United States EnvrronmentalProtectton Agency 

### Office of Prevention, Pesticides, and Toxic Substances



# Environmental Fate and Ecological Risk Assessment for the Reregistration of

# Antimycin A

I 1 

### inti-ol@ CAS 1397-94-0 PC Code 006314

Page 1 of 118 Prepared by: Dirk Young, Ph.D., Environmental Engineer

Thomas Steeger,Ph.D., Senior Biologist
Reviewedby: ElizabethBehl, BranchChief U.S. Environmental Protection Agency Office ofPesticidePrograms Environmental Fate andEffects Division Environmental Risk Branch IV

1200PennsylvaniaAve., NW Mail Code 7507P Washington, DC 20460

## EXECUTIVE SUMMARY
Nature ofthe ChemicalStressor Antimycin A (CAS 1397-94-0, under the tradename ~introp), is a restricted use pesticide derived as 

### a fermentation product

### from Strevtomvces mold and is registered for use as a piscicide. The chemical isprimarily used to renovate recreationaljshpopulations and to remove scaledjsh from catJish jingerling andfood-fish production ponds. Over thepast decade antimycin has been used to restore Federally-listed threatened/endangered (listed)jsh to their native habitats. Antimycin A is applied directly to water and is typically used at treatment concentrations of25pg/L or less; however, the current labels do not speczfjl a maximum application rate. Conclusions Regarding Exposure Antimycin is applied directly to water and is maintained at a targeted treatment concentration for specijied periods oftime. Although environmental fate data are limited, theprimary route ofdegradation appears to be base hydrolysis where under alkaline conditions the compound can degrade with half-lives ofroughly 30 minutes. However, in at least one study, the compound persisted in an aquatic environment with a half-life of from 20 to 47 days. Field studies on downstream movement also show that the compound can extend and remain active to distances ofat least 1.75 km beyond desired treatment areas when no effort is made to deactivate the compound. There are no direct measurements of antimycin sorption; however, indirect evidence suggests that antimycin will sorb significantly to soil and sediments and may reduce potential exposure outside ofthe immediate treatment area. Conclusions Regarding Effects On an acute exposure basis antimycin is moderately toxic to mammals, highly to very highly toxic to birds and very highly toxic to aquatic animals resulting in mortality. There are no data to evaluate the chronic toxicity of antimycin to animals and there are no data to evaluate the toxicity of antimycin to either terrestrial or aquatic plants. Conclusions Regarding Potential Risks to Non-target Organisms Based on the most sensitive species and typical treatment concentrations of 25pg/L, acute risk levels of concern are exceededfor aquatic animals by factors of roughly 6,000X Evenfor the least sensitiveJsh and aquatic invertebrates tested, treatment concentrations of 25 pg/L would exceed acute risk levels ofconcern. Although antimycin is moderately to very highly toxic to terrestrial animals on an acute oral exposure basis, treatment rates to water of25pg/L or less are expected topresent a low risk of acute mortalityfor birds and mammals through ingestion ofdead/dyingfish or antimycin-treated water. Because there are no chronic toxicity data, it is notpossible to estimate potential chronic risk to aquatic or terrestrial animals. In situations where antimycin is applied to flowing water, the chemical is typically deactivated using potassium permanganate; however, deactivation is not currently required by the label. Additionally, in flowing water, antimycin would eventually be flushed through the system and would also be diluted by untreated tributaries. Also, in both lentic and lotic environments, antimycin is typically only applied once per year. Thus, the likelihood of chronic exposure in a lotic environment is low; however, in the absence of data, potential risk to nontarget organisms cannot be precluded. In situations where antimycin is not deactivated with permanganate and there is not dilution by untreated water, such as in lentic environments, the chronic risk to non-target aquatic organisms is uncertain. Page 2 of 118Where antimycin is used to remove scaledfzsh from catfish fingerling andfood-fish production ponds, treatment rates are considerably lower than 25pg/L in order to not impair the health/suwival of catJsh. Therefore, the likelihood of either acute or chronic effects in non-target animals from the use of antimycin in aquaculture is low particularly since the treated water is typically retained within the aquaculture facility. To the extent that treated water is not retained but rather released into surrounding streams/rivers, the risk to non-target aquatic animals is uncertain. Based on laboratory data, aquatic invertebrates appear to be as sensitive to antimycin as3sh; however, field studies of antimycin in high-gradient mountain streams, where the compound was deactivated with potassium permanganate after the desired treatment time, suggests that aquatic invertebrates are not aflected or are at worst temporarilyajgected. Sampling conducted several months to up to a year after treatment indicate that aquatic invertebrate abundance and diversity were similar to pretreatment levels. Even in first-order streams where antimycin was not deactivated withparmangunate, invertebratednjl and survival did not appear to be significantlyafected during or immediately after 8-hr treatments at 10pg/L. Whether these studies are indicative of aquatic invertebrate communities in all antimycin use areas is uncertain. Effects on listed species may be an important consideration for site-specific applications. Antimycin is an important chemical used in recovery efforts for listed salmonids by eliminating competing species. The following table provides a summary ofpotential direct efects to listed taxa.

a~aw data were not provided sothe default valueof4.5 is used. *Dose-based value. Uncertainties The database on the environmental fate and transport properties of antimycin is incomplete. Since the compound is used exclusively as a piscicide, the terrestrial fate properties are relatively undocumented. Antimycin appears to degrade rapidly in the environment with half-lives on the order of RQ
2,778 3,125 0.40

-

- <0.01*

--- <O.Ola --

-
-

Page 3 of 118 Slopea

15.5 4.5 4.5

-

- 4.5

6.5

-
- Listed Species Taxonomic Group of Concern Freshwater Fish Freshwater Invertebrates Saltwater Mollusc Aquatic Plants: Vascular Non-vascular Birds

Mammals

Terrestrial Plants: Monocots Dicots Direct Effects

Acute: mortality Acute: mortality/immobilization Acute mortality

no data no data

Acute: mortality/sublethal

Acute: mortality

Acute: no data Acute: no dataminutes to several weeks. Abiotic mechanisms (hydrolysis) appear to be the primary route ofdissipation, but biodegradation may contribute to affecting the persistence of antimycin in the environment. The potential contribution ofbiotic routes ofdegradation to affecting the persistence is uncertain. The database on the ecological effects ofantimycin is incomplete. There are no chronic toxicity data for either terrestrial or aquatic animals; however, the likelihood of chronic exposure to antimycin following application to flowing water is likely low due to a combination ofdegradation, flow-through, deactivation and dilution. Additionally, there are no data for terrestrial oraquatic plants. The likelihood ofexposure to terrestrial plants is considered low; however, aquatic plants will likely be exposed and the potential risk to these plants is uncertain. The extent to which direct applications of antimycin to water as a fish control agent can be limited to the desired treatment area is dependent on the rigor ofthe protocols used to apply the chemical and the degree to which they are followed. Biological and chemical monitoring ofthe treatment area and deactivation ofantimycin where feasible could track and limit exposure ofnon-target organisms. While many state and federal agencies follow standard operating procedures (SOPs) that limit exposure, the SOPs are not uniformly applied across all potential users. In addition, since the environmental fate database is incomplete, the extent to which antimycin will persist and move outside treated areas is uncertain. Historically, efforts to monitor for antimycin in the environment have relied onfish bioassays. Quantitative analytical procedures capable of detecting toxic concentrations of antimycin have been limited, and those that exist are not practical in the field. Actual environmental concentrations during and after treatments with and without deactiviation with potassium permanganate are uncertain. While field bioassay data suggest that antimycin is no longer toxic following deactivation with potassium permanganate, there are no analytical data to confirm that all of the antimycin has been degraded or what transformation products result from the degradation. Although antimycin may sorb to sediment, the extent to which the partitioning limits the toxicity of the pesticide is uncertain. There are no laboratory data on benthic macroinvertebrates with which to evaluate potential risks. A field study ofaquatic invertebrates suggests, however, that both abundance and diversity ofaquatic invertebrates exhibited transitory effects and many of these taxa are associated with the benthos. This assessment evaluates potential risks from antimycin A treatment concentrations of25pg/L or less and primarily focuses on use inflowing water (lotic environments). To the extent to antimycin is used at higher treatment rates, the potential risks to non-target organisms are likely to increase. Additionally, the fate of antimycin in static water (lentic environments), e.g., high alpine lakes and aquaculture production ponds, is uncertain since the chemical is not typically deactivated with potassium permanganate and there isa potentialfor it to persist under certain environmental conditions such as low pH. To the extent that risk managers require additional information to address uncertainties regarding potential risks to non-target organisms, additional data on the environmental fate and eflects ofantimycin would be required.Acknowledgement The Environmental Fate and Effects Division would like to acknowledge the U. S. National Park Service,the U. S. Geological Survey Biological Resource Division, the U. S. Fish and Wildlife Service, the New Mexico Department of Game and Fish, and the U. S. EPA Office of Research and Development (Athens, GA) and Office of Pesticide Programs' Biological and Economic Assessment Division (Bay St. Louis, MS) for their efforts to address uncertainties surrounding the environmental fate and ecological effects of antimycin.

Page 5 of 118

## 2 TABLE OF CONTENTS



# ................................................................................................... 1 EXECUTIVE SUMMARY 2
2 TABLE OF CONTENTS

# ...................................................................................................... 6 PROBLEM FORMULATION

# .................................................................................................. 9



### .................................................................................... 2.1 Stressor Source and Distribution 9



### ................................................................................................... 2.1


### .1 Sourceand Intensity 9



### ................................................... 2.1.2 Physical/Chemical/Fateand Transport Properties 9



### .............................................................. 2.1.3 PesticideType, Class, and Mode ofAction 10 2.1.4 Overview ofPesticide Usage


### .................................................................................... 11



### ....................................................................................................................... 2.2 Receptors 

### II



### ..................................................................................................... 2.2.1 Ecological Effects 11 2.2.2 Ecosystems at Risk


### .................................................................................................... 12



### ................................................................................................... 2.3 Assessment Endpoints 13



### ......................................................................................................... 2.4 Conceptual Model 13



### ........................................................................................................ 2.4.1 Risk Hypotheses 13 2.4.2 Conceptual Model Diagram


### ...................................................................................... 14 3 USE CHARACTERIZATION

# ...........................................................................................15



### ................................................................................................................ 3.1 DetoxzJication 17



### ..................................................................................................... 3.2 Usageand UseRates 17 4 ENVIRONMENTAL FATE CHARACTERIZATION

# ................................................... 20



### ................................................................................................................... 4.1 Background 20



### .............................................................................................................. 4.2 Fate Properties 20



### ................................................................................... 4.3 UsageandFate in WaterBodies 23 4.4 Estimates of Water Concentration for RiskAssessments



### .............................................. 24 5 ECOLOGICAL EFFECT CHARACTERIZATION 

# ...................................................... 25



### ......................................................................................... 5.1 Categories of Acute Toxicity 25 5.2 Toxicity toAquaticAnimals



### .......................................................................................... 27 5.2.1 Freshwater Fish, Acute


### .............................................................................................. 27 5.2.2 FreshwaterFish. Chronic


### .......................................................................................... 31 Page6 of 118

### .......................................................................... Freshwater Invertebrate. Acute 3 1



### .............................................................................. FreshwaterInvertebrate. Chronic 32 EstuarineIMarine Fish. Acute


### .................................................................................... 32



### ............................................................................... 5.2.6 EstuarineJMarineFish. Chronic 32



### ..................................................................... 5.2.7 EstuarineIMarine Invertebrates. Acute 3 2



### ................................................................. 5.2.8 EstuarineIMarine Invertebrates. Chronic 3 3



### .................................................................................... 5.3 Toxicity to Terrestrial Animals 33



### ................................................................................. 5.3.1 Birds. Acute and Subacute 3 3



### ......................................................................................................... 5.3.2 Birds. Chronic 33



### ...................................................................................................... 5.3.3 Mammals. Acute 33



### ............................................................................................... 5.3.4 Mammals. Chronic 34 5.4 Aquatic Eflects Characterization of Potassium Permanganate Toxicity



### ...................... 34



### ........................ 5.4.1 Acute Toxicityof Potassium Pennanganateto AquaticVertebrates 34



### .......................... 5.4.2 Acute ToxicityPotassium Pemanganateto Aquatic Invertebrates 36 6 RISK CHARACTERIZATION

# ........................................................................................ 38



### .............................................................................................................. 6.1 Risk Estimation 38 6.1.1 Freshwater Fish and Invertebrates


### ............................................................................ 40



### ................................................................... 6.1.2 EstuarineIMarine Fish and Invertebrates 41 6.1.3 Birds


### .......................................................................................................................... 41 6.1.4 Mammals


### ................................................................................................................... 43 6.2 Risk Discussion



### ............................................................................................................. 44



### ............................................................................................... 6.3 Risks toAquatic Animals 46



### .......................................................................................... 6.4 Risks to Terrestrial Animals 51



### .......................................................................................... 6.5 Ecological Incident Reports 52



### ................................................................................................................. 6.6 Uncertainties 52 6.6.1 Analytical Method Development


### .............................................................................. 53 6.6.2 Assumptionsand Limitations Related to Effects Assessment


### .................................. 54 6.6.3 AssumptionsRelated to Exposure Assessment


### ......................................................... 55



### .............................. 6.7 Federally Threatenedand Endangered (Listed) Species Concerns 56



### ........................................................................................................... 6.7.1 Action Area 56



### ..................................................................... 6.7.2 Taxonomic Groups Potentiallyat Risk 57



# ................................................................................................................. 7 CONCLUSIONS 60
8 REFERENCES

# .................................................................................................................... 62



# ............................................................................. 9 APPENDIX A

# . ASTER Model Output 66 10 APPENDIX 

### B

# . PBT Profder Model Output

# .................................................................... 70

### I1 APPENDIX C

# . New Mexico Standard Operating Procedure for the Use of Piscicides. 75
12 APPENDIX D

# .In vitro Mammalian Metabolism

# ............................................................ 79
13 APPENDIX E

# . Standard Operating Procedure for the Use of Fintrolmto Remove Scaled Fish 

### in Catfish Ponds

# ...................................................................................................... 81
14 APPENDIX F

# . Review of SelectedPapers

# ....................................................................... 84 15 Appendix 

### G: Memo from John Kenneketo Thomas Steeger and "Summary of Antimycin Hydrolysis Research by John F

# .Kenneke" 

# .........................................................108

Page 8 of 118

## PROBLEM 

## FORMULATION The problem formulation establishes the direction and scope of an ecological risk assessment. According to the Guidelines for Ecological Risk ~ssessment', problem formulation consists of defining the problem and purpose for the assessment, and developing a plan for analyzing and characterizing risk. The critical components of the problem formulation are selection of the assessment endpoints, formulation of risk hypotheses, development of a conceptual model, and development of an analysis plan. The analysis plan and supporting rationale are aimed at determining whether the labeled uses of antimycin A as a piscicide could result in exposures that cause unreasonable adverse effects (risk) to non-target organisms including those Federally listed as threatened or endangered species (hereafter referred to as "listed"). This assessment was developed as part of the supporting informationto determine the eligibility of antimycin A for re-registration. The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) requires that registered pesticide uses do not pose unreasonable adverse effects to the environment, and the Endangered Species Act requires that regulatory actions are not likely to adversely affect listed speciesor their habitats. 

### 2.1 Stressor Source and Distribution

2.1.1 Source and Intensity The source of the stressor considered in this ecological risk assessment is the application of antimycin A according to its label. Antimycin A was discovered in 

### 1945 and is used as a general fish toxicant (piscicide); piscicidal applications are made directly to water. The label for ~introl@ Concentrate states that the effective treatment concentration is 25 pglL for control of short nose gar (Xenocara dolichopterus),bowfin (Amia calva), goldfish (Carrasius auratus) and catfish (Ictalurus spp.) when the pH is 

### 8.5 or more and when water temperatures arebelow 16OC (60°F);no maximum application rate is stated on the label however. Piscicidal applications, in general, are assumed to occur once 

### a year since this type of application typically results in a complete fish kill2. 

### 2.1.2 PhysicaYChemicaUFate and Transport Properties Antimycin A (3-methylbutanoic acid 

### 3[[3-(formylamino)-2-hydroxylbenzoyl]amino]-8- hexy1-2,6-dimethy1-4,9-dioxo-1,5-dioxonan-7-y1 ester) consists of four major components which in turn consist of a pair of compounds, for a total of eight significant hom~logues~~~-four "major" and four "minor"-that are distinguished by their chemical substituents. Preliminary estimates of the physical and chemical properties that determine the fate and transport of antimycin A are detailed in Table 1and were estimated using a quantitative structure activityU.S. EPA. 1998. Guidelines for Ecological Risk Assessment. Risk AssessmentForum, Washington,DC. EPA 630lR-951002FApril 1998. Finlayson,B.J., R.A. Schick,R.L. Cailteux,L. DeMong, W.D. Horton, W. McClay, C.W. Thompson, and G.J. Tichacek. 2000.Rotenone Use in Fisheries Management: Administrative and Technical Guidelines Manual. American Fisheries Society, Bethesda, Maryland. 

### 'Abidi, S.L. and B.R. Adams. 1987. 1Hand 13Cresonance designation of antimycin A1 by two-dimensional NMR spectroscopy. Magn. Reson. Chem. 25: 1078-1080.

Abidi, S.L. 1988. High-performance liquid chromatographic separation of subcomponents of antimycinA. J. Chromatogr. 447: 65-79. Page 9 of 118based calculator, open literature, and registrant-submitted studies. There is a high degree of uncertainty surrounding the quantitative estimation of the environmental properties of antimycin. However, antimycin appears to sorb to sediment (potentially at a high level), degrade primarily by hydrolysis,not volatilize,not readily dissolve in water, and not bioaccumulate.



### 2.1.3 Pesticide Type, Class, and Mode ofAction Antimycin 

### A is an antibiotic derived from the soil mold Streptomyces. Antimycin A uncouples oxidative phosphorylation by blocking the electron transport pathway to Complex 

### I11 within the mit~chondria.~

Table 1. Physical and chemical properties of antimycinA.

Ibid ASTER 2004. Bettennann, A.D., J.M. Lazo~hak and J.C. Domfi. 1996. Profile of toxic response to sediments using whole-animal and in vitro submitochondrialparticle (SMP) assays. Journal of Environmental Toxicology and Chemistry 15(3): 319 - 324. Page 10 of 118 CASNumber
-- -- 1397-94-0 SMILESNotation ~~C(=O)~(NC(=O)~~~~(O)~(N~=O)>~W:~)~(C)~~(=O)~(CCCCCC)~(OC(=O)~C(C)~)~~

Molecular formula:C~~H.(ONZO~ Property Molecular weight Melting point Koc

Water solubility (20 EC)

Vapor pressure (25 EC) Henry's Law constant
Hydrolysis half-life (25 EC)

Aerobic Aquatic Metabolism half-life

Bioconcentration Factor (BCF) Value 548.6 g mol-' 149- 150 "C 10- 10,000

69 mg L" to insoluble

2.31~10.'~ mm Hg 2.42 x10-" atm-m' mol-'

Minutesto months, some (but inconsistent) evidence suggests dependanceon pH; faster degradation as pH increases High uncertainty (possibly on the order of days)

350x Reference estimated- ASTER, 2004' MERCK Index estimated- ASTER, 2004; indirect estimationfrom submitted degradation study (MRD45895901) Estimation of 69 mg/L is based on ASTER, 2004; "insoluble" from Walker et al. (1971) estimated - ASTER, 2004 estimated- ASTER, 2004

estimated- ASTER, 2004; open literature; submittedstudies (MRID 46023101)

submitted study (MRID45895901)

estimated - PBT ProfilerAntimycin A is formulated as a liquid concentrate with a single formulation, ~introl~ concentrate7. Antimycin A is applied to water by drip-feed device as part of a drip station, backpack sprayer, boat bailer, and sprayer.8 Spraydrift from anitmycin A applications is not considered likely even when applied by backpack sprayers since hand-held wands are used to dispense the spray relatively close to the surfaceofthe water. 

### 2.1.4 Overview of Pesticide Usage Antimycin A is used as a piscicide. According to a survey of fishery management agencies in the United States and Canada conducted by the American Fisheries Society (AFS) between 1991 and 2001, a total of 1,138 kg of antimycin were used by StatelFederal resource management agencies9. 

### In the latter half of the decade, antimycin use increased 75% due primarily to increased use in flowing waters. The average annual use was 5.05 kg (1 14 units) with each unit capable of treating 0.009 hrn3 at 5 ppb. The survey reported that the private sector used roughly 529 kg (1,166 lbs) and that private aquaculture facilities accounted for roughly three-quartersofthe antimycin purchased by the private sector. Also, according to the AFS survey, the primary use of antimycin has been to restore listed species, accounting for 33% of the water treated and 49% of the antimycin used. Renovation of fish populations was the second most frequent use of antimycin accounting for 29% of the waters treated and 27% of the antimycin applied. The third largest use was restoration of native fishes accounting for 26% of the waters treated and 19% of the antimycin used. In standing waters, 48% of the total antimycin used was to manipulate fish populations; in flowing waters, the majority (99%) of antimycin used was to restore native or listed specieslO. The primary use of antimycin in private aquaculture is for removal of scaled fish from catfish fingerling and food fishproductionponds. 

### 2.2 

### Receptors

2.2.1 Ecological Effects Table 2 provides taxonomic groups and test species used to indicate the potential for ecological effects in screening-level risk assessment. Within each ofthese very broad taxonomic groups, an acute andlor chronic endpoint is selected from the availabletest data (see Section 2.3).

EPA RegistsationNo. 39096-2 SpecialReview and ReregistrationDivision Use ClosureMemo dated October26,2005. 9. F~nlayson, B.J., R.A. Schnick,RL. Cailteux. L. DeMong, W.D. Horton, W. McClay, and C.W. Thompson. 2002. Assessment of AntimycinA Use in Fisheries and its Potential for Reregistration. Fisheries27(6): 10 -18 I0Ibid Finlaysonet al.2002. Page 11of 118pp -- - - - Fable2. Taxonomicgmupsand most sensitivetest species evaluatedfor ecological effects. 

# I 

# I Birdss 

# I Mallard duck (Anusplatyrhynchos) 

# I Chronic,NOAEC 

# I Taxonomicgroup 

# I Mammals 

# I Example(s) of representative species 

# I Laboratoryrat (Rattusnorvegicus) Acute LDso and Chronic 

# I Endpoint used

Bobwhite quail (Colinusvirginianus) Acute, LDsdK50 

# I Freshwater fishh 

# I Rainbow trout (Oncorhynchusmykiss) 

# I Acute K50 

# I I I 

# I

Terrestrial insects I I 

# I Honeybees (Apis mellifera)

Fathead ~~MOW (Pimephales promelas)


## 1 Estuarindmarine fish 

## I Sheepsheadminnow (Cyprinodonvariegatus) ( Acute LC50 

## 1 Acute Oral LD5o

ChronicNOAEC Freshwater invertebrates 

# I Estuarindmarine invertebrates 

# I Mysid shrimp(Mysidopsisbahia) 

# I Acute LC50 

# I I I 

# I
Water flea (Daphnia magna) Midge (Chironomusriparius) Acute LC50 and ChronicNOAEC ChronicNOAEC (Benthic) 

# I Eastern oyster (Crassostrea virginica)

Terrestrial plantsC



### 2.2.2 Ecosystems atRisk Acute ECso

Aquatic plants and algae

Antimycin A may be applied directlyto both static (lentic) and flowing (lotic) waters in a manner that disperses the compound throughout the water column for maximum efficacy. Residues can then be managed to prevent release from the treated portion ofthe water body. I I 

# I
Monocots -corn (Zea mays) Dicots - soybean(Glycinemax)

When antimycin A is applied directly to water, the ecosystem at risk consistsprimarily of that particular water body, but also includes the downstream outlet of the water body, if one exists. Steps may be taken to accelerate the natural degradation of antimycin A in order to reduce the risks of exposures occurring outside of the treatment area. Within the water body, compartments of concern are the water column, sediments, and pore water. Organisms of concern include fish, aquatic invertebrates, aquatic-phase amphibians, aquatic plants (algae and vascular), and birds or mammals that may rely on the water body as a habitat andlor drinking water source. Given the apparent propensity of antimycin to sorb to organic matter and depending 

### on the flow regime and water quality characteristics, deposition of antimycin Acontaminated sediment into downstream flood plains, marshes, or estuaries may pose a concern. While antimycin A likely sorbs to sediment, it appears to have a low potential to bioaccumulate in aquatic organisms. Acute (Tier I, no ECso estimated) 

# I 'Birds areused as sumgates forterrestrialphase amphibiansand reptiles (US EPA, 2004). 'Freshwater fish are used as surrogatesfor aquatic phase amphibians (US EPA, 2004). 'Four species of two familiesofmonocots, ofwhich one is corn; six speciesofat least four dicot families, ofwhich one is soybeans. Green algae (Selenastrumcapricornutum)

Page 12 of 118 Acute EC50

### 2.3 

### Assessment Endpoints Agency guidelines define assessment endpoints as "explicit expressions of the actual environmental value that is to be protected."" Operationally, the environmental value is represented by an ecological entity and associated attributes or characteristics. The assessment endpoints for this ecological risk assessment are survival, growth, and reproduction of terrestrial and aquatic animals and plants. Specifically, this assessment addresses birds, mammals, reptiles, amphibians, fish, terrestrial and aquatic invertebrates, and terrestrial and aquatic plants. These endpoints are meant to reflect population sustainability and community diversity within ecosystems. These are standard assessment endpoints that are considered for the majority of pesticides with agricultural uses. Measures of effect and measures of exposure are explicit toxicity and exposure measurements or estimates that are used to identify risks. Measures of effect are used to relate pesticide exposure to potential effects on the assessment endpoints, which are not explicitly evaluated in the assessment. Measures of exposure are typically values derived from chemical use information and standard fate, transport and exposure models. The toxicity and exposure values areused to generate a risk quotient, which is considered a screening-level estimate ofrisk only. Additional refinements may be triggered if the screen identifies potentially unacceptable levels of risk. Refinements may include the selection of assessment endpoints more directly relevant to populations or communities, spatially-explicit identification of risks, and the use of probabilistic methods for risk estimation. Assessingrisks to reptiles and amphibians represents unique challenges. Currently, data on direct toxicity to reptiles and amphibians are not required as part of the standard dataset submitted to the Agency for pesticide registration or re-registration and these data are only occasionally available from the literature. For ecological risk assessments in the Office of Pesticide Programs (OPP), if risks to birds are below the level of concern, then risks to reptiles are also assumed to be low. For amphibians, freshwater fish are considered a suitable surrogate for the aquatic life-stage and birds a suitable surrogatefor the terrestrial life-stage. 

### 2.4 

### ConceptualModel

2.4.1 

### Risk Hypotheses The risk hypothesis that is presumed for the ecological risk assessment of antimycin A is based on the use and characteristics of this compound. Based on the fact that antimycin A is applied directly to water, it is presumed that aquatic environments are at risk. The following risk hypothesis is presumed for this screening-levelassessment: The use ofantimycinA in accordance with the label results in adverse effects on survival and/or fecundity to non-target aquatic plants and animals. 

### To the extent that terrestrial animals may feed offofdead or dyingJish in the treatment area and/or ingest antimycintreated water, terrestrial animals may experience adverse eflects as well.

11 U.S. EPA. 1998.Guidelines for EcologicalRisk Assessment.Risk Assessment Forum, Washington,DC. EPA 630lR-951002FApril 1998. Page 13 of 118

### 2.4.2 Conceptual Model Diagram A conceptual model was developed for antimycin A to reflect its uses as a piscicide (direct application to water). The diagram in Figure 1 includes the stressor (antimycin A), the source of the pesticide and/or transport pathway, exposure media, exposure points, biological receptors, and attribute changes (effects). When used as a piscicide, antimycin A is deposited directly into a receiving water, which may be a stream, pond, or lake/reservoir. Once in the water column, non-target aquatic organisms may be exposed to antimycin A via three main exposure pathways and two minor pathways. 1. Antimycin A will initially be distributedin the water column of the receiving water, and organisms in the water column (aquatic invertebrates, fish, amphibians and plants) may be directly exposed through contact. Organisms can take up soluble antimycin A through the gills or integument. If sufficient concentrationsexist, then individual toxic effects in aquatic invertebrates, fish, aquatic-phase amphibians and/or plants can result. This may result in reductions in populations of species within these taxa. This is expected to be the main route of exposure for antimycin A. 

### 2. Antimycin A released into water will likely sorb to sediment and suspended solids. Receptors such as aquatic invertebrates, fish, and aquatic-phase amphibians may be exposed to sorbed antimycin A through incidental ingestion of sediment and/or suspended solids. This can result in changes in populations based on reduced survival, growth and reproduction. 3. Antimycin A may sorb to plant surfaces. Ingestion of plant materials with sorbed antimycin A can result in 

### a similar exposure pathway as described for sediment and suspended solids. Nonvascular or vascular plants may also be exposed to antimycin A through direct contact or root uptake. This could result in changes in plant populations due to direct toxicity. However, since no plant toxicity data are available for antimycin A, potential risks to aquatic plants cannotbe evaluated. 4. Antimycin A residues may accumulate in target and non-target organisms, although this is not considered likely given the low estimated bioconcentration factor. Other organisms that live in or around the treatment area may be exposed to antimycin A by consumingtreated organisms. 5. Direct exposure of terrestrial animals/plantsto antimycin is not likely since the chemical is only applied to water bodies. Indirect exposureto both mammals and birds is possible through the consumption of dead and dying fish and larger invertebrates and through ingestion of antimycin-treated water.

Page 14 of 118SedimenUSuspendedSolids DIRECT
Contacr/GiIllntegument

Fish,Aquatic-phase Amphibians,Aquatic Invertebrates
INDIRECT EXPOSURE PATHWAY

Invertebrates

EFFECTS Chronic Reduced Growth SOURCE

Chronic Reduced Figure 1. Conceptual model for antimycin A depicting source, direct and indirect routes of exposure, biological receptorsand possible effects. Antimycin Applied Directlyto Water as a Pesticide



## 3 USE CHARACTERIZATION 

## I I

Antimycin A is registered for use as a piscicide. As stated in the use closure memo (dated October 26, 2005), there is a single supported registration (Fintrolm; EPA Registration number: 39096-2) that contains antimycin A as an active ingredient. Antimycin A is applied to water by a Page 15 of 118drip-feed device as part of a drip station, backpack sprayer, boat bailer, and sprayer. Drip stations are typically used in streams and rivers inaccessible to boat traffic; a photograph of a drip station is shown in Figure 2. Backpack sprayersmay be used to supplement drip stations or other application devices in areas with poor water circulation (e.g., stagnant pools that the chemical may not reach through natural stream flow). The ~introl@ label recommends that backpack sprayersbe used in areas where water depth is 0.3 meters (1 foot) or less. Boat bailers are used in larger water bodies such as ponds and rivers. Deeper water bodies may require the use of a pump mechanism (to ensure adequate mixing throughout the water column) where antimycin is dispensed through a perforated hose stretching the length of the water column or is deliveredthrough the propeller wash.12

19-liter(5-gallon)bucket containing formulated product (i.e.,water, antimycin A and the

Drip-feed device calibrated to deliver 3.8 liters (1 gallon)per
Figure 2. Antimycin A drip station used for applying product to high gradient (mountain) streams. Environmental factors such as water body size, flow rate, pH, temperature, and stream gradient may affect the quantity of antimycin 

### A and the number of application stations that must be used to achieve the desired concentration in the treatment area. For example, antimycin A is more effective in warm water; thus, less antimycinA may be required duringthe summer months than the winter months for the same treatment site, assuming other factorsremain constant. There are two broad uses for antimycin A as a piscicide: complete kill and selective kill. In a completekill, the water body is treated at roughly 5 to 25 pglL antimycin A to eliminate all fish in the treatment area. A common objective of a complete kill is to eliminate invasive or non-native speciesin an areato restore listed or indigenous species.13 In a selective kill, the water body is treated at 0.5 to 1.0pgfLof antimycin A to eliminate only small, scaled fish. A common objective of a selectivekill is to eliminate smaller fish to free up food and other resources for larger fish. Selective kills at higher concentrations are also used in catfish production to eliminate scaled fish that commonly reduce the catfish yields of

l2 Wormell, L. 2005. Use Closure Memo. Special Review and Reregistration Division Memo dated October 26.2005. Ibid Wormell 2005. Page 160f 118commercial catfish farmers. According to the FintrolBlabel, scaled fish in aquaculture ponds succumbto treatment at 5 to 10pg/L of antimycin A, whereas catfish generally tolerate up to 20 pg/~.14 Based on information provided by the registrantI5 the majority of ~introl@ use in [catfish] aquaculture is for food-fish production and not finger-production. According to aquaculture use information collected by the Southern Regional Aquaculture centerI6,Fintrolm use is roughly equally divided between food-fish and fingerling-production ponds to eliminate scaled fish at various points within theproduction cycle. According to the FintrolBlabel, in both complete and selective kills, dead fish may be collected and disposed of or left to biodegrade. Additionally, areas downstream of the treatment area may or may not be neutralized with an oxidizing agent such as potassium permanganate to intentionally inactivate antimycinA. 

### 3.1 

### Detoxification In situations where fish kills cannot extend beyond a certain point downstream, the ~introl~ label provides instructions for detoxifying antimycin A with potassium permanganate (KMn04) at a treatment concentration of 1 mg/L (part per million). Potassium permanganate (CAS No. 7722-64-7) is a strong oxidizing agent commonly used to purifjr drinking water and kill pond algae. According to the product label, antimycin may be considered detoxified when fingerling rainbow trout (Oncorhynchus mykiss) or juvenile bluegill sunfish (Lepomis macrochirus) survive for at least 48 hours in holding tanks (live-cars) placed 91 meters (100 yards) downstream from the site of potassium permanganate application. Depending on environmental conditions (e.g. total organic carbon) higher KMn04treatment rates (e.g.3 - 4 mg/L) may be required to deactivateantimycin A'~. 

### 3.2 

### Usageand Use Rates There is currently one active antimycin A registration (Section 3). There is currently no emergency exemption (Section 18)use or special local need (Section 24c) use. Accordingto the registrant, less than 45 kg (100 lbs.) of antimycin A are used annually in the US. EPA's Screening Level Usage Analysis returned no data on agricultural or non-agricultural uses of antimycin A. Antimycin A is available as a soluble concentratelliquid. Retreatment and reentry intervals are not specified on the current label. According to the registrant and based on sales data, antimycin is typically used for fishery management purposes between August and September while it is in aquaculture primarilyin May through september18. Table 3 presents the antimycin A labeled use rates that are being considered for reregistration. The risk assessments will be based on the formulations, use rates, retreatment

15

Personalcomrnuniction, Mary Romeo, President,Aquabiotics Corporation.2006. 16 personalcommunicationDr. CraigTucker, Director, National Warmwater AquacultureCenterand SouthemRegional Aquaculture Center, 2006 17 Personalcommunication, Mr. SteveMoore, Supervisory Fishery Biologist, Great SmokyMountain National Park, 2006. l8 Personal communication,Ms. Mary Romeo, Presidient, AquabioticsCorporation, 2006. Page 17 of 118

### intervals, and application methods presented in the table below.19 As stated previously, the Fintrol" label indicates that rates as high has 25 yg/L are needed to be effective for control of certain fish when pH is 8.5 

### or more and water temperatures arebelow 16°C. 

### Although maximum treatment rates are not stated on the label, this risk assessment is based on an upper-bound treatment rate of 25 yglL 

### applied onceper year.

a Use Site: The use siterefers to the entity (crop, building, surfaceor article)where apesticide is appliedand/orwhich isbeing protected.

Max.Rateper App: Maximumdose for a single applicationto a singlesite. System calculated. Max.Rate UnitIArea: Units and Area associatedwiththe maximumdose (UG: UseGroup codes) Form: Thephysical form ofthe end use product found in the container. Max. # Apps cc &yr: maximum number of applications.

19/bid Wormell 2005 20~bid Wormell 2005 Table 3. Supported uses of antimycin A on use groups: aquatic food crops (Dl), aquatic nonfood outdoors (El)and aquatic non-food industrial (FI).''

Page 18 of 118 Application ~~uiptnen?

l~~~~e~ Mia. App Interval Formd Max.# Apps cc & yre NON-FOODINON-FEED Max. App Ratd ~c & Yr Max. Rate UniUArea UGc Use SiteB Max. Rate per Appb

(PP~)

Backpack sprayer1 Boat bailer1DripFeed device/

sprayer //Water treatment Backpack sprayer1Boat bailer1Dripfeed device/

Sprayer //Water treatment

- Backpack sprayer1 Boat bailer1Dripfeed device/

Sprayer //Water treatment SCIL~

SCIL

SCL lakes/ponds/rese rvoirs (without human or wildlife use)

streams/rivers/ channeled water

swampslmarshe s/ wetlands/stagna nt water

FOODIFEEDUSES NS

NS

NS USES

NS~

NS

NS NS

NS

NS 25 "roughly"

25 ~~roughly,,

25 'sroughly" ppb15 gal F1

ppb/5 gal El

ppb15 gal El

commercial fishery water

systems Backpack sprayerl Boat bailed Dripfeed device/

Sprayer //Water treatment NS 25 "roughly" ppb15 gal D 1 SCIL NS NSax. App Ratdcc & yr: maximumamount ofpesticide product thatcan be appliedto a site in onegrowingseason(lcc) or during the span of one year (Iyr). Min. App Interval (days):minimumretreatmentintervalbetweenapplications in days (aggregated). h~pplication Equipment: equipmentused to applypesticide (aggregated). iApplicationType: type ofpesticide application (aggregated). 

# 'SCIL: SolubleConcentmtdliquid

NS: Not Specified(onlabel). The extent to which direct applications of antimycin A to water as a fish control agent can be limited to the desired treatment area is dependent on the rigor of the protocols used to apply the chemical and the degree to which they are followed. Biological and chemical monitoring of the treatment area and deactivationof antimycin A where feasible could track and limit exposure of non-target organisms. While many state and federal agencies follow standard operating procedures (SOPs) that limit exposure, the SOPs are not uniformly applied across all potential users. Also, since the environmental fate database is incomplete for antimycin A, the extentto which antimycinA will persist and potentially move outsidetreated areas is uncertain. Although not required by the label, relatively rigorous application procedures are typically followed by resource managers using this restricted use pesticide;

### Appendix 

### B contains an example of a standard operating procedure followed by the State of New Mexico Department of Game and Fish. Additionally, state and federal resource managers participate in training courses such as Rotenone andAntimycin Use in Fish Management, offered by the U.S. Fish and Wildlife Service National Conservation Training Center (Shepherdstown, WV) to familiarize staff members with procedures for planning and executing fish management objectives using piscicides. Although not required by the label, antimycin is typically deactivated followingtreatment of lotic systems. The label recommends that potassium permanganate be applied at a rate of 1 ppm to the outflow and that additional permanganate may be needed if the stream has a high permanganate demand, i.e., organic carbon content. Drip systems of hose-and-clamp or carburetor types are recommended on the label to continuously disperse a solution of potassium permanganate into the water at the discharge outlet. Deactivation is maintained until all of the Fintrol-treated water has passed through the station and fish in holding containers (livecars) placed 91 meters (100 yds) downstream of the detoxification station remain alive for at least 48 hours.

Page 19of 118

## ENVIRONMENTALFATE CHARACTERIZATION
4.1 

### Background Antimycin A is a relatively minor-use piscicide used primarily for direct applicationsto remote mountain streams, lakes and ponds and fishery operations. Several factors complicate the estimation of environmental concentrations of antimycin, among them non-strict application rates, sparse fate data, and insensitive detection methods. Because of these uncertainties quantification of antimycin exposure is speculative. With regard to labeled use rates, the antimycin label provides only guidance and is not prescriptive with regard to the maximum amount of antimycin that may be applied to a water body. The label only refers to "rough" estimatesof effective dosesthat are to be applied to water bodies, and it is left to the user to determine the actual dose. The label recommends that the amount of antimycin used should be determined by bioassays using the specific water to be treated. Such labeling makes it difficult to estimate how much antimycin may enter the environment since there is no maximum label rate. Another difficulty is that there are comparatively little EPA-guideline fate data available for evaluationofthe fate of antimycin A in the environment. The registrant has submitted only a hydrolysis and an aerobic aquatic metabolism study, both of which the EPA classified as providing only supplemental information and not fulfilling the data requirement. There are, however, non-guideline studies (primarily concerning hydrolysis rates) that offer some insight into the fate of antimycin in the environment. The combination of results from the submitted studies and the open literature studies gives a wide range of degradation rates for antimycin, further complicating the estimation ofpotential antimycin environmental concentrations. Finally, analytical techniques for determination of antimycin are very limited for the concentration levels that are used in the environment. The only method currently available is a bioassay, in which detections would be non-specific (unknown if antimycin or some other toxicant is present) and would only be detected after detrimental effects occur (fish death). Although the above issues hinder the ability to calculate "standard" EECs in a manner similar to other pesticide assessments, rough estimates that include the uncertainties associated with fate properties and application rates are possible. By considering the uncertainties, a large range of possible EECs and dissipationtimes are calculated as described below. 

### 4.2 

### Fate 

### Properties Antimycin A is a complex of 4 structures that is reportedly soluble in polar organic solvents (e.g. ethanol, acetone, chloroform) and relatively insoluble in water (Walker et al., 1964). Most of the available fate studies in the open literature are hydrolysis studies, and there are no direct studies that measure the sorption properties of antimycin. There are only two

### registrant-submitted studies-a hydrolysis study and an aerobic aquatic metabolism study. When the fate studies are viewed together, some conflictinginformation results in uncertainty. One ossible mechanism of antimycin degradation is base hydrolysis as suggested in the literature. 21, 2, 23 

# '
The supplementary registrant-submitted hydrolysis stud J4 

### using H)-labeled antimycin also suggests that base hydrolysis is a mechanism of degradation, but hydrolysis rates reported by the registrant's study are significantlyhigher than the earlier open literature studies (Table4). 

### The studJ5 indicates that antimycin in an aquatic environment at 25°C

### has a half-life of about 15 days at a pH of 5,3 days at pH 7, and 3 hours at pH 9. 

### Lee et 

### al. (1971)~~ reported much faster degradation rates using yeast assays, with half-lives of about 5.5 

### hours at pH 7, and 20 minutes at pH 9.5. ~ussain~~ found degradation half-lives of about 46 hours for antimycin (Al) at pH 7.55 and about 

### 2 minutes at pH 9. However, a recent study by Kenneke (2006)~', which suggests that hydrolysis is an important mechanism of degradation, does not clearly show that the degradationrate is related to pH. Kenneke (2006)~' estimated half lives ofaround 4 to 10 hours in unmixed systems; whereas in mixed systems half lives ranged from 1 to 10 hours. Hydrolysis rates fi-omthe literature are summarized in Table 1. 

### In all cases hydrolysis half lives are on the order ofminutesto hours. Transformation products were not identified in the registrant-submitted study3' ,Lee et 

### al.31 or ~enneke'~. In 

### an anecdotal report, Walker et 

### al.') claimed that antimycin degrades by base hydrolysis and produced a diagram showing the mechanism of degradation along with assumed breakdown products. ~ussain~~ made similar claims as to the degradation products. These products are blastrnycic acid, antimycin lactone, and antimycic acid. No quantitative information regarding the relative concentrationsofthe transformation productsis available. The only other registrant-submitted environmental fate study is an aerobic aquatic transformation study3' which indicated that antimycin A degrades with a half-life in the range of

21 Walker, C.R., RE. Lennon, and B.L. Berger. 1964.Preliminary observationson the toxicity of antimycin A to fish and other aquatic animals. U.S. Bureau of Sport Fisheriesand Wildlife, Investigations in Fish Control No. 2 (CircularNo. 186). 18pp. 22 Hussain, A 1969.Kinetics and mechanisms ofhydrolysisofantimycin A in solution.Journal ofPharmaceutical Sciences58: 

### 316-320. 91 L-' Lee, T.H., Dme, P.H., and Morton, S. 1971. Effects of Physical and Chemical Conditionson the Detoxification of Antimycin. Transactions %the American Fisheries Society 1971; 100: 13-17 L4 Heim, D. 2003. Hydrolysis Determination forAntimycin A Complex. Project Number 47132. Unpublishedstudyprepared by Analytical Bio-Chemistry Labs, Inc. 63 p. (MRID460231-01) 25 Ibid Heim 2003. 261bid Lee et 01. 1971. 27 Ibid Hussain 1969. 28 See Appendix G for Kenneke 2006 29~bid Kenneke 2006. 30 Ibid Heim 2003. -. 31 IbidLeeetal. 1971. 32~bid Kenneke 2006 33 Ibid Walker et al. 1964. 341bidHussain 1969. 35 Heim, D. 2003. DeterminationofAerobic Aquatic Metabolism forAntimycin A Complex: Amended Final Report. Lab Pmjst No. 473 13. Unpublishedstudy preparedby ABC Laboratories,Inc. 79p (MRD458959-01). Page21 of 11823 to 

### 47 days in pH 6.5 water. This half-life appears to be substantially longer than the half-life due to hydrolysis alone at this pH (see above and Table 4), which adds some uncertaintyto the half-lives reported for the hydrolysis study. However, since the aerobic aquatic metabolism study system included sediment, the longer half-life could be due to high sorption of antimycin to sediment which may shield antimycin from hydrolytic degradation (although this is purely speculative since sorption studies areunavailable). Although the registrant did not provide sorption studies for antimycin, indications from the aerobic aquatic metabolism study are that antimycin A does sorb significantlyto sediment. Rough estimates of sorption can be made from the aerobic aquatic metabolism study by considering the relative distribution of antimycin in the water and the sediment 

### as measured in that study. If equilibrium between the water and the sediment were assumed throughout the aerobic aquatic metabolism study then 

### K, values would range from about 1 to 88 mllg or K, values in the range of 84to 10000mllg, which amounts to significant sorption. In order to gain additionalinsight into antimycin, fateproperties were also estimated with ASTER. ASTER (Assessment Tools for the Evaluation of Risk) was developed by the U.S. EPA Mid-Continent Ecology Division, Duluth, MN (MED-Duluth) to assist regulators in performing ecological risk assessments. ASTER is an integration of the AOUlRE (AQUatic toxicity Information REtrieval) toxic effects database and the QSAR (Quantitative Structure Activity Relationships) system, a structure-activity-based expert system. When empirical data are not available, mechanistically-based predictivemodels can be used to estimateecotoxicology endpoints, chemical properties, biodegradation, and environmental partitioning. ASTER is designed to provide high quality data for discrete chemicals (when available in the associated databases), and QSAR-based estimates (when data are lacking). The QSAR system includes a database of measured physicochemical properties such as melting point, boiling point, vapor pressure, and water solubility as well as more than 56,000 molecular structures stored as SMILES (Simplified Molecular Input Line Entry System) strings for specific

## chemical^.^^ Based on ASTER model estimates, antimycin is not expected to be mobile in soil and sediment (log K,= 3.41) and the chemical has a relatively low potential for bioconcentrating in aquatic organisms (bioconcentrationfactor (BCF=350X). From ASTER, it appears that Antimycin A is not likely to be persistent in the environment and its low vapor pressure 

### (2.3 1x10-l5 

### mm Hg) and Henry's Law constant (2.42 x10-l7atm-m3mol-I)limit its volatility

36 ASTER 2004 httn wu u cod ro, ~ncd f'rodr I'ub .wet httl, Page 22 of 118

### 4.3 

### Usage and Fate in WaterBodies Table4. Antimycinfate Property Molecular Wt HydrolysispH 3 Hydrolysis pH 4 Hydrolysis pH 5 Hydrolysis pH 6 Hydrolysis pH 7 Hydrolysis pH 8 Hydrolysis pH 9 Hydrolysis pH 5 Hydrolysis pH 7 

## -Hydrolysis pH 9

HydrolysispH 4.5 - 5.5 Hydrolysis pH 7 - 8 HydrolysispH 8.5 HydrolysispH 9.5 Hydrolysis pH 10 Hydrolysis pH 7.55 (A1 only) Hydrolysis pH 9 (A1 only) Hydrolysis pH 10.1 (A1 only) Hydrolysis (25") Water Solubility (20 "C) Koc KOC Aerobic Aquatic Degradation Bioconcentration Factor (BCF) Henry's Law Constant Vapor Pressure (25 EC)

The antimycin label only gives "rough estimates" of the target concentration at which antimycin is to be used, and the label gives no upward limit on what concentration may be used. Rough estimates for target concentrations range from 25 ppb (for pHX3.5 and temperatures <60°C) to 5 ppb (for pHc8.5 and temperatures >60°C). The label suggests that the actual concentrationthat is to be used be confirmed with a bioassay. Because of the great latitude that the label gives, it cannot be determined apriori what amount of antimycin may be applied to a water body. For both stream and lake applications of antimycin, downstream movement of antimycin will occur; however, the extent is unknown. In many cases, application of antimycin Page 23 of 118 properties Value 548.7
9 hours 8.3hours 10.5 hours 11hours 7.1 hours 10hours 3.4 hours

15days 3 days 3 hours >7 hours 5.5 hours 40 minutes 20 minutes 6 minutes 46 hours 2.2 hours 1 minute 190 days 69 mg L-' 2500 ml/g 84 - 10000mVg

20 to 47 days 350x 2.42 x10-17atm-m3mol-' 2.31~1~'~ mm Hg Source

Kenneke (2006) Kenneke (2006) Kenneke (2006) Kenneke (2006) Kenneke (2006) Kenneke (2006) Kenneke (2006) MRID 46023101 MRID 46023101 MRID 46023101 Lee et al. (1971) Lee et al. (1971) Lee et al. (1971) Lee et al. (1971) Lee et al. (1971) Hussain (1969) Hussain (1 969) Hussain (1969) Estimated -ASTER, 2004 Estimated - ASTER, 2004 Estimated 

### - ASTER, 2004 Estimated fiom aerobic study MRID 458959-01 (see text) MRID 458959-01 Estimated -PBT Profiler Estimated - ASTER, 2004 Estimated - ASTER, 2004may coincide with the use of live-cars containing sensitive speciesthat areplaced downstream of the antimycin application, although this is not a label requirement. Use of live-cars with sensitive species would allow monitoring of the dissipation of antimycin effectiveness but is does not prevent antimycin from proceeding downstream, and there is some evidence that such downstream movement canbe significant37. In one study, Tiffan and Bergersen3*observed that antimycin was 100% effective at fish kills to at least 1.75

### krn downstream of a Colorado creek (pH = 6.3, 9-15°C). Because there was a 100% fish kill at 1.75 

### km and no live-cars placed downstream of this distance, the effect of antimycin probably proceeded much farther downstream. In this creek, antimycin was applied to the creek at 8 ppb for 8 hours, which is a typical application. The reason that antimycin remained effective in this stream system is unclear (although the authors speculate stream gradient had an effect). 

### In other streams examined by Tiffan and ~er~ersen~~, antimycin was effectiveto at least 0.5 

### km downstream. 

### 4.4 

### Estimates of Water Concentrationfor Risk Assessments When antimycin is applied to a water body (whether a lake, pond, or stream) the acute concentration that should be used for ecological assessments is the application concentration. The maximum application concentration is ambiguous but the label states that it is "roughly" 

### 25 ppb. However, because of the significant uncertainties regarding the persistence and sorption properties of antirnycin, predicted temporal concentration trends and chronic concentrations are also full ofuncertainty. With this in consideration,ranges of estimates canbe made by using the fullrange ofpossible degradation (hydrolysis) rates reported in Table4. Table

### 5 gives the range of chronic concentrationsthat may result following a 

### 25 ppb application of antimycin to a water body (and with no consideration for sorption or other means of dissipation). Chronic concentrations vary considerably dependingon the half-life assumed for antimycin. Such values apply to both static water bodies (e.g., lakes) as well as flowing streams. In the case of streams, however, the chronic concentrationwould apply only if an organism were traveling downstream at the stream speed. Also in the case of streams, antimycin likely will dissipate due to hydrodynamic dispersion, sorption, and mixing with sidechannels; however, these factors cannot be readily determined. One other factor that may be useful in assessing antimycin environmental exposure is the time that it would take antimycin concentrationsto degradebelow a certain level oftoxicity. For example, Table 6 presents the times that it would take antimycin to degrade from 25 ppb down to a concentration of 0.004 ppb (concentration where the most sensitive aquatic risk quotient would be equivalent to the acute risk level of concern of 0.5). These values apply to both streams and water bodies. For the case of streams the time would be associated with the downstream movement of antimycin, and if stream velocity were known these values could be associated with distance downstream of antimycin applications where antimycin remains toxic.

37 Tiffan, K.F. and E.P. Bergenen. 1996. Performanceofantimycen in high-gradient streams.North American Journal ofFisheries Management 16: 465-468. 38 lbid Tiffan and Bergmen. 1996. 39 lbid Tiffan and Bergmen 1996 Page 24 of 118In this regard, Tiffan and ~er~ersen~' showed that antimycin can remain toxic in some circumstance for over 2 kilometers downstream ofthe antimycin application.



## I Table 6. Time for antimycin to get below LOC concentration (0.004 ppb) when applied at 25 

### )

Table 5. Ranges of chronic concentrations depending on assumed half-life. Values in ppb. Initial concentration assumed to be 25 ppb. Higher initial concentrations would result in proportionally higher chronic concentrations.

5.5 hours 

### I 2.9 days 20 minutes 4 hours 1 year 4.6 2.3 1.5 0.49 0.022 0.0013

Assumed half-life 47 days 15 days 5 days



## 5 

## ECOLOGICALEFFECT 

## CHARACTERIZATION Time required to reach0.004 ppb 592 days 189 days 63 days

Toxicity testing reported in this section does not represent all species of bird, mammal, or aquatic organism. Only a few surrogate species for both freshwater fish and birds are used to represent all freshwater fish (2000+) and bird (680+) speciesin the United States. For mammals, acute studies are usually limited to Norway rat or the house mouse. Estuarine/marinetesting is usually limited to a crustacean, a mollusk, and a fish. Also, neither reptiles nor amphibians are tested. The assessment of risk or hazard assumes that avian toxicity is similar to that of terrestrial-phase amphibians and reptiles. The same assumption is made for fish and aquaticphase amphibians. 60 day 17 12 8.4 3.0 0.14 0.0083 21 day

2 1 19 16 8.1 0.39 0.023 Half-life 47 days 23 day 15 days 5 days 5.5 hours 20 minutes



### 5.1 

### Categories ofAcute Toxicity In general, categories of acute toxicity ranging fkom "practically nontoxic" to "very highly toxic" have been established for aquatic organisms based on LC,, values (Table 

### 7), terrestrial organisms based on LD,, values (Table 8), and avian species based on LD,, values (Table 

### 9). Subacute dietary toxicity for avian speciesisbased on the LC,, values (Table 10). 90 day 14 8.6 5.9 2.0 0.09 0.0055 4 day

24 24 23 19 2.1 0.13

40 IbidTiffanand Bergmen 1996. Page 25 of 118Table 7. Categories for aquatic animal acute toxicity based on median lethal concentration in mg per liter (parts per million). LC50 @~m) <O. 1 0.1-1 >1-10 >10-100

>lo0

Table 8. Categories for mammalian acute toxicity based on median lethal dose in mg per kilogram body weight (parts per million).

Page 26 of 118 Toxicity Category Very highly toxic Highly toxic Moderately toxic

Slightlytoxic Practicallynon-toxic

LDW (mg a.i./kg)

<lo 10-50 5 1-500 501-2000

>2000



# .

Table 9. Categories of avian acute oral toxicity based on median lethal dose in milligrams per

kilogram body weight (parts per million). ToxicityCategory Very highly toxic Highlytoxic Moderately toxic

Slightlytoxic Practically non-toxic

LDw, @~m) <10 10-50 5 1-500 50 1-2000

>2000 Toxicity Category Very highly toxic

Highly toxic Moderatelytoxic

Slightlytoxic Practically non-toxic

### 5.2 

### ToxicitytoAquaticAnimals Table 10. Categories of avian subacute dietary toxicity based on median lethal concentrationin milligramsper kilogramdietper day (partsper million).



### 5.2.1 FreshwaterFish, Acute A total of 15 acute toxicity studies of technical grade (>95% active ingredient) on freshwater fish are contained in the EFED ecotoxicity database for antimycin A (Table 11). Paddlefish (Polyodon spathula) are the most sensitive (LC,,=0.001 pg/L) freshwaterfish species tested. To ensurethe risk assessment is as protective as possible of non-target species, the lowest scientifically defensible toxicity value available is used to evaluate acute risks to freshwater fish. Although paddlefish are the most sensitive species reported, the raw data used to support this endpoint could not be evaluated and therefore this value cannot be used to quantitatively assess risk. The most sensitive species for which raw data could be reviewed is coho salmon (Oncorhynchus kisutch); therefore, the freshwater fish acute toxicity endpoint (96-hr LC,,) is 0.009 pg/L. Based on the sensitivityof freshwater fish to antimycin, the piscicide is classified as very highly toxic to fish on an acute exposurebasis. LCso @~m) 40 50-500 501-1000 1001-5000

>5000

Based on the available data, fish in the family Ictaluridae (catfish), like black bullhead (Ictalurus melas) and channel catfish (Ictaluruspunctatus), are the least sensitive species tested which accounts for how antimycin can be used to selectively remove scaled fish from catfish aquacultureponds. Although paddlefish do not have scales and yet are the most sensitive species to antimycin, this phylogenic primitive fish is not related to the catfish but is instead distantly related to sturgeon. Toxicity Category Very highly toxic Highlytoxic Moderately toxic

Slightly toxic Practicallynon-toxic

Toxicity testing of formulated product (1 to 10% active ingredient) indicates that formulated products are less toxic than the technical grade active ingredient; the 96-hr LC,, for bluegill sunfish is 1.18 pg/L for the formulated product (Table 12) whereas the technical grade active ingredient has a 96-hr LC,, of 0.034 pg/L. Similarly, rainbow trout had LC,, values ranging from 0.63 to 185 pg/L for formulated product while technical grade antimycin had an LC,, averaging0.011pgJL.

Page 27 of 118OPP utilized the ECOTOX (Ecotoxicology Database system)" on-line database to conduct an open literature review in an attempt to supplement the registrant-submitted data. ECOTOX is a comprehensive computer-based system that provides single chemical toxic effect data for aquatic life, terrestrial plants and terrestrial wildlife derived predominately from peerreviewed literature. The literaturerelevant to the exposure and toxic effects of antimycin A and the metabolites was collected, reviewed and evaluated for inclusion into this chapter. Citations and abstracts were obtained by searching the following commercial or publicly available databases: TOXLINE, MEDLINE, BIOSIS previews, AGRICOLA, and AQUIRE, as well as Dissertation Abstracts. For a more in-depth discussion of the ECOTOX on-line database see htt~:iiwww.e~a.~oviecotox:'. While a number of acute toxicity studies using various fish species are available through ECOTOX, none ofthe toxicity values obtained from the open literature are more sensitivethan that ofthe coho salmon. Additional data were submitted by the registrant and represented a compendium of unpublished studies42; however, information on the toxicity of antimycin was not of sufficient qualityto use quantitatively in this ecological risk assessment.

41ECOTOX (ECOTOXicologydatabase). 2005. Maintained by the U.S. EPA ORDand the National Health and Environmental Effects Research Laboratory's(NHEERL's)Mid-ContinentEcology Division. httn \+ lr H cn't xo\ CLOIOX 42 Ayerst Laboratories. 1964. Eficacy ofAntimycin. (Compilation: unpublished study received in 1994under 8991-51 CDL: 005666-A (Accession No. 1 00135924)) Page 28 of 11843 Mayer,F. and M. Ellersieck. 1986. ManualofAcute Toxicity: Interpretation and Data Base for410 Chemicals and 66 Species of Freshwater animals. U. S. Fish and Wildlife Service. Resource Publication 160. 579p. (MRID 400980-01) Page 29 of 118 Study Classification
Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental

- - Supplemental MRIDl Accession No.
400980-01" 400980-01 400980-01
400980-01
400980-01
400980-01
400980-0 1
400980-01
400980-01
400980-01
400980-01
400980-01
400980-01
400980-01
400980-01
400980-01 sntimycinA.

Toxicity Category
very highly toxic highly toxic veryhighly toxic very toxic Very highly toxic 

# "" toxic very 'ghly toxic highly toxic
very toxic highly toxic highly toxic highly toxic highly toxic very highly toxic highly toxic 

## ---- very toxic of technical grade 9dhour LCSO

(pg a.in) (95%C.I.)

0.034 (0.008

## - 0.141) 0.22 (0.128 

### - 0.416) 0.012 (0.0066 - 0.023) 0.010 (0.0056

### - 0.019) 0.057 (0.019

### - 0.166) 0.009 (0.006 

### - 0.014) 0.053 (0.045 

### - 0.063) 0.180 (0.099

### - 0.348) 0.025 (0.008 - 0.074) 4.8 (3.4

### - 6.8) 1.36 (1.02 - 0.82) 0.19 (0.114 

### - 0.324) 0.24 (0.16

### - 0.35) 0.04 (0.031

### - 0.052) 0.34 (0.27

### - 0.42) 0.001 (0.0004 - 0.003) Table 11. Freshwaterfish
Species Bluegill (Lepomismacrochirus) Green Sunfish (Lepomiscyanellus)

Rainbow Trout (Oncorhynchusmykiss) Rainbow Trout (Oneorhynchusmykiss) Cutthroat Trout (Oncorhynchusclarki) Coho Salmon (Oncorhynchuskisutch) Lake Trout (Salvelinus namaycush) Goldfish (Carassiusauratus)

Fathead Mimow (Pimephales promelas) Black Bullhead (Ictalurusmelas) Channel Catfish (Ictalurus punctatus) Mosquitofish (Gambusiaasnis) Largemouth Bass (Micropterus salmoides) Yellow Perch (Perca Jlavescens) White Crappie (Pomoxisannularis) Paddlefish (Polyodon spathula) acutetoxicity

%ai

95'5
95'5
95'5
95'5
95'5
95'5
95'5
95'5
95.5
95.5
95'5
95.5
95'5
95'5
95'5
95'5The toxicity of antimycin to fish and the efficacy of treatments have been related to pH, temperature4', and the gradient (velocity/flow rate) of water being treated. Increased temperatures and more alkalinepH levels tend to increase the toxicity of antimycin while cooler water temperatures and more acidic conditions tend to decrease the toxicity/effectiveness of antimycin. Figure 3 depicts that at 12"C, 0.8 pg/L did not kill any green sunfish after 24 hours; however, at 22OC and the same treatment concentration, all of the sunfish were killed. Similarly at 12°Cno bluegill were killed after 24 hrs followingtreatment at 0.2 pg/L; however, at 22OC all of thebluegill were dead after 24 hours after exposureto 0.2 pg/~~~. Table 12. Freshwaterfish acute toxicity of formulated antimycinA

44 Berger, B.L., R.E. Lennon and J.W. Hogan. Laboratory Studies of Antimycinas a Fish Toxicant. Pages 1 -21 45 Walker, C.R., R.E. Lemon and B.L. Berger. 1964. Preliminary observations on the toxicity of antimycin A to fish and otheraquatic animals. Pages 1 - 18 in Investigations in Fish ControlNo. 2. Bureau of Sport Fisheries and Wildlife Circular 186 Page 30 of 118 Species Bluegill (Lepomis macrochirus) Rainbow Trout (Oncorhynchusmykiss) Rainbow Trout (Oncorhynchusmykiss) Bluegill (Lepomismacrochirus) Bluegill (Lepomismacrochirus) %ai

lo
10

1

1 96-h0ur LCa (pg a.i./L) (95YoC.I.) 1.18 (0.9

### - 1.51) 185 (134 - 255) 0.63 (0.58 - 0.68) 48-hr LcsO=29.5 (26 -33)

48-hr LcsO=29 .5 (20.1 -25.2) Toxicity Category
very highly toxic highlytoxic VerY highly toxic very highly toxic
very toxic MRIDI Accession No.
TN 901 TN 944 TN 35
TN 1533

TN 153 Study Classification
supplemental supplemental supplemental supplemental
supplementalFigure3 Complete survivaloffish treated with antimycinA at 12,17 and 22'C



### 5.2.2 Freshwater Fish, Chronic No chronic toxicity data on antimycin A are available for freshwater fish. A searchofthe open literature did not identify any additional data on the chronic toxicity of antimycin A to fish. 

### 5.2.3 Freshwater Invertebrate,Acute Although an acute toxicity test measuring effects of technical grade antimycin A to waterfleas 

### (D. magna) was submitted 

### (MRID 400980-Ol), the study failed to establish 

### a definitive EC,, value 

### (Table 13) since greater than 50% mortality was observed in all of the treatment groups. However, a 96-hr EC,, value is available for the scud, Gammarus fasciatus (EC,,=0.008 pglL). Based on the sensitivity of Gammarus to antimycin A, the compound is classified as very highly toxic to freshwater invertebrateson an acute exposure basis. A search ofthe open literature did not identify other studies that would provide additional information concerning the acute toxicity of freshwater invertebratesexposed to antimycin A.

Page 31of 118

### 5.2.4 Freshwater Invertebrate, Chronic No chronic toxicity data on antimycin A are available for freshwater invertebrates. A search of the open literature did not identify other studies that would provide additional information concerning the chronic toxicity of antimycin A to freshwater invertebrates.

Table 13. Freshwater invertebrate acutetoxicity oftechnical grade antimycinA



### 5.2.5 EstuarineMarine Fish, Acute A single estuarinelmarine fish acute toxicity study of technical grade antimycin A with the spot (Leiostomusxanthurus)resulted in a 48-hr LC,, of 0.23 pg1L; therefore, antimycinA is classified as very highly toxic to estuarine/marine fish on an acute exposure basis. 

### 5.2.6 EstuarineIMarine Fish, Chronic No chronic estuarinelmarine fish toxicity data were submitted and no useable data were located in the open literature for antimycin A; therefore, chronic risks associated with estuarine/marinefish exposureto antimycin A areunknown. 

### 5.2.7 EstuarineMarine Invertebrates,Acute 96-hourECw a.W (95XC.I.) 48 hr EC50 <lo (5 - 10)

0.008 (0.0058

### - 0.011) >1.0 Species
Waterflea (Daphnia magna) Scud (Garnmarus fasciatus) Aquatic Isopod (Asellusbrevicaudus)

Antimycin A is very highly toxic to estuarinelmarineinvertebrates on 

### an acute exposure basis (pink shrimp 96-hr LC,,=24 pg/L) (Table 14). Acute toxicity data were also available on the Eastern oyster, Crassostrea virginica, showing that antimycin is very highly toxic to mollusks as well. Study Classification
Supplemental Supplemental Supplemental Toxicity Category
very toxic very highly toxic very toxic %ai

95.5 95'5 95.5

Page 32 of 118 MRIDI Accession No.
400980-01 400980-01 400980-01

### 5.2.8 EstuarineIMarine Invertebrates, Chronic No chronic estuarinelmarine invertebrate toxicity data were submitted and no useable data were located in the open literature for antimycin A; therefore, chronic toxicity associated with estuarine/marineinvertebrate exposureto antimycin A isunknown. Table 14. Estuarinelmarineinvertebrate acutetoxicity oftechnical grade antimycinA



### 5.3 

### Toxicity to TerrestrialAnimals 5.3.1 Birds, Acute and Subacute Study Classification
Supplemental Supplemental Supplemental

No toxicity data are available for technical grade antimycin. However, based on acute oral (14-day) toxicity studies using formulated product of unspecified strength (MRID 135924), antimycin is classified as very highly toxic to water fowl (mallard duck LD,,=2.9 mgkgbw) and highly toxic to upland game birds (bobwhitequail LD,,=39 mg/kg bw) (Table 15). No data are availableto evaluate the subacute dietary toxicityof antimycin A to birds. Species
Pink Shrimp (Panaeusduorarum) Blue Crab (Callinectessapidus) Eastern Oyster (Crassostrea virginica) Toxicity Category
veryYghly toxic highly toxic very toxic



### 5.3.2 Birds, Chronic No chronic toxicity data are available for birds. MRIDI Accession No.
402284-0 1 402284-01 402284-01 %

95.5 95.5 95.5

Table 15. Avian acute oraltoxicity.



### 5.3.3 Mammals, Acute 96-hour ECSo (~b a.W
48-hr
48-hr LC,,> 100 62

Species
Mallardduck (Anas platyrhynchos) Bobwhite quail (Colinusvirginianus)

No toxicity data are available on technical grade antimcyin A; however, the acute oral LD,, for ~introl@ Concentrate (20% solution) is 286 mg/kg for male and 361 mg/kg for female
Page 33 of 118 " Not stated. %a.i.
NS'

NS" Toxicity Value LDSo: 2.9 rngikg LD5(): 39 mgkg Toxicity Category Very highly toxic Highly toxic MRID No. Author, Year 135924
135924 Study Classification Supplemental Supplementalrats46(MRID 455279-01). Based on these data, the formulated endproduct is classified as moderatelytoxic to mammals on an acute oral exposure basis. 

### 5.3.4 Mammals, Chronic No chronic toxicity data are available formammals. 

### 5.4 

### Aquatic Effects Characterization of Potassium Permanganate Toxicity Potassium permanganate (CAS No. 7722-64-7) is a strong oxidizing agent47 and is commonly used as a treatment for deactivating antimycin in water bodies treated to control or eradicate undesirable fish, quantify fish populations, or restore listed species48.Given that potassium permanganate is typically applied at 1 ppm to deactivate antimycin, permanganate may be present at exposure concentrations that present a secondary risk to non-target aquatic organisms. Presumably though, the permanganate reacts with antimycin and most organic matter in the aquatic environment and is therefore short-lived. A search for toxicity data on potassium permanganate in U.S. EPA's ECOTOX database49(ECOTOX, 2005) yielded a total of 57 toxicity values (96-hour LC,,s) for freshwater fish. More than 30 ofthe LC,, values in ECOTOX are reported5' and include toxicity values for bluegill sunfish (Lepomis macrochirus) and rainbow trout, the recommended test species for assessing acute toxicity to warm water and cold water fish species, respectively5' (OPPTS Guideline 850.1075). Twelve 96-hour LC,, values for estuarinelmarine fish are described in ECOTOX; however, none of the studies included guideline-recommended surrogate estuarinelmarine species. Five toxicity values for potassium permanganate toxicityto freshwater invertebrates,i.e.,daphnids, are availablein ECOTOX. 

### 5.4.1 Acute Toxicity ofPotassium Permanganate to Aquatic Vertebrates Table 16 summarizes examples of 96-hour LC,, values presented by Marking and ~ills'~ and Birdsong and 

### b vault^^; however, the studies from which these results were obtained were not evaluated and verified. The data suggest that potassium permanganate can be categorized as highly to moderately toxic to fish on an acute exposure basis. Channel catfish appear to be more sensitive to potassium permanganate compared to other fish species. Although the LC,, values presented in Table 16 vary across species by almost three orders of magnitude, intra-species

46 Kuhn. J.O. 2001. ~introl@ Concentrate(Antimycin) Final Report Acute Oral Toxicity in Rats. Stillmeadow, Inc. RepoltNo. 6025-00. Submittedto Aquabiotics Corporation, Bainbridge Island, WA. (MRID455279-01). 47 Marking, L.L. and T.D. Bills. 1975.Toxicity ofpotassium permanganate to fish and its effectivenessfor detoxifying antimycin.Trans. Am. Fish. Soc. 104(3): 579-583. 48 Finlayson, B.J., R.A. Schnick. R.L. Cailteux,L. DeMong,W.D. Horton, W. McClay, C.W. Thompson, and G.J. Tichacek 2000. Rotenone Use in Fisheries Management:Administrativeand Technical Guidelines Manual. American Fisheries Society, Bethesda, Maryland. 49 ECOTOX (ECOTOXicologydatabase).2005. Maintainedby the U.S. EPA ORDand the National Health and Environmental Effects Research Laboratory's (NHEERL's) Mid-Continent Ecology Division. Iltra \\ \v w CIII <I)\CLI!to\ 50 IbidMarkingand Bills 1975. <I J 1 OPPTS 850.1075. 1996. Ecological effectstest guidelines: fish acutetoxicity test, freshwater and marine. U. S. Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances: EPA712-C-96-118. htu~: ,www.cna.zov olfisl~a ~~uhl~catlons. OPPTS Hn~.~nol~l~etl 850 kcolo~~cdi Ellcct\ I cbr Ciuldcl~tlc\, UwIb 850-1075 ~tll 52 Ibid Marking and Bills 1975. C? JJBirdsong, C.L. and J.W. Avault Jr. 1971.Toxicity ofcertain chemicals toJuvenilepompano. Pmg. Fish-Cult. 33(2): 76-80. Page34of 118variability is relatively low for all species except striped bass (Morone saxatilis). This variation may partly reflect temperature and salinity effects on toxicity. Reardon and ~arre11'~ demonstrated that the toxicity of potassium permanganate to striped bass is inversely related to salinity of the test waters. However, studies with Florida Pompano (Trachinotus carolinus) suggest that toxicity increases with increasing salinity5'. As shown in 

### Figure 4, studies with rainbow trout and channel catfish both suggest that acute toxicity of potassium permanganate is inversely related to water temperature.56 Additionally, in a recent study by Hobbs et a~.~~, the 96-hr LC,, for fathead minnows (Pimephalespromelas) in synthetic and pond water was 2,130 and 11,280 pg/L, respectively. The difference in toxicity estimates between the synthetic and pond water is attributedby the study authorsto increased organic carbon in the pond water. The toxicity estimates for potassium permanganate in synthetic water from the Hobb's study are consistentwith the 96-hrLC,, values reported in Marking and ~ills'~ for freshwater fish.

"referred surrogate warm water fish species. Preferred surrogatecold water fish species. Bluegill Sunfish (Lepomis macrochirus) Common Carp (Cyprinuscarpio) Channel Catfish (Ictalurus punctatus) Rainbow Troutc (Oncorhynchus mykiss) StripedBass (Moronesaxatilis) Florida Pompano (Trachinotus carolinus)

54~eardon, I.S.and R.M.Hanell. 1994.Effects ofvarying salinities onthe toxicity ofpotassium pennanganateto larval andjuvenilestriped bass,Movone saratilis (Walbaum). Aquacult. Fish. Manag.25(6): 571-578. 55 Bid Bindsong andAvault 1971. 56fbid Marking and Bills 1975. 57~obbs. M. S., R S. Grippo, l.L Fams. 8.R. Griffinand L. L. Handing. 2006. CompamUveToxicity ofPotassium Permanganateto Nontarget Aquatic Organisms. Environmental Toxicologyand Chemistry25(1): 3046 - 3052. 58~bid Marking and Bills 1975. Page 35 of 118 'All data obtained fromstudies summarizedin ECOTOX (2005). 2300 - 3600 3050 - 3450 750 1220- 1800 960 -4920 1600 - 2900 Warm Water Warm Water Warm Water ColdWater

--

-- Freshwater Freshwater Freshwater Freshwater Freshwater; brackish;marine Brackish; marine Marking and Bills, 1975 Marking andBills, 1975 Marking and Bills, 1975 Marking andBills, 1975 Reardon and Harrell, 1994 Birdsong and Avault, 1971

## &

- - I
1 0.5 1 I .~ainbowtrout 10 Channel catfish,

- -- - -- -- - I I I
I 0.0

- 0 5 10 15 20 25 1 I I

- - - - - - - - - - - - - ---- Figure 4. Effect of temperature CC) on potassium permanganate toxicity to rainbow trout at pH 7.5 and 40- 48 mg/L. CaCO,. Source:Marking and Bills (1975).


### 5.4.2 Acute Toxicity Potassium Permanganate to Aquatic Invertebrates Only two 48-hour ECsovalues for potassium permanganate in D. magna were available in ECOTOX. These values are 84 and 3500 pg/L. 

### The studies reporting these toxicity values were not available for evaluation and verification of the results. The lower value suggests that potassium permanganate is very highly toxic to aquatic organisms on 

### an acute exposure basis. Sreekala et a1j9and hderson60(1944) reported 2-hour ECsovalues for potassium permanganate of 540 and 1200 pg/L, respectively, in Daphnia spp. ~nderson~' also showed a dose-related increase in toxicity of potassium permanganate to 

### D. magna with a NOAEL of 630 pg/L. In a recent study, Hobbs et aLb2 reports 96-hr LC,, values for 

### D. magna and Ceriodaphnia dubia of 53 and 58 pg/L in synthetic water; however, in pond water under similar testing conditions the 96-hr LC,, is 1980 and 2390 pg/L, respectively. Hobbs et ~1.~~ also report that Chironomous tentans and Hyallela azteca tested with artificial substrates have 96-hr LC,, values of 4,430 and 4,740 pg/L, respectively,in syntheticwater versus 13,550and 12,300pg/L, respectively, in pond

59 Sreekala,K.G., J. Jennita,and V.R Prakasam. 1991.Toleranceand healtrate of Daphnia sp. (crustacean) in responseto disinfectants, bleachingpowder and potassium pennanganate. Pollut. Res. lO(1): 33-36. 60 Anderson, B.G. 1944.Thetoxicity thresholds of various substances found in industrial wastes asdeterminedby the use ofDophnia rnngna. SewageWorks J. 16(6): 1156-1165. IbidAnderson 1944. 62 Ibid Hobbs et al. 2006. 63~bid Hobbs et a/.2006. Page 36 of 118

### water. The decreased toxicity of potassium permanganate to aquatic animals in pond water compared to the toxicity in synthetic water is attributed to the higher organic carbon content of the pond water.

Page 37 of 118

## RISK CHARACTERIZATION
6.1 

### Risk Estimation A means of integrating the results of exposure and ecotoxicity data is called the quotient method. For this method, risk quotients (RQs) are calculated by dividing exposure estimatesby ecotoxicity values, both acute and chronic.

RQs arethen compared to OPP's levels of concern (LOCs). TheseLOCs are criteriaused by OPP to indicate potential risk to non-target organisms and the need to consider regulatory action. The criteria indicate that a pesticide used as directed has the potential to cause adverse effects on non-target organisms. LOCs currently address the following risk presumption categories: (1) acute high 

### - potential for acute risk to Federally non-listed species is high, regulatory action may be warranted in addition to restricted use classification(2) acute restricted use 

### - the potential for acute risk is high, but this may be mitigated through restricted use classification (3) acute endangered species - the potential for acute risk to endangered (Federally listed) speciesis high, regulatory action may be warranted, and (4) chronic risk
- the potential for chronic risk is high, regulatory action may be warranted. Currently, EFED does not perform assessments for chronic risk to plants, acute or chronicrisks to non-target insects, or chronicrisk from granularhait formulations to mammalian or avian species. The ecotoxicity test values (i.e., measurement endpoints) used in the acute and chronic risk quotients are derived from the results of required studies. Examples of ecotoxicity values derived fiom the results of short-term laboratory studies that assess acute effects are: (1) median lethal concentrations (LCZo)(fish and birds) (2) median lethal doses (LD50) (birds and mammals) (3) median effects concentrations(ECS0) (aquatic plants and aquatic invertebrates)and (4) first quartile effects concentration (EC25)(terrestrial plants). Examples of toxicity test effect levels derived from the results of long-term laboratory studies that assess chronic effects are: (1) lowest observed adverse effect concentration (LOAEC) (birds, fish, and aquatic invertebrates) and (2) no observed adverse effect concentration (NOAEC) (birds, fish and aquatic invertebrates). For birds, mammals, and all aquatic organisms, the NOAEC is the ecotoxicity test value used in assessing chronic risk. Other values may be used when justified. Risk presumptions, along with the corresponding RQs and LOCs, are summarized in Tables 17 through 19.

Table 17. Risk presumptionsfor terrestrial animals (birds and wild mammals) I I

Acute Restricted Use 

# I EEC/LCwor ~~~dft~ or LDdday (orLD5o < 50 mgikg) 

# I 0.2 I I 

# I Risk Presumption Acute High (Non-listed) Risk
Acute Endangered (Listed) Species 

## I EEC/LC50 or LDSdft2 or LDSdday 

# I 0.1 I 

# I RQ EEC'/LC~~ or ~D~dft~ or LD5dda$

Page 38 of 118 LOC 0.5


# I Chronic Risk 

## I EECMOAEC 1

abbreviation for Estimated Environmental Concentration (ppm)on avian/mammalian food items 

# I Zmp/ft2 LDSO * wt. ofbird LDso * wt. ofbirdTable 18. Risk presumptions for aquatic animals I I 

# I Risk Presumption 

# I RQ 

# I LOC

Acute High (Non-listed) Risk 

# 1 EEC~ILC~~ or ECso 

# I 0.5

Acute Endangered (Listed) Species 

## I EEC/LCSo or ECso 

## I 0.05 Acute Restricted Use


### 1 Chronic Risk 

### I EECMOAEC 

### I 1 1 -

EEC = @pm orppb) in water I I EECILCSo or ECso 0.1

Terrestrialand Semi-AquaticPlants I I 

# I i Table 19. Riskpresumptions for plants

Acute High (Non-listed)Risk 

# I EEC'/EC~~ 

# I 1 

# I Acute Endangered (Listed) Species 

## I EEC/ECoS or NOAEC 

## I 1 

# I LOC Risk Presumption



# I Aquatic Plants 

# I RQ

Acute Endangered (Listed) Species 

# I EEC/ECoS orNOAEC 

## I 1 

# I EEC = Ibs ai/A Acute (Non-listed) Risk

EEC = (ppblppm) in water 

### For the assessment of antimycin risks, the deterministic risk quotient (RQ) 

### method is used to compare exposure and toxicity values. A summary of toxicity values used to calculate RQs is provided in Table 20; 

### a more detailed discussion of antimycin toxicity can be found in Section 5. EEC~/EC~~ 1

Page 39 of 118 Table 20. Summary of toxicity data for most sensitivetest species used to calculate risk quotients to evaluate ecological risks of antimycin A use Taxonomic Group

Birdsa

Mammals

Terrestrial insects

7 ExposureCategory Acute (LDjO/LCs0) Chronic (NOAEC) Acute LDS0 Chronic NOAEL (NOAEC)
Acute LDso Most Sensitive Species Mallard duck (Anasplatyrhynchos) Not available Laboratoryrat (Rattusnorvegicus) Not available Honey bees (Apis mellifera) Toxicity Value LDS0 =2.9 mgkg Not available LDso =286 mgkg bw
Not available
Not availableTable 20. Summary of toxicity datafor most sensitive test species used to calculate risk quotients to evaluate ecological

### risks ofantimycinA use Taxonomic Group

Freshwater fishb Exposure Category

ChronicNOAEC Acute LC50

Freshwater invertebrates Most SensitiveSpecies

Rainbow trout

Estuarinelmarine fish Toxicity Value

I (Oncorhynchusmykiss) Coho Salmon Not available
Acute ECSo ChronicNOAEC

Estuarinelmarine
Terrestrial plants 

# 1 Acute 

# I Not available 

# 1 Not available 96-h LCSo = 0.009 pg/L

Acute LCs0 ChronicNOAEC

invertebrates Water flea (Daphnia magna) Water flea

Acute EC50



### 6.1.1 Freshwater Fish and Invertebrates 48-h ECm= 0.008 pg/L. Not available Spot (Leiostomusxanthurus) Not available

I I I Chronic NOAEC

Aquatic plants and algae

At a treatmentrate of 25 pg/L, the acute estimated environmental concentration (EEC) of antimycin is expected to be equivalent to the application rate. At this exposure concentration, RQ values (RQ=EECILC,,) for freshwater fish and invertebrates are 2,778 (2510.009) and 3,125 (25/0.008), respectively. Acute high risk levels of concern (RQLO.5) for fish and invertebrates are exceeded by factors of 5,556 and 6,250X for fish and invertebrates, respectively (Table 21). Even if RQ values were based on the least sensitive fish, i.e., black bullhead 96-hr LC,,=4.8 pglL, the RQ (5.6) would exceed the acute high risk LOC by a factor of 11X. It should be noted, however, that RQ values in this screening-level assessment compare peak concentrations to toxicity values based on a 96-hr exposure; it is likely that that shorter-duration toxicity studies would have lower LC,, values and in 

### turn, RQ values would be lower. Additionally, at the lower treatment concentration (5 pgl~)~ recommended to remove scaled fish from aquaculture ponds, the RQ is roughly equal to unity (5 pg/L +- 4.8 pg/L=1.04) and exceed the acutehigh risk LOC. 96-h LCso=0.23 pg/L Not available Pink Shrimp (Panaeusduorarum)

Avety, J.L. 2006. Use of ~intm? to remove scaled fishin catfishponds. ne Ca@h Journal.April 2006 Page40 of 118 48-hrLC,,=24 pg/L
Not available

aBirdsareused as surrogates forterrestrial phase amphibians andreptiles (US EPA, 2004). '~reshwater fish are used as surrogates for aquatic phase amphibians (US EPA, 2004). Acute ECS0 Not available

Not available Not available

## 1 Table 21. Acute risk quotients for freshwater fish and invertebrates based on a targeted (

No acceptable toxicity data are available with which to evaluate the chronic risk to aquatic animals associated with the use of antimycin; however, given that the treatment concentrations exceed acute median lethal concentrations by several orders of magnitude, it is reasonableto believe that most aquatic animalswill be killed by currenttreatment concentrations of antimycin A. In situations where antimycin is applied to flowing water, it is typically deactivated with potassium permanganate; however, deactivation is not required by the label. The combinationof flow through, dilution with untreated water and deactivationwith potassium perrnanganate makes chronic exposure to antimycin unlikely. Additionally, the acute toxicity of antimycin makes it likely that few biological receptors would be present to exhibit effects. However, to the extent that treated water is not deactivated using permanganate nor diluted by untreated water, thepotential for chronic risks is uncertain. 

## .antimycinA treatmentAte of25 pfl.



### 6.1.2 

### EstuarineIMarine Fish and Invertebrates At the maximum application rate, the acute RQ values for estuarine/marine fish and invertebrates are 109 and 1.0, respectively (Table 

### 22). Both fish and invertebrate RQ values exceed the acute high risk LOC by factors of 

### 218X and 

### 2X, respectively. Although the toxicity value for estuarine/marinemollusks (62 pg/L) is less sensitive than that of pink shrimp, an RQ value for mollusks (RQ=0.40) exceeds the acuterestricted use LOC (RQ1O.l). Acute RQ Value
2,778" 3 125" Species
Coho Salmon (Oncorhynchus kisutch) Scud (Gammams fasciatus)



## I Table 22. Acute risk quotients j Exceedsacuterisk (RQ?0.5), restricted use (RQa.I), and endangered species (RQ20.05) levels ofconcern.

targeted antimycin A treatment ra 

### I I Estimated Exposure Concentration 

## (rcnn)

25 25

Species Toxicity LC, (Irgn) 0.009 0.008

for estuarinelmarine fish and invertebrates based on a te of25 pgL. Estimated
n-- _ -. . . 

# I Toxicity 

# I hxposure 

# I I Acute RQ Value 

### (

6.1.3 Birds The use of antimycin to achieve fishery management objectives, where the compound is applied directly to water, is not likely to represent a means of exposure to birds relative to Page 41 of 118consumption of antimycin residues on terrestrial forage items since the compound is applied directly to water. On the chance that birds forage on dead or dying fish, it is possible to estimate the potential amount of antimycin in their diet. For example, the weight of food ingested (FI) by a great blue heron Ardea herodias (body weight=2.576 kg) is given by taking the antilog of the value estimate using formula (EQ1) recommended by the Wildlife Exposure Factors 

### andb book^'. Loglo (FI) =0.966 loglo (body weight in grams) -0.640 (EQ 1) Based on this relationship, a blue heron will consume 452 g of fish per day. Piscivorous birds are likely to consume small to medium-sized fish and thereforerainbow trout are used as a surrogate. Fish tissue residue data for fish killed with antimycin indicate that rainbow trout contained as much as 172 pg antimycin/kg66.Therefore, a heron eating 0.45 kg would consume 77 pg (0.45kglday x 172pgfkg)of antimycin. Extrapolations from one bird to another need to consider differences in the scaling of toxicity for differences in body weight. The LDSo can be adjusted for body weight based on the formula 

### (EQ 2) recommended by Mineau et at7: Adjusted LDso =LDSo (AW/TW)'~" ) (EQ 2) where adjusted LDsois the median 50% lethal dose for the species being assessed, LDSO is the median lethal dose in the test organism, AW is the body weight ofthe assessed organism, TW is the body weight for the test organism, and a is the slope of the regression line for estimating the assessed species LDsOfrom the test species LDso (EFED default value of 1.15). 

### In the case of assessing a blue heron, 2576 g is a suitable value (adult bird) for AW. The test organism is a mallard duck which weighs about 1230 g. With a test LDsoof 2.9 mg kg-', the adjusted LDsois 3.2 mg kg-'. The actual dose is then the LDsO times the weight of the heron (2.576 kg) which equals 8.246 mg. Therefore,the amount of antimycin potentially consumed by herons is several orders of magnitude less than the acute median lethal dose; the dose-based RQ value is less than 0.01 and is below acute risk levels of concern for non-listed and listed species (Table 23). Accordingto the Wildlife Exposure Factors

## andb book^^ the mean food ingestionrate for a bald eagle (Haliaeetus leucocephalus) is 0.12 kgkg day. A bald eagle weighmg 5.089-kg bird would consume 610 g of fish per day representing a potential antimycin exposure of 105 pg (0.610 kglday 

### x 172 pgkg). The adjusted LDsofor bald eagles is 3.6 mg kg'' and the equivalent dose for a 5.089-kg bird is 18.32 mg; therefore, the dose-based RQ value is less than acute risk levels of concern (Table 23).

65 ORD 1993. WildlifeExposure FactorsHandbook. of Health and EnvironmentalAssessment, OfficeofResearch and Development. EPA/600/R-931187. htto:,wwu.cna.trc~v ncca b~t.cls.lxil 66 Ritter, P.O. and F.M. Strung. 1966. Residues in tissues offishkilled by antimycin. Journal ofAgricultural Food Chemistry 14(4): 403 -407 MRID 461534-01) 67 Mineau, P., B.T. Collins,and A. Baril. 1996. Onthe use of scaling factors to improveinterspeciesextrapolationof acute toxicity in bids. Regulatory ToxicologyandPharmacology.24: 24-29. 68 [bid ORD 1993. Page 42 of 118Similarly, the mean for food ingestion rate for a belted kingfisher (Ceryle alcyon) is 0.1 1 g/g-day69.Based on this rate, an adult kingfisherweighing 148 g would consume 16g of fish per day representing a potential antimycin exposure of 2.8 pg (0.016 kg/day x 172 pglkg). The adjusted LD,, for kingfishers is 2.1 mg kge' and once again, the estimated exposureis well below the effects threshold.

Although ecological risk assessments do not typically take drinking water exposure into account, birds could potentially drink water from treated areas. The Wildlife Exposure Factors 

## andb book^^ estimates that bald eagles ingest 0.037 g waterlg bw day. Therefore, a 5-kg bird would ingest 188g waterlday. At a treatment rate to water of25 pglL (25 pgkg), an eagle could consume 4.7 pg/day. The potential contribution from drinking water (4.7 pg) and diet (105 pg) is still two orders ofmagnitudebelow the adjusted LDSo. Table 23. Acute dose-based risk quotients for piscivorous birds based on potential residues in dead or dyingfish.



### 6.1.4 

### Mammals Similarly, the use of antimycin as a piscicide is not likely to represent a means of exposure to wild mammals relative to consumption of antimycin residues on terrestrial forage items (plants, seeds, insects) since the compound is applied directly to water. Based on the chance that different-sized mammals forage on dead or dying fish, it is possible to estimate the potential amount of antimycin in their diet. Using daily food intake as estimated by ~a~~~~ (EQ3), a 350-g mammal will consumeabout 68 g of food based on the allometricequation: Species Blue Heron (ArdeaHerodias) Bald Eagle (Haliaeetusleucocephalus) Belted Kingfisher (Ceryle alcyon)

where F is the food intake in grams of fresh weight, BW is the body mass of the organism in grams, and W is the mass fraction of water in the food. For this assessment W is assumed to be 0.75~~. Piscivorous mammals are not confined to eating small fish and may feed opportunistically on any sized fish that may succumb to antimycin; therefore, common carp (Cyprinus carpio) are used as surrogates. Based on the data on antimycin tissue residues in Estimated Daily Dose (Icg) 77 105 2.8 Adjusted LD, bp) 8246 18,320

3 11

69 Ibid ORD 1993 70 Ibid ORD 1993 71 Acute RQ Value <0.01 <0.01 <0.01

I I Nagy, K.A. 1987.Field metabolic late and food requirement scaling in mammalsand birds. EcologicalMonographs 57: 111-128. 72 Ibid Nagy 1987. Page 43 of 118common carp with a body weight of 99 grams, a small mammal would only consume 68% (68/99) of the total carp body mass. According to the data for common carp, maximum total body residues of antimycin in carp tissue amounted to 261 A 350-8 mammal consuming 68 grams represents an equivalent dose of 18mg of antimycin; this value isbelow the median lethal dose of antimycin (286 mg/kg*0.350 kg=100 mg) for similarly sized mammals. Similar to birds, extrapolations from one size of mammal to another must consider differences in the scaling oftoxicity for differencein body weight. The LDSo can be adjusted for body weight based on the formularecommendedby Mineau et (EQ4): Adjusted LD50=LDSo(TW/AW) (0.25) (EQ4) where adjusted LDS0 is the median 50% lethal dose for the species being assessed, LDsois the median lethal dose in the test organism, AW is the body weight of the assessed organism and TW is the body weight for the test organism. Sincewe are assessing a largemammal, 1000 g is a suitable value for AW. The test organism is a rat weighing about 350 g. With a test LDSo of 286 meg,the adjusted LD,, is 220 mag. Using the daily intake equation, a 1000-gmammal will consume about 122 g of food. If the animal fed exclusively on carp killed by antimycin, the equivalent dose would be 0.122 kg *261 pg/kg or 32 pg of antimycin. Once again, this value is well below the estimated median lethal equivalent dose adjusted for body weight (220 mgkg

## * 1 kg220mg) and the RQ value is less than 0.01 (Table 24). Therefore, even if fish are available for consumption by mammals scavenging along the shoreline for dead or dying fish, it is not likely that the mammals would consume sufficient quantities of antimycin to result in acute toxicity.



### 6.2 

### Risk 

### Discussion Table 24. Acute dose-based risk quotients for piscivorous mammals based on potential residues in dead or dyingfish.

Antimycin A (~introl")~ applied directly to water and as a piscicide; it is intended to kill fish. Niclosarnide (CAS No. 50-65-7), 3-trifluoromethyl-4-nitrophenol(TFM; CAS No. 99-30- 2) and rotenone (CAS No. 83-79-4) are other piscicides that have recently been evaluated by OPP for ecological effects; however, of these piscicides, antimycin is one of the most toxic to fish (Table 25). Antimycin's high toxicity to fish accounts for the low treatment concentrations (typically 25 pg/L or less) needed and may account for why antimycin does not evoke an avoidance response in most fish. Based on the available informationit is reasonable to believe that one component of the initial risk hypothesis discussed in the problem formulation cannot be rejected, i.e.,the use of antimycin A in accordance with the label will likely result in adverse Species Large Mammal (1000 g)

73 Ibid Ritterand Strong. 1966. 74 lbid Mineau etal. 1996. Page 44 of 118 Acute RQ Value <O.O 1 EstimatedDaily Dose (~g) 8.9 Adjusted LD50 @& 220,000effects, such as acute mortality, to aquatic animals when the chemical is used at typical rates of 25 pg/L as identified in the use closure memo (dated October 26, 2005). The extent to which nontarget mortality will occur is uncertain; field studies suggest that while aquatic invertebrate populations are immediately affected in terms of decreased numbers, invertebrate communities recover to pretreatment levels in terms ofboth abundance and diversity within several months7'. While the initial risk hypothesis included potential risk to terrestrial animals feeding on dead or dying fish or through the ingestion of antimycin-treated water, the likelihood of adverse effects on these taxa is consideredlow based on available data.

Although there are data indicating that antimycin A is subject to abiotic degradation and, under alkaline conditions, may degrade rapidly by hydrolysis, the susceptibility of antimycin to biotic degradation is unknown. Preliminary in vitro tests with rat hepatocytes76 indicate that antimycin in vitro Phase 1 metabolism leads to hydrolysis of the acyloxy group along with deforrnylation of the N-formylaniline (Figure 5; see Appendix D for further discussion). Deformylation of antimycin has been hypothesized as resulting in its deactivation as a fish toxicant." There are data to support that the toxicity of antimycin is related to both pH and temperature and label application rates are adjusted accordingly; at higher pH levels, the degradation rate increases and thus larger amounts of antimycin are required for treatment. At lower water temperatures, i.e., below 15.5"C (60°F), the label recommends higher treatment concentrationsand likely reflects the decreased toxicity of antimycin at lower water temperatures dueto decreased fishmetabolicrates. Table 25. Acute toxicity values for freshwater fish 

### (LC3and invertebrates 

### (LC3for selected piscicides based on most sensitive endpoints used in 

### OPP Ecological Risk Assessment chapters in supportofthe Reregistration EligibilityDecisions.

75 Moore, S.E.,M.A. Kulp,J. Hamrnonds,and B. Rosenlund. 2005. RestorationofSamsCreek and an AssessmentofBrook Trout Restomtion Methods,Great Smoky Mountain National Park. National Park Setvice,U. S.Departmentofthe Interior. Technical Report NPS/NRWRD/NRTR-2005. Chemical AntimycinA Rotenone" ~iclosamide~ TFI@

76 personal communicationJohn Kenneke, Chemist,U.S. EPA ORD 2006; these data areconsidered preliminary and have not undergone extensivepeer review or quality assurancethat would permit their use in a more quantitativefashion. 77 Ritter, P.O. andF.M.Strong. 1966. Residues in tissuesoffishkilled by antimycin. Journal ofAgticultu~al Food Chemistry 14(4):403 -407. Page 45 of 118 ' Rotenone RED ChapterDP Barcode D307382 ' NiclosarnideRED ChapterDP Barcode D255595 ' TFM 3-trifluoromethyl-4-nitrophenol RED ChapterDP Barcode D2 19182 Freshwater Fish 96-hr LC,

6fi) 0.009

1.94 30 600 Freshwater Invertebrate 48-hr EC, bdL) 0.008

3.7 34 3800

### 6.3 

### Risks toAquatic Animals Antimycin A is very highly toxic to fish on an acute exposure basis. With fish LC50 values as low as 

### 1nanogram (n) per liter (part per trillion (pptr)) and treatment rates 25 hundred times higher than this LC,, value, the direct application of antimycin to flowing and static water to eliminate undesirable fish species is likely to kill many of the fish species present. Figure 6 depicts the distribution of 96-hr LC,, values in parts per trillion across fish genera and shows that roughly 80% of the genera have LC,, values of less than or equal to 20 ng/L; 95% of the genera are at or below 79 nglL. Therefore, at treatment rates of 25 pg/L, the concentrationof antimycin exceeds the 96-hr LC,, for 95% ofthe speciesreported. Since fish serve as surrogatesfor aquatic phase amphibians,risk is presumed for aquatic-phaseamphibians as well.

R = n-butyl; n-hexyl Figure 5. Metabolites tentatively identified from the phase 1 in vitro metabolism of antimycin A at pH 7.4, 37OC, with rat hepatic microsomes.Rrh

# - SpeciesMtivity DCsbiMon~~cal)

Figure 6. Distribution of acute 96-hr median lethal concentrations &C,J for fish genera exposed to antimycinA.
Although typical treatment rates are likely to kill most fish species, the chemical is used to selectively remove scaled fish from catfish (fingerling and food-fish) aquacultureproduction ponds by taking advantage ofthe differential toxicity of antimycin to scaled and non-scaled fish. 

### In studies of channel catfish conducted by the 

### U.S. Fish and Wildlife (see Appendix 

### F for further study details), exposure of catfish fry to 0.75 pglL resulted in 100% mortality; however, fingerling catfish in the same study were roughly an order of magnitude less sensitive with a 96-hr LC,?value of 8.4 pg/L. Additionally, studyresults are consistent with data reported in Section 5 ind~cating that catfish are several orders of magnitude less sensitive to antimycin than scaled species (Table26). Antimycin's use in aquacultureto remove scaled fish from catfishproduction ponds does not typically involve subsequent deactivation with potassium permanganate; rather, the antimycin is allowed to degrade naturallyin the production pond water. Fingerlingcatfish would not be harvested as food fish for roughly 1 - 1.5 years post-treatment. 

### Theuse of antimycin to remove scaled fish from food-fish production ponds is conducted relatively closeto the time ofharvest; however, again the antimycin is not typically deactivated. Water from harvested aquaculture ponds is frequently reused for fish production. To the extent

78 Hogan, J. 1966. Antirnycin as a Fish Toxicant in Catfish Culhlre. Presented at the 20" Annual Meeting of the Southeastern Association of Game and Fish Commissioners, Oct 24 - 26, 1966. Bureau of Sport Fisheries and Wildlife, U.S. Fish and Wildlife Service. (AccessionNo. 00045801;MRID 2400798-13) Page 47 of 118that recently treated water is released to adjoining waterbodies, nontarget mortality of both fish and invertebratescould occur.

Although no data were submitted with which to evaluate the chronic toxicity of antimycin to fish, quantitative structure-activity relationship models can be used to estimate the potential toxicity of antimycin. The Ecological StructureActivity Relationships (ECOSAR)~~ is a component of the Estimation Program Interface (EPI) suite""80 developed by EPA's Office of Pollution Prevention and Toxics and Syracuse Research Corporation. ECOSAR is a model which predicts acute and chronic aquatic animal/plant toxicity. Based on this model (see Appendix B for model output), the 90-day chronic toxicity value (ChV) for freshwater fish is 67 pg/L; however, this value is higher than all of the 96-hr median lethal dose estimates reported in this assessment and therefore is not considered a realistic estimateof a chronic toxicity endpoint. Table26. Fingerling fish bioassay ofantimycin96-hr LC, values (nominal concentrations).

The available laboratory data indicate that fieshwater invertebrates have roughly similar sensitivitiesas fish to antimycin on an acute exposure basis and many of these species may die as well at treatment rates of 25 pg/L. Although there are anecdotal reportss1 suggestingthat plants and invertebrates are relativelyunaffected by antimycin treatments, no laboratory data have been reviewed by EPA that can substantiate these claims. To the extent that application rates are less than 25 pg/L and less sensitive animals are in the treatment areas, aquatic invertebrate mortality could be substantially reduced. Although invertebrates are less conspicuous members of the aquatic community, they are a major component of aquatic ecosystems and food webs. Any significant effects on invertebrates would most likely influence other components of the ecosystem. Effects may not be limited to merely a change in total biomass as a result of widespread mortality but any changes associated with differential sensitivity could bring about significant changes in the communitystructure, which could alter system function. Species Goldfish Common Carp FatheadMinnow

7 Green Sunfish Bluegill Sunfish LargemouthBass Channel Catfish

There are no data on freshwater mollusks; however, data on estuarine/marinemollusks indicate that they are less sensitive to antimycin than other invertebrates although risk quotients 96-hrLC, ~gn 0.137 0.200 0.074 0.060 0.051 0.104

8.4

79 ECOSAR. Ecological Structure Activity Relationshipsilllo M r\ w u~.i a,,\ onn~1n11 ~lc\r~llcln- toc)1\ 2 ic~c,\,o rltn, Qn "" EPI Suite ht~nwww.c-.vu.ro\ oonllnll.,cxnosurc docs cn~su~rc.llu~i 81 Wisconsin Alumni ResearchFoundation. 1965. Antimycin as a Fish Toxicant: aresume of informationand data pertaining to the use ofantimycin in fish management proceduresin ponds and lakes. (AccessionNo. 00135924) Page 48 of 118based on mollusk toxicity data exceed acute restricted use and endangered species levels of concern. Based on PBT profilers2 modeling (Appendix 

### B), which generated the estimated bioconcentration factor for antimycin (350X), the compound does not appear likely to bioconcentrate in fish. In general, chemicals that have the potential to bioconcentrate also have the potential to bioaccumulate. Since a bioconcentration factor (BCF) in fish can be readily measured in the laboratory and bioaccumulation is much more complicated to determine, the BCF is fiequently used to predict the importance of bioaccumulation. Because of antimycin's relatively low BCF, the chemical is not considered likely to bioaccumulate in aquatic food chains. 

### In addition to acute risks, there is a potential for chronic effects to freshwater invertebrates when antimycin is applied directly to water. The limited data on freshwater invertebrates suggest that they are as sensitive to antimycin as fish, and it is possible that many invertebrates will not survive antimycin treatment concentrations in the targeted area at the typical application rate; therefore, chronic effects will be limited by a lack of receptors. Additionally, at the maximum recommended treatment rate, a single application of antimycin directly to water is intended to kill all fish in the treated area; therefore, it is unlikely that repeated applications of antimycin will occur within the same year. Also, antimycin degrades rapidly in the environment, particularly at warmer temperatures in alkaline waters and can be deactivated with potassium permanganate to prevent its movement to non-target areas, thus further reducing the likelihoodof chronic exposure. A field study conducted by the National Park as part of Southern Appalachian brook trout (Salvelinusfontinalis) restoration effort in the Great Smoky Mountain National Park evaluated the impacts of antimycin A (~introl? treatment at 8 pg/L for 

### 8 hours in high gradient streams. Pre- and post-treatment monitoring of freshwater invertebrates indicated an 18 - 25% decline in total taxa in two of the three sites treated with antimycin roughly two weeks posttreatment; mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddis flies (Trichoptera), collectively referred to as EPT, being the most sensitive families. Sampling conducted approximately one year after treatment indicated no statistical difference (ANOVA) between pre- and post-treatment for total taxa and EPT taxa. Furthermore, crayfish were observed feeding on dead fish in the antimycin treatment area and there was no difference in the numbers of crayfish observed pre- and post-treatment. In this study, potassium permanganate (1 ppm) was used to inactivatethe antimycin A and some ofthe effects noted on macroinvertebrates soon aftertreatment were believed to result from the permanganate.

82 PBT ProfilerModel htto WHH ~blniolii~i rict 83 Moore, S.E.,M.A. Kulp, J. Hammonds,and B.Rosenlund. 2005. RestorationofSamsCreek and an AssessmentofBmkTrout RestorationMethods,Great Smoky Mountain National Park. National Park Service, U.S. Departmentofthe Interior. TechnicalReport NPSMRWRDINRTR-2005.Similarly, in a study of Bear Creek in Great Srnokey Mountain National parkg4 benthic macroinvertebrate communitieswere sampled before and after treatment with antimycin A with subsequent deactivation with potassium permanganate to determine whether aquatic invertebrates were impacted and if so, how. Based on Jaccard and Morisita-Horn similarity indicesn5 the report concluded that benthic [invertebrate] communities were at pre-treatment conditions within 4 months post-treatment. Where decreased numbers of invertebrates were noted 2 weeks post-treatment, it was uncertain whether the reductions were due to antimycin or potassium permanganate. It is also uncertain though how representative these study results are for the recovery of aquatic invertebrates in other areastconditions in which antimycin may be used in both lotic and lentic environments. In Great Basin National Park, Nevada, efforts to restore the native Bonneville cutthroat trout (Oncorhynchus clarki utah) also relied on antimycin A'~. Macroinvertebrates were surveyed in Snake Creek following the EPA Rapid Bioassessment of Creeks and Small Rivers protocols87 three years prior to treatment and followingtreatment at one week, one month, nine months and one year. Roughly 7.6 kilometers of Snake Creek were treated over a 6 day period. Segments of the creek were maintained at 

### 8 pg/L for 

### 8 hours and were later deactivated with permanganate ( 4 mg/L). The efficacy oftreatment was monitored by using brook trout (Salvelinus fontinalis) in live cars maintained throughout and downstream of the treatment areas. Overall

numbers of aquatic invertebrates declined by 61% and 54% for EPT taxa one month after treatment; macroinvertebrate diversity declined by 29% one month post-treatment. However, by 9 months post-treatment, invertebratepopulations had returned to pretreatment conditions and in some cases exceeded pre-treatment abundance by over 300% and diversity was within 95% of pre-treatment levels. The deactivation of antimycin with potassium permanganate was considered effective;however, there was a gradient of effect. Brook trout in live cars at 100,200 and 500 m downstream of the deactivation station experienced 90%, 25% and 0% mortality, respectively. The downstream mortality was attributed to the permanganate treatment (personal communicationN. Darby, Biologist,Great Basin National Park, NV, 2006). Although antimycin is likely to partition to sediments containing organic matter, given 

### KO, estimates ranging from 2500 - 10000 mglg, there are no data available to quantify the extent to which antimycin may be available to benthic invertebrates. Additionally, no laboratory data are available to assess the toxicity of antimycin to benthic invertebrates; however, the National Park Service studies discussed above only show transient effects in invertebratecommunities and many of these taxa are associated with the benthic environment.

84 Etnier, D. a.and C. D. Hulsey. 2005. EffectsofAntimycin A treatmenton benthic macroinvertebratesin Bear Creek tributaries to Fomey Creek, Great SmokeyMounthain National Park, Swain Couty, North Carolina. University ofTennesseeFinal report prepared under cooperativeOa,greement CA-5460-A1-005.
OJ - - Krebs, D. J. 1999. Ecological Methodology. Addison-WelseyEducational Publishers, Inc.,Menlo Park, CA, 620p. 86 Baker, G. M.,N. W. Darby and T. B. Williams. 2004. Balancing Bonneville cutthroattrout with non-nativesalmonidsin Great Basin NatioQ~l Park. Wild Trout 111 Symposium: Working Together to Ensure the Future ofWild Trout: PI41 - 150. 0 1 Barbour,M.T.,J. Gemtsen, B. D. Snyder,andJ. B. Stribling. 1999. Rapid BioassessmentProtocols for Use in Streamsand Wadeable Rivers: Periphyton, Benthic Macroinvertebrates,and Fish, Second Edition. EPA 841-B-99-002. U. S.Environmental Protection Agency; OficeofWater, Washington D.C. httn wwu cn,~ r?lo owow monltoiliir rbn Page 50 of 118Studies conducted by cerretogg in conjunction with the Wyoming Game and Fish Department indicated that benthic invertebrate communities were not significantly (p>0.05) affected in terms of invertebrate biomass, richness or diversity by 8-hr treatments of first-order streams with antimycin A at 10 pg/L relative to reference streams. In situ bioassays with mayflies (Cinygmula spp.) and caddisflies showed no effect on survival (Brachycentrus spp.). Although there was high variability in invertebrate drifi estimates the studyhad a low a abilityto detect treatment effects (poor statistical power), the results indicate that invertebrate communities were not eliminated during and immediately aRer a 8-hour treatment with antimycin A at 10pg/L. As mentioned earlier, potassium permanganate is sometimes used to detoxifL antimycin following stream treatments. Permanganate is typically applied at a treatment concentrationof 1 mg/L. Based on the most sensitive fish (channel catfish 96-hr LC5,=750 pg/L; 

### Table 16) and invertebrate (waterflea EC5,=84 pglL) species reported, RQ values for fish and invertebrates would exceed the acute high risk level of concern. 

### In the National Park Service Sams Creek surveyg9, total taxa declined by 34% following treatment with 1 mg/L KMnO, for 

### 9 days; EPT taxa declinedby as much as 11%90.However, similarto what was observed followingantimycin treatment, the number and diversity of aquatic insects in the ermanganate-treated stream had returned to pretreatment levels 4 months afier treatment! 

### In the same study, higher permanganate concentrations, i.e., 

### 9 days at 4 mg/L, were lethal to 40% of the trout; however, fish were not affected at lower concentrations. Also, as mentioned earlier, deactivation with permanganate is not required by the label.


### 6.4 

### Risks to TerrestrialAnimals Only acute oral toxicity data are available for birds and these indicate that antimcyin is highly to very highly toxic to birds on an acute oral exposure basis. Risk quotients are calculated using the dose-based acute toxicity values and result in values below acute risk levels of concern. The dose-based approach considers the uptake and absorption kinetics of a gavage toxicity study to approximate exposure associated with uptake from a dietary matrix. Toxic response is a function of duration and intensity of exposure. For many compounds, a gavage dose represents a very short-term, high intensity exposure. Although the dose-based estimates may not reflect reality in that animals do not receive a gavage while feeding, it is possible that a short-duration, high-intensity exposure could occur associated with feeding along side treated surface waters since many birds may gorge themselves when food items are available; this would be particular true 

### as birds opportunisticallyfeed on dead and dying fish. No chronic avian toxicity data were submitted or located in the open literature for antimycin; therefore, chronic risks associated with avian exposure to antimycin are unknown. However, the likelihood of chronic exposure is considered low since dead fish would soon sink

88 Cemto, K. M. 2004. Antimycinand rotenone: short-termeffectson invertebrates in first order,high-elevationstreams. A master's thesis submitted to the DepartmentofZoology and Physiology andthe Graduate School ofthe University ofWyoming. 89 Ibid Mooreet al. 2005. 90 Personal communication: S. Moore. Supervisory Fishery Biologist, Great Smoky Mountains National Park, June 2006 91 Ibid Mooreet 01.2005 Page 51of 118to the bottom of treated water. While exposure is possible through ingestion of antimycin A residues in drinking water, the likelihood is considered low due to flow-through. Ecological risk assessments typically evaluate the potential acute and chronic effects of pesticides to terrestrial and aquatic organisms. As indicatedby Table 20, there are a number of toxicity studies (avian sub-acute dietary, avian chronic, mammalian chronic, aquatic vertebratehnvertebrate chronic and terrestrial/aquaticplant) that are not available for antimycin A. In determining whether any of these taxa would be at risk from the use of antimycin, it must first be determined whether exposure is likely. As indicated by acute risk quotients, acute exposure estimates based on recommended treatment rates are more than sufficient to exceed acute high risk level of concern for aquatic animals. However, given the uncertainty regarding the degradation of antimycin, chronic exposure estimatesvary widely. The extent to which nontarget animals will be affected will depend on the extentto which antimycinmoves outside ofthe targeted treatment area. Although two studies were submitted by the registrant examining the potential effects on mammals from the ingestion of antimycin-treated water9' and fish93,there were insufficient details included in the studies to substantiate their conclusions regarding no effect (see Appendix F for additional study details). 

### 6.5 

### Ecological Incident Reports There are no incidents related to the use of antimycin A reported in the Ecological Incident Information System database. However, the fact that incidents have not been formally reported does not mean that incidents have not occurred. It is noteworthy that there are incidents reported for the other piscicides mentioned in Table 25. The primary use of antimycin in high gradient streams and the low overall amount of antimycin used nationally may limit opportunitiesto observe adverse effects on non-target organisms. 

### 6.6 

### Uncertainties Although the ecological risks assessed in this chapter are based on a treatment rate of 

### 25 pg/L maintained for roughly 8 hours, the label does not specify a maximum application rate. At this treatment rate, though, the available toxicity data suggest that a substantial number of aquatic species will be subject to acute mortality. Higher application rates can be reasonably expected to cause acute mortality on a largerproportion ofthe aquatic community. Resource management agencies have developed guidance, such as that depicted in Appendix C, for the use of piscicides in New Mexico. Additionally, professional fishery organizations such as the Fish Management Chemicals Subcommittee of the American Fisheries

-- -- -- 92 Arslaneglau, L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration of Water Treated with Antimycin to Rats and Dogs. Ayerst Research Laboratories. (MRID 2400798-10)
93 Arslaneglau. L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration of Fish Killed with Antimycin to Rats and Dogs. Ayerst Research Laboratories. (MRID 2400798-08)Society have completed monographs for the use of antimycin in streams9' and in lakes~reservoirs~~; however, the label does not currently requirethat these application methods be followed. Mr. Stephen Moore and Mr. Matt Kulp with the U.S. National Park Service in collaboration with Mr. James Brooks and Mr. Bruce Rosenlund of the U.S. Fish and Wildlife Service and Dr. David Probst with the New Mexico Department of Game and Fish are currently developing a standard operatingprocedure (SOP) for the use of antimycin. While this manual is intended to serve National Park Service biologists in the proper use of antimycin on National Park Service lands, it is also being developed on behalf of the registrant96and will serve as a SOP to accompany the label for the use of antimycin in streams, lakes and reservoirs. Mississippi State University (MSU) Agricultural Research Extension Service has completed a SOP for the use of antimycin in catfish aquaculture (Appendix E). These documents are intended to assure the consistent use of antimycin in targeted treatment areas and minimize non-target species mortality. 

### 6.6.1 Analytical Method Development The available data on both the environmental fate and ecological effects of antimycin have been limited by the lack of an analytical method for detecting antimycin. The low application rates, typically less than 25 pg/L, coupled with the fact that antimycin is composed of 4 subunits that may readily dissociate from one another, would require fairly sensitive methods of detection. During the problem formulation phase ofthis risk assessment, there were relatively frequent discussions with the registrant and stakeholders;EPA emphasized the need to develop an analytical method that could be applied in the field. Through these discussions and with funding provided by the U.S. Fish and Wildlife Service, methodology is currently under development by the U.S. Geological Survey in La Crosse, Wisconsin. Research thus far has resulted in levels of quantification (LOQ) at 50 ng/L. The methodology requires that a sample size of 50 mL is first concentrated on a solid phase extraction (SPE) column then eluted with and stored in an organic solvent to stabilize the antimycin A. Increased sensitivity can be achieved through higher sample sizes (a 10pptr quantification limit has been achieved by extracting 250- mL water samples); however, 50 mL is considered a reasonable sample size to process through an SPE columnunder field 

## condition^^^. The extracted sample is then eluted off the SPE column with acetone and is minimally stable for 2 weeks and may be stablefor several months. In the lab, the extracted sample is then analyzed using reversed-phase high pressure liquid chromatography (HPLC) with a mass spectroscopy (MS) detector. Using a gradient elution method, antimycin A resolves into four quantifiablepeaks (A,,A,, A, and A,). The limit of detection (LOD)is approximately 15pptr when using a sample size of 50 mL.

94 American Fisheries Society. 2006. ~intml'Stream and River Use Monographfor CompleteRemoval of Fish. Use in Streams and Rivers. httn. ~www.tishcnch.o~rht~nI.Anr~it~~n~ n~n~rv~~anlr~.~l~~~irn~ 95 American Fisheries Society. 2006. Fintml' Pond, Lake and Reservoir Use Monograph for Complete Removal of Fish: Use in Ponds,Lakes and Reservoirs. htta, www.tishencs.c1l~~11I1t1111~ Ant~nvcln ~no~ior~anh~.sl~ml 96 Memo from Mary Romeo; President, Aquabiotics Corporation; dated February 13,2006 Pmonal communication I.B-dy and T Hub&. Biological Resources Division ofthe U.S. Geological Survey, May 2006 Page 53 of 118

### 6.6.2 Assumptions and LimitationsRelated to EffectsAssessment 

### 6.6.2.1 Age Class and Sensitivity ofEffects Thresholds Test organism age may have a significantimpact on the observed sensitivityto a toxicant. The screening risk assessment acute toxicity data for fish are collected onjuvenile fish weighing between 0.1 and 5 grams. Aquatic invertebrate acute testing is performed on recommended immature age classes (e.g., first instar for daphnids, second instar for amphipods, stoneflies and mayflies, and third instar for midges). Similarly, acute dietary testing with birds is also performed on juveniles, with mallard being 5-10 days old and quail 10-14 days old. The screening risk assessmenthas no current provisions for a generally applied method that accounts for uncertainty associated with study organism age. In so far as the available toxicity data may provide ranges of sensitivity information with respect to age class, the risk assessment uses the most sensitive life-stage information as the screeningendpoint. Additionally, this assessment does not evaluate whether antimycin A could be more toxic for organisms that have lower metabolic activity. This may occur in more sensitive life stages and may render these organisms more vulnerable to chronic effects.


### 6.6.2.2 Lack of EffectsData for Amphibians and Reptiles Currently, toxicity studies on amphibians and reptiles are not required for pesticide registration. Since these data are lacking, the Agency uses fish as surrogates for aquatic-phase amphibians and birds as surrogates for terrestrial-phase amphibians and reptiles. These surrogates are thought to be reflective of or protective (more sensitive) of herpetofauna. Amphibians are characterized by a permeable skin. The most important route of exposure for aquatic amphibianswould likelybe the dermal route. Freshwater fish may be suitable surrogates since exposure would likely be surface area-dependent and the gill surface area of many fish is fairly large. Also, both fish and amphibians are ectothermic so metabolic rates and demands would likelybe similar. For terrestrial species,however, the differencebetween amphibiansand birds and reptiles and birds is quite large. Terrestrial amphibians and reptiles are both ectothermic while birds are endothermic; birds have a higher basal metabolic rate required to maintain constant body temperature. The higher metabolic demands of birds may predispose birds to higher relative exposures. However, this does not address any potential differences in toxicity. To date, there are few controlled studies on reptile species that could be used to compare to similar studies on birds. A priori, there is no strongreason to think that one taxon is more or less sensitive than another. Further research is required to determine whether, in general, reptiles and terrestrial-phase amphibians are suitably represented by bird species in assessing risks.


### 6.6.2.3 Use of the Most Sensitive Species Tested Although the screening risk assessment relies on a selected toxicity endpoint from the most sensitive species tested, it does not necessarily mean that the selected toxicity endpoints reflect sensitivity of the most sensitive species existing in a given environment. The relative Page 54 of 118position of the most sensitive species tested in the distributionof all possible speciesis a function of the overall variability among species to a particular chemical. The relationship between the sensitivity of the most tested species versus wild species (including listed species) is unknown and a source of significant uncertainty. The use of laboratory species has historically been driven by availability and ease ofmaintenance. A widespread comparison of species is lacking; however, even variation within a speciescan be quitehigh.


### 6.6.2.4 EffectivenessofDeactivation with Potassium Permanganate There are no data to evaluate the effect of potassium permanganate on antimycin A. Although information from the National Park suggests that permanganate is an effective means of deactivating antimycin and there is similar information for other piscicides such as rotenone99, no data have been submitted to identify the degradation products from mixing antimycin A with potassium permanganate.


### 6.6.3 Assumptions Related to Exposure Assessment In this exposure assessment, a wide range of possible EECs were derived, due primarily to the lack of fate data and the lack of use limits on the antimycin label. Chronic exposure concentrations are especially affected by these uncertainties because these values are influenced by both the uncertainty in application rates as well as the uncertainty in the degradation rate. Acute concentrations uncertainties are due primarilyto the label rate ambiguitiesonly. Uncertainties due to application rates were not directly addressed here, but it should be noted that any change in application rate would result in directly proportional changes in EECs. The label reports a concentration of "roughly 25 ppb" as the highest in a list of recommended starting concentrations. It is unknown if there would be an application substantially higher than 25 ppb.Chronic concentrations in a stream would likely be affected by dissipation due to hydrodynamic dispersion, degradation, mixing, and sorption as the antimycin flows downstream. Thus, antimycin chronic concentrations in streams are likely to be lower than calculated here. However, the dissipation mechanisms of antimycin in a stream are not well understood and will likely be site-specific, depending on such highly localized factors as stream velocity and turbulence; thus, no further attempts at refining stream concentrations were made in this assessment. The label describes a method of detoxifllng antimycin with 1 ppm of potassium permanganate,but the label does not require that this detoxificationbe performed. Furthermore, EFED has no information regarding the effectiveness of potassium permanganate at removing

98 Ibid Moore et al. 2006. 99 EPA. 2006. Environmental Fate and EcologicalRisk Assessment for the Reregistration of Rotenone. (DP Barcode 307380); Docket Number OPP-EPA-HQ-2005-0494 hu~ u~ w lcculailuriz KO\ Page 55 of 118antimycin and thus EFED cannot assume that potassium permanganate actually removes antimycin from a water body. EFED did not attempt to adjust recommended estimated environmental concentrationswith consideration forpotassium permanganate use.


### 6.7 

### Federally Threatenedand Endangered (Listed) Species Concerns Section 7 of the Endangered Species Act, 16 U.S.C. Section 1536(a)(2), requires all federal agencies to consult with the National Marine Fisheries Service (NMFS) for marine and anadromous listed species, or the United States Fish and Wildlife Services (FWS) for listed wildlife and freshwater organisms,ifthey areproposing an "action" that may affect listed species or their designated habitat. Each federal agency is required under the Act to insure that any action they authorize, fund, or carry out is not likely to jeopardize the continued existence of a listed species or result in the destruction or adverse modification of designated critical habitat. To jeopardize the continued existence of a listed species means "to engage in an action that reasonablywould be expected, directly or indirectly,to reduce appreciably the likelihood of both the survival and recovery of a listed species in the wild by reducing the reproduction, numbers, or distribution of the species."100 To facilitate compliancewith the requirements of the Endangered Species Act subsection (a)(2), the Environmental Protection Agency Office of Pesticide Programs has established procedures to evaluate whether a proposed registration action may directly or indirectly reduce appreciably the likelihood of both the survival and recovery of a listed species in the wild by reducing the reproduction,numbers, or distribution of any listed species (U.S. EPA 

### 2004). After the Agency's screening-levelrisk assessment is conducted, if any of the Agency's listed species LOCs are exceeded for either direct or indirect effects, an analysis is conducted to determine if any listed or candidate species may co-occur in the area of the proposed pesticide use or areas downstream or downwind that could be contaminated from drift or runofVerosion. 

### If determined that listed or candidate species may be present in the proposed action areas, further biological assessment is undertaken. The extent to which listed species may be at risk then determines the need for the development of a more comprehensive consultation package as required by the Endangered SpeciesAct. The federal action addressed herein is the proposed re-registration of pesticide product that contains the active ingredient antimycin.


### 6.7.1 

### Action Area For listed species assessment purposes, the action area is considered to be the area affected directly or indirectlyby the Federal action and not merely the immediate area involved in the action. At the initial screening level, the risk assessment considers broadly described taxonomic groups and so conservatively assumes that listed species within those broad groups

loo 50 C.F.R. f 402.02 Page 56 of 118are collocated with the pesticide treatment area. This means that terrestrial plants and wildlife are assumed to be located adjacent to the treated site and aquatic organisms are assumed to be located in a surface water that is the treated site. The assessment also assumes that the listed species are located within an assumed area which has the relatively highest potential exposureto the pesticide, and that exposures are likely to decrease with distance from the treatment area. The use characterization section of this risk assessment presents the pesticide use sites that are used to establish initial collocationof specieswith treatment areas.


### 6.7.2 Taxonomic GroupsPotentially at Risk If the assumptions associated with the screening-level action area result in RQs that are below the listed species LOCs, a "no effect" determination conclusion is made with respect to listed species in that taxa, and no furtherrefinement ofthe action area is necessary. Furthermore, RQs below the listed species LOCs for a given taxonomic group indicate no concern for indirect effects upon listed species that depend upon the taxonomic group covered by the RQ as a resource. However, in situations where the screening assumptions lead to RQs in excess of the listed species LOCs for a given taxonomic group, a potential for a "may affect" conclusion exists and may be associated with direct effects on listed speciesbelonging to that taxonomic group or may extend to indirect effects upon listed species that depend upon that taxonomic group as a resource. In such cases, additional information on the biology of listed species, the locations of these species, and the locations of use sites could be considered to determinethe extent to which screening assumptions regarding an action area apply to a particular listed organism. These subsequent refinement steps could consider how this information would impact the action area for a particular listed organism and may potentially include areas of exposure that are downwind and downstream ofthe pesticide use site. Assessment endpoints, exposure pathways, the conceptual model addressing proposed antimycin re-registration uses, and the associated exposure and effects analyses conducted for the antimycin screening-levelrisk assessment are in Sections 4 and 5. The assessment endpoints used in the screening-level risk assessment include those defined operationally as reduced survival, reproduction, and growth for both aquatic and terrestrial animal species from direct acute and direct chronic exposures. These assessment endpoints address the standard set forth in the Endangered Species Act requiring federal agencies to ensure that any action they authorize does not reduce appreciably the likelihood ofboth the survival and recovery of a listed speciesin the wild by reducing the reproduction, numbers, or distribution of the species. Risk estimates (i.e., RQs integrating exposure and effects) are calculated for broad-based taxa groups for the screening-level risk assessment and presented in Section 6. Both acute endangered species and chronic risk LOCs are considered in the screeninglevel risk assessment to identify direct and indirect effects to taxa of listed species. This section identifies direct effect concerns, by taxa, triggered by exceeding listed species LOCs in the screening-level risk assessment with an evaluation of the potential probability of individual effects for exposuresthat may occur at the established listed speciesLOC. Data on exposure and effects collected under field conditions are evaluated to make determinations on the predictive utility of the direct effect screening assessment findings to listed species. Additionally, the Page 57 of 118results of a screen for indirect effects to listed species, using direct effect acute and chronic LOCs for each taxonomic group, arepresented and evaluated. 

### 6.7.2.1 Listed Species Risk Quotients A description of the potential direct effects associated with exposure to antimycin is discussed for each of the taxonomic groups below. Table 27 provides a summary of the direct effects for Federally listed threatenedendangered species, including the range of RQ values and the acute dose-response slopes used in evaluating the probability of individual effects on listed species.



### 6.7.2.1.1 Freshwater Fish and Amphibians Listed species acute risk LOCs for direct effects on freshwater fish and amphibians are exceeded (RQ = 2,778) for antimycin when used at the typical label rate for piscicidal use in warm waters where degradationwould be fastest.

Table27. Summary ofdirect effects for Federally listed speciesfrom pisicidal uses.

It is noteworthy that piscicides such as antimycin are used in efforts to restore listed species. For example, the Gila trout (Oncorhynchus gilaes) was first listed in 1967. It is currently designated as endangered in the entire range; the published range of this species Listed Species Taxonomic Group of Concern FreshwaterFish Freshwater Invertebrates SaltwaterMollusc Aquatic Plants: Vascular Non-vascular Birds

Mammals

Terrestrial Plants: Monocots Dicots

Page 58 of 118 a~aw data were not provided sothe default value of4.5is used. *Dose-based value. Direct Effects

Acute: mortality Acute: mortality/immobilization Acute mortality

no data no data
Acute:mortality/sublethal

Acute: mortality

Acute: no data Acute: no data Slopea

15.5 4.5 4.5

-

- 4.5

6.5

- - RQ
2,778 3,125 0.40

-

- <0.0l8

--- <0.018

--

-
-includes Arizona and New ~exico.'~' Stocking and naturalization of non-native trout with the range of Gila trout and ensuing hybridization, predation and competition are considered by the U.S. Fish and Wildlife Service to be major causes of the imperiled status of the species. The revised recovery plan for this species focuses on chemically treating non-native salmonidsto reestablish viable populations of Gila trout in its historic rangelo2. A vital component of the recovery and long-term survival of Gila trout is removal of non-native trout and hybrids; to that end, antimycin A has been used to eradicate the non-nativetrout.


### 6.7.2.1.2 Freshwater Invertebrates Listed species acuterisk LOCs for direct effects on fi-eshwaterinvertebrates are exceeded (RQ =3,125) for antimycin when used at the typical treatment rate for piscicidal use in flowing and static waters. No chronic freshwater invertebrate toxicity data were submitted and no useable data were located in the open literature; therefore, chronic effects associated with freshwater invertebrate exposureto antimycin areunknown.


### 6.7.2.1.3 EstuarineMarine Fish and In vertebrates Listed species acute risk LOCs for direct effects on estuarinelmarine fish and invertebrates are exceeded with RQ values of 109 and 1.0, respectively. No chronic estuarinelmarine fish or invertebrate toxicity data were submitted and no useable data were located in the open literature for antimycin; therefore, acute and chronic effects associated with estuarinelmarinefish and invertebrateexposure to antimycin areunknown. 

### 6.7.2.1.4 Aquatic Plants No aquatic plant toxicity data were submitted and no useable data were located in the open literature for antimycin; therefore, risks associated with aquaticplant exposureto antimycin are unknown. 

### 6.7.2.1.5 Birds No acute risk LOCs are exceeded for birds. No chronicbird toxicity data were submitted and no useable data were located in the open literature for antimycin; therefore, chronic risks associated with avian exposureto antimycin areunknown. 

### 6.7.2.1.6 Mammals No acute risk LOCs are exceeded for mammals. No chronic mammalian toxicity data were submitted and no useable data were located in the open literature for antimycin; therefore, chronic risks associatedwith mammalian exposureto antimycin areunknown.

lolu.S.Fish andWildlife Service Endangered Species Status. htt~.; ccos.lwa.euvsncclcs nlr)lilc scmlct.EOL Clol.blxcICa ~)rolilc.ac~\ IcIs.S~CCIC~PIT)I~~C'.'~~C~I~C EOOtiis~i~tu~ lo2 Pittenger, .I. 1993. Gila Trout (Oncorhynchusgilae) (Third Revision) Recovery Plan. U. S.Fishand Wildlife Service, Albuquerque,NM htui): ,cco~.lws.tro~ cloca. I.CCOLCIV i~lilli:, 2003 03001O.n~lt

Page 59 of 118

### 6.7.2.2 Probit 

### Dose Response Relationship 6.7.2.2.1 Aquatic Listed Species Probability of Effects on Individuals The probability ofindividual effects at estimated acute RQs above the listed species acute risk LOC was calculated. The probit slopes used in this analysis were obtained from doseresponse relationships used in calculating RQs. For freshwater fish, the probit dose-response slope is 15.5based on the coho salmon acute toxicity test. Should exposure to listed freshwater fish occur at the typical treatment rate of 25 pg/L, the probability of one individual being affectedis 1 in 1.OO (i.e., 100%). The probability of individual effects to listed freshwater invertebrates should exposure occur at the typical treatment rate is again 100%. The probit dose-response slope used for freshwater invertebrateswas 4.5 (the default value used in OPP assessments). 

## 7 CONCLUSIONS There are a number of uncertainties associated with the environmental fate and potential ecological effects of antimycin A. Although there are data indicating that the chemical is shortlived in warm, alkaline waters, the persistence of antimycin under cold, acidic conditions is unknown. Furthermore, the extent to which antimycin partitions to sediments is unknown. In spite of these uncertainties, available literature suggests that through a combination of environmental factors (pH, temperature, flow velocity, dilution), antimycin does not appear to persist in the environment; however, the exact mechanism underlying its limited persistence is unclear. Relative to other piscicides and other pesticides in general, antimycin is extremelytoxic to some species of fish; however, field studies indicate that through the use of rigorous application procedures, the extent of acute mortality can be limited to the targeted treatment area. Although the current label does not require that standard operating procedures be followed, the registrant has indicatedthat it is committed to developing such procedures and linkingthem to the labeled uses of antimycin. Available data indicate that aquatic invertebratescan be as sensitiveto antimycin as fish; however, field studies have indicated that effects on aquatic macroinvertebrates are transitory. Aquatic invertebrate populations appear to recover to pretreatment levels in terms of both abundanceand diversity; this likely occursthrough immigration from upstream, untreated areas. Although terrestrial animals may consume dead or dying fish or ingest antimycin-treated drinking water, current data indicate that antimycin residues would not be sufficient from either sourceto constitute a risk of acute mortalityto these animals. There is uncertainty regarding the chronic effects of antimycin particularly for animals with low metabolic rates such as may exist in certain life stages; however, the potential for Page 60 of 118

### chronic exposure appears limited. 

### In lotic environments, antimycin is expected to move out ofthe treatment area by wash through and dilution by untreated tributaries. Additionally, although the current label does not require deactivation using potassium perrnanganate, the oxidizing agent is commonly used in lotic environments to prevent the movement of antimycin outside of the targeted treatment area. The potential for chronic effects in lentic environments, though, continues to be uncertain; however, antimycin use in aquaculture does not appear to have any reported adverse effects on fingerling catfish that are stocked afterwards. Additionally, in alpine lakes, aquatic communities apparently recover enough to sustain restocking efforts. While there are potential risks to Federally-listed threatenedlendangered aquatic species, antimycin is used in the recovery of some species. It is incumbent of the users to conduct the necessary monitoring to establishwhether listed species arepresent; however, given the treatment rates and the vulnerability of aquatic animals to antimycin, acute mortality may result from exposure. Finally, the lack of a quantitative method for measuring antimycin in water has been a limiting factor in being able to conduct detailed environmental fate and ecological effect studies. The current methodologies being developedby the U.S. Geological Survey are expected to vastly improvethe analytical detection limits for antimycin A.

Page 61 of 118

## 8 REFERENCES American Fisheries Society. 2006. ~introl~ Stream and River Use Monograph for Complete Removal of Fish. Use in Streams and Rivers. http:/!www.fisheries.~~r~html/Ant~~nyc~n monor~raphs.stitn~l American Fisheries Society. 2006. ~introl@ Pond, Lake and Reservoir Use Monograph for Complete Removal of Fish: Use in Ponds, Lakes and Reservoirs http:~/www.fisheries.or~htmliAnt~mvc~n mononraphs.shtm1 Anderson, B.G. 1944.Thetoxicity thresholds of various substances found in industrial wastes as determined by the use ofDaphnia magna. Sewage Works J. 16(6): 1 156-1165. Arslaneglau, L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration of Water Treated with Antimycin to Rats and Dogs. Ayerst Research Laboratories. (MRID 2400798-10). Arslaneglau, L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration of Fish Killed with Antimycin to Rats and Dogs. Ayerst Research Laboratories. (MRID 2400798-08) Ayerst Laboratories. 1964. Efficacy of Antimycin. (Compilation: unpublished studyreceived in 1994 under 8991-51 CDL: 005666-A (AccessionNo. 

### 100135924) Bettermann, A.D., J.M. Lazorchak and J.C. Dorofi. 1996. Profile of toxic responseto sediments using whole-animal and in vitro submitochondrial particle (SMP) assays. Journal of Environmental Toxicologyand Chemistry 15(3): 

### 3 19 - 324. Birdsong, C.L. and J.W. Avault Jr. 1971. Toxicity of certain chemicals to Juvenilepompano. Prog. Fish-Cult. 33(2): 76-80. Cerreto, K. M. 2004. Antimycin and rotenone: short-term effects on invertebrates in first order, high-elevation streams. A master's thesis submitted to the Department of Zoology and Physiology and the Graduate School ofthe University ofWyoming ECOSAR. Ecological Structure Activity Relationships http:1/www.epa.~viopptintr1newche~ns1toc~Isi3 1ecosar.htm ECOTOX (ECOTOXicology database). 2005. Maintained by the U.S. EPA ORD and the National Health and Environmental Effects Research Laboratory's (NHEERL's) Mid-Continent Ecology Division. http://www.em. rrov!ecc~tox. EPA. 2006. Environmental Fate and Ecological Risk Assessment for the Reregistration of Rotenone. (DP Barcode 307380); Docket Number OPP-EPA-HQ-2005-0494 http:/~www.re~ulati~~~~,~~v~ Page 62 of 118EPI Suite http:/~www.epa.~oviopptintr/exposureldt Finlayson, B.J., R.A. Schnick, R.L. Cailteux, L. DeMong, W.D. Horton, W. McClay, and C.W. Thompson. 2002. Assessment of Antimycin A Use in Fisheries and its Potential for Reregistration. Fisheries 27(6): 10-18. Finlayson, B.J., R.A. Schnick., R.L. Cailteux., 

### L. DeMong., W.D. Horton 

### ,W. McClay 

### ,C.W. Thompson, and G.J. Tichacek.. 2000. Rotenone Use in Fisheries Management: Administrative and Technical GuidelinesManual. American Fisheries Society, Bethesda, Maryland. Heim, D. 2003. Determination of Aerobic Aquatic Metabolism for Antimycin A Complex: Amended Final Report. Lab Project No. 47313. Unpublished study prepared by ABC Laboratories, Inc. 79p (MRID 458959-01). Heim, D. 2003. Hydrolysis Determination for Antimycin A Complex. Project Number 47132. Unpublished studyprepared by Analytical Bio-Chemistry Labs, Inc. 63p. (MRID 46023 1-01). Hobbs. M. S., R. S. Grippo, J. L. Farris, B. R. Griffin and L. L. Harding. 2006. Comparative Toxicity of Potassium Permanganate to Nontarget Aquatic Organisms. Environmental Toxicologyand Chemistry25(1): 3046 - 3052. Hogan, J. 1966. Antimycin as a Fish Toxicant in Catfish Culture. Presented at the 20' Annual Meeting of the southeastern Association of Game and Fish Commissioners, Oct 24 

### - 26, 1966. Bureau of Sport Fisheries and Wildlife, U.S. Fish and Wildlife Service. (Accession No. 00045801;MRID 2400798- 13). Hussain, A. 1969.Kinetics and mechanismsof hydrolysis of antimycin A in solution.Journal of Pharmaceutical Sciences 58: 3 16-320. Kenneke, J.F. Summary ofAntimycin Hydrolysis Research, US EPA, unpublished report (see Appendix G) Kuhn, 

### 1.0.2001. ~introl" Concentrate (Antimycin) Final Report Acute Oral Toxicity in Rats. Stillmeadow, Inc. Report No. 6025-00. Submitted to Aquabiotics Corporation, Bainbridge Island,WA. (MRID 455279-01). Lee, T.H., Derse, P.H., and Morton, S. 1971. Effects ofPhysical and Chemical Conditionson the DetoxificationofAntimycin. Transactionsofthe American Fisheries Society 1971; 100: 13-1 7 Liu, W.C., E.E. van Tamelan, F.M. Strong. 1960. The chemistry of antimycen A. VIII DegradationofAntimycen A. Journal ofthe American Chemical Society 82: 1652-1654 Marking, L.L. and T.D. Bills. 1975. Toxicity of potassium permanganate to fish and its effectiveness for detoxifying antimycin. Trans.Am. Fish. Soc. 104(3):579-583. Page 63 of 118Mayer, F. and M. Ellersieck. 1986. Manual ofAcute Toxicity: Interpretation and Data Base for 410 Chemicals and 66 Species of Freshwater Animals. U.S. Fish and Wildlife Service. Resource Publication 160. 579p. (MRID 400980-01) Mineau, P, B.T. Collins, and A. Baril. 1996.On the use of scaling factorsto improve interspecies extrapolation of acute toxicity in birds. Regulatory Toxicologyand Pharmacology. 24: 24-29. Moore, S.E., M.A. Kulp, J. Hammonds, and B. Rosenlund. 2005. Restoration of SamsCreek and an Assessment of Brook Trout Restoration Methods, Great SmokyMountain National Park. National Park Service, U.S. Department ofthe Interior. Technical Report NPS/NRWRD/NRTR2005. Nagy, K.A. 1987. Field metabolic rate and food requirement scaling in mammals and birds. Ecological Monographs 

### 57: 1 1 1 

### -128. OPPTS 850.1075. 1996. Ecological effects test guidelines: fish acute toxicity test, freshwater and marine. U.S. Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances: EPA7 12-C-96- 1 1 8. http:l/www.epa.~ov1ot'ftsfrsipubl1cations/OPP1'S Harnionizedi8.50 Ecolog~calEffects Test Gu 1delines1Drafts1850- 1075.pdf ORD. 1993. Wildlife Exposure Factors Handbook. Office of Health and Environmental Assessment, Office of Research and Development. EPA/600/R-93/1 87. http://www.epa.~ovinceaipdfs/b~rds.pdf PBT Profiler http:/iwww.pbtprofiler.net ' Pittenger, J. 1993. Gila Trout (Oncorhynchusgilae) (Third Revision) Recovery Plan. U.S. Fish and Wildlife Service, Albuquerque, NM http:/~ecos.fws.~ovidocs~recoverv plansi20031030~) 10.pdt' Reardon, I.S. and R.M. Harrell. 1994. Effects of varying salinities on the toxicity of potassium permanganate to larval andjuvenile striped bass, Morone saxatilis (Walbaum). Aquacult. Fish. Manag. 25(6): 571-578. Ritter, P.O. and F.M. Strong. 1966. Residues in tissues of fish killed by antimycin. Journal of Agricultural Food Chemistry, 14(4): 403 -407. (MRID 461534-01) Sreekala, K.G., J. Jennita, and V.R. Prakasam. 1991. Tolerance and heart rate of Daphnia sp. (crustacean) in response to disinfectants,bleaching powder and potassium permanganate. Pollut. Res. lO(1): 33-36. Tiffan, K.F. and E.P. Bergersen. 1996. Performance of Antimycen in high-gradient Streams. North American Journal ofFisheriesManagement 16: 465-468. Page 64 of 118

### U.S. Fish and Wildlife ServiceEndangered Species Status. 2006. http:~/ecos.fws.~ov/species - profile/servlet/gov.do~.spec~es ~rotile.servlets.SpcclesProtile?spcod e=EOOE#status 

### Walker, C.R., R.E. Lennon, and B.L. Berger. 1964. 

### Preliminary observations on the toxicity of antimycin A to fish and other aquatic animals. U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control No. 2 

### (CircularNo. 186). 18 

### pp. Wisconsin Alumni Research Foundation. 1965. 

### Antimycin as a Fish Toxicant: a resume of information and data pertaining to the use of antimycin in fish management procedures in ponds and lakes. (AccessionNo. 00135924)

Page 65 of 118

## APPENDIXA. ASTER 

## Model Output

ASTER ECOTOXICITY PROFILE
U.S. Environmental Protection Agency

Office of Research and Development National Health and Environmental Effects Research Laboratory Mid-Continent Ecology Division (formerly the Environmental Research Laboratory-Duluth)
Contact: Scientific Outreach Program

218-529-5225 or FAX 218-529-5003 E-mail: ecotox.support@epa.gov

CHEMICAL IDENTIFICATION
Name Antimycin A
CAS number 1397-94-0 SMILES OlC (=O)C (NC(=O)C-C~C (0) c (NC=O) ccc2)C (C)OC (=O)C (CCCCCC)C(OC (= O)CC(C)C)Cl Formula C28 H40 N2 09

ENVIRONMENTAL EXPOSURE ASSESSMENT
Parameter Value Source Reference Molecular Weight (g/mole) 548.6 Calc
. Melting Point (C) not available for this chemical Boiling Point (C) 550. Calc 

### . Vapor Pressure (rnrn of Hg) 2.313-15 Calc. Ht Vaporization (cal/mole) 1.683+04 Calc. Solubility in Water (mg/L) 69.0 Calc
. Log P 3.81 CLogP 17934 PKa 9.92 Calc. Adsorption Coef (log Koc) 3.41 Calc 

### . Henry's Constant (atm-m**3/mole) 2.423-17 Calc. LoglO(Henry1s Constant) (atm-mx*3/mole) -16.6 Calc
. Hydrolysis Half-life (days) 190. Calc 

### . BOD Half-life: Calc. HALF-LIFE > 100 DAYS [E.G., SEE ALEXANDER (1965,1973), AND MCKENNA Page 66 of 118AND HEATH (1976) FOR REVIEWS ON THE RECALCITRANCE OF POLYAROMATIC COMPOUNDS

### I Mackay Level 1 Environmental Partitioning @25 C Fugacity = 1.7533-17 Pa 0.00 % into air 25.81 % into soil 50.04 % into water

0.04 % into suspended solids 0.02 % into aquatic biota 24.09 % into sediment

ECOTOXICOLOGICAL HAZARD ASSESSMENT
Aquatic Hazard Identification Researchers and managers using AQUIRE data for analysis or summary projects should consult with the original scientific paper to ensure an understanding of the content of the data retrieved from AQUIRE.

BIOCONCENTRATION DATA

.............................................................................. Species Common Name I~x(~uratio(~ndpoint (~on~/ BCF ]Source 

## I Ref

Species Latin Name 

## ITY[ (days) 

### 1 ~ff ect (~ype ( 

## I NO. 

### I

-- FRESH WATER 184207 : Fathead minnow F 2.00- BCF RSD 410 QSAR Pimephales promelas 304 calculated

Human Health Hazard Identification There is no information in the QSAR SYSTEM which would suggest that this chemical is a potential carcinogen or mutagen.

IV. ECOLOGICAL RISK CHARACTERIZATION

A. Environmental Exposure Assessment Henry's Constant = 2.423-17 atm-m**3/mole Page 67 of 118Log10 (Henry'sConstant) = -16.6 atm-m**3/mole Lyman et al. 1982. would conclude that a chemical with these properties is non-volatile. See page 15-15. The hydrolysis half-life of the amide moeity is expected to be greater than six months. Hydrolysis Half-Life = 190 days

B. Ecotoxicological Hazard Assessment Genetic/Mutagenic Assessment There is no information in the QSAR SYSTEM which would suggest that this chemical is a potential carcinogen or mutagen. NEUROTOXICANT: PYRETHROIDS The acute mode of toxic action for pyrethroid insecticides is generally attributed to an interaction with sodium channels in nerve membranes that ultimately results in tremors and seizures [94031.

When sufficient data is available from fathead minnow early life stage (ELS) tests (32-d exposures) completed at ERL-Duluth, QSAR models have been developed to predict chronic values for either survival or growth, which ever is the most sensitive endpoint. A chronic value is defined as the geometric mean of the LOEC(1owest observable effect concentration) and the NOEC (no observable effect concentration). These models have been developed for groups of xenobiotics that have been classified based on their acute modes of toxic action. Empirical observations suggest that when a statistically robust ELS QSAR can be established and when 96-h LC50/32-d ELS chronic value ratios are within a factor of 20 it is reasonable to assume that adverse effects are elicited through the same mode of toxic action in both 4-d and 32-d exposures. If during a chronic exposure a different mode of action is involved, or if metabolic activation is significant, the ratios between acute and chronic endpoint values for a group of xenobiotics are generally quite variable and typically exceed two orders of magnitude. In addition, the statistical strength of ELS QSARs in these instances are poor.
A chronic value cannot be calculated for the fathead minnow for pyrethroid and organochlorine type insecticides. 96-h LC50/32-d ELS chronic value ratios for neurotoxicants tested at ERL-Duluth using the fathead minnow range from 3.98 to 22.2 (log P range of 6.06 to 6.50).

C. Other Information
V. CITATION INFORMATIONREFERENCE NUMBER: 7 Veith,G.D. and P.Kosian 1983 Estimating Bioconcentration Potential from Octanol/Water Partition Coefficients In: D-Mackay,et al., (Eds.), Physical Behavior of PCBs in the Great Lakes, Ann Arbor Sci. Publ., Ann Arbor, MI:269- 282
REFERENCE NUMBER: 9403 Coats,J.R. 1990 Mechanisms of Toxic Action and Structure-Activity Relationships for Organochlorine and Synthetic Pyrethroid Insecticides Environ. Health Perspect. 87:255-262

REFERENCE NUMBER: 17934 Leo,A. and D.Weininger 1997. Daylight Software CLogP Version 3.15+ for Unix Pomona Medical Chemistry Project, Pomona College, Claremont, CA. Distributed by Daylight Chemical Information Systems, Inc., 3952 Claremont St., Irving, CA 92714

Page 69 of 118

## 10 

## APPENDIX 

## B. PBT ProfilerModel 

## Output
Persistence Summary Partitioning The PBT Profiler uses three environmental compartments (water, soil, and sediment) to determine the persistence of a chemical in the environment. If released to the environment, antimycin A is expected to be found predominantly in soil. It is also expected to be found in water and sediment. The PBT Profiler does not explicitly consider a chemical's fate in the atmosphere in its persistence estimate. It also does not consider a chemical's potential to enter groundwater. Important P2 considerations in these media may be discussed on a chemical by chemical basis in the sectionsthat follow. Transformation and Persistence The PBT Profiler has estimated that antimycin A is expected to be found predominantly in soil and its persistence estimate is based on its transformation in this medium. Its half-life in soil, 75 days, exceeds the EPA criteria of 

## > 2 months (and 

### I 6 months). Therefore, antimycin A is estimated to be persistent in the environment. The PBT Profiler calculates a chemical's atmospheric half-life fiom the estimated gas-phase reaction rate with hydroxyl radicals and ozone. The vapor pressure of antimycin A, 0.000000000001 

### mrn Hg, suggests that it will exist as a gas/particulate mixture in the atmosphere. Since particulates react slower with hydroxyl radicals and ozone (relative to a gasphase reaction), the atmospheric lifetime of antimycin A is expected to be longer than that predicted by the PBT Profiler. As a result, the distribution of antimycin A in the various environment compartmentsmay be differentthan that predicted by the PBT Profiler. This should be considered when identifying P2 opportunities. Overall Persistence The overall persistence is a calculated term that allows the persistence of different chemicals to be compared using a singlevalue. Even though the units of the overall persistence are the same as those used for a chemical's half-life (hrs), these two terms are not inter-convertible. The overall persistence takes into account both a chemical's media-specific half-life as well as itsrate of transport into (and out of) that compartment. Because the overall persistence takes into account transport, its value will likelybe differentthan any ofthe media-specifichalf-lives. The overall persistence can only be calculated in a mass-balance multimedia model. These models calculate the overall persistence by determining the weighted average of the residence time in each compartment. The overall persistence for antimycin A is 96 days using the default emission scenario of the level I11 multimedia model. The overall persistence using different release scenarios is provided in the following section. Page 70 of 118Release Scenarios The PBT Profiler estimates persistence based on a standard release scenario emitting equal amounts to soil, water, and air. A more in-depth 

### P2 assessment may utilize a release scenario that is more representative of 

### an individual chemical's life cycle. This section ofthe PBT Profiler provides seven different release scenariosto help identify P2 opportunitiesfor antimycin A. The seven release scenarios arebased on a more realistic total release of 300 

### kghrto the environment and not the 1,000 

### kghr shown on the PBT Profiler results page. Since the fugacity model is linear, the percent in each compartment does not change based on the total release to the environment, but only on the relative amount released to air, water, and soil. The following table provides the percent estimated in each environmental compartment using different release scenarios. The media (water, soil, and sediment)the chemical is expected to be found in predominantly (thepredominant compartment) is underlined. The color of each estimate indicates if the EPA criteria have been exceeded in that specific medium. Therefore, by determining the color of the underlined value in each row, the persistence ranking for each different scenario can be compared directly to the default persistence value, 

### P, estimated by the PBT Profiler. The overall persistence, Po(days), calculated for each release scenario is also provided. Release to each medium (Kg/hr) Percent 

### in each medium Air Water Soil Air Water Soil Sed Po

BioaccumulationSummary Bioconcentration Bioaccurnulation is the process by which the chemical concentration in an aquatic organism achieves a level that exceeds that in the water, as a result of chemical uptake through all possible routes of exposure. Biomagnification refers to the concentration of a chemical to a level that exceeds that resulting from its diet. Bioaccurnulation includes both biomagnificationand bioconcentration.
Page 71of 118In general, chemicals that have the potential to bioconcentrate also have the potential to bioaccumulate. Since a bioconcentration factor (BCF) in fish can be readily measured in the laboratory and bioaccumulation is much more complicated to determine, the BCF is frequently used to predict the importanceofbioaccumulation. The estimated bioconcentration factor (BCF) of 350 for antimycinA does not exceed the EPA bioconcentration criteria. Bioaccumulation Estimate The PBT Profiler estimatesthat antimycin 

### A is not expected to bioaccumulate in the food chain because it does not exceed the BCF criteria. Toxicity Summary 

### Fish Chronic Toxic PBT chemicals are those that persist in the environment, bioconcentrate in aquatic organisms,and may bioaccumulate in humans, birds, and wild mammals. Exposure to PBT chemicalswill result in chronic exposureswhich, in 

### turn, can lead to chronictoxicity. The PBT Profileruses an estimated fish chronic toxicity value (ChV) to allow organic chemicals lacking experimental data to be screenedfor P2 opportunities. A more in-depth 

### P2 assessment requires that the potential toxicity of antimycinA to other aquaticorganisms (and at other durations of exposure) be determined. The PBT Profiler estimates that antimycin A is chronically toxic to fish. It is important to note that antimycin A may also be toxic to other aquaticorganisms. Some aquaticorganisms, such as daphnids, may be more sensitive to both acute and chronic exposures to antimycin A. To help assess the toxicity of antimycin A to other aquatic organisms, the PBT Profiler provides the completeECOSAR~~ estimatesfor this compound, as discussed below. Other Toxicity Information Unlike persistence and bioaccumulation, there is a wide range of different aquatic toxicity endpoints that may be of concern when assessing a chemical for P2 opportunities. The PBT Profiler determines the fish chronic aquatic toxicity for its toxicity ranking. Endpoints specific to humans, avian and terrestrial species, benthic organisms, and other aquatic animals are not included in the PBT Profiler toxicity ranking that appears on the initial results page. Other endpoints associated with the acute, sub chronic, and chronic toxicity of antimycin A should be considered for the above organisms in light of its persistence, potential for bioaccumulation, release to the environment,and life cyclewhen performing an in-depth P2 Assessment. To help address some of these toxicity issues, the following information may be useful when identifying P2 opportunities for antimycin A:

Page 72 of 118

# I ECOSAR~~ is a personal computer program developed by EPA's Office of Pollution Prevention and Toxics to estimatethe acute and chronictoxicity of chemicals discharged into water. 

## ECOSP predicts a chemical's effects on aquatic organisms, although its results should be evaluated together with information on persistence and biodegradation. The ECOSAR~~ program, as well as more information on its use, is available from EPA's web slte. The followingis the completeECOSAR~ output for antimycinA: SMlLES :01 

### C(=O)C(NC(=O)Cc2c(O)c(NC(=O))ccc2)C(C)OC(=O)C(CCCCCC)C(OC(=O)CC(C)C )C 1 MOL FOR: C28 H40 N2 09 MOL WT :548.64 Log Kow: 4.21 (KowWinestimate) Melt Pt: Wat Sol: 13.59mg/L (calculated) ECOSAR v0.99g Class(es)Found



### .............................. Esters Phenols Predicted ECOSAR Class Organism Duration End Pt mgL(ppm)

........................... ........................... ------------------ -------- ------ Neutral Organic SAR: Fish 14-day LC50 8.757 (Baseline Toxicity) Esters :Fish 96-hr LC50 5.457 Esters :Daphnid 48-hr LC50 4.248 Esters :Green Algae 96-hr EC50 0.471 Esters :Green Algae ChV 0.382 Esters :Fish ChV 0.473 Phenols :Fish 96-hr LC50 3.507 Phenols :Daphnid 48-hr LC50 3.685 Phenols :Green Algae 96-hr EC50 3.016 Phenols :Fish 30-day ChV 0.509 Phenols :Fish 90-day ChV 0.067 Phenols :Daphnid 21-day ChV 0.381 Phenols :Green Algae 96-hr ChV 1.082 Note: 

### * =asterisk designates: Chemicalmay not be solubleenough to measure this predicted effect. Esters:

Fish and daphnid acute toxicity log Kow cutoff: 5.0 Green algal EC50 toxicity log Kow cutof2 6.4 Chronic toxicity log Kow cutoff: 8.0 MW cutoff: 1000 Page 73 of 118Phenols: Fish and daphnid acute toxicity log Kow cutoff: 7.0 Green algal EC50 toxicity log Kow cutoff: 7.0 Chronic toxicity log Kow cutoff: 9.0 MW cutoff 1000
CloseWindow Developed by the Environmental Science Centerunder contract to the Ofice ofPollution Preventionand Toxics U. S. Environmental Protection Agency Computer Resources Donated by Syracuse Research Corporation Ver 1.203 Last Updated August 27, 2004

Page 74 of 118

## 11 

## APPENDIX C. 

## New Mexico Standard Operating Procedure for the Use of Piscicides.

PROCEDUREFOR DEPLOYMENTOF PISCICIDEDURING RESTOWTION OF LENTIC WATERS Introduction: New Mexico Department of Game and Fish (NMDGF) manages fisheries in waterbodies (lakes, reservoirs, and ponds) to support the recreational fishing and conservation of native fishes. Natural lakes and anthropogenic impoundsments are imprtant components to the state's fisheries program. They provide biological diversity and geographically dqmsed oppo~ties for recreation. In addition, they are complex systems 

### that may be inhabited by nuisance species or unwanted normative species. To effectively manage watdesfor public's recreational interest andlorspecies conservation,thedeploymentofa piscicide into lenticwaters is a valuabletool. Tomeet management plan objectives,NMDGFwill consider piscicideuse in appropriatewatdes. Deployment ofapiscicideis a complextask requiring guidanceby specificobjectives,public scoping, interagency cooxdhation, andidentified benefits tothepublic andtheresource. Pre-treatment information gathering will include input hm appropriate agencies, diverse public representatives, private landowners, and other interested parties. This investigation process will address public concerns for the deploymentofapiscicideinto surfacewaters ofthe state. 

### Purpose: Because of the complexities involved with piscicide deployment, a stan* procedure is essential to provide guidance during the treatment of any waterbody in the state. This document provides general methods that will facilitatesuccessll use ofpiscicideshi lentic systemsforfishery management activities inNew Mexico. ProjectDevelopmentandPrehhry Assessments 1. The use of a piscicide in waterbodies is an important fishery management tool 

### that will support the programs

. . admmstmtedby NMDGF. Use of a piscicide hi a lentic system hi New Mexico must be supported by objectives listedin a fishery management plan. A Fcularmanagement plan shouldprovide objective criteriaforprioritizing and selectingwaterbodies forchemical reclamation. 2. Iden% IssuesandPotential Conflicts Identifypublic opinionsand attitudeshithe community Identifjl land status Id- recreationalandcommercialinterests Page 75 of 118Iden* water developmentanduser groups 3. Descriptionofwaterbodyproposed fortreatment. Physical character- surface acres, waterbody volume, depth profiles, thd stratiiication, water retention time, springs, inflow and outflow channels, water quality, waterbody longevity, barrier presence, barrier function and stability.

Biological character- iden* khery, natural repdction, conduct genetics testing if appropriate, charact& macroinvertebratecommunity. Determine accessto mtmbodyandpotentialtributaries. 4. Completeallstate andfederalpermit requirements. e.g.NEPA, SHPO,ESA, CWA404 5. ARer a comprehensive evaluation of the proposed project by all planning entities, a decision on whether to continuewiththeproposed project willbe made. 6. Staff will develop a treatment plan to include piscicide application plan, staff needs, safety protocol, and emergencymponseprotocol. Statecertifiedpesticide applicato~s will supervisechemicalrestomtionprojects in New Mexico. In addition, personnel supervising restoration projects in New Mexico will have successllly completed the National Conservation Training Center (USFWS) course, Rotenone and Antimycin Use in FisheriesManagement. Wtment Activities 1. Determinewaterbody boundaries, basin profile, inflows and outflows and otherperhnent infonnation (e.g. fishmovements). 2. Determinesurface acres and volume ofwaterbody. 

### Ifappropriate, calculate streamdischarge oftributaries. Calculateflowthroughtime 

### ifappropriate.Conduct water @ty monitoring. 3. Determinekh species present (targetandnon-target species).

4. Determinebestmethodfor treating lakeand, ifnecessary, laketributaries in accordancewithproductlabel. Methods selectedshould consider pH, temperature, organic load, plantmaterial, andbasinmrpho10gy. Logisticalconsiderations includecost and accessibility. 5. Determine concentration of piscicidetoremove target species. 6. Calculateamountof piscicideneededtotreat waterbody including tributaries ifreqw.Iftreatment of tributariesis reqmed, followtheappropriateprovisions in theProtocol for Deploymentof Piscicides during Restoration ofNativeFisheries inLoticWaters. 7. Ifreqmd, locate aneutralization siteon theoutflowfiomthewaterbody. 8. Supervisorypersonnel will reviewdeployment schedule, personnel responsibilities, methods ofcommunication, and outlinepmcedulpsforhandlingunexpectedissueswithfield staff. Page 76 of 1189. Personnel willpost noticesofthe application atpointsofentry intothetreatment

### areasaswell as inappropriate offices andwebsites. Suchnotices willbepostedatleast48 hoursbefore deployment. Deployment ofPiscicide Boat deploymentofliquid rotenone or antimycin 1. Determineamountof piscicidetobe deployedin thewaterbodyusingdirections onthe label, water volume and inflow hm tributaries. Ifnecessat-y,tributaries canbetreatedin accordancewiththeProtocol for Deployment of Piscicides duringRestorationofNative Fisheries inLoticWaters. 2. Safetyequipmentwillbeworn according to label recommendations. 3. Setup livecarswithtarget 

### fishto assesstreatment effectivenessatvaryingpoints anddepthsinthewatabody. 4. Determineifit isnecessaryto divide lakeinto sections for deployment.Markerbuoys should beused for guidanceto ensuretotal coverage.Deploy thepiscicide in a mannerto effectively coverthetreatmentarea, e.g. in a grid-likepattem. Backpacksprayers shouldbeusedtotreatthe shoreline and smallinletswhereboat access islimited. Drip stationscanbe deployedonlargerinletsconcurrently with lakeapplication. 5. Shallow waters canbetreated using venturi apparatusthatpulls diluted piscicides intotheprop washoftheboat motor. Deeper sections ofthewaterbody shouldbe treated withbilgepumps andhoses. Toensuretoxicity overlapbetweenthe sections,theentirevolumeofpiscicide shouldbe deployed within48 hours. 6. Suspectedproblem areasmaybetreated with severalofthese methods. Sandmix formulas maybe useMfor underwater spring

### areas. 7. Treatmenttimes willdependonturnovertimeinthe lakeandthetypeofpiscicide charm 8. Wherefeasible, dead fishwillbe collected and buried in appropriate locations. 9. 

### All deployment equipmentwillbe emptied, rinsed with watertoremove residue, disassembled, collected and storedinaccordancewiththeproduct label. Boat deployment of powdered rotenoneproduct 1. Determine amountofpiscicidetobe deployed inthewaterbodyusing directions onthe label, water volume, and inflow hm tributaries. Ifnecessary,tributaries canbe treated in accordance withthe Protocolfor DeploymentofPiscicidesduringRestorationofNativeFisherieshi LoticWaters. 2. Safetyequipmentwill be worn accordingto the labelingrequirements. 3. Powdered rotenone should be applied using a closed system to limit 

### driR. An example of this type of systemis an aspirator system that suctions the powdered rotenone hm the containers into water that is pumped throughthe system. 4. Setup livecarswith target fishto assesstreatmenteffectiveness at varyingpoints and depthsinthewaterbody. Page 77 of 1185. Detamineifitisnecessaryto dividelakeinto sectionsfordeploynent.Marker buoys shouldbeused for guidanceto ensuretotal coverage. Deploythepiscicidein amannerto effectively coverthetreatment area, e.g. in a grid-likepattern Backpacksprayers should beusedtotseat shorelineand smallinletswhere boat accessis limited. Drip stationscanbe deployedonlargerinletsconcurrently withlakeapplication using a liquid rotenone formulation. 6. To ensure toxicity overlap betweenthe sections, the entirevolume ofpiscicide shouldbe deployed within 48 hours. 7. Wherefeasible, dead fish willbe collectedand buried in appropriate locations. 8. All deployment equipmentwillbe emptied,rinsedwith watertoremove residue, disassembled,collected and stored in accordancewiththeproduct label. Application ofsandmixrotenone Maybeeffectiveinshallow impoundments. 1. Determine amountofpiscicidetobe deployedhithewaterbodyusing directions onthe label,watervolume and results frombioassays. Idowrates need to be factored into treatment concentrationand duration. 2. Staffmixing and applying sand formulationwill wear safetyequipment accordingto label -en&. 3. Sandmixture canbe appliedusingburlap sacksorothertypesofpesticideapplication equipment that would be appropriate fordrymaterials. Equipment should be chosento assure evenapplication. Aria1applicationofliquidrotenone Aerialapplicationisdesirablein situationswhere accessto lakesislimited. Gendy,it works forlakes <20 feet deepwhere piscicide distributionat greaterdepthsis not aumcem.

Page 78 of 118

## 12 APPENDIX D. In vitro 

## Mammalian Metabolism.

Dr. John Kenneke with the Processes and Modeling Branch in the U.S. EPA National Exposure Research Laboratory (NERL) Ecosystems Research Division has generated preliminary information on an in vitro metabolic pathway for antimycin A, As preliminary information, these data have not undergone rigorous internal review nor have they been subject to quality assurance measuresthat may permit a more quantitativeuse ofthe data. Based on preliminary analyses using liquid chromatography/mass spectroscopy of an antimycin sample from the OPP Biological and Economic Assessment Division repository, Antimycin is composed of 8 components that group into two major categories: ones that have a butyl chain and the others that have a hexyl chain. Within each of these groups, four different structures existbased upon differences in the acyloxy side chain as indicated with the red circles (butyl and propyl structural isomers). The theoretical masses of 1 

### ah,2ah, 3&, 4ah are: 549, 535,521, and 507 glmole respectively. The mass spectral data we collected from our antimycin a standard shows 5 peaks with the same masses. In vitro phase 1 metabolism leads to hydrolysis of the acyloxy group (along with deformylation of the N-formylani1ine)--all four of the hexyl compounds (1 a, 1 b, 2a, 2b) lead to the same metabolite--and all four of the butyl compounds (3a, 3b, 4a, 4b) lead to a second metabolite.

Page 79of 118Hexyl Components

In vitro Phase 1 Metabolism

Butyl Components

Page 80 of 11813 

## APPENDIX E. Standard Operating Procedure for the Use of I?introlBto Remove Scaled Fish in Catfish Pondslo3

Fish other than catfish are sometimes intentionally stocked into catfish ponds for a specific purpose. For example, fathead minnows might be added to brood ponds to provide forage for broodfish. However, most catfishponds are operated as monocultures, and fish otherthan catfish are a nuisance. 

### In fingerlings ponds, wild fish may eat fry or compete for food. In foodfish ponds, wild fish may compete for food, interfere with harvest, or cause economic losses as "weigh-backs" at the processing plant. Accordingly, there are times when it may be necessary to remove wild fish from catfishponds. Two fish toxicants areregistered by the Environmental Protection Agency for use in commercial catfish production. Antimycin is a non-selective toxicant and is typically used to eliminate the few remaining fingerlings fi-omnursery ponds prior to stocking fry. FintrolB(active ingredient antimycinA) is effective in removing scaled fish from a pond without harming catfish. FintrolBhas been in use since the late 1960s.The chemical is absorbed through the fish's gills where it then interferes with respiration leading to death. The attributes that make Fintrolma desirable management tool are a wide difference in toxicity to catfish and scaled fish, a low toxicity to mammals, and how quickly it degrades. Fintrolmcan selectively remove scaled fish because they are much more susceptible to low concentrations of the toxicant. Problematic scaled fish (usually shad, carp, or sunfish) can be eliminated at concentrations of 5 to 10parts per billion (ppb), while it may take in excess of 20 ppb to kill catfish under normal conditions. It should be pointed out that concentrations high enough to remove unwanted bullheads would alsobe fatal to catfish. To determine an effective concentration of Fintrolm, you need to know the species of fish to be killed as well as the pH and temperature of the pond water. The toxicant is most effective when the pH ofthe pond water is 8.5 or lower and at water temperatures above 60" F (a rough estimate of the required concentrations are given in Table 1). 

### In moderately buffered catfish ponds (total alkalinity above 50 ppm), the ideal application time would be early in the morning (becausepH is lowest at that time) after pond temperatures have stabilized above 60" 

### F. Avoid treatingponds when catfish may be stressed due to high water temperatures orperiods of low dissolved oxygen. To determine the amount ofFintrolBneeded to treat the pond, you will need to estimatethe pond volume, in acre-feet of water. First, measure the surface area ofthe water to be treated. Multiply the surface area in acres by the average depth of the pond in feet. The result is the number of acre-feetto be treated.


## '03 Avey, I.L.2006. Use ofFintm? to xmove scaled fish in catfish ponds. Zhr Calfsh Journal April ZOO6 (reproduced with permissionof author). Page 81 of 118Fintrol" is sold in a metal can containing two bottles. The bottle with the green label contains 240 cubic centimeters (cc) of Fintro1"-concentrate (20% solution of active ingredient) while the blue label bottle contains 240 cc of diluent (inactive solution). These two liquids are mixed together in equal amounts to obtain a solution used to treat the pond. Treatment rates are therefore expressed as the amount of final mixture needed to treat a certainpond volume. To get a correct volume of concentrate and diluent, use Table 2 to determine the amount of final solution needed to achieve a desired concentration per acre-foot. Multiply that number by the number of acre feet to be treated. Then thoroughly mix together half that amount of ~introl@- concentrate and half that amount of diluent. For example, if you need 80 ounces of mixture to treat a pond, mix together 40 ounces of Fintrolm-concentrateand 40 ounces of diluent to obtain the 80 ounces you need. The final mixture retains potency for seven days. However, once this solutionis mixed with water, it must be applied within eight hours. As an example, let's say a catfish farmer wants to remove shad from a 10-acre catfish pond that is 4 feet deep with a water temperature of 75" F and a pH of 7.9. From Table 1, we see that a concentration of 5 ppb should selectively remove the shad without harming catfish. Table 2 indicates that we need to apply 2 ounces of FintrolB solution per acre-foot to achieve a 5 ppb concentration. Therefore, 10 acres 

### x 4 feet 

### x 2 ounces per acre-foot = 80 ounces of FintrolB solutionneeded to treat the pond. You would then mix 40 ounces of Fintro1"-concentrate and 40 ounces ofdiluentto obtain the 80 ouncesyou need. After dilution with water, the Fintrol" solution can be applied by drip tubes, spray equipment, or mixing tanks on a chemical boat. During mixing, handling, or applying FintrolB,workers should wear protective goggles and protective gloves. If any contact occurs with eyes or skin, flush immediatelywith water. Treat ponds with Fintrol" only when ponds are not overflowing. Do not treat during rainy weather or when water is being added to ponds. This will prevent the chemical from being diluted or flushed out of the pond, thereby assuring a more effective treatment. This also helps protect the environment by preventing the chemical from being discharged into other water bodies. If the chemical is fiesh and treatment rates are calculated correctly, FintrolBis one of the most dependable pond-management tools available to the catfish farmer. Fintrolmis, however, a relatively slow-acting chemical so be patient when waiting to see results. Depending on water temperature, it may be a day or two before you start to see signs of an effective treatment. As with all pesticides, read and follow all label recommendations.

Page 82 of 118Table 1. Rough estimation of concentrationsof Fintrol

@ needed for eradication of different fish speciesunder various combinationsofpH and water temperature. Effective Concentration ofFintrol (in ppb of active ingredient) pH is 8.5 or less pH is 8.5 or higher Water temp. Water temp. Water temp. Water temp. Target Species above 60" F below 60" F above 60" F below 60" F Gizzard shad, carp, minnows, 5 7.5 sunfish Catfish, bullheads 15 20

Table 2. Amount of ~introl@ solutionto achievedesired concentration. Amount of ~introl@ solution Desired (~introl@-concentrate 

### +Diluent) Concentration per acre-foot (ppb active ingredient) (ml>' (ounces)



### ............................................. 1ppb 12.3 0.50 2 ppb


### ............................................. 24.6 0.75 3 ppb


### ............................................. 36.9 1.25 4 ppb


### ............................................. 49.2 1.50 5 ppb


### ............................................. 61.5 2.00 6 ppb


### ............................................. 73.8 2.50 7 ppb


### ............................................. 86.1 2.75 8 ppb


### ............................................. 98.4 3.25 9 ppb


### ............................................. 110.7 3.75 10ppb


### ............................................. 123.0 4.00

## 14 APPENDIX 

# E Review 

## of Selected Papers
Ritter, P.O. and F.M. Strong. 1966. Residues in Tissues of Fish Killed by Antimycin. (Magnitude of Residue Study). Aquabiotics Corporation.Case No. 4121 (MRID461534-01) DeformylantirnycinA3 hydrochloride was mixed with H3-formicacid to yield ~~-antim~icin. While the resulting antimycin was as active as unlabeled (cold) antimycin in inhibiting reduced coenzyme Qcytochrome c reductase, the deformylated starting material only showed trace activity. The tritiumlabeled antimycin did not show a significant exchange of its tritium label. Small (40 - 115 g) rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinusfontinalis) and common carp (Cyprinuscalpio) were placed individually in 19-literglassjars containing 15 liters of water treated with antimycinA. After the fish died, the fish were removed and the water was discarded.Larger (230 - 320 g) brook trout and common carp (100 g) were placed individually in metal tanks containing 43 liters of water treated with antimycinA. After a trout was exposed and died, it was followedby a carp and then a trout; aRer 3 fish had been exposed successively using the same water, the water was discarded. Fish were then frozen. Afterward small trout and carp were divided into 3 factions: head plus viscera, gills, and the remainder of the body. All tissue samples were homogenized except the gills. The gills were divided as equally as possible into left and right halves. Samples were then lyophilized and the radioactivity in each freeze-dried sample was determined by combusting the sample in oxygen and collecting and counting the water sample. Spiked (H3-antimycin) samples showed recoveries of 96%. Heart, liver, kidney, muscle, gills and skin from each of the large trout were sampled separately. The remainder of the carcass was divided into two fractions: head plus remaining viscera and the balance of the carcass. Small carp (90 

### - 200 g) were collected from West Salem, Wisconsin and poisoned with 10 ppb of nonradioactive antimycin (presumably in the lab). Tissues were homogenized and then extracted with chloroform six times, the extracts combined and dried and then redissolved in ethanol. The antimycin content of the extracts was then estimated by determining the toxicity to goldfish. Glass beakers (3 L) containing2 goldfish (1 - 3 g) in 1.5 L of distilledwater. The volume of ethanol introduced with the test solution was not over 2 mL. Controls were run with 2.5 mL ethanoV1.5 L; responses were gauged relative to a standardbioassay of goldfish with antimycin at known concentrations. Spikedhomogenates were also extracted to determine the extractionefficiency. Results: Radioactive residues in tissues were assumed to be intact antimycin A. Table 1 depicts estimated antimycin tissue concentrations of fish killed by antimycin at two different treatment concentrations (5 and 10ppb). As might be expected, the time required to kill fish with antimycin tended to be shorter at higher antimycin A concentrations. Tissue residues in fish also tended to be higher at higher treatment concentrations. Overall, of the fish studied (16), tissue residues averaged 203 pglkg fish weight. Tissue residues in larger trout killed with antimycin at 10ppb were similarto residues in small trout killed with antimycinat 5 ppb; the lowest tissue concentrationswere identified in larger trout killed with antimycin at 5 ppb. Ofthe fish studied, carp appeared to be the least sensitive to antimycin A and required the longest time to kill; carp requiredroughly 5 times longerto die from antimycin than the trout. Page 84 of 118Table 1. Whole body H~-antimycin levels in fish killed with antimycin. Number Used Species Survival Time Antimycin Tissue ~esidue' (hrs) Dosage (PP~) pglkg fish weight 4 carp 15-22 5 219,238,247,286 4 carp 12 - 16 10 323,334,355,450 3 Small Trout 2.5 - 16 5 109, 135, 137 3 Small Trout 2-4 10 152,160,172 2 Larger Trout 2-5 5 59.2,92.3 2 Larger Trout 2-3 10 109,79.3 ITissueresidues based on radioactivity and presume radioactivity reflects 100% antimycin.
Fish

Brook Trout AntimycinResiduespglkg fresh weight Body Survival Number Weight (g) Time (hrs)

Carp 1 106 22.2 5 299 460,480 139,146 2 112 20.3 5 279 473 167,164 3 107 20.8 5 238,242 362,417 218,203 4 98.5 15.3 10 33 1 700,780 261,248 5 100 14.3 10 386,406 413 290 6 90.2 16.7 10 298,275 751,811 190,181 Antimycin Concentration Head and @pb) Gills Viscera Remainder

Brook trout and carp treated with 5 ppb antimycin tended to take longer to die than when fish were treated at 10ppb. Although trout treated with 5 ppb showed that the highest tissue residues (pg/kg fish weight) based on radioactivity were associated with the head and viscera, trout treated with 10ppb tended to show the highest residues in the gills (Table 2). Once again, carp treated with antimycin took roughly 5 times longer to die than trout; however, the carp were roughly 2 times as large as the trout. For carp, the highest tissue residues of antimycin were associated with the head and viscera. Page85 of 118Table 3 summarizes the tissue distribution of H) antimycin [equivalents in pgtkg] and indicates that residues appear to be predominately located in the liver and kidney. In this case, survival time was not clearly correlated with dose, as fish treated with 5 and 10 pg/L took roughly equivalent periods of time to die. The authors conclude from Tables 2 and 3 that the lowest antimycin concentrationsoccurred in the body. Average residues inthemuscle were 79 i 14.4 pglkg (mean k std error). The authors concludethat the ability of the fish to absorb and accumulate antirnycin against a steep concentration gradient, e.g., 5 ppb in the water versus up to 900 ppb in certain tissues, points to a firm binding of the toxicity with some body constituents which may be the antimycin-sensitive site ofthe electron transport system. ion of antimycin in rainbow trout killedwith II3-antimycin. Table 3. Tissue Fish No. Body Wt. Survival Time Dose 1 319 g 2.7 hrs 5 PP~ 2 263 g 2.3 hrs 10 PP~ 3 230 g 5.0hrs 5 PP~ 4 271 g 2.7 hrs 10 PP~ Antimycin Equivalentto Radioactivity@@kg Fresh Weigh I I I I distribul

Liver

470 475 563 502 954 790 762 855 702 795 797

670 538 Head plus Kidney Gills Muscle Viscera

300 143 74.4 38.1 74.6 Remainder of Body

The authors attemptedto quantify the tissue concentrationsof intact antimycin A by extractingthe tissues with solvent and then exposing carp to the concentrated extract in a mixture of acetone and ethanol and compare the results to survival times at known concentrations of antimycin. Fish survival times after treatment at 2, 5 and 10pg/L were 9.5 hrs, 2.7 to 6.8 hrs, and 4.6 hrs, respectively. Based on this response and the survival time of carp treated with the tissue extracts, estimates of antimycin ranged from 30 pglkg to less than 15pg/Kg (Table 4). Recovery of antimycinfrom spiked tissuesranged between 8.5 and 21%. While these data suggest that fish killed with antimycin have sufficient antimycin in their tissues to be toxic to other fish, the poor recoveries fiom spiked samplesmake it difficult to even qualitatively estimate tissue residues. The authors conclude,though, that given the oral LD50 values reported for mammals (10 - 55 mgkg) and birds (2.9 mgkg), coupled with the results from feeding studies where rats were fed 50% of their diet composed of fish killed with 5 to 10 ppb of antimycin, no ill effects are likely from the consumption of fishkilled with antimycin.Table4. Goldfish assay of antimycin extracted from homogenized carp tissue.

100-g Carp

95-g control carp with 35.5 pg 

# I 6.5, 8.5 

# I 7.5 pfig 

# I Qualitative Estimate of Activity Sample Tested
200-g Carp 93-g control carp with 35.5pg

Uncertainties: It is unclear whether the 96% recovery of labeled material following combustion and counting also included the lyophilizationprocedure. It's not clear from the study to what concentration of antimycin (labeled and cold combined) the fish were actuallyexposed. Survival Time (hours) 8.1, 9.7

The goldfish bioassay for estimating antimycin A residues in tissues contained roughly 1.3 mL/L (2 W1.5L) and exceeds the EPArecommended maximum of 0.1 mL/L. 30 clg/Kg 13.7, 14.7 8.8, 10.1

Water quality conditions are not reported for the bioassays. According to the methods section, distilled water was used. Distilled water can be very damaging to fish and is contrary to EPA's recommended use ofreconstitutedwater or natural waters that have been characterized. 45 ~flg 3.5 ~flg

The assumption that radioactive residues in tissues are representative of parent antimycin A was not adequately substantiated. The authors acknowledge the questionable validity of their assumption since antimycin was labeled in its formyl group and this group isrelatively labile. The authors conclusions that the ability of the fish to accumulate antimycin against a steep concentration gradient points to a firm binding of the toxicant with some body constituent that may be the antimycin sensitive site of the electron transport system is not supported by the data. Rather, these data suggest that antimycinis lipophillicand that it is partitioning to tissues where lipids are more likely.Thompson, C.M., J. Griffen, P. Boudreau, W. Cranor, and K. Lawsin. 1980. Acute Toxicity of BOLERO 10G (SX-1252) to Rainbow Trout (Oncorhynchusmykiss formerly Salmogairdneri). ABC Analytical Bio Chemistry Laboratories, Lnc., P.O. Box 1097, Columbia, MO. ABC Report #26078. (Accession No. 00050664; MRID 2401255-02). Although the study focused on ~olero~ 10G (Thiobencarb), the positive control for this static study was antimycinA (Sigma Chemical Co.; Type 111, crystalline,Lot 12%-0152). Report cites following Standard Methods for Examination of Water and Wastewater and Methods of Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians. Trout (mean wt = 1.1 g; mean length 41 mm) obtained from Mt. Lassen Trout Hatchery (Red Bluff, CA); 14-day acclimationperiod. Animals fasted 48 hrs prior to study. Study conducted in 19-liter glass vessels with 15 liters of soft, reconstituted water (DO 8.9 mg/L, pH 7.6, hardness 45 mg/L at CaC03; 12°C; 16-hr light: 8-hr dark cycle). Each treatment consisted of 10 fish. Study conducted in compliance with Good Laboratory Practice Regulations. DO dropped as low as 5.5 ma. Uncertainties: Procedure for measuring concentrations of thiobencarb are reported; however, it is not stated whether the concentration of antimycin was measured. Purity of antimycin A is not specified. Test concentrations used to determine the LCSo of antimycin A are not reported and no raw data on mortality resulting from exposure to antimycin A are provided. At normal atmospheric pressure (760 mm Hg) and at 12"C,DO saturation is 10.8mg/L; however, with readings of 5.5 mg/L at 96 hrs, the DO is at roughly 51% saturation and is below the recommended limit of 60% saturation. 48 hr LCSo= 0.00014 mg/L (95% CI: 0.000075 

### - 0.00024mg/L) 96hr LC50= 0.000062 mg/L (95% CI: 0.000042 

### - 0.00014 mg/L)
Hogan, J. 1966. Antimycin as a Fish Toxicant in Catfish Culture. Presented at the 20' Annual Meeting of the southeastern Association of Game and Fish Commissioners, Oct 24 - 26, 1966. Bureau of Sport Fisheries and Wildlife, U.S. Fish and Wildlife Service. (AccessionNo. 00045801; MRlD 2400798-13) Liquid formulation: crystalline antimycin dissolved in acetone diluted to obtain antimycin concentrations ranging from 0.0 1ppb to 500 ppb. Sand formulation: Fintrol 5 (antimycin incorporated in Carbowax and coated on 40-mesh sand; sand to carbowaxweight ratio 1:99) Twelve species of fish utilized in the study: paddlefish (Polyodon spathula), gizzard shad (Dorosoma cepedianus), goldfish (Carassius auratus), carp (Cyprinus calpio), fathead minnow (Pimephales promelas), buffalo hybrid (Ictiobus spp.), white catfish (Ictalurusfurcatus), channel catfish (I. punctatus), green sunfish (Lepomis cyanellus), bluegill (L. macrochirus), and largemouth bass (Micropterus salmoides). Eggs of carp and channel catfish obtained from Marion National Fish Hatchery,AL; goldfish eggs obtained fkom Southeastern Fish Control Laboratory. Channel catfish eggs treated with acriflavineto inhibit hgalgrowth. All eggs less than 48-hrs old when exposed to antimycin. Prolonged exposure consisted of exposing eggs until all had visibly hatched; 2-hr exposures and prolonged exposures conducted according to Berger, B.L., R.E. Lennon, and J. Hogan. Investigations in Fish Control: Laboratory studies on antimycin as a fish toxiciant. Bureau of Sport Fisheries and Wildlife, Resource Publication. Fry obtainedby hatching channel catfisheggs at laboratory. Adult fish treated in 0.25-acre pond at the Fish Farming Experimental Station, Stuttgart, Arkansas. Page 88 of 118Four vinyl wading pools containing 3,785 liters of water stocked with 80 channel catfish fingerlings from the same lot. A bioassay cage containing 10 bluegillswas placed in each pool. Two weeks later, Fintrol5 (10 ppb) was added to two pools while the other two pools served as controls. Cage containing dead bluegills was emptied and an additional 10bluegills were added to cage. Catfish in each pool fed daily at a rate of 3% of body weight; however, because of decreasedwater temperatures and ice formation, feeding was reduced to once or twice a week. Four months after stocking, the pools were drained and the surviving channel catfish were counted,weighed and measured. Field Trial: 0.25-acrepond treated in mid-December (watertemp=ll°C); by Day 8 water temperatureshad declined to 4.4OC. Seining indicated the presence of paddlefish, shad, carp, buffalo, white catfish, blue catfish, and channel catfish. Pond treated 10ppb antimycin (400 g Fintrol 5). Stability of the antimycin tested by placing cages containing 10 fingerlingrainbow trout into treated pond. ResultsEgg Studies Control mortality in goldfish study was 37%. Among antimycin-treated goldfish, there was 83% mortality at 1 ppb and 100% mortality at 2.5, 5.0 and 10 ppb. Control mortality in the carp study was 81.3%; average mortality in antimycin-treated carp was 73% at 1.0ppb and 100% at 5 and 10ppb. For channel catfish, mortality was 4%; 100% of the eggs treated with antimycin at 27.5 ppb or higher died (failed to hatch); at 25 ppb, there was 74% mortality; antimycin concentrations of 20 ppb or less did not appear to affect the hatchability of the eggs. Treatment of channel catfish eggs with antimycin for 2 hours at 500 ppb resulted in 100% mortality while treatment with 250 ppb resultedin 81%mortality. Concentrationsat or less than 100ppb for two hours did not affect total hatch; control fish exhibited 95% hatching success. Fry Studies Channel catfish control mortalitywas 1 1 %; for fingerlings treated with 0.75ppb there was 100%mortality within 96 hours. Fingerling Bioassay 96-hr LCSo Values goldfish: 0.137 ppb carp: 0.200 ppb fatheadminnow: 0.074 ppb green sunfish: 0.060 ppb bluegill: 0.051 ppb largemouthbass: 0.104 ppb channel catfish: 8.4 ppb Based on these data, the authors conclude that on average channel catfish are 42 to 165 times less sensitive to antimycin than fingerlings of 6 other species. Tests in VinylPools After 4 months of acclimatization,control survival averaged 88% while survival in treated pools averaged 97%. Page 89 of 118Uncertainties: Purity of the antimycin liquid formulation is not specified;purity of the sand formulationis not specified. Presumably, all 7 species of adult fish tested (paddlefish, shad, carp, buffalo, white catfish, blue catfish and channel catfish) were tested concurrently in the same pond. Static bioassays were conducted in indoor and outdoor facilities. Indoor tests with eggs, fry and fingerling fish conducted in accord with Berger et al. Water temperatures in catfish study presumably fluctuated considerably and there was ice formation in the water. Feeding rates varied because of fluctuating water temperatures. Presumably, mortality in the egg study is referringto hatching success. Control mortality in the 0.25-acre ponds (37% for goldfish and 8 1%for carp) was above the EPA-recommendedmaximum of 20%.

Kuhn, J.O. 200 1

. Acute Oral Toxicity in Rats. Stillmeadow, Inc. 12852 Park One Dr., Sugar Land, TX. Laboratory Study No. 6025-00. Sponsor: Aquabiotics Corporation, 10750 Arrow Point Dr., Bainbridge Island, WA. (MRID455279-01) Study is GLP-compliant. Study conducted according to OPPTS 870.1 100. Fintrol Concentrate(20%); certificate of analysisnot provided. Dark brown liquidmaintained at room temperature. Sprague-Dawley rats (approximately 2 months old) from Texas Animal Specialists, Humble, TX, acclimated for 5 days. Twenty males (234 

### - 350 g) and twenty females (158 

### - 210 g) housed individually in suspended, wire bottom stainless steel cages. Animals fed Formulab #5008 (PMI Feeds Inc) ad libitum except 16hrs before dosing. Drinking water was from municipal water supply. Test substance mixed with acetone to produce 10% vlv concentration. Treatment levels consisted of 50, 200, 350 and 500 mglkg. Presumably, the highest treatment level received 5 rnL/kg of the test mixture. Body weights measured just prior to dosing and on Days 7 and 14post-dosing. Observations for mortality and clinicalibehavioral signs of toxicity were made 3 times the day of dosing and at least once daily thereafter for 14days. On Day 14, animals were euthanizedby C02and necropsied. Table 1. Summary of 14-day male, female and combined rat mortality following oral administration of antimycinA. I 1

The authors calculated the 14-day LD50 as follows: mean (95% confidence interval) male 286 (258 

### - 3 12mglkg) female 36 1 (344 - 3 8 1mgkg) Dose Level mgfkg

50 200 350 500 Number Deadmumber Treated rnL/kg

0.5 2.0 3.5 5.O Males 015 115 315 515 Females 015 015 215 515 Combined 0110 1110 5/10 10110combined 3 16 (29 1 -343mglkg) Clinical signs included decreased activity, ataxia, circling, diarrhea, hunched posture, ocular discharge, piloerection, respiratory chirplgurgle, soft feces, splayed legs and walking on tiptoe. No clinical signs were apparent in surviving animals after 12 days. Gasping, lateral recumbency, muscle tremors, ptosis, salivation and swollenlredpenile area was observed only in animals that died on test. No effect onbody weight was reported except in one animal treated with 50 mgkg. Reviewer comments: No solvent control was run. Given the increasing amount of acetone administered to the animals with increasing concentration of antimycin, it would be helphl to know whether the highest dose of acetone alonehad any effect on the animals. Probit Analysis: using the Probit procedure of the Statistical Analysis System (Release 8.2; SAS Institute, Inc., Cary, NC), the following LD50 values were calculated: Males: 285 mglkg (95% CI: 152

### - 419 mgkg) Slope: 6.506 Females: 354mglkg (95% CI: -) Slope: 46.92 Combined: 3 1 7 mglkg (95% CI: 253 

### - 386 mag) Slope: 7.759PROBIT ANALYSIS (LOG10) OF Male Rat MORTALITY AFTER 14-Days Probit Procedure Iteration History for Parameter Estimates
Iter Ridge Loglikelihood Intercept LoqlO(D0SE)

Model Information
Data Set Events Variable Trials Variable Number of Observations Number of Events Number of Trials Name of Distribution Log Likelihood WORK. MALES RESPONSE N 4 9 20 Normal -6.350107015

Parameter Information

Parameter Effect Intercept Intercept DOSE DOSE

Last Evaluation of the Negative of the Gradient Intercept LoglO(D0SE)

Last Evaluation of the Negative of the Hessian Intercept Log10 (DOSE) Intercept 6.2699709446 15.556556047 LoqlO(D0SE) 15.556556047 38.732077222 PROBIT ANALYSIS (LOG10) OF Male Rat MORTALITY AFTER 14-Days Probit Procedure Algorithm converged.

Goodness-of-Fit Tests Statistic Value
Pearson Chi-Square L.R. Chi -Square
Response-Covariate Profile Page 92 of 118Response Levels 2 Number of Covariate Values 4 Since the chi-square is small (p > 0.1000), fiducial limits will be calculated using a t value of 1.96.

Type I11 Analysis of Effects

Effect Wald DF Chi-Square Pr > ChiSq Log10 (DOSE) 1 5.6883 0.0171
Analysis of Parameter Estimates Standard 95% Confidence ChiParameter DF Estimate Error Limits Square Pr >. ChiSq Intercept 1 -15.9747 6.7801 -29.2634 -2.6860 5.55 0.0185 LoglO(D0SE) 1 6.5061 2.7279 1.1595 11.8527 5.69 0.0171

Estimated Covariance Matrix Intercept LoglO(D0SE) Intercept 45.969367 -18.463379 LoglO(D0SE) -18.463379 7.441548

PROBIT ANALYSIS (LOG10) OF Male Rat MORTALITY AFTER 14-Days Probit Procedure Probit Model in Terms of Tolerance Distribution
MU SIGMA 2.45533211 0.15370139

Estimated Covariance Matrix

for Tolerance Parameters

MU SIGMA
MU 0.003885 -0.000697 SIGMA -0.000697 0.004153 PROBIT ANALYSIS (LOG101 OF Male Rat MORTALITY AFTER 14-Days

Probability Probit Procedure Probit Analysis on LoglO(D0SE) LoglO(D0SE) 95% Fiducial Limits 2.09777 0.30737 2.13967 0.53976 2.16625 0.68684 2.18625 0.79726 2.20252 0.88689 2.21636 0.96304 2.22850 1.02967 2.23937 1.08922 2.24926 1.14327 2.25836 1.19291 2.29603 1.39717

Page 93 of 1180.20 2.32597 1.55740 2.44912 0.25 2.35166 1.69257 2.47219 0.30 2.37473 1.81124 2.49563 0.35 2.39611 1.91781 2.52074 0.40 2.41639 2.01450 2.54900 0.45 2.43602 2.10217 2.58223 0.50 2.45533 2.18069 2.62269 0.55 2.47465 2.24940 2.67295 0.60 2.49427 2.30788 2.73537 0.65 2.51456 2.35663 2.81157 0.70 2.53593 2.39730 2.90259 0.75 2.55900 2.43214 3.00986 0.80 2.58469 2.46352 3.13672 0.85 2.61463 2.49387 3.29082 0.90 2.65231 2.52638 3.49039 0.91 2.66141 2.53360 3.53922 0.92 2.67129 2.54124 3.59248 0.93 2.68216 2.54941 3.65127 0.94 2.69430 2.55830 3.71716 0.95 2.70815 2.56818 3.79257 0.96 2.72441 2.57948 3.88147 0.97 2.74441 2.59300 3.99114 0.98 2.77100 2.61046 4.13743 0.99 2.81290 2.63710 4.36889 PROBIT ANALYSIS (LOG10) OF Male Rat MORTALITY AFTER 14-Days Probit Procedure Probit Analysis on DOSE Probability DOSE 95% Fiducial Limits 0.01 125.24754 2.02940 196.51730 0.02 137.93296 3.46543 208.50207 0.03 146.63968 4.86233 216.66029 0.04 153.54979 6.26987 223 .I2724 0.05 159.41009 7.70713 228.62391 0.06 164.57401 9.18411 233.48878 0.07 169.23920 10.70710 237.91027 0.08 173.52844 12.28056 242.00518 0.09 177.52364 13.90803 245.85151 0.10 181.28249 15.59247 249.50439 0.15 197.71101 24.95572 266.01352 0.20 211.82331 36.09130 281.26891 0.25 224.73054 49.26892 296.61507 0.30 236.99055 64.75072 313.06020 0.35 248.94753 82.75820 331.69889 0.40 260.85088 103.39622 354.00085 0.45 272.90895 126.52444 382.14562 0.50 285.31993 151.59697 419.45988 0.55 298.29532 177.58408 470.92802 0.60 312.08429 203.17883 543.71895 0.65 327.00651 227.31810 647.99672 0.70 343.50511 249.63332 799.07375 0.75 362.24477 270.48260 1022.95451 0.80 384.31777 290.74757 1370.00320 0.85 411.74977 311.79589 1953.51927 0.90 449.06413 336.03323 3093.04746 0.91 458.57252 341.66721 3461.14703 0.92 469.13039 347.72631 3912.74395 0.93 481.02012 354.33209 4479.88125 0.94 494.65565 361.66008 5213.84388 0.95 510.67947 369.97876 6202.55507 0.96 530.16981 379.73188 7611.56116 0.97 555.15302 391.73958 9798.07301 0.98 590.19588 407.81322 13722.4898 0.99 649.97255 433.60996 23382.7088 PROBIT ANALYSIS (LOG10) OF Female Rat MORTALITY AFTER 14-Days Page 94 of 118Probit Procedure

Iter

0

PROBIT ANALYSIS Iteration History for Parameter Estimates Ridge Loglikelihood Intercept LoglO(M)SE)

Model Information
Data Set Events Variable Trials Variable Number of Observations Number of Events Number of Trials Name of Distribution Log Likelihood WORK.FEMALES RESPONSE N 4 7 20 Normal -3.365058335
lLOG10) OF Female Rat MORTALITY AFTER 14-Days Probit Procedure
Parameter Information

Parameter Effect Intercept Intercept DOSE DOSE

Last Evaluation of the Negative of the Gradient Intercept LoglO(D0SE)

Last Evaluation of the Negative of the Hessian Page 95 of 118Intercept LoglO(D0SE) Intercept 3.1095948584 7.9110209101 LoglO(D0SE) 7.9110209101 20.126175496
Algorithm converged.

Goodness-of-FitTests Statistic Value DF Pr > ChiSq
Pearson Chi-Square L.R. Chi-Square
Response-Covariate Profile Response Levels 2 Number of Covariate Values 4 Since the chi-square is small (p > 0.1000), fiducial limits will be calculated using a t value of 1.96.
Type I11 Analysis of Effects Wald Effect DF Chi-square Pr > ChiSq LoglO(D0SE) 1 0.0000 0.9999 PROBIT ANALYSIS (LOG10) OF Female Rat MORTALITY AFTER 14-Days Probit Procedure Analysis of Parameter Estimates Standard 95% Confidence Chi- Parameter DF Estimate Error Limits Square Pr 7 ChiSq Intercept 1 -119.625 966847.0 -1895105 1894866 0.00 0.9999 LoglO(D0SE) 1 46.9217 380039.8 -744817 744911.2 0.00 0.9999

Estimated Covariance Matrix Intercept LoglO(D0SE) Intercept 934793186240 -3.674403311 LoglO(D0SE) -3.674403311 144430226507

Probit Model in Terms of Tolerance Distribution
MU SIGMA

Estimated Covariance Matrix

for Tolerance Parameters

MU SIGMA MU 1912.467729 7548.803851 SIGMA 7548.803851 29796.290457 PROBIT ANALYSIS (LOGLO)OF Female Rat MORTALITY AFTER 14-DaysProbit Procedure Probit Analysis on LoglO(D0SE) Probability LoglO(D0SE) 95% Fiducial Limits 0.01 2.49989 0.02 2.50570 0.03 2.50938 0.04 2.51216 0.05 2.51441 0.06 2.51633 0.07 2.51802 0.08 2.51952 0.09 2.52089 0.10 2.52215 0.15 2.52738 0.20 2.53153 0.25 2.53509 0.30 2.53829 0.35 2.54126 0.40 2.54407 0.45 2.54679 0.50 2.54947 0.55 2.55215 0.60 2.55487 0.65 2.55768 0.70 2.56064 0.75 2.56384 0.80 2.56740 0.85 2.57156 0.90 2.57678 0.91 2.57804 0.92 2.57941 0.93 2.58092 0.94 2.58260 0.95 2.58452 0.96 2.58678 0.97 2.58955 0.98 2.59324 0.99 2.59905 PROBIT ANALYSIS (LOG10)OF Female Rat MORTALITY AFTER 14-Days

Probability Probit Procedure Probit Analysis on DOSE DOSE 95% Fiducial Limits 316.14627 320.40385 323.13482 325.20456 326.89790 328.34615 329.62126 330.76716 331.81278 332.77819 336.80524 340.04054 342.84089 345.37534 347.74061 350.00000 352.19995 354.37853 356.57058 358.81183 Page 97 of 1180.65 361.14315 0.70 363.61641 0.75 366.30444 0.80 369.32109 0.85 372.86873 0.90 377.38092 0.91 378.47892 0.92 379.67536 0.93 380.99528 0.94 382.47484 0.95 384.16931 0.96 386.16969 0.97 388.64317 0.98 391.95579 0.99 397.23429 PROBIT ANALYSIS (LOG10) OF Male and Female Rats Combined MORTALITY AFTER 14-Days 11 Probit Procedure Iteration History for Parameter Estimates Iter Ridge Loglikelihood Intercept LoglO(D0SE)

Model Information
Data Set Events Variable Trials Variable Number of Observations Number of Events Number of Trials Name of Distribution Log Likelihood WORK.COMBINED RESPONSE N 4 16 40 Normal -11.29675287

Parameter Information

Parameter Effect Intercept Intercept DOSE DOSE
Last Evaluation of the Negative of the Gradient Intercept LoglO(D0SE)

Last Evaluation of the Negative of the Hessian Intercept LoglO(DOSE) Intercept 11.260491197 28.307489577 LoglO(D0SE) 28.307489577 71.367913469 PROBIT ANALYSIS (LOG10)OF Male and Female Rats Combined MORTALITY AFTER 14-Days 12Probit Procedure Algorithm converged.

Goodness-of-Fit Tests Statistic Value DF Pr > ChiSq
Pearson Chi-Square L .R . Chi-Square
Response-Covariate Profile Response Levels 2 Number of Covariate Values 4 Since the chi-square is small (p > 0.1000), fiducial limits will be calculated using a t value of 1.96.
Type 111 Analysis of Effects

Effect Wald DF Chi-Square Pr > ChiSq

Analysis of Parameter Estimates Standard 95% Confidence Chi- Parameter DF Estimate Error Limits Square Pr > ChiSq Intercept 1 -19.4043 5.5418 -30.2660 -8.5425 12.26 0.0005 LoglO(D0SEl 1 7.7594 2.2013 3.4450 12.0739 12.43 0.0004

Estimated Covariance Matrix Intercept LoglO(D0SE) Intercept 30.711791 -12.181577 LoglO(D0SE) -12.181577 4.845733

PROBIT ANALYSIS (LOG10) OF Male and Female Rats Combined MORTALITY AFTER 14-Days Probit Procedure Probit Model in Terms of Tolerance Distribution
MU SIGMA

Estimated Covariance Matrix

for Tolerance Parameters

MU SIGMA
MU 0.001489 -0.000136 SIGMA -0.000136 0.001337 PROBIT ANALYSIS (LOG10) OF Male and Female Rats Combined MORTALITY AFTER 14-Days Probit Procedure Probit Analysis on LoglO(D0SE) Page 99 of 118Probability LoglO(D0SE) 95% Fiducial Limits

Page 100of 118PROBIT ANALYSIS (LOG10) OF Male and Female Rats Combined MORTALITY AFTER 14-Days 15 Probit Procedure Probit Analysis on DOSE Probability DOSE 95% Fiducial Limits

Page 101 of 118Arslaneglau, L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration ofWater Treated with Antimycin to Rats and Dogs. Ayerst Research Laboratories.(MRID 2400798-10) Singlenominal concentration of antimycin (R-17,001-17, Lot P 13326)at 125 ppb Antimycin of unknown purity (12.5 mg) was dissolved in 200 ml of 70% ethanol and added to 100 liters to yield a final concentration of 125 ppb. Since the purity of antimycin is not stated, the actual concentration of antimycin A in solution is unknown. Co-solvent concentration is 2 mVL and exceeds EPA-recommended concentrationof 0.1 mVL. "Certified" food coloring (unspecified quantity or quality) added to distinguish treatment groups. Stock solution of drinking water stored in plastic bottles and aerated once weekly throughout the test. The conditions under which the stock solutions were stored were not specified. Four-month-old beagles (4 males and 4 females) were exposed to either control or antimycin-treated water. Although the study reported that control animal water was treated in the same way it is unclear whether there was a solvent control. Animals provided 1L of treated or control water per day along with 400 g of Purina dog chow. Daily food and water consumption measured along with body weight. Prior to start of study and afterwards at 4-wk intervals, hematology (blood cell counts, white blood cell differential, hemoglobin, prothrombin time, sedimentation rate and hematocrit), blood chemistry (alkaline phosphatase, serum glutamic-oxaloacetic transaminase, blood urea nitrogen, lactic dehydrogenase and glucose) and urinalysis (albumin, ketone bodies, sugar; appearance) measured. Afterwards, dogs were sacrificed and the following organs measured; thymus, thyroid, adrenals, pituitary gland, spleen, heart, liver, brain, kidneys, testes, prostate, uterus, ovaries, lung, pancreas and submaxillary salivary glands. Tissue samples collected for histology; additional tissues for histology included: eye, trachea, lymph node, esophagus, skin, stomach, small intestine, large intestine, sternal bone, urinary bladder, aorta, gall bladder, muscle and mammary gland. Additionally 50 

### - 60 g Charles River strain rats (20 males and 20 females) received treated or control water for 3 months. Animals were housed 6 - 7 ratslcage at 74 - 76°C and 46 

### - 48% humidity. Prior to and at regular intervals during treatment, the following data were collected: weekly individual body weights; daily food and water intake [for each group housed together], hematology (red and white blood cell counts, differential white blood cell count and hemoglobin determination at 4-wk intervals), plus daily clinical observations. At the end of the study, the animals were sacrificed and the following organs measured: thymus, thyroid, adrenals, pituitary gland, spleen, heart, liver, brain, kidneys, testes, prostate, seminal vesicles, uterus, ovaries and submaxillary glands. Additional samples were taken for histology and included: eye, trachea, lymph node, esophagus, skin, stomach, small intestine, large intestine, sternal bone, urinary bladder, aorta, muscle, lung, pancreas and mammary gland. Results According to the authors, with one exception, there was a decrease in the intake of treated water; the treated water tasted bitter [to man] and the authors speculated that the bitter taste may have "caused" the lower water intake during the first month of the study. The authors also stated that lower than normal water intake will result in lower food intake and that this will be reflected in the body weight gainbut that the growth pattern is considered normal. Only line graphs are presented to depict water and food intake and no raw data are available for analysis in the report. The graphs suggest that for male dogs, food and water intake was reduced for the first 5 weeks of the study. For female dogs, both water and feed consumption were considerably more erratic;however, there appeared to be a trend where both water and feed consumption were reduced for treated animals during the first 5 weeks of the study. Body weights Page 102of 118among treated male dogs were also reduced relative to controls; however, the same pattern was not apparent for females. For rats, there was no apparent difference in either food or water intake between control and treated males or females. The study authors did not observe any differencesbetween control or treated male or female dogs or rats for any of the other measurement endpoints. However, it is unclear whether any statistical analysis had been conducted. The study concludes that water treated with 125ppb of antimycin is not toxic per se and does not degrade to products which aretoxic when consumedby rats and dogs for three months. Reviewer's comments: This study is classified asunacceptable since the purity ofthe test substance is not stated. Exposure has not been verified through measurements. The study does not contain sufficient detail to determine whether controls were negative controls (water only) or solvent controls and therefore whether the decreased water intake observed among dogs was due to a solvent effect or whether it was due to antimycin. Rejection Factors: Purity ofthe test compound is not stated and nominal concentration was not verified. Treatment solution was aerated; however, concentration was not verified. Co-solvent (ethanol) concentration2 mVL exceeded the recommended concentrationof 0.1 ml/L and no solvent control was included. Raw data are not available to test whether there are statisticaldifferences for food and water consumption and forbody weights.

Page 103of 118Arslaneglau, L. and V. Korths. 1967. Antimycin Toxicity Studies: Administration of Fish Killed with Antimycin to Rats and Dogs. Ayerst ResearchLaboratories. (MRID 2400798-08)

Frozen rainbow trout (0.6 - 1 lb each), killed by antimycin at 125 ppb or by suffocation. Cooked fish mixed with standard laboratoryanimal diets in a preparation of 1:1. RejectionFactors: Treated diets were prepared by first cookingthe fish. The effectsof heat on antimycin areunknown. Review discontinued since quality ofthephotocopy was so poor.

Page 104of 118Hobbs. M. S., R. S. Grippo, J. L. Farris, B. R. Griffin and L. L. Harding. 2006. Comparative Toxicity of Potassium Permanganate to Nontarget Aquatic Organisms. Environmental Toxicology and Chemistry 25(1): 3046 -3052.
In-house cultures of cladocerans (Daphnia magna, Ceriodaphnia dubia), fathead minnow (Pimephales promelas), a midge (Chironomustentans) and an amphipod (Hyalella azteca) at Arkansas State University Ecotoxicology Research Facility used in 96-hr acute toxicity tests. Treatment solutions were renewed at 48 hours for the cladocerans and the fathead minnow following U.S. EPA procedures; however, amphipod studies were conductedwith a substrate (spiked water) and were static 96-hrtoxicity tests without renewal and reportedly conducted according to U. S. EPA and American Public Health Association water-only sediment test procedures. Cladocerans were <25 hours old, midges were third instars, amphipods were 7 - 14 days old and fatheadminnows were 4 - 10days old. Water temperature for cladoceran and fish was 25 

### + 1°Cwhile midges and amphipods were tested at 23 

## + 1°C. Substrate for toxicity tests with Hyalella 

### - consisted of two 2.5-cm diameter maple leaf discs; substrate for toxicity tests with chironomids consisted of glass beads (150 - 212 p). Testing consisted of 5 cladocerans per replicate with 4 replicates per treatment with six treatment concentrations. For the remaining test species, there were 10 organisms per replicate with 4 replicates per treatment and six treatment concentrations. Organisms were not fed during treatments. Tests were conducted with "moderately hard" syntheticwater (hardnessrange: 89 - 100mg/L as CaCO,) and experimentalpond water water (hardness range: 87 - 104mg/L as CaCO,) obtained from H.K. Dupree Stuttgart National Aquaculture Research Center in Stuttgart, AR, with historical records of previous chemicaltreatments. Total organic carbon in synthetic and pond water was 24.3

# + 1.04 and 34.8 + 26.7 ma, respectively. Dissolved oxygen ranged between 7.5 to 8.1 mg/L. Measured concentrations 

### - of MnO, ranged between 86.5 to 109% of nominal in syntheticwater; however, measured concentrations ranged between 5.5 - 65% of nominal in pond water. In syntheticwater, the pH ranged between 7.95 to 8.14 and in pond waterpH rangedbetween 8.06 - 8.63. Table 1 summarizesthe 96-hr LC,, and no-observed effect concentrations (NOEC) for aquatic animals in synthetic water and in pond water. The study authors and the data suggestthat potassium pennanganate is less toxic to the aquatic speciestested when studies were conducted in pond water and they conclude that pond water offers an ameliorating effect on the toxicity of potassium pennanganate. The decreased toxicity of permanganate in the pond water is attributed to higher "potassium permanganate demand" (PPD) related to the higher organic carbon. Table 1. Acute toxicity estimates (96-hr LC,, and NOEC) forpotassium permanganate in moderately hard

This study provides useful information on the acute toxicity of potassium permanganate to aquatic organisms. Mean measured concentrations of permanganate were unreasonably low in the pond water; therefore, exposurein this portion ofthe study is uncertain. synthetic and pond water based on nominal treatment concentrations.

Page 105of 118 Test Organism Daphnia rnagna Ceriodaphnia dubia Pimephalespromelas Chironornustentans Hyafellaazteca SyntheticWater 96-hr LC,

mdL 0.053 0.058 2.13 4.43 4.74 SyntheticWater 96-hr NOEC mgk 0.049 0.047 1.36 <3.2 3.56 Pond Water 96-hr LC,

mp%

1.98 2.39 11.28 13.55 12.30 Pond Water 96-hr NOEC mg/L 1.75 2.25 9.45 9.45 7.83Cerreto, K. M. 2004. Antimycin and rotenone: short-term effects on invertebratesin first order, highelevation streams. A master's thesis submitted to the Department of Zoology and Physiology and the Graduate School ofthe University ofWyoming. In conjunction with the Wyoming Game and Fish Department, this study examined the effect of antimycin and rotenonelantimycintreatments of two streams within the LaBarge Creek watershed in the Bridger-Teton National Forest of western Wyoming. Both streams are first-order, ground water-fed tributaries of LaBarge Creek and are located at an average elevation of 2,400 m. Cabin Creek was treated with antimycin alone in the summer of 2002 and 2003 while Indian Creek was treated with both antimycin and a combination of antimycin and rotenone in 2001, 2002 and 2003. Both streams were treated during the summer; drift and bioassays were conducted during the treatments in 2002 and 2003. An insect bioassay was also conducted in a third stream (Crystal Creek) that was treated for the first time with antimycin alone in 2003. The headwater portions of the streams had summer temperatures at or below 5°C while the lower reaches had water temperatures as high as 20°C during the day; pH ranged between 8.7 and 9.0 in the afternoon. Higher water temperatures in the lower reaches were considered due to decreased tree canopy comparedto headwater portions. Antimycin A in an acetone carrier was added to streams over an 8-hr period at an estimated treatment concentration of 10 pgL using multiple drip stations. In addition to the antimycin A treatment, Indian Creek was also treated with a 1 mg/L solution of rotenone in kerosene carrier applied using a backpack sprayerto backwater areas and groundwater seeps yielding an approximated rotenone concentration in the water of 0.1 - 0.3 pa. Untreated headwater portions of each of the streams served as reference sites; however, these sites were smaller in terms of channel width and length relative to the treatment reaches. A potassium permanganate detoxification station was established near the confluence of each treatment stream and LaBarge Creek. Potassium permanganate was used at 2 mgL over a 20-hr period to deactivate the piscicides. Bioassays using brook trout were used to monitor whether treatments were killing fish. In Cabin and Indian Creeks invertebrate drift was measured over a 12 - 14 hour period starting 1 hour before treatment and ending approximately3 hours after treatment stopped using 250-pm mesh drift nets. In 2002, 30-min drift samples were collected every 3 hrs and in 2003, drift sampling time was reduced to 20 minutes. Benthic samples were collected from Cabin and Indian Creek before and after treatment using a 250-p Hess net which covered an area of 0.086 m2; benthic samples were approximately 250 m apart on rifflelrun streamreaches; however, due to the narrowness of the reference site channels, benthic samples could not be collected at reference sites. Drift and benthic samples were sieved in nested 1 -mm 250-pm sieves and then subsampled; invertebrates were then identifiedto family or genus depending on the taxon. Bioassays using mayflies (Cinygmula spp.) were conducted in Cabin and Crystal Creeks and bioassays using the caddisfly (Brachycentrus spp.) were conducted in Indian Creek. Nine bioassay cages (500-ml Nalgene bottles with 3-mm mesh screen covered windows on the sides, top and bottom), each containing 10 individuals, were spaced 120-m apart and tethered to cobbles in the substrate in Cabin Creek. In Indian Creek, cases were spaced 30-m apart in the antimycin-treated reach and 75-m apart in the antimycinlrotenonetreated reach the evening before treatment. The morning after treatment the bioassay cages were collected for analysis. Page 106of 118Results According to the study author, antimycin treatments in Cabin and Indian Creeks had no detectable effects on invertebrate drift relative to control reaches; however, invertebrate drift increased nearly 40-fold in Indian Creek after rotenone addition in the antimycin plus rotenone treatment. Antimycin treatment in Cabin Creek had no statistically significant effects on invertebrate density, biomass, or taxa richness. Mean invertebrate density was 22,000 invertebratesper m2less and mean invertebratebiomass was 1.2 g ash-free dry mass per m2 than before treatment; however, there differences were not statistically significant. The author acknowledgesthough that the power of the study to detect statistical differences was low; a minimum reduction of 81,000 invertebrates per m2would have been necessary for a significant difference. Similar to Cabin Creek, antimycin treatment of Indian Creek had no statistically significant effect on invertebrate density, biomass or taxa richness. Rotenone and antimycin treatment of Indian Creek significantly (p=0.024) reduced invertebrate density from 84,000 to 47,000 invertebrates/m2and the number of taxa in the benthos was reduced from 46 pretreatment to 35 post-treatment. Invertebrate biomass was significantly(p=0.047) reduced from 9.4 to 5.8 g ash-freedryweight. Antimycin treatments had no observed toxic effect on insects in in-situ bioassays; mean survival of mayflies was 82% + 9.8 and was not significantlydifferent than survival at reference sites (87% +18.9). Similarly, survival of caddisflies in antimycin treated areas (96%

### + 5.5) was not statistically differentthan in reference sites (94.3% + 7.9); however, caddisfly survival was 58% lower in areas treated with both antimycin and rotenone compared to reference sites. This study provides useful information on invertebrate responses to antimycin and antimycin plus rotenone treatments; however, the ability of the studies to detect treatment effects appears to be relatively low. Additionally, the areas selected as reference sites do not appear to be suitable controls given the reduced size and presumably lower invertebratepopulations; this likely contributed to the low statistical power of the study. Also, the significant effects attributed to antimycin A in combination with rotenone would be difficult to attribute to rotenone alone since it was formulated with kerosene. In spite ofthe low statistical power of the study, the in-situ bioassays indicate that at least the species of mayflies and caddisflies used are not particularly sensitive to antimcyin A treatment concentrations of 10 pg/L for 8 hours. Also, although there was high variability in estimates of invertebrate biomass, richness and diversity, the results indicate that invertebratecommunities were not eliminated during and immediately afier a 8-hourtreatment with antimycin A at 10pa.

Page 107of 118

## 15 

## Appendix G: 

## Memo from John Kenneke to Thomas Steeger and "Summary ofAntimycin Hydrolysis Research by John F. Kennekess

Page 108 of 118UNITED STATES ENVIRONMENTAL PROTECTION AGENCY NATIONAL EXPOSURE RESEARCH LABORATORY ECOSYSTEMS RESEARCH DIVISIOh 960 COLLEGE STATION ROAD ATHENS. (iA 30605-2700 November 20,2006
(.IFFICE OF RESEARCH AND DEVELOPMENT
Subject: Antimycin Report From: John F. Kenneke, Ph.D. Is/ Research Scientist Thru: J. MacArthurLong, Ph.D. Is/ Chief, Processes and Modeling Branch EricWeber, Ph.D. Is/ Acting Director To: Thomas 

### M. Steeger, Ph.D. SeniorBiologist Environmental Fate and Effects Division Please find attached a copy ofthe report, ''Summary ofAntimycin HydrolysisResearch." Thereport has undergoneinternal review for technical merit and quality assurance

### and isbeing submittedto OPP at theirrequest to aid in the re-registrationassessment of antimycin A. Antimycin A is a bacteria fermentationproduct containinga nine member dilactone ring (i.e., potential for hydrolysisto occur) and exists as a mixture of at least eight structuralisomers. Antimycin A is one ofthe most potent known inhibitorsofmitochondria1respiration, and in spite ofthe fact that it has been used for over fifty years as a piscicide, little information exists on its environmental fate. Several reports on the hydrolysis of antimycin A were published in the late 1960sand early 1970s; however, the results are incompleteand inconsistent. In response to OPP's request for additionalhydrolysisdata, laboratory studieswere conducted over a seven-monthperiod using certifiedbuffers at eleven different pHs between pH 1 and pH 9. Both static and mixed batch reactors were used, and for the first time first-order hydrolysis half-lives were measured for individual antimycin isomers in additionto total antimycin. These results indicatethat antimycin A hydrolyzes the quickest at pH 9, and more slowlywith little noticeable differencebetween pH 1 and pH 

### 8. Additionally, the 3.b isomerhyrolyzes more slowlythan the la,b isomer under basic conditions. Page 109 of 118As expected, the mixed batch systemsexhibited faster hydrolysis rates than the static systems (i.e., one order ofmagnitude); however, only at pH 

### <8 (i.e., at pH 8 and above the rates were nearly identical). The accelerated rates of antimycin hydrolysisunder acidic conditions relative to those measured under basic conditions is inconsistent with the static results and literaturevalues. Thereason for this discrepancy is uncertain, but sinceproduct formation could not be followed dueto analytical constraints, it is possiblethat under the mixed acidic conditions antimycin is depleted via a non-hydrolytic route. Further studies are being conducted with the static and mixed systems, and therefore, the results presented in this report should be viewed as preliminaryuntil those studies are completed. Attachment

Page 110 of 118

### SUMMARY 

### OF ANTlMYClN HYDROLYSIS RESEARCH

John F. 

### Kenneke, Ph.D. 

### U.S. EPA Office of Research 

### & Development National Exposure Research Laboratory Athens, GA
November 20,2006

BACKGROUND An analytical methodwas developed for the determination of antimycin 4a,b1 3a,b, 2al 2b, and 1 a,b 

### in aqueous samples. Antimycin hydrolysis experimentswere conducted between pH 1 and pH 9 using both static and mixed batch systems. MATERIALS AND METHODS Chemicals. Hydrolysis studies were conducted using certified pH buffers purchased from Fisher Scientific. Buffer pHs were: 1.00, 2.00, 4.00, 4.63, 5.00, 6.00, 6.86, 7.00, 7.40, 8.00, and 9.00. The buffers were 0.05 

### M and comprised of different buffer mixtures; some containedadditives to prevent microbial growth. Antimycin stocks were prepared in acetonitrile (ACN) using neat antimycin obtained from the EPA Repository and were assumedto be 100% pure. Experimental Approach. Antimycin hydrolysiswas measured under both static and mixed conditions. Static studies were conducted by placing 1732 VL of pH buffer and 18 

### pL of antimycin stock in a 

### 2 mL HPLC autosamplervial. The contents of the vial were sealed with a screw cap and mixed. The sample was placedon an HPLC autosampler tray heatedto 25OC (+/-I) and sampled every 28 minutes over a period of 14 hours. The initial concentration of antimycin was 5 ppm. All hydrolysis studies had a final concentration of 1 % 

### ACN. Studies were run in triplicate. Mixed batch studies were conducted in 40 mL vials containing 9.9 mL of buffer and 100 pL of appropriate antimycin stock 

### (5 ppm final concentration). All buffer solutions were heatedto 25 OCpriorto the addition of the antimycin stock. After spiking, the vial was placed in a temperature controlled incubatorat 25OC (+/0.5) and shaken at 200 rpm. After 5 minutes a 0.5 mL sample was removedand transferred to a 2 

### mL HPLC vial.
Page 111of 118

### The sample was analyzed immediately by HPLC. Additional 0.5 mL samples were periodically removed from the 40 mL vial and analyzed immediately by HPLC. 

### Analysis 

### of Antimycin. Antimycin was analyzed using an HPLC equipped with a photodiode array detector. The eluent was 75-80%ACN and 20-25%, 0.02M sodium acetate (pH 5) 

### run isocratic at 0.4 mUmin. Sample injection volume was 50 pL. All antimycin componentswere monitored at 230 and 350 nm. 

### Calibration. Initialcalibrationcurves (ICAL) containing a minimum of 5 concentrations of antimycin were prepared using pH 5, 6, 7, and 8 buffers. ICALs were calculated using total antimycin peak area. ICALswere calculated at both 230 nm and 350 nm and covered the entire concentration range observed in the hydrolysis studies. 

### Quality Assurance. Quality assurance/quality control (QNQC) samples were prepared inACN from neat antimycin and were analyzed by HPLC-UV prior to sample analysis and after every 10thsample. Antimycin 3a,b, 2a, Zb, laTb 

### and total antimycin peak areas and retentiontimes were followed using control charts based upon mean peak areas determined from a cumulative historic average. QNQC samples having peak areas deviating by less than *I 

### 0% from the historical average were considered valid. 

### Data Analysis. All peak integrations were subjected to internal QNQC review. Peaks that were found to be integrated incorrectly during the original HPLC analysis and data processingwere re-integrated. All data was exported to an electronic data base for sorting and QNQC sample validation. Samples which were not bracketed by a valid QNQC sample were flagged. Hydrolysis data was imported into a custom report and analysis template developed for antimycin hydrolysis studies to minimize calculation errors. All experimental parameters and ICAL parameterswere also imported into the template. Hydrolysis ratesfor antimycin qa,b,3a,b, 2a, 2b, 1 a,b 

### and total antimycinwere determined using a least-squaresregression analysis of In[A]t/[A]o as a function of time, where [AIt is the concentration of antimycin at time, t, and CAIois the initial concentration of antimycin. Ratherthan assuming [A10 based upon the amount of antimycin stock spiked, 

### [Ale was determined experimentallyfor total antimycin as well as each component, from the intercept of the ordinate at time 0 minutes (determined from an iterative least-squares regression). The experimental data was considered valid if 

### the determined value of [Ale 

### for total antimycin was 70% to 135%the theoretical value spiked. Additionally, the component ratios of 4a,b, 2a,Zb, Ia,b

### for [A10 had to be (0.033) : 

### (0.367) : 

### (0.058) : 

### (0.072) : 

### (0.471) respectively

### +/-I 0%, which correspondsto experimentally measured ratios in the ICAL, for the data set to be considered valid. Jackknife residualswere usedto detect and reject outliers in the least-squares regression. Where possible, only data between 20% and 70% antimycin depletion was used in the least-squares Page 112of 118

### regression; at a minimum at least 45% total antimycin degradation hadto have occurred for the data to be considered usable. The slope from the least-square regression is the pseudo-first order rate constant ((bbs) and was considered valid if it was based upon at least 5 

### data points and had an ?>0.7, in additionto meeting the previously described criteria. Half-lives(tin) 

### were determined for each component and total antimycin using: tl12= 

### 0.693/kbs. RESULTS 

### Antimycin Analysis. An isocratic HPLC methodwas developed utilizing20%, 0.02 M 

### sodium acetate (pH 5) and 80% 

### ACN as the eluent. It provided reproducible results, relatively good sensitivity, and relatively consistent peak retentiontimes. Additionally, the 4a,b, 3a,b, 2a, 2br la,b~~mp~nent~ could be separated in less than 20 minutes (Figure 1). Although 230 nm was significantly more sensitive than 350 nm (approx. 4.5 times) the 230 nm wavelength exhibited much greater baseline variability with some pH buffers; the 350 nm was not significantly affected in any of the studies. All antimycin componentsyielded similar absorbance spectrafrom 200 to 800 nm and were assumed to have similar extinctioncoefficients for purposes of quantitation. Hydrolysis. Ideally, samples would be collected from a mixed batch reactor over a period of hoursto days, however, this could only be done in some cases due to the unavoidable constraints that aqueous samples were expected to be unstable and would have to be collected and analyzed immediately, and although this could be done during the day, samples could not be collected during non-working hours. The ideal solution would have beento have the HPLC automaticallysample a mixed batch reactor, however, the appropriate equipment for this was not available. Consequently, a static batch reactor was employed for a majority of these studies. Hydrolysis results are shown inTable 1. Mean values (n=3)with 95% 

### confidence intervalsare depicted in Figure 2 along with several values from mixed batch reactor studies and literature values. The static batchvalues should be viewed as a conservative estimate of antimycin hydrolysis half-lives. A majority of the mixed batch studies that were performedsupport this position as evidenced by their shorter halflives. Further, studies that were conducted to determine the effect of sampling frequency on antimycin half-life show that for the same pH buffer, the more frequently the HPLC sampled the static batch reactor (and consequently liftedand moved it via the autosampler) the shorter the measured half-life.These observations may help explain some of the variability observed in published literature values for antimycin hydrolysis (i.e., mixing does not appear to have been used in at least one report, while in others it is not clear at all). Although only total antimycin hydrolysis half-livesare presented (Table 1)

### for sake of brevity, additionalsets of data were compiled for the individual components4a,br3a,b, Page 113 of 118

### 2, 2b, and la,b 

### (i.e.l over 200 tln values). Figure 3 shows the average half-life as a function of 

### pH for 3aPb and la,b, which makes up 370h and 47% of the total antimycin respectively. It appears that 3a,b hydrolyzes significantlyslower than la,bl 

### above pH 6 while laYb 

### hydrolyzes more slowly than 3a,bbelow pH 6. The same trend in relative hydrolysis rateswas also observed for mixed batch studies (not shown). The information in this document has been funded (wholly) or (in part) by the United States Environmental Protection Agency. Although it has been subjected to theAgency=speer and administrative review, it may not necessarily reflect ofJicialAgency policy. Mention oftrade names ofcommercial products does not constitute endorsementor recommendation for use.

Page 114 of 118

## Antimycin HPLC Chromatogram at 350 

## nm

Time, minutes Figure 1. 

### HPLC chromatogram of antimycin hydrolysis sample at pH 7.4. 

### Experimental conditions are described in the text.

Page 115 of 118

### Table 1. 

### Total Antimycin Hydrolysis Half-Life as a Functionof pHfor Static and Mixed Batch Systems

Page 116 of 118

Method

Static Batch

Mixed Batch (a) All values have least-squareslinear regressionrf>0.7, n>5, antimycin (total) recoverybetween 70% and 135%, ~45% total antimycindegradation; data only regressed between 20-70%total antirnycin degradation Total Antimycin ~alf-~ife(') (minutes) n

1

. 2 3 Avg PH 1 672 561 807 680



### -- 3 458 619 543 540



### -- 4 497 462 526 495 56 4.63 646 665 851 721

-- 5 619 681 594 631 36 7.4 517 473 439 476



### -- 6 700 608 678 662 43 8 589 630 586 602 603 6.86 542 537 456 512

-- 9 268 200 137 202 186 7 391 442 444 426 139

## Total Antimycin Hydrolysis

Static Batch (+I-95%CI) Lee eta/.(1971) Hussain,A, only (1969) Mixed Batch Figure 

### 2. Half-lifefor total antimycin hydrolysis as a function of pH. Average values shown with 95% confidence intervals (CI) for laboratory studies conducted using static and mixed batch reactors. Literaturevalues are shown for comparison.

Page 117of 118

## Antimycin A Hydrolysis 

## Static Batch



# ri

Total Antirnycin 

### Figure

### 3. Average hydrolysis half-lifewith standard error (S.E.) for individual components of antimycin as a function of pH. Values determined using a static batch reactor.

Page 118 of 118