# Risks of Diflubenzuron Use to Federally Threatened California Red-legged Frog ( Rana aurora draytonii )

Pesticide Effects Determination

Environmental Fate and Effects Division Office of Pesticide Programs Washington, D.C. 20460

October 19, 2009

### Primary Authors: Stephen Wente Fred Jenkins Lewis Brown

Secondary Review: Brian Anderson Jim Lin Ed Odenkirchen

Branch Chief, Environmental Risk Assessment Branch 1: Nancy AndrewsTable of Contents

1.0 Executive Summary .......................................................................................... 4 2.0 Problem Formulation ..................................................................................... 13 2.1 Purpose .............................................................................................................. 13 2.2 Scope................................................................................................................. 15 2.3 Previous Assessments ....................................................................................... 17 2.4 Stressor Source and Distribution ...................................................................... 18 2.4.1 Environmental Fate Assessment ................................................................... 18 2.4.2 Mechanism of Action .................................................................................... 21 2.4.3 Use Characterization ..................................................................................... 21 2.5 Assessed Species ............................................................................................... 26 2.5.1 Distribution ................................................................................................... 26 2.5.2 Reproduction ................................................................................................. 29 2.5.3 Diet ................................................................................................................ 29 2.5.4 Habitat ........................................................................................................... 30 2.6 Designated Critical Habitat ............................................................................... 31 2.7 Action Area ....................................................................................................... 33 2.8 Assessment Endpoints and Measures of Ecological Effect .............................. 34 2.8.1 Assessment Endpoints for the CRLF ............................................................ 35 2.8.2 Assessment Endpoints for Designated Critical Habitat ................................ 37 2.9 Conceptual Model ............................................................................................. 39 2.9.1 Risk Hypotheses ............................................................................................ 39 2.9.2 Diagram ......................................................................................................... 39 2.10 Analysis Plan .................................................................................................... 41 2.10.1 Measures to Evaluate the Risk Hypothesis and Conceptual Model.............. 42 3.0 Exposure Assessment ...................................................................................... 46 3.1. Label Application Rates and Intervals.............................................................. 46 3.2. Aquatic Exposure Assessment.......................................................................... 51 3.2.1. Modeling Approach ...................................................................................... 51 3.2.2. Model Inputs ................................................................................................. 52 3.2.3. Results ........................................................................................................... 53 3.2.4. Existing Monitoring Data ............................................................................. 57 3.3. Terrestrial Animal Exposure Assessment ......................................................... 57 3.5 Terrestrial Plant Exposure Assessment ............................................................. 60 4.0 Effects Assessment .......................................................................................... 60 4.1. Evaluation of Aquatic Ecotoxicity Studies ....................................................... 62 4.1.1. Toxicity to Freshwater Fish .......................................................................... 64 4.1.2. Toxicity to Freshwater Invertebrates ............................................................ 65 4.1.3. Toxicity to Aquatic Plants ............................................................................ 67 4.2. Toxicity of diflubenzuron to Terrestrial Organisms ......................................... 72 4.2.1. Toxicity to Birds ........................................................................................... 72 4.2.3. Toxicity to Mammals .................................................................................... 73 4.2.4. Toxicity to Terrestrial Invertebrates ............................................................. 74 4.2.5. Toxicity to Terrestrial Plants ........................................................................ 764.3. Use of Probit Slope Response Relation ship to Provide Information on the Endangered Species Levels of Concern ............................................................ 77 4.4. Incident Database Review ................................................................................ 78 5.0 Risk Characterization ..................................................................................... 79 5.1. Risk Estimation ................................................................................................. 79 5.1.1. Exposures in the Aquatic Habitat ................................................................. 79 5.1.2. Exposures in the Terrestrial Habitat ............................................................. 83 5.1.3. Primary Constituent Elements of Designated Critical Habitat ..................... 87 5.2. Risk Description ............................................................................................... 87 5.2.1. Direct Effects ................................................................................................ 91 5.2.2. Indirect Effects (via Re ductions in Prey Base) ............................................. 94 5.2.3. Indirect Effects (via Habitat Effects) ............................................................ 97 5.2.4. Modification to Designa ted Critical Habitat ................................................. 98 5.2.5. Spatial Extent of Potential Effects ................................................................ 99 6.0 Uncertainties .................................................................................................. 104 6.1 Exposure Assessment Uncertainties ............................................................... 104 6.1.1 Maximum Use Scenario .............................................................................. 104 6.1.2 Crops Not Grown in California (include if you had crops that are not grown in CA) .......................................................................................................... 104 6.1.3 Aquatic Exposure Modeling of Diflubenzuron........................................... 104 6.1.4 Usage Uncertainties .................................................................................... 106 6.1.5 Terrestrial Exposure Modeling of Diflubenzuron ....................................... 106 6.1.6 Spray Drift Modeling .................................................................................. 107 6.2 Effects Assessment Uncertainties ................................................................... 108 6.2.1 Age Class and Sensitivity of Effects Thresholds ........................................ 108 6.2.2 Use of Surrogate Species Effects Data ....................................................... 108 6.2.3 Sublethal Effects ......................................................................................... 109 6.2.4 Location of Wildlife Species ....................................................................... 109 7.0 Risk Conclusions ........................................................................................... 110 8.0 References ...................................................................................................... 113List of Tables

Table 1-1 Effects Determination Summa ry for Diflubenzuron Use and the CRLF ........... 7

Table 1-2. Effects Determination Summar y for Diflubenzuron Use and CRLF Critical Habitat Impact Analysis .................................................................................... 8 Table 1-3 Diflubenzuron Use-specifi c Direct Effects Determinations 1 for the CRLF ....... 9 Table 1-4 Diflubenzuron Use-specific Indirect Effects Determinations 1 Based on Effects to Aquatic Prey ............................................................................................... 10 Table 2-1 Summary of Diflubenzur on Environmental Fate Properties ............................ 19

Table 2-2 Diflubenzuron Uses Assessed for the CRLF .................................................... 22 Table 2-3 Summary of California Department of Pesticide Registration (CDPR) Pesticide Use Reporting (PUR) Data from 1999 to 2006 for Currently Registered Diflubenzuron Uses ........................................................................................ 25 Table 2-4. Assessment Endpoints and Measures of Ecological Effects .......................... 36 Table 2-5. Summary of Asse ssment Endpoints and Measures of Ecological Effect for Primary Constituent Elements of Designated Critical Habitat a ...................... 38
Table 3-1 Diflubenzuron Uses, Scenarios, and Application Information for the CRLF risk

assessment 1 ..................................................................................................... 48 Table 3-2 Summary of PRZM/EZAMS Envi ronmental Fate Data Used for Aquatic Exposure Inputs for Diflubenzuron Endangered Species Assessment for the CRLF 1 ............................................................................................................. 53 Table 3-3 Aquatic EECs ( μ g/L) for Diflubenzuron Uses in California ............................ 54 Table 3-4 Input Parameters for Foliar Applic ations Used to Derive Terrestrial EECs for Diflubenzuron with T-REX ............................................................................ 58 Table 3-5. Upper-bound Kene ga Nomogram EECs for Dietary- and Dose-based Exposures of the CRLF and its Prey to Diflubenzuron. ................................. 59 Table 3-6 EECs (ppm) for Indirect Effects to the Terrestrial-Phase CRLF via Effects to Terrestrial Invertebrate Prey Items ................................................................. 60 Table 4-1 Freshwater Aquatic To xicity Profile for Diflubenzuron .................................. 63 Table 4-2 Categories of Acute Toxicity for Fish and Aquatic Invertebrates .................... 63 Table 4-3. Diflubenzuron Effects Reporte d in a Littoral Enclosure Study (MRID 443862- 01). .................................................................................................................. 69

Table 4-4 Terrestrial Toxici ty Profile for Diflubenzuron ................................................. 72 Table 4-5 Categories of Acute Toxic ity for Avian and Mammalian Studies ................... 72 Table 4-6. Probit slopes used for taxonomi c groups evaluated in this assessment .......... 77 Table 5-1 Summary of Direct Effect RQ LOCs for the Aquatic-phase CRLF ................. 80Table 5-2 Summary of RQs Used to Estimate In direct Effects to the CRLF via Effects to Non-Vascular Aquatic Plants (diet of CRLF in tadpole li fe stage and habitat of aquatic-phase CRLF) ...................................................................................... 80 Table 5-3 Summary of Acute and Chronic RQs Us ed to Estimate Indirect Effects to the CRLF via Direct Effects on Aquatic Inve rtebrates as Dietary Food Items (prey of CRLF juveniles and adu lts in aquatic habitats) .......................................... 82 Table 5-4 Summary of RQs Used to Estimate In direct Effects to the CRLF via Effects to Vascular Aquatic Plants (hab itat of aquatic-phase CRLF) a ............................ 83 Table 5-5 Summary of RQs Used to Estimate Direct Effects to the Terrestrial-phase CRLF ............................................................................................................... 84 Table 5-6 Summary of RQs Used to Estimate Indirect Effects to the Terrestrial-phase CRLF via Direct Effects on Terrestrial Invertebrates as Dietary Food Items based on an LC50 of 234 ppm (30 μg per bee / 0.128 grams per bee) ........... 85 Table 5-7 Summary of Chronic RQs Used to Es timate Indirect Effects to the Terrestrialphase CRLF via Direct Effects on Sm all Mammals as Dietary Food Items .. 86

Table 5-8 Risk Estimation Summ ary for Diflubenzuron - Direct and Indirect Effects to CRLF ............................................................................................................... 88 Table 5-9 Risk Estimation Summary for Difl ubenzuron – PCEs of Designated Critical Habitat for the CRLF ...................................................................................... 89

Table 5-10. Results from T-HERP S for the Barnyard/Mushroom Use. .......................... 93 Table 5-11. Summary of AgDRIFT* Predic ted Terrestrial Spray Drift Distances ........ 102

Table 7-1. Effects Determination Summa ry for Diflubenzuron Use and the CRLF ...... 110

Table 7-2. Effects Determination Summar y for Diflubenzuron Use and CRLF Critical Habitat Impact Analysis ................................................................................ 111List of Figures

Figure 2-1. Diflubenzuron use in total pounds per square mile of agricultural land in county. ............................................................................................................. 24 Figure 2-2 Recovery Unit, Core Area, Critic al Habitat, and Occu rrence Designations for CRLF ............................................................................................................... 28 Figure 2-3 CRLF Reproductive Events by Month........................................................... 29 Figure 2-4 Conceptual Model for Pesticid e Effects on Terrestrial Phase of the CRLF .. 40 Figure 2-5. Conceptual Model for Pestic ide Effects on Aquatic Phase of the CRLF. .... 41 Figure 3-1. Identification of application date s based on analysis of the average pounds of diflubenzuron applied in California to citrus via ground application methods based on CDPR PUR data (1990-2007).......................................................... 52 Figure 3-2. Variation in di flubenzuron EECs (peak, 21-day, and 60-day) as function of application date (first application date for scenarios with multiple applications). ................................................................................................... 56 Figure 3-3. Variation in difl ubenzuron EECs (peak, 21-day, and 60-day) as function of application date (first application date for scenarios with multiple applications) compared to variation in the amount of diflubenzuron applied (lbs. ai/day) per day in California for fruit use. .............................................. 56

Appendices

Appendix A Summary of Registra nt Submitted Ecotoxicity Data Appendix B Multi-ai Product Analysis Appendix C RQ Method and LOC Description Appendix D Exposure Data Appendix E Human Health Assessment on Diflubenzuron Appendix F Manure Use Assessment Method Appendix G1 Accepted ECOTOX Da ta Table (sorted by effect) Appendix G2 Diflubenzuron Bibliography of Acceptable Papers Appendix G3 Diflubenzuron Bibliography of Rejected Papers Appendix G4 Papers Rejected by Ecotox Becau se they were Tested Using Mixtures Appendix H Bibliography of all Registrant Submitted Data Appendix I Description of Ecotox Rejection Codes Appendix J Residential Use Assessment Method Appendix K Right-of-Way Use Assessment Method

Attachment I. Status and Life Hist ory of the California Red-legged Frog Attachment II. Baseline Status and Cumu lative Effects for the California Red-legged Frog

### 1.0 Executive Summary

1.1. Purpose

The purpose of this assessment is to evaluate potential direct and i ndirect effects on the California red-legged frog (Rana aurora draytonii ) (CRLF) arising from Federal Insecticide, Fungicide, Roden ticide, Act (FIFRA) regulator y actions regarding use of diflubenzuron on agricultural and non-agricul tural sites. In ad dition, this assessment evaluates whether these actions can be expect ed to result in modifi cation of the species’ designated critical habitat. This assessme nt was completed in accordance with the U.S. Fish and Wildlife Service (U.S. FWS) and National Marine Fisheries Service (NMFS) Endangered Species Consultation Handbook (U.S. FWS/NMFS 1998) and procedures outlined in the Agency’s Overview Document (U.S. EPA 2004).

The CRLF was listed as a threatened speci es by U.S. FWS in 1996. The species is endemic to California and Baja California (Mex ico) and inhabits both coastal and interior mountain ranges. A total of 243 streams or drainages are believed to be currently occupied by the species, with the greatest numbers in Monterey, San Luis Obispo, and Santa Barbara counties (U.S. FWS 1996) in California.

1.2. Chemical Assessed

Diflubenzuron is a benzoylphenylurea insecticide that kills immature insects by disrupting the molting process. Formulati on types registered include liquid, water dispersible granules, wettable powder, effervescent tablets (for ornamental pond treatment), and termite bait (in bait stations).

1.3. Use Characterization

Labeled uses of diflubenzuron include citrus , cotton, forestry, mushrooms, ornamentals, pastures, soybeans, standing water, sewage sy stems, termite bait stations, and wide-area general outdoor treatment sites. All of these uses are considered as part of the federal action evaluated in this assessment with the ex ception of termite bait stations (in which a solidified form of the pesticide is placed in an enclosed bait station to minimize loss to the environment) and soybeans, which are not grown in California.

1.4. Environmental Fate and Exposure Summary

Diflubenzuron appears to be relatively non- persistent and immobile under normal use conditions. Diflubenzuron is re latively stable to hydrolys is and photolysis. The major route of dissipation appears to be biotic processes (half-life of approximately 2 days for aerobic soil metabolism). Available data indi cate that it is unlik ely that diflubenzuron will contaminate ground water or appreciably volatilize and be transported through longrange atmospheric transport processes. However, a degradate of diflubenzuron, 4- chlorophenylurea (CPU), has a much greater potential to l each through soil. Spray drift

4and transport with eroded soil in runoff ar e thought to be the main mechanisms of transport from the site of application.

Since CRLFs exist within aquatic and terrestria l habitats, exposure of the CRLF, its prey and its habitats to diflubenzuron are assessed separately for the two habitats. Tier-II aquatic exposure models are used to estim ate high-end exposures of diflubenzuron in aquatic habitats resulting from runoff and sp ray drift from different uses. Peak modelestimated environmental concentrations resul ting from different diflubenzuron uses range from 0.01 to 113 μg/L. Diflubenzuron is typically not measured by surface and groundwater monitoring programs, therefore, no monitoring data are available for comparison with the model estimated diflubenzuron concentrations.

1.4. Toxicity Summary

Diflubenzuron is very highly toxic to aquatic invertebrates. The most sensitive LC 50 identified for diflubenzuron was 0.0028 μg/L (E cotox no. 16591). Toxicity is influenced by species and life-stage of the test or ganism. The most sensitive NOAEC was 0.00025 μg/L (Ecotox No. 9397). Diflubenzuron is less toxic to fish, with a 96-hr LC 50 of 129,000 μg/L (MRID 00056150) and a reprodu ction NOAEC of 110 μg/L (MRID 00099755). Diflubenzuron was not toxic to aq uatic plants at up to 200 μg/L (MRID 45252205 and 42940103).

Diflubenzuron is also practically non-toxic to birds on an acute basis. The most sensitive study available produced an LD 50 value of 3763 mg/kg-bw in the Red-Winged Blackbird (MRID 00038614).

Using the technical grade active ingredient (TGAI), diflubenzuron is categorized as practically non-toxic to avia n species on a subacute dietary toxicity basis based on an LC 50 value of >4640 ppm for the bobwhite quail and mallard duck (MRID 00039080). A 1 % Granular formulation was also categorized as practically non-toxic to bobwhite quail and mallard ducks based on an LC 50 value of >20,000 ppm (MRID 00060381). The most sensitive NOAEC from a reliable re production study was 500 mg/kg-diet (MRID 4166800102). The NOAEC of 500 mg/kg-food wa s based on effects on eggshell thickness in mallard ducks and reduced egg production in bobwhite quail at 1000 mg/kgdiet.

The available mammalian acute toxicity da ta demonstrate that diflubenzuron is practically nontoxic to mammals (LD 50 > 5000 mg/kg; MRID 00157103). In a 2- generation reproduction study no effects on re productive performance were observed at any dose level in F0 or F1 males or fema les. Litter and mean pup weights decreased slightly from birth to 21 days postpartum in F1 offspring at 2500 mg/kg/day. The NOEL for reproductive performance in parental adults is 2500 mg/kg/day. The NOEL for developmental toxicity in progeny is 250 mg /kg/day and the LOAEL is 2500 mg/kg/day, based on decreased body weights in F1 pups fr om birth to 21 days postpartum. (MRID 43578301). A NOAEL of 250 mg/kg-bw is used in this assessment.

5Currently, there are no guideline terrestrial plan t toxicity data for di flubenzuron for use in this assessment. However, given the mode of toxicity of diflube nzuron as an insect growth regulator, the long history of use on numerous agricultural commodities without incidence of effects to terrestri al plants that are clearly a ssociated with diflubenzuron, and the available efficacy data that reported mini mal phytotoxic effects, the available data do not suggest that terrestrial plan ts are likely to be impacted to an extent that indirect effects will result to species that depe nd on them for survival or reproduction.

This assessment was based on potential risks from diflubenzuron. Several degradates have been identified that are of equal or greater to xicity than the parent chemical. These degradates were considered in this assessm ent; however, analysis of potential exposure to and toxicity of these degradat es suggests that potential risks to these degradates is negligible relative to risk s posed by parent diflubenzuron.

1.5. Effects Determination

The effects determination assessment endpoints for the CRLF include direct toxic effects on the survival, reproduction, and growth of th e CRLF itself, as well as indirect effects, such as reduction of the prey base or modifi cation of its habitat. Direct effects to the CRLF in the aquatic habitat are based on toxic ity information for freshwater fish, which are used as a surrogate for aquatic-phase amphibians. In the terrestrial habitat, direct effects are based on toxicity information for birds, which are used as a surrogate for terrestrial-phase amphibians. Given that th e CRLF’s prey items and designated critical habitat requirements in the aquatic habitat ar e dependant on the availability of freshwater aquatic invertebrates and aquatic plants, toxi city information for these taxonomic groups is also discussed. In the terrestrial habitat, indirect effects due to depletion of prey are assessed by considering effects to terrestrial in sects, small terrestrial mammals, and frogs. Indirect effects due to modifi cation of the terrestrial habitat are characterized by available data for terrestrial monocots and dicots. Ho wever, given the lack of guideline toxicity studies on terrestrial plants, additional lines of evidence we re used to determine if terrestrial plants may be impacted to an extent that could result in indirect effects to the CRLF.

Risk quotients (RQs) are derived as quantita tive estimates of potential high-end risk. Acute and chronic RQs are compared to the Agency’s levels of concern (LOCs) to identify instances where diflubenzuron use w ithin the action area ha s the potential to adversely affect the CRLF and its designated critical habitat via direct toxicity or indirectly based on direct effects to its food supply ( i.e. , freshwater invertebrates, algae, fish, frogs, terrestrial invertebra tes, and mammals) or habitat ( i.e. , aquatic plants and terrestrial upland and riparian vegetation). Wh en RQs for each particular type of effect are below LOCs, the pesticide is determined to have “no effect” on the CRLF. Where RQs exceed LOCs, a potential to cause adve rse effects is identified, leading to a conclusion of “may affect.” If a determin ation is made that use of diflubenzuron use within the action area “may affect” the CRLF and its designated critical habitat, additional information is consid ered to refine the potential for exposure and effects, and the best available information is used to distinguish those acti ons that “may affect, but are

6not likely to adversely affect” (NLAA) from those actions that are “likely to adversely affect” (LAA) the CRLF. Similarly for critical habitat, additional information is considered to refine the potential for exposure and effects to distingu ish those actions that do or do not result in modificati on of its critical habitat.

Based on the best available information, the Ag ency makes a Likely to Adversely Affect determination for the CRLF from the use of diflubenzuron. Additionally, the Agency has determined that there is a potential for modi fication of CRLF designa ted critical habitat from the use of the chemical. A summary of the risk conclusions and effects determinations for the CRLF and its critical habitat is presented in Table 1-1 and 1-2.

Table 1-1 Effects Determina tion Summary for Diflube nzuron Use and the CRLF

Assessment

Endpoint Effects Determination 1 Basis for Determination

Potential for Direct Effects Aquatic-phase (Eggs, Larvae, and Adults) :

There was an LOC exceedance for ri sk to fish (CRLF surrogate) from chronic exposures to diflubenzuron posed by the rice use. However, the risk was determined to be discountable because effects were considered to be unlikely to occur for reasons discussed in Section 5.2. LOCs were not exceeded for any other use. The effects determination for direct effects to aquatic phase CRLFs was NE for all uses except rice and NLAA for rice. Terrestrial-phase (Juveniles and Adults) : Direct effects to CRLFs could occur from diflubenzuron’s use on barn yards based on acute and chronic LOC exceedances for birds and refined analysis for frogs. Direct effects to CRLFs are not expected to occur for any other use. The effects determination is LAA for barnyard/mushroom use and NE for all other uses. Potential for Indirect Effects Aquatic prey items, aquatic habitat, cover and/or primary

p roductivit y Aquatic prey of the CRLF may be impacted based on potential impacts to aquatic invertebrate for all uses. Several lines of evidence support the conclusion that aquatic invertebrates may be impacted by labeled diflubenzuron uses including laboratory studies and field studies. The effects determination is LAA for all uses based on potential impacts to aquatic invertebrates. Survival, growth, and/or reproduction of CRLF individuals LAA, all uses

Terrestrial prey items, riparian habitat

Terrestrial prey items of the CRLF are expected to be impacted based on potential impacts to terrest rial invertebrates. The effects determination is LAA for all uses based on potential impacts to terrestrial invertebrates.

1 No effect (NE); May affect, but not likely to adversely affect (NLAA) ; May affect, likely to adversely affect (LAA)

7Table 1-2. Effects Determination Summary for Diflubenzuron Use and CRLF Critical Habitat Impact Analysis

Assessment

Endpoint Effects Determination 1 Basis for Determination

Modification of aquatic-phase PCE There are LOC exceedances for ri sk to terrestrial and aquatic invertebrate prey of the CRLF.

Modification of terrestrial-phase PCE HM

There are LOC exceedances for ri sk to terrestrial and aquatic invertebrate prey of the CRLF.

1 Habitat Modification or No effect (NE)

Use-specific determinations for direct and in direct effects to the CRLF are provided in Table 1-3, 1-4, and 1-5. Further information on the results of the effects determination is included as part of the Risk Description in Section 5.2.

8Table 1-3 Diflubenzuron Use-specific Direct Effects Determinations 1 for the CRLF Aquatic Habitat Terrestrial Habitat Use(s) Acute Chronic Acute Chronic Barnyard/Mushroom NE NE LAA LAA Spray on manure, Manure to field NE NE NE NE Beech Nut NE NE NE NE Brassica (head and stem) vegetables NE NE NE NE Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines NE NE NE NE Broccoli raab, cabbage - Chinese, collards, and kale NE NE NE NE Cotton (unspecified) NE NE NE NE Christmas tree plantations and forest trees (softwoods - conifers) NE NE NE NE Apricot, nectarine, peach, pe ar, plum, prune, and stone fruits NE NE NE NE Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (hazelnut), hickory nut, macada mia nut (bushnut), pecan, tree nuts, and walnut (English/black) NE NE NE NE

Barley, canola\rape, oats, tritical e, and wheat NE NE NE NE Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings NE NE NE NE

Grass forage/fodder/hay, pastures, and rangeland NE NE NE NE Pistachio NE NE NE NE Household/domestic dwellings outdoor premises NE NE NE NE Rice/Ornamental Pond NE NLAA NE NE Agricultural rights-of-way/fencerows/hedgerows,fencerows/hedgerows, and nonagricultural rights-ofway/fencerows/hedgerows NE NE NE NE

Artichoke, mustard, peanuts (unspecified), and pepper NE NE NE NE Commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) NE NE NE NE

Butternut NE NE NE NE Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils NE NE NE NE
Ornamental sod farm (turf) and re creational areas NE NE NE NE Turnip (greens) NE NE NE NE

1 NE = No effect; NLAA = May affect, not likely to adversely affect; LAA = Likely to adversely affect

9Table 1-4 Diflubenzuron Use-specific Indirect Effects Determinations 1 Based on Eff ects to Aquatic Prey

Aquatic Invertebrates Aquatic-phase frogs and fish Use(s) Algae Acute Chronic Acute Chronic Barnyard/Mushroom NE LAA LAA NE NE Spray on manure, Manure to field NE LAA LAA NE NE Beech Nut NE LAA LAA NE NE Brassica (head and stem) vegetables NE LAA LAA NE NE Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines NE LAA LAA NE NE Broccoli raab, cabbage - Chinese, collards, and kale NE LAA LAA NE NE Cotton (unspecified) NE LAA LAA NE NE Christmas tree plantations and forest trees (softwoods - conifers) NE LAA LAA NE NE Apricot, nectarine, p each, pear, plum, prune, and stone fruits NE LAA LAA NE NE

Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (h azelnut), hickory nut, macadamia nut ( bushnut), pecan, tree nuts, and walnut (English/black) NE LAA LAA NE NE

Barley, canola\rape, oats, triticale, and wheat NE LAA LAA NE NE Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings NE LAA LAA NE NE

Grass forage/fodder/hay, pastures, and rangeland NE LAA LAA NE NE

Pistachio NE LAA LAA NE NE Household/domestic dwellings outdoor premises NE LAA LAA NE NE

Rice/Ornamental Pond NE LAA LAA NE NLAAAgricultural rights-ofway/fencerows/hedgerows, fencerows/hedgerows, and nonagricultural rights-of-way/fencerows/hedgerows NE LAA LAA NE NE

Artichoke, mustard, peanuts (unspecified), and pepper NE LAA LAA NE NE

Commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) NE LAA LAA NE NE

Butternut NE LAA LAA NE NE Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils NE LAA LAA NE NE
Ornamental sod farm (turf) and recreational

areas NE LAA LAA NE NE Turnip (greens) NE LAA LAA NE NE

1 NE = No effect; NLAA = May affect, not likely to adversely affect; LAA = Likely to adversely affect

10Table 1-5. Diflubenzuron Use-speci fic Indirect Effects Determinations 1 Based on Effects to Terrestrial Prey Terrestrial-phase frogs Small Mammals Use(s) Terrestrial Inverts. (Acute) Acute Chronic Acute Chronic Barnyard/Mushroom LAA LAA LAA NE NE Spray on manure, Manure to field LAA NE NE NE NE Beech Nut LAA NE NE NE NE Brassica (head and stem) vegetables LAA NE NE NE NE Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines LAA NE NE NE NE Broccoli raab, cabbage - Chinese, collards, and kale LAA NE NE NE NE Cotton (unspecified) LAA NE NE NE NE Christmas tree plantations and forest trees (softwoods - conifers) LAA NE NE NE NE Apricot, nectarine, p each, pear, plum, prune, and stone fruits LAA NE NE NE NE

Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (h azelnut), hickory nut, macadamia nut ( bushnut), pecan, tree nuts, and walnut (English/black) LAA NE NE NE NE

Barley, canola\rape, oats, triticale, and wheat LAA NE NE NE NE Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings LAA NE NE NE NE

Grass forage/fodder/hay, pastures, and rangeland LAA NE NE NE NE

Pistachio LAA NE NE NE NE Household/domestic dwellings outdoor premises LAA NE NE NE NE

Rice/Ornamental Pond LAA NE NE NE NEAgricultural rights-ofway/fencerows/hedgerows, fencerows/hedgerows, and nonagricultural rights-of-way/fencerows/hedgerows LAA NE NE NE NE

Artichoke, mustard, peanuts (unspecified), and
pepper LAA NE NE NE NE

Commercial/institutional/industrial
p remises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalk s), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) LAA NE NE NE NE

Butternut LAA NE NE NE NE Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils LAA NE NE NE NE
Ornamental sod farm (turf) and recreational

areas LAA NE NE NE NE Turnip (greens) LAA NE NE NE NE

1 NE = No effect; NLAA = May affect, not likely to adversely affect; LAA = Likely to adversely affect

11Based on the conclusions of this assessment, a formal consultation with the U. S. Fish and Wildlife Service under Sect ion 7 of the Endangered Specie s Act should be initiated. When evaluating the significance of this ri sk assessment’s direct /indirect and adverse habitat modification effects determinations , it is important to note that pesticide exposures and predicted risks to the species and its resources ( i.e. , food and habitat) are not expected to be uniform across the action ar ea. In fact, given the assumptions of drift and downstream transport ( i.e. , attenuation with distance) , pesticide exposure and associated risks to the species and its resour ces are expected to decrease with increasing distance away from the treated field or site of application. Evaluation of the implication of this non-uniform distribution of risk to the species would re quire information and assessment techniques that are not currently avai lable. Examples of such information and methodology required for this type of an alysis would include the following:

• Enhanced information on the density and distribution of CRLF life stages within specific recovery units and/or designated critical habitat within the action area. This information would allow for quantitative extrapolation of the present risk assessment’s pred ictions of individual effects to the proportion of the population extant with in geographical areas where those effects are predicted. Furthermore, such population information would allow for a more comprehensive eval uation of the significance of potential resource impairment to individuals of the species.

• Quantitative information on prey base requirements for individual aquaticand terrestrial-phase frogs. Wh ile existing information provides a preliminary picture of the types of food sources util ized by the frog, it does not establish minimal requirements to sustain healthy individuals at varying life stages. Such informa tion could be used to establish biologically relevant thresholds of ef fects on the prey base, and ultimately establish geographical limits to those effects. This information could be used together with the density data discussed above to characterize the likelihood of adverse effects to individuals.

• Information on population responses of prey base organisms to the pesticide. Currently, methodologies are limited to predicting exposures and likely levels of direct mortalit y, growth or reproductive impairment immediately following exposure to the pesticide. The degree to which repeated exposure events and the inherent demographic characteristics of the prey population play into the ex tent to which prey resources may recover is not predictable. An e nhanced understanding of long-term prey responses to pesticide exposure would allow for a more refined determination of the magnitude and duration of resource impairment, and together with the information descri bed above, a more complete prediction of effects to individual frogs and potential modificati on to critical habitat.

12

### 2.0 Problem Formulation

Problem formulation provides a strategic framework for the risk assessment. By identifying the important components of the problem, it focuses the assessment on the most relevant life history st ages, habitat components, chemical properties, exposure routes, and endpoints. The structure of th is risk assessment is based on guidance contained in U.S. Environmental Protecti on Agency's (EPA’s) Guidance for Ecological Risk Assessment (U.S. EPA 1998), the Se rvices’ Endangered Species Consultation Handbook (U.S. FWS/NMFS 1998) and is consis tent with procedures and methodology outlined in the Overview Document (U.S. EP A 2004) and reviewed by the U.S. Fish and Wildlife Service and National Marine Fisheries Service (U.S. FWS/NMFS 2004).

2.1 Purpose

The purpose of this endangered species assessme nt is to evaluate potential direct and indirect effects on individuals of the fede rally threatened California red-legged frog ( Rana aurora draytonii ) (CRLF) arising from FIFRA regul atory actions regarding use of diflubenzuron on agricultural uses (nuts, co tton, rice, row crops, citrus, fruits, etc.), forestry, nursery, residential, right-of-ways, and turf uses. In addition, this assessment evaluates whether use on these sites is expect ed to result in modification of the species’ designated critical habitat. This ecological risk assessment has been prepared consistent with a settlement agreement in the case Cent er for Biological Dive rsity (CBD) vs. EPA et al. (Case No. 02-1580-JSW(JL) settlement entere d in Federal District Court for the Northern District of California on October 20, 2006.

In this assessment, direct a nd indirect effects to the CRLF and potential modification to its designated critical habitat are evaluated in accordance w ith the methods described in the Agency’s Overview Document (U.S. EP A 2004). Screening level methods include use of standard models such as PRZM-EXAM S, T-REX, and AgDRIFT, all of which are described at length in the Ov erview Document. Additional refinements were made to PRZM/EXAMS scenarios to account for heter ogeneous land cover within treatment areas in order to more accurately model reside ntial and right-of-way uses. Use of such information is consistent with the met hodology described in the Overview Document (U.S. EPA 2004), which specifi es that “the assessment process may, on a case-by-case basis, incorporate additional methods, models, and lines of evidence that EPA finds technically appropriate for risk management objectives” (Section V, page 31 of U.S. EPA 2004).

In accordance with the Overview Document, provisions of the ESA, and the Services’ Endangered Species Consultation Handbook, the a ssessment of effects associated with registrations of diflubenzuron is based on an action area. The action area is the area directly or indirectly affect ed by the federal action, as indi cated by the exceedance of the Agency’s Levels of Concern (LOCs). It is acknowledged that the action area for a national-level FIFRA regulatory decision asso ciated with a use of diflubenzuron may potentially involve numerous areas throughout the United States and its Territories. However, for the purposes of this assessment, attention will be focused on relevant

13sections of the action area including those ge ographic areas associated with locations of the CRLF and its designated critical habitat with in the state of California. As part of the “effects determination,” one of the following three conclusions will be reached regarding the potential use of diflubenzuron on the CRLF in accordance with current labels:

• “No effect”;

• “May affect, but not likely to adversely affect”; or

• “May affect and likely to adversely affect”.

Designated critical habitat identifies specific areas that have the phys ical and biological features, (known as primary constituent elem ents or PCEs) essential to the conservation of the listed species. The PCEs for CRLFs ar e aquatic and upland areas where suitable breeding and non-breeding aquatic habitat is located, interspe rsed with upland foraging and dispersal habitat.

If the results of initial scr eening-level assessment methods show no direct or indirect effects (no LOC exceedances) upon individual CRL Fs or upon the PCEs of the species’ designated critical habitat, a “no effect” determination is made for use of diflubenzuron as it relates to this species and its designated cr itical habitat. If, how ever, potential direct or indirect effects to individual CRLFs are an ticipated or effects ma y impact the PCEs of the CRLF’s designated critical habitat, a pre liminary “may affect” determination is made for the FIFRA regulatory ac tion regarding diflubenzuron.

If a determination is made that use of difl ubenzuron within the acti on area(s) associated with the CRLF “may affect” this species or its designated critical habitat, additional information is considered to refine the poten tial for exposure and fo r effects to the CRLF and other taxonomic groups upon which these species depend ( e.g. , aquatic and terrestrial vertebrates and invertebrates, aquatic plan ts, riparian vegetation, etc.). Additional information, including spatial analysis (to de termine the geographical proximity of CRLF habitat and diflubenzuron use sites) and further evaluation of the potential impact of diflubenzuron on the PCEs is also used to determine whether modification of designated critical habitat may occur. Based on the re fined information, the Agency uses the best available information to disti nguish those actions that “may affect, but are not likely to adversely affect” from those actions that “may affect and are likely to adversely affect” the CRLF or the PCEs of its designated critical habitat. This information is presented as part of the Risk Characterization in Section 5 of this document.

The Agency believes that the analysis of di rect and indirect effects to listed species provides the basis for an analys is of potential effects on the designated critical habitat. Because diflubenzuron is expected to directly impact living organisms within the action area (defined in Section 2.7 ), critical habitat analysis for diflubenzuron is limited in a practical sense to those PCEs of critical habitat that are biological or that can be reasonably linked to biological ly mediated processes ( i.e. , the biological resource requirements for the listed species associated with the critical habitat or important physical aspects of the habitat that may be reasonably influenced through biological processes). Activities that ma y modify critical habitat are th ose that alter the PCEs and

14appreciably diminish the value of the habitat. Evaluation of actions related to use of diflubenzuron that may alter the PCEs of the CR LF’s critical habitat form the basis of the critical habitat impact analysis. Actions that may affect the CRLF’s designated critical habitat have been identifie d by the Services and are di scussed further in Section 2.6

.

2.2 Scope

Diflubenzuron is an insecticide that is registered for both agricultural and nonagricultural uses in Californ ia and can be applied through ground, aerial, and airblast methods.

The end result of the EPA pesticide registration process ( i.e. , the FIFRA regulatory action) is an approved product label. The la bel is a legal document that stipulates how and where a given pesticide may be used. Product labels (also known as end-use labels) describe the formulation type ( e.g. , liquid or granular), accepta ble methods of application, approved use sites, and any rest rictions on how applications may be conducted. Thus, the use or potential use of diflubenzuron in accordance with the approved product labels for California is “the action” relevant to this ecol ogical risk assessment.

Although current registrations of diflubenzuron allow for use nationwide, this ecological risk assessment and effects determination addresses currently registered uses of diflubenzuron in portions of the action area that are reasonably assumed to be biologically relevant to the CRLF an d its designated critical habitat.

There are two reasons for considering the acti on area of diflubenzuron use to include the entire state of California. First, because of the large variety of label uses for diflubenzuron in the state of California, it would be difficult to exclude any portions of this state from potential for diflubenzuron us e. Second, a degradate of diflubenzon is a suspected carcinogen and is genotoxic. Becau se there is no concentration threshold defined that would result in no genotoxic effect s to exposed organisms, it is not possible to define a threshold below which effects can occur. Therefore, the action area is assumed to be the entire state of California. Further discussion of the action area for the CRLF and its critical habitat is provided in Section 2.7

.

2.2.1. Chemicals Assessed

This assessment evaluates the potential for diflubenzuron to adversely affect the CRLF. Several degradates of diflubenzuron have b een identified and tested for toxicity. The available toxicity data are summarized belo w and are further described in Section 4 and Appendix A.

Degradates of diflubenzuron include 2,6-diflubenzoic acid (DFBA), 4-chlorophenylurea (CPU), 4-chloroaniline (PCA), and 2,6-diflubenzamide (DFBAM). The available data suggest that the degradates are several orde rs of magnitude less toxic than diflubenzuron to invertebrates. For example, LC 50 s in midge for PCA and DFBA are 43 mg/L and >100 mg/L, respectively, compared with an LC 50 of 0.07 mg/L for parent chemical in midge.

15Lower toxicity to invertebrates of degradates is consistent with the specific mode of action of diflubenzuron as a chitin inhibitor.

Several degradates have been shown to be of similar toxicity to fish compared with parent difbluenzuron. In pa rticular, PCA has been shown to be more toxic than diflubenzuron to fish with LC 50 values ranging from 2 mg/L to 23 mg/L. DFBA and PCPU appear to have similar toxicity rela tive to parent diflub enzuron with 96-hr LC 50 values of approximately 70 mg/L to >100 mg/L in fish. The most sensitive LC 50 in fish was 127 mg/L for diflubenzuron.

Although PCA has been shown to be more toxic to fish, it has not formed more than 10% of parent compound in available degradation studies. Potential risks resulting from exposure to PCA or other degr adates were not quantified. It was determined that quantifying risks from the degradates woul d not affect risk conclusions because comparing EECs for parent diflubenzuron (whi ch would represent a highly conservative exposure estimate for any of the degradates) with the lowest toxi city value across all degradates still results in no LOC exceedances for fish. Therefore, degradates were not further considered.

One diflubenzuron formulated product toxicity study provided a lower LC 50 than values from studies on the TGAI. The lowest LC 50 for formulated products was 57 mg/L (see Appendix A). EFED evaluates potential risk s from formulated produc ts resulting from drift only exposure. The highest drift fraction estimated to be deposited into water bodies was approximately 4% from aerial spray. The highest application rate for diflubenzuron is 8.5 lbs a.i./Acre (b arnyard/mushroom use). The resulting drift only EEC is 0.003 mg a.i./L assuming a volume of 20,000,000 L in th e receiving water. Based on an LC 50 of 57 mg/L, the resulting RQ would be orders of magnitude below concern levels (approximately 10 -5 ). Therefore, toxicity values from formulated products were not further considered as part of this assessment.

2.2.2. Evaluation of Mixtures

The Agency does not routinely include, in its risk assessments, an evaluation of mixtures of active ingredients, either those mixtur es of multiple active ingredients in product formulations or those in the applicator’s ta nk. In the case of the product formulations of active ingredients (that is, a registered product containing mo re than one active ingredient), each active ingredient is subj ect to an individual risk assessment for regulatory decision regarding the active ingredient on a particular use site. If effects data are available for a formulated product contai ning more than one active ingredient, they may be used qualitatively or quantitatively in accordance with the Agency’s Overview Document and the Services’ Evalua tion Memorandum (U.S. EPA 2004; U.S. FWS/NMFS 2004).

Diflubenzuron has one registered product that contains multiple active ingredients (diflubenzuron and permethrin). Analysis of the available open literature and acute oral

16mammalian LD 50 data for the diflubenzuron and pe rmethrin products relative to the single active ingredient is provided in Appendix B. There are no product LD 50 values, with associated 95% Confidence Interv als (CIs) available for diflubenzuron.

As discussed in USEPA (2004) a quantitati ve component-based evaluation of mixture toxicity requires data of appr opriate quality for each compon ent of a mixture. In this mixture evaluation an LD 50 with associated 95% CI is needed for the formulated product. The same quality of data is al so required for each component of the mixture. Given that the single formulated product for diflubenzur on does not have a 95% CI associated with the oral LD 50 value, it is not possible to undertake a quantitative or qua litative analysis for potential interactive e ffects (See appendix B). Ho wever, because the active ingredients are not expected to have similar mechanisms of action, metabolites, or toxicokinetic behavior, it is reasonable to conclude that an assu mption of dose-addition would be inappropriate. Consequently, an assessment based on the toxicity of diflubenzuron is the only reas onable approach that employs th e available data to address the potential acute risks of the formulated pr oducts. Studies on mixt ures located in the open literature are incl uded in Appendix B.

2.3 Previous Assessments

A large number of risk assessments have been performed on diflubenzuron by EFED. Two recent, comprehensive assessments are described below.

The Reregistration Eligibility Decision (RED) Science Chapter (6/30/95) identified risks to aquatic invertebrates as the major envir onmental threat of diflubenzuron. Acute and chronic levels of concern (LOCs) were exc eeded for both freshwater and estuarine and marine aquatic invertebrates, for the use of diflubenzuron on cotton, citrus, forest trees and forest plantings.

In an assessment entitled “Section 3 Registration Request for Uses of Diflubenzuron on Pears, Stone Fruits, Tree Nuts, Peppers , and Pasture Grass (Chemical # 108201, DP Barcode D279732, D279735, D278770)” from 2/5/02, EFED identified acute concerns for freshwater invertebrates, but not for fish, birds, or mammals for the uses assessed. The document indicates concern for sediment dwelling aquatic organisms due to the expectation of diflubenzuron accumulation in se diments, but did not quantify this risk due to the lack of chronic t oxicity data for these organism s. This document developed the critical life stage hypothesis for diflubenzur on effects which is discussed in Section 4.1.2 of this document.

A major difference between this assessment a nd the previous assessments concerns the inclusion of open literature to xicity values in the current assessment. This assessment is the first to include open liter ature toxicity data from the Ecotox database (See Appendix G). Inclusion of this data resulted in much lower acute and chronic freshwater invertebrate endpoints, which result in much greater risk estimates.

172.4 Stressor Source and Distribution

2.4.1 Environmental Fate Assessment Diflubenzuron (DFB) is a member of a la rger group of insecticides known as benzoylphenylureas. Based on acceptable and supplemental registrant-submitted data, diflubenzuron appears to be relatively non-persistent a nd relatively immobile under normal use conditions. The major route of di ssipation for diflubenzuron appears to be biotic mediated processes (t 1⁄2 ≈ 2-14 days for aerobic soil metabolism). Anaerobic aquatic metabolism was reported to be slower (t1⁄2 = 34 days). Aerobic aqua tic metabolism halflife in total water sediment system was 26 da ys. Other laboratory data indicate that diflubenzuron is stab le to hydrolysis (t1⁄2 ≈ 30-433 days for pHs 5-9) and photolysis (t1⁄2 = 80 days for aquatic; t1⁄2 for control < light e xposed for soil) and is relative immobile in soil (Rf values = 0.01, 0.07, 0.14, and 0.34 for silt y clay loam, clay loam, and two sand loam soils, respectively). Supplemental and ac ceptable field data (including forestry dissipation data) confirm the la boratory data with reported half-lives of 5.8 to 60 days, and diflubenzuron detectable only in the 0-15 cm soil depth segments. However, calculated half-lives for CA and OR orchard applicati ons were higher (t1⁄2 = 68.2-78days). Diflubenzuron has not been detected in well monitoring data (National SummaryPesticides in Ground Water Data base-A Compilation of Monitoring Studies: 1971- 1991), as well.

Under aerobic conditions diflubenzuron appears to degrade to 4-chlorophenyl urea (CPU) which reached a maximum concentration of 37% of applied at 14 days post-treatment. The other major degradate, CO 2 , was reported to reach a maximum concentration of 26.3% of applied by day 21 post-treatment. Three minor degradates, 2,6-difluorobenzoic acid, 2,6-difluorobenzamide, and p-chloroaniline, each reaching a maximum concentration of <10% of applied, were identified in the aerobic soil study. These metabolites were detectable in an anaer obic metabolism study, as well. Due to the stability of diflubenzuron to abiotic pr ocesses, limited data are available on the persistence and mobility of diflubenzuron metabolites. However, CPU was reported in leachate of a column leaching study (approximately 15 to 30% of applied in MRIDs 00039477 & 00040777). Even though CPU appears to be mobile under laboratory conditions, it has not been reported below the 0 to 15 cm soil depth segment in any field data.

Diflubenzuron does appear to accumulate at low le vels and depurate rapidly in fish tissue. The reported bioconcentration factors ranged from 34 to 200X for fillet, 78 to 360X for whole fish, and 100 to 500X for viscera. In addition, the depuration rate indicates a rapid decrease (99%) of accumulated residues in tissue during the 14 day depuration period.

Table 2-1 lists the environmental fate properties of diflubenzuron, along with the major and minor degradates detected in the submitted environmental fate and transport studies.

18Table 2-1 Summary of Diflubenzuron Environmental Fate Properties

Study Value (units) Major Degradates Minor Degradates MRID # Study Status

14 C label 433 days @ pH 5 117 days @ pH 7 42 days @ pH 9

3 H Label 187 days @ pH 5 158 days @ pH 7 40 days @ pH 9

Unlabeled Stable @ pH 5 Stable @ pH 7 44 days @ pH 9

14 C and 3 H label 7.5 days @ pH 12 CPU – Not Quantified DFBA – Not Quantified 00143355 Supplemental

223.6 days @ pH 5 247 days @ pH 7 32.5 days @ pH 9 CPU – Not Quantified DFBA – Not Quantified 40859801 41087801 Acceptable Hydrolysis

Dark aqueous photolysis control: Stable @ pH 7 None Identified 40816301 Supplemental (15 days only)

Direct Aqueous Photolysis 40 days (continuous light) 80 days (assuming 12 hr. light/dark cycle CPU – 8% DFBA – 4% DFBAM – 1% 2,6-Difluorobenzene – not quantified Unknown – 6% 40816301 41087802 Acceptable

144 days Not Quantified 40941601 Supplemental

Soil Photolysis 11.3 days CPU – 12.9 % (Day l0) DFBA – 3.0% (Day 7) Unknown SP1 – 0.6% (Day 10) Unknown PK1 – 0.1% (Day 16) 42251201 Acceptable

00039473 00039474 00040154 Supplemental (39474 same

as 40154) 00099875 Supplemental Aerobic Soil Metabolism 2 to 14 days CPU – 37 % (Day l4) DFBA – <10% DFBAM – <10% PCA – <10% 41722801 Same as 46888708 Acceptable

Anaerobic Soil Metabolism 2 to 14 days CPU – 37 % (Day 2-l4: depending on temperature) DFBA – 23% (Day 35) 00040782 41837601 Acceptable

Anaerobic Aquatic Metabolism 34 days CPU – 31 % (Day 42) DFBA – 31% (Day 42) PCA – 0.4% (Day 18) 41837601 Acceptable

26 days CPU – 30 % (Day l6) DFBA – 9% (Day 1) DFBAM – 1.6% (Day 2) PCA – 2% (Day 16) 44895001 Acceptable Aerobic Aquatic Metabolism 5.4 days 3.7 days DFBA – 17% (Day 4) CPU – 48 % (Day l6) 46888707 Supplemental

19Major Degradates Study Value (units) Minor Degradates MRID # Study Status

K oc 2878 3008 3401 6918 6801 2780 1938 Not Applicable 00039476 00039477 00040777 00157842 46888704 46888705 46895401 Acceptable

Terrestrial Field Dissipation The orchard and bare ground halflives (5.8 to 13.2 days).

CA citrus and the OR apple orchards half-life of approximately 68.2 to 78 days. DFBA – up to 0.01 ppm CPU – up to 0.06 ppm 00040156 40660601 40660602 41816502 41816503 41821002 41826701 42290401 42290402 42441101 Acceptable

Forestry Field Dissipation 30 to 35 days None Detected 41077201 41077202 41922201 42197701 Supplemental

Aquatic Field Dissipation 2 to 6 days CPU 00040155 00161945 00161947 40598601 44399309 45009601 45191001 45197601 Acceptable

Accumulation Bioconcentration factors of 34 to 200× for fillet, 78 to 360× for whole fish, and 100, to 550× for viscera.

99% depuration within 14 day DFBAM – 16% (0.28,μg/g) in Fillet; 12% (0.37 μg/g) in Whole Fish; and 11% (0.55 μg/g) in Viscera.

6 unidentified minor metabolites 42258401 42258402 Acceptable

2.4.2 Environmental Transport Assessment

Surface water runoff and spray drift are expected to be the major routes of exposure for diflubenzuron.

In general, deposition of drifting pesticides is expected to be greatest close to the site of application. Computer models of spray drif t (AgDRIFT) are used to determine potential exposures to aquatic and terres trial organisms via spray drift. The distance of potential impact away from the use sites (action area) is determined by the distance required to fall below the LOC for aquatic invertebrates.

202.4.2 Mechanism of Action

Diflubenzuron is an insect growth regulator and works by preventing the formation of chitin, a molecule necessary to the formation of an insect’s cuticle or outer shell. Insects exposed to a sufficient dose of diflubenzuron cannot form their prot ective outer shell and die during molting. It is most e ffective against insect larva, but also acts as an ovicide, killing insect eggs (U.S. EPA 1997).

2.4.3 Use Characterization

Currently, labeled uses of diflubenzuron incl ude citrus, cotton, forestry, mushrooms, ornamentals, pastures, soybeans, standing water, sewage systems, termite bait stations, and wide-area general outdoor trea tment sites. All of these uses are considered as part of the federal action evaluated in this assessment with the exception of termite bait stations (in which a solidified form of the pesticide is placed in an enclosed bait station to minimize loss to the environment) and soyb eans, which are not grown in California (NASS 2002). If use patterns i ndicate that soybeans are grown in CA in the future, the conclusions of this assessment may need to be revisited.

Analysis of labeled use information is the cr itical first step in evaluating the federal action. The current label for diflubenzuron represents the FIFRA regulatory action; therefore, labeled use and app lication rates specified on the label form the basis of this assessment. The assessment of use information is critical to the deve lopment of the action area and selection of appropriate modeling scenarios and inputs.

Table 2-2 presents the uses and corresponding application rates and methods of application considered in this assessment.

21Table 2-2 Diflubenzuron Uses Assessed for the CRLF

Use (Application Method – Ground (G), Airblast (AB), or Air (A)) Max. Single Appl. Rate

(lb ai/A) Max. Number of Application per Year Application Interval Spray on manure, Manure to field G 8.508 NS (17) NS (21) G 0.0408 3 7 Beech Nut A 0.0408 3 7 Brassica (head and stem) vegetables G 0.0313 1 NA G 0.3125 3 21 Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines A 0.125 3 7 Broccoli raab, cabbage - Chinese, collards, and kale G 0.25 4 21

G 0.3125 3 7 Cotton (unspecified) A 0.3125 3 90 G 0.22048 NS (4) 14 Christmas tree plantations and forest trees (softwoods - conifers) A 0.25 1 NA G 0.3125 3 7 AB 0.3125 3 7 Apricot, nectarine, peach , pear, plum, prune, and stone fruits A 0.3125 3 7 G 0.3125 3 7 AB 0.3125 3 7 Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (hazelnut), hickory nut, macadamia nut (bushnut), pecan, tree nuts, and walnut (English/black) A 0.3125 3 90

G 0.25 4 21 Barley, canola\rape, oats , triticale, and wheat A 0.125 3 NS (21) G 0.25 1 NA Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings A 0.25 1 NA

G 0.25 1 NA AB 0.25 1 NA Grass forage/fodder/hay, pastures, and rangeland A 0.25 1 NA Pistachio G 0.75 2 14 G 0.25 1 NA Household/domestic dwellings outdoor premises A 0.25 6 10 Rice A 0.625 6 5 G 0.25 2 5 AB 0.25 2 5 Agricultural rights-of-way/fencerows/hedgerows,fencerows/hedgerows, and nonagricultural rightsof-way/fencerows/hedgerows A 0.25 2 5 G 0.3125 3 21 Artichoke, mustard, peanuts (unspecified), and
pepper A 0.3125 3 14 Commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) A 0.25 6 NS (10)

G 0.25 4 15 Butternut A 0.25 4 15 G 0.25 2 14 AB 0.25 2 14 Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils A 0.25 2 5 Ornamental sod farm (turf) and recreational areas G 0.0313 3 7

22Use (Application Method – Ground (G), Airblast (AB), or Air (A)) Max. Single Max. Number of Application Appl. Rate Application per

(lb ai/A) Year Interval AB 0.0313 3 7 A 0.0313 3 7 Turnip (greens) G 0.25 2 21 NS = Not specified on label.

Several of the agricultural crop uses for diflubenzuron may have more than one crop cycle per year. Because many diflubenzuron la bels specify applica tion limits based on crop cycles rather than an annual limitation, th e potential exists for more diflubenzuron to be applied than is reflected in Table2-2. However, because the persistence of diflubenzuron is relatively short compared to the length of a crop cy cle, there would be little accumulation of the parent compound acro ss crop cycles. Instead the greatest impact from multiple crop cycles would likely result from the extension of the time that diflubenzuron would be applied both earlier in the spring and later in the fall when more runoff would occur. This extension of the time that diflubenzuron may be used is addressed in this assessme nt in Section 3.2.1, which evaluates both early and late application dates on diflubenzuron exposure.

Figure 2-1 provides a visual depiction of the sp atial distribution of diflubenzuron use across the United States in terms of pounds of diflubenzuron applied per square mile of agricultural land in each county. The map was downloaded from a U.S. Geological Survey (USGS), National Water Quality Assessment Program (N AWQA) website. Many simplifying assumptions were made in order to produce this map. Non-agricultural uses of diflubenzuron such as forestry, residentia l and right-of-way uses are not included in this depiction.

23Figure 2-1. Diflubenzuron use in total po unds per square mile of agricultural land in county.

The Agency’s Biological and Economic Analysis Division (BEAD) provides an analysis of both national- and county-l evel usage information (U.S. EPA 2009) using state-level usage data obtained from USDA-NASS 1 , Doane ( www.doane.com ; the full dataset is not provided due to its proprietary nature) and the California’s Department of Pesticide Regulation Pesticide Use Re porting (CDPR PUR) database 2 . CDPR PUR is considered a more comprehensive source of usage da ta than USDA-NASS or EPA proprietary databases, and thus the usage data repor ted for diflubenzuron by county in this California-specific assessment were genera ted using CDPR PUR data. Eight years (1999-2006) of usage data were included in th is analysis. Data from CDPR PUR were obtained for every pesticide application made on every use site at the section level (approximately one square mile) of the public land survey system. BEAD summarized these data to the county level by site, pesticide, and unit trea ted. Calculating county-level usage involved summarizing across all applica tions made within a section and then across

1 United States Depart of Agriculture (USDA), Natio nal Agricultural Statistics Service (NASS) Chemical Use Reports provide summary pesticide usage statistic s for select agricultural use sites by chemical, crop and state. See http://www.usda.gov/nass/pubs/estindx1.htm#agchem

. 2 The California Department of Pesticide Regulation’s Pesticide Use Reporting database provides a census of pesticide applications in the state. See http://www.cdpr.ca.gov/docs/pur/purmain.htm

.

24all sections within a county for each use site and for each pesticide. The county level usage data that were calculated include: average annual pounds applied, average annual area treated, and average and maximum applica tion rate across all eight years. The units of area treated are also provided where available.

A summary of diflubenzuron us age for all California use sites is provided below in Table 2-3

.

Table 2-3 Summary of California Department of Pesticide Registration (CDPR) Pesticide Use Reporting (PUR) Data from 1999 to 2006 for Currently Registered Diflubenzuron Uses

Site Name, Application Method ( A erial, G round, or O ther), and Years of Data Average Pounds Applied by Use Average App. Rate by Use Maximum App. Rate by Use A (3) 988.30 0.24 4.05 Almond G (3) 8007.51 0.22 3.74 A (1) 17.46 0.25 0.25 Apricot G (3) 100.25 0.19 0.24 Aquatic Areas, Wate r Areas (All Or Unspec) O (7) 15.82 0.22 0.25

A (8) 1737.13 0.14 2.23 Artichoke (Globe) (All Or Unspec) G (8) 1982.66 0.14 2.97 Beets, General G (1) 3.50 5.00 5.00 A (1) 0.92 0.02 0.02 Broccoli G (1) 2.10 0.02 0.02 Brussels Sprouts G (2) 1.36 0.41 0.70 Cherry G (1) 3.75 0.25 0.25 Christmas Tree Plantations G (6) 0.71 0.08 0.13 A (3) 45.42 0.12 0.14 Cotton, General G (2) 54.38 0.10 0.13 Endive (Escarole) G (1) 26.64 0.19 0.19 A (1) 2.44 0.13 0.13 Grapefruit G (4) 73.25 0.17 0.78 Landscape Maintenance O (8) 23.11 NR NR Nectarine G (3) 38.51 0.09 0.25 A (3) 0.06 0.32 0.69 G (8) 18.14 0.46 24.16 N-Outdr Container/Fld Grwn Plants O (1) 0.00 0.00 0.00 A (1) 0.03 0.00 0.00 N-Outdr Grwn Cut Flwrs Or Greens G (8) 2.82 0.29 3.57 A (1) 2.55 0.21 0.59 N-Outdr Grwn Trnsplnt/Prpgtv Mtrl G (6) 9.23 0.20 2.08 A (2) 31.03 0.13 0.14 Orange (All Or Unspec) G (4) 762.37 0.19 2.50 Pastures (All Or Unspec) A (1) 0.95 0.02 0.02 Peach G (4) 16 7.31 0.20 6.07 Pear G (3) 75.48 0.22 0.26 Peppers (Chili Type) (Flavoring And Spice Crop) G (1) 0.75 0.13 0.13
Peppers (Fruiting Vegetable), (Bell,Chili, Etc.) G (1) 141.96 0.12 0.13

Pistachio (Pistache Nut) G (2) 36.87 0.24 0.25

25Site Name, Application Method ( A erial, G round, or O ther), and Years of Data Average Pounds Applied by Use Average App. Maximum App. Rate by Use Rate by Use A (1) 5.95 0.20 0.20 Plum (Includes Wild Plums For Human Consumption) G (3) 94.31 0.19 0.25 A (1) 5.56 0.25 0.25 Prune G (3) 66.94 0.25 0.95 Public Health Pest Control O (8) 527.15 NR NR A (2) 3.08 0.03 0.03 Rangeland (All Or Unspec) G (1) 2.21 0.03 0.03 Regulatory Pest Control O (5) 42.22 NR NR A (8) 789.74 0.14 3.93 Rice (All Or Unspec) G (1) 4.08 0.09 0.09 Rights Of Way O (6) 9.98 NR NR Soil Application, Preplant-Outdoor (Seedbeds,Etc.) G (3) 213.56 11.28 12.61
Strawberry (All Or Unspec) G (1) 23.50 0.25 0.25 Structural Pest Control O (8) 284.86 NR NR Tangerine (Mandarin, Satsuma, Murcott, Etc.) G (2) 30.73 0.16 0.26
Uncultivated Agricultural Areas (All Or Unspec) G (2) 1.39 0.05 0.07
Uncultivated Non-Ag Areas (All Or Unspec) O (2) 8.15 0.06 0.13

A (4) 37.22 0.24 0.38 Walnut (English Walnut, Persian Walnut) G (8) 1406.79 0.26 2.02

2.5 Assessed Species

The CRLF was federally listed as a threatened species by U.S. FWS effective June 24, 1996 (U.S. FWS 1996). It is one of two subspe cies of the red-legged frog and is the largest native frog in the western United St ates (U.S. FWS 2002). A brief summary of information regarding CRLF distribution, repr oduction, diet, and habitat requirements is provided in Sections 2.5.1 through 2.5.4 , respectively. Further information on the status, distribution, and life history of and speci fic threats to the CRLF is provided in Attachment I.

Final critical habitat for the CRLF was de signated by U.S. FWS on April 13, 2006 (U.S. FWS 2006; 71 FR 19244-19346). Further informa tion on designated cri tical habitat for the CRLF is provided in Section 2.6

.

2.5.1 Distribution

The CRLF is endemic to California and Baja California (Mexico) and historically inhabited 46 counties in Ca lifornia including the Central Valley and both coastal and interior mountain ranges (U.S. FWS 1996). It s range has been reduced by about 70%, and the species currently resides in 22 c ounties in California (U .S. FWS 1996). The species has an elevational range of near sea level to 1,500 meters (5,200 feet) (Jennings and Hayes 1994); however, nearly all of the known CRLF populations have been documented below 1,050 meters (3,500 feet) (U.S. FWS 2002).

26Populations currently exist along the northern California coast, northern Transverse Ranges (U.S. FWS 2002), foothills of the Si erra Nevada (5-6 populations), and in southern California south of Santa Barbara (two populations) (Felle rs 2005a). Relatively larger numbers of CRLFs are located be tween Marin and Santa Barbara Counties (Jennings and Hayes 1994). A total of 243 st reams or drainages are believed to be currently occupied by the sp ecies, with the greatest numbers in Monterey, San Luis Obispo, and Santa Barbara counties (U.S. FWS 1 996). Occupied drainages or watersheds include all bodies of water that support CRLFs ( i.e. , streams, creeks, tributaries, associated natural and artificial ponds, and adjacent drainages), and habitats through which CRLFs can move ( i.e. , riparian vegetation, upl ands) (U.S. FWS 2002).

The distribution of CRLFs with in California is addressed in this assessment using four categories of location including r ecovery units, core areas, desi gnated critical habitat, and known occurrences of the CRLF reported in the California Natural Diversity Database (CNDDB) that are not included within core ar eas and/or designated critical habitat. Recovery units, core areas, and other known occurrences of the CRLF from the CNDDB are described in further detail in Attachment I, and designated critical habitat is addressed in Section 2.6 . Recovery units are large areas defi ned at the watershed level that have similar conservation needs and management strate gies. The recovery un it is primarily an administrative designation, and land area wi thin the recovery unit boundary is not exclusively CRLF habitat. Core areas are sm aller areas within the recovery units that comprise portions of the species ’ historic and current range and have been determined by U.S. FWS to be important in the preservation of the species. Designate d critical habitat is generally contained within the core areas, although a number of critical habitat units are outside the boundaries of core areas, but w ithin the boundaries of the recovery units. Additional information on CRLF occurrences from the CNDDB is used to cover the current range of the species not included in co re areas and/or designa ted critical habitat, but within the recovery units.

Other Known Occurrences from the CNDBB

The CNDDB provides location and natural history information on species found in California. The CNDDB serves as a repositor y for historical and cu rrent species location sightings. Information regarding known occurrences of CRLFs outside of the currently occupied core areas and designated critical ha bitat is considered in defining the current range of the CRLF. See: http://www.dfg.ca.gov/bdb/html/cnddb_info.html for additional information on the CNDDB.

27Recovery Units 1. Sierra Nevada Foothills and Central Valley 2. North Coast Range Foothills and Western Sacramento River Valley 3. North Coast and North San Francisco Bay 4. South and East San Francisco Bay 5. Central Coast 6. Diablo Range and Salinas Valley 7. Northern Transverse Ranges and Tehachapi Mountains 8. Southern Transverse and Peninsular Ranges

Core Areas

1. Feather River 2. Yuba River- S. Fork Feather River 3. Traverse Creek/ Middle Fork/ American R. Rubicon 4. Cosumnes River 5. South Fork Calaveras River* 6. Tuolumne River* 7. Piney Creek* 8. Cottonwood Creek 9. Putah Creek – Cache Creek* 10. Lake Berryessa Tributaries 11. Upper Sonoma Creek 12. Petaluma Creek – Sonoma Creek 13. Pt. Reyes Peninsula 14. Belvedere Lagoon 15. Jameson Canyon – Lower Napa River 16. East San Francisco Bay 17. Santa Clara Valley 18. South San Francisco Bay 19. Watsonville Slough-Elkhorn Slough 20. Carmel River – Santa Lucia 21. Gablan Range 22. Estero Bay 23. Arroyo Grange River 24. Santa Maria River – Santa Ynez River 25. Sisquoc River 26. Ventura River – Santa Clara River 27. Santa Monica Bay – Venura Coastal Streams 28. Estrella River 29. San Gabriel Mountain* 30. Forks of the Mojave* 31. Santa Ana Mountain* 32. Santa Rosa Plateau 33. San Luis Ray* 34. Sweetwater* 35. Laguna Mountain*

* Core areas that were historically occupied by the California red-legged frog are not included in the map Recovery Units 1. Sierra Nevada Foothills and Central Valley 2. North Coast Range Foothills and Western Sacramento River Valley 3. North Coast and North San Francisco Bay 4. South and East San Francisco Bay 5. Central Coast 6. Diablo Range and Salinas Valley 7. Northern Transverse Ranges and Tehachapi Mountains 8. Southern Transverse and Peninsular Ranges

Core Areas

1. Feather River 2. Yuba River- S. Fork Feather River 3. Traverse Creek/ Middle Fork/ American R. Rubicon 4. Cosumnes River 5. South Fork Calaveras River* 6. Tuolumne River* 7. Piney Creek* 8. Cottonwood Creek 9. Putah Creek – Cache Creek* 10. Lake Berryessa Tributaries 11. Upper Sonoma Creek 12. Petaluma Creek – Sonoma Creek 13. Pt. Reyes Peninsula 14. Belvedere Lagoon 15. Jameson Canyon – Lower Napa River 16. East San Francisco Bay 17. Santa Clara Valley 18. South San Francisco Bay 19. Watsonville Slough-Elkhorn Slough 20. Carmel River – Santa Lucia 21. Gablan Range 22. Estero Bay 23. Arroyo Grange River 24. Santa Maria River – Santa Ynez River 25. Sisquoc River 26. Ventura River – Santa Clara River 27. Santa Monica Bay – Venura Coastal Streams 28. Estrella River 29. San Gabriel Mountain* 30. Forks of the Mojave* 31. Santa Ana Mountain* 32. Santa Rosa Plateau 33. San Luis Ray* 34. Sweetwater* 35. Laguna Mountain*

* Core areas that were historically occupied by the California red-legged frog are not included in the map Figure 2-2 Recovery Unit, Core Are a, Critical Habitat, and Occurrence Designations for CRLF

282.5.2 Reproduction

CRLFs breed primarily in ponds; however, they may also breed in quiescent streams, marshes, and lagoons (Fellers 2005a). Accord ing to the Recovery Plan (U.S. FWS 2002), CRLFs breed from November through late April. Peaks in spawning activity vary geographically; Fellers (2005b) re ports peak spawning as early as January in parts of coastal central California. Eggs are fertili zed as they are being laid. Egg masses are typically attached to emergent vegetation, such as bulrushes ( Scirpus spp.) and cattails ( Typha spp.) or roots and twigs, and float on or near the surface of the water (Hayes and Miyamoto 1984). Egg masses contain appr oximately 2000 to 6000 eggs ranging in size between 2 and 2.8 mm (Jennings and Hayes 1994). Embryos hatch 10 to 14 days after fertilization (Fellers 2005a) depending on water temperature. Egg predation is reported to be infrequent and most mortality is associated with the larval stage (particularly through predation by fish); however, predation on eggs by newts has also been reported (Rathburn 1998). Tadpoles re quire 11 to 28 weeks to me tamorphose into juveniles (terrestrial-phase), typica lly between May and Septem ber (Jennings and Hayes 1994, U.S. FWS 2002); tadpoles have been observed to over-winter (delay metamorphosis until the following year) (Fellers 2005b; U.S. FWS 2002). Males reach sexual maturity at 2 years, and females reach sexual maturity at 3 years of age; adults have been reported to live 8 to 10 years (U.S. FWS 2002). Figure 2-3 depicts CRLF annual reproductive timing.

D N O S A J J M A M F J

Light Blue = Breeding/Egg Masses Green = Tadpoles (except those that over-winter) Orange = Young Juveniles Adults and juveniles can be present all year D N O S A J J M A J F M

Light Blue = Breeding/Egg Masses Green = Orange = Young Juveniles Adults and juveniles can be present all year Tadpoles (except those that over-winter)

Figure 2-3 CRLF Reproduc tive Events by Month

2.5.3 Diet

Although the diet of CRLF aquatic-phase la rvae (tadpoles) has not been studied specifically, it is assumed that their diet is si milar to that of other frog species, with the aquatic phase feeding exclusiv ely in water and consuming diatoms, algae, and detritus (U.S. FWS 2002). Tadpoles filter and entrap suspended algae (Seale and Beckvar 1980) via mouthparts designed for effective grazing of periphyton (Wassersug 1984; Kupferberg et al. 1994; Kupferberg 1997; Altig and McDiarmid 1999).

Juvenile and adult CRLFs forage in aquatic a nd terrestrial habitats, and their diet differs greatly from that of larvae. The main food source for juvenile aquatic- and terrestrialphase CRLFs is thought to be aquatic a nd terrestrial invert ebrates found along the shoreline and on the water surface. Hayes a nd Tennant (1985) report, based on a study

29examining the gut content of 35 juvenile a nd adult CRLFs, that the species feeds on as many as 42 different invertebrate taxa , including Arachnida, Amphipoda, Isopoda, Insecta, and Mollusca. The most commonly obs erved prey species were larval alderflies ( Sialis cf. californica ), pillbugs ( Armadilliadrium vulgare ), and water striders ( Gerris sp). The preferred prey species, however, was the sowbug (Hayes and Tennant 1985). This study suggests that CRLFs forage primarily above water, although the authors note other data reporting that adults also feed under water, are cannibalistic, and consume fish. For larger CRLFs, over 50% of the prey mass may c onsists of vertebrates such as mice, frogs, and fish, although aquatic and terrestrial inve rtebrates were the most numerous food items (Hayes and Tennant 1985). For adults, feeding activity takes place primarily at night; for juveniles feeding o ccurs during the day and at ni ght (Hayes and Tennant 1985).

2.5.4 Habitat

CRLFs require aquatic habitat for breeding, but also use other habitat types including riparian and upland areas throughout their lif e cycle. CRLF use of their environment varies; they may complete their entire life cycl e in a particular habitat or they may utilize multiple habitat types. Overall, populations are most likely to exist where multiple breeding areas are embedded within varying ha bitats used for dispersal (U.S. FWS 2002). Generally, CRLFs utilize habitat with perennial or near-perennial water (Jennings et al. 1997). Dense vegetation close to water, shadin g, and water of modera te depth are habitat features that appear especially impor tant for CRLF (Hayes and Jennings 1988). Breeding sites include streams, deep pools, b ackwaters within streams and creeks, ponds, marshes, sag ponds (land depressions between fault zones that have filled with water), dune ponds, and lagoons. Breeding adults have be en found near deep (0.7 m) still or slow moving water surrounded by dense vegetation (U.S. FWS 2002); however, the largest number of tadpoles have been found in sh allower pools (0.26 – 0.5 m) (Reis 1999). Data indicate that CRLFs do not fre quently inhabit vernal pools, as conditions in these habitats generally are not suitable (Hayes and Jennings 1988).

CRLFs also frequently breed in artificial impoundments such as stock ponds, although additional research is needed to identify habitat requirement s within artificial ponds (U.S. FWS 2002). Adult CRLFs use dense, shrubby, or emergent vegetation closely associated with deep-water pools bordered with cattail s and dense stands of overhanging vegetation ( http://ecos.fws.gov/speciesProfi le/SpeciesReport.do?spcode=D02D ).

In general, dispersal and habitat use depends on climatic conditions, habitat suitability, and life stage. Adults rely on riparian vegetation for resti ng, feeding, and dispersal. The foraging quality of the riparian habitat depends on moisture, composition of the plant community, and presence of pools and backwa ter aquatic areas for breeding. CRLFs can be found living within streams at distances up to 3 km (2 miles) from their breeding site and have been found up to 30 m (100 feet) from water in dense riparian vegetation for up to 77 days (U.S. FWS 2002).

During dry periods, the CRLF is rarely found far from water, although it will sometimes disperse from its breeding hab itat to forage and seek othe r suitable habitat under downed

30trees or logs, industrial debr is, and agricultural features (U.S. FWS 2002). According to Jennings and Hayes (1994), CRLFs also use sm all mammal burrows and moist leaf litter as habitat. In addition, CRLFs may also use larg e cracks in the bottom of dried ponds as refugia; these cracks may provide moisture for individuals avoidi ng predation and solar exposure (Alvarez 2000).

2.6 Designated Critical Habitat

In a final rule published on April 13, 2006, 34 se parate units of critical habitat were designated for the CRLF by U.S. FWS (U.S. FWS 2006; FR 51 19244-19346). A summary of the 34 critical habitat units rela tive to U.S. FWS-desi gnated recovery units and core areas (previously discussed in Section 2.5.1 ) is provided in Attachment I.

‘Critical habitat’ is defined in the ESA as th e geographic area occupi ed by the species at the time of the listing where the physical and biological features necessary for the conservation of the species exis t, and there is a need for special management to protect the listed species. It may also include ar eas outside the occupied area at the time of listing if such areas are ‘essential to the c onservation of the species.’ All designated critical habitat for the CRLF was occupied at the time of listing. Cr itical habitat receives protection under Section 7 of the ESA (Section 7) through prohibition against destruction or adverse modification with regard to actions carried ou t, funded, or authorized by a federal Agency. Section 7 requires consultation on federal actions that are likely to result in the destruction or adverse modification of critical habitat.

To be included in a critical habitat desi gnation, the habitat must be ‘essential to the conservation of the species.’ Critical hab itat designations identify, to the extent known using the best scientific a nd commercial data available, habitat areas that provide essential life cycle needs of the species or areas that contai n certain primary constituent elements (PCEs) (as defined in 50 CFR 414.12(b )). PCEs include, but are not limited to, space for individual and population growth a nd for normal behavior; food, water, air, light, minerals, or other nutritional or physiolo gical requirements; cove r or shelter; sites for breeding, reproduction, rearing (or developmen t) of offspring; a nd habitats that are protected from disturbance or are represen tative of the histor ic geographical and ecological distributions of a species. The de signated critical habitat areas for the CRLF are considered to have the following PCEs that justify critical habitat designation:

• Breeding aquatic habitat;

• Non-breeding aquatic habitat;

• Upland habitat; and

• Dispersal habitat.

Further description of thes e habitat types is provided in Attachment I.

Occupied habitat may be include d in the critical habitat only if essential features within the habitat may require special management or protection. Therefore, U.S. FWS does not include areas where existing management is su fficient to conserve the species. Critical

31habitat is designated outside the geographic area presently occupied by the species only when a designation limited to its present ra nge would be inadequa te to ensure the conservation of the species. For the CRLF, all designated critical habi tat units contain all four of the PCEs, and were occupied by th e CRLF at the time of the Final Rule (FR) listing notice in April 2006 (71 FR 19243, 2006) . The FR notice designating critical habitat for the CRLF includes a special ru le exempting routine ranching activities associated with livestock ranc hing from incidental take prohi bitions. The purpose of this exemption is to promote the conservation of ra ngelands, which could be beneficial to the CRLF, and to reduce the rate of conversion to other land uses that are incompatible with CRLF conservation. Please see Attachment I fo r a full explanation on this special rule.

U.S. FWS has established adverse modificati on standards for designated critical habitat (U.S. FWS 2006). Activities that may destr oy or adversely modify critical habitat are those that alter the PCEs and jeopardize th e continued existence of the species. Evaluation of actions related to use of di flubenzuron that may alter the PCEs of the CRLF’s critical habitat form the basis of the critical habitat impact analysis. According to U.S. FWS (2006), activities that may aff ect critical habitat and therefore result in adverse effects to the CRLF include, but are not limited to the following:

(1) Significant alteration of water chemistr y or temperature to levels beyond the tolerances of the CRLF that result in di rect or cumulative adverse effects to individuals and their life-cycles. (2) Alteration of chemical characteristics n ecessary for normal growth and viability of juvenile and adult CRLFs. (3) Significant increase in sediment deposition within the stream channel or pond or disturbance of upland fora ging and dispersal habitat that could result in elimination or reduction of habitat necessary for the growth and reproduction of the CRLF by increasing the sediment deposit ion to levels that would adversely affect their ability to co mplete their life cycles. (4) Significant alteration of ch annel/pond morphology or geometry that may lead to changes to the hydrologic functioning of the stream or pond and alter the timing, duration, water flows, and levels that would degrade or eliminate the CRLF and/or its habitat. Such an effect coul d also lead to increased sedimentation and degradation in water quality to levels that are beyond the CRLF’s tolerances. (5) Elimination of upland foraging and/or aes tivating habitat or dispersal habitat. (6) Introduction, spread, or augmentation of non-native aquatic species in stream segments or ponds used by the CRLF. (7) Alteration or elimination of the CRLF ’s food sources or prey base (also evaluated as indirect effects to the CRLF).

As previously noted in Section 2.1, the Agency believes that the analysis of direct and indirect effects to listed species provides the basis for an analysis of potential effects on the designated critical habitat. Because di flubenzuron is expected to directly impact living organisms within the action area, critical habitat analysis for diflubenzuron is limited in a practical sense to those PCEs of cr itical habitat that are biological or that can be reasonably linked to biol ogically mediated processes.

322.7 Action Area

For listed species assessment purposes, the action area is considered to be the area affected directly or indirec tly by the federal action and not merely the immediate area involved in the action (50 CFR 402.02). It is recognized th at the overall action area for the national registration of difl ubenzuron is likely to encompass considerable portions of the United States based on the large array of ag ricultural uses. However, the scope of this assessment limits considerati on of the overall action area to those portions that may be applicable to the protection of the CRLF and its designated critical ha bitat within the state of California. The Agency’s approach to defining the action area unde r the provisions of the Overview Document (U.S. EPA 2004) cons iders the results of the risk assessment process to establish boundaries for that action area with the understanding that exposures below the Agency’s defined Levels of Concern (LOCs) constitute a no-effect threshold. For the purposes of this assessment, attenti on will be focused on the footprint of the action ( i.e. , the area where pesticide application occurs), plus all areas where offsite transport ( i.e. , spray drift, downstream dilution, et c.) may result in potential exposure within the state of California th at exceeds the Agency’s LOCs.

Deriving the geographical extent of this portion of the action area is based on consideration of the types of effects that diflubenzuron may be expected to have on the environment, the exposure levels to diflubenzuron that are as sociated with those effects, and the best available information concerni ng the use of diflubenz uron and its fate and transport within the state of California. Speci fic measures of ecological effect that define the action area include any direct and indirect toxic effect and any potential modification of its critical habitat, includi ng reduction in survival, growth, and fecundity as well as the full suite of sublethal effects available in the effects literature. Th erefore, the action area extends to a point where environmental expos ures are below any m easured lethal or sublethal effect threshold for any biological entity at the whole organism, organ, tissue, and cellular level of or ganization. In situations where it is not possible to determine the threshold for an observed effect, the action ar ea is not spatially limite d and is assumed to be the entire state of California.

The definition of action area requires a stepwise approach that begins with an understanding of the federal act ion. The federal action is de fined by the currently labeled uses for diflubenzuron. An analysis of la beled uses and review of available product labels was completed. Several of the currently labeled uses are special local needs (SLN) uses or are restricted to specific states and are excluded from this assessment. In addition, a distinction has been made betw een food use crops a nd those that are nonfood/non-agricultural uses. For those uses relevant to the CRLF, the analysis indicates that, for diflubenzuron, all uses are considered as part of th e federal action evaluated in this assessment except soybeans and termite bait station uses.

Following a determination of the assessed uses , an evaluation of the potential “footprint” of diflubenzuron use patterns ( i.e. , the area where pesticide application occurs) is determined. This “footprint” represents the initial area of concern, based on an analysis of available land cover data fo r the state of California. The initial area of concern is defined as all land cover types and the stream reaches within the land cover areas that

33represent the labeled uses described above. Because di flubenzuron has a diverse set of uses that encompass all land cover classes used to define the initial “footprint”, the initial area of concern is considered to be statewide.

Once the initial area of concern is defined, the next step is to define the potential boundaries of the action area by determining the ex tent of offsite tran sport via spray drift and runoff where exposure of one or more ta xonomic groups to the pe sticide exceeds the listed species LOCs.

As previously discussed, the action area is defined by the most sensitive measure of direct and indirect ecological toxic effects including re duction in survival, growth, reproduction, and the entire suite of sublethal effects from valid, peer-reviewed studies.

The action area is determined by the footprint of the action plus all offsite areas where exposure of one or more taxonomic groups to diflubenzuron exceeds the Agency’s LOCs. The spatial extent at which the Agency’s LO Cs are not exceeded is based on the potential exposure level and the most sensitive eff ects endpoint. The most sensitive effects endpoint is the acute aquatic i nvertebrate endpoint. It is exp ected that invertebrates would be the most sensitive since this chemical is an insecticide. This endpoint and all other effects endpoints used in this asse ssment are discussed in Section 4.

Because diflubenzuron has a relatively short pe riod of persistence relative to seasonal variations in rainfall in California, the major diflubenzuron exposure route to the organisms of concern (aquatic invertebrates) varies throughout th e year. During the dryseason (late spring, summer, and early fall) when little runoff occurs, most of the exposure will occur via spray dr ift. During the wet season (l ate fall, winter, and early spring) when much more runoff occurs, runo ff will be a major contributor to aquatic EECs and risks.

The initial footprint for diflubenzuron is th e entire state of Calif ornia given the large various types of uses (e.g., agri culture, residential, rights of ways, forestry). In addition, given the genotoxicity of a diflubenzuron degr adates, defining a threshold below which effects can occur is not possible. Therefor e, the action area is the entire state of California.

2.8 Assessment Endpoints and Measures of Ecological Effect

Assessment endpoints are defined as “explicit expressions of the ac tual environmental value that is to be protected.” 3 Selection of the assessment endpoints is based on valued entities ( e.g. , CRLF, organisms important in the life cycle of the CRLF, and the PCEs of its designated critical habitat), the ecosystems potentially at risk ( e.g. , waterbodies, riparian vegetation, and upla nd and dispersal habitats), the migration pathways of diflubenzuron ( e.g. , runoff, spray drift, etc.), and the routes by which ecological receptors are exposed to diflubenzuron ( e.g. , direct contact, etc.).

3 U.S. EPA (1992). Framework for Ecological Risk Assessment. EPA/630/R-92/001.

342.8.1 Assessment Endpoints for the CRLF

Assessment endpoints for the CRLF include direct toxic effects on the survival, reproduction, and growth of the CRLF, as well as indirect effects, such as reduction of the prey base or modification of its habitat. In addition, potential m odification of critical habitat is assessed by evaluating potential eff ects to PCEs, which are components of the habitat areas that provide essential life cycle needs of the CRLF. Each assessment endpoint requires one or more “measures of ecological effect,” defined as changes in the attributes of an assessment endpoint or cha nges in a surrogate entity or attribute in response to exposure to a pesticide. Specific measures of ecological effect are generally evaluated based on acute and chronic toxicity information from registrant-submitted guideline tests that are performed on a limited number of organisms. Additional ecological effects data from the open literature are also considered. It should be noted that assessment endpoints are limited to dir ect and indirect effects associated with survival, growth, and fecundity, and do not incl ude the full suite of sublethal effects used to define the action area. According th e Overview Document (U.S. EPA 2004), the Agency relies on acute and chronic effects endpoi nts that are either direct measures of impairment of survival, growth, or fec undity or endpoints for which there is a scientifically robust, peer reviewed relationship that ca n quantify the impact of the measured effect endpoint on the assessment endpoi nts of survival, growth, and fecundity.

A complete discussion of all the toxicity data available for this risk assessment, including resulting measures of ecologica l effect selected for each ta xonomic group of concern, is included in Section 4.0 of this document. A summary of the assessment endpoints and measures of ecological effect selected to characterize po tential assessed direct and indirect CRLF risks associated with exposur e to diflubenzuron is provided in Table 2-4.

35Table 2-4. Assessment Endpoints and Measures of Ecological Effects Assessment Endpoint Measures of Ecological Effects 4 Aquatic-Phase CRLF (Eggs, larvae, juveniles, and adults) a Direct Effects

1. Survival, growth, and reproduction of CRLF 1a. Most sensitive fish acute LC 50 (guideline study; test species: bluegill sunfish) 1b. Most sensitive fish chronic NOAEC (guideline; test species: fathead minnow)

Indirect Effects and Critical Habitat Effects

2. Survival, growth, and reproduction of CRLF individuals via indirect effects on aquatic prey food supply ( i.e., fish, freshwater invertebrates, nonvascular plants) 2a. Most sensitive fish (guideline study; test species: bluegill sunfish), aquatic invertebrate (ECOTOX study; test species: mosquito larvae ), and aquatic plant EC 50 (guideline study; test species: Duck weed) or LC 50 2b. Most sensitive aquatic invertebrate and fish chronic NOAEC (ECOTOX study and guideline respectively) 3. Survival, growth, and reproduction of CRLF individuals via indirect effects on habitat, cover, food supply, and/or primary productivity ( i.e., aquatic plant community) 3a. Vascular plant acute EC 50 (duckweed guideline test) 3b. Non-vascular plant acute EC 50 (guideline study; test species: green algae) 4. Survival, growth, and reproduction of CRLF individuals via effects to riparian vegetation Currently no acceptable, gu ideline terrestrial plant toxicity data are available. Terrestrial-Phase CRLF

(Juveniles and adults) Direct Effects

5. Survival, growth, and reproduction of CRLF individuals via direct effects on terrestrial phase adults and juveniles 5a. Most sensitive bird b or terrestrial-phase amphibian acute LC 50 or LD 50 (guideline study; test species: Redwinged blackbird) 5b. Most sensitive bird b or terrestrial-phase amphibian chronic NOAEC (guideline study; test species: Bobwhite quail) Indirect Effects and Critical Habitat Effects 6. Survival, growth, and reproduction of CRLF individuals via effects on terrestrial prey ( i.e., terrestrial invertebrates, small mammals , and frogs) 6a. Most sensitive terrestrial invertebrate and vertebrate acute EC 50 (guideline study; test species: Bobwhite quail)

7. Survival, growth, and reproduction of CRLF individuals via indirect effects on habitat ( i.e., riparian and upland vegetation) Currently no acceptable, gu ideline terrestrial plant toxicity data are available.

a Adult frogs are no longer in the “aquatic phase” of the amphibian life cycle; however, submerged adult frogs are considered “aquatic” for the purposes of th is assessment because exposure pathways in the water are considerably different that exposure pathways on land.

b Birds are used as surrogates for terrestrial phase amphibians.

4 Citations for all registrant-submitted and open literatu re toxicity data reviewed for this assessment are included in Appendix A.

362.8.2 Assessment Endpoints for Desi gnated Critical Habitat

As previously discussed, designa ted critical habitat is assessed to evaluate actions related to the use of diflubenzuron that may alter the PCEs of the CRLF’s critical habitat. PCEs for the CRLF were previously described in Section 2.6 . Actions that may modify critical habitat are those that alter the PCEs and jeopa rdize the continued existence of the CRLF. Therefore, these actions are id entified as assessment endpoints. It should be noted that evaluation of PCEs as assessment endpoints is limited to those of a biological nature ( i.e.

, the biological resource requirements for the list ed species associated with the critical habitat) and those for which diflubenzuron effects data are available. Adverse modification to the critical habitat of the CRL F includes, but is not limited to, those listed in Section 2.6

. Measures of such possible effects by labele d use of diflubenzuron on critical habitat of the CRLF are described in Table 2-5. Some components of these PCEs are associated with physical abiotic features ( e.g. , presence and/or depth of a water body, or distance between two sites), which are not expected to be measurably altered by use of pesticides. Assessment endpoints used for the analysis of designated critical ha bitat are based on the adverse modification standard established by U.S. FWS (2006).

37Table 2-5. Summary of Assessment Endpoints and Measures of Ecological Effect for Primary Constituent Elements of Desi gnated Critical Habitat a

Assessment Endpoint Measures of Ecological Effect

Aquatic-Phase CRLF PCEs (Aquatic Breeding Habitat and Aquatic Non-Breeding Habitat) Alteration of channel/pond morphology or geometry and/or increase in sediment deposition within the stream channel or pond: aquatic habitat (including riparian vegetation) provides for shelter, foraging, predator avoidance, and aquatic dispersal for juvenile and adult CRLFs. a. Most sensitive aquatic plant EC 50 (guideline study: test species: green algae) b. Currently no guideline terrestrial plant toxicity data is available.

Alteration in water chemistry/quality including temperature, turbidity, and oxygen content necessary for normal growth and viability of juvenile and adult CRLFs and their food source. a. Most sensitive aquatic plant EC 50 b. Currently, no guideline terrestrial plant toxicity data is available.

Alteration of other chemical characteristics necessary for normal growth and viability of CRLFs and their food source. a. Most sensitive EC 50 or LC 50 values for fish and aquatic invertebrates (guideline and ECOTOX studies respectively; respective test species: bluegill sunfish and mosquito larvae) b. Most sensitive NOAEC values for fish and aquatic invertebrates (guideline and ECOTOX respectively; respective test species: fathead minnow and water flea) Reduction and/or modification of aquatic-based food sources for pre-metamorphs ( e.g., algae) a. Most sensitive aquatic plant EC 50 (guideline study; test species: green algae)

Terrestrial-Phase CRLF PCEs (Upland Habitat and Dispersal Habitat) Elimination and/or disturbance of upland habitat; ability of habitat to support food source of CRLFs: Upland areas within 200 ft of the edge of the riparian vegetation or dripline surrounding aquatic and riparian habitat that are comprised of grasslands, woodlands, and/or wetland/riparian plant species that provides the CRLF shelter, forage, and predator avoidance Elimination and/or disturbance of dispersal habitat: Upland or riparian dispersal habitat within designated units and betw een occupied locations within 0.7 mi of each other that allow for movement between sites including both natural and altered sites which do not contain barriers to dispersal Reduction and/or modification of food sources for terrestrial phase juveniles and adults Alteration of chemical ch aracteristics necessary for normal growth and viability of juvenile and adult CRLFs and their food source. a Currently, no guideline terrestrial plant toxicity data are available. b. c. Most sensitive food source acute EC 50 /LC 50 and NOAEC values for terrestrial vertebrates (mammals) and invertebrates, birds or terrestrial-phase amphibians, and freshwater fish.

a Physico-chemical water quality parameters such as salinity, pH, and hardness are not evaluated because these processes are not biologically mediated and, therefore, are not re levant to the endpoints included in this assessment.

382.9 Conceptual Model

2.9.1 Risk Hypotheses

Risk hypotheses are specific assumptions about potential adverse effects ( i.e. , changes in assessment endpoints) and may be based on theory and logic, empirical data, mathematical models, or probability models (U.S. EPA 1998). For this assessment, the risk is stressor-linked, where the stresso r is the release of diflubenzuron to the environment. The following risk hypotheses are presumed for this endangered species

assessment:

The labeled use of diflubenzuron within the action area may:

• directly affect the CRLF by causing mortal ity or by adversely a ffecting growth or fecundity;

• indirectly affect the CRLF by reducing or changing the composition of food supply;

• indirectly affect the CRLF or modify designated critical habitat by reducing or changing the composition of the aquatic plant community in the ponds and streams comprising the species’ current rang e and designated critic al habitat, thus affecting primary productivity and/or cover;

• indirectly affect the CRLF or modify designated critical habitat by reducing or changing the composition of the terrestrial plant community ( i.e. , riparian habitat) required to maintain acceptable water qual ity and habitat in the ponds and streams comprising the species’ current range and designated critical habitat;

• modify the designated critical habita t of the CRLF by reducing or changing breeding and non-breeding aqua tic habitat (via modifi cation of water quality parameters, habitat morphology, and/or sedimentation);

• modify the designated critical habita t of the CRLF by reducing the food supply required for normal growth and viabili ty of juvenile and adult CRLFs;

• modify the designated critical habita t of the CRLF by reducing or changing upland habitat within 200 ft of the edge of the riparian vegetation necessary for shelter, foraging, and pr edator avoidance;

• modify the designated critical habita t of the CRLF by reducing or changing dispersal habitat within designated units and between occupied locations within 0.7 mi of each other that allow for moveme nt between sites including both natural and altered sites which do not c ontain barriers to dispersal; or

• modify the designated critical habi tat of the CRLF by altering chemical characteristics necessary for normal growth and viability of juvenile and adult CRLFs.

2.9.2 Diagram

The conceptual model is a graphic representation of the structure of the risk assessment. It specifies the diflubenzuron release mechanisms, biological receptor types, and effects endpoints of potential concern. The conceptual models for all of the uses other than fly

39control at livestock operations for both terr estrial and aquatic exposures are shown in Figure 2-4 and Figure 2-5 , respectively, which include th e conceptual models for the aquatic and terrestrial PCE components of cri tical habitat. Exposure routes shown in dashed lines are not quantitatively considered because the contributi on of those potential exposure routes to pote ntial risks to the CRLF and modifi cation to designated critical habitat is expected to be negligible.

Diflubenzuron applied to use site Stressor

Source

Receptors

Attribute Change Direct application Spra y drift

Birds/terrestrialphase amphibians/ reptiles/mammals

Terrestrial

insects

Individual organisms Reduced survival Reduced growth Reduced reproduction Food chain Reduction in prey Modification of PCEs related to prey availability Habitat integrity Reduction in primary productivity Reduced cover Community change Modification of PCEs related to habitat Terrestrial/riparian plants grasses/forbs, fruit, seeds (trees, shrubs) Runoff

Mammals/ birds Exposure Media Soil

Ingestion In g estion

Ingestion In g estion Dermal u p take/In g estion Long range atmospheric

transport

Root uptake

Wet/dr y de p osition

Terrestrial-phase amphibians Ingestion

Figure 2-4 Conceptual Mode l for Pesticide Effects on Terrestrial Phase of the CRLF

4041 Diflubenzuron applied to use site Stre ssor

Source

Receptors

Attribute Change Spra y drift

Fish/aquatic-phase amphibians Exposure Media

Piscivorous mammals and birds

Individual organisms Reduced survival Reduced growth Reduced reproduction Food chain Reduction in algae Reduction in prey Modification of PCEs related to prey availability Habitat integrity Reduction in primary productivity Reduced cover Community change Modification of PCEs related to habitat Surface water/ Sediment Runoff

Aquatic Animals Invertebrates Vertebrates Uptake/gills or integument

In g estion In g estion Uptake/gills or integument

A quatic Plants Non-vascular Vascula r Uptake/cell, roots

, leaves Riparian plants terrestrial exposure pathways see

Figure 2.4 Long range atmospheric

transport

Wet/dry deposition

Groundwate r Soil

Figure 2-5. Conceptual Mode l for Pesticide Effects on Aquatic Phase of the CRLF.

Because the assessment methods are distinctly different for fly control at livestock operations, a separate conceptual model di agram is included al ong with the detailed description of these methods for this use in Appendix F. Other uses (right-of-ways in Appendix J and residential uses in Appendix K) have separate a ppendices to describe there unique assessment methods, but concep tually, are similar enough to use Figure 2-4 and 5 as the conceptual model diagram for these uses.

2.10 Analysis Plan

In order to address the risk hypot hesis, the potential for direct and indirect effects to the CRLF, its prey, and its habitat is estimat ed. In the following sections, the use, environmental fate, and ecological effects of diflubenzuron are characterized and integrated to assess the risks. This is accomplished using a risk quotient (ratio of exposure concentration to effect s concentration) approach. Although risk is often defined as the likelihood and magnitude of adverse ecological effect s, the risk quotient-based approach does not provide a quantitative estim ate of likelihood and/or magnitude of an adverse effect. However, as outlined in the Overview Document (U.S. EPA 2004), the likelihood of effects to indivi dual organisms from particular uses of diflubenzuron is estimated using the probit dose-response slope and either the level of concern (discussed below) or actual calculated risk quotient value.2.10.1 Measures to Evaluate the Risk Hypothesis and Conceptual Model

2.10.1.1 Measure s of Exposure

The environmental fate properties of diflube nzuron along with available monitoring data indicate that runoff and spra y drift are the principle potential transport mechanisms of diflubenzuron to the aquatic and terrestrial habitats of the CRLF. In this assessment, transport of diflubenzuron through runoff a nd spray drift is considered in deriving quantitative estimates of diflubenzuron expos ure to CRLF, its prey and its habitats. Long-range atmospheric transport of diflube nzuron is not considered due to this chemical’s physical properti es related to volatilization.

Measures of exposure are based on aquatic and terrestrial models that predict estimated environmental concentrations (EECs) of diflubenzuron using maximum labeled application rates and methods of application. The models used to predict aquatic EECs are the Pesticide Root Zone Model coupled with the Exposure Analysis Model System (PRZM/EXAMS) and GEN eric E xpected E nvironmental C oncentration (GENEEC). The model used to predict terrestrial EECs on food items is T-REX. These models are parameterized using relevant reviewed registrant-submitted environmental fate data.

PRZM (v3.12.2, May 2005) and EXAMS (v2.98.4.6, April 2005) are screening simulation models coupled with the input shell pe5.pl (Aug 2007) to generate daily exposures and 1-in-10 year EECs of diflube nzuron that may occur in surface water bodies adjacent to application sites receiving diflubenzu ron through runoff and spray drift. PRZM simulates pesticide applic ation, movement and transformation on an agricultural field and the result ant pesticide loadings to a receiving water body via runoff, erosion and spray drift. EXAMS simulates the fate of the pesticide and resulting concentrations in the water body. The standa rd scenario used fo r ecological pesticide assessments assumes application to a 10-hectar e agricultural field that drains into an adjacent 1-hectare water body, 2-meters deep (20,000 m 3 volume) with no outlet. PRZM/EXAMS was used to estimate screeni ng-level exposure of aquatic organisms to diflubenzuron. The measure of exposure for a quatic species is the 1-in-10 year return peak or rolling mean concentration. The 1-in -10 year peak is used for estimating acute exposures of direct effects to the CRLF, as well as indirect effects to the CRLF through effects to potential prey items, including: alg ae, aquatic invertebrate s, fish and frogs. The 1-in-10-year 60-day mean is used for assess ing chronic exposure to the CRLF and fish and frogs serving as prey items; the 1-in-1 0-year 21-day mean is used for assessing chronic exposure for aquatic invertebrates, which are also potential prey items.

The EFED GENEEC ( GEN eric E xpected E nvironmental C oncentration) model (version 1.2) is used to estimate pesticide concentra tions in a 1 hectare by 2 meter deep pond with no outlet draining an adjacent 10 hectare fi eld. It provides an upper-bound screening concentration value for most types of su rface water for up to 56 days after runoff. GENEEC is a single runoff event but accounts for spray drift from single or multiple applications. The pond receives a pesticide load from spray drif t for each application plus loading from a single-runoff event, in this cas e, two days after the last application. The

42runoff event transports a maximum of 10% of the pesticide remaining in the top 2.5 cm of soil. This amount can be reduced thr ough soil adsorption. The amount of pesticide remaining on the field in the top 2.5 cm of so il depends on the application rate, number of applications, interval between applications, incorporation depth, and degradation rate in the soil.

Exposure estimates for the terrestrial-phase CRLF and terrestria l invertebrates and mammals (serving as potential prey) assumed to be in the target area or in an area exposed to spray drift are derived usi ng the T-REX model (version 1.3.1, 12/07/2006). This model incorporates the Kenega nomograph, as modified by Fletcher et al. (1994), which is based on a large set of actual field residue data. The upper limit values from the nomograph represented the 95t h percentile of residue values from actual field measurements (Hoerger and Ke nega 1972). For modeling pur poses, direct exposures of the CRLF to diflubenzuron through contaminated food are estimated using the EECs for the small bird (20 g) which consumes small insects. Dietary-based and dose-based exposures of potential prey (small mammals) are assessed using the small mammal (15 g) which consumes short grass. The small bird (20g) consuming small insects and the small mammal (15g) consuming short grass are used because these categ ories represent the largest RQs of the size and dietary categorie s in T-REX that are appropriate surrogates for the CRLF and one of its prey items. Es timated exposures of terrestrial insects to diflubenzuron are bound by usi ng the dietary based EECs for small insects and large insects.

Birds are currently used as surrogates for te rrestrial-phase CRLF. However, amphibians are poikilotherms (body temperat ure varies with environmenta l temperature) while birds are homeotherms (temperature is regulate d, constant, and largely independent of environmental temperatures). Therefore, am phibians tend to have much lower metabolic rates and lower caloric intake requirements than birds or mammals. As a consequence, birds are likely to consume more food than amphibians on a daily dietary intake basis, assuming similar caloric content of the f ood items. Therefore, the use of avian food intake allometric equation as a surrogate to amphibians is likely to result in an overestimation of exposure and risk for reptiles a nd terrestrial-phase amphibians. Therefore, T-REX (version 1.3.1) has been refined to th e T-HERPS model (v. 1.0), which allows for an estimation of food intake for poikilotherms using the same basic procedure as T-REX to estimate avian food intake.

AgDRIFT was used to assess aquatic exposures of aquatic phase CRLF and its prey to diflubenzuron deposited in aquatic habitats by spray drift and to determine the maximum distance from a treated area that effects may occur.

2.10.1.2 Measure s of Effect

Diflubenzuron is a chitinase inhibitor, impair ing the ability of in sects to synthesize a critical component of their exoskeleton (U.S. EPA, 1997). Chitin synthesis is particularly important in the early life stages of insects, as they molt and form a new exoskeleton in

43various growth stages. Thus, aquatic guideli ne tests, which typically run for 48 hours for the aquatic invertebrate ( Daphnia magna ), may not capture a molting stage, and are not an appropriate “most sensitive” acute endpoint for assessments. Endpoints derived from studies that test the toxic effects of diflubenzuron on th e larval/molting stages of freshwater invertebrates and th at test the chronic exposure of freshwater invertebrates to diflubenzuron more appropriately assess the toxicity of this ty pe of chemical. Diflubenzuron is fairly persistent in both a quatic and terrestrial environments. For this reason, data that evaluated the toxic effects of diflubenzuron at life stages (larval/molting stages) most vulnerable to this chemical we re used as both acute and chronic endpoints for aquatic invertebrate RQs. Likewise, guideline tests on honeybees frequently run for only 48 hours and are conducted on a dult bees, and, therefore, ma y not reflect the toxicity of diflubenzuron.

Data identified in Section 2.8 are used as measures of effect for direct and indirect effects to the CRLF. Data were obtained from regi strant submitted studies or from literature studies identified by ECOTOX. The ECOTOXicology database (ECOTOX) was searched in order to provide more ecological effects data and in an attempt to bridge existing data gaps. ECOTOX is a source for locating single chemical toxicity data for aquatic life, terrestrial plants, and wildlife. ECOTOX was created and is maintained by the U.S. EPA, Office of Research and Devel opment, and the National Health and Environmental Effects Research Laboratory's Mid- Continent Ecology Division.

The assessment of risk for direct effect s to the terrestrial-phase CRLF makes the assumption that toxicity of diflubenzuron to bird s is similar to or less than the toxicity to the terrestrial-phase CRLF. The same assu mption is made for fish and aquatic-phase CRLF. Algae, aquatic invertebrates, fish, and amphibians represent potential prey of theCRLF in the aquatic habitat. Terrestrial invertebrates, small mammals, and terrestrialphase amphibians represent potential prey of th e CRLF in the terrestrial habitat. Aquatic, semi-aquatic, and terrestrial plants represent habitat of CRLF.

The acute measures of effect used for animal s in this screening level assessment are the LD 50 , LC 50 and EC 50 . LD stands for "Lethal Dose", and LD 50 is the amount of a material, given all at once, that is estimated to cause the death of 50% of the test organisms. LC stands for “Lethal Concentration” and LC 50 is the concentration of a chemical that is estimated to kill 50% of the test organisms. EC stands for “Effective Concentration” and the EC 50 is the concentration of a chemical that is estimated to produce a specific effect in 50% of the test organisms. Endpoints for chronic measures of exposure for listed and non-listed animals are the NOAEL/NOAEC an d NOEC. NOAEL stands for “No Observed-Adverse-Effect-Level” and refers to the highest tested dose of a substance that has been reported to have no harmful (adve rse) effects on test organisms. The NOAEC ( i.e. , “No-Observed-Adverse-Effect -Concentration”) is the highest test concentration at which none of the observed effects were statis tically different from the control. The NOEC is the No-Observed-Effects-Concentra tion. For non-listed plants, only acute exposures are assessed ( i.e. , EC 25 for terrestrial plants and EC 50 for aquatic plants).

44It is important to note that the measures of effect for direct and indirect effects to the CRLF and its designated critical habitat are as sociated with impacts to survival, growth, and fecundity, and do not include the full suite of sublethal effects used to define the action area. According the Overview Docu ment (USEPA 2004), the Agency relies on effects endpoints that are either direct measures of impairme nt of survival, growth, or fecundity or endpoints for which there is a scientifically robust, peer reviewed relationship that can quantify the impact of the measured effect endpoint on the assessment endpoints of survival, growth, and fecundity.

2.10.1.3 Integra tion of Exposure and Effects

Risk characterization is the integration of exposure and ecological e ffects characterization to determine the potential ecolo gical risk from agricultural and non-agricultural uses of diflubenzuron, and the likelihood of direct and indi rect effects to CRLF in aquatic and terrestrial habitats. The expos ure and toxicity effects data are integrated in order to evaluate the risks of adverse ecological effects on non-target species. For the assessment of diflubenzuron risks, the risk quotient (R Q) 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 Agen cy’s levels of con cern (LOCs) (U.S. EPA 2004) (see Appendix C).

For this endangered species assessment, listed species LOCs are used for comparing RQ values for acute and chronic exposures of diflubenzuron directly to the CRLF. If estimated exposures directly to the CRLF of diflubenzuron resulting from a particular use are sufficient to exceed the listed species LOC, then the effects determination for that use is “may affect”. When considering indirect effects to the CRLF due to effects to animal prey (aquatic and terrestrial invertebrates, fish, frogs, and mice), the listed species LOCs are also used. If estimated exposures to CRLF prey of diflubenzuron resulting from a particular use are sufficient to exceed th e listed species LOC, then the effects determination for that use is a “may affect.” If the RQ being considered also exceeds the non-listed species acute risk LOC, then the eff ects determination is a LAA. If the acute RQ is between the listed species LOC and th e non-listed acute risk species LOC, then further lines of evidence ( i.e ., probability of individual effects, species sensitivity distributions) are considered in distinguishing between a determination of NLAA and a LAA. When considering indir ect effects to the CRLF due to effects to algae as dietary items or plants as habitat, the non-listed sp ecies LOC for plants is used because the CRLF does not have an obligate relationship with any pa rticular aquatic and/or terrestrial plant. If the RQ being considered for a particul ar use exceeds the non-listed species LOC for plants, the effects determination is “may affect”. Further information on LOCs is provided in Appendix C.

452.10.1.4 Data Ga ps No guideline terrestrial plant toxicity data ha ve been submitted to the Agency. However, open literature data and other lines of eviden ce will be used to evaluate the potential for diflubenzuron to indirectly affect CR LFs via impacts to terrestrial plants.



### 3.0 Exposure Assessment

Diflubenzuron formulations include liquid, wate r dispersible granules, wettable powder, effervescent tablets (for orna mental pond treatment), and term ite bait (in bait stations). Application equipment includes ground, aerial, and airblast applic ators and directed sprayers. Risks from ground boom and aerial ap plications are expected to result in the highest off-target levels of diflubenzuron due to generally higher spray drift levels. Ground boom and aerial modes of application te nd to use lower volumes of application applied in finer sprays than applications coin cident with sprayers and spreaders and thus have a higher potential for off-targ et movement via spray drift.

3.1. Label Application Rates and Intervals

Diflubenzuron labels may be categorized into two types: labels for manufacturing uses (including technical grade diflubenzuron a nd its formulated products) and end-use products. While technical products which c ontain diflubenzuron of high purity are not used directly in the environment, they are used to make formulated products, which can be applied in specific areas to control insects. The formulated product labels legally limit diflubenzuron’s potential use to only those sites that are specified on the labels.

Currently registered agricu ltural and non-agricultural uses of diflubenzuron within California include: almond, Brazil nut, cashew, chestnut, chinquapin, filbert (hazelnut), hickory nut, macadamia nut (bushnut), pecan, tree nuts, walnut (English/black), citrus hybrids other than tangelo, grapefruit, ora nge, pummelo (shaddock), tangerines, broccoli raab, cabbage - Chinese, collards, kale, s oybeans (unspecified), cotton (unspecified), Christmas tree plantations, forest trees (softw oods - conifers), apricot, nectarine, peach, pear, plum, prune, stone fruits, commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (out door), wide area/general outdoor treatment (quarantine/eradication use), but ternut, forest nursery planting s (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, shelterbelt plantings, turnip (greens), household/domestic dwellings outdoor premises, artichoke, mustard, peanuts (unspecified), pepper, agricult ural fallow/idleland, golf course turf, nonagricultural uncultivated areas /soils, barley, canola\rape, oa ts, triticale, wheat, grass forage/fodder/hay, pastures, rangeland, agri cultural rights-of-way /fencerows/hedgerows, nonagricultural rights-of-way/fencer ows/hedgerows, agricultural/farm structures/buildings and equipment, barns/ barnyards/auction barns, manure, brassica (head and stem) vegetables, ornamental sod farm (turf), recreational areas, rice, drainage systems, forest plantings (reforestation progr ams)(tree farms - tree plantations - etc.), forest trees (all or unspecified), forest trees (softwoods - conifers), intermittently flooded areas/water, lakes/ponds/reservoirs (with human or wildlife use), lakes/ponds/reservoirs

46(without human or wildlife use), ornamental herbaceous plants, ornamental ponds/aquaria, ornamental woody shrubs and vines, shelterbelt plantings, streams/rivers/channeled water, swimming pool water systems, wide area/general outdoor treatment (quarantine/er adication use), beech nut, pist achio. The uses being assessed are summarized in Table 3-1.

47Table 3-1 Diflubenzuron Uses, Scenarios, and Application Information for the CRLF risk assessment 1

Scenario Uses Represented by Scenario Application Rate Number of Applications Application Interval Application Method 1. Barnyard Spray on manure, Manure to field 8.508 NS (17) NS (21) Ground 0.0408 3 7 Ground 2. Beech Nut Beech Nut 0.0408 3 7 Air 3 . Brassica Brassica (head and stem) vegetables 0.0313 1 NA Ground 0.3125 3 21 Ground 4 . Citrus Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines 0.125 3 7 Air

5 . Cole Crop Broccoli raab, cabbage - Chinese, collards, and kale 0.25 4 21 Ground

0.3125 3 7 Ground 6 . Cotton Cotton (unspecified) 0.3125 3 90 Air 0.22048 NS (4) 14 Ground 7 . Forest Christmas tree plantations and forest trees (softwoods - conifers) 0.25 1 NA Air 0.3125 3 7 Ground 0.3125 3 7 Airblast 8 . Fruit Apricot, nectarine, peach, pear, plum, prune, and stone fruits 0.3125 3 7 Air
0.3125 3 7 Ground

0.3125 3 7 Airblast 9 . General Nuts Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (hazelnut), hickory nut, macadamia nut (bushnut), pecan, tree nuts, and walnut (English/black) 0.3125 3 90 Air
0.25 4 21 Ground 10 . Grains Barley, canola\rape, oats, triticale, and wheat 0.125 3 NS (21) Air

0.25 1 NA Ground

11 . Nursery Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings 0.25 1 NA Air

0.25 1 NA Ground 0.25 1 NA Airblast 12 . Pasture Grass forage/fodder/hay, pastures, and rangeland 0.25 1 NA Air 13 . Pistachio Pistachio 0.75 2 14 Ground 0.25 1 NA Ground 14

. Residential Household/domestic dwellings outdoor premises 0.25 6 10 Air 15 . Rice Rice 0.625 6 5 Air

0.25 2 5 Ground

0.25 2 5 Airblast 16 . Rightsof-way Agricultural rights-ofway/fencerows/hedgerows, fencerows/hedgerows, andnonagricultural rights-ofway/fencerows/hedgerows 0.25 2 5 Air

0.3125 3 21 Ground 17 . Row Crop Artichoke, mustard, peanuts (unspecified), and pepper 0.3125 3 14 Air

18 . Urban Commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) 0.25 6 NS (10) Air

0.25 4 15 Ground 19 . Squash Butternut 0.25 4 15 Air

48Scenario Uses Represented by Scenario Application Rate Number of Applications Application Application Interval Method 0.25 2 14 Ground 0.25 2 14 Airblast 20 . Turf (High Appl. Rate) Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils 0.25 2 5 Air 0.0313 3 7 Ground 0.0313 3 7 Airblast 21 . Turf (Low Appl. Rate) Ornamental sod farm (turf) and recreational areas 0.0313 3 7 Air 22 . Turnip Turnip (greens) 0.25 2 21 Ground

1 Uses assessed based on memorandum from SRRD dated 2/24/09. NS = Not specified on the label.

The large number of scenarios is mainly a re flection of the diverse uses of diflubenzuron. However, some uses were split from other similar uses because they had much higher or lower application rates.

Some of the scenarios require additional explanation. The barnyard s cenario is a direct spray of diflubenzuron on manure. This scen ario, methods of analysis, and resulting EECs are discussed in detail in Appendix F. Similarly, the rationales for the residential and right-of-way scenarios are de scribed in appendices J and K.

In addition, diflubenzuron can be administered to cattle with a gun-like device that shoots a “bolus” impregnated with diflubenzuron dow n the throats of the cattle. This bolus slowly dissolves and releases diflubenzuron w ith the waste. One bolus treats an animal for as long as 21 weeks. Similarly, diflubenzuron can be administ ered through cattle and swine feed.

EFED does not have data indicating the diflubenzuron concentration in the manure resulting from all three methods (direct spray on manure, bolus, or feed-through treatments). However, the desired result from all three methods is to produce manure that has a concentration of diflube nzuron that inhibits the growth of the same species of nuisance flies. Therefore, EFED assumes for purposes of this assessment that the exposure levels from all three methods of manure treatment would be similar and considers the barnyard EECs to apply to all three manure treatment methods.

The urban scenario provides EECs for the quarantine/eradica tion use of diflubenzuron if used in urban areas. It assumes 100% impe rvious surfaces in the watershed treated.

Another non-standard diflubenzuron use is or namental pond care. Because this use is a direct application to water (ornamental ponds) and results in EECs that are roughly equivalent to the EECS produced by the ti er 1 rice model for the rice uses of diflubenzuron, the ornamental pond care us e is assessed using the rice model EECs. However, an important difference between the rice use and ornamental pond care is ornamental ponds are required to not be connect ed to natural drainage-ways, while rice paddy water is assumed to be discharged to waters off of the use site.

As an example, the ornamental pond care EEC for label registration # 400-469 is calculated as:

49Lg galLgal ggg

Volume Amount pecent EEC

Treated Applied ron Diflubenzu

μ μ 66 8.31000 101%25 6

= × ××

= ×

=

where: EEC is the ornamental pond concentration after treatment in μg/L; Amount Applied is the amount of diflubenzuron applied to each 1000 gallons of pond water treated (converted to μg); and volume treated is 1000 gallons (converted to liters).

The last non-standard use is ap plication of diflubenzuron to th e material (often compost) in which mushrooms are grown. The compost is treated with diflubenzuron to keep nuisance flies from harming the mushroom cr op. As mushrooms are grown, the ability of the compost material to grow mushrooms decr eases. Therefore, the compost material that was treated with diflubenzuron must be peri odically replaced and the old material discarded. Often this material is land applied on farm fields.

Mushrooms can be grown outdoors or in mu shroom houses. When grown outdoors, there is potential for exposure to CRLFs while the di flubenzuron-treated compost is in use for growing mushrooms as well as after it has been land applied. Alternatively when mushrooms are grown indoors, exposure to the diflubenzuron-tre ated compost would only occur after the compost is land applied on farm fields.

The mushroom use of diflubenzuron is very similar to the barnyard or manure use described previously in terms of both exposure at the site of applicati on and at the site of disposal through land applicati on as well as the concentratio n in the compost and manure. For the barnyard or manure use (Appendix F) , the resultant diflubenzuron concentration in the manure is approximately 10 to 60 ppm (Appendix F, Figure F3). For the mushroom use, the desired diflubenzuron concentration in the compost is approximately 30 to 50 ppm. Therefore, mushroom uses will be assessed using the EECs generated for the barnyard use.

An important assumption in the barnyard an alysis (appendix F) is that degradation behavior and rate of diflubenz uron in manure is similar to the degradation behavior and rate of diflubenzuron in soil. This assump tion is made because EFED does not have diflubenzuron fate data in manure. Similarl y, the assumption is made that the behavior and rate of diflubenzuron degradation in mush room compost is similar to the degradation behavior and rate of diflubenzuron in soil because EFED does not have diflubenzuron fate data in mushroom compost.

503.2. Aquatic Exposure Assessment

3.2.1. Modeling Approach

Aquatic exposures are quantitatively estimated for all of assessed uses using scenarios that represent high exposure si tes for diflubenzuron use. Each of these sites represents a 10 hectare field that drains into a 1-hectare pond that is 2 meters deep and has no outlet. Exposure estimates generated using the sta ndard pond are intended to represent a wide variety of vulnerable water bodi es that occur at the top of watersheds including prairie pot holes, playa lakes, wetlands, vernal pools, man-made and natural ponds, and intermittent and first-order streams. As a group, there are factors that make these water bodies more or less vulnerable than the standa rd surrogate pond. Stat ic water bodies that have larger ratios of drainage area to water body volume would be expected to have higher peak EECs than the standard pond. These water bodies will be either shallower or have large drainage areas (or both). Shallow water bodies te nd to have limited additional storage capacity, and thus, tend to overflow a nd carry pesticide in the discharge whereas the standard pond has no discharge. As wa tershed size increases beyond 10 hectares, at some point, it becomes unlikely that the enti re watershed is planted to a single crop, which is all treated with the pesticide. Headwater streams can also have peak concentrations higher than the standard pond, but they tend to persist for only short periods of time and are then carried downstream.

Crop-specific management practices for all of the assessed uses of diflubenzuron were used for modeling, including application ra tes, number of applications per year, application intervals, buffer widths and re sulting spray drift values modeled from AgDRIFT, and the first application date for each crop. The date of first application was developed based on the use information provi ded by BEAD and a summary of individual applications by use from the CDPR PUR data. Because diflubenzuron persists in fields for only a short time after application (aerobic soil metabolism half-life of 2 to 14 days) and most runoff in the semi-arid portions of California occur mainly in the winter and early spring, application date will greatly affect the estima ted exposure to diflubenzuron.

As an example of identifying scenario appl ication dates based on PUR data, Figure 3-1 depicts variability in diflubenzuron applicati on by date within a calendar year for ground citrus applications based on CDPR PUR data from 1990 through 2007. The curvy indigo (dark blue) line indicates the average pounds of diflubenzuron applied on each day. This average is calculated as a 15-day moving aver age of the average total pounds applied for each day of the year. The solid vertical li ne indicates the date on which this 15-day average is highest. The dashed vertical line s the first and last application dates which were calculated to bracket the highest daily average. Similar analyses for other uses are included in Appendix D, Figure D3. Because PU R data only identifies applications as ground or air, airblast app lication dates are based on th e highest 15-day average of ground applications.

510 1 2 3 4 5 6

J J MAM J J A S OND J

Calender DateAverage Diflubenzuron Applied perDay (lbs. ai/Day)

F

Figure 3-1. Identification of application dates based on analysis of the average pounds of diflubenzuron applied in Calif ornia to citrus via ground application methods based on CDPR PUR data (1990-2007).

This method of selecting application dates works well for those uses that are wellrepresented in the PUR data set. However many of the uses that can be legally applied in California (and therefore, must be assessed in this document), do not appear in the PUR data set. For those uses that do not occu r in the PUR, the model was run for each potential date of application a nd the date that resulted in the highest exposure was used in order to ensure a conservative exposure estimate. This method is described at the end of Section 3.2.3.

3.2.2. Model Inputs

Diflubenzuron is an insecticide used on a wide variety of food and non-food uses. Diflubenzuron environmental fate data used fo r generating model parameters is listed in Table 2-2 . The input parameters for PRZM and EXAMS are in Table 3-2.

52Table 3-2 Summary of PRZM/EZAMS Envi ronmental Fate Data Used for Aquatic Exposure Inputs for Diflubenzuron Endangered Species Assessment for the CRLF 1
Fate Property Value (unit) MRID (or source) Molecular Weight 311 g/mol Product Chemistry Henry’s constant 1.87×10 -09 atm-m 3 /mol Product Chemistry Vapor Pressure 9.00×10 -10 torr Product Chemistry Solubility in Water 0.2 mg/L Product Chemistry

Photolysis in Water 80 days 40816301 41087802

Aerobic Soil Metabolism Half-lives 4.7 days 00039473 00039474 41722801

Hydrolysis Stable @ pH 5 119 days @ pH 7 32 days @ pH 9 00143355 40859801 41087801 40816301

Aerobic Aquatic Metabolism (water column) 23.4 days Combined Data Mean = 11.70 days SD = 12.41 days, n=3

t 90, n-1 = 1.638
t input = (mean of t 1⁄2 ) + (t 90,n-1 ×SD)/n 1⁄2 Anaerobic Aquatic Metabolism (benthic) 102 days 34 days×3

Koc 3961 Mean from Table 2-1 Kocs

Application rate and frequency (See Table 3-1) Label or Maximum from LUIS Report

Application intervals (See Table 3-1) Label or Minimum from LUIS Report Chemical Application Method (CAM) 2 Input Parameter Guidance 1 Application Efficiency 0.99 (Ground/Airb last) or .95 (Air) Input Parameter Guidance 1

Spray Drift Fraction 2 0.0007 (Ground), .015 (Airblast), or .039 (Air) AgDRIFT Model

1 Inputs determined in accordance with EFED “Guidance for Chemistry and Management Practice Input Parameters for Use in Modeling the Environmental Fate and Transport of Pesticides” dated February 28, 2002.

2 Spray drift fraction assumed a buffer of 150 feet. The fraction was calculated using AgDrift and default Tier 1 parameters.

3.2.3. Results

The aquatic EECs for the various scenarios and application me thods are listed in Table3- 3 based on the first application date indi cated in column three of this table

. The highest EECs (113 μg/L) are for rice/ornamental pond (direct application to water). Because rice/ornamental pond EECs were calculated using the rice model which assumes no degradation, the highest pea k, 1-in-10-year 21-day averag e, and 1-in-10-year 60-day average EECs are all the same. Excluding the rice/ornamental pond EECs: peak EECs varied by a factor of 5400 between uses from 0.01 to 54 μg/L; 1-in-10-year 21-dayaverage EECs varied by a factor of 6900 between uses from 0.006 to 42 μg/L; and 1-in10-year 60-day average EECs varied by a factor of 7500 between uses from 0.004 to 27 μg/L.

53Table 3-3 Aquatic EECs ( μ g/L) for Diflubenzuron Uses in California

Scenario ( G round, A ir b last, or A ir) Appl. Rate 1 st – last Appl. Date 1 Crops Represented Peak EEC 21-day

avera g e

EEC 60-day
average

EEC Spray on manure 54.20 42.00 27.30 1. Barnyard/ Mushroom G 8.508 Year-round 17 appl. Treated manure applied to field 7.29 5.63 3.65 G 0.0408 NP (1/12) 0.11 0.06 0.03 2. Beech

Nut A 0.0408 NP (1/12) Beech Nut 0.21 0.13 0.08 3 . Brassica G 0.0313 NP (12/30) Brassica (head and stem) vegetables 0.07 0.04 0.02 G 0.3125 4/23 – 5/14 0.24 0.10 0.05 Citrus hybrids, grapefruit, orange, pummelo (shaddock), and tangerines 4 . Citrus A 0.125 4/23 – 5/14 0.50 0.30 0.18 Broccoli raab, cabbage - Chinese, collards, and kale 5

. Cole Crop G 0.25 9/11 – 12/4 0.36 0.20 0.14

G 0.312 5 5/14 – 6/4 0.15 0.11 0.06 6 . Cotton Cotton (unspecified) A 0.3125 3/14 – 12/9 0.82 0.47 0.30 G 0.22048 6/14 – 7/5 0.58 0.31 0.19 7 . Forest A 0.25 3/10 Christmas tree plantations and forest trees (softwoods - conifers) 0.80 0.45 0.26 G 0.3125 2/20 – 3/12 0.26 0.14 0.08 AB 0.3125 2/20 – 3/12 0.66 0.43 0.26 8 . Fruit A 0.3125 3/24 – 4/14 Apricot, nectarine, peach, pear, plum, prune, and stone fruits 1.36 0.86 0.54

G 0.3125 1/13 – 4/6 0.90 0.46 0.25

AB 0.3125 1/13 – 4/6 1.26 0.70 0.42 9 . General Nuts
A 0.3125 1/26 – 3/29 Almond, Brazil nut, cashew, chestnut, chinquapin, filbert (hazelnut), hickory nut, macadamia nut (bushnut), pecan, tree nuts, and walnut (English/black) 0.85 0.49 0.28

G 0.25 NP (1/19) 1.34 0.78 0.56 10 . Grains A 0.125 NP (1/19) Barley, canola\rape, oats, triticale, and wheat 0.93 0.52 0.41

G 0.25 7/4 0.20 0.11 0.08

11 . Nursery

A 0.25 12/5 Forest nursery plantings (for transplant purposes), ornamental and/or shade trees, ornamental woody shrubs and vines, and shelterbelt plantings 0.65 0.38 0.30

G 0.25 2/28 0.12 0.08 0.04 AB 0.25 2/28 0.29 0.17 0.09 12 . Pasture A 0.25 6/6 Grass forage/fodder/hay, pastures, and rangeland 0.55 0.25 0.13 13 . Pistachio G 0.75 3/30 Pistachio 0.15 0.08 0.06 G 0.25 NP (1/10) 0.08 0.05 0.03 14

. Residential A 0.25 NP (1/10) Household/domestic dwellings outdoor premises 0.40 0.30 0.25 15 . Rice/ Ornamental Pond A 0.625 5/14 – 6/13 Rice/Ornamental Pond 112.92 112.92 112.92

G 0.25 NP (1/12) 5.55 3.04 1.61

AB 0.25 NP (1/12) 8.13 4.39 2.32 16 . Rightsof-way

A 0.25 NP (1/12) Agricultural rights-ofway/fencerows/hedgerows, fencerows/hedgerows, andnonagricultural rights-ofway/fencerows/hedgerows 5.59 3.07 1.63

17 . Row Crop G 0.3125 8/14 - 10/16 Artichoke, mustard, peanuts (unspecified), and pepper 0.02 0.01 0.01

54Scenario ( G round, A ir b last, or A ir) Appl. Rate 1 st – last Appl. Date 1 Crops Represented Peak EEC 21-day

avera g e

EEC 60-day
average

EEC A 0.3125 2/1 – 3/14 1.15 0.77 0.58

18 . Urban A 0.25 NP (1/10) Commercial/institutional/industrial premises/equipment (outdoor), nonagricultural outdoor buildings/structures, paved areas (private roads/sidewalks), refuse/solid waste sites (outdoor), and wide area/general outdoor treatment (quarantine/eradication use) 34.13 21.98 15.28

G 0.25 NP (1/10) 1.15 0.65 0.38 19 . Squash A 0.25 NP (1/10) Butternut 1.87 1.18 0.79 G 0.25 7/5 – 8/2 0.01 0.01 0.00 AB 0.25 7/5 – 8/2 0.21 0.13 0.08 20 . Turf (High Appl. Rate) A 0.25 5/26 – 6/5 Agricultural fallow/idleland, golf course turf, and nonagricultural uncultivated areas/soils 0.61 0.37 0.20 G 0.0313 7/5 – 8/2 0.02 0.01 0.01 AB 0.0313 7/5 – 8/2 0.07 0.04 0.02 21 . Turf (Low Appl. Rate) A 0.0313 5/26 – 6/5 Ornamental sod farm (turf) and recreational areas 0.13 0.08 0.05 22 . Turnip G 0.25 NP (1/18) Turnip (greens) 0.72 0.35 0.19

1 NP = Not in CDPR Pesticide Use Reporting data set. Typically, the date of first application that yielded the highest peak concentration (indicated in parentheses) was used as the first application date in the scenario modeled (see text for date selection for residential and right-of-way uses).

The EECs for several of the uses are based on application dates that were selected specifically because that application date would yield the highest EECs. These highest dates were used to produce a conservative es timate EECs because there was no PUR data available to better indicate when diflubenz uron would be applied for that use (Section 3.2.1). Figure 3-2 depicts the variation in PRZM/EXAMS EECs based on the day of the year chosen as the first app lication date. As shown in Fi gure 3.2a, using the date that yields the highest EECs (typically in the wint er) could be extremely conservative (high EECs) if the actual application of diflubenzur on would only occur in the summer. This is not always the case though as is shown in Figure 3.2b. Typically, ground applications (Figure 3.2a) will show large va riations between winter a nd summer diflubenzuron EECs because ground application EECs are dominated by runoff contributions which vary with rainfall. Aerial applications (Figure 3.2b) resu lt in a much larger spray drift contribution, which does not vary with application date and therefore, results in less variation in EECs between summer and winter. Appendix D, Figur e D4, provides similar graphs for all of the diflubenzuron uses for which PUR data was not available.

55Ground Applications Aerial ApplicationsDiflubenzuron EECs (μg/L)

0 0.02 0.04 0.06 0.08 0.1 0.12

J FM AM J J A S O N D J peak 21day 60day Beech Nut

0 0.05 0.1 0.15 0.2 0.25

J FM AM J J A S O N D J peak 21day 60day Beech Nut

Calendar Date Calendar Date a b

Figure 3-2. Variation in diflubenzuron EECs (peak, 21-day, and 60-day) as function of application date (first application date for scen arios with multiple applications).

However, even for those uses for which the PU R data suggest a specific date is typically used for diflubenzuron applications (as in Figur e 3-1), it is important to consider that diflubenzuron is applied over a range of dates. In Figure 3-3, the dist ribution of PUR data application dates is compared to the diflube nzuron EECs for each first application date. According to the PUR data, March 2 nd is the most typical app lication date, while January 25 th generates the highest EECs. It is important to consider that diflubenzuron is applied on the date that yields the highest EECs, but because of the procedure used to select application dates, applica tion dates surrounding March 2 nd were used in this assessment. Appendix D, Figure D5, provides similar gra phs for all of the diflubenzuron uses for which PUR data were available.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

J FM AM J J A S O N D J

Calendar DateDiflubenzuron EECs (μg/L)

0 1 2 3 4 5 6 7Pounds Applied (lbs. ai/day) peak 21day 60day

PUR

Figure 3-3. Variation in diflubenz uron EECs (peak, 21-day, and 60-d ay) as function of application date (first application date for scenarios with mu ltiple applications) compared to variation in the amount of diflubenzuron applied (lbs. ai/d ay) per day in California for fruit use.

563.2.4. Existing Monitoring Data

A critical step in the process of characteri zing EECs is comparing the modeled estimates with available surface water monitoring data . EFED was unable to locate diflubenzuron monitoring for surface and groundwater and atmospheric data (Fellers et al 2004, LeNoir et al 1999, and McConnell et al 1998). The sources checked included the USGS National Water-Quality Assessment (NAWQA) Program a nd California Department of Pesticide Regulation (CDPR) data sets.

3.3. Terrestrial Animal Exposure Assessment

T-REX (Version 1.4.1) is used to calculate dietary and dose-based EECs of diflubenzuron for the CRLF and its potential prey ( e.g . small mammals and terrestrial insects) inhabiting terrestrial areas. EECs used to re present the CRLF are also used to represent exposure values for frogs serving as potential prey of CRLF adults.

Terrestrial EECs for foliar formulations of diflubenzuron were derived for the uses summarized in Table 3-4. Foliar dissipati on half-lives were reported in Willis and McDowell (1987) for diflubenzuron of 12, 27, and 35 days. Therefore, a 35-day half-life was used in this assessment. Use specific input values, including number of applications, application rate and application in terval are provided in Table 3-4.

57Table 3-4 Input Parameters for Foliar Applications Used to Derive Terrestrial EECs for Diflubenzuron with T-REX

Maximum Application Rate (lbs/A) Number of Applications (Re-treatment Interval in Days) Scenario Name a Aerial Ground Other Aerial Ground Other Barnyard/Mushroom 8.508 17 (21) Beech nut 0.0408 0.0408 2.3 (7) 2.3 (7) Brassica 0.0313 1 (NA) Citrus 0.125 0.3125 3 (7) 3 (21) Cole Crop 0.25 4 (21) Cotton 0.3125 0.3125 3 (90) 3 (7) Forest 0.25 0.22048 1 (NA) NS 1 (14) Fruit 0.3125 0.3125 3 (7) 3 (7) General Nuts 0.3125 0.3125 3 (90) 3 (7) Grains 0.125 0.25 3 (NS 2 ) 4 (21) Nursery 0.25 0.25 1 (NA) 1 (NA) Pasture 0.25 0.25 1 (NA) 1 (NA) Pistachio 0.75 1.333 (14) Residential 0.25 0.25 1 (NA) 6 (10) Rice/Ornamental Pond 0.0625 6 (5) Rights-of-way 0.25 0.25 1.5 (5) 1.5 (5) Row Crop 0.3125 0.3125 3 (14) 3 (21) Squash 0.25 0.25 NS 3 (15) 4 (15) Turf (High App. Rate) 0.25 0.25 1.5 (5) 1.5 (14) Turf (Low App. Rate) 0.0313 0.0313 3 (7) 3 (7) Turnip 0.25 2 (21) NA = Not applicable (there is no re-treatment interval if only 1 application is allowed). NS = Not specified on any label.

a No terrestrial assessment was performed for bait, pond, and soybean exposure scenarios (see text).

1 The number of applications was assumed to be 4 as a conservative assumption because 4 applications were the most applications allowed on any non-residential label.

2 A re-treatment interval of 21 days is assumed because 21 days is the smallest re-treatment interval allowed on the ground application to grains label.

3 The number of applications was assumed to be 4 as a conservative assumption because 4 applications were the most applications allowed on any non-residential label.

T-REX is also used to calculate EECs for terrestrial insects expos ed to diflubenzuron. Dietary-based EECs calculated by T-REX for sma ll and large insects (units of a.i./g) are used to bound an estimate of exposure to te rrestrial insects. Av ailable acute contact toxicity data for bees exposed to diflubenzuron (in units of μg a.i./bee), are converted to μg a.i./g (of bee) by multiplying by 1 bee/0.128 g. The EECs are later compared to the adjusted acute contact toxicity data fo r bees in order to derive RQs.

For modeling purposes, exposures of the CRL F to diflubenzuron through contaminated food are estimated using the EECs for the small bird (20 g) which consumes small insects. Dietary-based and dose-based exposur es of potential prey are assessed using the small mammal (15 g) which consumes short grass. Upper-bound Kenega nomogram values reported by T-REX for these two organi sm types are used for derivation of EECs for the CRLF and its potential prey (Table 3-5). Dietary-based EECs for small and large

58insects reported by T-REX as well as the resulting adjusted EECs are available in Table 3-6.

Table 3-5. Upper-bound Kenega Nomogram EECs for Dietary- and Dose-based Exposures of the CRLF and its Prey to Diflubenzuron. EECs for Prey (short grass food item) EECs for CRLF (small insect food item) (small mammals) Dose-based EEC Dose-based EEC Use Dietary-based EEC (ppm) (mg/kg-bw) Dietary-based EEC (ppm) (mg/kg-bw) Barnyard/Mushroom 3369 3840.66 5990 5690.5 Beech nut 10.3 11.742 18.32 17.404 Brassica 4.2 4.788 7.5 7.125 Citrus 88 100.32 157 149.15 Cole Crop 80 91.2 143 135.85 Cotton 111 126.54 197 187.15 Forest 34 38.76 60 57 Fruit 111 126.54 197 187.15 General Nuts 111 126.54 197 187.15 Grains 80 91.2 143 135.85 Nursery 34 38.76 60 57 Pasture 34 38.76 60 57 Pistachio 101 115.14 180 171 Residential 131 149.34 232 220.4 Rice/Ornamental Pond 40 45.6 71 67.45 Rights-of-way 34 38.76 60 57 Row Crop 88 100.32 157 149.15 Squash 91 103.74 162 153.9 Turf (High App. Rate) 34 38.76 60 57 Turf (Low App. Rate) 11 12.54 20 19 Turnip 56 63.84 100 95

59Table 3-6 EECs (ppm) for Indirect Effects to the Terrestrial-Phase CRLF via Effects to Terrestrial Invertebrate Prey Items

Use Small Insect Large Insect Barnyard/Mushroom 3369 374 Beech nut 10.3 1.1 Brassica 4.2 0.47 Citrus 88 9.8 Cole Crop 80 8.9 Cotton, fruit, nuts 111 12 Forest 34 3.8 Grains 80 8.9 Nursery 34 3.8 Pasture 34 3.8 Pistachio 101 11 Residential 131 15 Rice/Ornamental Pond 40 4.4 Rights-of-way 34 3.8 Row Crop 88 9. 8 Squash 91 10 Turf (High App. Rate) 34 3.8 Turf (Low App. Rate) 11 1.2 Turnip 56 6.2

3.5 Terrestrial Plant Ex posure Assessment

Exposures to terrestrial plants were not quant ified because no guideline toxicity data are available for diflubenzuron. Therefore, RQs we re not calculated for terrestrial plants. See Section 5.2 for discussion of potential risks to terr estrial plants in the context of this effects determination.



### 4.0 Effects Assessment

This assessment evaluates the potential for diflubenzuron to directly or indirectly affect the CRLF or modify its designated critical habi tat. As previously discussed in Section 2.7 , assessment endpoints for the CRLF effects de termination include direct toxic effects on the survival, reproduction, and growth of CR LF, as well as indirect effects, such as reduction of the prey base or modification of its habitat. In addition, potential modification of critical habitat is assessed by evaluating effects to the PCEs, which are components of the critical habitat areas that provide essential lif e cycle needs of the CRLF. Direct effects to the aquatic-phase of the CRLF are based on toxicity information for freshwater fish because no aquatic phase amphibian data have been submitted or were located in the open literature. Terrestrial-p hase effects assessments are based on avian toxicity data because no open literature or registrant-submitted studies on terrestrial phase amphibians were located. Because the frog’s prey items and habitat requirements are dependent on the availability of freshwat er fish and invertebrates, small mammals, terrestrial invertebrates, and aquatic and terrestrial plants, toxicity information for these taxa are also discussed. Acute (short-term) and chronic (long-term) toxicity information

60is characterized based on registrant-submitted studies and a comprehensive review of the open literature on diflubenzuron.

As described in the Agency’s Overview Do cument (U.S. EPA 2004), the most sensitive endpoint for each taxon is used for risk estimation. For this assessment, evaluated taxa include aquatic-phase amphibians, freshwater fish, freshwater i nvertebrates, aquatic plants, birds (surrogate for terrestria l-phase amphibians), mammals, terrestrial invertebrates, and terrestrial plants.

Toxicity endpoints are esta blished based on data gene rated 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/Office of Research and Development (ORD) (U.S. EPA 2004). A comp lete list of all av ailable registrantsubmitted studies is in Appendix H. Open lite rature data presented in this assessment were obtained from the ECOTOX database se arched on April 28, 2009. In order to be included in the ECOTOX database, papers must meet the following minimum criteria:

(1) the toxic effects are related to single chemical exposure; 5 (2) the toxic effects are on an aquatic or terrestrial plant or animal species; (3) there is a biological effect on live, whole organisms; (4) a concurrent environmental chemical concentration/dose or application rate is reported; and (5) there is an explicit duration of exposure.

Data that pass the ECOTOX screen are ev aluated along with the registrant-submitted data, and may be incorporated qualitatively or quantitatively into this endangered species assessment. In general, effects data in the open literature that are mo re conservative than the registrant-submitted data are considered. The degree to which open literature data are quantitatively or qualitatively characterized for the effects determination is dependent on whether the information is releva nt to the assessment endpoints ( i.e. , maintenance of CRLF survival, reproduction, and gr owth) identified in Section 2.8 . For example, endpoints such as behavior modifications are likely to be qualitativ ely evaluated, because quantitative relationships between modificat ions and reduction in species survival, reproduction, and/or growth ar e not available. Although the effects determination relies on endpoints that are relevant to the asse ssment endpoints of survival, growth, or reproduction, it is important to note that the full suite of subletha l endpoints potentially available in the effects literature (regardle ss of their significance to the assessment endpoints) are considered to define the action area for diflubenzuron.

Citations of all open literature not considered as part of this assessment because they were either rejected by the ECOTOX scr een or accepted by ECOTOX but not used ( e.g. , the endpoint is less sensitive) are included in Appendix G1 to G4. Appendix G also includes a rationale for rejec tion of those studies that di d not pass the ECOTOX screen

5 The studies that have information on mixtures are listed in the bibliography as rejected due to the presence of mixtures. These studies are evaluated by EFED when applicable to the assessment; however, the data is not used quantitatively in the assessment.

61and those that were not evalua ted as part of this endangere d species risk assessment. Rejection Codes are described in Appendix I.

A detailed spreadsheet of the available EC OTOX open literature data, including the full suite of lethal and sublethal endpoints is pr esented in Appendix G1. Appendix E includes a summary of the human health effects data for diflubenzuron.

In addition to registrant-submitted and open lite rature toxicity information, other sources of information, including use of the acute pr obit dose response rela tionship to establish the probability of an individual effect and re views of the Ecological Incident Information System (EIIS), are conducted to further refi ne the characterization of potential ecological effects associated with exposure to diflubenz uron. A summary of the available aquatic and terrestrial ecotoxicity information, use of the probit dose res ponse relationship, and the incident information for diflubenzur on are provided in Sections 4.3 through 4.4 respectively. A detailed summary of the available registrant-submitted ecotoxicity information is presented in Appendix A, and open literature information is presented in Appendix G.

4.1. Evaluation of Aquati c Ecotoxicity Studies

Table 4-1 summarizes the most sensitive aquatic toxicity endpoints for the CRLF, based on an evaluation of both the submitted studies and the open literature, as previously discussed. A brief summary of submitted and open literature data considered relevant to this ecological risk assessment for the CRLF is presented below. Additional information is provided in Appendix A and Appendix G.

62Table 4-1 Freshwater Aquatic Toxi city Profile for Diflubenzuron Freshwater Aquatic Toxicity Prof ile for Diflubenzuron (TGAI) Assessment

Endpoint Surrogate Species Toxicity Value Used Source Citation Study Classification Acute Toxicity toFish and Aquaticphase Amphibians Bluegill sunfish ( Lepomis macrochirus ) 129 mg/L (CI: 116 to 142) Slope: 4.7 (3.5 to 5.9) MRID 00056150 Supplemental Note 1

Chronic ToxicityFish and Aquaticphase Amphibians Fathead minnow NOAEC = 0.100 mg/L a.i. LOAEC = >0.100 mg/L a.i. MRID 00099755 Acceptable Note 2

Acute Toxicity to Aquatic Invertebrates 2 nd instar Aedes albopictus (mosquito larvae) EC 50 was 0.0028 μg/L Slope: Not available . Ho et al. (1987) (Open literature study:ECOTOX # 16591) Quantitative Note 3

Chronic Toxicity to Aquatic Invertebrates Water flea (Daphnia magna) NOAEC = 0.00025 μg/L Kashian et al (2002) (Open literature study; ECOTOX 93397) Quantitative Note 4

Acute Toxicity to

non-vascular Aquatic Plants Green algae ( Selanustrum capricornutum ) EC 50 = > 0.20 mg/L a.i. NOAEL = 0.20 mg/L a.i. 45252205 Supplemental Note 5

Acute Toxicity to vascular Aquatic Plants Duckweed EC 50 = >0.190 mg/L a.i. NOAEC = 0.190 mg/L MRID 42940103 Supplemental Note 6

Note 1: The available da ta evaluation record does not specify whether the LC 50 was based on the mean measured or nominal concentration. It is important to note that given the persiste nce of the compound, nom inal concentrations could potentially undere stimate the toxicity. Note 2: Although the data evaluation r ecord (DER) deems this study as accept able, the DER does not specify whether the LC 50 was based on the mean measured or nominal concentration. It is important to note that given the persistence of the compound, nominal concentrations could potentially underes timate the toxicity. Note 3: The study Authors do not specify whether the EC 50 was based on the mean measured or nominal concentration. It is important to note that given the persistence of the co mpound, nominal concentra tions could potentially underestimate the toxicity. Note 4: The NOAEC is based on the nominal concentration. It is important to note that given the persistence of the compound, nominal concentra tions could potentially unde restimate the toxicity. Note 5: The study was deem ed supplementa l because th e test procedures deviated from guidelines in the following manner: The study was not continued after th ree days because of an une xpected high growth of the algae, which caused the pH to increase to a value of 9 after 3 days. Additionally t he NOAEC is based on the nominal concentration. It is important to note that given the limited persistenc e of the compound, nominal concentrations coul d potentially underes timate the toxicity. Note 6: The study was deemed supplem ental because 20% of the initial measured concentration. Based on the mean measured concentration of 190 μg/l, growth of L. gibba exposed to diflubenzuron was not reduced in comparison to the solvent control over the 14-day study period.

Toxicity to fish and aquatic invertebrates is categorized us ing the system shown in Table 4-2 (U.S. EPA 2004). Toxicity categories fo r aquatic plants have not been defined.

Table 4-2 Categories of Acute Toxicity for Fish and Aquatic Invertebrates

LC 50 (ppm) Toxicity Category < 0.1 Very highly toxic > 0.1 - 1 Highly toxic > 1 - 10 Moderately toxic

63LC 50 (ppm) Toxicity Category > 10 - 100 Slightly toxic > 100 Practically nontoxic

4.1.1. Toxicity to Freshwater Fish

Given that no diflubenzuron t oxicity data are available fo r aquatic-phase amphibians, freshwater fish data were used as a surrogate to estimate direct acute and chronic risks to the CRLF. Freshwater fish toxicity data were also used to assess poten tial indirect effects of diflubenzuron to the CRLF fr om reduction in available food. As discussed in Section 2.5.3 , over 50% of the prey mass of the CRLF ma y consist of vertebrates such as mice, frogs, and fish (Hayes and Tennant 1985).

A summary of acute and chr onic freshwater fish data, including data from the open literature, is provided belo w in Sections 4.1.1.1 through 4.1.1.3.

4.1.1.1. Freshwater Fish: Acute Ex posure (Mortality) Studies There are numerous registrant submitted freshwater fish acute toxicity studies testing the technical grade active ingredient of diflube nzuron. These studies tested 7 different species of freshwater fish and demonstrate th at the technical grade active ingredient of diflubenzuron is slightly to practically non-toxic to freshwater fish. The LC 50 from these studies range from 129 ppm to >500 ppm. Further details about these studies are provided in Appendix A. The most sens itive, acceptable, freshwater fish LC 50 of 129 ppm (MRID 00056150) will be use to assess the acu te risk of the technical grade active ingredient to the a quatic-phase CRLF and to freshwat er fish prey of the CRLF. Appendix A provides further deta ils regarding all the fish acu te toxicity studies of the TGAI.

There are 16 registrant submitted studies testing several formulated products of diflubenzuron. The toxicity results of these studies range from slightly to practically nontoxic (LC 50 = 57 ppm to >1000 ppm), which suggests that the toxicity of formulated products are not dramatically greater than the toxicity of the technical grade material. Further details about these studi es are also provided in Appendix A. Potential risks from exposure to formulated products were not quantified as discussed in Section 2. No studies were located in the ope n literature that reported LC 50 s that were lower than those reported in the registrant-subm itted studies (see Appendix G).

Degradate toxicity data are summarized in A ppendix A. These data suggest that PCA is more toxic than diflubenzuron to fish with LC 50 values ranging from 2 mg/L to 23 mg/L. DFBA and PCPU appear to have similar toxi city relative to parent diflubenzuron. As discussed in Section 2, potential risks from degradates were not quantified, and were considered to have negligible impact on conclusions of this assessment.

644.1.1.2. Freshwater Fish: Chronic Exposure (Early Life Stage and Reproduction) Studies There is one available registrant submitte d freshwater fish ch ronic toxicity study (Appendix A). Based on the resu lts of this study, there we re no effects to survival, reproduction, or growth at concentrations up to 0.1 mg/L in a full lif e-cycle study (egg to egg exposure). The LOAEC was > 0.1 mg a.i./L and the NOAEC was 0.1 mg a.i./L (MRID 00099755). The NOAEC of 0.1 mg/L will be used to assess the chronic risk of diflubenzuron to the aquatic-phase CRLF and to freshwater fish prey of the CRLF since this endpoint is the most sens itive chronic toxic endpoint avai lable for freshwater fish. However, because a LOAEC was not establis hed (no effects occurred at any test concentration), use of 0.1 mg a.i./L as a chronic endpoint is likely conservative.

4.1.1.3. Freshwater Fish: Sublethal E ffects and Additional Open Literature Information

The available freshwater fish toxicity da ta do not indicate that diflubenzuron caused sublethal effects to freshwater fish at levels below those used to calculate RQs in this

assessment.

Currently, there are no available guideline acu te or chronic aquatic-phase amphibian toxicity studies testing difl ubenzuron. However, one field study was located in ECOTOX in Pacific chorus frogs that did not observe any effects at 0.28 kg a.i./hectare (0.25 lbs a.i./Acre) (Ecotox No. 111232). No other test levels were evalua ted, and a LOAEC was not established. Therefore, fish are used in this assessment as a surrogate for aquatic phase amphibians.

4.1.2. Toxicity to Freshwater Invertebrates

Freshwater aquatic invertebrate toxicity data were used to as sess potential indirect effects of diflubenzuron to the CRLF via prey reduction. As discussed in Section 2.5.3 , the main food source for juvenile aquatic- and terres trial-phase CRLFs is thought to be aquatic invertebrates found along the shoreline and on the water surface, including aquatic sowbugs, larval alderflies, and water striders.

A summary of acute and chronic freshwater invertebrate data , including data published in the open literature, is provided below in Sections 4.1.2.1 through 4.1.2.3. Diflubenzuron is a chitinase inhibitor, impairing the ability of insects to synthesi ze a critical component of their exoskeleton (U.S. EPA, 1997). Chitin synthesis is particularly important in the early life stages of insects, as they molt and form a new exoskeleton in various growth stages. Thus, aquatic guideline tests, wh ich typically run for 48 hours for the aquatic invertebrate ( Daphnia magna ), may not capture a molti ng stage, and are not an appropriate “most sensitive” acute endpoint for assessments. Endpoints derived from studies that test the toxic effects of diflubenzuron on th e larval/molting stages of freshwater invertebrates and th at test the chronic exposure of freshwater invertebrates to diflubenzuron more appropriately assess the toxicity of this ty pe of chemical.

65Diflubenzuron is fairly persistent in both a quatic and terrestrial environments. For this reason, data that evaluated the toxic effects of diflubenzuron at life stages (larval/molting stages) most vulnerable to this chemical we re used as both acute and chronic endpoints for aquatic invertebrate RQs. Results from acute studies are also presented. Likewise, guideline tests on honeybees frequently run for only 48 hours and ar e conducted on adult bees, and, therefore, may not refl ect the toxicity of diflubenzuron.

4.1.2.1. Freshwater Invertebrates: Acut e Exposure (Mortality) Studies from Registrant-Submitted Studies

There are 8 registrant submitted freshwater invertebrate aquatic toxicity studies testing the technical grade diflubenzuron. Base d on the registrant submitted freshwater invertebrate toxicity studie s diflubenzuron is very highly toxic to slightly toxic to freshwater invertebrates (EC 50 ranges from 0.0026 ppm to > 100 ppm). Further details about these studies are provided in Appendix A.

There are also several diflubenzuron formul ated product studies (Appendix A). The toxicity values of these st udies range from 1300 μg/L to >1,000,000 μg/L. These data suggest that the toxicity of the formulated products is not greater than toxicity of the technical grade material.

Degradate toxicity data are also summarized in Appendix A. The degradates were orders of magnitude less toxic to invertebrates th an parent diflubenzuron. For example, LC 50 s in midge for PCA and DFBA are 43 mg/L and >100 mg/L, respectively, compared with an LC 50 of 0.07 mg/L for parent chemical in midge (See Appendix A for additional details).

4.1.2.2. Freshwater Invertebrates: Chronic Exposure (Reproduction) Studies from Registra nt-Submitted Studies

Registrant-submitted chronic studies in a quatic invertebrates are summarized in Appendix A. The most sensitive endpoint produced in these st udies was a NOAEL of 0.05 μg/L and a LOAEL of 0.075 μg/L. In registrant submitted studies, no sublethal effects were observed at levels lower than the NOAEC used to calculate RQs. There is uncertainty in the most sensitive NOAEC becau se diflubenzuron was not detected in 4 of the 7 test samples analyzed. Therefore, the mean measured values assumed a test concentration of the detection limit when m easured values were below the detection limit. Appendix A contains more detail on registrant-submitted studies. Registrantsubmitted toxicity studies were not used to calculate RQs because an open literature study was located that produced a lower toxicity value. See Section 4.1.2.3.

664.1.2.3. Freshwater Invertebrates: Sublethal Effects and Open Literature Data

Several open literature studies have been identified that produced more sensitive endpoints than any of the registrant submitted studies. Ho et al. (1987) reported 30% and 44% mortality in 2 nd instar Aedes albopictus (mosquito larvae) at diflubenzuron concentrations of 0.00000025 mg/L (0.00025 μg/L) and 0.0000025 mg/L (0.0025 μg/L), respectively, in a 1 day study. Pairwise comp arisons between contro l and treated groups were not made; however, no larval mortality occurred in controls. The EC 50 was 0.0000028 mg/L (0.0028 μg/L) in this study as determined by probit analysis. A concentration dependent reduction in norma l pupation and adult emergence was observed in this study in 2 nd , 3 rd , and 4 th instar animals, with younger animals being more sensitive. A number of sublethal effects were ob served in this study including physical abnormalities and biochemical changes. Howe ver, these sublethal effects were only observed at levels that were also asso ciated with increased mortality. The EC 50 of 0.0000028 mg/L will be used to calculate the acute risk of diflubenzuron to aquatic invertebrates because this value is based on a larval/molting stage of mosquito and because it is the most sensitive freshwater invertebrate acute toxicity value.

. In addition, Kashian et al ( 2002) observed significant (p<0.05 ) reduction in survival at diflubenzuron concentrations of 0.01 μg/L (NOAEC = 0.00001 mg/L) in a 6-day semistatic study in daphnia magna (40 per test level, replicated test). Test concentrations were not measured; however, the study was repl icated. Mortality rate was approximately 30% at 0.01 μg/L (estimated from Figure 2).

The toxicity of 0.00025 μg/L from Ho et al. (1987) will be used to calculate the chronic risk of diflubenzuron to aquatic invertebrate s because it is the mo st sensitive toxicity value available for freshwater invertebrates impacting vulnerable life stage that this chemical is expected to impact.

4.1.3. Toxicity to Aquatic Plants

Aquatic plant toxicity studies were used as one of the measures of effect to evaluate whether diflubenzuron may affect primary pr oduction and the availa bility of aquatic plants as food for CRLF tadpoles. Primary productivity is essen tial for indirectly supporting the growth and abundance of the CRLF.

Two types of studies were used to evaluate the potential of diflubenzuron to affect aquatic plants. Laboratory and field studies were us ed to determine whether diflubenzuron may cause direct effects to a quatic plants. A summary of the laboratory data and freshwater field st udies for aquatic plants is provided in Sections 4.1.3.1 and 4.1.3.2

674.1.3.1. Aquatic Plants: Laboratory Data

There are four available aquati c plant toxicity studies incl uding three nonvascular aquatic plant studies and 1 vascular plant st udy. In all of these studies the EC 50 values were greater than the highest diflubenzuron c oncentrations tested. The most sensitive endpoints are summarized in the fo llowing table and produced an EC 50 and NOAEL of >0.20 ppm and 0.20, respectively, in algae and duckweed (MRID 45252205 and 42940103). Since these are the most sensitive en dpoints, these values will be used to assess the risk of diflubenzuron to aquatic plants. Appendix A pr ovides further details regarding these studies.

Freshwater Aquatic Toxicity Prof ile for Diflubenzuron (TGAI) Assessment

Endpoint Surrogate Species Toxicity Value Used Source Citation Study Classification Acute Toxicity to

non-vascular Aquatic Plants Green algae ( Selanustrum capricornutum ) EC 50 = > 0.20 mg/L a.i. NOAEL = 0.20 mg/L a.i. 45252205 Supplemental Note 1

Acute Toxicity to vascular Aquatic Plants Duckweed EC 50 = >0.190 mg/L a.i. NOAEC = 0.190 mg/L 42940103 Supplemental Note 2

Note 1: The study was deem ed supplementa l because th e test procedures deviated from guidelines in the following manner: The study was not continued after th ree days because of an une xpected high growth of the algae, which caused the pH to increase to a value of 9 after 3 days. Additionally t he NOAEC is based on the nominal concentration. It is important to note that given the limited persistenc e of the compound, nominal concentrations coul d potentially underes timate the toxicity. Note 2: The study was deemed supplem ental because 20% of the initial measured concentration. Based on the mean measured concentration of 190 ug/l, growth of L. gibba exposed to diflubenzuron was not reduced in comparison to the solvent control over the 14-day study period.

4.1.4. Freshwater Field Studies

The USEPA Mid-Continent Ecology Divisi on, Duluth, MN conducted a study of the biological effects, persistence, and distribu tion of diflubenzuron in littoral enclosures (MRID 44386201). The Duluth study investig ated the distribution of diflubenzuron (water, sediments and biota) a nd effects (plants, i nvertebrates, and fish) of diflubenzuron applied to oligotrophic littora l enclosures (2 applications 30 days apart). The following table (4-3) describes the obs erved effects in this study.

68Table 4-3. Diflubenzuron Effects Reported in a Littoral Enclosure Study (MRID 443862-01). Endpoint LOEC NOEC Comments Aquatic plant Effects phytoplankton (chlorophyll a: numbers, and biovolume) Not Applicable (highest dose tested produced no statistical effects) 30 μg/L The littoral enclosures were determined by the study authors to be nutrient limited with respect to
p hytoplanktonic growth. Therefore, the experiment was suitable for measuring direct effects on phytoplankton by diflubenzuron only. It could not determine any indirect effects related to adverse impacts to herbivorous zooplankton, macr oinvertebrates, insects, or fish. periphyton (reduced biomass and chlorophyll a) 7 μg/L 2.5 μg/L --

macrophytes (standing crop of submerged and floating plants, species composition) Not Applicable (highest dose tested produced no statistical effects) 30 μg/L --

decompos~t~ornat e of plant l~tter Not Applicable (highest dose tested produced no statistical effects) 30 μg/L --

Zooplankton Effects Cladocera (total numbers) 0.7 μg/L 0.6 μg/L Though the effects observed at 0.7 μg/L were not statistically significant, they were very severe, with a reduction in total cladoceran population of >89%. 0 6 μg/L is the study authors' extrapolated MATC value Copepoda (adults, juveniles, and total number) 0.7 μg/L 0.3 μg/L At the lowest does tested, reductions in the numbers of adult, juvenile, and total copepod numbers reached 71%, 83%, and 81%, respectively. 0 3 μg/L is the study authors' extrapolated MATC value Rotifera (total number) Not Applicable (highest dose tested produced no statistical effects) --

Ostracoda (total number) 7 μg/L 2.5 μg/L The control variability was very large, thereby severely limiting the sensitivity of the test. Reductions in ostracods at the 7.0 and 30 μg/L doses were 59 to 98% lower than controls

69Endpoint LOEC NOEC Comments Macroinvertebrate Effects Chironomidae abundance) 7 μg/L 2.5 μg/L The high CV among treatment blocks severely limits the sensitivity of this endpoint. The LOEC for this endpoint equates to an approximate 50% reduction In Abundance. Epherneroptera (abundance) 2.5 μg/L 0.7 μg/L The high CV among treatment blocks severely limits the sensitivity of this endpoint. The LOEC for this endpoint equates to an approximate 50% reduction in Abundance total adjusted macroinvertebrate community biomass 2.5 μg/L 0.7 μg/L --

Mean macroinvertebrate taxa richness 0.7 μg/L Not Applicable (lowest dose tested produced adverse effects) --

Insect Emergence Effects Insect emergence (all insects) 0.7 μg/L Not Applicable (lowest dose tested produced adverse effects) The power of the test was severely limited by the number of replicates within treatments. Power analysis conducted by the study authors concluded that mean differences between treatments and control for insect emergence would have to be greater than 93% before the test would show statistically significant results. Emergence reductions of 17.7 to 29.9% were observed at the lowest concentration tested. Chironomidae (emergence) 0.7 μg/L Not Applicable (lowest dose tested produced adverse effects) The power of the test was severely limited by the number of replicates within treatments. Power analysis conducted by the study authors concluded that mean differences between treatments and control for insect emergence would have to be greater than 93% before the test would show statistically significant Emergence reductions of 17. 4 to 31.1% were observed at the lowest concentration tested

70Endpoint LOEC NOEC Comments Fish Bluegill sunfish young or the year (weight, length, growth rate) 2.5 0.7 μg/L The authors concluded that young of the year bluegill growth rates were directly correlated to the density of several invertebrate taxa (notably Cladocera and Copepoda) that are preferred prey items for the species The authors concluded that the study clearly demonstrated the indirect effect of the pesticide on fish population without direct toxic effects on the fish Indigenous fish (mean size, population numbers, biomass) Not Applicable (highest dose tested produced no statistical effects) 30 μg/L There is considerable uncertainty with respect to endpoints for indigenous fish. No measurements of effects variables were made immediately following treatments. Instead, measurements were made 70 days after application of the first treatment of pesticide and 37 days after the second application.

The results indicate that reductions in the abundance of zooplankton (particularly cladocerans and copepods) can be expected to occur at in itial nominal concentrations lower than 0.7 μg/L. Macroinvertebrate bi omass and abundance of selected insect families are reduced at initial nominal di flubenzuron concentrations greater than 0.7 μg/L. Aquatic insect emergence and the richne ss of macroinvertebrate taxa are reduced at concentrations of diflubenzur on at or below an initial nom inal concentration 0.7 μg/L diflubenzuron. These impacts to the aquati c community were also observed to be correlated to reductions in the growth rate of introduced bluegill sunfish, with reductions in length, weight, and growth rate occurring at initial nominal concen trations of 2.5 μg/L and above. It is important to note that MRID 44386201 fails to empirically demonstrate a NOEC for a number of endpoints, namely ef fects on cladocerans, copepods, aquatic insect emergence and richness of macroinvert ebrate taxa. Therefor e, the study does not allow EFED to evaluate risks to these orga nisms for diflubenzuron concentrations below 0.7 μg/L with any certainty.

Results of several field studies and meso cosm studies were presented in MRID 44460702. The results of these other studies do not materially differ from results reported for the Duluth Study discussed above. Ali et al.(1 988) reported no observable effects on benthos and zooplankton in a pond adjacent to citrus treated with diflubenzuron. Concentrations were re ported to range from a high of 0.197 pg/L immediately after diflubenzur on application to the orange groves to <0.027 pg/L by 14 days after pesticide application. Booth and Fe rrell (1977) reported si gnificant (up to 97%) reductions in aquatic invertebrates for up to 30 days in a pond with diflubenzuron at 36 μg/L immediately after treatment to trace (4 μg/L) after 23 days. Boyle et al. (1996) exposed mesocosms to 5 monthly or 9 biweekly treatments of diflubenzuron at a targeted rate of10 pg/L. The authors observed signifi cant reductions in zooplankton and insect numbers, insect diversity, and grow th of bass and bluegill recruit.

714.2. Toxicity of diflubenzuron to Terrestrial Organisms

Table 4-4 summarizes the most sensitive terres trial toxicity endpoints used to evaluate toxicity of diflubenzuron to terrestrial pha se CRLFs, based on an evaluation of both the submitted studies and the open literature. A brief summary of submitted and open literature data considered rele vant to this ecological risk assessment for the CRLF is presented below. Additional inform ation can be found in Appendix A.

Table 4-4 Terrestrial Toxicity Profile for Diflubenzuron

Assessment

Endpoint Acute/ Chronic Species Toxicity Value Used in Risk Assessment MRID Classification

Acute oral LD 50 Red-winged blackbird LD 50 = 3763 mg/kg a.i. bwt MRID 00038614 Supplemental Acute Toxicity to Birds Acute dietary LC 50 Bobwhite quail LC 50 >4640 mg/kg a.i. bwt MRID 00039080 Acceptable

Reproduction Toxicity to Birds NOAEC: Bobwhite quail NOAEC= 500 mg/kg a.i. diet LOAEC= 1000 mg/kg a.i. diet MRID 41668001 Supplemental

Acute Toxicity to Mammals LD 50 : Rat LD 50 >5,000 mg/kg a.i. bwt MRID 00157103 Acceptable
Reproduction Toxicity to Mammals NOAEC: Rat NOAEC = 250 mg/kg-bw MRID 43578301 Acceptable

Acute toxicity to Terrestrial Invertebrates LD 50 : Honeybee LD 50 > 30 μg a.i./bee a.i. MRID 05001991 Acceptable

Risk to Terrestrial Plants No data submitted N/A N/A N/A N/A

Acute toxicity to terrestrial animals is categ orized using the classi fication system shown in Table 4-5 (U.S. EPA 2004). Toxicity catego ries for terrestrial pl ants have not been defined.

Table 4-5 Categories of Acute Toxicity for Avian and Mammalian Studies

Toxicity Category Oral LD 50 Dietary LC 50 Very highly toxic < 10 mg/kg < 50 ppm Highly toxic 10 – 50 mg/kg 50 - 500 ppm Moderately toxic 51 - 500 mg/kg 501 - 1000 ppm

Slightly toxic 501 - 2000 mg/kg 1001 - 5000 ppm Practically non-toxic > 2000 mg/kg > 5000 ppm

4.2.1. Toxicity to Birds

As specified in the Overview Document, the Agency uses birds as a surrogate for terrestrial-phase amphibians when amphibian toxicity data are not available (U.S. EPA

722004). No terrestrial-phase amphibian data ha ve been submitted or were located in the open literature for diflubenzuron; therefore, acute and chronic avian toxicity data are used to assess the potential direct effects of diflubenzuron to terrestrial-phase CRLFs.

4.2.1.1. Birds: Acute Exposure (Mortality) Studies

Diflubenzuron is categorized as practically non-toxic to avian speci es on an acute oral toxicity basis. Studies in bobwhite quail and mallard duck did not produce toxic effects at any dose tested (MRID 00073935). The most sensitive study available produced an LD 50 value of 3763 mg/kg-bw in the Red- Winged Blackbird (MRID 00038614). All three studies used technical grad e material (Appendix A).

Using the TGAI, diflubnezuron is categorized as practically non-toxic to avian species on a subacute dietary toxicity basis based on an LC50 value of >4640 ppm for the bobwhite quail and mallard duck (MRID 00039080). A 1 % Granular formulation was also categorized as practically non-toxic to bobw hite quail and mallard ducks based on an LC50 value of >20,000 ppm (MRID 00060381). Th ese studies were classified as "Acceptable". Appendix A provides furt her details regarding these studies.

4.2.1.2. Birds: Chronic Exposure (Growth, Reproduction) Studies

The most sensitive NOAEC from a reliable/ verifiable study was 500 mg/kg-diet (MRID 4166800102). Other studies are available th at produced lower NOAECs. However, these studies either did not produce a LOAE C (no effects were obs erved at any test concentration) or the NOAECs were consider ed unreliable. In particular, a NOAEC of <10 mg/kg-diet has been reported in a non-guideline study in mallard ducks (MRID 99862, Reinert ett al, 1975) based on a reducti on in the number of eggs embryonated at 10 mg/kg-diet. However, this effect was not observed at higher test concentrations in the same study and has not been observed in more recent guideline studies. Therefore, a NOAEC of <10 mg/kg-diet was not considered re liable and was not chosen for use in risk estimation.

The NOAEC of 500 mg/kg-food was based on e ffects on eggshell thickness in mallard ducks and reduced egg production in bobwhite quail at 1000 mg/kg-diet . Further details about the avian reproducti on studies are summarized in Appendix A.

4.2.2. Terrestrial-phase Amphibian Acute and Chronic Studies

No terrestrial-phase amphibian acute or chronic studies have been submitted by registrants or were located in the open literature.

4.2.3. Toxicity to Mammals

73Mammalian toxicity data are used to assess po tential indirect effect s of diflubenzuron to the terrestrial-phase CRLF. Effects to small mammals resulting from exposure to diflubenzuron may also indirectly affect the CRLF via reduction in available food. As discussed in Section 2.5.3 , over 50% of the prey mass of the CRLF may consist of vertebrates such as mice, frogs, and fish (Hayes and Tennant 1985).

4.2.3.1. Mammals: Acute Exposure (Mortality) Studies The available mammalian acute toxicity da ta demonstrate that diflubenzuron is practically nontoxic to mammals (LD 50 > 5000 mg/kg; MRID 00157103). Appendix A provides further details regarding this data.

4.2.3.2. Mammals: Chronic Exposure (Growth, Reproduction) Studies

In a 2-generation reproduction study technical grade diflube nzuron was administered in the diet to rats at dose levels of 0 (con trol), 500, 5000 or 50000 ppm (equivalent to about 0, 25, 250 or 2500 mg/kg/day).

No effects on reproductive performance were observed at any dose level in F0 or F1 males or females. Litter and mean pup weights decreased slightly from birth to 21 days postpartum in F1 offspring at 2500 mg/kg/da y. The NOEL for reproductive performance in parental adults is 2500 mg /kg/day. The NOEL for developmen tal toxicity in progeny is 250 mg/kg/day and the LEL is 2500 mg/kg/day, based on decreased body weights in F1 pups from birth to 21 days postpartum. (MRID 43578301). A NOAEL of 250 mg/kg-bw is used in this assessment.

4.2.4. Toxicity to Terrestrial Invertebrates

Terrestrial invertebrate toxici ty data are used to assess pot ential indirect effects of diflubenzuron to the terrestria l-phase CRLF. Effects to terrestrial invertebrates resulting from exposure to diflubenzuron may indirectly affect the CRLF via reduction in available food.

4.2.4.1. Terrestrial Invertebrates: Acut e Exposure (Mortality) Studies

Guideline studies in honeybees have been submitted and accepted by the Agency. These tests produced acute oral and contact LD 50 s of >30 μg/bee (MRID 05001991), resulting in a practically non-toxic classification. Howe ver, given the short dur ation of these tests (typically 48-72 hours), a nd the typical age of organisms used in the tests, it is unlikely that the contact / ingestion coincided with a molting stage, and, thus, the results may not accurately reflect the toxicity of difl ubenzuron to terrestrial invertebrates.

74A number of field studies regarding effects on terrestrial invertebrates were submitted to the Agency and reviewed in 1984 to 1985 (MRIDs 00099743, 000171816, 00071207, 00095416, 00071215, 00070179, 00071212, 00095407, and 00071210). The following information is based on the reviews of thes e studies, which evaluated a number of different organisms and exposure routes.

Three honey bee studies resulted in no eff ects on colonies near fields treated with application rates of up to 0.5 oz Dimi lin W-25/A or 0.06 lbs a.i./A (MRIDs 0071816, 00099743, 00071212). Another study that exposed honey bees to 100 ppm ai in their water source determined that the exposure "almost eliminated production of a sealed brood" (MRID 0095407). A study on a stream receiving runoff from a treated field indicated that amphipod and aquatic beetle larvae populations were reduced and copepods and ostracods may also have been im pacted" at an applic ation rate of 0.08 lb ai/A (MRID 00071210). A study evaluating the e ffects of diflubenzuron (2.24 kg ai/ha) on soil arthropods when the chemical was in corporated into the soil showed some decrease in numbers of springtails at 2 months post-treatment and on soil mites at 6 weeks, but little effect on other soil or ganisms (MRID 0007121 5). Differences between control and treatment plots we re not distinguishable at 4 months post-treatment. The Agency reviewer concluded diflubenzuron had "l ittle adverse effect on the soil fauna." A study on beneficial arthropods showed some sp ecies were severely affected, while other species were not affected at all (MRID 00070179) . Aerial application of granular Dimilin to salt marsh mosquito habitat significantly reduced numbers and diversity of aquatic invertebrates (MRID 00095416). Measured water concentrations of Dimilin (diflubenzuron) ranged from 0.24-1.8 μg/L. Based on a study on soil arthropods, Dimilin appears to have "little or no effect on so il microarthropods, but may adversely affect insect parasites" (MRID 007 1207).

Overall, studies on terrestrial invertebrates illu strate that the effect of diflubenzuron is highly dependent on the life stage of the orga nism when it is exposed. Diflubenzuron is very highly toxic to invertebra tes that rely on chitin as an exoskeleton at the critical life stage ( i.e. , a molting event). Available data are not sufficiently robust to estimate an assessment endpoint (e.g, LC50 or NOAEC) for this taxon, but adverse effects on nontarget arthropods should be an ticipated following use of diflubenzuron given its mode of action as an insecticide.

4.2.4.2. Terrestrial Invertebrates: Open Literature Studies

A number of open literature stud ies that evaluated effects to terrestrial invertebrates were identified in the ECOTOX database (Appendix G). These studies support the conclusions presented in Section 4.2.4.1 a nd do not add additional insight into the potential effects of diflubenzuron to te rrestrial invertebrates (Ecotox Nos. 65290, 110971, and 111061).

754.2.5. Toxicity to Terrestrial Plants

Terrestrial plant toxicity data are used to evaluate the potential for diflubenzuron to affect riparian zone and upland vegetation within the action area for the CRLF. Impacts to riparian and upland ( i.e. , grassland, woodland) vegetation may result in indirect effects to both aquatic- and terrestrial-phase CRLFs, as well as modification to designated critical habitat PCEs via increased sedimentation, a lteration in water quali ty, and reduction in upland and riparian habitat that provides shelter, foraging, predator avoidance and dispersal for juvenile and adult CRLFs.

Plant toxicity data from both registrant-subm itted studies and studies in the scientific literature are typically reviewed for this type of assessment. Registrant-submitted studies are conducted under conditions and with species defined in EPA toxicity test guidelines. Sub-lethal endpoints such as plant growth, dry weight, and biomass are evaluated for both monocots and dicots, and effects are evaluated at both seedling emergence and vegetative life stages. Guideline studies genera lly evaluate toxicity to ten crop species. A drawback to these tests is that they ar e conducted on herbaceous crop species only, and extrapolation of effects to other species, such as the woody shrubs and trees and wild herbaceous species, contributes uncer tainty to risk conclusions.

Commercial crop species have b een selectively bred, and may be more or less resistant to particular stressors than wild herbs and forb s. The direction of this uncertainty for specific plants and stressors, including diflubenzuron, is largel y unknown. Homogenous test plant seed lots also lack the genetic vari ation that occurs in na tural populations, so the range of effects seen from tests is likely to be smaller than would be expected from wild populations.

Currently, there are no available guideline terres trial plant toxicity data for use in this assessment. However, Cooper et al. (1990) di d not observe differences in characteristics of vegetation when comparing plots treated with diflubenzur on to untreated controls. The study evaluated 10 circular 0.04 ha plots (5 treated and 5 untreated). Vegetative characteristics evaluated included percent litte r cover, litter dept h, percent herbaceous cover, percent shrub cover, percent canopy cover in each of 5 canopy height classes, percent deciduous canopy cover, percent coni ferous canopy cover, percent total canopy cover, and number of trees and number of snags in 6 dbh classes. Diflubenzuron was applied at 0.06 lbs a.i./Acre.

Similarly, Seidel and Whitmore did not observe differences in vegetative characteristics including litter depth, number of saplings, num ber of 2 classes of tree snags, and presence of herbaceous growth, shrubs. Diflubenzuron wa s applied to test plots at 140 g/ha by air (0.12 lbs a.i./Acre).

In addition, a number of efficacy studies have been submitted to the Agency that also evaluated phytotoxic effects to target species . These studies have typically noted no or minimal phytotoxicity (See Appendix H for citati ons). Last, diflubenz uron is labeled for

76use on numerous commodoties without reports of incidents that have been clearly associated with difblubenzuron toxicity.

4.3. Use of Probit Slope Response Relati onship to Provide Information on the Endangered Species Levels of Concern

The Agency uses the probit dose response rela tionship 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 th e listed species of concern (U.S. EPA 2004). As part of the risk characterization, an inte rpretation of acute RQ for listed species is discussed. This interpretation is presented in terms of the chance of an individual event ( i.e. , mortality or immobilization) should exposur e at the EEC actually occur for a species with sensitivity to diflubenzu ron on par with the acute toxici ty endpoint selected for RQ calculation. To accomplish this interpretati on, 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 relationshi p. In addition to a single effects probability es timate based on the mean, upper and lower estimates of the effects probability are also provi ded to account for variance in the slope, if available.

Individual effect probabilitie s are calculated based on an Ex cel spreadsheet tool IECV1.1 (Individual Effect Chance Model Version 1.1) developed by the U.S. EPA, OPP, Environmental Fate and Effects Division (J une 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 spread sheet. In addition, the acute RQ is entered as the desired threshold.

Probit slopes used in this analysis are summarized in Table 4-6.

Table 4-6. Probit slopes used for taxonomic groups evaluated in this assessment Taxonomic Group Probit Slope 95% CI MRID Species Fish 4.7 3.5 to 5.9 00056150 Bluegill Aquatic Invertebrates 0.41 0.3 to 0.5 Ho et al., 1987 (Ecotox No. 16591) Mosquito larvae

Birds Default of 4.5: Data do not allow for calculation of reliable slope 2 to 9 00038614 Red-winged blackbird

Mammals Default of 4.5: No mortality occurred in acute mammal studies 2 to 9 00157103 Rat

774.4. Incident Database Review

One incident (I009846-002) had been reported for diflubenzuron when the EIIS database was queried (10/2/2009). Damage to ora nges in Polk County, Florida was reported following direct application. Legality of the a pplication was classified as registered use, and certainty that diflubenzuron caused the dama ge was rated as possible. However, the incident was also associated with petroleum distillates, which have a number of other terrestrial plant incidents asso ciates with its use. Given that diflubenzuron has a long history of use on citrus trees without other incidents and th at petroleum distillates has been associated with several similar incidents on terrestrial plants, it is more likely that the damage to oranges was caused by use of petroleum distillates and not by diflubenzuron. A lack of reported incidents s hould not be taken as an indicator of little or no ecological effects, especially in the cas e of a chemical like diflubenz uron, which acts primarily to inhibit growth of insects. Insects and other invertebrates that are vulnerable to chitinase inhibitors are generally not a highly visible component of the ecosystem, and even largescale die-offs may go unreported.

78

### 5.0 Risk Characterization

Risk characterization is the integration of the exposure and effects characterizations. Risk characterization is used to determine the potential for direct and/or indirect effects to the CRLF or for modification to its designated critical habita t from the use of diflubenzuron in CA. The risk characteri zation provides an estim ation (Section 5.1) and a description (Section 5.2) of the likelihood of adverse effects; articulates risk assessment assumptions, limitations, and uncertainties; and synthesizes an overall conclusion regarding the likelihood of adverse effects to the CRLF or its designated critical habitat ( i.e. , “no effect,” “likely to adve rsely affect,” or “may affect , but not likely to adversely affect”).

5.1. Risk Estimation

Risk is estimated by calculating the ratio of e xposure to toxicity. Th is ratio is the risk quotient (RQ), which is then compared to pr e-established acute a nd chronic levels of concern (LOCs) for each category evaluated (A ppendix C). For acute exposures to the CRLF and its animal prey in aquatic habitats, as well as terrestrial invertebrates, the LOC is 0.05. For acute exposures to the CRLF and mammals, the LOC is 0.1. The LOC for chronic exposures to CRLF and its prey, as well as acute exposures to plants is 1.0.

Risk to the aquatic-phase CRLF is estimated by calculating the ratio of exposure to toxicity using 1-in-10 year EECs based on the label-recommended diflubenzuron usage scenarios summarized in Table 3-3 and the a ppropriate aquatic toxicity endpoint from Section 4. Risks to the terrestrial-phase CRLF and its prey ( e.g . terrestrial insects, small mammals and terrestrial-phase frogs) are es timated based on exposures resulting from applications of diflubenzuron (Section 3) a nd the appropriate toxi city endpoint from Section 4.

5.1.1. Exposures in the Aquatic Habitat

5.1.1.1 Direct Effects to Aquatic-Phase CRLF

Direct effects to the aquatic-phase CRLF are based on EECs in the standard pond and the lowest acute toxicity value for freshwater fis h. In order to assess di rect chronic risks to the CRLF, 60-day EECs and the lowest chronic toxicity value for freshwater fish are used. Based on the RQ calculations none of the LOCs were exceeded for risk to freshwater water fish or aquatic phase amphibians except for the aerial use of diflubenzuron on rice crops (See Table 5-1). The LOC was exceeded for chronic risk to freshwater fish or aquatic phase amphibians for this use. Based on these results only the diflubenzuron aerial use on rice “may affect ” aquatic-phase CRLFs. No LOCs were exceeded for other uses. Therefore, all other registered uses are expected to have “no effect” on the aquatic-phase CRLF. RQs presen ted in Table 5-1 are further discussed in the context of an effects determination in Section 5.2.

79Table 5-1 Summary of Dir ect Effect RQ LOCs for the Aquatic-phase CRLF

Direct Effects to

CRLF a Use Surrogate Species Toxicity Value (mg/L) EEC (mg/L) b RQ Probability of Individual

Effect at ES LOC Probability of Individual Effect at RQ

Direct Application to rice 60-day: 0.112 1.12 c

Manure 0.027 0.27 Chronic Direct Toxicity All other uses Fathead minnow NOAEC = 0.100

<0.015 <0.15 Not calculated for chronic endpoints

Acute direct toxicity All uses All species tested 129 <0.11 <0.01 1 in 3.7E20 (1 in 7.8E11 to 1 in 5E30)

a RQs associated with acute and ch ronic direct toxicity to the CRLF are also used to assess potential indirect effects to the CRLF based on a reduction in freshwater fish and frogs as food items.

b The highest EEC based on aerial use of diflubenzuron on rice paddies (see Table 3-3 ).

c RQ > chronic LOC of 1.0.

5.1.1.2. Indirect Effects to Aquatic- Phase CRLF via Reduction in Prey (non-vascular aquatic plants, aquatic invertebrates, fish, and frogs)

Non-vascular Aquatic Plants

Indirect effects of difluben zuron to the aquatic-phase CRL F (tadpoles) via reduction in non-vascular aquatic plants in its diet or via habitat modi fication are based on peak EECs and the lowest toxicity value (EC 50 ) for aquatic non-vascular plants. Based on the RQ calculations there were no LOC exceedances of diflubenzuron to aquatic plants. Therefore, diflubenzuron is expected to have “no effect ” to CRLFs by affecting nonvascular aquatic plants. These RQs are furt her discussed in the c ontext of an effects determination in Section 5.2.

Table 5-2 Summary of RQs Used to Estimate I ndirect Effects to the CRLF via Effects to NonVascular Aquatic Plants (diet of CRLF in tadpol e life stage and habitat of aquatic-phase CRLF)

Uses Toxicity Value (μg/L) Peak EEC (μg/L) Indirect effects RQ (food and habitat)

All Uses 200 (MRID 45252205) <113 <0.6

Aquatic Invertebrates

Indirect acute effects to the aquatic-phase CRLF via effects to prey (invertebrates) in aquatic habitats are based on peak EECs in the standard pond and the lowest acute toxicity value for freshwater invertebrates. Due to the m ode of action of diflubenzuron, results from chronic toxicity st udies are also considered to be acute effects. Therefore,

80chronic RQs are calculated using peak EECs. However, chronic RQs are also presented using 21-day EECs.

A summary of the acute and chronic RQ values for exposure to aquatic invertebrates (as prey items of aquatic-phase CRLFs) is provide d in Table 5-3. Based on RQ calculations diflubenzuron exceeds either the acute or chronic LOC for risk to freshwater invertebrates for all the mode led uses of diflubenzuron. The acute RQs ranged from 4 to 40,000 and the chronic LOC exceedances range from 40 to 450,000. Based on these results, diflubenzuron “may affect” CRLFs vi a reduction in freshwater invertebrates prey items. These RQs are further discussed in th e context of an effects determination in Section 5.2.

81Table 5-3 Summary of Acute and Chronic RQs Used to Estimate In direct Effects to the CRLF via Direct Effects on Aquatic Invertebra tes as Dietary Food Items (prey of CRLF juveniles and adults in aquatic habitats)

Scenario Group Application Type Water Column Peak EEC

(μg/L) Acute RQ Based on a 6- day LC50 of 0.0028 μg/L) Chronic RQ Based on a 6-Day NOAEC of 0.00025 μg/L and

a 21-Day EEC Chronic RQ Based on a 6-Day NOAEC of 0.00025 μg/L and a peak EEC Ground 0.11 39.07 227.21 437.56* Beech Nut Air 0.21 73.94 521.28 828.16* Brassica Ground 0.07 23.49 156.96 263.12* Ground 0.24 87.48 393.05 979.76* Citrus Air 0.50 178.06 1204.76 1994.32* Cole Crop Ground 0.36 128.46 817.76 1438.72* Ground 0.15 54.34 429.96 608.60* Cotton Air 0.82 293.71 1874.28 3289.56* Ground 0.58 205.99 1221.32 2307.08* Forest Air 0.80 285.44 1786.60 3196.96* Ground 0.26 94.15 556.48 1054.48* Airblast 0.66 236.88 1702.68 2653.00* Fruit Air 1.36 485.86 3424.92 5441.60* Ground 0.90 322.81 1859.68 3615.44* Airblast 1.26 450.56 2794.92 5046.24* General Nuts Air 0.85 303.31 1965.88 3397.04* Ground 1.34 477.32 3117.32 5346.00* Grains Air 0.93 333.08 2084.80 3730.48* Ground 0.20 71.60 449.60 801.96* Nursery Air 0.65 231.78 1504.92 2595.88* Ground 0.12 41.64 302.73 466.40* Airblast 0.29 102.95 665.00 1153.04* Pasture Air 0.55 195.64 1003.56 2191.20* Pistachio Ground 0.15 52.45 331.80 587.40* Ground 0.08 29.37 184.23 328.96* Residential Air 0.40 143.71 1207.47 1609.50* Rice/Ornamental Pond Air 112.92 40327.13 451663.90 451663.90*

Ground 5.55 1981.66 12178.53 22194.60* Airblast 8.13 2903.57 17541.57 32520.00* Rights-of-way Air 5.59 1997.41 12268.73 22371.00* Ground 0.02 6.86 59.72 76.82* Row Crop Air 1.15 409.57 3098.08 4587.20* Urban Air 34 12187.86 87924.00 136504.00* Ground 1.15 409.11 2610.32 4582.00* Squash Air 1.87 666.75 4736.00 7467.60* Ground 0.01 3.60 24.39 40.26* Airblast 0.21 76.57 506.32 857.60* Turf (High Application Rate) Air 0.61 216.57 1489.40 2425.56* Ground 0.02 7.67 32.30 85.88* Airblast 0.07 23.89 146.85 267.52* Turf (Low Application Rate) Air 0.13 46.49 326.01 520.68* Turnip Ground 0.72 256.26 1402.12 2870.12* * Exceeds the chronic LOC of 1 for risk to aquatic invertebrates

82Fish and Frogs

Fish and frogs also represent potential prey items of adult aquatic-phase CRLFs. RQs associated with acute and chronic direct toxicity to the CRLF (Table 5-1) are used to assess potential indirect effects to the CRLF based on a reduction in freshwater fish and frogs as food items. Based on the RQ calcula tions none of the LOCs were exceeded for risk to freshwater fish or aquatic phase amphibians except for the aerial use of diflubenzuron on rice (See Ta ble 5-1). The LOC was exceeded for chronic risk to freshwater fish for this use. These RQs are further discussed in the context of an effects determination in Section 5.2.

5.1.1.3. Indirect Effects to CRLF via Reduction in Habitat and/or Primary Productivity (Freshwater Aquatic Plants)

Indirect effects to the CRLF vi a direct toxicity to aquatic plants are estimated using the most sensitive non-vascular and vascular plant toxicity endp oints. Because there are no obligate relationships between the CRLF and any aquatic plant species, the most sensitive EC 50 values, rather than NOAEC values, were us ed to derive RQs. Based on the RQ calculations there were no LOC exceedances for risk of diflubenzuron to aquatic plants. Therefore, the effects determination is “no effect” for all uses. These RQs are further discussed in the context of an eff ects determination in Section 5.2.

Table 5-4 Summary of RQs Used to Estimate Indi rect Effects to the CRLF vi a Effects to Vascular Aquatic Plants (habitat of aquatic-phase CRLF) a

Uses Peak EEC (μg/L) EC50 (μg/L) Indirect effects RQ (habitat) All Uses <113 190 <0.6

a RQs used to estimate indirect effects to the CRL F via toxicity to non-vascular aquatic plants are summarized in Table 5-2

5.1.2. Exposures in the Terrestrial Habitat

5.1.2.1. Direct Effects to Terrestrial-phase CRLF

Potential direct acute effect s to the terrestrial-phase CRL F are derived by considering dose- and dietary-based EECs modeled in T- REX for a small bird (20 g) consuming small invertebrates (Table 5-5) and acute oral and subacute dietary toxicity endpoints for avian species.

Potential direct chronic effects of diflubenzuron to the terrestrial-phase CRLF are derived by considering dietary-based exposures m odeled in T-REX for a small bird (20g) consuming small invertebrates. Chronic eff ects are estimated using the lowest available toxicity data for birds. EECs are divided by toxicity values to estimate chronic dietarybased RQs. Subacute dietary based RQs were not calculated because the dietary toxicity data indicated that no toxicolo gical effects were observed at dietary concentrations that were equivalent to the limit concentr ation for acute dietary toxicity.

83Based on the acute and chronic RQ calculations there are only LOC exceedances for risks to the terrestrial-phase of the CRLF for use of diflubenzuron on barnyard manure. Thus, the preliminary determination is a “may e ffect” only for the use on barnyard manure and “no effect” for all other modeled uses. Tabl e 5-5 lists only dose based acute RQs because no dietary LC 50 study produced a definitive toxicity value (all LC 50 s were above the limit dose).

Table 5-5 Summary of RQs Used to Estimate Direct Effects to the Terrestrial-phase CRLF Chronic RQ Acute RQ

Model Use EEC RQ Dose-based EEC (based on 20-

gram bird) RQ Barnyard (Manure) 3369 6.738* 3840.66 1.2** Beech nut 10.3 0.0 206 11.742 <0.01 Brassica 4.2 0.0084 4.788 0.00

Citrus 88 0.176 100.32 0.03 Cole Crop 80 0.16 91.2 0.03

Cotton 111 0.222 126.54 0.04 Forest 34 0.068 38.76 0.01 Fruit 111 0.222 126.54 0.04 General Nuts 111 0.222 126.54 0.04 Grains 80 0.16 91.2 0.03 Nursery 34 0.068 38.76 0.01 Pasture 34 0.068 38.76 0.01 Pistachio 101 0.202 115.14 0.04 Residential 131 0.262 149.34 0.05 Rice 40 0.08 45.6 0.01 Rights-of-way 34 0.068 38.76 0.01

Row Crop 88 0.176 100.32 0.03

Squash 91 0.182 103.74 0.03 Turf (High App. Rate) 34 0.068 38.76 0.01 Turf (Low App. Rate) 11 0.022 12.54 <0.01 Turnip 56 0.112 63.84 0.02 * Exceeds the chronic of LOC of 1. ** Exceeds the acute LOC of 0.5.

5.1.2.2. Indirect Effects to Te rrestrial-Phase CRLF via Reduction in Prey (terrestrial invertebrates, mammals, and frogs)

a) Terrestrial Invertebrates

In order to assess the potential risks of diflubenzuron to terrestrial invertebrates, which are considered prey of CRLF in terrestrial ha bitats, the honey bee is used as a surrogate for terrestrial invertebrates. The toxicity value for terrestrial invertebrates is calculated by multiplying the lowest available acute contact LD 50 of 30 μg a.i./bee by 1 bee/0.128g, which is based on the weight of an adult honey bee. EECs (μg a.i./g of bee) calculated by T-REX for small and large insects are divide d by the calculated toxicity value for

84terrestrial invertebrates, which is 30 μg a.i. /g of bee. Based on the RQ calculations, the modeled diflubenzuron uses except beechnut a nd brassica exceed the LOC for risks to terrestrial invertebrate (Table 5-6). Ba sed on these results, the preliminary effects determination is a “may effect” for the potenti al of diflubenzuron to indirectly affect the CRLF by reducing terrestrial invertebrate prey. Section 5.2 further discusses these RQs and additional terrestrial invertebrate toxicity information in the context of this effects determination.

Table 5-6 Summary of RQs Used to Estimate I ndirect Effects to the Terrestrial-phase CRLF via Direct Effects on Terrestrial Invertebrates as Dietar y Food Items based on an LC50 of 234 ppm (30 μg per bee / 0.128 grams per bee)

Uses Small Insects EEC Small Insect RQ Large Insects EEC Large Insect RQ Barnyard/Mushroom 3369 14.37 374 1.60 Beechnut 10.3 0.04 1.14 0.00 Brassica 4.2 0.02 0.47 0.00 Citrus 88 0.38 9.78 0.04 Cole 80 0.34 8.89 0.04 Cotton 111 0.47 12.33 0.05 Forest 34 0.15 3.78 0.02 Fruit 111 0.47 12.33 0.05 General Nut Crop 111 0.47 12.33 0.05 Grain 80 0.34 8.89 0.04 Impervious 34 0.15 3.78 0.02 Nursery 34 0.15 3.78 0.02 Pistachio 101 0.43 11.22 0.05 Residential 131 0.56 14.56 0.06 Rice/Ornamental Pond 40 0.17 4.44 0.02 Rights away 34 0.15 3.78 0.02 Row crops 88 0.38 9.78 0.04 Squash 91 0.39 10.11 0.04 Turf (high application rate) 34 0.15 3.78 0.02 Turf (low application rate) 11 0.05 1.22 0.01 Turnip 56 0.24 6.22 0.03

b) Mammals

Risks associated with reduction of small ma mmals as prey by large terrestrial-phase CRLFs are derived for dietary-based and dos e-based exposures modeled in T-REX for a small mammal (15g) herbivore and insectivore. Acute and chronic effects are estimated using the most sensitive mamma lian toxicity data. EECs are divided by the toxicity value to estimate acute and chronic dose-based RQ s as well as chronic dietary-based RQs. There were no mortalities in the mammalian ac ute toxicity studies at the limit dose of 5000 mg/kg-bw; therefore, acute RQs were not calculated and risk was presumed to be below levels that could result in indirect effects to CRLFs. Based on the chronic RQ calculations, there are no chronic LOC exceedan ces for risk to small mammal prey items except for the barnyard/mushroom use (Table 5-7). Based on these results, the preliminary effects determination is “no e ffect” regarding potential risks to the CRLF

85from reduction in mammalian prey except for the barnyard/mushroom use. The chronic RQ is 10 for the barnyard/mushroom use. Therefore, the preliminary effects determination for this use is “may effect.” These RQs are further discussed in the context of an effects determination in Section 5.2.

Table 5-7 Summary of Chronic RQs Used to Estimate Indirect Effect s to the Terrestrial-phase CRLF via Direct Effects on Small Mamma ls as Dietary Food Items Chronic RQ Model Use EEC RQ Barnyard/Mushroom 5690.5 10.37 (exceeds LOC of 1.0) Beech nut 17.404 0.03 Brassica 7.125 0.01 Citrus 149.15 0.27 Cole Crop 135.85 0.25 Cotton 187.15 0.34 Forest 57 0.10 Fruit 187.15 0.34 General Nuts 187.15 0.34 Grains 135.85 0.25 Nursery 57 0.10 Pasture 57 0.10 Pistachio 171 0.31 Residential 220.4 0.40 Rice/Ornamental Pond 67.45 0.12 Rights-of-way 57 0.10 Row Crop 149.15 0.27 Squash 153.9 0.28 Turf (High App. Rate) 57 0.10 Turf (Low App. Rate) 19 0.03 Turnip 95 0.17

c) Amphibians

An additional prey item of the adult terrestrial -phase CRLF is other amphibians. In order to assess risks to these organisms, dietar y-based and dose-based exposures modeled in TREX for a small bird (20g) consuming small invertebrates are used. See Section 5.1.2.1 and associated table (Table 5-5) for results. Based on LOC exceedances for the barnyard/mushroom use, this labeled use of diflubenzuron may affect the CRLF. Avian LOCs were not exceeded for any other us e; therefore, the preliminary effects determination for diflubenzuron for all other us es is “no effect.” These RQs are further discussed in the context of an eff ects determination in Section 5.2.

5.1.2.3. Indirect Effects to CRLF via Reduction in Terrestrial Plant Community (Riparian and Upland Habitat)

Potential indirect effects to the CRLF resul ting from direct effects on riparian and upland vegetation are assessed using RQs from te rrestrial plant seedling emergence and vegetative vigor EC 25 data as a screen. Currently there are no available guideline

86terrestrial plant toxicity data. Therefore, RQs were not calculated. Additional lines of evidence including open literature studies and non-guideline registrant-submitted studies regarding potential risks to terrestrial plants in the context of an effects determination for the CRLF are described in Section 5.2.

5.1.3. Primary Constituent Elements of Designated Critical Habitat

For diflubenzuron use, the assessment endpoint s for designated critical habitat PCEs involve a reduction and/or modi fication of food sources neces sary for normal growth and viability of aquatic-phase CRLFs, and/or a reduction and/or modification of food sources for terrestrial-phase juveniles and adults. Because these endpoints are also being assessed relative to the potential for indirect effects to aquatic- and terrestrial-phase CRLF, the effects determinations for indirect effects from the potential loss of food items are used as the basis of the effects determin ation for potential modi fication to designated critical habitat. As discussed in Sections 5.1.3 and 5.2.4, effects to a quatic and terrestrial plants are not expected to occur.

However, one PCE is “alteration of other ch emical characteristics necessary for normal growth and viability of CRLFs and their food source.” To assess the impact of diflubenzuron on this PCE ( i.e. , alteration of food sources), acute and chronic freshwater fish and invertebrate toxicity endpoints, as well endpoints for aquati c non-vascular plants, are used as measures of effects. RQs for these endpoints were calc ulated in Sections 5.1.1.1 and 5.1.1.2. Based on LOC exceedances for aquatic invertebrates, diflubenzuron may modify aquatic-phase PCEs of designated critical habi tat related to effects of alteration of other chemical characteristics necessary for normal growth and viability of CRLFs and their food source.

Two terrestrial phase PCEs are not evaluated using solely terr estrial plant toxicity data. The PCE of “reduction and/or modification of food sources fo r terrestrial phase juveniles and adults” is evaluated using acute and chr onic toxicity endpoints for birds, mammals, and terrestrial invertebrates as measures of effects. RQs for these endpoints were calculated in Section 5.1.2.2. Diflubenzuron has the poten tial for modifying critical habitat via this PCE because as an insecticide, terrestrial i nvertebrates are likely to be affected if diflubenzuron is us ed near critical habitat.

The fourth terrestrial-phase PCE is based on alteration of chemical characteristics necessary for normal growth and viability of juvenile and adult CRLFs and their food source. Based on potential impacts to terres trial invertebrates for all assessed uses, diflubenzuron may impact critical habitat by altering chemical characteristics necessary for normal growth and viability of juven ile and adult CRLFs for all uses assessed.

5.2. Risk Description

The risk description synthesizes an overall conclusion regarding th e likelihood of adverse impacts leading to an effects determination ( i.e ., “no effect,” “may affect, but not likely

87to adversely affect,” or “likely to advers ely affect”) for the CRLF and its designated critical habitat.

Based on the RQs presented in the Risk Es timation (Section 5.1) a preliminary effects determination is “may affect” for the CRL F and modification of critical habitat.

A summary of the results of the risk estim ation results are provided in Table 5-8 for direct and indirect effects to the CRLF a nd in Table 5-9 for the PCEs of designated critical habitat for the CRLF.

Table 5-8 Risk Estimation Summa ry for Diflubenzuron - Direct and Indirect Effects to CRLF

Assessment Endpoint LOC Exceedances (Y/N) Description of Results of Risk Estimation Aquatic Phase (eggs, larvae, tadpoles, juveniles, and adults)

Direct Effects Survival, growth, and reproduction of CRLF individuals via direct effects on aquatic phases Yes Direct application to rice only The chronic fish RQ was 1.12 for direct application to rice. All other chronic RQs were lower than the LOC of 1.0. Acute RQs were <0.01. The probability of an individual mortality at the acute RQs of 0.01 is <1 in 1,000,000 based on a slope of 4.7 (95% CI of 3.5 to 5.9 (MRID 56150). Indirect Effects Survival, growth, and reproduction of CRLF individuals via effects to food supply ( i.e., freshwater invertebrates, non-vascular plants) Yes All uses Acute RQs ranged from approximately 4 to 40,000. The probability of an individual effect at these RQs approaches 100% for reasonable lower and upper bound slopes of 2 to 9. Chronic RQs also exceeded LOCs and were 25 to 450,000.

Indirect Effects Survival, growth, and reproduction of CRLF individuals via effects on habitat, cover, and/or primary productivity ( i.e., aquatic plant community) No No LOCs were exceeded for vascular or nonvascular aquatic plants. RQs were less than 0.6.

Indirect Effects Survival, growth, and reproduction of CRLF individuals via effects to riparian vegetation, required to maintain acceptable water quality and habitat in ponds and streams comprising the species’ current range. No RQs were not calculated due to the lack of guideline terrestrial plant toxicity data; further discussion of potential risks to CRLFs resulting from impacts to terrestrial plants is presented in Section 5.2.

Terrestrial Phase (Juveniles and adults)

88LOC Exceedances Assessment Endpoint (Y/N) Description of Results of Risk Estimation

Direct Effects Survival, growth, and reproduction of CRLF individuals via direct effects on terrestrial phase adults and juveniles Yes Barnyard/mushroom use only Acute and chronic RQs exceeded LOCs. The acute RQ was 1.2 and the chronic RQ was 6.7. At an RQ of 1.2, the probability of an individual effect is 1 in 1.6 based on the default probit slope of 4.5. The range of probabilities of an individual effect is 1 in 1.4 to 1 in 1.8 based on lower and upper bound probit slopes of 2 to 8. LOCs were not exceeded for any other use. Indirect Effects Survival, growth, and reproduction of CRLF individuals via effects on prey ( i.e., terrestrial invertebrates, small terrestrial mammals and terrestrial phase amphibians) Yes All uses Diflubenzuron is an insecticide. Therefore, there is potential for terrestrial invertebrate abundance to be impacted. In add ition, LOCs were exceeded for all uses based on submitted data in the honey bee.

Indirect Effects Survival, growth, and reproduction of CRLF individuals via effects on habitat ( i.e., riparian vegetation) No RQs were not calculated for terrestrial plants based on the lack of terrestrial plants toxicity data available for diflubenzuron.

Table 5-9 Risk Estimation Summary for Diflubenzuron – PCEs of Designated Critical Habitat for the CRLF

Assessment Endpoint Habitat Modification (Y/N) Description of Results of Risk Estimation

Aquatic Phase PCEs (Aquatic Breeding Habitat and Aquatic Non-Breeding Habitat) Alteration of channel/pond morphology or geometry and/or increase in sediment deposition within the stream channel or pond: aquatic habitat (including riparian vegetation) provides for shelter, foraging, predator avoidance, and aquatic dispersal for juvenile and adult CRLFs. No There are no LOC exceedances for risk to aquatic plants.

Alteration in water chemistry/quality including temperature, turbidity, and oxygen content necessary for normal growth and viability of juvenile and adult CRLFs and their food source. No --

Alteration of other chemical characteristics necessary for normal growth and viability of CRLFs and their food source. Yes There are acute and chronic LOC exceedances for risk of diflubenzuron to aquatic invertebrate prey of the CRLF for all uses.

Reduction and/or modification ofaquatic-based food sources for premetamorphs ( e.g. , algae) No There were no LOC ex ceedances for risk to Algae for any use evaluated.

89Habitat Assessment Endpoint Description of Results of Risk Estimation Modification (Y/N) Terrestrial Phase PCEs (Upland Habitat and Dispersal Habitat) Elimination and/or disturbance of upland habitat; ability of habitat to support food source of CRLFs: Upland areas within 200 ft of the edge of the riparian vegetation or dripline surrounding aquatic and riparian habitat that are comprised of grasslands, woodlands, and/or wetland/riparian plant species that provides the CRLF shelter, forage, and predator avoidance No Currently, there are no guideline terrestrial plant toxicity data available to assess this risk. See Section 5.2 for additional description of potential risks to CRLFs resulting from impacts to terrestrial vegetation.

Elimination and/or disturbance of dispersal habitat: Upland or riparian dispersal habitat within designated units and between occupied locations within 0.7 mi of each other that allow for movement between sites including both natural and altered sites which do not contain barriers to dispersal No Currently, there are no guideline terrestrial plant toxicity data available to assess this risk. See Section 5.2 for additional description of potential risks to CRLFs resulting from impacts to terrestrial vegetation.

Reduction and/or modification of food sources for terrestrial phase juveniles and adults Yes There are LOC exceedances for risk terrestrial invertebrate prey for all uses, and potential magnitude of effect could be substantial such that CRLFs could be impacted. Alteration of chemical characteristics necessary for normal growth and viability of juvenile and adult CRLFs and their fo od source. Yes There are LOC exceedances for risk terrestrial invertebrate prey, and potential magnitude of effect could be substantial such that CRLFs could be impacted.

Following a “may affect” determination, additio nal information is considered to refine the potential for exposure at th e predicted levels based on th e life history characteristics ( i.e. , habitat range, feedi ng preferences, etc.) of the CRLF. Based on the best available information, the Agency uses the refined ev aluation to distinguish those actions that “may affect, but are not likely to adversely a ffect” from those actions that are “likely to adversely affect” the CRLF and its designated critical habitat.

The criteria used to make determinations that the effects of an acti on are “not likely to adversely affect” the CRLF and its designated critical habitat include the following:

• Significance of Effect : Insignificant effects are those that cannot be meaningfully measured, detected, or evaluated in the c ontext of a level of effect where “take” occurs for even a single individual. “T ake” in this context means to harass or harm, defined as the following:

 Harm includes significant habitat m odification or degradation that results in death or injury to listed species by significantly impairing behavioral patterns such as br eeding, feeding, or sheltering.

 Harass is defined as actions that crea te the likelihood of injury to listed species to such an extent as to significantly disrupt normal behavior

90patterns which include, but are no t limited to, breeding, feeding, or sheltering.

• Likelihood of the Effect Occurring : Discountable effect s are those that are extremely unlikely to occur.

• Adverse Nature of Effect : Effects that are wholly be neficial without any adverse effects are not considered adverse.

A description of the risk and effects determ ination for each of the established assessment endpoints for the CRLF and its designated crit ical habitat is provided in Sections 5.2.1 through 5.2.3.

5.2.1. Direct Effects

5.2.1.1. Aquatic-Phase CRLF

The aquatic-phase considers life stages of th e frog that are obligatory aquatic organisms, including eggs and larvae. It also considers submerged te rrestrial-phase juveniles and adults, which spend a portion of their time in water bodies that may receive runoff and spray drift containing diflubenzuron.

No acute LOCs were exceeded for fish based on the most sensitive species tested. No open literature studies were locat ed that reported lower toxicity values in either fish or amphibians than those used to calculate risk quotients. No incident s have been reported for diflubenzuron that associate its use with eff ects to aquatic animals. The probability of an individual mortality at the acute RQs of <0.01 is < 1 in 3.7E20 (<1 in 7.8E11 to <5.1E31) based on a slope of 4.7 (95% CI of 3.5 to 5.9, MRID 56150).

Therefore, the effect determination for poten tial acute direct effects to the CRLF is “no effect”.

The chronic LOC was exceeded only for direct application to rice. The RQ for this use was 1.12. RQs for other uses were <0.3. Howe ver, the RQ was based on results from a full life-cycle study that did not achieve a LOAEC (no eff ects were observed in this study at any concentration tested). In addition, the 60-day EEC used to calculate the rice exposure values did not allow for any degr adation over time. Because diflubenzuron degradates somewhat rapidly in the standard pond, similar ra tes of degradation would be expected to occur in rice paddies; therefor e, the EEC is likely an over-estimation of exposure. For these reasons (the close proxim ity of the RQ to the LOC, the lack of a LOAEC in the available study, and the l ack of degradation in the 60-day EEC calculation), the effects determination for dire ct application to rice is “not likely to adversely affect” because the potential for a direct effect is discountable (highly unlikely to occur).

The effects determination for all other uses is “no effect” based on the lack of LOC exceedances and the low probability of an individual mortality.

915.2.1.2. Terrestrial-Phase CRLF

Barnyard/Mushroom Use

Acute and chronic RQs were exceeded for CRLFs based on the avian assessment used as a surrogate species. The acute RQ was 1.2. Assuming a probit slope of 4.5, the probability of an individual e ffect is approximately 1 in 2. The reproduction RQ was 6.7. The reproduction RQ was based on re duced egg production at 1000 mg/kg-food (NOAEC was 500 mg/kg-food). At an RQ of 6. 7, exposure values were estimated to be approximately 3 times higher than levels asso ciated with reduced e gg production in birds (surrogate for CRLF). This assessment also assumed applications at the highest labeled application rate for 17 applica tions per year (21 days between applications). However, even assuming a single applica tion results in LOC exceedances (acute RQ = 0.74; chronic RQ = 2).

Potential effects to CRLFs depend on the pr oportion of its diet that may occur from contaminated food located at or near the use site. The barnyard/mushroom use is a spot treatment; therefore, the likelihood that a CRLF will consume contaminated food depends on the number and size of “spots” applie d to any given field, which may vary considerably from field to field. In addition, manure use is typically associated with feed lots, which are not attractive habitats for CRLFs due to high density of animals and resulting lack of habitat such as clean water and vegetation.

This assessment was based on food intake equations for birds. Birds ar e currently used as surrogates for reptiles and terrestrial-phase amphibians. However, reptiles and amphibians are poikilotherms (body temperature varies with environmental temperature) while birds are homeotherms (temperature is regulated, constant, and largely independent of environmental temperatures). Therefore, reptiles and amphibians (collectively referred to as herptiles) tend to have much lowe r metabolic rates and lower caloric intake requirements than birds or mammals. As a consequence, birds are likely to consume more food than amphibians or reptiles on a da ily dietary intake ba sis, assuming similar caloric content of the food items. This can be seen when comparing the estimated caloric requirements for free living i guanid lizards (Iguanidae) ( EQ 1 ) to passerines (song birds) ( EQ 2 ) (U.S. EPA, 1993):

iguanid FMR (kcal/day)= 0.0535 *(bw in g) 0.799 (EQ 1)

passerine FMR (kcal/day) = 2.123 *(bw in g) 0.749 (EQ 2)

With relatively comparable exponents (slopes) to the allometric functions, one can see that, given a comparable body weight, the free living metabolic rate of birds can be 40 times higher than reptiles, though the requi rement differences narrow with high body weights. Consequently, birds are conservative surrogates for terrestrial phase CRLFs because birds are expected to have higher pote ntial exposures relative to terrestrial phase amphibians.

92The potential differences in exposure of CRLFs and birds were quantified using a modified version of T-REX, called T-HER PS (version 1.0). T-HERPs incorporates food intake equations that are specific for te rrestrial phase amphibians and includes two additional food items of the CRLF that are not included in T-REX (CRLFs that consume small mammals and other amphibians).

The results of this analysis for the bar nyard/mushroom use are in Table 5-10. RQs remained above the endangered species LOC for the small mammal food item (CRLFs that consume small mammals). However, LOCs were not exceeded for other food items.

Table 5-10. Results from T-HERP S for the Barnyard/Mushroom Use. Amphibian/Reptile Acute RQs for Small, Medium, and Large Species (grams) Dose-based RQs (Dose-based EEC/adjusted LD50) 1.4 37 238 Short Grass 0.06 0.06 0.04 Tall Grass 0.03 0.03 0.02 Broadleaf plants/sm insects 0.03 0.03 0.02 Fruits/pods/seeds/lg insects 0.00 0.00 0.00 Small herbivore mammals N/A 0.97 0.15 Small insectivore mammals N/A 0.06 0.01 Small terrestrial phase amphibian N/A 0.00 0.00

Although barnyards and feedlots are not likely to typically pr ovide suitable habitat for CRLFs, the presence of CRLFs in such habita ts cannot be precluded. Therefore, based on acute and chronic LOC exceedances and th e likelihood of potential effects at the estimated exposure levels, the effects determination for direct effects to the CRLF is “likely to adversely affect” fo r the barnyard/mushroom use.

All Other Uses

No LOCs were exceeded. Acute RQs range d from <0.01 to 0.04. At these RQs, the probability of an individual effect is 1 in 6E9 based on the default probit slope of 4.5 (see Section 4). Based on reasonable lower a nd upper bounds on the slope of 2 to 8, the probability of an individual effect ranged from 1 in 4E2 to 1 in 7E35.

No incidents associating difl ubenzuron with effects to terrestrial animals have been reported to the Agency.

Also, as previously discussed, birds were used as a surrogate for the CRLF, which results in a conservative estimate of exposure and ri sk due to the greater energetic requirements and resulting greater food intake (and resulting potential exposures) of birds relative to terrestrial phase CRLFs.

Based on the lack of LOC exceedances in bird s, the low probability of an individual mortality, and the lack of reported incidents, diflubenzuron is e xpected to have “no effect” via direct toxic effects on terrestrial phase CRLFs.

935.2.2. Indirect Effects (via Reductions in Prey Base)

5.2.2.1. Algae (non-vascular plants)

As discussed in Section 2.5.3 , the diet of CRLF tadpoles is composed primarily of unicellular aquatic plants ( i.e. , algae and diatoms) and detritus. LOCs were not exceeded for aquatic plants. No effects were observed in any of the available toxicity studies in aquatic plants, and all RQs were less than 1 (the highest RQ was 0.6). Therefore, estimated diflubenzuron concentrations were lower than NOAEC values in all aquatic plant species tested.

Therefore, the effects determination for CRL Fs based on potential indirect effects from impacts to aquatic plants is “no effect.”

5.2.2.2. Aquatic Invertebrates

The potential for diflubenzuron to elicit i ndirect effects to th e CRLF via effects on freshwater invertebrate food items is depende nt on several factor s including: (1) the potential magnitude of effect on freshwater invertebrate individuals and populations; and (2) the number of prey species potentially affected relative to the expected number of species needed to maintain the dietary needs of the CRLF. Together, these data provide a basis to evaluate whether the number of indivi duals within a prey species is likely to be reduced such that it may indirectly affect the CRLF.

Based on the most sensitive species tested (mosquito), RQs exceeded LOCs and ranged from 4 to 40,000 (acute) and 25 to 450,000 (chronic ). At these acute RQs, the probability of an individual mortality approaches 100% , and potential impacts to abundance of aquatic invertebrates could be sufficien t to reduce available food of the CRLF.

As shown in Figure 5-1 below, based on open literature studies (Appendix G), the lowest EEC across all uses of diflubenzuron is above the average acute toxicity value for a majority of species tested (RQ >1). These values represent short- term toxicity studies, and may, therefore, underestimate potential impacts to aquati c invertebrates as discussed in Section 4.

The life stage of the exposed invertebrate is li kely to influence potential effects. Adults are less sensitive to diflubenzuron than larvae and juveniles. The results of littoral enclosure studies discussed in Section 4 sugge sted that some taxonomic groups of aquatic invertebrates could be impacted at diflubenzuron levels lower than 0.7 μg/L (NOEC not achieved). These impacts to the aquatic commu nity were also observed to be correlated to reductions in the growth rate of introduced bluegill sunfish, with reductions in length, weight, and growth rate occurring at initial nominal concentrations of 

### 2.5 μg/L and above. Growth reductions in fish were attr ibuted to reductions in food source (indirect

94effect). It is important to note that MRID 44386201 fails to empirically demonstrate a NOEC for a number of endpoints, namely ef fects on cladocerans, copepods, aquatic insect emergence and richness of macroinvertebrate taxa.

Based on the number of potential species that may be affected and the potential magnitude of effect, diflubenzuron is likel y to adversely affect the CRLF by reducing available aquatic prey. This effects determin ation applied to all labeled uses included in this assessment.

Based on AgDRIFT analysis (version 2.1.0.3, Tier 3 analysis using aerial and ground spray), spray drift could be sufficient to result in indirect effects at applications greater than 5000 feet from treated sites as eviden ced by LOC exceedances for the most sensitive species tested.

Mean Acute EC50 Values from Open Literature Data (Ecotox)

0 0.02 0.04 0.06 0.08

0.1 0.12 0.14 0.16 0.18

Blackfly Cyclopoid copepod Fairy Shrimp Insect class Midge Mosquito Water flea

SpeciesEC50 (ug/L)

Solid line represents minimum EEC Solid Line represents approx. 20th Percentile EEC

Figure 5-1. Mean acute EC50 values from open literature data according to the Ecotox data base. Figure note: Values for fairy shrimp and water flea ar e low such that they are not visible on the graph. EC50s for two species were not included in this figure due to the scale of the graph. A single EC50 for parasitic nematode (EC50: 4.4 μg/L) and a single EC 50 for backswimmer (EC 50 : 1.9 μg/L).

5.2.2.3. Fish and Aquatic-phase Frogs

As described in Section 5.2.1.1, the effects dete rmination for potential direct effects to the CRLF is “no effect” for acute effects and ch ronic effects for all uses except rice and is “not likely to adversely affect ” for chronic effects for the rice use. These conclusions are based on potential effects to the most sensitive fish species tested. The same conclusions are reached for the potential for diflubenzu ron to adversely affect the CRLF based on reduction in fish or frogs as food. See Section 5.2.1.1 for additional detail.

955.2.2.4. Terrestrial Invertebrates

When the terrestrial-phase CRLF reaches juveni le and adult stages, its diet is mainly composed of terrestrial invertebrates. RQs were based on acute studies in adult bees. Although RQs suggest that there is a potential for impacts to terres trial invertebrates, these RQs were based on a study that did not produce mortality at any concentration tested (30 μg/bee). However, as discussed in Section 4, acute studie s in adult bees are not considered to represent sens itive invertebrate life stages due to the mode of action of diflubenzuron as an insect growth regulator, which affects insects at molting. Results of field studies have been incons istent as discussed in Secti on 4; however, several field studies have shown reduced abundance in invert ebrates at currently approved application rates. Also, given the use of diflubenzuron as an insecticide, many insect species are presumably at risk if exposed at a sensitive life stage. Therefore, the effects determination from labeled use of diflubenzur on is “likely to adve rsely affect.” The potential impact to the diet of the CRLF will depend on the timing of application and predominance of invertebrates in sensitive life stages.

The distance from treated fields that effect s may occur has not been defined because the available data do not allow for quantitative measures of effects.

5.2.2.5. Mammals

Life history data for terres trial-phase CRLFs indicate that large adult frogs consume terrestrial vertebrates, including mice. No acute LOCs were exceeded for mammals. No effects were observed in the available acute oral toxicity study at doses up to 5000 mg/kg-bw. No reproduction RQs were exceeded for any use except the barnyard/mushroom use. Therefore, the effect s determination is “no effect” for all uses except barnyard/mushroom use based on the l ack of LOC exceedances and the resulting low magnitude of any potential effects to mammals.

Reproduction LOCs were exceeded for barnyard/mushroom use (RQ = 10). There is a 10-fold dose spacing in the available 2-genera tion reproduction toxicity study; therefore, at an RQ of 10, estimated exposures are equi valent to the LOAEC in mammals. At the LOAEC, there was a reduction in body weight in F1 pups. Frank reproductive effects were not observed. The EECs were based on the short grass food item and assumed that small mammal prey consume only contaminated short grass at high-end exposure levels. However, the barnyard/mushroom use is a sp ot treatment (directed spray at manure). Therefore, it is unlikely that a small mammal will obtain 100% of its diet as contaminated short grass in environments consistent with the barnyard/mushroom use. Nonetheless, such effects could not be precl uded. Therefore, the effects determination is “likely to adversely affect” based on poten tial reductions in mammalia n prey. These conclusions are highly uncertain.

965.2.2.6. Terrestrial-phase Amphibians

Terrestrial-phase adult CRLFs also consume ot her amphibians. RQ values representing direct exposures of diflubenz uron to terrestrial-phase CRL Fs are used to represent exposures of diflubenzuron to frogs in terres trial habitats. The dire ct effects assessment was “no effect” for all uses except the barnya rd/mushroom use. Therefore, the effects determination is also “no effect” via reduction in amphibian prey.

For the barnyard/mushroom use, the effect s determination was “likely to adversely affect” based on potential direct effects to the CRLF. However, the potential magnitude of effect is expected to be relatively low wh en considering dietary factors of terrestrial phase amphibians relative to th e surrogate avian species.

When incorporating food intake equations sp ecific for amphibians and reptiles using the T-HERPS model, LOCs were not exceeded fo r terrestrial phase amphibians that consume plants or insects. See Section 5.2.1.2 for additional information. Therefore, the effects determination for potential effects to the CRLF via reductions in available amphibian prey is “not likely to adversely affect.”

5.2.3. Indirect Effects (via Habitat Effects)

5.2.3.1. Aquatic Plants (Vascular and Non-vascular)

Aquatic plants serve several important functi ons in aquatic ecosystems. Non-vascular aquatic plants are primary producers and pr ovide the autochthonous energy base for aquatic ecosystems. Vascular plants provide structure as at tachment sites and refugia for many aquatic invertebrates, fish, and juvenile organisms, such as fish and frogs. In addition, vascular plants al so provide primary productivity and oxygen to the aquatic ecosystem. Rooted plants help reduce se diment loading and provide stability to nearshore areas and lower streambanks. In a ddition, vascular aquatic plants are important as attachment sites for egg masses of CRLFs.

Potential indirect effects to the CRLF ba sed on impacts to habitat and/or primary production were assessed using RQs from fres hwater aquatic vascul ar and non-vascular plant data. No LOCs were exceeded for va scular aquatic plants. No effects were observed in the available toxicity studies in a quatic plants at any te st level. EECs were lower than all vascular plant NOAECs. Theref ore, effects to vascular plants are not expected to occur. For this reason, the effects determination for the CRLF based on indirect effects resulting from impacts to vascular plants is “no effect.”

5.2.3.2. Terrestrial Plants

Terrestrial plants serve several important ha bitat-related functions for the CRLF. In addition to providing habitat and cover for inve rtebrate and vertebrate prey items of the

97CRLF, terrestrial vegetation also provides sh elter for the CRLF and cover from predators while foraging. Terrestrial plan ts also provide energy to th e terrestrial ecosystem through primary production. Upland vegetation incl uding grassland and woodlands provides cover during dispersal. Riparian vegetation he lps to maintain the integrity of aquatic systems by providing bank and thermal stability, serving as a buffer to filter out sediment, nutrients, and contaminants before they r each the watershed, and serving as an energy

source.

Impacts to terrestrial plants are not expected to occur at levels that may result in indirect effects to CRLFs. Although no suitable toxicity data are available in terrestrial plants, diflubenzuron is an insecticide that has b een used to treat numerous crops and non-crop plants for many years typically without incident. However, one incident has associated diflubenzuron with damage to oranges. This incident may or may not have been caused by diflubenzuron. It was assigned a certainty inde x of “possible”.

Although the data do not allow fo r quantification of potential ri sks to terrestrial plants, the weight of evidence suggests that effects to plants from labeled uses of diflubenzuron are minimal.

No guideline studies have been submitted that evaluated effects to terrestrial plants. Therefore, additional lines of evidence were used to determine if diflubenzuron may affect terrestrial plants at a magnitude that may result in indirect effects to the CRLF.

Diflubenzuron has a long history of use on num erous types of crops including grasses, trees, vines, and numerous fruit and vegeta ble crops without cl ear evidence of any adverse effects to terrestrial plants. Two fiel d studies were located in the open literature study that evaluated impacts to vegetation from use of difl ubenzuron that did not observe impacts to vegetation (discussed in Section 4) . No incidents have been reported that definitively linked use of diflubenzuron use to damage to terrestrial plants. One incident was associated with damage to orange tree s; however, that incident is more likely associated with use of petroleum distillate s as discussed in Section 4. In addition, numerous studies have been submitted to the Agency that evaluated efficacy of diflubenzuron, and no evidence of plant damage was reported in these studies.

For these reasons, use of diflubenzuron is not likely to affect the CRLF by impacting terrestrial vegetation.

The effects determination for the CRLF resulting from indirect effects from impacts to terrestrial plants is “no effect.”

5.2.4. Modification to Designated Critical Habitat

Risk conclusions for designate d critical habitat of the CRLF are the same as those for indirect effects. Critical habitat may be modified by imp acting availability of prey (terrestrial and aquatic i nvertebrates) as follows:

98• PCE: “reduction and/or modification of food sources for terrestrial phase juveniles and adults.” This PCE may be modified by diflubenzuron by potentially impacting invertebrate f ood sources. See Section 5.2.2.4.

• PCE: “alteration of chemical characteri stics necessary for normal growth and viability of juvenile and adult CRLFs a nd their food source.” This PCE may be impacted by effects to food sources such as terrestrial inverteb rates. See Section 5.2.2.4.

5.2.5. Spatial Extent of Potential Effects

Direct effects to the CRLF are limited to the barnyard/mushroom use. Locations of cattle feed lots (locations where diflubenzuron may be used on cattle manur e) are depicted in Figure 5.2. For this use, diflubenzuron is a pplied as a directed spray to manure. Based on the low magnitude of the RQ for this use and the low potential for spray drift, it is assumed that potential risks are limited to treated sites.

99Figure 5-2. Comparison of location of sites likely to use diflubenzuron for control of flies at livestock facilities (see te xt) and CRLF occupied core areas.

100RQs used to estimate potential for indirect e ffects to aquatic phase CRLFs were as high as approximately 500,000. The distance needed to reduce potential e xposures to levels that do not exceed LOCs is greater than di stances that can be predicted with any reliability. Potential indirect effects could occur at distan ces >5000 feet from the treated sites as described in Section 5.2.5.1

Indirect effects to the CRLF may also occur via impacts to terrestri al invertebrates. However, the data do not allow for quantification of the potential distance from treated sites to which risks extend primarily because th e toxicity data used to estimate potential impacts to terrestrial invert ebrates are based on adult honey bee studies. Sensitivity of adult honey bees to diflubenzuron is expected to be considerab ly lower than sensitivity of other insects, particularly inve rtebrates in early life stages. Therefore, potential impacts to terrestrial invertebrates ar e assumed to extend to distances greater than 1000 feet from treated fields.

An LAA effects determination applies to t hose areas where it is expected that the pesticide’s use will directly or indirectly affect the CRLF or its designated critical habitat. To determine this area, the footprint of di flubenzuron’s use pattern is identified, using land cover data that correspond to diflubenz uron’s use pattern. Areas of potential exposure also include areas beyond the initial area of concern ( e.g. , use footprint) that may be impacted by runoff and/or spray drift. The identified direct and indirect effects and modification to critical habitat are anticipated to occur only for those currently occupied core habitat areas, CNDDB occurrenc e sections, and designa ted critical habitat for the CRLF that overlap with the initial area of concer n plus >5000 feet from its boundary. It is assumed that non-flowing wa terbodies (or potentia l CRLF habitat) are included within this area. Direct eff ects are only expected to occur for the barnyard/mushroom use. For this use, risks are expected to be predominantly restricted to the treated site because of the mode of application (directed spray to manure).

The determination of the buffer distance and downstream dilution for spatial extent of the effects determination is described below.

5.2.5.1. Spray Drift

In order to determine terrestrial and aquati c habitats of concern due to diflubenzuron exposures through spray drift, it is necessary to estimat e the distance that spray applications can drift from the treated area and still be present at concentrations that exceed levels of concern. An analysis of spray drift distances was completed using AgDRIFT (version 2.01). AgDRIFT was run usi ng default parameters. Tier 3 was used to expand the distance evaluated to 5000 feet.

101Table 5-11. Summary of Ag DRIFT* Predicted Terrestrial Spray Drift Distances

Landcover Uses Represented Highest RQ Distance to reduce spray drift to level that is <LOC Forest Forestry 3200 >5000 feet Non-cropland agriculture Turf, nursery 2600 >5000 feet

Residential Residential, urban, rights of
ways 137,000 >5000 feet

Orchard Fruit, nuts, pistachio, Beech

nut 5400 >5000 feet

Pasture/Rangeland Barnyards, pastures + 217,000 (barnyard) 2200 (pasture) >5000 feet

Cropland Turnip, squash, row crops, rice, grains, nuts, cotton, cole crops, brassica, 452,000 >5000 feet

* AgDRIFT was run in Tier 3, with a maximum distance of 5 000 feet and RQs for a quatic invertebrates.

AgDRIFT has not been validated at distances >100 0 feet; therefore, there is uncertainty in the estimations presented in Table 5-11. +.Minimal drift is expected from the barnyard uses because of the mode of application (downward spray

directed at manure); therefore, the drift dist ance was based on the pastureland RQ of 2200.

5.2.6.2. Downstream Dilution Analysis

The downstream extent of exposure in streams and rivers where the EEC could potentially be above levels that would exceed the most sensitive LOC was not completed because of the large number of uses and large corresponding landcover area. The large geographic extent of potential use sites prec ludes quantification of dilution potential based on landcover data repr esenting non-use sites.

5.2.6.3. Overlap between CRLF habita t and Spatial Extent of Potential Effects

An LAA effects determination is made to t hose areas where it is expected that the pesticide’s use will directly or indirectly affect the CRLF or its designated critical habitat and the area overlaps with the core areas, crit ical habitat and available occurrence data for CRLF.

For diflubenzuron, the use pattern encompasses all land cover classes. Therefore, the initial area of concern is the entire state of CA. As described in Section 5, diflubenzuron could potentially impact the CRLF via indi rect effects to aquatic and terrestrial invertebrates. Effects could occur at distances >5000 feet fr om the site of application. Potential overlap between the CRLF and diflube nzuron uses is graphi cally represented in Figure 5.3.

102Recovery Units 1. Sierra Nevada Foothills and Central Valley 2. North Coast Range Foothills and Western Sacramento River Valley 3. North Coast and North San Francisco Bay 4. South and East San Francisco Bay 5. Central Coast 6. Diablo Range and Salinas Valley 7. Northern Transverse Ranges and Tehachapi Mountains 8. Southern Transverse and Peninsular Ranges

Core Areas

1. Feather River 2. Yuba River- S. Fork Feather River 3. Traverse Creek/ Middle Fork/ American R. Rubicon 4. Cosumnes River 5. South Fork Calaveras River* 6. Tuolumne River* 7. Piney Creek* 8. Cottonwood Creek 9. Putah Creek – Cache Creek* 10. Lake Berryessa Tributaries 11. Upper Sonoma Creek 12. Petaluma Creek – Sonoma Creek 13. Pt. Reyes Peninsula 14. Belvedere Lagoon 15. Jameson Canyon – Lower Napa River 16. East San Francisco Bay 17. Santa Clara Valley 18. South San Francisco Bay 19. Watsonville Slough-Elkhorn Slough 20. Carmel River – Santa Lucia 21. Gablan Range 22. Estero Bay 23. Arroyo Grange River 24. Santa Maria River – Santa Ynez River 25. Sisquoc River 26. Ventura River – Santa Clara River 27. Santa Monica Bay – Venura Coastal Streams 28. Estrella River 29. San Gabriel Mountain* 30. Forks of the Mojave* 31. Santa Ana Mountain* 32. Santa Rosa Plateau 33. San Luis Ray* 34. Sweetwater* 35. Laguna Mountain*

* Core areas that were historically occupied by the California red-legged frog are not included in the map Recovery Units 1. Sierra Nevada Foothills and Central Valley 2. North Coast Range Foothills and Western Sacramento River Valley 3. North Coast and North San Francisco Bay 4. South and East San Francisco Bay 5. Central Coast 6. Diablo Range and Salinas Valley 7. Northern Transverse Ranges and Tehachapi Mountains 8. Southern Transverse and Peninsular Ranges

Core Areas

1. Feather River 2. Yuba River- S. Fork Feather River 3. Traverse Creek/ Middle Fork/ American R. Rubicon 4. Cosumnes River 5. South Fork Calaveras River* 6. Tuolumne River* 7. Piney Creek* 8. Cottonwood Creek 9. Putah Creek – Cache Creek* 10. Lake Berryessa Tributaries 11. Upper Sonoma Creek 12. Petaluma Creek – Sonoma Creek 13. Pt. Reyes Peninsula 14. Belvedere Lagoon 15. Jameson Canyon – Lower Napa River 16. East San Francisco Bay 17. Santa Clara Valley 18. South San Francisco Bay 19. Watsonville Slough-Elkhorn Slough 20. Carmel River – Santa Lucia 21. Gablan Range 22. Estero Bay 23. Arroyo Grange River 24. Santa Maria River – Santa Ynez River 25. Sisquoc River 26. Ventura River – Santa Clara River 27. Santa Monica Bay – Venura Coastal Streams 28. Estrella River 29. San Gabriel Mountain* 30. Forks of the Mojave* 31. Santa Ana Mountain* 32. Santa Rosa Plateau 33. San Luis Ray* 34. Sweetwater* 35. Laguna Mountain*

* Core areas that were historically occupied by the California red-legged frog are not included in the map Figure 5-3 Recovery Unit, Core Are a, Critical Habitat, and Occurrence Designations for CRLF

103

### 6.0 Uncertainties

6.1 Exposure Assessment Uncertainties

6.1 .1 Maximum Use Scenario

The screening-level risk assessment focuses on characterizing poten tial ecological risks resulting from a maximum use scenario, which is determined from labeled statements of maximum application rate and number of appli cations with the shortest time interval between applications. The frequency at whic h actual uses approach this maximum use scenario may be dependant on pest resistance, timing of applications, cultural practices, and market forces.

Several fast-maturing crops may have multiple crop cycles within a single year. For these crops, it is often the case that labels do not specify a maximum a nnual application rate, but rather a maximum crop cycle rate (m aximum pounds per acre per crop cycle). Because diflubenzuron dissipates rapidly in the environment (aerobic soil metabolism rate is ~2 days), additional applications of diflubenzuron during following crop cycles are not expected to greatly increase terrestrial EEC s within the field where it is applied or in the terrestrial spray drift zone. However, aquatic EECs can be affected because the multiple crop cycles extend the growing season into those parts of the year when runoff is more likely (fall through early spring). To address this uncertainty, aquatic EECs were modeled across a large range of application dates (Section 3.2.3 and Appendix tables D4 and D5).

6.1.2 Crops Not Grown in California (include if you had crops that are not grown in CA)

Soybeans are the only crop listed on diflubenzuron labels that is not grown in California according to National Agricultural Statistical Service data. Because these crops are not currently grown in California a nd are not expected to be grow n in California in the future, use of diflubenzuron was not assessed for thes e crops. If use patte rns indicate soybeans are grown in California in the future, the conclusions of this assessment may need to be revisited.

6.1.3 Aquatic Exposure Modeling of Diflubenzuron

The standard ecological water body scenario (EXAMS pond) used to calculate potential aquatic exposure to pesticides is intended to represent conservative estimates, and to avoid underestimations of the actual exposure. The standard scenario consists of application to a 10-hectare field border ing a 1-hectare, 2-meter deep (20,000 m 3 ) pond with no outlet. Exposure estimates genera ted using the EXAMS pond are intended to represent a wide variety of vulnerable water bodies that occur at the top of watersheds

104including prairie pot holes, playa lakes, wetlands, vernal pools, man-made and natural ponds, and intermittent and lower order streams. As a group, there are factors that make these water bodies more or less vulnerable than the EXAMS pond. Static water bodies that have larger ratios of pesticide-treate d drainage area to water body volume would be expected to have higher peak EECs than the EXAMS pond. These water bodies will be either smaller in size or have larger drainage areas. Sm aller water bodies have limited storage capacity and thus may overflow and carry pesticide in the discharge, whereas the EXAMS pond has no discharge. As waters hed size increases beyond 10-hectares, it becomes increasingly unlikely that the entire watershed is planted w ith a single crop that is all treated simultaneously with the pesticid e. Headwater streams can also have peak concentrations higher than the EXAMS pond, but they likely persist for only short periods of time and are then car ried and dissipated downstream.

The Agency acknowledges that there are so me unique aquatic habitats that are not accurately captured by this modeling scenar io and modeling results may, therefore, under- or over-estimate exposure, depending on a number of variables. For example, aquatic-phase CRLFs may inhabit water bodies of different size a nd depth and/or are located adjacent to larger orsmaller drainage areas than the EXAMS pond. The Agency does not currently have sufficient informa tion regarding the hydrology of these aquatic habitats to develop a specific alternate scenar io for the CRLF. CRLFs prefer habitat with perennial (present year-round) or near-perennial water and do not frequently inhabit vernal (temporary) pools because conditions in these habitats are generally not suitable (Hayes and Jennings 1988). Therefore, the EXAMS pond is assumed to be representative of exposure to aquatic-phase CRLFs. In addi tion, the Services agree that the existing EXAMS pond represents the best currently available approach for estimating aquatic exposure to pesticides (U.S. FWS/NMFS 2004).

In general, the linked PRZM/EXAMS model produces estimated aquatic concentrations that are expected to be exceeded once within a ten-year period. The Pesticide Root Zone Model is a process or “simulation” model that calculates what happens to a pesticide in an agricultural field on a day-to-day basis. It considers factors such as rainfall and plant transpiration of water, as well as how and when the pesticide is applied. It has two major components: hydrology and chemical transport. Water movement is simulated by the use of generalized soil parameters, including fi eld capacity, wilting point, and saturation water content. The chemical transport com ponent can simulate pesticide application on the soil or on the plant foliage. Dissolved, adsorbed, and vapor-phase concentrations in the soil are estimated by simultaneously cons idering the processes of pesticide uptake by plants, surface runoff, erosion, decay, volatilization, foliar wash-off, advection, dispersion, and retardation.

Uncertainties associated with each of thes e individual components add to the overall uncertainty of the modeled concentrations . Additionally, model inputs from the environmental fate degradation studies ar e chosen to represen t the upper confidence bound on the mean values that are not expected to be exceeded in the environment approximately 90 percent of the time. M obility input values are chosen to be representative of conditions in the environment. The natural va riation in soils adds to the

105uncertainty of modeled values. Factors such as application date, crop emergence date, and canopy cover can also affect estimated c oncentrations, adding to the uncertainty of modeled values. Factors within the ambien t environment such as soil temperatures, sunlight intensity, antecedent soil moistu re, and surface water temperatures can cause actual aquatic concentrations to differ for the modeled values.

Unlike spray drift, tools are currently not av ailable to evaluate th e effectiveness of a vegetative setback on runoff and loadings. Th e effectiveness of vegetative setbacks is highly dependent on the condition of the vegetative strip. For example, a wellestablished, healthy vegetative setback can be a very eff ective means of reducing runoff and erosion from agricultural fields. Alternatively, a setbac k of poor vegetative quality or a setback that is channelized can be ine ffective at reducing loadings. Until such time as a quantitative method to estimate the effect of vegetative setbacks on various conditions on pesticide loadings becomes available, the aqua tic exposure predictions are likely to overestimate exposure where h ealthy vegetative setbacks exist and underestimate exposure where poorly developed, channelized, or bare setbacks exist.

6.1.4 Usage Uncertainties

County-level usage data were obtained from California’s Department of Pesticide Regulation Pesticide Use Reporti ng (CDPR PUR) database. Ei ght years of data (1999 – 2006) were included in this analysis b ecause statistical methodology for identifying outliers, in terms of area treated and pounds applied, was provided by CDPR for these years only. No methodology for removing out liers was provided by CDPR for 1998 and earlier pesticide data; therefore, this inform ation was not included in the analysis because it may misrepresent actual usage patterns. CDPR PUR documentation indicates that errors in the data may include the following: a misplaced decimal; incorrect measures, area treated, or units; and reports of diluted pesticide c oncentrations. In addition, it is possible that the data may contain reports for pesticide uses that have been cancelled. The CPDR PUR data does not include home owner applied pesticides; therefore, residential uses are not likely to be reported. As with all pesticid e usage data, there may be instances of misuse and misreporting. The Agency made use of the most current, verifiable information; in cases where ther e were discrepancies, the most conservative information was used.

6.1.5 Terrestrial Exposure Modeling of Diflubenzuron

The Agency relies on the work of Fletcher et al. (1994) for setting the assumed pesticide residues in wildlife dietary it ems. These residue assumptions are believed to reflect a realistic upper-bound residue estimate, alt hough the degree to which this assumption reflects a specific percentile estimate is difficult to quantify. It is important to note that the field measurement efforts used to devel op the Fletcher estimates of exposure involve highly varied sampling techniques. It is entirely possible that much of these data reflect residues averaged over entire above ground pl ants in the case of grass and forage sampling.

106It was assumed that ingestion of food items in the field occurs at rates commensurate with those in the laborato ry. Although the screening a ssessment process adjusts dryweight estimates of food intake to reflect the increased mass in fresh-weight wildlife food intake estimates, it does not allow for gross energy differences. Direct comparison of a laboratory dietary concentrati on- based effects threshold to a fresh-weight pesticide residue estimate would result in an underestimation of field exposure by food consumption by a factor of 1.25 – 2.5 for most food items.

Differences in assimilative efficiency betw een laboratory and wild diets suggest that current screening assessment me thods do not account for a potentially important aspect of food requirements. Depending upon species and dietary matrix, bird assimilation of wild diet energy ranges from 23 – 80%, and mamm al’s assimilation ranges from 41 – 85% (U.S. EPA 1993). If it is assumed that labo ratory chow is formulated to maximize assimilative efficiency ( e.g. , a value of 85%), a potential for underestimation of exposure may exist by assuming that consumption of food in the wild is comparable with consumption during laboratory testing. In the screening process, exposure may be underestimated because metabolic rates are not related to food consumption.

For the terrestrial exposure analysis of this risk assessment, a generic bird or mammal was assumed to occupy either the treated field or adjacent areas receiving a treatment rate on the field. Actual habitat requirements of any particular terrestrial species were not considered, and it was assumed that species occupy, exclusively and permanently, the modeled treatment area. Spray drift model predictions sugges t that this assumption leads to an overestimation of exposure to species that do not occupy the treated field exclusively and permanently.

6.1.6 Spray Drift Modeling

Although there may be multiple diflubenzuron app lications at a single site, it is unlikely that the same organism would be exposed to the maximum amount of spray drift from every application made. In order for an or ganism to receive the maximum concentration of diflubenzuron from multiple applications, each application of diflubenzuron would have to occur under identical atmospheric conditions ( e.g. , same wind speed and – for plants – same wind direction) and (if it is an animal) th e animal being exposed would have to be present directly downwind at the same distance after each application. Although there may be sites where the dominant wind direction is fairly consistent (at least during the relatively quiescent conditions that are most favorable for aerial spray applications), it is neverthe less highly unlikely that plants in any specific area would receive the maximum amount of spray drift repe atedly. It appears that in most areas (based upon available meteorological data) wind direction is tempora lly very changeable, even within the same day. Additionally, other factors, including variations in topography, cover, and meteorological conditio ns over the transport distance are not accounted for by the AgDRIFT model ( i.e ., it models spray drift from aerial and ground applications in a flat area with little to no ground cover and a steady, constant wind speed and direction). Therefore, in most cas es, the drift estimates from AgDRIFT may

107overestimate exposure even from single applica tions, especially as the distance increases from the site of application, since the m odel does not account for potential obstructions ( e.g. , large hills, berms, buildings, trees, etc .). Furthermore, conservative assumptions are often made regarding the droplet size di stributions being mode led (‘ASAE Very Fine to Fine’ for orchard uses and ‘ASAE Very Fi ne’ for agricultural uses), the application method ( e.g. , aerial), release heights and wind speeds. Alterations in any of these inputs would change the area of potential effect.

6.2 Effects Assessment Uncertainties

6.2.1 Age Class and Sensitivity of Effects Thresholds

It is generally recognized that test orga nism age may have a significant impact on the observed sensitivity to a toxicant. The acute toxicity data for fish are collected on juvenile fish between 0.1 and 5 grams. Aqua tic invertebrate acute testing is performed on recommended immature age classes ( e.g. , first instar for daphni ds, second instar for amphipods, stoneflies, mayflies, and third instar for midges).

Testing of juveniles may overe stimate toxicity at older age classes for pesticide active ingredients that act directly without me tabolic transformation because younger age classes may not have the enzymatic systems a ssociated with detoxifying xenobiotics. In so far as the available toxicity data may provide ranges of sensitivity information with respect to age class, this assessment uses th e most sensitive life-stage information as measures of effect for surrogate aquatic an imals, and is therefore, considered as protective of the CRLF.

For diflubenzuron, the most sensitive endpoints are for immature invertebrates that are molting. However, acute toxicity test procedur es typically select or ganisms that are not molting during the test. This may result in an underestimation of risk to molting individuals of the same species.

6.2.2 Use of Surrogate Species Effects Data

Guideline toxicity tests and open literature data on diflubenzuron are not available for frogs or any other aquatic-phase amphibian; therefore, freshwater fish are used as surrogate species for aquatic-phase amphi bians. Therefore, endpoints based on freshwater fish ecotoxicity data are assumed to be protective of potential direct effects to aquatic-phase amphibians including the CRLF, and extrapolation of the risk conclusions from the most sensitive tested species to the aquatic-phase CRLF is likely to overestimate the potential risks to those species. Efforts ar e made to select the organisms most likely to be affected by the type of compound and us age pattern; however, there is an inherent uncertainty in extrapolating across phyla. In addition, the Agency’s LOCs are intentionally set very low, and conservative estimates are made in the screening level risk assessment to account fo r these uncertainties.

1086.2.3 Sublethal Effects

When assessing acute risk, the screening risk assessment relies on the acute mortality endpoint as well as a suite of sublethal respon ses to the pesticide, as determined by the testing of species response to chronic expos ure conditions and subsequent chronic risk assessment. Consideration of additional sublet hal data in the effects determination t is exercised on a case-by-case basis and only after careful consideration of the nature of the sublethal effect measured and the extent and quality of available data to support establishing a plausible relationship between the measure of effect (sublethal endpoint) and the assessment endpoints. However, the fu ll suite of sublethal effects from valid open literature studies is considered fo r the purposes of defining the action area.

No open literature or registrant studies on s ublethal effects associated with exposure to diflubenzuron were identified. To the extent to which sublet hal effects are not considered in this assessment, the potential direct and indirect effects of di flubenzuron on CRLF may be underestimated.

6.2.4 Location of Wildlife Species

For the terrestrial exposure analysis of this risk assessment, a generic bird or mammal was assumed to occupy either the treated field or adjacent areas receiving a treatment rate on the field. Actual habitat requirements of any particular terrestrial species were not considered, and it was assumed that species occupy, exclusively and permanently, the modeled treatment area. Spray drift model predictions sugges t that this assumption leads to an overestimation of exposure to species that do not occupy the treated field exclusively and permanently.

109

### 7.0 Risk Conclusions

In fulfilling its obligations under Section 7(a)(2) of the Endangered Species Act, the information presented in this endangered specie s risk assessment represents the best data currently available to assess the potential risks of diflubenzuron to the CRLF and its designated critical habitat.

Based on the best available information, the Ag ency makes a Likely to Adversely Affect determination for the CRLF from the use of diflubenzuron. The Agency has determined that there is no potential fo r modification of CRLF designate d critical habitat from the use of the chemical. All the uses of diflube nzuron are likely to adversely affect the CRLF primarily because of a there is a signi ficant risk to terre strial and aquatic invertebrate prey of the CRLF. There is a L AA for risk of direct effect to the aquatic phase CRLF only for the diflubenzuron use on rice and there is a LAA for risk of direct effect to the terrestrial phase CRLF for di flubenzuron use in barnyard/mushroom sites.

The LAA effects determination applies to t hose areas where it is expected that the pesticide’s use will directly or indirectly affect the CRLF or its designated critical habitat. To determine this area, the footprint of di flubenzuron’s use pattern is identified, using land cover data that correspond to diflubenzuron’s use pattern. The spatial extent of the LAA effects determination also includes area s beyond the initial area of concern that may be impacted by runoff and/or spray drift. The identified indirect effects are anticipated to occur only for those currently occupied core habitat areas, CNDDB occurrence sections, and designated critical habitat for the CRLF that overlap with the initi al area of concern + 5000 feet from its boundary (refer to analysis in Section 5.1. 4). It is assumed that nonflowing waterbodies (or poten tial CRLF habitat) are incl uded within this area.

A summary of the risk conclusions and e ffects determinations for the CRLF and its critical habitat, given the un certainties discussed in Secti on 6, is presented in Table 7-1 and 7-2.

Table 7-1. Effects Determination Summar y for Diflubenzuron Use and the CRLF

Assessment

Endpoint Effects Determination 1 Basis for Determination

Potential for Direct Effects Aquatic-phase (Eggs, Larvae, and Adults) :

Direct effects are not expected fo r any labeled use. All effects determinations are either NE or NLAA.

Terrestrial-phase (Juveniles and Adults) : Direct effects are only expected for diflubenzuron’s use on barn yards based on acute and chronic LOC’s for birds.

Potential for Indirect Effects Survival, growth, and/or reproduction of CRLF individuals LAA, all uses

Aquatic prey items, aquatic habitat, cover and/or primary

p roductivit y

110Assessment Endpoint Effects Basis for Determination Determination 1 Aquatic prey of the CRLF are expected to be impacted to an extent that could adversely affect CRLFs.

Terrestrial prey items, riparian habitat

Terrestrial prey items of the CRLF are expected to be impacted based on LOC exceedances for risk to terrestrial invertebrates.

1 No effect (NE); May affect, but not likely to adversely affect (NLAA) ; May affect, likely to adversely affect (LAA)

Table 7-2. Effects Determination Summary for Diflubenzuron Use and CRLF Critical Habitat Impact Analysis

Assessment

Endpoint Effects Determination 1 Basis for Determination

Modification of aquatic-phase PCE There are LOC exceedances for ri sk to terrestrial and aquatic invertebrate prey of the CRLF.

Modification of terrestrial-phase PCE Habitat Modification

There are LOC exceedances for ri sk to terrestrial and aquatic invertebrate prey of the CRLF.

1 Habitat Modification or No effect (NE)

Based on the conclusions of this assessment, a formal consultation with the U. S. Fish and Wildlife Service under Sect ion 7 of the Endangered Specie s Act should be initiated. When evaluating the significance of this ri sk assessment’s direct /indirect and adverse habitat modification effects determinations , it is important to note that pesticide exposures and predicted risks to the species and its resources ( i.e. , food and habitat) are not expected to be uniform across the action ar ea. In fact, given the assumptions of drift and downstream transport ( i.e. , attenuation with distance) , pesticide exposure and associated risks to the species and its resour ces are expected to decrease with increasing distance away from the treated field or site of application. Evaluation of the implication of this non-uniform distribution of risk to the species would re quire information and assessment techniques that are not currently avai lable. Examples of such information and methodology required for this type of an alysis would include the following:

• Enhanced information on the density and distribution of CRLF life stages within specific recovery units and/or designated critical habitat within the action area. This information would allow for quantitat ive extrapolation of the present risk assessment’s predictions of individual effects to the proportion of the population extant within geographical areas where t hose effects are predicted. Furthermore, such population information would allow fo r a more comprehensive evaluation of the significance of potential resource impa irment to individuals of the species.

• Quantitative information on prey base requirements for individual aquatic- and terrestrial-phase frogs. While existing in formation provides a preliminary picture of the types of food sources utilized by the frog, it does not establish minimal

111requirements to sustain healthy individua ls at varying life stages. Such information could be used to establish bi ologically relevant thresholds of effects on the prey base, and ultimately establis h geographical limits to those effects. This information could be used together with the density data discussed above to characterize the likelihood of a dverse effects to individuals.

• Information on population responses of prey base organisms to the pesticide. Currently, methodologies are limited to pr edicting exposures and likely levels of direct mortality, growth or reproduct ive impairment immediately following exposure to the pesticide. The degree to which repeated exposure events and the inherent demographic characte ristics of the prey population play into the extent to which prey resources may recover is not predictable. An enhanced understanding of long-term prey responses to pesticid e exposure would allow for a more refined determination of the magnitude and durati on of resource impairment, and together with the information described above, a more complete prediction of effects to individual frogs and potential modi fication to critical habitat.

112

### 8.0 References

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Altig, R.; R.W. McDiarmid. 1999. Body Plan: Development and Morphology. In R.W. McDiarmid and R. Altig (Eds.), Tadpoles: The Biology of Anuran Larvae

. University of Chicago Press, Chicago. pp. 24-51.

Boyle, T. P., Fairchild, J. F., Robinson-Wils on, E. F., Haverland, P. S., and Lebo, J. A. (1996). Ecological Restructur ing in Experimental Aqua tic Mesocosms due to the Application of Diflubenzuron. Environ.Toxicol.Chem. 15: 1806-1814.

Cooper, R. J., Dodge, K. M., Martinat, P. J ., Donahoe, S. B., and Whitmore, R. C. (1990). Effect of Diflubenzuron Application on Eastern Deciduous Forest Birds. J.Wildl.Manag. 54: 486-493.

Fellers, G.M, L.L. McConnell, D. Pratt, S. Datta. 2004. Pesticides in Mountain YellowLegged Frogs (Rana Mucosa) from the Sie rra Nevada Mountains of California, USA. Environmental Toxicology & Chemistry 23 (9):2170-2177.

Fellers, G. M. 2005a. Rana draytonii Baird and Girard 1852. California Red-legged Frog. Pages 552-554. In : M. Lannoo (ed.) Amphibian Declines: The Conservation Status of United States Species , Vol. 2: Species Accounts. University of California Press, Berkeley, California. xxi+1094 pp. ( http://www.werc.usgs.gov/pt-rey es/pdfs/Rana%20draytonii.PDF )

Fellers, G. M. 2005b. California red-legged frog, Rana draytonii Baird and Girard. Pages 198-201. In : L.L.C. Jones, et al (eds.) Amphibians of the Pacific Northwest

. xxi+227.

Fletcher J.S.; J.E. Nellessen; T.G. Pfleeger . 1994. Literature Review and Evaluation of the EPA Food-chain (Kenaga) Nomogram, an Instrument for Estimating Pesticide Residues on Plants . Environmental Toxicology and Chemistry , 13:9 (1383-1391).

Hayes, M. P.; M. R. Jennings. 1988. Habitat correlates of distribut ion of the California redlegged frog ( Rana aurora draytonii ) and the foothill yellow-legged frog ( Rana boylii ): Implications for management. Pages 144-158 In: R. Sarzo, K. E. Severson, and D. R. Patton (technical coordinators). Proceedings of the Symposium on the Management of Amphibi ans, Reptiles, and small mammals in NorthAmerica. U.S.D.A. Forest Service Ge neral Technical Report RM-166.

113Hayes, M.P. and M.M. Miyamoto. 1984. Biochemical, behavioral and body size differences between Rana aurora aurora and R. a. draytonii

. Copeia 1984(4): 1018-22.

Hayes, M.P.; M.R. Tennant. 1985. Diet and feed ing behavior of the California red-legged frog. The Southwestern Naturalist 30(4): 601-605.

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Jennings, M.R.; M.P. Hayes. 1994. Amphibian and reptile species of special concern in California. Report prepared for the Californi a Department of Fish and Game, Inland Fisheries Division, Rancho Cordova, California. 255 pp.

Jennings, M.R.; S. Townsend; R.R. Duke. 1997. Santa Clara Valley Water District California red-legged frog distribution and status – 1997 . Final Report prepared by H.T. Harvey & Associates, Alviso, California. 22 pp.

Kashian, D. R. and Dodson, S. I. (2002) . Effects of Common-Use Pesticides on Developmental and Reproductive Processes in Daphnia. Toxicol.Ind.Health 18: 225-235.

Kupferberg, S. 1997. Facilitation of periphyt on production by tadpole gr azing: Functional differences between species. Freshwater Biology 37:427-439.

Kupferberg, S.J.; J.C. Marks and M.E. Power. 1994. Effects of varia tion in natural algal and detrital diets on larval anuran ( Hyla regilla ) life-history traits. Copeia 1994: 446-457.

LeNoir, J.S., L.L. McConnell, G.M. Fellers, T.M. Cahill, J.N. Seiber. 1999. Summertime Transport of Current-use pesticides from California’s Central Valley to the Sierra Nevada Mountain Range,USA. Environmental Toxicology & Chemistry 18(12): 2715-2722.

McConnell, L.L., J.S. LeNoir, S. Datta, J.N. Seiber. 1998. Wet deposition of current-use pesticides in the Sierra Nevada mountain range, California, USA. Environmental Toxicology & Chemistry 17(10):1908-1916.

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Rathburn, G.B. 1998. Rana aurora draytonii egg predation. Herpetological Review 29(3): 165.

114Reis, D.K. 1999. Habitat characteristics of California red-legged frogs (Rana aurora draytonii): Ecological differ ences between eggs, tadpoles, and adults in a coastal brackish and freshwater system . M.S. Thesis. San Jose State University. 58 pp.

Seale, D. B., and N. Beckvar. 1980. The comp arative ability of anuran larvae (genera: Hyla

, Bufo , and Rana ) to ingest suspended blue-green algae. Copeia 1980: 495- 503.

U.S. Environmental Protection Agency. (U.S. EPA). 1993. Wildlife Exposure Factors Handbook. EPA/600/R-13/187a, Office of Research and Development, Washington, DC.

U.S. EPA. 1997. Diflubenzuron. Reregistration Eligibility Decision. OPPT. EPA 738-R97-008. August, 1997

U.S. EPA. 1998. Guidance for Ecological Risk Assessment. Risk Assessment Forum. EPA/630/R-95/002F, April 1998.

U.S. EPA. 2004. Overview of the Ecological Risk A ssessment Process in the Office of Pesticide Programs. Office of Prevention, Pestic ides, and Toxic Substances. Office of Pesticide Programs. Washington, D.C. January 23, 2004.

U.S. Fish and Wildlife Service (U.S. FWS) . 1996. Endangered and threatened wildlife and plants: determination of threatened status for the California red-legged frog. Federal Register 61(101):25813-25833.

U.S. FWS and National Marine Fisherie s Service (NMFS). 1998. Endangered Species Consultation Handbook: Procedures for Conducting Consultation and Conference Activities Under Section 7 of the Endangered Species Act. Final Draft. March 1998.

U.S. FWS. 2002. Recovery Plan for the California Red-legged Frog ( Rana aurora draytonii ). Region 1, USFWS, Portland, Oregon. ( http://ecos.fws.gov/doc /recovery_plans/2002/020528.pdf )

U.S. FWS/NMFS. 2004. 50 CFR Part 402. Joint Counterpart Endangered Species Act Section 7 Consultation Regulations; Final Rule. 69 Federal Register 47732- 47762.

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115Wassersug, R. 1984. Why tadpoles love fast food. Natural History 4/84.

Willis, G. H.; L.L. McDowell. 1987. Pesticide persistence on foliage. Reviews of Environmental Contamination and Toxicology 100: 23–73.

Previous Assessments

U.S. EPA 2002a. Environmental Fate a nd Ecological Effects Assessment and Characterization for a Section 3 for Aeri al and Ground Spray Treatment to Pears, Stone Fruits, Almonds, Peppers, a nd Pasture Grass (DP 321153, DP 321278) Ecological Fate and Effects Division, Office of Pesticide Programs, U.S. Environmental Protection Agen cy, Washington D.C. May 2002.

U.S. EPA 2006. Environmental Fate and Ecolog ical Risk Assessment for the Registration of Diflubenzuron (Dimilin-2L) Use on Peanuts, Okra, Small Grains-(Winter wheat, Spring wheat, Durum, Barley, and Oats) , Pummelo, Mustard Greens, Broccoli, Raab, Cabbage, Chinese (bok c hoy); Collards; Kale; Mizuna; Mustard spinach; Rape, Greens and Turnip gr eens. (DP 321153, DP 321278). Ecological Fate and Effects Division, Office of Pe sticide Programs, U.S. Environmental Protection Agency, Washington D.C., September 2006.

U.S. EPA 2008. New Use of Dimilin®2L (act ive ingredient 22% Diflubenzuron) on Fly Breeding Areas in Livestock Opera tions. (DP 348755). Ecological Fate and Effects Division, Office of Pesticide Pr ograms, U.S. Environmental Protection Agency, Washington D.C., September 2008.

U.S. EPA 2009. County-Level Usage for Strychnidin; Strychnine; Triclopyr, butoxyethyl ester; Triclopyr, triethylamine salt; Diflubenzuron; Trifluralin ; Thiobencarb; Chlorpyrifos; Vinclozolin; Iprodione in California in Support of a Red Legged Frog Endangered Species Assessment, DP # TBD. Biological and Economic Analysis Divis ion , Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington D.C., June 8 th , 2009.

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