United States 

Envrronmental Protectton 

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 

Reviewed by: 

Elizabeth Behl, Branch Chief 

U.S. Environmental Protection Agency 

Office of Pesticide Programs 

Environmental Fate and Effects Division 

Environmental Risk Branch IV 

1200 Pennsylvania Ave., NW 

Mail Code 7507P 

Washington, DC 20460 
EXECUTIVE SUMMARY 

Nature of the Chemical Stressor 

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 is primarily used to renovate recreationaljsh populations and to remove scaledjsh from 

catJish jingerling and food-fish production ponds. Over the past 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 of 25 pg/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 of time. Although environmental fate data are limited, the primary route of degradation 

appears to be base hydrolysis where under alkaline conditions the compound can degrade with half-lives 

of roughly 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 of at 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 of the 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 25 pg/L, acute risk 

levels of concern are exceeded for aquatic animals by factors of roughly 6,000X Even for the least 

sensitive Jsh and aquatic invertebrates tested, treatment concentrations of 25 pg/L would exceed acute 

risk levels of concern. Although antimycin is moderately to very highly toxic to terrestrial animals on an 

acute oral exposure basis, treatment rates to water of 25 pg/L or less are expected to present a low risk of 

acute mortalityfor birds and mammals through ingestion of dead/dyingfish or antimycin-treated water. 

Because there are no chronic toxicity data, it is not possible 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 118 
Where antimycin is used to remove scaled fzsh from catfish fingerling and food-fish production 

ponds, treatment rates are considerably lower than 25 pg/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 temporarily ajgected. 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 with parmangunate, 

invertebrate dnjl and survival did not appear to be significantly afected during or immediately after 8-hr 

treatments at 10 pg/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 so the default value of 4.5 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 data 
minutes to several weeks. Abiotic mechanisms (hydrolysis) appear to be the primary route of dissipation, 

but biodegradation may contribute to affecting the persistence of antimycin in the environment. The 

potential contribution of biotic routes of degradation to affecting the persistence is uncertain. 

The database on the ecological effects of antimycin 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 of degradation, flow-through, 

deactivation and dilution. Additionally, there are no data for terrestrial or aquatic plants. The likelihood 

of exposure 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 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 

deactivation of antimycin 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. 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 on fish 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 of aquatic invertebrates suggests, however, that both abundance 

and diversity of aquatic invertebrates exhibited transitory effects and many of these taxa are associated 

with the benthos. 

This assessment evaluates potential risks from antimycin A treatment concentrations of 25 pg/L or 

less and primarily focuses on use in flowing 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 is a potential for 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 of antimycin 

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 Source and Intensity ................................................................................................... 9 

2.1.2 Physical/Chemical/Fate and Transport Properties ................................................... 9 

2.1.3 Pesticide Type, Class, and Mode of Action .............................................................. 10 

2.1.4 Overview of Pesticide Usage .................................................................................... 11 

2.2 Receptors ....................................................................................................................... I I 

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 Usage and Use Rates ..................................................................................................... 17 

4 ENVIRONMENTAL FATE CHARACTERIZATION ................................................... 20 

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

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

4.3 Usage and Fate in Water Bodies ................................................................................... 23 

4.4 Estimates of Water Concentration for Risk Assessments .............................................. 24 

5 ECOLOGICAL EFFECT CHARACTERIZATION ...................................................... 25 

5.1 Categories of Acute Toxicity ......................................................................................... 25 

5.2 Toxicity to Aquatic Animals .......................................................................................... 27 

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

5.2.2 Freshwater Fish. Chronic .......................................................................................... 31 

Page6 of 118 
Freshwater Invertebrate. Acute .......................................................................... 3 1 

Freshwater Invertebrate. Chronic .............................................................................. 32 

EstuarineIMarine Fish. Acute .................................................................................... 32 

5.2.6 EstuarineJMarine Fish. 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 Toxicity of Potassium Pennanganate to Aquatic Vertebrates ........................ 34 

5.4.2 Acute Toxicity Potassium Pemanganate to 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 to Aquatic 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 Assumptions and Limitations Related to Effects Assessment .................................. 54 

6.6.3 Assumptions Related to Exposure Assessment ......................................................... 55 

6.7 Federally Threatened and Endangered (Listed) Species Concerns .............................. 56 

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

6.7.2 Taxonomic Groups Potentially at 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 Fintrolm to Remove 

Scaled Fish in Catfish Ponds ...................................................................................................... 81 

14 APPENDIX F . Review of Selected Papers ....................................................................... 84 

15 Appendix G: Memo from John Kenneke to 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 information to 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 species or 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 are below 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 1 and were estimated using a quantitative structure activityU.S. EPA. 1998. Guidelines for Ecological Risk Assessment. Risk Assessment Forum, Washington, DC. EPA 630lR-951002F April 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. 1H and 13C resonance 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 antimycin A. J. Chromatogr. 447: 65-79. 

Page 9 of 118 
based 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 of Action 

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 antimycin A. 

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 

submitochondrial particle (SMP) assays. Journal of Environmental Toxicology and Chemistry 15(3): 319 - 324. 

Page 10 of 118 

CAS Number -- -- 1397-94-0 

SMILES Notation ~~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-' 

Minutes to months, some (but 

inconsistent) evidence suggests 

dependance on 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 

estimation from submitted degradation 

study (MRD 45895901) 

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; 

submitted studies (MRID 46023 101 ) 

submitted study (MRID 45895901) 

estimated - PBT Profiler 
Antimycin 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 surface of the 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-quarters of the 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 fish production ponds. 

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 of these very broad taxonomic 

groups, an acute andlor chronic endpoint is selected from the available test data (see Section 2.3). 

EPA Registsation No. 39096-2 

Special Review and Reregistration Division Use Closure Memo dated October 26,2005. 

9. F~nlayson, B.J., R.A. Schnick, RL. Cailteux. L. DeMong, W.D. Horton, W. McClay, and C.W. Thompson. 2002. Assessment of Antimycin A 

Use in Fisheries and its Potential for Reregistration. Fisheries 27(6): 10 -18 

I0Ibid Finlayson et al. 2002. 

Page 11 of 118 
pp -- - - - 

Fable 2. Taxonomic gmups and most sensitive test species evaluated for ecological effects. I 

I Birdss I Mallard duck (Anus platyrhynchos) I Chronic, NOAEC I 

Taxonomic group 

I Mammals 

I Example(s) of representative species 

I 

Laboratory rat (Rattus norvegicus) Acute LDso and Chronic I 

Endpoint used 

Bobwhite quail (Colinus virginianus) Acute, LDsdK50 

I Freshwater fishh I Rainbow trout (Oncorhynchus mykiss) I Acute K50 I 

I I I 

Terrestrial insects 

I I I Honeybees (Apis mellifera) 

Fathead ~~MOW (Pimephales promelas) 

1 Estuarindmarine fish I Sheepshead minnow (Cyprinodon variegatus) ( Acute LC50 1 

Acute Oral LD5o 

Chronic NOAEC 

Freshwater invertebrates 

I Estuarindmarine invertebrates I Mysid shrimp (Mysidopsis bahia) I Acute LC50 I 

I I I 

Water flea (Daphnia magna) 

Midge (Chironomus riparius) 

Acute LC50 and Chronic NOAEC 

Chronic NOAEC (Benthic) 

I Eastern oyster (Crassostrea virginica) 

Terrestrial plantsC 

2.2.2 Ecosystems at Risk 

Acute ECso 

Aquatic plants and algae 

Antimycin A may be applied directly to 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 of the water body. 

I I I 

Monocots - corn (Zea mays) 

Dicots - soybean (Glycine max) 

When antimycin A is applied directly to water, the ecosystem at risk consists primarily 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 are used as sumgates for terrestrial phase amphibians and reptiles (US EPA, 2004). 

'Freshwater fish are used as surrogates for aquatic phase amphibians (US EPA, 2004). 

'Four species of two families of monocots, of which one is corn; six species of at least four dicot families, of which one is soybeans. 

Green algae (Selenastrum capricornutum) 

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 are used to generate a risk quotient, which is considered a screening-level estimate of risk 

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. 

Assessing risks 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 surrogate for the terrestrial life-stage. 

2.4 Conceptual Model 

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-level assessment: 

The use of antimycin A 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 off of dead 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 Ecological Risk Assessment. Risk Assessment Forum, Washington, DC. EPA 630lR-951002F April 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 distributed in 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 concentrations exist, 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 cannot be 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 

consuming treated organisms. 

5. Direct exposure of terrestrial animals/plants to antimycin is not likely since the chemical 

is only applied to water bodies. Indirect exposure to 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 118 
SedimenUSuspended Solids 

DIRECT 

Contacr/GiI llntegument 

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 receptors and possible effects. 

Antimycin Applied Directly to 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 118 
drip-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 sprayers may 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 sprayers be 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 

delivered through 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 antimycin A may be required during the summer months 

than the winter months for the same treatment site, assuming other factors remain constant. 

There are two broad uses for antimycin A as a piscicide: complete kill and selective kill. 

In a complete kill, 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 species in an area to restore listed or indigenous species.13 

In a selective kill, the water body is treated at 0.5 to 1.0 pgfL of antimycin A to eliminate 

only small, scaled fish. A common objective of a selective kill 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 118 
commercial catfish farmers. According to the FintrolB label, scaled fish in aquaculture ponds 

succumb to treatment at 5 to 10 pg/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 the production cycle. 

According to the FintrolB label, 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 antimycin A. 

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 KMn04 treatment rates (e.g. 3 - 4 

mg/L) may be required to deactivate antimycin A'~. 

3.2 Usage and 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. According to 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 primarily in 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 Personal comrnuniction, Mary Romeo, President, Aquabiotics Corporation. 2006. 

16 personal communication Dr. Craig Tucker, Director, National Warmwater Aquaculture Center and Southem Regional Aquaculture Center, 

2006 

17 Personal communication, Mr. Steve Moore, Supervisory Fishery Biologist, Great Smoky Mountain National Park, 2006. 

l8 Personal communication, Ms. Mary Romeo, Presidient, Aquabiotics Corporation, 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 are below 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 once per year. 

a Use Site: The use site refers to the entity (crop, building, surface or article) where a pesticide is applied and/or which is being protected. 

Max.Rate per App: Maximum dose for a single application to a single site. System calculated. 

Max.Rate UnitIArea: Units and Area associated with the maximum dose (UG: Use Group codes) 

Form: The physical form of the 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 

Formd Interval 

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 

bailer1 DripFeed device/ 

sprayer 

//Water 

treatment 

Backpack 

sprayer1 Boat 

bailer1 Dripfeed device/ 

Sprayer 

//Water 

treatment - 

Backpack 

sprayer1 Boat 

bailer1 Dripfeed 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 

FOODIFEED USES 

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 NS 
 ax. App Ratdcc & yr: maximum amount of pesticide product that can be applied to a site in one growing season (lcc) or during the span of 

one year (Iyr). 

Min. App Interval (days): minimum retreatment interval between applications in days (aggregated). 

h~pplication Equipment: equipment used to apply pesticide (aggregated). 

i Application Type: type of pesticide application (aggregated). ' SCIL: Soluble Concentmtdliquid 

NS: Not Specified (on label). 

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 deactivation of 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 

extent to which antimycin A will persist and potentially move outside treated 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 and Antimycin 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 following treatment 

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 (1 00 yds) downstream of the detoxification station remain alive for at least 48 

hours. 

Page 19 of 118 
ENVIRONMENTAL FATE CHARACTERIZATION 

4.1 Background 

Antimycin A is a relatively minor-use piscicide used primarily for direct applications to 

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" estimates of effective doses that 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 evaluation of the 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 of potential 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 dissipation times 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 conflicting information 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 significantly higher than the earlier open literature studies 

(Table 4). 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~~ founddegradation 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 degradation rate is related to pH. Kenneke (2006)~' estimated half lives of around 4 to 10 

hours in unmixed systems; whereas in mixed systems half lives ranged from 1 to 10 hours. 

Hydrolysis rates fi-om the literature are summarized in Table 1. In all cases hydrolysis half lives 

are on the order of minutes to 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~~ madesimilar claims as to the degradation products. 

These products are blastrnycic acid, antimycin lactone, and antimycic acid. No quantitative 

information regarding the relative concentrations of the transformation products is 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 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 (Circular No. 186). 18 pp. 

22 Hussain, A 1969. Kinetics and mechanisms of hydrolysis of antimycin A in solution. Journal of Pharmaceutical Sciences 58: 316-320. 91 L-' Lee, T.H., Dme, P.H., and Morton, S. 1971. Effects of Physical and Chemical Conditions on the Detoxification of Antimycin. Transactions 

%the American Fisheries Society 1971; 100: 13-17 L4 Heim, D. 2003. Hydrolysis Determination for Antimycin A Complex. Project Number 47132. Unpublished study prepared by Analytical 

Bio-Chemistry Labs, Inc. 63 p. (MRID 46023 1-01) 

25 Ibid Heim 2003. 

261bid Lee et 01. 197 1. 

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. 

341bid Hussain 1969. 

35 Heim, D. 2003. Determination of Aerobic Aquatic Metabolism for Antimycin A Complex: Amended Final Report. Lab Pmjst No. 473 13. 

Unpublished study prepared by ABC Laboratories, Inc. 79 p (MRD 458959-01). 

Page21 of 118 
23 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 uncertainty to 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 are unavailable). 

Although the registrant did not provide sorption studies for antimycin, indications from 

the aerobic aquatic metabolism study are that antimycin A does sorb significantly to 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 84 to 10000 mllg, which amounts to significant sorption. 

In order to gain additional insight into antimycin, fate properties 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 predictive models can be used to estimate ecotoxicology 

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 (bioconcentration factor (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-l7 atm-m3 mol-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 Water Bodies 

Table 4. Antimycin fate 

Property 

Molecular Wt 

Hydrolysis pH 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 

Hydrolysis pH 4.5 - 5.5 

Hydrolysis pH 7 - 8 

Hydrolysis pH 8.5 

Hydrolysis pH 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 

concentration that is to be used be confirmed with a bioassay. Because of the great latitude that 

the label gives, it cannot be determined a priori 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 

8.3 hours 

10.5 hours 

11 hours 

7.1 hours 

10 hours 

3.4 hours 

15 days 

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 - 10000 mVg 

20 to 47 days 

350x 

2.42 x10-17 atm-m3 mol-' 

2.31~1~'~ mm Hg 

Source 

Kenneke (2006) 

Kenneke (2006) 

Kenneke (2006) 

Kenneke (2006) 

Kenneke (2006) 

Kenneke (2006) 

Kenneke (2006) 

MRID 46023 10 1 

MRID 46023 10 1 

MRID 46023 10 1 

Lee et al. (1971) 

Lee et al. (1971) 

Lee et al. (1 97 1) 

Lee et al. (1971) 

Lee et al. (1971) 

Hussain (1969) 

Hussain (1 969) 

Hussain (1 969) 

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, 2004 
may coincide with the use of live-cars containing sensitive species that are placed 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 can be 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 

effective to at least 0.5 km downstream. 

4.4 Estimates of Water Concentration for 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 of uncertainty. With this in consideration, ranges of estimates can be made by using the 

full range of possible degradation (hydrolysis) rates reported in Table 4. Table 5 gives the range 

of chronic concentrations that 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 depending on 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 concentration would 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 side channels; 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 concentrations to degrade below a certain level of toxicity. 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. Performance of antimycen in high-gradient streams. North American Journal of Fisheries 

Management 16: 465-468. 

38 lbid Tiffan and Bergmen. 1996. 

39 lbid Tiffan and Bergmen 1996 

Page 24 of 118 
In this regard, Tiffan and ~er~ersen~' showed that antimycin can remain toxic in some 

circumstance for over 2 kilometers downstream of the 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 ECOLOGICAL EFFECT CHARACTERIZATION 

Time required to reach 0.004 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+) species in the United States. For mammals, 

acute studies are usually limited to Norway rat or the house mouse. Estuarine/marine testing 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 of Acute 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 species is based 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 Ibid Tiffan and Bergmen 1996. 

Page 25 of 118 
Table 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 

Slightly toxic 

Practically non-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). 

Toxicity Category 

Very highly toxic 

Highly toxic 

Moderately toxic 

Slightly toxic 

Practically non-toxic 

LDw, @~m) 

<10 

10-50 

5 1-500 

50 1-2000 

>2000 

Toxicity Category 

Very highly toxic 

Highly toxic 

Moderately toxic 

Slightly toxic 

Practically non-toxic 
5.2 Toxicity to Aquatic Animals 

Table 10. Categories of avian subacute dietary toxicity based on median lethal concentration in 

milligrams per kilogram diet per day (parts per million). 

5.2.1 Freshwater Fish, 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) freshwater fish species 

tested. To ensure the 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 sensitivity of freshwater fish to antimycin, the piscicide is classified as 

very highly toxic to fish on an acute exposure basis. 

LCso @~m) 

40 

50-500 

501-1000 

100 1-5000 

>5000 

Based on the available data, fish in the family Ictaluridae (catfish), like black bullhead 

(Ictalurus melas) and channel catfish (Ictalurus punctatus), are the least sensitive species tested 

which accounts for how antimycin can be used to selectively remove scaled fish from catfish 

aquaculture ponds. 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 

Highly toxic 

Moderately toxic 

Slightly toxic 

Practically non-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,, averaging 0.01 1 pgJL. 

Page 27 of 118 
OPP 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 literature relevant 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 of the toxicity values obtained from the open literature are 

more sensitive than that of the 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 

quality to use quantitatively in this ecological risk assessment. 

41 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. httn \+ lr H cn't xo\ CLOIOX 

42 

Ayerst Laboratories. 1964. Eficacy of Antimycin. (Compilation: unpublished study received in 1994 under 8991-51 CDL: 

005666-A (Accession No. 1 00135924)) 

Page 28 of 1 18 
43 Mayer, F. and M. Ellersieck. 1986. Manual of Acute Toxicity: Interpretation and Data Base for 410 Chemicals and 66 Species of Freshwater 

animals. U. S. Fish and Wildlife Service. Resource Publication 160. 579 p. (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" 

400980-0 1 

400980-01 

400980-0 1 

400980-01 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-0 1 

400980-01 

400980-0 1 

sntimycin A. 

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 

(0.1 14 - 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. Freshwater fish 

Species 

Bluegill 

(Lepomis macrochirus) 

Green Sunfish 

(Lepomis cyanellus) 

Rainbow Trout 

(Oncorhynchus mykiss) 

Rainbow Trout 

(Oneorhynchus mykiss) 

Cutthroat Trout 

(Oncorhynchus clarki) 

Coho Salmon 

(Oncorhynchus kisutch) 

Lake Trout 

(Salvelinus namaycush) 

Goldfish 

(Carassius auratus) 

Fathead Mimow 

(Pimephales promelas) 

Black Bullhead 

(Ictalurus melas) 

Channel Catfish 

(Ictalurus punctatus) 

Mosquitofish 

(Gambusia asnis) 

Largemouth Bass 

(Micropterus salmoides) 

Yellow Perch 

(Perca Jlavescens) 

White Crappie 

(Pomoxis annularis) 

Paddlefish 

(Polyodon spathula) 

acute toxicity 

% 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 

95'5 
The 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 alkaline pH 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°C no bluegill were killed after 24 hrs following treatment at 0.2 pg/L; however, at 22OC all 

of the bluegill were dead after 24 hours after exposure to 0.2 pg/~~~. 

Table 12. Freshwater fish acute toxicity of formulated antimycin A 

44 Berger, B.L., R.E. Lennon and J.W. Hogan. Laboratory Studies of Antimycin as 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 other aquatic animals. 

Pages 1 - 18 in Investigations in Fish Control No. 2. Bureau of Sport Fisheries and Wildlife Circular 186 

Page 30 of 118 

Species 

Bluegill 

(Lepomis macrochirus) 

Rainbow Trout 

(Oncorhynchus mykiss) 

Rainbow Trout 

(Oncorhynchus mykiss) 

Bluegill 

(Lepomis macrochirus) 

Bluegill 

(Lepomis macrochirus) 

% 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 

highly toxic 

VerY highly 

toxic 

very highly 

toxic 

very 

toxic 

MRIDI 

Accession 

No. 

TN 90 1 

TN 944 

TN 35 

TN 1533 

TN 153 

Study 

Classification 

supplemental 

supplemental 

supplemental 

supplemental 

supplemental 
Figure 3 Complete survival of fish treated with antimycin A 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 search of the 

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 invertebrates on an acute exposure basis. 

A search of the open literature did not identify other studies that would provide additional 

information concerning the acute toxicity of freshwater invertebrates exposed to antimycin A. 

Page 31 of 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 acute toxicity of technical grade antimycin A 

5.2.5 EstuarineMarine Fish, Acute 

A single estuarinelmarine fish acute toxicity study of technical grade antimycin A with 

the spot (Leiostomus xanthurus) resulted in a 48-hr LC,, of 0.23 pg1L; therefore, antimycin A 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/marine fish exposure to antimycin A are unknown. 

5.2.7 EstuarineMarine Invertebrates, Acute 

96-hour ECw 

a.W 

(95XC.I.) 

48 hr EC50 <lo 

(5 - 10) 

0.008 

(0.0058 - 0.01 1) 

>1.0 

Species 

Waterflea 

(Daphnia magna) 

Scud 

(Garnmarus fasciatus) 

Aquatic Isopod 

(Asellus brevicaudus) 

Antimycin A is very highly toxic to estuarinelmarine invertebrates 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 

400980-0 1 

400980-0 1 
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/marine invertebrate exposure to antimycin A is unknown. 

Table 14. Estuarinelmarine invertebrate acute toxicity of technical grade antimycin A 

5.3 Toxicity to Terrestrial Animals 

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 mgkg bw) and 

highly toxic to upland game birds (bobwhite quail LD,,=39 mg/kg bw) (Table 15). No data are 

available to evaluate the subacute dietary toxicity of antimycin A to birds. 

Species 

Pink Shrimp 

(Panaeus duorarum) 

Blue Crab 

(Callinectes sapidus) 

Eastern Oyster 

(Crassostrea virginica) 

Toxicity 

Category 

very Yghly 

toxic 

highly toxic 

very 

toxic 

5.3.2 Birds, Chronic 

No chronic toxicity data are available for birds. 

MRIDI 

Accession 

No. 

402284-0 1 

402284-0 1 

402284-01 

% 

95.5 

95.5 

95.5 

Table 15. Avian acute oral toxicity. 

5.3.3 Mammals, Acute 

96-hour ECSo 

(~b a.W 

48-hr 

48-hr LC,,> 100 

62 

Species 

Mallard duck 

(Anas platyrhynchos) 

Bobwhite quail 

(Colinus virginianus) 

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 

Supplemental 
rats46 (MRID 455279-01). Based on these data, the formulated endproduct is classified as 

moderately toxic to mammals on an acute oral exposure basis. 

5.3.4 Mammals, Chronic 

No chronic toxicity data are available for mammals. 

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 of the 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 toxicity to freshwater invertebrates, i.e., daphnids, are available in ECOTOX. 

5.4.1 Acute Toxicity of Potassium 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. Repolt No. 6025-00. 

Submitted to Aquabiotics Corporation, Bainbridge Island, WA. (MRID 455279-01). 47 

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. 

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: Administrative and Technical Guidelines Manual. American Fisheries Society, Bethesda, Maryland. 

49 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. Iltra \\ \v w CIII <I)\ CLI !to\ 

50 Ibid Marking and Bills 1975. <I 

J 1 

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: 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? 

JJ Birdsong, C.L. and J.W. Avault Jr. 1971. Toxicity of certain chemicals to Juvenilepompano. Pmg. Fish-Cult. 33(2): 76-80. 

Page34of 118 
variability 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 (Pimephales promelas) 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 attributed by the study authors to increased organic carbon in the pond water. The 

toxicity estimates for potassium permanganate in synthetic water from the Hobb's study are 

consistent with the 96-hr LC,, values reported in Marking and ~ills'~ for freshwater fish. 

"referred surrogate warm water fish species. 

Preferred surrogate cold water fish species. 

Bluegill Sunfish 

(Lepomis macrochirus) 

Common Carp 

(Cyprinus carpio) 

Channel Catfish 

(Ictalurus punctatus) 

Rainbow Troutc 

(Oncorhynchus mykiss) 

Striped Bass 

(Morone saxatilis) 

Florida Pompano 

(Trachinotus carolinus) 

54~eardon, I.S. and R.M. Hanell. 1994. Effects of varying salinities on the toxicity of potassium pennanganate to larval and juvenile striped 

bass, Movone saratilis (Walbaum). Aquacult. Fish. Manag. 25(6): 571-578. 

55 Bid Bindsong and Avault 1971. 

56 fbid Marking and Bills 1975. 

57~obbs. M. S., R S. Grippo, l. L Fams. 8. R. Griffin and L. L. Handing. 2006. CompamUve Toxicity of Potassium Permanganate to 

Nontarget Aquatic Organisms. Environmental Toxicology and Chemistry 25(1): 3046 - 3052. 

58~bid Marking and Bills 1975. 

Page 35 of 118 

' All data obtained from studies summarized in ECOTOX (2005). 

2300 - 3600 

3050 - 3450 

750 

1220 - 1800 

960 - 4920 

1600 - 2900 

Warm Water 

Warm Water 

Warm Water 

Cold Water 

-- 

-- 

Freshwater 

Freshwater 

Freshwater 

Freshwater 

Freshwater; 

brackish; marine 

Brackish; marine 

Marking and Bills, 

1975 

Marking and Bills, 

1975 

Marking and Bills, 

1975 

Marking and Bills, 

1975 

Reardon and Harrell, 

1994 

Birdsong and 

Avault, 1971 
& - - 

I 

1 0.5 1 

I .~ainbow trout 

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 ECso values 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 a1j9 and hderson60 (1 944) reported 2-hour ECso values 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 synthetic water versus 13,550 and 12,300 pg/L, respectively, in pond 

59 Sreekala, K.G., J. Jennita, and V.R Prakasam. 1991. Tolerance and healt rate of Daphnia sp. (crustacean) in response to disinfectants, 

bleaching powder and potassium pennanganate. Pollut. Res. lO(1): 33-36. 

60 Anderson, B.G. 1944. The toxicity thresholds of various substances found in industrial wastes as determined by the use of Dophnia rnngna. 

Sewage Works J. 16(6): 1 156-1 165. 

Ibid Anderson 1 944. 

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 estimates by ecotoxicity values, 

both acute and chronic. 

RQs are then compared to OPP's levels of concern (LOCs). These LOCs are criteria used 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) species is 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 chronic risks to non-target insects, or chronic risk 

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 presumptions for terrestrial animals (birds and wild mammals) 

I I 

Acute Restricted Use I EEC/LCw or ~~~dft~ or LDdday (or LD5o < 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. of bird LDso * wt. of bird 
Table 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 

Terrestrial and Semi-Aquatic Plants 

I I I 

i 

Table 19. Risk presumptions 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 

Risk Presumption LOC 

I Aquatic Plants I 

RQ 

Acute Endangered (Listed) Species I EEC/ECoS or NOAEC 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 sensitive test species used to calculate risk quotients 

to evaluate ecological risks of antimycin A use 

Taxonomic Group 

Birdsa 

Mammals 

Terrestrial insects 

7 

Exposure Category 

Acute (LDjO/LCs0) 

Chronic (NOAEC) 

Acute LDS0 

Chronic NOAEL 

(NOAEC) 

Acute LDso 

Most Sensitive Species 

Mallard duck 

(Anas platyrhynchos) 

Not available 

Laboratory rat 

(Rattus norvegicus) 

Not available 

Honey bees 

(Apis mellifera) 

Toxicity Value 

LDS0 = 2.9 mgkg 

Not available 

LDso = 286 mgkg bw 

Not available 

Not available 
Table 20. Summary of toxicity data for most sensitive test species used to calculate risk quotients 

to evaluate ecological risks of antimycin A use 

Taxonomic Group 

Freshwater fishb 

Exposure Category 

Chronic NOAEC 

Acute LC50 

Freshwater 

invertebrates 

Most Sensitive Species 

Rainbow trout 

Estuarinelmarine 

fish 

Toxicity Value 

I (Oncorhynchus mykiss) 

Coho Salmon 

Not available 

Acute ECSo 

Chronic NOAEC 

Estuarinelmarine 

Terrestrial plants 1 Acute I Not available 1 Not available 

96-h LCSo = 0.009 pg/L 

Acute LCs0 

Chronic NOAEC 

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 

(Leiostomus xanthurus) 

Not available 

I I I 

Chronic NOAEC 

Aquatic plants and 

algae 

At a treatment rate 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 acute high risk LOC. 

96-h LCso=0.23 pg/L 

Not available 

Pink Shrimp 

(Panaeus duorarum) 

Avety, J.L. 2006. Use of ~intm? to remove scaled fish in catfish ponds. ne Ca@h Journal. April 2006 

Page40 of 118 

48-hr LC,,=24 pg/L 

Not available 

aBirds are used as surrogates for terrestrial phase amphibians and reptiles (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 

reasonable to believe that most aquatic animals will be killed by current treatment 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 combination of flow through, dilution with untreated water and deactivation with 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, the potential for chronic risks is uncertain. 

. antimycin A treatment Ate of 25 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/marine mollusks (62 pg/L) is less sensitive than that of pink shrimp, an RQ 

value for mollusks (RQ=0.40) exceeds the acute restricted 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 

Exceeds acute risk (RQ?0.5), restricted use (RQa. I), and endangered species (RQ20.05) levels of concern. 

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 of 25 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 118 
consumption 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 therefore rainbow 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 (0.45 kglday x 172 pgfkg) 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 LDso is 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 of the 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 LDsO from 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 LDso of 2.9 mg kg-', the adjusted LDso is 

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). 

According to the Wildlife Exposure Factors andb book^^ the mean food ingestion rate 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 LDso for 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. Wildlife Exposure Factors Handbook. of Health and Environmental Assessment, Office of Research 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 of fish killed by antimycin. Journal of Agricultural Food Chemistry 14(4): 

403 - 407 MRID 461 534-01 ) 67 Mineau, P., B.T. Collins, and A. Baril. 1996. On the use of scaling factors to improve interspecies extrapolation of acute toxicity in 

bids. Regulatory Toxicology and Pharmacology. 24: 24-29. 

68 [bid ORD 1993. 

Page 42 of 118 
Similarly, 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 kingfisher weighing 148 g would consume 16 g of fish per 

day representing a potential antimycin exposure of 2.8 pg (0.01 6 kg/day x 172 pglkg). The 

adjusted LD,, for kingfishers is 2.1 mg kge' and once again, the estimated exposure is 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 188 g waterlday. At a treatment rate to water of 25 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 of magnitude below the adjusted LDSo. 

Table 23. Acute dose-based risk quotients for piscivorous birds based on potential residues 

in dead or dying fish. 

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 consume about 68 g of food based on the allometric equation: 

Species 

Blue Heron 

(Ardea Herodias) 

Bald Eagle 

(Haliaeetus leucocephalus) 

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 7 1 

Acute RQ Value 

<0.01 

<0.01 

<0.01 

I I 

Nagy, K.A. 1987. Field metabolic late and food requirement scaling in mammals and birds. Ecological Monographs 57: 11 1-128. 

72 Ibid Nagy 1987. 

Page 43 of 118 
common 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 18 mg of antimycin; this value is below 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 of toxicity for difference in body weight. The LDSo can be adjusted for 

body weight based on the formula recommended by 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, LDso is 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. Since we are assessing a large mammal, 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-g mammal 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 

kg220 mg) 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 dying fish. 

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 information it 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 Ritter and Strong. 1966. 

74 lbid Mineau et al. 1996. 

Page 44 of 118 

Acute RQ Value 

<O.O 1 

Estimated Daily 

Dose (~g) 

8.9 

Adjusted LD50 

@& 

220,000 
effects, 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 of both 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 considered low 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 

concentrations and likely reflects the decreased toxicity of antimycin at lower water temperatures 

due to decreased fish metabolic rates. 

Table 25. Acute toxicity values for freshwater fish (LC3 and invertebrates (LC3 for selected 

piscicides based on most sensitive endpoints used in OPP Ecological Risk Assessment chapters in 

support of the Reregistration Eligibility Decisions. 

75 Moore, S.E., M.A. Kulp, J. Hamrnonds, and B. Rosenlund. 2005. Restoration of Sams Creek and an Assessment of Brook Trout 

Restomtion Methods, Great Smoky Mountain National Park. National Park Setvice, U. S. Department of the Interior. Technical Report 

NPS/NRWRD/NRTR-2005. 

Chemical 

Antimycin A 

Rotenone" 

~iclosamide~ 

TF I@ 

76 personal communication John Kenneke, Chemist, U.S. EPA ORD 2006; these data are considered preliminary and have not undergone 

extensive peer review or quality assurance that would permit their use in a more quantitative fashion. 

77 Ritter, P.O. and F.M. Strong. 1966. Residues in tissues of fish killed by antimycin. Journal of Agticultu~al Food Chemistry 14(4): 403 - 407. 

Page 45 of 118 

' Rotenone RED Chapter DP Barcode D307382 

' Niclosarnide RED Chapter DP Barcode D255595 

' TFM 3-trifluoromethyl-4-nitrophenol RED Chapter DP Barcode D2 191 82 

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 to Aquatic Animals 

Antimycin A is very highly toxic to fish on an acute exposure basis. With fish LC50 

values as low as 1 nanogram (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 concentration of antimycin 

exceeds the 96-hr LC,, for 95% of the species reported. Since fish serve as surrogates for aquatic 

phase amphibians, risk is presumed for aquatic-phase amphibians 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 - Species Mtivity DCsbiMon ~~cal) 

Figure 6. Distribution of acute 96-hr median lethal concentrations &C,J for fish genera exposed to 

antimycin A. 

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) aquaculture production 

ponds by taking advantage of the 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, study results are consistent with data reported 

in Section 5 ind~cating that catfish are several orders of magnitude less sensitive to antimycin 

than scaled species (Table 26). 

Antimycin's use in aquaculture to remove scaled fish from catfish production ponds does 

not typically involve subsequent deactivation with potassium permanganate; rather, the 

antimycin is allowed to degrade naturally in the production pond water. Fingerling catfish would 

not be harvested as food fish for roughly 1 - 1.5 years post-treatment. 

The use of antimycin to remove scaled fish from food-fish production ponds is conducted 

relatively close to the time of harvest; 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. 

(Accession No. 00045801; MRID 2400798-13) 

Page 47 of 118 
that recently treated water is released to adjoining waterbodies, nontarget mortality of both fish 

and invertebrates could 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 Structure Activity 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 estimate of a chronic toxicity endpoint. 

Table 26. Fingerling fish bioassay of antimycin 96-hr LC, values (nominal concentrations). 

The available laboratory data indicate that fieshwater invertebrates have roughly similar 

sensitivities as 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 suggesting that plants 

and invertebrates are relatively unaffected 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 community structure, which could alter system function. 

Species 

Goldfish 

Common Carp 

Fathead Minnow 7 

Green Sunfish 

Bluegill Sunfish 

Largemouth Bass 

Channel Catfish 

There are no data on freshwater mollusks; however, data on estuarine/marine mollusks 

indicate that they are less sensitive to antimycin than other invertebrates although risk quotients 

96-hr LC, 

~gn 

0.137 

0.200 

0.074 

0.060 

0.05 1 

0.104 

8.4 

79 ECOSAR. Ecological Structure Activity Relationships illlo M r\ w u~.i a,,\ onn~1n11 ~lc\r~llcln- toc)1\ 2 ic~c,\,o rltn, Qn "" EPI Suite ht~n www.c-.vu.ro\ oonllnll.,cxnosurc docs cn~su~rc.llu~i 

81 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. (Accession No. 00135924) 

Page 48 of 118 
based 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 likelihood of chronic exposure. 

A field study conducted by the National Park as part of Southern Appalachian 

brook trout (Salvelinus fontinalis) 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 inactivate the antimycin A and some of the effects noted on macroinvertebrates soon 

after treatment were believed to result from the permanganate. 

82 PBT Profiler Model htto WHH ~blniolii~i rict 

83 Moore, S.E., M.A. Kulp, J. Hammonds, and B. Rosenlund. 2005. Restoration of Sams Creek and an Assessment of Bmk Trout 

Restoration Methods, Great Smoky Mountain National Park. National Park Service, U.S. Department of the Interior. Technical Report 

NPSMRWRDINRTR-2005. 
Similarly, in a study of Bear Creek in Great Srnokey Mountain National parkg4 benthic 

macroinvertebrate communities were 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 following treatment 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 of treatment 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, invertebrate populations 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 

communication N. 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 invertebrate communities and 

many of these taxa are associated with the benthic environment. 

84 Etnier, D. a. and C. D. Hulsey. 2005. Effects of Antimycin A treatment on benthic macroinvertebrates in Bear Creek tributaries to 

Fomey Creek, Great Smokey Mounthain National Park, Swain Couty, North Carolina. University of Tennessee Final report prepared under 

cooperativeOa,greement CA-5460-A 1-005. OJ 

- - Krebs, D. J. 1999. Ecological Methodology. Addison-Welsey Educational Publishers, Inc., Menlo Park, CA, 620p. 86 Baker, G. M., N. W. Darby and T. B. Williams. 2004. Balancing Bonneville cutthroat trout with non-native salmonids in Great 

Basin NatioQ~l Park. Wild Trout 111 Symposium: Working Together to Ensure the Future of Wild Trout: PI41 - 150. 0 1 Barbour, M. T., J. Gemtsen, B. D. Snyder, and J. B. Stribling. 1999. Rapid Bioassessment Protocols for Use in Streams and 

Wadeable Rivers: Periphyton, Benthic Macroinvertebrates, and Fish, Second Edition. EPA 841-B-99-002. U. S. Environmental Protection 

Agency; Ofice of Water, Washington D.C. httn wwu cn,~ r?lo owow monltoiliir rbn 

Page 50 of 118 
Studies 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 study had a low a ability to 

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 10 pg/L. 

As mentioned earlier, potassium permanganate is sometimes used to detoxifL antimycin 

following stream treatments. Permanganate is typically applied at a treatment concentration of 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 declined by as much as 1 1%90. However, similar to what was observed following antimycin 

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 Terrestrial Animals 

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 opportunistically feed 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. 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 of the University of Wyoming. 

89 Ibid Moore et al. 2005. 

90 Personal communication: S. Moore. Supervisory Fishery Biologist, Great Smoky Mountains National Park, June 2006 

91 Ibid Moore et 01.2005 

Page 51 of 118 
to 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 indicated by Table 20, there are a number of 

toxicity studies (avian sub-acute dietary, avian chronic, mammalian chronic, aquatic 

vertebratehnvertebrate chronic and terrestrial/aquatic plant) 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 estimates vary widely. The extent to which nontarget animals will be affected will depend on the extent to which antimycin moves outside of the 

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 

opportunities to 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 larger proportion of the 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 require that 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 operating procedure (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 registrant96 and 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 of this 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 10 pptr 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 column under field condition^^^. The extracted sample is then eluted off the SPE column 

with acetone and is minimally stable for 2 weeks and may be stable for 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 quantifiable peaks (A,, A,, A, and A,). The limit 

of detection (LOD) is approximately 15 pptr when using a sample size of 50 mL. 

94 American Fisheries Society. 2006. ~intml' Stream and River Use Monograph for Complete Removal 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 of the U.S. Geological Survey, May 2006 

Page 53 of 118 
6.6.2 Assumptions and Limitations Related to Effects Assessment 

6.6.2.1 Age Class and Sensitivity of Effects Thresholds 

Test organism age may have a significant impact on the observed sensitivity to a toxicant. 

The screening risk assessment acute toxicity data for fish are collected on juvenile 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 assessment has 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 screening endpoint. 

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 Effects Data 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 amphibians would likely be 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 likely be similar. For terrestrial species, however, the difference between amphibians and 

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 strong reason 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 118 
position of the most sensitive species tested in the distribution of all possible species is 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 of maintenance. A widespread comparison of species is lacking; 

however, even variation within a species can be quite high. 

6.6.2.4 Effectiveness of Deactivation 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 primarily to the label rate ambiguities only. 

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 detoxification be 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 Ecological Risk Assessment for the Reregistration of Rotenone. (DP Barcode 307380); 

Docket Number OPP-EPA-HQ-2005-0494 hu~ u~ w lcculailuriz KO\ 

Page 55 of 118 
antimycin and thus EFED cannot assume that potassium permanganate actually removes 

antimycin from a water body. EFED did not attempt to adjust recommended estimated 

environmental concentrations with consideration for potassium permanganate use. 

6.7 Federally Threatened and 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, if they are proposing 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 

reasonably would 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 compliance with 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-level risk 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 Species Act. 

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 indirectly by the Federal action and not merely the immediate area involved 

in the action. At the initial screening level, the risk assessment considers broadly described 

taxonomic groups and so conservatively assumes that listed species within those broad groups 

loo 50 C.F.R. f 402.02 

Page 56 of 118 
are 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 exposure to 

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 collocation of species with treatment areas. 

6.7.2 Taxonomic Groups Potentially 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 further refinement of the action area is necessary. Furthermore, 

RQs below the listed species LOCs for a given taxonomic group indicate no concern for indirect 

effects upon listed species that depend upon the taxonomic group covered by the RQ as a 

resource. However, in situations where the screening assumptions lead to RQs in excess of the 

listed species LOCs for a given taxonomic group, a potential for a "may affect" conclusion exists 

and may be associated with direct effects on listed species belonging to that taxonomic group or 

may extend to indirect effects upon listed species that depend upon that taxonomic group as a 

resource. In such cases, additional information on the biology of listed species, the locations of 

these species, and the locations of use sites could be considered to determine the extent to which 

screening assumptions regarding an action area apply to a particular listed organism. These 

subsequent refinement steps could consider how this information would impact the action area 

for a particular listed organism and may potentially include areas of exposure that are downwind 

and downstream of the pesticide use site. 

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-level risk 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 of both the survival and recovery of a listed species in 

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 exposures that may occur at the established listed species LOC. 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 118 
results of a screen for indirect effects to listed species, using direct effect acute and chronic 

LOCs for each taxonomic group, are presented 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 degradation would be fastest. 

Table 27. Summary of direct effects for Federally listed species from 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 

Freshwater Fish 

Freshwater Invertebrates 

Saltwater Mollusc 

Aquatic Plants: 

Vascular 

Non-vascular 

Birds 

Mammals 

Terrestrial Plants: 

Monocots 

Dicots 

Page 58 of 118 

a~aw data were not provided so the default value of 4.5 is 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 salmonids to 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-native trout. 

6.7.2.1.2 Freshwater Invertebrates 

Listed species acute risk LOCs for direct effects on fi-eshwater invertebrates 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 exposure to antimycin are unknown. 

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 

estuarinelmarine fish and invertebrate exposure to antimycin are unknown. 

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 aquatic plant exposure to antimycin 

are unknown. 

6.7.2.1.5 Birds 

No acute risk LOCs are exceeded for birds. No chronic bird toxicity data were submitted 

and no useable data were located in the open literature for antimycin; therefore, chronic risks 

associated with avian exposure to antimycin are unknown. 

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 associated with mammalian exposure to antimycin are unknown. 

lolu. S. Fish and Wildlife Service Endangered Species Status. 

htt~.; ccos.lwa.euv sncclcs 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 (Oncorhynchus gilae) (Third Revision) Recovery Plan. U. S. Fish and 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 of individual 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 15.5 based 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 

affected is 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 invertebrates was 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 extremely toxic 

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 indicated that it is committed to developing such procedures and linking them to the 

labeled uses of antimycin. 

Available data indicate that aquatic invertebrates can be as sensitive to 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 

abundance and diversity; this likely occurs through 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 

source to constitute a risk of acute mortality to 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 of the 

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 establish whether listed species are present; 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 developed by the U.S. Geological Survey are expected to vastly 

improve the analytical detection limits for antimycin A. 

Page 61 of 118 
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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 

Toxicology and Chemistry 25(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 mechanisms of hydrolysis of antimycin A in solution. Journal of 

Pharmaceutical Sciences 58: 3 16-320. 

Kenneke, J.F. Summary of Antimycin 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 of Physical and Chemical Conditions on the 

Detoxification of Antimycin. Transactions of the 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 

Degradation of Antimycen A. Journal of the 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 118 
Mayer, F. and M. Ellersieck. 1986. Manual of Acute Toxicity: Interpretation and Data Base for 

410 Chemicals and 66 Species of Freshwater Animals. U.S. Fish and Wildlife Service. 

Resource Publication 160. 579 p. (MRID 400980-01) 

Mineau, P, B.T. Collins, and A. Baril. 1996. On the use of scaling factors to improve interspecies 

extrapolation of acute toxicity in birds. Regulatory Toxicology and Pharmacology. 24: 24-29. 

Moore, S.E., M.A. Kulp, J. Hammonds, and B. Rosenlund. 2005. Restoration of Sams Creek 

and an Assessment of Brook Trout Restoration Methods, Great Smoky Mountain National Park. 

National Park Service, U.S. Department of the 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~cal Effects 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 (Oncorhynchus gilae) (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 and juvenile 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 of Fisheries Management 16: 465-468. 

Page 64 of 118 
U.S. Fish and Wildlife Service Endangered 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 (Circular No. 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. (Accession No. 00 13 5924) 

Page 65 of 118 
APPENDIX A. 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 118 
AND 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 3 04 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 118 
Log10 (Henry's Constant) = -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 INFORMATION 
REFERENCE 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- 

2 82 

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 Profiler Model 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 sections that 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 compartments may be different than 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 single value. 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 its rate 

of transport into (and out of) that compartment. Because the overall persistence takes into 

account transport, its value will likely be different than any of the media-specific half-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 118 
Release 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 of the PBT Profiler 

provides seven different release scenarios to help identify P2 opportunities for antimycin A. The 

seven release scenarios are based on a more realistic total release of 300 kghr to 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 (the predominant 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 

Bioaccumulation Summary 

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 

biomagnification and bioconcentration. 

Page 71 of 118 
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 frequently 

used to predict the importance of bioaccumulation. The estimated bioconcentration factor (BCF) 

of 350 for antimycin A does not exceed the EPA bioconcentration criteria. 

Bioaccumulation Estimate 

The PBT Profiler estimates that 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 

chemicals will result in chronic exposures which, in turn, can lead to chronic toxicity. The PBT 

Profiler uses an estimated fish chronic toxicity value (ChV) to allow organic chemicals lacking 

experimental data to be screened for P2 opportunities. A more in-depth P2 assessment requires 

that the potential toxicity of antimycin A to other aquatic organisms (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 aquatic organisms. Some aquatic organisms, 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 

complete ECOSAR~~ estimates for 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 cycle when 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 estimate the acute and chronic toxicity 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 

following is the complete ECOSAR~ output for antimycin A: 

SMlLES : 0 1 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 (KowWin estimate) 

Melt Pt: 

Wat Sol: 13.59 mg/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: Chemical may not be soluble enough 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 118 
Phenols: 

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 

Close Window 

Developed by the Environmental Science Center under contract to the Ofice of Pollution Prevention and 

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. 

PROCEDURE FOR DEPLOYMENT OF 

PISCICIDE DURING 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 watdes for public's recreational 

interest andlor species conservation, the deployment of a piscicide into lentic waters is a valuable tool. 

To meet management plan objectives, NMDGF will consider piscicide use in appropriate watdes. Deployment 

of a piscicide is a complex task requiring guidance by specific objectives, public scoping, interagency cooxdhation, 

and identified benefits to the public and the resource. 

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 

deployment of a piscicide into surface waters of the 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 

facilitate successll use of piscicides hi lentic systems for fishery management activities in New Mexico. 

Project Development and Prehhry Assessments 

1. The use of a piscicide in waterbodies is an important fishery management tool that will support the programs . . admmstmted by NMDGF. Use of a piscicide hi a lentic system hi New Mexico must be supported by objectives 

listed in a fishery management plan. A Fcular management plan should provide objective criteria for prioritizing 

and selecting waterbodies for chemical reclamation. 

2. Iden% Issues and Potential Conflicts 

Identify public opinions and attitudes hi the community 

Identifjl land status 

Id- recreational and commercial interests 

Page 75 of 118 
Iden* water development and user groups 

3. Description of waterbody proposed for treatment. 

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& macroinvertebrate community. 

Determine access to mtmbody and potential tributaries. 

4. Complete all state and federal permit requirements. 

e.g. NEPA, SHPO, ESA, CWA 404 

5. ARer a comprehensive evaluation of the proposed project by all planning entities, a decision on whether to 

continue with the proposed project will be made. 

6. Staff will develop a treatment plan to include piscicide application plan, staff needs, safety protocol, and 

emergency mponse protocol. State certified pesticide applicato~s will supervise chemical restomtion projects 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 

Fisheries Management. 

Wtment Activities 

1. Determine waterbody boundaries, basin profile, inflows and outflows and other perhnent infonnation (e.g. 

fish movements). 

2. Determine surface acres and volume of waterbody. If appropriate, calculate stream discharge of tributaries. 

Calculate flow through time if appropriate. Conduct water @ty monitoring. 

3. Determine kh species present (target and non-target species). 

4. Determine best method for treating lake and, if necessary, lake tributaries in accordance with product label. 

Methods selected should consider pH, temperature, organic load, plant material, and basin mrpho10gy. 

Logistical considerations include cost and accessibility. 

5. Determine concentration of piscicide to remove target species. 

6. Calculate amount of piscicide needed to treat waterbody including tributaries if reqw. If treatment of 

tributaries is reqmed, follow the appropriate provisions in the Protocol for Deployment of Piscicides during 

Restoration of Native Fisheries in Lotic Waters. 

7. Ifreqmd, locate a neutralization site on the outflow fiom the waterbody. 

8. Supervisory personnel will review deployment schedule, personnel responsibilities, methods of communication, 

and outline pmcedulps for handling unexpected issues with field staff. 

Page 76 of 118 
9. Personnel will post notices of the application at points of entry into the treatment areas as well as in appropriate 

offices and websites. Such notices will be posted at least 48 hours before deployment. 

Deployment of Piscicide 

Boat deployment of liquid rotenone or antimycin 

1. Determine amount of piscicide to be deployed in the waterbody using directions on the label, water volume and 

inflow hm tributaries. Ifnecessat-y, tributaries can be treated in accordance with the Protocol for Deployment 

of Piscicides during Restoration of Native Fisheries in Lotic Waters. 

2. Safety equipment will be worn according to label recommendations. 

3. Set up livecars with target fish to assess treatment effectiveness at varying points and depths in the watabody. 

4. Determine if it is necessary to divide lake into sections for deployment. Marker buoys should be used for 

guidance to ensure total coverage. Deploy the piscicide in a manner to effectively cover the treatment area, e.g. 

in a grid-like pattem. Backpack sprayers should be used to treat the shoreline and small inlets where boat access 

is limited. Drip stations can be deployed on larger inlets concurrently with lake application. 

5. Shallow waters can be treated using venturi apparatus that pulls diluted piscicides into the prop wash of the boat 

motor. Deeper sections of the waterbody should be treated with bilge pumps and hoses. To ensure toxicity 

overlap between the sections, the entire volume of piscicide should be deployed within 48 hours. 

6. Suspected problem areas may be treated with several of these methods. Sand mix formulas may be useM for 

underwater spring areas. 

7. Treatment times will depend on turnover time in the lake and the type of piscicide charm 

8. Where feasible, dead fish will be collected and buried in appropriate locations. 

9. All deployment equipment will be emptied, rinsed with water to remove residue, disassembled, collected 

and stored in accordance with the product label. 

Boat deployment of powdered rotenone product 

1. Determine amount of piscicide to be deployed in the water body using directions on the label, water 

volume, and inflow hm tributaries. If necessary, tributaries can be treated in accordance with the 

Protocol for Deployment of Piscicides during Restoration of Native Fisheries hi Lotic Waters. 

2. Safety equipment will be worn according to the labeling requirements. 

3. Powdered rotenone should be applied using a closed system to limit driR. An example of this type of 

system is an aspirator system that suctions the powdered rotenone hm the containers into water that is 

pumped through the system. 

4. Set up livecars with target fish to assess treatment effectiveness at varying points and depths in the waterbody. 

Page 77 of 118 
5. Detamine if it is necessary to divide lake into sections for deploynent. Marker buoys should be used for 

guidance to ensure total coverage. Deploy the piscicide in a manner to effectively cover the treatment area, e.g. 

in a grid-like pattern Backpack sprayers should be used to tseat shoreline and small inlets where boat access is 

limited. Drip stations can be deployed on larger inlets concurrently with lake application using a liquid rotenone 

formulation. 

6. To ensure toxicity overlap between the sections, the entire volume of piscicide should be deployed within 48 

hours. 

7. Where feasible, dead fish will be collected and buried in appropriate locations. 

8. All deployment equipment will be emptied, rinsed with water to remove residue, disassembled, collected 

and stored in accordance with the product label. 

Application of sand mix rotenone 

May be effective in shallow impoundments. 

1. Determine amount of piscicide to be deployed hi the water body using directions on the label, water volume 

and results from bioassays. Idow rates need to be factored into treatment concentration and duration. 

2. Staff mixing and applying sand formulation will wear safety equipment according to label -en&. 

3. Sand mixture can be applied using burlap sacks or other types of pesticide application equipment that would 

be appropriate for dry materials. Equipment should be chosen to assure even application. 

Aria1 application of liquid rotenone 

Aerial application is desirable in situations where access to lakes is limited. Gendy, it works for lakes <20 feet 

deep where piscicide distribution at greater depths is not a umcem. 

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 measures that may permit a more quantitative use of the 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 

exist based 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 79 of 118 
Hexyl Components 

In vitro Phase 1 Metabolism 

Butyl Components 

Page 80 of 118 
13 APPENDIX E. Standard Operating Procedure for the Use of 

I?introlB to 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 catfish ponds are operated as monocultures, and fish other than 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 catfish ponds. 

Two fish toxicants are registered 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-om nursery ponds prior to stocking fry. FintrolB (active ingredient 

antimycin A) is effective in removing scaled fish from a pond without harming catfish. 

FintrolB has 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 Fintrolm a 

desirable management tool are a wide difference in toxicity to catfish and scaled fish, a low 

toxicity to mammals, and how quickly it degrades. 

Fintrolm can 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 10 parts 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 also be 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 of the 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 (because pH 

is lowest at that time) after pond temperatures have stabilized above 60" F. Avoid treating ponds 

when catfish may be stressed due to high water temperatures or periods of low dissolved oxygen. 

To determine the amount of FintrolB needed to treat the pond, you will need to estimate the pond 

volume, in acre-feet of water. First, measure the surface area of the 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-feet to be treated. 

'03 Avey, I.L. 2006. Use of Fintm? to xmove scaled fish in catfish ponds. Zhr Calfsh Journal April ZOO6 (reproduced with permission of 

author). 

Page 81 of 118 
Fintrol" 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 certain pond 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-concentrate and 40 ounces of diluent to obtain 

the 80 ounces you need. The final mixture retains potency for seven days. However, once this 

solution is 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 

solution needed to treat the pond. You would then mix 40 ounces of Fintro1"-concentrate and 40 

ounces of diluent to obtain the 80 ounces you 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 

immediately with 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, FintrolB is one of the most 

dependable pond-management tools available to the catfish farmer. Fintrolm is, 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 118 
Table 1. Rough estimation of concentrations of Fintrol @ needed for eradication of different fish 

species under various combinations of pH and water temperature. 

Effective Concentration of Fintrol 

(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@ solution to achieve desired concentration. 

Amount of ~introl@ solution 

Desired (~introl@-concentrate + Diluent) 

Concentration per acre-foot 

(ppb active ingredient) (ml>' (ounces) 

1 ppb ............................................. 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 

10 ppb ............................................. 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. 41 2 1 (MRID46 1534-01) 

Deformylantirnycin A3 hydrochloride was mixed with H3-formic acid 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 (Salvelinus fontinalis) and common 

carp (Cyprinus calpio) were placed individually in 19-liter glass jars containing 15 liters of water treated 

with antimycin A. 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 antimycin A. After a trout was exposed and died, it was followed by 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) 

containing 2 goldfish (1 - 3 g) in 1.5 L of distilled water. 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 standard bioassay of goldfish with antimycin at known concentrations. Spiked homogenates 

were also extracted to determine the extraction efficiency. 

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 10 ppb). 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 (1 6), tissue residues averaged 203 pglkg fish weight. Tissue 

residues in larger trout killed with antimycin at 10 ppb were similar to residues in small trout killed with 

antimycin at 5 ppb; the lowest tissue concentrations were identified in larger trout killed with antimycin at 

5 ppb. Of the fish studied, carp appeared to be the least sensitive to antimycin A and required the longest 

time to kill; carp required roughly 5 times longer to die from antimycin than the trout. 

Page 84 of 118 
Table 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 

I Tissue residues based on radioactivity and presume radioactivity reflects 100% antimycin. 

Fish 

Brook Trout 

Antimycin Residues pglkg 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 10 ppb. 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 10 ppb 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 118 
Table 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 concentrations occurred in the 

body. Average residues in the muscle were 79 i 14.4 pglkg (mean k std error). The authors conclude that 

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 of the electron transport system. 

Table 3. Tissue ion of antimycin in rainbow trout killed with II3-antimycin. 

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 

5.0 hrs 

5 PP~ 

4 

271 g 

2.7 hrs 

10 PP~ 

Antimycin Equivalent to 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 attempted to quantify the tissue concentrations of intact antimycin A by extracting the 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 10 pg/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 15 pg/Kg (Table 4). Recovery of antimycin from spiked tissues ranged 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 samples make 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 fish killed with antimycin. 
Table 4. 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 

Sample Tested Activity 

200-g Carp 

93-g control carp with 35.5 pg 

Uncertainties: It is unclear whether the 96% recovery of labeled material following combustion and 

counting also included the lyophilization procedure. It's not clear from the study to what concentration of 

antimycin (labeled and cold combined) the fish were actually exposed. 

Survival Time (hours) 

8.1, 9.7 

The goldfish bioassay for estimating antimycin A residues in tissues contained roughly 1.3 mL/L (2 

W1.5 L) and exceeds the EPA recommended maximum of 0.1 mL/L. 

3 0 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 

of reconstituted water 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 is relatively 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 

antimycin is lipophillic and 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 (Oncorhynchus mykiss formerly Salmo gairdneri). 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 

antimycin A (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 acclimation period. 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 10.8 mg/L; however, with readings of 5.5 mg/L at 96 hrs, the DO is at roughly 

5 1% saturation and is below the recommended limit of 60% saturation. 

48 hr LCSo= 0.00014 mg/L (95% CI: 0.000075 - 0.00024 mg/L) 

96 hr 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. (Accession No. 00045801; MRlD 2400798-1 3) 

Liquid formulation: crystalline antimycin dissolved in acetone diluted to obtain antimycin concentrations 

ranging from 0.0 1 ppb to 500 ppb. 

Sand formulation: Fintrol 5 (antimycin incorporated in Carbowax and coated on 40-mesh sand; sand to 

carbowax weight 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 (Ictalurus furcatus), 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 acriflavine to 

inhibit hgal growth. 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 obtained by hatching channel catfish eggs at laboratory. 

Adult fish treated in 0.25-acre pond at the Fish Farming Experimental Station, Stuttgart, Arkansas. 

Page 88 of 118 
Four vinyl wading pools containing 3,785 liters of water stocked with 80 channel catfish fingerlings from 

the same lot. A bioassay cage containing 10 bluegills was 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 10 bluegills were added to cage. Catfish in each pool fed daily at 

a rate of 3% of body weight; however, because of decreased water 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-acre pond treated in mid-December (water temp=ll°C); by Day 8 water temperatures had 

declined to 4.4OC. Seining indicated the presence of paddlefish, shad, carp, buffalo, white catfish, blue 

catfish, and channel catfish. Pond treated 10 ppb antimycin (400 g Fintrol 5). Stability of the antimycin 

tested by placing cages containing 10 fingerling rainbow trout into treated pond. 

Results 

Egg 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 1.0 ppb and 100% at 5 and 10 ppb. 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 resulted in 81 % mortality. Concentrations at 

or less than 100 ppb for two hours did not affect total hatch; control fish exhibited 95% hatching success. 

Fry Studies 

Channel catfish control mortality was 1 1 %; for fingerlings treated with 0.75 ppb there was 100% mortality 

within 96 hours. 

Fingerling Bioassay 

96-hr LCSo Values 

goldfish: 0.137 ppb 

carp: 0.200 ppb 

fathead minnow: 0.074 ppb 

green sunfish: 0.060 ppb 

bluegill: 0.05 1 ppb 

largemouth bass: 0.1 04 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 Vinyl Pools 

After 4 months of acclimatization, control survival averaged 88% while survival in treated pools averaged 

97%. 

Page 89 of 118 
Uncertainties: Purity of the antimycin liquid formulation is not specified; purity of the sand formulation is 

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 referring to hatching success. Control mortality in the 0.25-acre 

ponds (37% for goldfish and 8 1% for carp) was above the EPA-recommended maximum 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. (MRID 455279-01) 

Study is GLP-compliant. Study conducted according to OPPTS 870.1 100. 

Fintrol Concentrate (20%); certificate of analysis not provided. 

Dark brown liquid maintained 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 16 hrs 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 14 post-dosing. Observations for mortality 

and clinicalibehavioral signs of toxicity were made 3 times the day of dosing and at least once daily 

thereafter for 14 days. On Day 14, animals were euthanized by C02 and necropsied. 

Table 1. Summary of 14-day male, female and combined rat mortality following oral administration 

of antimycin A. 

I 1 

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

male 286 (258 - 3 12 mglkg) 

female 3 6 1 (344 - 3 8 1 mgkg) 

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 

10110 
combined 3 16 (29 1 - 343 mglkg) 

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 swollenlred penile area was observed only in animals that died on test. 

No effect on body 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 alone had 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: 354 mglkg (95% CI: -) 

Slope: 46.92 

Combined: 3 1 7 mglkg (95% CI: 253 - 386 mag) 

Slope: 7.759 
PROBIT 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 118 
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 

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 118 
0.20 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 118 
Probit Procedure 

I ter 

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 

2 0 

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 118 
Intercept LoglO(D0SE) 

Intercept 3.1095948584 7.9110209101 

LoglO(D0SE) 7.9110209101 20.126175496 

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 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-Days 
Probit 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 118 
0.65 361.14315 

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 12 
Probit 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 118 
Probability LoglO(D0SE) 95% Fiducial Limits 

Page 100 of 118 
PROBIT 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 118 
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) 

Single nominal 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 concentration of 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 1 L 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 gain but 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 102 of 118 
among 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 differences between 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 125 ppb of antimycin is not toxic per se and does not degrade 

to products which are toxic when consumed by rats and dogs for three months. 

Reviewer's comments: This study is classified as unacceptable since the purity of the 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 of the test compound is not stated and nominal concentration was not verified. 

Treatment solution was aerated; however, concentration was not verified. 

Co-solvent (ethanol) concentration 2 mVL exceeded the recommended concentration of 0.1 ml/L and no 

solvent control was included. 

Raw data are not available to test whether there are statistical differences for food and water consumption 

and for body weights. 

Page 103 of 118 
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) 

Frozen rainbow trout (0.6 - 1 lb each), killed by antimycin at 125 ppb or by suffocation. Cooked fish 

mixed with standard laboratory animal diets in a preparation of 1 : 1. 

Rejection Factors: 

Treated diets were prepared by first cooking the fish. The effects of heat on antimycin are unknown. 

Review discontinued since quality of the photocopy was so poor. 

Page 104 of 118 
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 Toxicology and Chemistry 

25(1): 3046 - 3052. 

In-house cultures of cladocerans (Daphnia magna, Ceriodaphnia dubia), fathead minnow (Pimephales 

promelas), a midge (Chironomus tentans) 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 conducted with a substrate (spiked water) and were static 96-hr toxicity 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 fathead minnows were 4 - 10 days old. Water temperature for cladoceran and fish was 25 

- + 1°C while 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" synthetic water (hardness range: 89 - 100 mg/L 

as CaCO,) and experimental pond water water (hardness range: 87 - 104 mg/L as CaCO,) obtained from 

H.K. Dupree Stuttgart National Aquaculture Research Center in Stuttgart, AR, with historical records of 

previous chemical treatments. 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 synthetic water; however, measured concentrations 

ranged between 5.5 - 65% of nominal in pond water. In synthetic water, the pH ranged between 7.95 to 

8.14 and in pond water pH ranged between 8.06 - 8.63. 

Table 1 summarizes the 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 suggest that potassium pennanganate is 

less toxic to the aquatic species tested 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) for potassium 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, exposure in this portion of the study is uncertain. 

synthetic and pond water based on nominal treatment concentrations. 

Page 105 of 118 

Test Organism 

Daphnia rnagna 

Ceriodaphnia dubia 

Pimephales promelas 

Chironornus tentans 

Hyafella azteca 

Synthetic Water 

96-hr LC, 

mdL 

0.053 

0.058 

2.13 

4.43 

4.74 

Synthetic Water 

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 

7.83 
Cerreto, K. M. 2004. Antimycin and rotenone: short-term effects on invertebrates in first order, highelevation streams. A master's thesis submitted to the Department of Zoology and Physiology and the 

Graduate School of the University of Wyoming. 

In conjunction with the Wyoming Game and Fish Department, this study examined the effect of 

antimycin and rotenonelantimycin treatments 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 compared to 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 

sprayer to 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 approximately 3 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 stream reaches; 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 identified to 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 

antimycinlrotenone treated reach the evening before treatment. The morning after treatment the bioassay 

cages were collected for analysis. 

Page 106 of 118 
Results 

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 invertebrates per m2 less and mean invertebrate biomass was 1.2 g 

ash-free dry mass per m2 than before treatment; however, there differences were not statistically 

significant. The author acknowledges though that the power of the study to detect statistical differences 

was low; a minimum reduction of 81,000 invertebrates per m2 would 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/m2 and 

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-free dry weight. 

Antimycin treatments had no observed toxic effect on insects in in-situ bioassays; mean survival of 

mayflies was 82% + 9.8 and was not significantly different than survival at reference sites (87% +18.9). 

Similarly, survival of caddisflies in antimycin treated areas (96% + 5.5) was not statistically different than 

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 invertebrate populations; 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 of the 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 invertebrate communities were not eliminated during and immediately 

afier a 8-hour treatment with antimycin A at 10 pa. 

Page 107of 118 
15 Appendix G: Memo from John Kenneke to Thomas Steeger and 

"Summary of Antimycin Hydrolysis Research by John F. 

Kennekess 

Page 108 of 1 18 
UNITED 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. MacArthur Long, Ph.D. Is/ 

Chief, Processes and Modeling Branch 

Eric Weber, Ph.D. Is/ 

Acting Director 

To: Thomas M. Steeger, Ph.D. 

Senior Biologist 

Environmental Fate and Effects Division 

Please find attached a copy of the report, ''Summary of Antimycin Hydrolysis Research." 

The report has undergone internal review for technical merit and quality assurance and is being 

submitted to OPP at their request to aid in the re-registration assessment of antimycin A. 

Antimycin A is a bacteria fermentation product containing a nine member dilactone ring (i.e., 

potential for hydrolysis to occur) and exists as a mixture of at least eight structural isomers. 

Antimycin A is one of the most potent known inhibitors of mitochondria1 respiration, and in spite 

of the 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 

1960s and early 1970s; however, the results are incomplete and inconsistent. 

In response to OPP's request for additional hydrolysis data, laboratory studies were 

conducted over a seven-month period using certified buffers 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 addition to total 

antimycin. These results indicate that antimycin A hydrolyzes the 

quickest at pH 9, and more slowly with little noticeable difference between pH 1 and pH 8. 

Additionally, the 3.b isomer hyrolyzes more slowly than the la,b isomer under basic conditions. 

Page 109 of 118 
As expected, the mixed batch systems exhibited faster hydrolysis rates than 

the static systems (i.e., one order of magnitude); however, only at pH <8 (i.e., at pH 8 and above 

the rates were nearly identical). The accelerated rates of antimycin hydrolysis under acidic 

conditions relative to those measured under basic conditions is inconsistent 

with the static results and literature values. The reason for this discrepancy is uncertain, but 

since product formation could not be followed due to analytical constraints, it is possible that 

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 preliminary until 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 method was developed for the determination of antimycin 4a,b1 3a,b, 2al 2b, 

and 1 a,b in aqueous samples. Antimycin hydrolysis experiments were 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 contained additives to prevent microbial growth. Antimycin stocks were 

prepared in acetonitrile (ACN) using neat antimycin obtained from the EPA Repository 

and were assumed to be 100% pure. 

Experimental Approach. Antimycin hydrolysis was 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 autosampler vial. The contents of the vial 

were sealed with a screw cap and mixed. The sample was placed on an HPLC 

autosampler tray heated to 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 

heated to 25 OC prior to the addition of the antimycin stock. After spiking, the vial was 

placed in a temperature controlled incubator at 25OC (+/0.5) and shaken at 200 rpm. 

After 5 minutes a 0.5 mL sample was removed and transferred to a 2 mL HPLC vial. 

Page 111 of 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 components were monitored at 230 and 350 nm. 

Calibration. Initial calibration curves (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. ICALs were 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 

in ACN from neat antimycin and were analyzed by HPLC-UV prior to sample analysis 

and after every 10th sample. Antimycin 3a,b, 2a, Zb, laTb and total antimycin peak 

areas and retention times 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 

processing were 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 parameters were also imported into the 

template. 

Hydrolysis rates for antimycin qa,b, 3a,b, 2a, 2b, 1 a,b and total antimycin were determined 

using a least-squares regression analysis of In[A]t/[A]o as a function of time, where [AIt is 

the concentration of antimycin at time, t, and CAIo is the initial concentration of antimycin. 

Rather than assuming [A10 based upon the amount of antimycin stock spiked, [Ale was 

determined experimentally for 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 corresponds to experimentally measured 

ratios in the ICAL, for the data set to be considered valid. Jackknife residuals were 

used to 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 112 of 118 
regression; at a minimum at least 45% total antimycin degradation had to 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 addition to 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 method was developed utilizing 20%, 0.02 M 

sodium acetate (pH 5) and 80% ACN as the eluent. It provided reproducible results, 

relatively good sensitivity, and relatively consistent peak retention times. 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 

components yielded similar absorbance spectra from 200 to 800 nm and were assumed 

to have similar extinction coefficients for purposes of quantitation. 

Hydrolysis. Ideally, samples would be collected from a mixed batch reactor over a 

period of hours to 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 been to have the HPLC automatically sample 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 in Table 1. Mean values (n=3) with 95% confidence 

intervals are depicted in Figure 2 along with several values from mixed batch reactor 

studies and literature values. The static batch values should be viewed as a 

conservative estimate of antimycin hydrolysis half-lives. A majority of the mixed batch 

studies that were performed support 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 lifted and 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-lives are presented (Table 1) for sake of 

brevity, additional sets of data were compiled for the individual components 4a,br 3a,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 significantly slower than la,bl above pH 6 

while laYb hydrolyzes more slowly than 3a,b below pH 6. The same trend in relative 

hydrolysis rates was 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 the Agency=s peer and 

administrative review, it may not necessarily reflect ofJicial Agency policy. Mention of trade 

names of commercial products does not constitute endorsement or 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 Function of pH for Static and 

Mixed Batch Systems 

Page 116 of 118 

Method 

Static 

Batch 

Mixed 

Batch 

(a) All values have least-squares linear regression rf>0.7, n>5, antimycin (total) recovery between 70% 

and 135%, ~45% total antimycin degradation; 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/. (1 971) 

Hussain, A, only (1 969) 

Mixed Batch 

Figure 2. Half-life for 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. Literature values are shown for comparison. 

Page 117of 118 
Antimycin A Hydrolysis 

Static Batch 

ri 

Total Antirnycin 

Figure 3. Average hydrolysis half-life with 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