Factors Handbook (USEPA 1993). The concentration of diphacinone in bait is also used to estimate initial dietary exposure (mg a.i. per kg in bait) which in turn is used to calculate avian and mammalian dietary RQs.   
                 Estimating Secondary Terrestrial Exposure

Secondary exposure analysis (from consumption of contaminated prey and/or scavenged carcasses) requires consideration of residues in the bodies of organisms that are commonly consumed by predators and scavengers.  Details of the analysis are included in Section 3.
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.  

                 Integration of Exposure and Effects

Risk characterization is the integration of exposure and ecological effects characterization to determine the potential ecological risk from the use of diphacinone to control Black-tailed Prairie Dogs, and the likelihood of direct and indirect effects to the assessed species in terrestrial habitats.  The exposure and toxicity effects data are integrated in order to evaluate the risks of adverse ecological effects on non-target species.  The RQ method is used to compare exposure and measured toxicity values.  EECs are divided by acute and chronic toxicity values.  The resulting RQs are then compared to the Agency's LOCs (USEPA 2004a).  

Use of Probit Slope Response Relationship to Provide Information on the Endangered Species Levels of Concern
As part of the risk characterization, an interpretation of acute RQs for listed species is discussed.  This interpretation is presented in terms of the chance of an individual event (i.e., mortality or immobilization) should exposure at the EEC actually occur for a species with sensitivity to diphacinone on par with the acute toxicity endpoint selected for RQ calculation.  To accomplish this interpretation, the Agency uses the slope of the dose response relationship available from the toxicity study used to establish the acute toxicity measures of effect for each taxonomic group that is relevant to this assessment.  The individual effects probability associated with the acute RQ is based on the mean estimate of the slope and an assumption of a probit dose response relationship.  In addition to a single effects probability estimate based on the mean, upper and lower estimates of the effects probability are also provided to account for variance in the slope, if available.  

Individual effect probabilities are calculated based on an Excel spreadsheet tool IECV1.1 (Individual Effect Chance Model Version 1.1) developed by the U.S. EPA, OPP, Environmental Fate and Effects Division (June 22, 2004).  The model allows for such calculations by entering the mean slope estimate (and the 95% confidence bounds of that estimate) as the slope parameter for the spreadsheet.  In addition, the acute RQ is entered as the desired threshold. 
Data Gaps
Ecological
A data gap is identified for Field Testing for Terrestrial Wildlife (850.2500). 
The purpose of this study is to evaluate the availability of carcasses containing diphacinone residues to carnivores and scavengers. This study would also be useful for estimating diphacinone concentrations in prey.  The purpose of this requested study is to provide quantitative data that are currently unavailable for KAPUT-D Prairie Dog Bait to assess the availability of carcasses. More specifically, the study should be designed to evaluate:
   * the availability of poisoned/incapacitated prairie dogs and non-target animals to predators and scavengers,
   * residue levels in dead and dying prairie dogs,
   * the effect of carcass collection on mitigating exposure, and
   * the carcass collection efficacy (per label directions).

This study has a potential to add considerable value to the ecological risk assessment.  Because there is no Agency protocol for this study, a protocol for the carcass availability study should be submitted to EPA for approval prior to study initiation.

A data gap is identified for the Avian Reproduction Study (850.2300). 
Both the waterfowl and upland game bird reproduction studies have potential to add value to the ecological risk assessment. No acceptable data are available to assess possible reproductive effects to avian species from primary or secondary/tertiary exposure to diphacinone.  Due to the increased use area and the fact that poisoned and moribund Prairie Dogs are easy prey and scavenge items for raptors and other birds, avian reproduction data are needed to adequately quantify the risks this proposed use poses.

An additional data gap is identified for an Avian Acute Oral Toxicity Test (850.2100) conducted using a passerine species. Under 40 CFR Part 158, data are required for one passerine species and either one waterfowl species or one upland game bird species for terrestrial, aquatic, forestry, and residential outdoor uses.  The current method of calculating a weight-adjusted LD50 using bobwhite quail or mallard duck data may over- or under-estimate risks to passerines because these birds may metabolize the chemical differently. Because of the documented high toxicity to birds through submitted studies and the reported incidents, information regarding relative toxicity to passerines is essential for further characterization of the avian risks of diphacinone. This study has a potential to add value to the ecological risk assessment. Because the 850.2100 guideline has not yet been finalized, protocols for the study of passerine species should be submitted to EPA for approval prior to study initiation.

Environmental Fate
As cited in the RED (USEPA, 1998b), the only available environmental fate data from studies for diphacinone are 1) physicochemical properties, 2) hydrolysis, 3) aerobic soil metabolism, and 4)  aged column leaching.  However, because of the specific results of studies and because the specific use and application rates for diphacinone relative to established end-point toxicities is low, EFED can satisfactorily evaluate/screen limits of potential environmental concentrations and ecological effects without additional environmental fate data.
 
Exposure Assessment
Label Application Rates and Intervals
For this assessed use (i.e., control of BPD), diphacinone is formulated as a bait composed of 50 mg a.i./kg-bait. The proposed Section 3 label submitted to EPA describes methods of application as detailed previously. It must be hand applied on rangeland and adjacent non-crop areas at least 6" into active burrows.

Aquatic Exposure Assessment

Aquatic exposure from the use of diphacinone is expected to be negligible, and thus aquatic routes of exposure are not predicted to make significant contributions to total exposure.  Also, indirect risk from potential effects on aquatic organisms was assumed to be negligible due to lack of direct effects to aquatic organisms.  
Aquatic EECs
There are no aquatic monitoring data available for the use of diphacinone.  For diphacinone usage as bait application or animal burrow treatment, it is possible to approximate a `high-end' exposure scenario with two tier 1 models: GENEEC model for surface water exposure and SCIGROW model for ground water exposure.  The peak surface water concentration (EEC) predicted by GENEEC is 483 ng/L.  The peak ground water concentration (EEC) predicted by SCIGROW is 4.75 ng/L (concentrations taken from previous assessment).  Thus, aquatic routes of exposure are not predicted to make significant contributions to total exposure

Terrestrial Exposure Assessment

For this assessment, it was assumed that terrestrial animals could be exposed in two different pathways. Animals may directly consume bait (primary consumption), or animals may consume contaminated carcasses either killed or scavenged by the consumer (secondary consumption). Both approaches and the expected exposure levels are detailed below. 
Expected Diphacinone Ingestion through Bait Consumption
Exposure through bait consumption will be calculated using two methodologies. For the first method, diphacinone exposure is calculated as mg a.i./kg-bwt, where kg-bwt is the kilograms of the consuming individual for three standard weight classes of passeriforms and rodents. Exposure (food dry weight consumption) estimates were derived using allometric equations from USEPA (1993). The allometric equations for passeriform birds and rodent mammals were used as these would best approximate those individuals with high potential for consuming grain and they would give the most conservative exposure estimates. Food dry weight was converted to wet weight assuming the bait contained 10% water, similar to the assumption that seeds for wildlife consumption contain 10% water (USEPA 1993). Formulas for calculation of dose estimates are provided in Table 3.1, and exposure estimates (on a dose basis) are provided in Table 3.2. RQs are generated by dividing these exposure estimates (mg a.i./kg-bwt) for a given weight class by the most conservative toxicity endpoint for the relevant taxa adjusted for the default body weights.  Acute RQs using these exposure estimates were generated for birds and mammals (using LD50 data).

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

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

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

      Food intake (g, wet weight):	FI (g wet-wt/day) = FI (g dry-wt/day) / 0.90	
      	
      Diphacinone intake (mg a.i./kg-bwt/day) =  FI (g wet-wt/day) * 50 mg a.i./kg-bait  /  Wt(g)

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

Table 3.2 Expected Diphacinone Intake for Default Bird and Mammal Weights based on Consumption of Bait.

                                  Weight (g)
                                 Food intake 
                                (g dry-wt/day)
                                 Food intake 
                                (g wet-wt/day)
                              Dphacinone intake 
                             (mg a.i./kg-bwt/day)
Birds*
                                      20
                                      5.1
                                      5.6
                                     14.1

                                      100
                                     19.9
                                     22.2
                                     11.1

                                     1000
                                     141.2
                                     156.9
                                      7.8
Rodent Mammals
                                      15
                                      2.9
                                      3.2
                                     10.6

                                      35
                                      4.6
                                      5.1
                                      7.3

                                     1000
                                     30.6
                                     34.0
                                      1.7
*surrogate for terrestrial-phase amphibians

The second exposure method for primary consumption of bait results in a diet concentration of 50 mg a.i./kg-bait (concentration of diphacinone as packaged in bait). RQs are calculated using this estimate of dietary concentration and the LC50 (mg a.i./kg-diet) available from dietary toxicity studies. Acute RQs using these exposure estimates were generated for birds and mammals.

            Expected Diphacinone Ingestion through Consumption of Contaminated Carcasses

Mammals and birds have the potential to consume diphacinone bait.  Therefore, if they consume bait and are taken as prey, then secondary exposure may result.  The determination of diphacinone intake for individual secondary consumers of diphacinone poisoned animals or carcasses are calculated in a manner similar to the approach for individuals consuming bait directly. Calculated and empirical residue data are used instead of bait concentration of diphacinone (Table 3.4).  Diphacinone body burdens in studies were also determined in mammals after exposure to diphacinone bait. 

Measured residue data are available for the CA ground squirrel, which were used as the basis for exposure estimates for secondary exposure.  It was assumed the concentrations were reported using wet weights of the mammals.  The mean concentration was reported as 1.4 mg ai/kg bw with a maximum concentration of 3.4 mg ai/kg bw (Baroch 1994a, MRID# 43534601).  Field collected data are subject to a number of uncertainties including, but not limited to:
 
   * possible partial decomposition of bodies in field, 
   * collection may miss carcasses with highest body burdens, and
   * collection may miss individuals that were rapidly predated, died off site, or died underground.

An additional uncertainty with the use of these whole-body exposure estimates is that it may underestimate exposure to animals that consume specific tissues, such as the liver, that may have more concentrated diphacinone levels relative to the whole-body estimates.  

Diphacinone exposure through carcass consumption is calculated as mg a.i./kg-bw, where kg-bw is the kilograms of the consuming individual for three weight classes of birds and mammals. For this analysis, bird weight classes of 50, 1000, and 2000 g individuals and mammal weight classes of 50, 1000, and 3000 g individuals were used to better represent the larger size of carnivores and scavengers relative to the full range of bird and mammal weights. Exposure (food dry weight consumption) estimates were derived using the generic bird and mammal allometric equations from (EPA 1993). Food dry weight was converted to wet weight assuming the consumed prey/carcass contained 68% water (USEPA 1993). Formulas for calculation of dose estimates are provided in Table 3.3, and exposure estimates (on a dose basis) are provided in Table 3.4. RQs are generated by dividing these exposure estimates (mg a.i./kg-bwt) for a given weight class by the most conservative toxicity endpoint for the relevant taxa adjusted for the default body weights.  RQs using these exposure estimates were generated for acute bird and mammal (using LD50 data).

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

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

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

Snake food intake (g dry weight):    FI (g dry-wt/day) = FI = 0.621 W 0.564
	
Food intake (g, wet weight):	FI (g wet-wt/day) = FI (g dry-wt/day) / 0.32	
      	
Diphacinone intake (mg a.i./kg-bwt/day) =  FI (g wet-wt/day) * 3.4 mg a.i./kg-carcass  /  Wt(g)

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

Table 3.4 Expected Diphacinone Intakes for Carnivorous Birds and Mammals based on Consumption of Contaminated Carcasses

                                  Weight (g)
                                 Food intake 
                                (g dry-wt/day)
                                 Food intake 
                                (g wet-wt/day)
                              Diphacinone intake 
                            (mg a.i./kg-bwt/day)[1]
Birds*

                                      50
                                      8.3
                                     25.8
                                     1.75

                                     1000
                                     58.2
                                     181.7
                                     0.62

                                     2000
                                     91.3
                                     285.4
                                     0.49
Mammals
                                      50
                                      5.9
                                     18.3
                                     1.24

                                     1000
                                     68.7
                                     214.7
                                     0.73

                                     3000
                                     169.5
                                     529.8
                                     0.60
Snake
                                      400
                                      N/A
                                     1.36
                                     3.40
*surrogate for terrestrial-phase amphibians
[1] See Table 3.8 for derivation.

The maximum measured concentration of diphacinone in carcasses was 3.4 mg a.i./kg-carcass.  This empirical measurement was also used to estimate exposures from consumption of contaminated carcasses. RQs are calculated using this estimate of dietary concentration and the LC50 (mg a.i./kg-diet) available from dietary toxicity studies. RQs using these exposure estimates were generated for acute bird and mammal (using LC50 data).
Ecological Effects Characterization
Toxicity endpoints are established based on data generated from guideline studies submitted by the registrant, and from open literature studies that meet the criteria for inclusion into the ECOTOX database maintained by EPA/ORD.  EFED policy is to use the effects endpoint from the most sensitive tested species for each taxa evaluated.  In aquatic systems, taxa evaluated include aquatic plants, invertebrates, and fish.  Freshwater fish serve as a surrogate for aquatic-phase amphibians when amphibian data are unavailable or unacceptable for quantitative use.  Where data are available, separate endpoints are used for freshwater and estuarine/marine organisms.  In terrestrial systems, taxa evaluated include birds, mammals and honey bees.  Bird endpoints are generally derived from guideline studies on bobwhite quail and/or mallard duck.  Bird data are used as a surrogate for reptiles and terrestrial-phase amphibians when data for these taxa are unavailable or unacceptable for quantitative use.  Mammal data are derived from guideline studies typically conducted on laboratory rats, mice, or rabbits. 

In addition to registrant-submitted and open literature toxicity information, other sources of information, including use of the acute probit dose response relationship to establish the probability of an individual effect and reviews of ecological incident data, are considered to further refine the characterization of potential ecological effects associated with exposure to diphacinone.  A summary of the available ecotoxicity information and the incident information for diphacinone are provided below.

Toxicity of Diphacinone to Aquatic Organisms

Table 4.1 summarizes the most sensitive aquatic toxicity endpoints, based on an evaluation of both the registrant-submitted studies and the open literature.   Data on toxicity of diphacinone to aquatic plants are not available.
Table 4.1.  Aquatic Toxicity Profile for Diphacinone
Assessment Endpoint 
Acute/ Chronic
Species
TGAI % a.i.
Toxicity Value Used in Risk Assessment
Citation  or MRID # 
(Author,  Date)[1]
Classification
Freshwater fish (surrogate for aquatic-phase amphibians)
Acute

Bluegill sunfish Lepomis macrochirus
TGAI 95.8%

Rainbow Trout
Oncorhynchus mykiss
TGAI 95.8%
96-hr LC50 = 7.5 mg/L

96-hr LC50 = 2.6 mg/L

MRID 432495-01
(Machado 1994)

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

Acceptable (moderately toxic)
Freshwater invertebrates
Acute
Daphnia Daphnia manga
TGAI 98.7%
EC50 = 1.8 mg/L

MRID 422822-01 (Putt 1992)
Acceptable (moderately toxic)
1-ECOTOX references are designated with an E followed by the ECOTOX reference number. 
Note: There were many other studies submitted but they were not listed here because they were classified as INVALID. 

Toxicity of Diphacinone to Terrestrial Organisms	

Table 4.2 summarizes the most sensitive terrestrial toxicity endpoints, based on an evaluation of both the registrant-submitted studies and the open literature.   Data on toxicity of diphacinone to terrestrial plants are not available.

Birds
Diphacinone is acutely moderately toxic to avian species.  The most sensitive avian endpoint that corresponded to a standard test species (bobwhite quail or mallard duck) was an LD50 = 1630 mg a.i./kg-bwt from a bobwhite quail study (MRID 42245201). This endpoint is uncertain because the 95% confidence interval ranged from 0 to .  Visual inspection of the data indicates that the LD50 is less than 2000 mg/kg but greater than 400 mg/kg. A second bobwhite quail study was available in the open literature (Rattner et al., 2010), which reported an LD50 of 2014 mg a.i./kg-bwt. In addition, an LD50 = 3158 mg a.i./kg-bwt was determined for mallard ducks (MRID 00128411). Also an LD50 of 96.8 mg a.i./kg-bwt was available for one raptor species (American kestrel, Falco sparverius) (Rattner et al. 2011). This study suggests that American kestrels may be more sensitive than the standard test species (i.e., based on the available studies, 17 to 21 times more sensitive than bobwhite quail and 33 times more sensitive than mallard ducks). Subacutely, diphacinone is considered moderately toxic (Mallard Duck LC50 = 906 ppm) to practically non-toxic (Bobwhite Quail LC50 = 5000 ppm).

Mammals
For mammals, the acute oral and dietary toxicity of diphacinone is classified as very highly toxic.  This is expected, given the target of this rodenticide is mammals. The acute oral LD50 for males was calculated as 2.50 mg/kg, with 95% confidence limits of 1.82-3.44 mg/kg; for females it was 2.10 mg/kg, with 95% confidence limits of 1.55-2.86 mg/kg. The combined oral LD50 for both sexes was calculated as 2.31 mg/kg (95% confidence limits of 1.86 to 2.88 mg/kg).  Dietary LC50s ranged from 2.08 to 2.5 ppm. Symptoms occurred at all doses, and were not necessarily associated with subsequent mortality. These included clear or colored nasal discharge, soft stool and/or diarrhea (possibly associated with the corn oil vehicle used), decreased motor activity and occasional drying of the corneal surface. Symptoms at higher dose levels included lacrimation, ataxia, cyanosis and bloody exudate from nose and eyes. Hemorrhage into the body cavities and of various organs was observed in animals which died. 

For diphacinone, the most sensitive laboratory rat data reported an LD50 = 1.9 mg a.i/kg-bwt (MRID 05002272). Toxicity tests conducted on pine voles and meadow voles (Microtus pennsylvanicus) resulted in LD50s of 67.7 and 11.7 mg a.i/kg-bwt (Byers, 1978).  LD50 values for dogs and cats ranged from 0.88 to 15 mg a.i/kg-bwt (LiphaTech, Inc, 1997). LD50s for the coyote (Canus latrans) and mongoose (Herpestes spp) were 0.6 and 0.18 mg a.i/kg-bwt, respectively (Savarie et al. 1979 and MRID 405655-01). These data suggest that diphacinone may be more toxic to carnivores than those animals experiencing primary exposure to diphacinone. This increased sensitivity of secondary exposure animals relative to primary exposure animals was also observed in the avian acute oral toxicity studies. 

Reptiles
Brooks et al. (1998) conducted a study to determine the acute oral and dermal toxicity of 18 chemicals, including diphacinone, to Brown Tree Snakes (Boiga irregularis).  Acute exposure to diphacinone resulted in LD50s of 20.75 mg/kg (95% CI=10.1 to 44.9) with a slope of 4.2 (ethanol used as a carrier) and 32.2 mg/kg (95% CI=18.8 to 55.9) with a slope of 5.0 (propylene glycol used as a carrier).  Five snakes of either sex were used in each treatment group. All snakes were observed for 7 days after administration of Diphacinone.  Diphacinone produced mortalities within 24 hrs of administration of doses of 10-40 mg/kg.  At necropsy, there was no evidence of hemorrhaging.  Diphacinone produced no mortalities from dermal application.
Table 4.2.  Terrestrial Toxicity Profile for Diphacinone
Species
                                Acute/ Chronic
                                    Species
                                Toxicity Value 
                                   Citation
                           MRID/ECOTOX reference No.
                                Classification
Birds (surrogate for terrestrial-phase amphibians and reptiles)
Acute oral
LD50
Bobwhite Quail

Kestrel
>400<2000 mg/kg (reported as >1630 mg/kg)

96.8 mg/kg
422452-01

Rattner et al 2011
Supplemental

Supplemental

Acute Dietary 

LC50[1]
Mallard Duck

Bobwhite Quail
906 ppm[3] (LOAEC=1.6 ppm)

>5000 ppm[2]
424088-02

424088-01
Core

Core
Mammals
Acute
Rat
2.5 mg/kg M
2.1 mg/kg F
00060605
Core

Acute
Rat
1.9
05002272
Supplemental

Acute Dietary LC50

Rat
Rat
2.08 ppm
2.5 ppm
TMN 075
TMN 051
Supplemental
Supplemental

Acute oral LD50
Coyote
Mongoose
0.6
0.2
Savarie et al. 1979[4]
DWRC[5]  
405655-01
Supplemental
Supplemental
Reptile
Acute 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. 

Elimination of Diphacinone
Available data on the retention times for diphacinone are provided in the table below. Yu et al. (1982) studied the metabolism and disposition of diphacinone in rats and mice. Rats given a single oral dose of radiolabeled diphacinone at either 0.18 or 0.4 mg a.i./kg, about 70% of the dose was eliminated in feces and 10% in urine within 8 days, whereas about 20% of the dose was retained in body tissues. Mice given a single dose of 0.6 mg a.i./kg eliminated most diphacinone within 4 days, and only 7% was retained in body tissues. In both rats and mice, most radioactivity (59 to 69%) was detected in the liver and the kidneys (9 to 12%). Radioactivity was also detected in the brain, heart, spleen, lungs, blood, muscle, fat, and gonads. Several major metabolites were identified, and parent diphacinone in excreta and liver accounted for only about 20% of the dose. In another study, cattle that received a single injection of 1 mg a.i./kg had almost constant residue concentrations in liver and kidney at 30, 60, and 90 days after dosing (Bullard et al. 1979). 

Table 4.3.  Retention of Diphacinone in Blood and Liver
                                  Rodenticide
                                    Species
                               Dose (mg a.i./kg)
                                    # doses
                             Blood t1/2[a] (days)
                             Liver retention[a,b]
                                    (days)
                                   Reference
                                    (MRID)
Diphacinone
Pig
                                     12.5
                                    3 or 5
                                      NA
                                  5.43 (t1/2)
Fisher 2006
Diphacinone
Rat
                                      1.5
                                       1
                                      NA
                                   3 (t1/2)
Fisher et al. 2003 (48190801)
Diphacinone
Cattle
                                      1.0
                                       1
                                      NA
                                    > 90
Bullard et al. 1976
[a] t1/2 for plasma and liver is the elimination half-life
[b] liver retention is expressed as either the time period for which residues persist or as the elimination half-life

Diaz and Whitacre (1976) orally dosed rats with diphacinone (0.32 mg a.i./kg/day) for 1 or 2 days. Rats dosed for 2 days were sacrificed 72 hours after the second dose and those dosed for 1 day were sacrificed after 48 hours. In rats dosed for 2 days, about 45% of the total dose administered was excreted (86% in feces, 14% in urine) and 25% was retained in body tissues 72 hours after the last dose. The remaining 30% of the dose was not recovered. The body tissues retaining the most diphacinone at 96 hours were the hide and tail, liver, intestine, blood, and the carcass. In rats dosed for 1 day and sacrificed after 48 hours, about 5% of the dose was excreted and 61% retained; the remainder was not recovered. 

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

 Birds and Mammals, Secondary Toxicity Tests
Available secondary poisoning studies involving diphacinone indicate that some mammals are susceptible to secondary poisoning from consuming diphacinone residue in animal tissue. The toxicant loads of the rats, mice, and nutria fed to raptors, mustelids, and dogs are not known but were sufficiently high to cause mortality in most species. The relationship between target loads in poisoned target species and the levels presented in coyote and sheep muscle was not established.  Moreover, the amount of toxicant present in the coyote muscle was found to be considerably lower than that contained in the small intestine, liver, kidney, and heart tissues. The data are adequate to demonstrate that avian and mammalian predators may be killed from consuming target species poisoned with 0.01% a.i. bait and that mammals may be killed from consuming target animals exposed to either 0.01% or 0.005% bait.  

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 toxicity[a]
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 toxicity[a]
Great horned owl
                    mice fed choice of 0.01% bait or untreated food for 10 days
                                                                              2
                                                                              5
                                                                              3
                                                                              2
                                                                         1 (ct)
Saw-whet owl
                    mice fed choice of 0.01% bait or untreated food for 10 days
                                                                              2
                                                                              5
                                                                              1
                                                                              1
                                                                   no survivors
Barn owl
                    rats fed choice of 0.005% bait or untreated food for 5 days
                                                                        ad lib.
                                                                             10
                                                                              2
                                                                              0
                                                                              0
American crow
                                               rats fed 0.005% bait until death
                                                                              1
                                                                         1-2[b]
                                                                              1
                                                                              6
                                                                             10
                                                                             11
                                                                              0
                                                                              0
                                                                              0
                                                                      5 (eb/ct)
Golden eagle
                                                       meat laced at 2.7 ppm ai
                                                                          454 g
                                                                               
                                                                              5
                                                                             10
                                                                              4
                                                                              3
                                                                              0
                                                                              0
                                                                      4 (eb/ct)
                                                                   3 (eb/ct)[c]
[a] eb = external bleeding; ih = internal hematoma; bl = bleeding (unspecified); ct = increased blood coagulation time; nr = not reported
[b] offered 1 rat per crow for 5 days and 2 rats per crow on day 6
[c] general weakness of all eagles was observed after 5 days

 Incident Database Review

A review of the Ecological Incident Information System (EIIS, version 2.1), the `Aggregate Incident Reports' (v. 1.0) database, and the Avian Monitoring Information System (AIMS) for ecological incidents involving diphacinone was completed.  The results of this review for terrestrial, plant, and aquatic incidents are discussed below.   

There were a total of 33 incidents listed in the database. Eleven (33.5%) were deemed "highly probable", eight (24%) were deemed as "probable", eleven (33.5%) were deemed "possible", two (6%) were deemed "unlikely" and one (3%) was deemed "unrelated".  Descriptions of the incidents that are most likely associated with diphacinone are described below.  These incidents confirm that exposure to and effects from diphacinone exposure can occur from both primary consumers and secondary poisoning.  

Interpretation of incident data is difficult and may be confounded by a number of factors.  The report of an incident can demonstrate that a complete exposure pathway resulting in an effect (i.e., mortality) has occurred.  Only a small fraction of wildlife incidents are believed to be reported to the Agency.  The lack of incidents or the presence of fewer incidents in and of itself does not necessarily suggest that fewer incidents are occurring or that one chemical is safer than another.  However, incident data can corroborate results of deterministic risk assessments.  

 There were a number of terrestrial incidents reported for diphacinone and involved both primary and secondary toxicity.  Eleven incidents were deemed "highly probable". These included mortality to a mountain lion, a raccoon and a snowy owl through secondary poisoning, as well as nineteen gray squirrels, two white-tailed deer, a ground squirrel, a kit fox and a rat through bait consumption.  Listed below are descriptions of "highly probable" incidents as well as those that resulted in measurable residues. Incidents located in EIIS are summarized below.  

Terrestrial Incidents

   * Testing found diphacinone residue concentrations of 45 ppm and 55 ppm from blood and liver samples in a mountain lion and raccoon found dead in Northern CA respectively (B000641-001). 
   * Diphacinone was also found in a dead adult kit fox (found in Bakersfield CA and weighing 5.75 lbs) by immunoassay in liver and blood at a concentration of 0.18 ppm (B000640-001).
   * A kangaroo rat was found in CA in a moribund state. An analysis of the liver detected diphacinone residues at 3.5 ppm (R000-02-001) 
   * A male deer fawn (found approximately 2 weeks post mortem) and an adult buck (weighing 202 lbs) was found dead on Fire Island NY and contained 0.2 ppm and 0.93 ppm diphacinone in liver samples respectively (I004768-001 and I004769-001).  
   * An adult male gray squirrel was found dead in Brookhaven NY and contained 2.0 ppm diphacinone (I005353-001).  
   * A male snowy owl was found dead in Rhinebeck NY of likely secondary poisoning (I006306-01).  Diphacinone was found in the liver but the concentration was not reported.  
   * A number of gray squirrels were found dead in Richmond VA and pooled liver samples from two individuals (one weighed 490g and the other weighed 567g) contained 4.94 and 3.4 ppm diphacinone respectively (I007488-004).  
   * Although listed as a "probable" incident, a red-tailed hawk was found moribund (dying the next day) in Roslyn NY and contained 0.34 ppm diphacinone in a liver sample (I009270-001).  
   * A gray squirrel was found dead in Albany NY of apparent blunt impact trauma (listed as a "possible" incident).  However, the liver contained 1.02 ppm diphacinone (I011518-001).  
   * Eight squirrels were found dead in Conyers GA and 3.6 ppm diphacinone was found in the liver of one (410g) of the squirrels that was sampled (I019839-001).  
   * A rat and a ground squirrel were found dead in Ventura CA and contained 4.12 and 1.99 ppm diphacinone in liver samples respectively (I0I4886-001).  
   * Two deer found dead in Suffolk NY contained 0.2 and 0.93 ppm diphacinone in liver samples respectively (I013810-007 and I013810-008, both incidents deemed as "probable").  
   * A radio-collared yearling female coyote was found dead in Ventura CA and contained diphacinone residues of 1.3 ppm in liver, 0.1 ppm in blood and 0.16 ppm in stomach contents but also contained 0.083 ppm Brodifacoum in the liver (I007107-014, I007165-003).   
     
Plant Incidents

There were no incidents reported for diphacinone involving plants. 
 
Aquatic Incidents

There were no aquatic incidents reported for diphacinone.  
Risk Characterization
Risk characterization is the integration of exposure and effects characterization to determine the ecological risk from the use of diphacinone for BPD removal and the likelihood of effects on aquatic life, wildlife, and plants based on different pesticide-use scenarios. The risk characterization provides estimation and description of the likelihood of adverse effects; articulates risk assessment assumptions, limitations, and uncertainties; and synthesizes an overall conclusion regarding the likelihood of adverse effects. In the risk estimation section, RQs are calculated using standard EFED procedures explained in the following section.  In the risk description section, additional analyses may be conducted to help characterize the potential for risk.

Risk Estimation: Integration of Exposure and Effects Data

Results of the exposure and toxicity effects data are used to evaluate the likelihood of adverse ecological effects on non-target species. For the assessment of diphacinone risks, the risk quotient (RQ) method is used to compare exposure and measured toxicity values:

      RQ = EEC / (Toxicity Endpoint)

The RQs are compared to the Agency's levels of concern (LOCs). These LOCs are the Agency's interpretive policy and are used to analyze potential risk to non-target organisms and the need to consider regulatory action. These criteria are used to indicate when a pesticide's use as directed on the label has the potential to cause adverse effects on non-target organisms. 
Aquatic Risk Estimation
Based on the GENEEC estimated EECs, risk estimates for aquatic organisms are lower than concern levels. EECs in previous assessments were in pptr (ng/L), which is orders of magnitude lower than available toxicity reference values. Therefore, effects to aquatic organisms from the proposed diphacinone use are unlikely. 
Bird and Mammal Risk Estimation
             Risks from Direct Bait Consumption

In the case of primary exposure, it is assumed the bait containing diphacinone is ingested by 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. Toxicity is measured by the LD50 obtained from the single-gavage studies for birds and mammals. The LD50's are adjusted for the weight of the assessed animals (birds: 20, 100, 1000 g; mammals: 15, 35, 1000 g).  Avian and mammalian RQs based on acute single-dose gavage studies are provided in table 5.2. For birds, the LOCs were not exceeded. For mammals, the Listed Species, Restricted Use, and Acute LOCs were exceeded for all three weight classes.

Table 5.1 Formulas for Calculation of Weight-adjusted Avian and Mammalian Diphacinone LD50s.
Adjusted avian LD50:       where:
            Adj. LD50 = adjusted LD50 (mg/kg-bw) 
            LD50 = endpoint reported from bird study (mg/kg-bw)
            TW = body weight of tested animal (178g bobwhite quail)
                  AW = body weight of assessed animal (20g, 100g, and 1000g)
            x = Mineau scaling factor for birds; EFED default 1.15

Adjusted mammalian LD50:  where:
            Adj. LD50 = adjusted LD50 (mg/kg-bw)
             LD50 = endpoint reported from mammal study (mg/kg-bw)
            TW = body weight of tested animal (350g)
                  AW = body weight of assessed animal (15g, 35g, 1000g)

Table 5.2 Bird and Mammal Acute RQs based on a Single-dose of Diphacinone through consumption of bait
Taxonomic Group
                                  Weight (g)
                              Diphacinone intake 
                            (mg a.i./kg-bwt/day)[1]
                                Adjusted LD50 
                              (mg a.i./kg-bw)[2]
                                     RQ[3]
Birds*
                                      20
                                      13
                                     1163
                                     0.01

                                      100
                                      10
                                     1480
                                     0.01

                                     1000
                                      7.1
                                     2091
                                   <0.01
Mammals
                                      15
                                      10
                                      4.8
                                    2.63***

                                      35
                                      6.6
                                      3.9
                                    2.24***

                                     1000
                                      1.5
                                      1.7
                                    1.20***
*surrogate for terrestrial-phase amphibians
[1] See Table 3.5 and 3.6 for derivation.
[2] See Table 5.2 for derivation.
[3] Bolded (***) RQs exceed Listed Species, Restricted Use, and Acute Risk LOCs. 

For toxic response elicited from the dietary exposure route extended over several days, exposure is measured as mg a.i./kg-bait. Toxicity is measured by the LC50 obtained from the dietary studies (5 days on treated diet) for birds and mammals. Avian and mammalian RQs based on dietary studies are provided in Table 5.3. For mammals, the Listed Species, Restricted Use, and Acute LOCs were exceeded (RQ = 24.04). 

Table 5.3 Bird and Mammal Acute RQs based on a 5-day Exposure to Diphacinone in the Diet (consumption of bait)
                                Taxonomic Group
              Diphacinone concentration in bait (mg a.i./kg-bait)
                                     LC50 
                               (mg a.i./kg-diet)
                                     RQ[1]
                                    Birds*
                                      50
                                      906
                                     0.06
                                    Mammals
                                      50
                                     2.08
                                   24.04***
*surrogate for reptiles and terrestrial-phase amphibians
[1] Bolded (***) RQs exceed Listed Species, Restricted Use, and Acute Risk LOCs.

Secondary Risks from Consumption of Diphacinone-contaminated Carcasses

To determine secondary exposure EECs, available literature concerning carcass residue concentrations were considered.  For this assessment, a maximum carcass concentration found in a field study using diphacinone of 3.4 mg a.i./kg-carcass was used to estimate exposure to birds and mammals. Estimated diphacinone intake for secondary consumers (mg a.i./kg-bwt/day) was calculated in Table 3.4. As in the direct consumption risk estimation, the LD50's are adjusted for the weight of the assessed animals (birds: 50, 1000, 2000 g; mammals: 50, 1000, 3000 g). These heavier weights are used to better represent carnivores and scavengers which tend to be larger individuals. For reptiles, an LD50 (20.75 mg/kg) was used to calculate the risk to a 400g snake eating a prey animal that has ingested diphacinone.

Carnivore/scavenger bird LOCs were not exceeded; however, RQs for carnivore and scavenger mammals exceeded Listed Species and Restricted Use LOCs while the snake RQ exceeded only the Listed Species LOC (Table 5.4).

Table 5.4 Carnivorous/Scavenger Bird and Mammal Acute RQs based on a Single-dose of Diphacinone by Gavage, consumption of contaminated carcasses
Taxonomic Group
                                  Weight (g)
                  Diphacinone intake (mg a.i./kg-bwt/day)[1]
                       Adjusted LD50 (mg a.i./kg-bwt)[2]
                                     RQ[3]
Birds+
                                      50
                                     1.75
                                     87.2
                                     0.02

                                     1000
                                     0.62
                                     136.7
                                     0.004

                                     2000
                                     0.48
                                     151.7
                                     0.003
Mammals
                                      50
                                     1.24
                                      3.4
                                    0.36**

                                     1000
                                     0.73
                                      1.6
                                    0.45**

                                     3000
                                     0.60
                                      1.2
                                    0.49**
Reptile (snake)
                                      400
                                      3.4
                                    20.75++
                                     0.16*
+surrogate for terrestrial-phase amphibians
++ not adjusted
[1] See Tables 3.8 and 3.9 for derivation.
[2] See Table 5.2 for derivation with assessed body weights used in this table.	
[3] Bolded (*) RQs exceed Listed Species LOCs. 
Bolded (**) RQs exceed Listed Species & Restricted Use LOCs.

For toxic response elicited from the dietary exposure route extended over several days, exposure is measured as the concentration of contaminant in the carcass, 3.4 mg a.i./kg-carcass.  Toxicity is measured by the LC50 obtained from the dietary studies (5 days on treated diet) for birds and mammals. Avian and mammalian RQs based on dietary studies are provided in Table 5.5.  Carnivore/scavenger bird LOCs were not exceeded; however, RQs for carnivore and scavenger mammals exceeded Listed Species, Restricted Use, and Acute LOCs.

Table 5.5 Bird and Mammal Acute RQs based on a 5-day Exposure to Diphacinone in the Diet, consumption of contaminated carcasses
                                Taxonomic Group
           Diphacinone concentration in carcass (mg a.i./kg-carcass)
                                     LC50 
                               (mg a.i./kg-diet)
                                     RQ[1]
                                    Birds*
                                      3.4
                                      906
                                     0.004
                                    Mammals
                                      3.4
                                     2.08
                                    1.63***
*surrogate for reptiles and terrestrial-phase amphibians
[1] Bolded (**) RQ exceed Listed Species & Restricted Use Risk LOCs. 

         Risk to Terrestrial Invertebrates

Toxicity data were not available to assess risk to terrestrial invertebrates.  
Risk Description	
The results of the screening-level risk assessment for the proposed uses of diphacinone for BPD control suggest that levels of diphacinone in the environment, when compared with toxicity values, may result in direct effects to listed and non-listed terrestrial animals. Although RQs only exceeded LOCs for mammals, incident data and secondary exposure studies also suggest that birds may be affected as well.  These direct effects may also result in indirect risk to non-target species. 
Risks to Aquatic Organisms
Based on the screening level EECs calculated in previous assessments, risk estimates for aquatic organisms are lower than concern levels. Therefore, effects to aquatic organisms from the proposed diphacinone use are unlikely.

EFED presumed risks were below concern levels to aquatic plants given the expected low concentrations in water and the mode of action of diphacinone. 
Risks to Terrestrial Organisms
      Risks to Birds directly consuming bait

Risk to birds (listed and non-listed species) are lower than concern levels from the proposed use of diphacinone. RQs for birds consuming a single-dose or doses over a 5 day period of diphacinone in bait do not exceed any LOCs.  On an acute basis (single gavage or 5-day dietary exposure), the calculated RQs ranged from <0.01 to 0.01, which do not exceed any LOCs.
      
      Risks to Carnivore and Scavenger Birds 
Carnivorous and scavenger birds may be exposed to diphacinone through consumption of carcasses that were contaminated with diphacinone. Based on available ground squirrel carcass data, carcass diphacinone concentration was estimated at 3.4 mg a.i./kg-carcass. On an acute basis (single gavage or 5-day dietary exposure), the calculated RQs did not exceed any LOCs.  The estimated concentrations were within a factor of 2 compared with the maximum carcass data.  Also, using the kestrel endpoint (96.8 mg/kg) to calculate quotients increased risk quotients slightly, but did not alter LOC exceedance conclusions.  

There is uncertainty associated with the RQs for carnivore and scavenger birds because the LD50 and LC50 used to calculate the RQs were based on granivorous species. These species prefer to eat a relatively consistent quantity of food on a daily basis. Carnivores and scavengers may consume a large meal (an entire carcass) once every several days. Given this feeding strategy, these birds may get a larger single-dose exposure of diphacinone than is currently estimated in the risk quotient methodology.  The kestrel data (Rattner et. al., 2011) indicate that diphacinone is 21x more toxic to a carnivorous bird compared to the standard test species (quail).

Incidents were reported in the United States of carnivorous/scavenger birds that died that of diphacinone poisoning. The known species include a red-tailed hawk and a snowy owl. The presence of confirmed incidents of diphacinone poisoning under the currently registered labels that have similar or more restrictive application rates and methods indicates that exposure at sufficient levels to cause poisoning of these predator birds does occur. In addition, two secondary exposure studies have been conducted that fed rats 0.005% diphacinone bait (American crows) or a choice of untreated or 0.005% diphacinone bait (barn owls) then fed the contaminated rats to birds.  Neither of the studies resulted in mortality of birds; however, in the study where rats were not given a choice of treated or untreated food, approximately half of the birds (American crows) showed signs of treatment-related effects including increased incidents of external bleeding and increased coagulation time (see table 4.5).  Together, the incident data and secondary exposure studies show that secondary exposure to birds can be sufficient to result in severe effects, including mortality.  
Risks to Mammals directly consuming bait
Mammals (listed and non-listed species) are at risk from this proposed use of diphacinone. Diphacinone presents primary exposure risk concerns for all mammal sizes assessed using all primary exposure assessment methods (Acute RQs were 1.20 to 2.63 and the acute dietary RQ was 24.04).  

Incidents were reported in the EPA database of non-carnivorous mammals that died of diphacinone poisoning. The known species include squirrels and deer. Omnivores such as the raccoon and San Joaquin kit fox were also cited in the reported incidents.
Risks to carnivore and scavenger Mammals
Carnivorous and scavenger mammals are exposed to diphacinone through consumption of carcasses that were contaminated with diphacinone. Based on available carcass data, carcass diphacinone concentration was estimated at 3.4 mg a.i./kg-carcass. This value represents the highest concentration of diphacinone found in any individual in all of the carcass residue studies.  On an acute basis for the single gavage exposure, the calculated RQs (<0.01) did not exceed the Listed Species and Restricted Use LOCs. The RQ=1.6 for mammals based on the 5-day dietary study does exceed Listed Species, Restricted Use, and Acute Risk LOCs. Therefore, mammals consuming contaminated carcasses over a several day period may expect mortality.

The LD50 and LC50 used to calculate the RQs were based on granivorous species. These species prefer to eat a relatively consistent quantity of food on a daily basis. Carnivores and scavengers may consume a large meal (an entire carcass) once every several days. Given this feeding strategy, these carnivorous mammals may get an even larger single-dose exposure of diphacinone than is currently estimated in the risk quotient methodology. 

Incidents were reported in the United States of carnivorous/scavenger mammals that died of diphacinone poisoning. The known species include San Joaquin kit fox, a mountain lion, a raccoon and a coyote. The presence of confirmed incidents of diphacinone poisoning under the currently registered labels that have similar or more restrictive application rates and methods indicates that exposure at sufficient levels to cause poisoning of these predator mammals does occur. The presence of confirmed incidents of diphacinone poisoning under the currently registered labels that have similar application rates and methods corroborates the risk estimation process that identified risks to carnivorous/scavenger mammals.  Although some uses have more or less restrictive use patterns.  In addition, a secondary feeding study that fed 0.005% diphacinone bait to rats, then fed contaminated rats to mongoose also observed high rates of mortality (Table 4.5).  
Risks to Reptiles
For reptiles, an LD50 (20.75 mg/kg) was used to calculate the risk to a 400g snake eating a prey animal that has ingested diphacinone.  The snake RQ (0.16) exceeded only the Listed Species LOC.  Diphacinone seems to have a different mode of action in reptiles.  Mortality occurs but does not result from anticoagulation and its mode of action in snakes is unknown.  Diphacinone produced mortalities within 24 hrs of administration of doses of 10-40 mg/kg.  At necropsy, there was no evidence of hemorrhaging.  Diphacinone produced no mortalities from dermal application.
Risks to Terrestrial Invertebrates
No data were found to allow for an assessment of risk to terrestrial invertebrates.
Terrestrial and Semi-aquatic Plants 
RQs were not calculated for terrestrial plants as toxicity data were not available. EFED presumed direct risks were low to terrestrial plants given the mode of action of diphacinone and the lack of any reported incidents.  Since diphacinone is not sprayed directly onto plants, and because so little is expected to leach from the bait and then be available for plant uptake, exposure is expected to be minimal. 

Terrestrial plants that rely on animals for pollination and seed dispersal do have the potential to experience indirect effects through reduction in the animals that perform those functions.
Threatened and Endangered Species Concern
Action Area
 For listed species assessment purposes, the action area is considered to be the area affected directly or indirectly by the Federal action and not merely the immediate area involved in the action.  At the initial screening-level, the risk assessment considers broadly described taxonomic groups and so conservatively assumes that listed species within those broad groups are 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 relatively highest potential exposure to the pesticide, and that exposures are likely to decrease with distance from the treatment area. This risk assessment presents the proposed new use of diphacinone to remove BPD and establishes initial co-location of listed species with treatment areas.  

If the assumptions associated with the screening-level action area result in RQs that are below the listed species LOCs, a "no effect" determination conclusion is made with respect to listed species in that taxa, and no further refinement of the action area is necessary.  Furthermore, RQs below the listed species LOCs for a given taxonomic group indicate no concern for indirect effects upon listed species that depend upon the taxonomic group covered by the RQ as a resource.  However, in situations where the screening assumptions lead to RQs in excess of the listed species LOCs for a given taxonomic group, a potential for a "may affect" conclusion exists and may be associated with direct effects on listed species belonging to that taxonomic group or may extend to indirect effects upon listed species that depend upon that taxonomic group as a resource.  In such cases, additional information on the biology of listed species, the locations of these species, and the locations of use sites could be considered to determine the extent to which screening assumptions regarding an action area apply to a particular listed organism.  These subsequent refinement steps could consider how this information would impact the action area for a particular listed organism and may potentially include areas of exposure that are downwind and downstream of the pesticide use site.
Taxonomic Groups Potentially at Risk
Based on available screening-level information for the proposed use of diphacinone, there is a potential for direct effects to listed terrestrial animals. Consequently, there is a potential concern for indirect effects upon the listed organisms by, for example, perturbing habitat, forage or prey availability. In conducting a screen for indirect effects, direct effect LOCs for each taxonomic group are used to make inferences concerning the potential for indirect effects upon listed species that rely upon non-endangered organisms in these taxonomic groups as resources critical to their life cycle. Terrestrial plants that rely on animals for pollination and seed dispersal do have the potential to experience indirect effects through reduction in the animals that perform those functions. 

Table 5.6. Listed species risks associated with direct or indirect effects due to proposed diphacinone use on the BPD
                                Listed Taxonomy
                                Direct Effects
                               Indirect Effects
Terrestrial and semi-aquatic plants  -  monocots
                                      No
                                      Yes
Terrestrial and semi-aquatic plants  -  dicots
                                      No
                                      Yes
Birds and terrestrial-phase amphibians 
                                      Yes
                                      Yes
Mammals
                                      Yes
                                      Yes
Reptiles
                                      Yes
                                      Yes
Aquatic vascular plants
                                      No
                                      No
Aquatic non-vascular plants[a]
                                      No
                                      No
Freshwater fish and aquatic-phase amphibians
                                      No
                                      No
Freshwater Invertebrates
                                      No
                                      No
Freshwater Benthic Invertebrates
                                      No
                                      No
Estuarine/Marine Fish
                                      No
                                      No
Estuarine/Marine Crustaceans
                                      No
                                      No
Estuarine/Marine Mollusks
                                      No
                                      No
[a] At the present time there are no Federally listed aquatic non-vascular plants.  The taxonomic group is included here for the purposes of evaluating potential contributions to indirect effects to other taxonomy and as a record of exceedances should future listings of non-vascular aquatic plants warrant additional evaluation of Federal actions.

The LOCATES database (version 2.9.7) identifies those U.S. counties in which a given crop is grown for which there are co-located listed species. For this analysis, it was presumed that all federally-listed endangered or threatened terrestrial animals in the proposed States (i.e., CO, KS, NE, NM, ND, SD, WY, TX, MT and OK ) may be directly or indirectly affected. With additional refinement by exploring more detailed use patterns and species biology (e.g., geographic location, specific feeding habits, time of year likely to utilize crop fields), some species listed may be determined to be not likely to be affected. 

The Fish and Wildlife Service (FWS) recently completed a Biological Opinion on a registration action that allows use of another anticoagulant rodenticide (chlorophacinone; trade name of Rozol black-tailed prairie dog bait -  hereafter referred to as Rozol) to control black tailed prairie dogs.  Although the Agency's endangered species risk assessment determined that 21 federally listed species may be affected, a number of conservation measures were developed with discussions with and agreement between the Fish and Wildlife Service, EPA, and the registrant to either reduce or preclude exposures to many of these species.  The Agency's effects determination for Rozol and resulting Biological Opinions are available on-line in the docket at regulations.gov and may be accessed using the following link:  

http://www.regulations.gov/#!docketDetail;dct=FR%252BPR%252BN%252BO%252BSR;rpp=25;so=ASC;sb=docId;po=0;D=EPA-HQ-OPP-2011-0909 

These conservation measures were part of the federal action and included label amendments to add specifications for a line-transect carcass search and reporting of dead/dying listed species and non-target animals in addition to prohibitions of Rozol use and timing restrictions via the Agency's Endangered Species Protection Program (ESPP) Bulletins Live system available at:  http://www.epa.gov/oppfead1/endanger/bulletins.htm.  The Biological Opinion on Rozol (http://epa.gov/espp/2012/borozol-final.pdf) found that the conservation measures adequately protected most of the endangered species, although FWS concluded that incidental take may still occur for the following three species:  (1) black-footed ferret; (2) gray wolf; and (3) northern aplomado falcon.  In order to minimize incidental take for these three species, FWS identified a number of additional reasonable and prudent measures (RPMs) including certified applicator training, development of website content to reduce the potential for secondary poisoning, requirements for the registrant to develop a stewardship training program and submit of Rozol distribution and production data, and additional bulletins for the northern aplomado falcon. Additional details are included in the Biological Opinion available on the docket at the aformentioned link.  

The exposure and toxicity profiles are similar for chlorophacinone and diphacinone (see table below).  In tests conducted in the same species, diphacinone is consistently less toxic to birds relative to chlorophacinone, and it is of similar toxicity to mammals (slight increases and decreases in median lethal doses/concentrations have been observed).  Therefore, because the area of potential use is identical for Rozol and Kaput black-tailed prairie dog baits given that they are both assumed to be applied anywhere within the range of black tailed prairie dogs, the listed species that may be exposed are also identical.  Also, given that the exposure and toxicity profiles are similar, the conservation measures and RPMs identified for Rozol should also be protective of endangered species if implemented on Kaput black tailed prairie dog bait.  In addition, labels for both Rozol and Kaput will stipulate that other anticoagulant rodenticides cannot be used if applying either of these products.  For this reason, the Biological Opinion issued on Rozol is expected to be applicable to both Rozol and Kaput black tailed prairie dog bait, and is, therefore, expected to provide adequate protections for listed species for use of both rodenticides.  

Table 5.7.  Comparison of Chlorophacinone and Diphacinone Exposure and Toxicity Parameters
Comparative Parameter
Chlorophacinone
Diphacinone
Comments
Exposure
Concentration in  Bait
0.005%
0.005%
                                      --

Food Consumption
Not chemical dependent
                                      --

Time to mortality
4-13 days
3-9 days
Time to mortality affects the amount of chemical that can be accumulated over time for target pests.

Liver Retention
35 days - mouse
3 days  -  Rat
5 days  -  Pig
>90 days - Cow
Lack of data in same species precludes definitive comparisons
Toxicity, Birds
BWQ LD50s (mg/kg-bw)
260
1630
                                      --

Other Avian LD50s (mg/kg-bw)
None available
Kestrel: 96.8
Mallard: 3200
                                      --

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

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

Other Species (mg/kg-diet)
60 (Japanese Quail)
Brown Tree Snake: 20.75 
                                      --
Toxicity Mammals
Lab Rat  -  Gavage (mg/kg-bw)
6
1.9
                                      --

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

Other Species  -  Gavage (mg/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-Footed Ferret
Of special concern is the federally listed Black-Footed Ferret, which has an obligate relationship with prairie dogs for food and the use of their borrows for shelter. The Black-Footed Ferret may consume poisoned prairie dogs or non-target animals that contain diphacinone residue in body tissues (secondary exposure) and decreases in prairie dogs may decrease the availability of suitable habitat for the ferret.  Historically, where Prairie Dogs were found, so were Black-Footed Ferrets.  A major cause for the decline in Black-Footed Ferrets is the reduction of the range of Prairie Dogs (USFWS 1988).  This label targets a major food source of the Black Footed Ferret within much of its entire historic range, making recolonization and recovery problematic.  Similar labels for chlorophacinone, a rodenticide of the same class, states, "Do not use this product within Prairie Dog towns in the range of the Black-Footed Ferret without first contacting U.S. Fish and Wildlife Service..." States/US Territories in which KAPUT-D is proposed for use and populations of Black-footed Ferrets are known to or are believed to occur include Colorado, Kansas, Montana, Nebraska, New Mexico, North Dakota, South Dakota, and Wyoming.

 Probit Slope Response Analysis 		
The Agency uses the probit dose-response relationship as a tool for providing additional information on the potential for acute direct effects to individual listed species and aquatic animals that may indirectly affect the listed species of concern (USEPA, 2004).  As part of this evaluation, the acute RQ for listed species is presented in terms of the chance of an individual event (i.e., mortality or immobilization) should exposure at the EEC actually occur for a species with sensitivity to diphacinone on par with the acute toxicity endpoint selected for RQ calculation.  To accomplish this interpretation, the Agency uses the slope of the dose-response relationship available from the toxicity study used to establish the acute toxicity measures of effect for each taxonomic group that is relevant to this assessment.  The individual effects probability associated with the acute RQ is based on the mean estimate of the slope and an assumption of a probit dose-response relationship.  In addition to a single effects probability estimate based on the mean, upper and lower estimates of the effects probability are also provided to account for variance in the slope, if available.  Based on the available acute toxicity for diphacinone, a summary of the probit dose-response analysis is provided below.  If no dose response information is available to estimate a slope for this analysis, a default slope assumption of 4.5 (with lower and upper bounds of 2 to 9) (USEPA 1986) is used. 

Individual effect probabilities are calculated based on an Excel spreadsheet tool IECv1.1 (Individual Effect Chance Model Version 1.1) developed by the U.S. EPA (OPP, Environmental Fate and Effects Division, June 22, 2004).  The spreadsheet performs these calculations by entering the mean slope estimate (and the 95% confidence bounds of that estimate) as the slope parameter for the spreadsheet.  The desired threshold for the probability of an individual effect is entered as the listed species LOC.  In addition, the probability of an individual effect is also derived based on the calculated acute RQ. For these calculations, bird and mammal RQs from the primary exposure to bait (direct consumption) were used.

Summary of Diphacinone Probit Dose Response Analysis for Listed Species*
                               Taxa (study type)
                                 Acute Effect
                               Slope (95% C.I.)
        Chance of Individual Effect at Listed Species LOC [1](95% C.I.)
          Chance of Individual Effect at Derived Acute RQ (95% C.I.)
Snake oral dose
(max RQ  = 1.08)
                                   Mortality
                                  Slope = 4.2
                                 1 in 7.49E+04
         Approximately 1 in 2 assuming a range of slopes from 2 to 9.
Bird dietary
(max RQ = 0.06-0.11)
                                   Mortality
                                  Slope = 4.5
                                 1 in 2.94E+05
                                       
                                <1 in 2.9E5
Mammal dietary fox
(max RQ = 6.1)
                                   Mortality
                                  Slope = 4.5
                                 1 in 2.94E+05
                                       
         Approximately 1 in 1 assuming a range of slopes from 2 to 9.
Mammal dietary mouse
(max RQ = 21.7-43.5)
                                   Mortality
                                 Slope = 4.23
                                 1 in 8.56E+04
         Approximately 1 in 1 assuming a range of slopes from 2 to 9.
[1] Listed Species LOC = 0.10 for terrestrials, 0.05 for aquatic species
*Taken from the 2011 Endangered Species Assessment 
Indirect Effects Analysis
The Agency acknowledges that pesticides have the potential to exert indirect effects upon the listed organisms by, for example, perturbing forage or prey availability, altering the extent of nesting habitat, etc. In conducting a screen for indirect effects, direct effect LOCs for each taxonomic group are used to make inferences concerning the potential for indirect effects upon listed species that rely upon non-endangered organisms in these taxonomic groups as resources critical to their life cycle.
		
Based on direct risk to terrestrial animals, there may be potential indirect effects to terrestrial animals that depend on these organisms (including their surrogates) as a source of food or habitat. Reduction in bird, reptile and mammal populations will reduce the food base for any obligate or opportunistic carnivores and scavengers. In addition, many of the potentially affected species (including the target species, BPD) provide unique habitats through the digging of burrows. Often these burrows are used concurrently with other species or other species may utilize abandoned burrows. If the populations of these burrowing animals are significantly affected, a shift in the entire ecosystem structure in the targeted region may occur. 

Terrestrial plants that rely on animals for pollination and seed dispersal may have the potential to experience indirect effects through reduction in the animals that perform those functions.
Critical Habitat for Listed Species
In the evaluation of pesticide effects on designated critical habitat, consideration is given to the physical and biological features (primary constituent elements) of a critical habitat identified by the U.S Fish and Wildlife and National Marine Fisheries Services as essential to the conservation of a listed species and which may require special management considerations or protection.  The evaluation of impacts for a screening level pesticide risk assessment focuses on the biological features that are primary constituent elements and is accomplished using the screening-level taxonomic analysis (RQs) and listed species' levels of concern (LOCs) that are used to evaluate direct and indirect effects to listed organisms.
      
The screening-level risk assessment for diphacinone has identified potential concerns for direct effects on listed terrestrial animals and indirect effect to those organisms dependent upon them for food and/or habitat.  In light of the potential for indirect effects, the next step for EPA and the Service(s) is to identify which listed species and critical habitat are potentially implicated.

Analytically, the identification of such species and critical habitat can occur in either of two ways.  First, the Services could determine whether the action area overlaps critical habitat or the occupied range of any listed species.  If so, EPA would examine whether the pesticide's potential impacts on non-endangered species would affect the listed species indirectly or directly affect a primary constituent element of the critical habitat.  Alternatively, the Services could determine which listed species depend on biological resources, or have constituent elements that fall into the taxa that may be directly or indirectly impacted by a pesticide.  Then EPA would determine whether or not use of the pesticide overlaps the critical habitat or the occupied range of those listed species.  At present, the information reviewed by EPA is not sufficient to permit use of either analytical approach to make a definitive identification of species that are potentially impacted indirectly or critical habitats that are potentially impacted directly by the use of pesticides.  EPA and the Service(s) are working together to conduct the necessary analysis.

This screening-level risk assessment for critical habitat provides a listing of potential biological features that, if they are primary constituent elements of one or more critical habitats, would be of potential concern.  These correspond to the taxa identified above as being of potential concern for indirect effects and include birds, reptiles, terrestrial phase amphibians, mammals, terrestrial plants and aquatic organisms.  This list should serve as an initial step in problem formulation for further assessment of critical habitat impacts outlined above, should additional work be necessary.
Uncertainties
Fate and Transport Properties
The most significant uncertainty regarding the fate and transport properties of diphacinone are major metabolite, diphenylglycolic acid.  Since the main exposure route is from consumption of the baits, this soil degradate is not expected to be digested.  Therefore, the impact of this uncertainty will be minimal.
Effects
Uncertainty in the exposure assessment stems mainly from assumption made in the assessment related to the consumption of bait by various types of animals.  Animals were assumed to consume an amount of bait equal to their predicted daily food ingestion rate.  Ingestion of bait is most certain for omnivorous mammals because the bait is designed to be attractive to rodents and other mammals.  However, small mammals could eat less bait than their average daily ingestion rate, either because they are also feeding on other food sources, or because they exhibit bait shyness.  Alternatively, if other food is scarce and they find the bait to be very attractive, then they could exhibit gorging behavior, consuming bait in excess of their daily average daily intake rate.  Incidents have shown that species other than small mammals have been killed due to diphacinone poisoning.  Animals which feed predominantly on live prey, including snakes, may not consume the bait.  The use of allometric equations to estimate daily food intake rate introduces additional uncertainty.  The food intake rate was estimated from the body weights of the animals using allometric equations.  How well the generic allometric equations used predict the specific food intake rate of the assessed species is an uncertainty.  For example, the relationship for the snake was based on an equation developed for insectivores, whereas snakes can consume a wide variety of vertebrate prey in addition to terrestrial invertebrates. 

The assessment of secondary exposure involves additional uncertainties. A conservative assumption was made that the entire amount of active ingredient consumed by the prey is present in the prey animal when it is consumed by the carnivorous or scavenger animal.  In reality, the amount of active ingredient in the prey may decrease between the time it consumes the bait by the prey and the prey is consumed by the carnivorous or scavenger animal as the result of elimination and detoxification.  Also, the amount of and rate of assimilation of diphacinone from the consumed prey into the secondary consumer is uncertain.  Assimilation efficiency may be considerably less than the assumed 100%.  Also, snakes and other reptiles typically digest large prey slowly over numerous days.  Thus, the toxic residues in the ingested prey may be released and assimilated in some reptiles slowly over numerous days.  In addition, it would seem that the mode of action that produces mortality in mammals is different than that in reptiles.  Since diphacinone is an anticoagulant, mammals die through hemorrhaging.  However, Brown Tree snakes dosed with diphacinone did not die of hemorrhage but by an unknown mechanism (Brooks et.al. 1998).

The dose of diphacinone from secondary exposure is dependent on the size of the prey and its intake rate.  The size of prey that a snake was predicted to be able to consume (100% of its BW) is uncertain.  These equations may overestimate the maximum size prey which it may consume.  

Lastly, chronic risk is an uncertainty in part because no data is available but also because sublethal adverse effects to reproduction are not known. It is assumed that ingestion of enough diphacinone would induce mortality where reproduction would not be an issue. However, non-lethal effects to reproduction in these species are unknown.  For example, foxes whelping pups do not hunt.  Instead, the male fox will bring food to the female.  In this case, the female may be killed if the prey brought by the male had ingested Diphacinone.  In addition, since Diphacinone does not kill immediately, it is not known if Diphacinone may be excreted in mammalian milk, thus exposing whelping young. 

Overall, the reported incident data indicates pathways are complete and include species mortalities under the current assessment.  Exposure assumptions are plausible and may result in mortality events.
Bibliography
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APPENDIX A
Risk Quotient Method and Levels of Concern

Risk characterization integrates the results of the exposure and ecotoxicity data to evaluate the likelihood of adverse ecological effects.  The means of this integration is the quotient method.  Risk quotients (RQs) are calculated by dividing exposure estimates (EECs) by acute and chronic ecotoxicity values.  

  RQ = EXPOSURE/TOXICITY

RQs are then compared to OPP's levels of concern (LOCs).  LOCs are used by OPP to determine potential risk to non-target organisms and identify the need to consider regulatory action to mitigate risk.  The criteria indicate that a pesticide used as directed has the potential to cause adverse effects on non-target organisms.  LOCs currently address the following risk presumption categories: 

      ·	acute risks - regulatory action may be warranted in addition to restricted use classification
      ·	acute restricted use - the potential for acute risk is high, but may be mitigated through restricted use classification
      ·	acute endangered species - endangered species may be adversely affected, and 
      ·	reproductive/chronic risk - the potential for chronic risk is high regulatory action may be warranted.  

EFED does not perform assessments for chronic risk to plants, acute or chronic risks to insects or chronic risk from granular/bait formulations to birds or mammals.

The ecotoxicity test values (measurement endpoints) used in the acute and reproductive/chronic RQs are derived from required studies.  Examples of ecotoxicity values derived from short-term laboratory studies that assess acute effects are: 

      ·	LC50 (fish and birds)
      ·	LD50 (birds and mammals)
      ·	EC50 (aquatic plants and aquatic invertebrates), and 
      ·	EC25 (terrestrial plants - nonlisted species)  
      ·	NOAEC or EC05 (terrestrial plants - listed species)

Examples of toxicity test effect levels derived from the results of longer-term laboratory studies that assess reproductive/chronic effects are: 

      ·	LOAEC (birds, fish, and aquatic invertebrates)
      ·	NOAEC (birds, fish and aquatic invertebrates)

Other toxicity values may be used when justified. 

Risk presumptions and the corresponding RQs and LOCs, are tabulated below.

Risk presumptions for terrestrial animals based on risk quotients (RQ) and levels of concern (LOC)

                               Risk Presumption

                                      RQ

                                      LOC

Birds:

  Acute Risk 

EEC[1]/LC50 or LD50/ft[2] or LD50/day[3]

                                      0.5

  Acute Restricted Use

EEC/LC50 or LD50/ft[2] or LD50/day (or LD50 < 50 mg/kg)

                                      0.2

  Acute Endangered
  Species

EEC/LC50 or LD50/ft[2] or LD50/day 

                                      0.1

  Chronic Risk

EEC/NOAEC

                                       1

Wild Mammals:

  Acute Risk 

EEC/LC50 or LD50/ft[2] or LD50/day	

                                      0.5

  Acute Restricted Use

EEC/LC50 or LD50/ft[2] or LD50/day (or LD50 < 50 mg/kg)

                                      0.2

  Acute Endangered
  Species

EEC/LC50 or LD50/ft[2] or LD50/day	

                                      0.1

  Chronic Risk 

EEC/NOAEC

                                       1
 [1]  Estimated Environmental Concentration (EEC, ppm) on avian and mammalian food items
 [2]  mg/ft[2]
 [3]  mg of toxicant consumed/day
  LD50 * wt. of bird
  LD50 * wt. of bird  

Risk presumptions for aquatic animals based on risk quotients (RQ) and levels of concern (LOC)

                               Risk Presumption

                                      RQ 

                                      LOC

  Acute Risk

EEC[1]/LC50 or EC50

                                      0.5

  Acute Restricted Use

EEC/LC50 or EC50

                                      0.1

  Acute Endangered Species

EEC/LC50 or EC50

                                     0.05

  Chronic Risk

EEC/NOAEC

                                       1
 [1]  EEC = (ppm or ppb) in water

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]/EC25

                                       1

  Acute Endangered Species

EEC/EC05 or NOAEC

                                       1

Aquatic Plants:

  Acute Risk

EEC[2]/EC50

                                       1

  Acute Endangered Species

EEC/EC05 or NOAEC 

                                       1
[1]  EEC = lbs ai/A 
[2]  EEC = (ppb/ppm) in water 

APPENDIX B
Listed Species Occurrence in KAPUT-D Use States for Selected Terrestrial Taxa:  Mammal, Bird, Amphibian, Reptile by Use Site 

Species Occurrence in Selected States and Selected Taxa
	No species were excluded
	All Medium Types Reported
	Mammal, Bird, Amphibian, Reptile
	CO, KS, MT, NE, NM, ND, OK, SD, TX, WY
	Colorado		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	Owl, Mexican Spotted	Strix occidentalis lucida	Threatened	Terrestrial	Yes
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	Mouse, Preble's Meadow Jumping	Zapus hudsonius preblei	Threatened	Terrestrial	Yes
	Wolf, Gray	Canis lupus	Endangered	Terrestrial	Yes

Kansas		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	
Montana		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	
Nebraska		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	
	  New Mexico		CH
	Amphibian
	Frog, Chiricahua Leopard	Rana chiricahuensis	Threatened	Freshwater, Terrestrial	No
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	Falcon, Northern Aplomado	Falco femoralis septentrionalis	Endangered	Terrestrial	No
	Owl, Mexican Spotted	Strix occidentalis lucida	Threatened	Terrestrial	Yes
	Mammal
Ferret, Black-Footed	Mustela nigripes	Endangered			Terrestrial			    No
Jaguar	Panthera onca	Endangered	Terrestrial	No
	Reptile
	Rattlesnake, New Mexican Ridge-nosed	Crotalus willardi obscurus	Threatened	Terrestrial	Yes
	 
	North Dakota		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	Mammal
	Wolf, Gray	Canis lupus	Endangered	Terrestrial	Yes
	
Oklahoma		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
		
	
	South Dakota		CH
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	Wolf, Gray	Canis lupus	Endangered	Terrestrial	Yes
		
Texas		CH
	Amphibian
Toad, Houston	Bufo houstonensis	Endangered	Terrestrial, Freshwater	Yes
	Bird
	Crane, Whooping	Grus americana	Endangered	Terrestrial, Freshwater	Yes
	Falcon, Northern Aplomado	Falco femoralis septentrionalis	Endangered	Terrestrial	No
	Owl, Mexican Spotted	Strix occidentalis lucida	Threatened	Terrestrial	Yes
	Prairie-chicken, Attwater's Greater	Tympanuchus cupido attwateri	Endangered	Terrestrial	No
	Warbler (=Wood), Golden-cheeked	Dendroica chrysoparia	Endangered	Terrestrial	No
	Mammal
  
	Jaguarundi, Gulf Coast	Herpailurus (=Felis) yagouaroundi 	Endangered	Terrestrial	No
	cacomitli
	Jaguarundi, Sinaloan	Herpailurus (=Felis) yagouaroundi 	Endangered	Terrestrial	No
	tolteca
	Ocelot	Leopardus (=Felis) pardalis	Endangered	Terrestrial	No
	Reptile
	Snake, Concho Water	Nerodia paucimaculata	Threatened	Freshwater, Terrestrial	Yes
	
	Wyoming		CH
	Amphibian
	Toad, Wyoming	Bufo baxteri (=hemiophrys)	Endangered	Freshwater, Terrestrial	No
	Mammal
	Ferret, Black-Footed	Mustela nigripes	Endangered	Terrestrial	No
	Mouse, Preble's Meadow Jumping	Zapus hudsonius preblei	Threatened	Terrestrial	Yes