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### SUBJECf: Re g i stra tion Rev iew

- Problem F ormulation for d -Limonene (CAS R eg . N o

. 59 89-27-5)

Jim Goo dyear

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### TO : Rus se ll W asem , C hemical R eview Mana ger Ri s k M anagement and Impl ementation B ranch 1

P e s ticide R e-evaluation D iv isio n

Th e Environm enta l Fa te an d Effec t s Divi s ion (EF ED) h as co mpleted t he prelim inary prob lem

fo rmulation (a ttached ) for th e e co logica l ri sk, e n v ironmental fate

, e ndangered s peci es, an d

drinkin g wa ter assess ments to be condu cted a s p art of th e R egi strati on Rev iew o f d-limonene

. T he pr obl e m C annul ation draws i nf orma tion fr o m both open li terature and fro m s t ud ie s

s ub m i tt ed by the t echnical r egi str ants i n re s po nse to d ata req uireme nt s

. Thi s docu ment i s intended to prov ide an overview of w hat i s c u rrently known re garding th e env ironmental f at e and

ec olog ical effec ts as soc iated with d -Iim o nene and o utlines unce rtaintie s regarding a ttributes o f

th e par ent compo und and i t s tran sform ation p rodu c t s

. It de s cribes th e prelimi nary eco logica l ri s k

h ypothe s is and th e p rocesses t hat wi ll b e u sed du r in g th e co mple tion of d rinking wa ter an d

eco logical r isk as sess ments i n s upport of r e gistr ation re view.

### 1 

## REGISTRATION REVIEW

PROBLEM FORMULATION FOR:



# Limonene ( d- Limonene)

PC Code 079701; CAS Reg No.5989-27-5

PREPARED BY:

Jim Goodyear, Ph.D., Biologist

Silvia Termes, Ph.D., Chemist

APPROVED BY:

Dana Spatz, Branch Chief
Environmental Risk Branch 3
Environmental Fate and Effects Division Office of Pesticide Programs

### 2 The purpose of this problem formulation is to provide the foundation for the ecological risk assessment being conducted for d -Limonene (“limonene,” unless otherwise specified). As such, it articulates the purpose and objectives of the risk assessment, evaluates the nature of the problem, and provides a plan for analyzing the data and characte rizing the risk (EPA, 1998). This problem formulation is intended to identify data gaps, uncertainties, a nd potential assumptions
used to address those uncertainties.

Nature of Regulatory Action

As part of the implementation of the new Regist ration Review program purs uant to Section 3(g) of the Federal Insecticide, Fungi cide Act (FIFRA), the Agency is beginning the re-evaluation of limonene as a pesticide active ingredient to de termine whether limonene continues to meet FIFRA standard for registration. This problem formulation for the environmental fate and ecological risk assessment chapter in support of the registration review of d- limonene (limonene) is intended for the initial doc ket opening phase of the review process.

Stressor Source and Distribution

Nature of the Chemical Stressor

Limonene ( d -limonene) is a natura lly occurring chemical obtained from the rind of citrus fruits. Limonene is a hydrocarbon classified as a cyc lic terpene. Specifically, it is a monoterpene. Limonene has a chiral (stereog enic) carbon. Thus, limonene occurs as an enantiomeric pair (i.e., as two non-superimposable mirror-image chemical structures and therefore, each enantiomer is optically active 1 

### . The limonene enantiomer under review is the d -enantiomer (i.e., the dextrorotatory enantiomer). This dextrorotatory enantiomer has the R- absolute configuration. Unless otherwise specified, the term “lim onene” is used interchangeably with “ d -limonene.”

The mode of action of limonene is believed to be inhibition of acetylc holine esterase and phosphatase activity 2 

### . As an herbicide, d - limonene is a non-systemic , contact herbicide that strips the waxy rind or cuticle from weeds, causing death of the plant 3



### .

1 The chirality of limonene arises from an asymmetric carbo n atom (i.e., carbon is the chiral/stereogenic center).

The odors of l- limonene and d -limonene are different; l -limonene smells piney and turpentine like and d-limonene has a pleasing orange scent. This is associated with enan tiomer-specific interactions w ith chiral olfactory receptors

.

2 Kostyukovsky M, Rafaeli A., Gileadi C., Demchenko N. and Shaaya ,E. 2002. Activation of octopaminergic receptors by essential oil cons tituents isolated from aromatic plants: possi ble mode of action against insect pests. Pest Management Science, Vol. 58 (11), pp 1101-1006

3 Stephen O. Duke, Joanne G. Romagni, Franck E. Dayan 2000. Natural products as sources for new mechanisms of herbicidal action. Crop Protection 19 (2000) 583-589

### 3 Overview of Pesticide Usage

Limonene is used as an acaricide, herbicide, insecticide, and repe llent/feeding depressant. Its use patterns are food/feed and non-food/ non- feed. A summary of use sites and groups for food/feed patterns are show n in Table 1 and in Table 2 for non-food/non-feed.

Limonene is not applied aeriall y. It can be applied using spra yers (ready-to-use containers, hooded sprayers, pump sprayers, and pressurized containers); aerosol containers (cans); impregnated strips; injection equipment; or with a sponge. There is a wi de range of limonene concentrations and application rates in formulated products.

Table 1. Food/Feed Use Sites and Groups
Use Sites Groups Terrestrial Citrus fruits, cole crops, flavoring/spice crops, fruiting vegetables, grapes, leafy vegetables, legume vegetables, pome fruits, root and tuber vegetables, small fruits, tree nuts Indoor Household/domestic dwellings Food handling areas Commercial egg packing plants Food processing plant pr emises (non-food contact) Food Stores/markets/supermarket premises Food/grocery/marketing storage/distribution
facilities Meat processing plant pr emises (non-food contact) Greenhouses Not specified



### Table 2. Non-food/Non-feed Use Sites and Groups
Use Sites Groups Terrestrial Citrus; Christmas trees; Forest trees; Grapes; Non agricultural outdoor buildings; Nonagriculturalrights-of-ways/fencerows,hedgerows; Nonagricultural cultivated arrears/soil; Nursery stock; Ornamental/shade trees/herbaceous plants/lawn and turf; nonflowering plants/ woody shrubs and vines; Pome fruits; Small fruits; Stone fruits Tree nuts Indoor Automobiles, taxis, limousines, recreational vehicles, tires
Commercial/institutional/industrial premises/equipment Commercial storage/warehouse premises Dogs/canine (adults/puppies) Hospitals/medical institutions premises (human/veterinary) Household/domestic dwellings/premises/handling

areas Human body/clothing while being worn Pet living/sleeping quarters Refuse/solid waste containers (garbage cans); Wide area/general indoor treat ment; Wood protection treatment to buildings/products

### 4 Outdoor Cats (adults/kittens commercial transportation
facilities
Commercial/institutional/industrial premises/equipment Household/domestic dwellings/premises

Manure Outdoor buildings and structures; Paths/patios;
Private roads/sidewalks Ships and boats; Recreati onal areas; Refuse/solid waste sites; Sewage systems; Ships and boats; Wide area/general indoor treatme nt (public health use; Wood protection treatment to buildings/products) Greenhouse Not specified



### The highest application rate (34.6 lb ai/A) co rresponds to use as a terrestrial burn-down herbicide. The herbicide is applied to terres trial food/feed crops as well as non-food/non-feed crops and outdoor premises. The he rbicide can be applied by broad cast or as a spot treatment. When the application rate of 34.6 lb ai/A is us ed, the herbicide is broadcasted over the target vegetation.

Integration of Available Information

Regulatory History of Limonene

Limonene ( d- limonene) has been registered in the Unite d States as an ins ecticide since 1958. In 1971, limonene was registered as an antimicrobial and in 1983 as a dog and cat repellent. In 1988, the Agency considered d -limonene as an inert when us ed in antimicrobial products (40CFR 180.001 (c) (2)). Limonene was exempted for the requirement of a tolerance in 1994 when it is used as a solvent or fragrance in pesticide formulations. Limonene is currently registered as an acaricide, herbicide, in secticide, and repellent/feeding depressant.

In September 1994, the Agency issued a “Re-re gistration Eligibility Decision” (RED; EPA-738- F-94-030) for d -Limonene. It was concluded that pestic ide products containing limonene would not pose unreasonable risks or adverse effects to humans and the environment. Therefore, the 1994 RED concluded that all products containing limonene were eligible for re-registration. Because the 1994 RED was issued prior to the 1996 enactment of the Food Quality Protection Act (FQPA), no drinking water assessment was pe rformed at that time. However, the TRED concluded that given its ra pid volatilization from wate r, it is unlikely that d -limonene will occur in drinking water sources resulting from any of the registered uses.

Limonene is also regulated by the U.S. Food a nd Drug Administration (FDA) as a food additive or flavoring and as a fragran ce additive. The FDA recognizes d -limonene as a Generally Recognized as Safe (GRAS) chemical (21 CFR 182.60).

Other non-pesticide uses of limonene include: (a) solvents (substitute for chlorinated hydrocarbons, chlorofluorocarbons, and other solvents); (b) degr easing of metals; (c) cleaning agent in the printing industry and househol d cleaning products; (d) solvent for paint.

### 5 Previous Ecological Risk Assessments

The ecological risk assessment in the Re-registra tion Eligibility Decision (RED) completed in September 1994 (EPA 738-R-94-034) is based on a limited number of environmental fate and ecological toxicity studies. Expos ure concentrations were estimate d using simple screening level exposure models. The risk assessment concluded that minimal risk is anticipated for birds, mammals, and aquatic animals for the pesticidal uses of limonene. EFED believed at that time that applications of limon ene did not pose any undue ri sks to endangered species.

Exposure Characteristics

Chemical identification and phys ical-chemical properties of l imonene are shown in Table 3. Whenever possible, experimental physic al-chemical properties are reported.

Table 3. Identity and Physical -Chemical Properties of the Stressor Type of Information Information

Common name d -Limonene

Chemical name IUPAC: ( R )-4-isopropenyl-1- methylcyclohexene

or p -mentha-1,8-diene CAS (4 R )-1-methyl-4-(1- methylethenyl)cyclohexene

Other Names : D -Limonene; Cyclohexene, 1-methyl-4- (1-methylethenyl)-, ( R )-; p-Mentha-1,8-diene, (R)-(+)-; (+)-( R )-Limonene; (+)-(4R)-Limonene; (+)-p-Mentha1,8-diene; ( R )-(+)-Limonene; Carvene; D -(+)-Limonene; Dipentene; Limonene; Limonene, (+)-; ( R )-1-methyl-4- (1-methylethenyl)cyclohexene; Dextro-limonene; ( γ )- Carvene; ( R )-4-Isopropenyl-1-methyl-1-cyclohexene; pMentha-1,8-diene; R ( γ )-Limonene; 4-Isopropenyl-1- methyl-1-cyclohexene; ( R )-limonene. For other names consult EPA’s Substance Registry
Service

Pesticide type Acaricide, herbicid e, insecticide, repellent/feeding depressant Chemical class Terpene (monoterpene) CAS Reg. No. d - limonene: 5989-27-5

Racemate : 7705-14-8
l - Limonene : 5989-54-8
US. EPA Chemical Code Number 079701 Empirical Formula 

### C 10 

### H 16 Smiles String C(=CCC(C(=C)C)C1)(C1)C

Chemical structure

### 6 Molecular mass (g/mol) 136.23 Physical State Liquid (colorless) Specific gravity 0.841 g/cm 3
Melting point,  ̊ C -74.35 Boiling point,  ̊ C 175.5-176 Vapor pressure 20  ̊ C 2 mmHg; 2 torr; 2.66 x 10 3 Pa (Product chemistry data (465111-03)

25  ̊ C 1.4 mmHg; 1.4 torr; 1.92 x 10 3 Pa (Estimated at 25  ̊ C (EPISuite) Henry’s Law Constant 3.19 x 10 -1 atm-m 3 /mole; 3.85 x 10 4 Pa-m 3 /mole Solubility in water (mg/L) Insoluble (465111-03) 13.8 mg/L (Massaldi and Kin,1973)

Massaldi and Kin as cited by Sm ith, T.G. and LeMaguer, M. (1980)

Solubilities of terpenic essential o il components in aqueous solutions.

J. Chem. Eng. Data

, 1980

, 25 (2), pp 150–152.

Log K

ow ( n -octanol/water partition coeffi cient) 4.8 (Experimental) Log K o-air (octanol-air partition coefficient) 4.26 (estimated; EPISuite)

pK

a /pK b Not applicable Optical Rotation (Technical Product) > + 98 (465111-03)



### Environmental Fate

Persistence Limonene does not hydrolyze. Its cy clohexene ring and ethylene groups are resistant to hydrolysis. Therefore, hydrolys is is not expected in soils or aquatic
systems.

Under normal environmental conditions, limone ne is not likely to be metabolized by soil microorganisms. Limonene has been reported to metabolize in some pure
microbial cultures. 4



### In the atmosphere, volatilized limonene undergoes rapid bimolecular gas-phase reactions (i.e., the reactants are gase s) with ozone, nitrate radicals, and photochemically generated hydroxyl radicals.

Transport Limonene binds strongly to soil (K
oc 

### > 1000 L/kg), therefore it is expected to be relatively immobile in soils. However, the vapor pressure (1.4 mmHg at 25  ̊ C) and Henry’s Law Constant 3.19 x 10 -1 

### atm-m 3 

### /mole indicate that limonene readily volatilizes from soil and water. However, th is dissipation process competes with binding.

4 Amaral, J.A., Keins, A., Richards, S.R., and Knowles, R. 1998. Effect of Selected Monoterpenes om Methane Oxidation, Denitrification, and Aerobic Metabolism by Bacteria in Pure Culture. Applied and Environmental Microbiology, Vol 64(2), pp 520-525.

### 7 Transformation The products formed by gas-phase reactions depend on the type of reactant (i.e., ozone, hydroxyl and nitrate free radicals). The products fo rmed by the nitrate radical are simpler than those formed by the hydroxyl radical.

Dissipation Pathways Volatilization from soil, other surfaces, and water are the major dissipation pathways for limonene.

Bioaccumulation The Log K
ow 

### of 4.83 indicates potential bioaccumulation.

Detections in Environmental Media (Environmental Levels)

Limonene residues have been detected in rural, urban, suburban, and forest areas in the U.S. and in other countries (CICADS document, 1998; Footnot e 3). It has been detected in air, surface water, ground water, seawater, wa stewater, and ice as well as in soils and sediments. Limonene has been detected in solid waste composting fac ilities. Levels of limonene are varied, but the
limited data from 1998 do not allow correlation w ith usage or geographical region. However, it appears that there is a temporal variab ility in the number of detections.

EFED has data from 270 samples collected in ambient surface of wa ter. Only one had a
concentration of limonene above 0.5 μ g/L (which is believed to be the limit of detection). That sample was 1.2 μ g/L.

Ecological Effects

Consistent with the process described in th e Overview Document (EPA, 2004), this risk assessment will use a surrogate species approach in its evaluation of d -limonene. Toxicological data generated from surrogate test species, wh ich are intended to be representative of broad taxonomic groups, are used to extrapolate to poten tial effects on a variety of species (receptors)
included under these ta xonomic groupings.

Acute and chronic toxicity data from studies submitted by pesticide registrants along with the available open literature are used to ev aluate the potential direct effects of d -limonene to the aquatic and terrestrial receptors identified in this section. This includes toxicity data on the technical grade active ingredient, degradates, and when available, formulated products (e.g. “SixPack” studies).

The open literature studies are iden tified through EPA’s ECOTOX database ( http://cfpub.epa.gov/ecotox/ ), which employs a literature search engine for locating chemical toxicity data for aquatic life, terrestrial plants , and wildlife. The evalua tion of both sources of data can also provide insight into the direct and indi rect effects of d -limonene on biotic communities from loss of species that are sensitive to the chemical and from changes in structure
and functional characteristics of the affected communities.

No ecological incidents were found in the Agency ’s Ecological Incident Information System
EIIS database.

### 8 Table 4 

### summarizes the available data used to eval uate the ecological toxicity and risk of
limonene in the 1994 assessment.

Table 4. Summary of data used to eval uate ecological effects of limonene.

Measures of effect Species Toxicity Value Toxicity
Classification Toxicity Category (Acute) MRID (Year) Notes

Single dose avian toxicity Bobwhite 1 >2,000 mg/kg Core Practically
Nontoxic 109340 (1982)

Mallard 2 >5,000 mg/kg Core Practically
Nontoxic 109341 (1982) Dietary avian toxicity Bobwhite 1 >5,000 mg/kg Core Practically
Nontoxic 109341 (1982)

96-hr Freshwater fish, cold water Rainbow Trout 3 205 ppm Supplemental Practically
Nontoxic 109343 (1982)

96-hr Freshwater fish, warm water Fathead minnow 4 595 ppm Supplemental Practically
Nontoxic 109344 (1982)

Freshwater
invertebrates Daphnia magna 17-20 ppm Supplemental Slightly
Toxic 109345 (1982)Formulated product(dog repellent)

Avian Dietary toxicity Bobwhite 1 >5,600 mg/kg Core Practically
Nontoxic 146988 (1984)

96-hr Freshwater fish, cold water Rainbow Trout 3 80 ppm Core Slightly
Toxic 146085 (1984)

Freshwater
invertebrates Daphnia magna 39 ppm Core Slightly
Toxic 146085 (1984)

Mammalian
Acute oral Laboratory rat 5 >5,000 mg/kg Core Practically
Nontoxic EFED
Science Chapter (1994)Technical Grade

1 Colinus virginianus, 2 Anas platyrhynchos, 3 Oncorhynchus mykiss, 4 Pimephales promelas,

5 Rattus norvegicus



### Avian and Aquatic Effects

The toxicity effects for avian species are “Practica lly Nontoxic” for both the technical and formulated products. Formulated limonene is “Practically Nont oxic” to aquatic vertebrates and “Slightly Toxic” to freshwater invertebrates. Te chnical limonene is “Sligh tly Toxic” to aquatic species.

### 9 Mammalian Acute Effects

The acute oral LD 50 

### value for the Norway rat is great er than 5000 mg/kg (5 g/kg), which classifies it as practically nont oxic. Granular limonene is regi stered as a mammalian repellent and is categorized as “Practically Nontoxic.” Th e “granules” are diatomaceous earth; therefore, their size is very small (typically 10 to 200 μ m). It is not anticipated that “granules” would be consumed in sufficient quantitie s to be a risk to mammals.

Ecosystems Potentially at Risk

In general, the ecosystems at risk are those in close proximity to the use areas. These include any water bodies and terrestrial ecosystems adjacen t to use areas that may receive runoff. Risks are considered for aquatic animals (fish, inverteb rates, amphibians) and plants, assumed to occur in small, static ponds and marine water bodies in areas adjacent to the us e site and terrestrial animals (birds, reptiles, terrestrial amphibians, mammals and insects) and plants, assumed to exclusively occur in the treated area.

Organisms of potential concern broadly include fi sh, aquatic invertebrate s and plants, birds, amphibians, reptiles, mammals and terrestrial invertebrates and plants . Based on the known effects of limonene, the organisms of concern coul d be terrestrial and aq uatic invertebrates and plants.

The assessment endpoints are intended to reflec t population sustainabi lity and community structure within ecosystems and hence relate back to ecosystems at risk. If risks are expected for given species/taxa based on the screening-level assessment, then risks might be expected to translate to higher levels of biological organization.

Assessment Endpoints

Assessment endpoints are defined as “explicit expressions of the actual envir onmental value that is to be protected.” Defining an assessment e ndpoint involves two step s, 1) identifying the
valued attributes of th e environment that are considered to be at risk, and 2) operationally defining the assessment endpoint in terms of an ecological entity (i.e., a community of fish and aquatic invertebrates) and its attr ibutes (i.e., survival and reproduc tion). Therefore, selection of the assessment endpoints is based on valued entit ies (i.e., ecological receptors), the ecosystems potentially at risk, the migration pathways of pesticides, and the routes by which ecological receptors are exposed to pesticide-related cont amination. The selection of clearly defined assessment endpoints is important because they provide direction and bou ndaries in the risk assessment for addressing risk management issues of concern. Changes to assessment endpoints are typically estimated from the available toxici ty studies, which are used as the measures of effects to characterize potential ec ological risks associated with e xposure to a pesticide, such as
limonene.

The most sensitive toxicity endpoints are used fro m surrogate test species to estimate treatmentrelated direct effects on acute mortality a nd chronic reproductive, growth and survival assessment endpoints. Toxicity tests are intende d to determine effects of pesticide exposure on

### 10 birds, mammals, fish, terrestrial and aquatic inve rtebrates, and plants. These tests include shortterm acute, sub-acute, and reproduc tion studies and are typically a rranged in a hierarchical or tiered system that progresses from basic laboratory tests to applie d field studies. The toxicity studies are used to evaluate the potential of a pe sticide to cause adverse effects, to determine whether further testing is required, and to determ ine the need for precautionary label statements to minimize the potential adverse effects to non-target animals and plants.

Conceptual Model

For a pesticide to pose an ecological risk, it mu st reach ecological receptors in biologically significant concentrations. An exposure pathway is the means by which a pesticide moves in the environment from a source to an ecological recepto r. For an ecological pathway to be complete, it must have a source, a release mechanism, an environmental transport medium, a point of exposure for ecological receptors, and a feasible route of exposure.

Risk Hypothesis

Based on an examination of the physical/chemical properties of d -limonene, the fate and disposition in the environment, and method of application, the follo wing ecological risk hypothesis is being employed for this baseline risk assessment: Limonene will affect the growth, reproduction, and survival of non-ta rget species when it is used as per label directions. The major transport pathway for d -limonene is volatilization.

### 11 Conceptual Diagram

Analysis Plan Options

In Registration Review, pesticide ecological risk assessments will follow the Agency’s Guidelines for Ecological Risk Assessment, will be consistent with the paper titled “Overview of the Ecological Risk Assessment Process in the O ffice of Pesticide Program s, U.S. Environmental Protection Agency” (“Overview Document”) (J anuary 2004), and will be done in accordance with Section 7 of the Endangered Species Act.

The ecological risk assessment for limonene will be performed only for uses of limonene as a pesticide. Non-pesticide uses will not be assesse d, but published data can be included in the overall environmental exposure of limonene. Exposure will be evalua ted using modeling and monitoring data. In the ecological risk reassessment for limonene, the Agency will include data previously used in the 1994 RED, as well as open literature identified using EPA’s ECOTOX database. Data on terrestrial and aquatic plants ar e needed and the results w ill be used in the risk assessment for plants.

Limonene is volatile. The use of atmospheric tr ansport models may be necessary because offtarget deposition of limonene on vegetation and aquatic ecosystems could potentially affect nonApplication of Limonene Stressor

Direct Ground Deposition Volatilization/ Wind Suspension Leaching

Non-Target Plants: •Plant growth abnormalities •Lethality •Developmental/reproductive
effects Source:

Transport Pathway

Terrestrial Animals: •Lethality •Reproductive/developmental
effects Aquatic Animals and Plants: •Effects to survival, growth, and reproduction •Reductions in cell density/ biomass in aquatic plants

Altered Terrestrial Community Assemblages: Reduced food supply and edge habitat availability Altered Aquatic Community Assemblages: Effects to riparian habitat availability Effects to species abundance/diversity Changes in physical/chemical parameters Source:

Exposure Media

Exposure Point

Receptors

Direct Effects

Indirect Effects Terrestrial Food Residues (foliage, fruit, insects)

Direct Contact & Ingestion

Terrestrial
Vertebrates and

Invertebrates Direct Contact/ Root Uptake

Terrestrial

Upland
Plants Surface Water
Sediment Groundwater

Gill/Integument Uptake

Aquatic
Invert &
Vertebrates Upland Soil and Foliage Riparian/Wetland Foliage/Soil Spray
Drift Runoff/
Erosion

Uptake/ Adsorption

Riparian/
Wetland
Plants Aquatic
Plants

Plant Population Reduced Population growth

### 12 target plants. Volatilization rates will be esti mated using volatilization models recommended by EFED and will be integrated in the models used to estimate concentrations of limonene in surface water. If necessary, published gas-phase kinetics data will be used to estimate the degradation of gaseous limonene in the atmo sphere (i.e., reactions with photochemically generated hydroxyl radicals, ozone, and nitrogen oxide ra dicals). A source for these data is the
National Institute of Science and Technology (NIST) Kinetics Database.

The Health Effects Division (HED) concl uded in 1994 that it is unlikely that d -limonene will occur in drinking water sources (surface; ground) resulting from any of the registered and proposed uses as an active ingredient or when used as an inert ingred ient. Residues in drinking
water were thus not included in the dietary risk assessments. Given the current use pattern, EFED plans to conduct a drinking water assessment in registration review.

Exposure concentrations in aquatic ecosystems will consider the use of the Tier I GENEEC2 and/or Tier II PRZM-EXAMS models using actu al or estimated environmental fate/physicalchemical data.

Endangered Species

Consistent with the Agency’s responsibility und er the Endangered Species Act (ESA), EPA will evaluate risks to federally-listed threatened and/ or endangered (listed) sp ecies from registered
uses of limonene. This assessment will be conducted in accordance with the Overview Document, provisions of the ESA, and the Se rvices’ Endangered Species Consultation
Handbook .

The assessment of effects associat ed with registrations of limonene is based on an action area. The action area is considered the area directly or indirectly affected by the federal action, as indicated by the exceedance of Agency LOCs used to evaluate direct or indirect effects. The Agency’s approach to defining the action area un der the provisions of the Overview Document considers the results of the risk assessment pr ocess to establish boundaries for that action area with the understanding that exposures below the Agency’s defined LOCs constitute a no-effect threshold. For the purposes of this assessment, attention will be focused on the footprint of the action (i.e., the area where limonene application o ccurs), plus all areas where offsite transport (i.e., spray drift and runoff) may result in potential exposure that exceeds the Agency’s LOCs. Specific measures of ecological effect that defi ne the action area for listed species include any
direct and indirect effects and/or potential modification of its cri tical habitat, including reduction in survival, growth, and reproductio n as well as the full suite of s ublethal effects available in the effects literature. Therefore, the action area ex tends to a point where environmental exposures are below any measured lethal or sublethal effect threshold for any biological entity at the whole organism, organ, tissue, and cellula r level of organization. In situ ations where it is not possible to determine the threshold for an observed effect, the action area is not spatially limited and is
assumed to be the entire United States.

### 13 Clean Water Act

Limonene is not identified as a cause of impairment for any wa ter bodies listed as impaired under section 303(d) of the Clean Water Act, based on information provided at http://iaspub.epa.gov/tmdl_waters10/attains_ nation_cy.cause_detail_303d?p_cause_group_id=88

5.

In addition, no Total Maximum Daily Loads (TMDL) have been developed for limonene, based on information provided at http://iaspub.epa.gov/tmdl_waters10/attains_ nation.tmdl_pollutant_de tail?p_pollutant_group_id =885&p_pollutant_group_name=PESTICIDES. More information on impaired water bodies and TMDLs can be found at http://www.epa.gov/owow/tmdl/.

EPA invites submission of water quality data for this pesticide. To the extent possible, data should conform to the quality standards in Appendix A of the OPP Standard Operating Procedure: Inclusion of Impaired Water B ody and Other Water Quality Data in OPP’s Registration Review Risk Assessment and Management Process (see: http://www.epa.gov/oppfead1/cb/ppdc/2006/november 06/session1-sop.pdf), in order to ensure they can be used quantitatively or qual itatively in pesticide risk assessments.

Endocrine Disruptor Screening Program

As required under FFDCA section 408(p), EP A has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (inc luding pesticide active and other ingredients) may have an effect in humans or wildlife si milar to an effect produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP employs a two-tiered ap proach to making the statutorily required
determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where EPA will determine which, if any, of th e Tier 2 tests are necessary base d on the available data. Tier 2 testing is designed to identify a ny adverse endocrine related eff ects caused by the substance, and establish a dose-response relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticid e active ingredients and 9 inert ingredients.
This list of chemicals was selected based on th e potential for human exposure through pathways such as food and water, residen tial activity, and certain post-applic ation agricultural scenarios.
This list should not be construed as a lis t of known or likely endocrine disruptors.

Limonene is not among the group of 58 pesticide active ingredients on th e initial list to be
screened under the EDSP. Under FFDCA sec. 408(p) the Agency must screen all pesticide chemicals. Accordingly, EPA anticipates issuin g future EDSP test orders/data call-ins for all pesticide active ingredients.

### 14 For further information on the status of the ED SP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, pleas e visit our website: http://www.epa.gov/endo/

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Preliminary Identification of Data Gaps

Environmental Fate

No guideline environmental fate studies have been submitted. Submission of guideline studies was waived in 1992. For the reassessment, up-to-d ate open literature and databases will be used to generate an integrated environmental fate as sessment (persistence, transformation, transport, and dissipation routes) of limonene. EFED believe s that the behavior of limonene in the
environment is well documented and that guideline studies are not necessary.

Ecological Effects

The 1994 assessment did not include a terrestrial or aquatic plant assessment because limonene was not registered as an herbicid e at that time. Terrestrial and a quatic plant toxicity data are not
available. The data are needed to evaluate the risk of limonene to non-target plants.

### 15 Guideline Number: 850.4100 and 850.4150
Test Substance: Limonene TEP
Title: Seedling emergence and vegetative vigor studies (Tier 2) Test Species: Terrestrial plant species as specified by 850.4100 and 850.4150 Rationale for Requiring the Data

There are no toxicity data available for terrestrial monocots and dicots. A risk assessment for exposure to
monocots and dicots is needed based on limonene’s use pattern. The data will be utilized to determine whether the Agency’s current measures are effective for protecting terrestrial plants.

Risk to terrestrial plants has not been quantified in previous assessments, and no terrestrial plant seedling emergence or vegetative vigor toxicity data are curren tly available for limonene. Based on the updated data requirements in 40 CFR part 158 (O ctober 2007), these data are requir ed to support all terrestrial use patterns, including those relevant to limonene. These phytotoxicity studies should be conducted using the maximum label application rate of limonene typical end-use product (TEP). Without these studies, the Agency would have to presume risk to listed and non-listed terrestrial plants and to organisms that depend on terrestrial plants for sustenance, shelter, or reprodu ction. Potential risks, however, would not be able to be quantified. Practical Utility of the Data

How will the data be used? Tier II seedling emergence and vegetative vigor toxi city data will be used to quantitatively estimate potential risks to terrestrial plants associated with the use of limonene. These data will be used to define an action area for endangered species and explore risk mi tigation strategies. The data will reduce uncertainties
associated with the risk assessment for terrestrial plants and will impr ove the Agency’s understanding of the potential effects of limonene on terrestrial plants. In the absence of data, risk to terrestrial plants will be presumed.

How could the data change the Agency’s decision or impact the Agency’s future decision-making? The value of these seedling emergence and vegetative vigor studies would be in quantifying and potentially refining risks to non-target terrestrial plants. Defining an action area for endangered species and exploring risk mitigation strategies cannot be evaluated without these data. Without terrestrial plant toxicity data for limonene, the Agency cannot determine if the current application rates of limonene may result in direct adverse effects to terrestrial plants and/or indirect e ffects (e.g., via loss of habitat/cover, riparian vegetation effects, etc.). If future endangered species risk a ssessments are performed without these data, the Agency will assume that limonene “may affect” terrestrial plants directly and listed species from other taxa indirectly, and use of limonene may need to be restri cted in areas where listed species could be exposed. The lack of these data will limit the flexibility the Agency and registrants have in coming into compliance with the Endangered Species Act and could result in us e restrictions for limonene that are unnecessarily

severe.

### 16 Guideline Number: 850.4400 and 850.5400
Test Substance: Limonene (TEP)
Title: Aquatic vascular and non-vascular plant toxicity tests; Tier II Test Species: Aquatic plant species as specified by 850.4400 and 850.5400 Rationale for Requiring the DataRisk to aquatic plants has not been considered in previous assessments, and no aquatic vascular or nonvascular plant toxicity data are currently available fo r limonene. Based on the updated data requirements in 40 CFR part 158 (October 2007), these data are required to support all terrestrial use patterns, including those relevant to limonene. (Tier II phytotoxicity tests woul d be required if a 50 percent or greater detrimental effect is observed in the Tier I test). Without this st udy, the Agency would have to presume risk to listed and non-listed aquatic vascular plants, but would not be able to quantify the risk.

Practical Utility of the Data

How will the data be used? Aquatic plant toxicity data will be used to quantitatively estimate the risk of limonene to aquatic plants and to define an action area for endangered species and explor e risk mitigation strategies. These data will greatly
reduce uncertainties associated with the risk assessment for aquatic plants. In the absence of aquatic plant data for limomene, quantitative risk estimation will not be possible, and risk will be presumed. Determination
of the distance downstream from a treated field to which risk may extend and application rate reductions resulting in no LOC exceedances cannot be quantified with out these data.

How could the data impact the Ag ency’s future decision-making? Because aquatic plants form the basi s of the food chain for aquatic hab itats and significantly contribute to overall water quality, a solid understanding of the poten tial risks to aquatic plants is essential for sound environmental risk management decision-making. Aquatic plant toxicity data will allow the Agency to quantitatively characterize the potential risks of limone ne to aquatic ecosystems. If future endangered species risk assessments are performed without these da ta, the Agency would have to assume that limonene "may affect" listed aquatic plants directly and other e ndangered species indirectly via reduction in the prey
base and/or loss of habitat or cover. The lack of these data will limit the flexibility the Agency and registrants have in complying with the Endangered Species Act and could result in unnecessary use
restrictions.