National Park Service 

U.S. Department of Interior 

Natural Resource Program Center 

A Field Manual for the Use of Antimycin A for 

Restoration of Native Fish Populations 

Natural Resource Report NPS/NRPC/NRR—2008/033 
ii

ON THE COVER 

Clockwise from top left: Pictures of a small stream in Great Smoky Mountains NP (NPS photo), a high elevation lake in Great 

Sand Dune NP (NPS photo), an antimycin detoxification site in Great Smoky Mountains NP (NPS photo), and boat application of 

antimycin A via a Venturi device on a high elevation lake in Rocky Mountain National Park (USFWS photo).
iii

A Field Manual for the Use of Antimycin A for 

Restoration of Native Fish Populations 

Natural Resource Report NPS/NRPC/NRR—2008/033 

Steve Moore1, Matt Kulp2, Bruce Rosenlund3, Jim Brooks4, and David Propst, Ph.D.5

1 Supervisory Fishery Biologist, National Park Service 

Great Smoky Mountains National Park 

107 Park Headquarters Road 

Gatlinburg, Tennessee 37738 

2 Fishery Biologist, National Park Service 

Great Smoky Mountains National Park 

107 Park Headquarters Road 

Gatlinburg, Tennessee 37738 

3

 Fishery Biologist, U.S. Fish & Wildlife Service 

CO-FWMAO 

134 Union Blvd., Suite 675 

Lakewood, CO 80225-0486 

4

 Fishery Biologist, U.S. Fish & Wildlife Service 

New Mexico Fishery Resources Office 

3800 Commons Avenue NE, 

Albuquerque, NM 87109 

5

 Ichthyologist, New Mexico Department of Game and Fish 

Conservation Services Division 

New Mexico Department of Game and Fish

P.O. Box 25112 

Santa Fe, NM 87504 

January 2008 

U.S. Department of the Interior 

National Park Service 

Natural Resource program Center 

Fort Collins, Colorado 
iv

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Views and conclusions in this report are those of the authors and do not necessarily reflect 

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Internet, or by sending a request to the address on the back cover. 

Please cite this publication as: 

Moore, S.E, M.A. Kulp, B. Rosenlund, J. Brooks, and D. Propst. 2008. A Field Manual for the 

Use of Antimycin A for Restoration of Native Fish Populations. Natural Resource Report 

NPS/NRPC/NRR—2008/001. National Park Service, Fort Collins, Colorado. 

NPS D-1966 January 2008 
v

Contents 

Page 

Figures........................................................................................................................................x 

Tables...................................................................................................................................... xii 

Appendices............................................................................................................................. xiii 

Executive Summary.................................................................................................................xv 

Acknowledgements................................................................................................................ xvi 

Introduction................................................................................................................................1 

Fisheries Management in National Parks ......................................................................1 

Introduction of Non-Native Species ..............................................................................1 

Restoration Efforts.........................................................................................................2 

The NPS Antimycin A Manual......................................................................................3 

Purpose...............................................................................................................3 

Scope and Applicability.....................................................................................3 

Structure.............................................................................................................3 

Updates ..............................................................................................................4 

Deviations ..........................................................................................................4 

Antimycin A Overview..............................................................................................................4 

Background....................................................................................................................4

Approved Uses...............................................................................................................5 

Formulation....................................................................................................................6 

Environmental Fate........................................................................................................6 

Toxicity..........................................................................................................................6 
vi

Page 

Regulatory Compliance .............................................................................................................7 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) .....................................7 

Clean Water Act (CWA)................................................................................................8 

National Environmental Policy Act (NEPA).................................................................8 

Endangered Species Protection Act (ESA)....................................................................9 

No Effect Determination..................................................................................12 

Not Likely to Adversely Effect Determination................................................12 

Likely to Adversely Affect Determination ......................................................12 

Overview of Project Planning and Implementation.................................................................12 

Training........................................................................................................................12 

Project Manager Qualifications .......................................................................13 

Project Personnel Qualifications......................................................................13 

Reporting......................................................................................................................13 

Preliminary Project Plan ..................................................................................13 

Final Project Plan.............................................................................................14 

Treatment Records...........................................................................................14 

Protocol Deviations..........................................................................................14 

Final Report .....................................................................................................14 

Overview of Antimycin A Application....................................................................................15 

Before Application.......................................................................................................15 

 

 Planning and Public Involvment.......................................................16 

 

 Defining the Treatment Area and Project Area.....................................16 
vii

 Treatment Area Characterization.....................................................................17 

Pre- and Post-Treatment Bio-monitoring.........................................................19 

Public Notification and Closures .....................................................................20 

Safety Training and Use of Personal Protective Equipment............................20 

Regulatory Compliance ...................................................................................20 

During the Application Dates ......................................................................................21 

Storage, Transportation, Decontamination, and Spill Containment of 

Antimycin A and Equipment ...........................................................................21 

Pre-Treatment Bioassay...................................................................................22 

Pre-Treatment Site Preparation and Setup.......................................................23 

Applying Antimycin A ....................................................................................25 

Exposure Mitigation.........................................................................................26 

Deactivating with Potassium Permanganate....................................................26 

After Application .........................................................................................................28 

Re-Treatment ...................................................................................................29 

Restocking........................................................................................................29 

Post-Treatment Bio-monitoring.......................................................................29 

Final Report .....................................................................................................29 

Administrative Operating Procedures......................................................................................30 

AOP 1 Preliminary and Final Project Plan ..................................................................30 

AOP 2 Planning and Public Involvement ....................................................................32 

AOP 3 Environmental Law Compliance .....................................................................34 

AOP 4 Public Notification and Closures .....................................................................37 

AOP 5 Safety Training and Use of Personal Protective Equipment............................38 
viii

AOP 6 Quality Assurance (QA) Measures Utilized in Antimycin Applications.........39 

Page 

AOP 7 Storage, Transportation, and Spill Containment of Antimycin A and 

Antimycin A Application Equipment ..........................................................................41 

AOP 8 Procedure for Management of Treatment Records..........................................43 

AOP 9 Contingency Management for Environmental Changes..................................44 

AOP10 Final Report ....................................................................................................45 

AOP 11 Protocol Deviations........................................................................................47 

Technical Operating Procedures..............................................................................................48 

TOP 1 Determination and Characterization of Treatment Area for a Stream .............48 

TOP 2 Determination and Characterization of Treatment Area for a Lake.................53 

TOP 3 Validation of A Potential Fish Barrier .............................................................54 

TOP 4 Pre- and Post-Treatment Aquatic Macroinvertebrate and Fish Monitoring.....55 

TOP 5 Conducting Stream Discharge Measurements .................................................57 

TOP 6 Determination of Stream Water Travel Rate Using Fluorescent Dyes 

(Fluorescene & Rhodamine)........................................................................................60 

TOP 7 Closure of Treatment Area and Public Notification of Closure.......................62 

TOP 8 Conducting a Pre-Treatment Safety Meeting...................................................63 

TOP 9 Setup of a Bioassay to Determine a Working Antimycin A Concentration.....65 

TOP 10 Collection, Maintenance, and Disposal of Sentinel Fish................................69 

TOP 11 Sentinel and Wild Fish Observations for Daily Treatment Evaluation..........71 

TOP 12 Determining Distance between Antimycin Dispensing Stations ...................73 

TOP 13 Operation of a Dispensing Station to Apply Antimycin A in Streams ..........75 

TOP 14 Application of Antimycin A to a Lake...........................................................79 

TOP 15 Use of a Backpack or Hand-held Sprayer to Apply Antimycin A .................82 
ix

Page 

TOP 16 Deactivation of Antimycin A in a Stream......................................................84 

TOP 17 Equipment and Safety Gear Cleaning ............................................................87 

TOP 18 Documenting Field Deviations from the SOP Manual ..................................88 

Literature Cited ........................................................................................................................89 
x

Figures 

Page 

Figure 1. Chemical structure of antimycin A molecule............................................................5 

Figure 2. A conceptual model outlining the interaction processes of working with the 

Services under the Endangered Species Act (ESA).................................................................11 

Figure 3. Example of a typical antimycin A project implementation planning and 

application chronology beginning with project conception through implementation.. ...........15 

Figure 4. An antimycin A drip station in operation during a small stream treatment 

project. .....................................................................................................................................24 

Figure 5. A cascade barrier to up-stream movement of salmonids in Great Smoky Mountains 

National Park. ....................................................................................... 48 

Figure 6. An example of a project area map for a stream slated for treatment. ...................49 

Figure 7. A graphical representation of a stream system targeted for antimycin A 

treatment including stream discharge, mean stream gradient (%), elevation, key study 

area identifiers and application rate information. ....................................................................51 

Figure 8. Mean stream gradient profile of Sams Creek, Great Smoky Mountains National 

Park. .........................................................................................................................................52 

Figure 9. Dr. David Etnier sorting a pre-treatment aquatic macro-invertebrate collection from 

Sams Creek, Great Smoky Mountains National Park.............................................. 56 

Figure 10. Measurement of stream discharge using the cross sectional area method. ........ 58 

Figure 11. Fluorescene dye traveling downstream during a flow study to establish project 

timelines.................................................................................................. 60 

Figure 12. A pretreatment antimycin A application safety meeting to discuss project 

supervision, safety, technique, assignments, and timelines.....................................................63 

Figure 13. Setup of streamside bioassay tubs and buckets used to determine the target 

concentration of antimycin (ppb) and potassium permanganate (ppm)................ ...........66 

Figure 14. A backpack fish transporter and aerator used to transport sentinel fish to and 

from holding cages and the treatment area... ...........................................................................70 

Figure 15. A operational antimycin dispensing station on Sams Creek, GRSM during 

treatment in 2000. .......................................................................................75 
xi

Figures (continued) 

Page 

Figure 16. An operational antimycin drip station on Bear Creek, GRSM during the 2003 

treatment...................................................................................................76 

Figure 17. A backpack sprayer applies antimycin to back water eddies during an antimycin 

project. Note the operator is wearing all required safety gear for safe application of 

antimycin A with a backpack sprayer .................................................................83 

Figure 18. The potassium permanganate deactivation station used to neutralize antimycin 

during a native fish restoration project in GRSM. ..................................................84 

Figure 19. A Potassium permanganate deactivation station configuration used to 

detoxify antimycin during native fish restoration project in Crater Lake NP. .......................85 

Figure 20. An example of the drip box system used to deliver potassium permanganate to 

a stream to neutralize antimycin A. Note the green dye upstream of the saw horses and 

the purple coloration indicative of the potassium permanganate entering the stream.............85 
xii

Tables 

Page 

Table 1. Necessary components of the preliminary and final project plan for a typical 

antimycin A project..................................................................................................................31 

Table 2. Results of a field bioassay conducted on rainbow trout in a GRSM stream. ...........67 

Table 3. Antimycin and potassium permanganate application rate based on stream 

discharge (ft3

/sec) and desired application concentration. ......................................................86 
xiii

Appendices 

Page 

Appendix A. Appendix A. Additional literature concerning the use, chemistry, 

detoxification, toxicity, and other data concerning antimycin A and potassium 

permanganate. ..........................................................................................................................91 

Appendix B.1. Integrated Pest Management Legislation and Policy........................... 106 

Appendix B.2. 11 Step Process to Developing and Implementing an Integrated Pest 

 Management Strategy...............................................................107 

 

Appendix B.3. Example of an antimycin project managers planning and implementation 

checklist.. ...............................................................................................................................108 

Appendix B.4. Example of a National Park Service close order sign including federal 

authorities and Superintendents signature under which such closures are authorized.. ........109 

Appendix B.5. Example of Great Smoky Mountains National Park stream discharge 

sheet.. .....................................................................................................................................110 

Appendix B.6. An example of a pre-treatment safety meeting checklist and personnel 

signup datasheet.....................................................................................................................111 

Appendix B.7.Antimycin station equipment checklist and vendor contact information.......112 

Appendix B.8. Potassium permanganate equipment checklist and vendor contact 

informatio...............................................................................................................................113 

Appendix B.9. An example of an Antimycin A daily application form.. ..............................114 

Appendix B.10. An example of a potassium permanganate daily application form.. ...........115 

Appendix B.11. An example of a antimycin and potassium permanganate application 

summary sheet. ......................................................................................................................116 

Appendix B.12. Protocol deviation form...............................................................................117 

Appendix C. A summary report of the results of antimycin A toxicity studies using 

various species, concentrations, and exposure times.............................................................118 

Appendix D. The antimycin toxicity summary report for Sams Creek, Great Smoky Mountains 

National Park in 1998..................................................................................120 

Appendix E. A copy of the Fintrol® (antimycin) product label. ....................................... 123 
xiv

Appendix F The Material Safety Data Sheet (MSDS) for Fintrol® (antimycin)............. 124 
xv

Executive Summary

“The national park system was created to conserve unimpaired many of the world’s most 

magnificent landscapes, places that enshrine our nation’s enduring principles, and places that 

remind us of the tremendous sacrifices Americans have made on behalf of those principles” 

(NPS, 2006). To accomplish this, an important component of the current management strategies 

calls for the National Park Service to maintain as parts of the natural ecosystems of parks all 

plants and animals native to park ecosystems and to restore, where appropriate, native plant and 

animal species (NPS, 2006), While management actions today make every attempt to meet this 

mandate, during the formative years of the National Park Service, managers sometimes promoted 

parks by managing scenery and enhancing tourism activities such as recreational fishing, 

sometimes to the detriment of natural systems. Oftentimes, these recreational activities were 

readily endorsed by the public and they clamored for more opportunities including the stocking 

of non-native fishes. In its early years, the new agency did not have knowledgeable staff to 

manage the fishery; this function was often turned over to state agencies. The states followed 

accepted recreational fishery management actions of the day and stocked non-native fish in 

National Park units across the nation. Only in more recent years have biologists in state agencies 

and national parks began to realize the irreparable damage that had occurred due to the loss of 

genetic integrity and the displacement or elimination of native species by non-native species. 

National Park Service fishery managers and fishery managers across the nation are now faced 

with the problem of protecting and preserving native fish and, if possible, restoring them to 

segments of their historic range. Currently, only two techniques (backpack electrofishing and 

piscicides) are available to successfully remove non-native fishes in order to restore native fish to 

segments of their historic range. Projects in Great Smoky Mountains National Park (GRSM) 

used electrofishing techniques to successfully remove non-native rainbow trout (Oncorhynchus 

mykiss) from segments of small streams with natural barriers to upstream migration of nonnative salmonids. Data from these projects demonstrate that they are labor intensive and are 

subject to failure if all of the right conditions are not met. 

In general, the use of piscicides (fish poisons) is the only way to ensure complete eradication of 

entire non-native fish populations short of dewatering habitats. Historically, Fintrol® (antimycin 

A) and Rotenone® have been used by fisheries biologists throughout the United States (U.S.) for 

restoring native fish species in streams and lakes. 

The purpose of this manual is to provide fisheries managers’ with protocols for conducting a 

safe, effective, and lawful restoration project with antimycin A. Using the protocols specified in 

this document, fisheries managers will be able to apply antimycin A consistently to remove 

unwanted fish species from streams, ponds, or small lakes and impoundments (<50ha) with 

subsequent re-establishment of natural aquatic communities. This standardized and consistent 

use of antimycin A will also help reduce potential for human exposure and unintended adverse 

ecological effects. 
xvi

Acknowledgements

The production of this manual would not have been possible without the support of the National 

Park Service, the U.S. Fish and Wildlife Service and the New Mexico Department of Game and 

Fish. The authors wish to acknowledge Jim Long, National Park Service, Carter Kruse, Turner 

Enterprises and Lance Wormell, U. S. Environmental Protection Agency for their constructive 

and professional review of the manual. Their comments served to greatly improve the quality 

and readability of the final product.
1

Introduction 

This report, A Field Manual for the Use of Antimycin A for Restoration of Native Fish 

Populations, provides protocols for the use of antimycin A which have been successfully used in 

restoration efforts within units of the national park system and in national forest and state 

projects. This document is based upon experiences of biologists from the National Park Service 

(NPS), the U.S. Fish and Wildlife Service (USFWS) and New Mexico Department of Game and 

Fish. The authors’ hope that the information included in the manual will be applicable to any 

application of antimycin for the restoration of native fish populations and that it provides 

fisheries managers with established protocols for safe, effective, and lawful restoration projects 

utilizing antimycin A. 

Fisheries Management in National Parks

The National Park Service (NPS) was established by Congress in 1916. This action formally 

recognized the significance of setting aside and preserving areas in the United States because of 

their important and unique natural and cultural resources. The Organic Act was passed in 1916 

and directs the National Park Service to protect and conserve the resources of these areas 

“unimpaired for both present and future generations.” This is a unique mandate among federal 

land management agencies and it directs park managers to be concerned with the conservation of 

natural diversity, the functioning of native ecosystems, and the maintenance of natural processes. 

While today NPS effectively meets these mandates, recognition of the actions required to comply 

with these mandates developed slowly in the National Park Service due mainly to the incomplete 

understanding of ecological processes during the formative years (Zuble 1996, Sellers 1997). 

By 1916, over 25 National Parks and monuments had been established primarily in remote 

western areas of the continental United States (Tilmant 2004). Some of these units were 

relatively pristine initially and others were not. Regardless of the condition, NPS managers were 

challenged to attract people and build a constituency for the fledgling agency (Tilmant 2004). 

Management actions during this time included providing easy access to the parks, programs to 

concentrate wildlife for easy viewing, and promoting recreational fishing, other outdoor sports, 

and sometimes even cattle grazing within the parks. Early managers appeared to interpret the 

Organic Act of 1916 to mean managing scenery and wildlife, accommodating tourists, and 

promoting parks (Tilmant 2004). One product of this early management philosophy was 

introduction of non-native fish species to support recreational angling and has subsequently led 

to ongoing restoration efforts to correct these actions. 

Introduction of Non-Native Species

The management of natural resources during the formative years of the NPS was often delegated 

to state agencies and their management actions followed the then current fishery management 

practices of the day, despite potential inconsistencies with NPS management policies. All of 

these actions reflected the anthropocentric view of the period that resources were to be managed 

for the benefit of people. This, in turn, led to the widespread and purposeful introduction of nonnative fishes within National Parks. This “Johnny Apple-fish” mentality was so prevalent that in 

some cases non-native fish rearing facilities were established within National Parks (Tilmant 

2004). These efforts aimed primarily to provide popular non-native sport fish and to diversify 

recreational angling opportunities (Schullery 1979, Rahel 1997). Many of these programs 
2

included stocking numerous fishless lakes and streams (Franke 1996) because a “vacant niche” 

was thought to exist and thus needed to be filled with sport fish. During this time, little if any 

thought was given to the potential long-term consequences of these actions for indigenous 

species and their habitat. Even though this description focuses on the NPS, state fishery 

management agency programs as well as those on U.S. Forest Service, U.S. Bureau of Land 

Management, state and tribal lands are a mirrored what occurred on National Park lands. 

Early managers had few resources to properly understand ecosystem functions and most saw no 

harm in non-native introductions; rather, they believed their actions to be consistent with 

Congressional mandates. The impact of non-native sport fish on native fish and aquatic systems 

became apparent around 1928 when park managers became concerned about the potential 

adverse effects of stocking fish (Tilmant 1999). Still, the majority of resource managers 

continued to believe that the public benefits of stocking outweighed the “disadvantages which 

may incidentally occur” (Wright and Thompson 1933; Sellars 1997). 

Because the effects of “non-native” introductions on aquatic systems were less noticeable than in 

terrestrial systems, they are arguably the most altered and adversely affected resources in park 

units today. Alterations include native species displacement, population reduction, disease and 

parasite introduction, and hybridization. The magnitude and extent of these actions make it 

difficult for park managers to protect and conserve aquatic resources “unimpaired for the 

enjoyment of future generations” (NPS Organic Act 16 USC). In many cases the impacts on 

native fish populations have been so great that many species are now Federally listed as 

Endangered or Threatened (e.g. Gila trout, Rio Grande Cutthroat trout, bull trout, etc.) 

Restoration Efforts

In an effort to restore the ecological integrity of historically altered ecosystems and to comply 

with the intent of the 1916 Organic Act and NPS policies, biologists initiated efforts to restore 

native fishes to segments of their historic range and these efforts are continuing. It cannot be 

overemphasized that successful restoration projects depend on appropriate site selection. A 

natural or man-made waterfall or other barrier must be located at any outflow sites to prevent 

non-native species from re-entering the treatment area after restoration is complete. 

The first effort to protect native fish occurred in 1936 when NPS Director Cammerer issued 

written fish management policies with the intent of prohibiting the wider distribution of nonnative fish in NPS waters (Cammerer 1936). This Order stated that “exotic species were not to 

be introduced in waters where only native fish existed” and that where exotic and native fish 

existed, the native species were to be favored. Despite this early effort, fish stocking continued 

from the 1930s to the 1970s. A policy to phase out non-native fish stocking in National Parks 

was adopted in 1968. 

Currently, only two techniques (backpack electrofishing and piscicides) are available to 

successfully remove non-native fishes in order to restore native fish to segments of their historic 

range. Kulp and Moore (2000) and Moore et al. (2005) demonstrated that electrofishing 

techniques were used successfully to remove non-native rainbow trout (Oncorhynchus mykiss) 

from segments of small streams in Great Smoky Mountains National Park (GRSM). While this 
3

technique is effective in segments of small streams with natural barriers, electrofishing is more 

time consuming and more costly than restoration projects using piscicides (Moore et al 2005). 

Restoration projects using piscicides typically rely on one of the four chemicals approved by the 

U.S. Environmental Protection Agency (EPA) for use in fisheries management to eliminate nonnative fish: Fintrol®, Rotenone®, Lampricide® and Bayluscide®. In general, the use of fish 

poisons (piscicides) is the only way to ensure complete eradication of entire non-native fish 

populations short of dewatering habitats. Historically, Fintrol® (antimycin A) and Rotenone®

have been used by fisheries biologists throughout the United States (U.S.) for restoring native 

fish species in streams and lakes. Lamprecid® (3-trifluromethyl-4-nitrophenol) and Bayluscide®

(niclosamide) can only be used legally in the Great Lakes drainages and their tributaries to 

control sea lampreys (Petromyzon marinus). 

The NPS Antimycin A Manual

Purpose 

The purpose of this manual is to provide fisheries managers’ with protocols for conducting 

lowest risk, yet safe, effective, and lawful fisheries restoration projects with antimycin A. Using 

the protocols specified in this document, fisheries managers will be able to apply antimycin A to 

remove unwanted fish species from streams, impoundments, or small lakes (<50ha) with 

subsequent re-establishment of natural aquatic communities. The standardized and consistent 

use of antimycin A will minimize if not eliminate the potential for human exposure and 

unintended adverse ecological effects on non-target organisms. In addition to the literature cited, 

a list of other pertinent references is listed for readers seeking additional information on topics 

discussed in this manual.

Scope and Applicability 

This document provides a thorough understanding of antimycin A use with regard to permitting, 

legalities, history, chemistry, risk analysis, safety precautions, application materials, treatment 

concentrations, application techniques, deactivation procedures, reporting, and project 

monitoring protocols. The manual contains the information necessary to plan and implement a 

project using antimycin A, pending the completion of all pertinent certifications, bioassays, 

environmental planning, permitting, and compliance requirements. Equipment checklists are 

also provided to help the project manager/applicator purchase, build, and maintain all equipment 

needed for antimycin A treatments. 

It is a violation of Federal law to apply antimycin A in a manner inconsistent with its labeling. 

. 

Structure

This manual is divided into four sections: the main text (pages 1 - 29), administrative operating 

procedures (pages 30 – 47), technical operating procedures (pages 48 - 88), and appendices (91 – 

124. The main text provides an overview of the complexities involved in preparing for and 

conducting an antimycin A treatment. The administrative operating procedures (AOP) provide 

detailed instructions on how to meet the administrative requirements of an antimycin A treatment 

(e.g., obtaining the necessary permits). The technical operating procedures (TOP) provide 

detailed instructions on how to satisfy the technical requirements of an antimycin A treatment 
4

(e.g., recommendations on how to mix and apply the compound). The appendices provide 

additional resources for conducting antimycin A treatments. 

Updates

Every effort has been made to ensure that the most accurate and up-to-date information is 

contained in this manual. The National Park Service recognizes that, from time to time, this 

manual may require revisions based on new information (e.g., improved treatment techniques), 

changes in state and Federal regulations (e.g., changes to the EPA-approved product label), 

and/or other factors. As appropriate, NPS will revise this manual in accordance with the 

appropriate NPS procedures. 

Deviations 

Although every effort should be made to adhere to the protocols outlined in this document, the 

NPS recognizes the potential need to deviate from certain protocols under extenuating 

circumstances. Such circumstances include situations which, if left unchecked, may jeopardize 

the safety of an individual, result in exposure beyond the treatment area, violate Federal or other 

regulations, or render the treatment ineffective. In such instances, the project manager will 

document the deviation and rationale in accordance with AOP 11. 

It is a violation of Federal law to apply antimycin A in a manner inconsistent with its labeling. A 

protocol deviation that violates the antimycin A product label, while properly documented, does 

not absolve the violator(s) from potential administrative or legal action under FIFRA. 

Antimycin A Overview 

This section provides an overview of the piscicide antimycin A including its background, 

approved uses, formulation and application methods, environmental fate, and toxicity. 

Additional information can be found on the Fintrol® product label and the literature cited in 

Appendix A. 

Background

Antimycin A, discovered in 1945, is an antibiotic derived from the soil mold Streptomyces. 

Antimycin A is currently registered with the U.S. Environmental Protection Agency (EPA) as a 

restricted use pesticide under the trade name Fintrol® (EPA Registration number 39096-2). 

Restricted use chemicals may be applied only by certified pesticide applicators. 

Antimycin A applications are made directly to water. The putative mode of action for antimycin 

A is that the compound uncouples oxidative phosphorylation by blocking the electron transport 

pathway to Complex III within the mitochondria (Bettermann et al. 1996). Antimycin A (3-

methylbutanoic acid 3[[3-(formylamino)-2-hydroxylbenzoyl]amino]-8-hexyl-2,6-dimethyl-4,9-

dioxo-1,5-dioxonan-7-yl_ester) consists of four major components which in turn consist of a pair 

of compounds, for a total of eight significant homologues (Abidi and Adams 1987; Abidi 1988) 

— four “major” and four “minor”—that are distinguished by their chemical constituents. Figure 

1 presents the chemical structure of the A3 component of antimycin A which is believed to be 

the most toxic isomer.
5

This section provides an overview of the piscicide antimycin A including its background, 

approved uses, formulation and application methods, environmental fate, and toxicity. 

Additional information can be found on the Fintrol® product label and the literature cited in 

Appendix E. 

Figure 1. Chemical structure of antimycin A (A3) molecule. 

Source: http://toxnet.nlm.nih.gov/cgi-bin/sis/search

Approved Uses 

Antimycin A is a restricted use pesticide and therefore may only be applied by pesticide 

applicators certified in the aquatic category by the state in which the application will occur. 

Employees working under the direct supervision of the certified project manager may assist in 

antimycin A application. 

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 with antimycin A at water column concentrations ranging 

from 5 to 25 μg/L 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 in order to restore 

listed or indigenous species (Wormell 2005). 

In a selective kill, the water body is treated with antimycin A at 0.5 to 1.0 μg/L of antimycin A to 

eliminate only small, scaled fishes. A common objective of a selective kill is to eliminate 

smaller fishes to free up food and other resources for larger fish. Selective kills at higher 

concentrations are also used in catfish production (aquaculture) to eliminate scaled fish that 

commonly reduce the catfish yields of commercial catfish farmers. According to the Fintrol®

label, scaled fish in aquaculture ponds succumb to treatment with antimycin A at 5 to 10 μg/L of 

antimycin A, whereas catfish generally tolerate antimycin A at concentrations up to 20μg/L 

(Wormell 2005). Based on information provided by the registrant (Personal communication, 

Mary Romeo, president of AquaBiotics), the majority of Fintrol® use in [catfish] aquaculture is 

for food-fish production and not fingerling-production. According to aquaculture use information 
6

collected by the Southern Regional Aquaculture Center (Personal communication, Dr. Craig 

Tucker, U. S. Department of Agriculture), Fintrol® use is roughly equally divided between foodfish and fingerling-production ponds to eliminate scaled fish at various points within the 

production cycle. 

Formulation 

Antimycin A is produced as a fermentation product from Streptomyces mold and is sold as a 

“unit” of biological activity (480 ml). A “unit” of antimycin A consists of one bottle of active 

ingredient (240 ml) and one bottle of diluent (240 ml) that contains a surfactant. According to 

label instructions, the active ingredient and diluent must be mixed together prior to use of the 

product. Once the product is mixed, the label states it must be used within seven days (Ayerst, 

1970). The surfactant is intended to enhance the solubility of antimycin in water and to increase 

the transfer efficiency of antimycin across cell membranes (e.g. gill uptake). 

Environmental Fate

The primary route of antimycin A degradation is alkaline hydrolysis and the chemical becomes 

inactive approximately eight hours after being mixed with water (USEPA 2006). Antimycin 

breaks down more rapidly in warmer (i.e. >12C) and more alkaline (i.e. >pH 8.0) water. Field 

studies of downstream movement also show that the compound can extend and remain active for 

linear distances of 1.75 km beyond desired treatment areas when no effort is made to deactivate 

the compound (Tiffan and Bergersen 1996). However, field observations from projects in New 

Mexico, Tennessee, Nevada and South Carolina demonstrate that antimycin A is naturally 

neutralized in much shorter distances, often 500 m or less. There are no direct measurements of 

antimycin adsorption; however, field observations provide evidence that antimycin adsorbs 

significantly to soil, sediments and organic material in the stream thereby reducing potential 

exposure outside of the immediate treatment area (USEPA 2006). Field observations from NPS 

supervised projects in South Carolina provide evidence that adsorption to organic material also 

limits the linear and vertical distance the antimycin is effective (Moore and Kulp, personal 

observations). 

Environmental factors such as water body size, discharge, pH, water 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. As antimycin A travels 

downstream, aeration and agitation due to the physical movement of the water contribute to the 

degradation of antimycin and decrease the piscicidal activity of antimycin. In Sams Creek 

(GRSM), a 15-20 m (50-60 ft) drop in elevation effectively deactivated antimycin, whereas in 

Bear Creek (GRSM) a drop in elevation of 24-30 m (80-100 ft) was required to deactivate 

antimycin (Moore et al 2005). In some western parks such as Rocky Mountain and Crater Lake 

NP, effective vertical change in elevation for antimycin deactivation was 40-60 m (120-200 ft) 

Rosenlund, personal observation). These data provide an indication of the variability between 

project areas and provide insights into the necessity for on site bioassays described in TOP 9. 

Toxicity

When antimycin is applied according to the procedures outlined in this manual for native fish 

restoration, it is not toxic to terrestrial organisms. Information derived from studies in GRSM 

also demonstrates that it is not toxic to salamanders or crayfish (Moore et. al. 2005). The lethal 

toxicity of antimycin varies between families and species of fish. Appendix C provides 
7

information on the amount of antimycin required for a lethal dose for many fish species. 

Although fish eggs are susceptible to laboratory exposure of antimycin A (100% mortality using 

10 ppb for 1 hour), impacts to eggs in the gravel have not been observed in actual treatments. 

Instream studies by Berger et al. (1975) and Olson and Marking (1965) demonstrated that treated 

water does not completely mix with the water flowing through gravel where salmonid eggs 

incubate and therefore does not have an impact on this life phase. Some native greenback 

cutthroat trout (O. clarki stomias) restoration projects in Rocky Mountain National Park failed 

because treatment occurred while non-native trout eggs were incubating in the gravel (personal 

communication, B. Rosenlund, U. S. Fish and Wildlife Service 2006). 

Regulatory Compliance 

Applying antimycin A in a lawful manner may require users to comply with at least four Federal 

laws: the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Clean Water Act 

(CWA), the National Environmental Policy Act (NEPA), and the Endangered Species Act (ESA) 

(AOP 3). Note that adherence to the provisions of the laws discussed herein does not absolve the 

applicator from complying with appropriate state, tribal, or territorial regulations. The project 

manager is responsible for ensuring and, where appropriate, documenting legal compliance with 

all applicable regulations. 

Additionally, projects on federal lands must comply with The Department of Interior, 

Department Manual 517 Integrated Pest Management (DOI, 2007) requires use of an integrated 

pest management approach when managing pest species (Title 7 136r-1), (Appendix B.1). At the 

NPS and FWS bureau levels pesticide use proposals should be submitted through the respective 

bureau's IPM Program's Pesticide Use Proposal Process where all aspects of the project are 

reviewed for compliance with the IPM approach, insures the problem not the symptom is 

addressed, and plans for long term management. Pesticide requests are reviewed, approved, 

denied, or approved on a conditional basis. Every park project or project on federal lands is 

required to go through an annual review when requesting approval for the use of a pesticide. 

For the context of this document, pests are defined as "living organisms that interfere with the 

purposes or management objectives of a specific site within a park or that jeopardize human 

health or safety" (MP 2006 4.4.5.1). The procedures in this manual support the "11 Step the IPM 

Process" implemented by NPS and USFWS (Appendix B.2). This approach fosters sciencebased decision-making on a site specific basis as described in this manual. 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) 

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) require all pesticides sold or 

distributed in the United States (including imported pesticides) to be registered by the EPA. 

Registration typically requires submission of toxicity, environmental fate, and other data which 

EPA then evaluates to determine whether or not the pesticide will pose “unreasonable adverse 

effects” to human health or the environment. 

FIFRA also requires that pesticide products be labeled appropriately and that use is consistent 

with both the affixed product label and any accompanying or referenced materials, together 

making up the product’s labeling. Each product’s labeling provides users with all the necessary 
8

information to conduct a FIFRA-compliant application. This information includes directions for 

use (including storage and disposal), permitted and prohibited use sites, personal protective 

equipment that must be worn when handling or applying the product, and precautionary 

statements based on the product’s toxicity. 

It is a violation of FIFRA to apply a pesticide in a manner inconsistent with its label. To ensure 

compliance with FIFRA, any and all antimycin A applications must be made in strict accordance 

with the EPA-approved product labeling. 

Clean Water Act 

The Federal Water Pollution Control Act Amendments of 1972, (as amended in 1977 and 

commonly known as the Clean Water Act or CWA), established the basic structure for regulating 

discharges of pollutants into the waters of the United States. CWA gives EPA the authority to 

implement pollution control programs and to set water quality standards for all contaminants in 

surface waters, and makes it unlawful for any entity to discharge a pollutant from a point source 

into navigable waters unless a permit is previously obtained under the National Pollutant 

Discharge Elimination System (NPDES) program (Clean Water Act Section 402).

EPA issued a Final Rule (FRL-8248-1) on November 27, 2006 clarifying two specific 

circumstances in which a Clean Water Act permit is not required to apply pesticides to or around 

waters of the United States: (1) the application of pesticides directly to water in order to control 

pests and (2) the application of pesticides to control pests that are present over (including near) 

water, where a portion of the pesticides will unavoidably be deposited into the water in order to 

target the pests effectively. 

Antimycin A is an aquatic pesticide applied directly to water to control fish; therefore, provided 

that antimycin A is registered with EPA, an NPDES permit is not required to make a CWAcompliant treatment. Note that EPA’s Final Rule does not absolve the project manager from 

ensuring appropriate state, tribal, or other compliance with CWA. 

 

National Environmental Policy Act 

In 1969, Congress passed the National Environmental Policy Act (NEPA) and this legislation 

became effective January 1, 1970. NEPA established the environmental policies for the United 

States with the intent of ensuring that the physical environment is protected for present and 

future generations. This legislation requires that any action or activity funded, authorized, or 

accomplished by a federal agency adhere to NEPA. This requires that the action agency inform 

interested public of the proposed action, document the decision-making process to ensure that the 

proposed action is evaluated in context of reasonable alternatives, if any, and make public the 

final decision regarding the proposed action. 

Public interest in the management of natural resources in National Parks and other federal lands 

has increased as has public knowledge of management efforts. With water resources, 

management actions related to the removal of non-native fish with piscicides is controversial, 

considered a major Federal action, and is not categorically excluded under NEPA. Because of 

this, an environmental assessment (EA) or environmental impact statement (EIS) must be 

prepared for a native fish restoration project using piscicides in National Parks or other federal 
9

lands. The EA is a concise public document that explains the need for the proposed action, 

alternatives to the proposed action, the environmental impact of each alternative and a list of the 

agencies and people consulted. The EA is intended to provide sufficient evidence to determine if 

an EIS or finding of no significant impact (FONSI) should be prepared (Director’s Order #12, 

2001). If the park or federal land owner has multiple projects in the same area, it is 

recommended that all proposed streams be listed in the EA or EIS. By following this 

recommendation, NEPA compliance will only need to be done once. Native fish restoration 

projects on other federal lands require NEPA compliance but the requirements may vary slightly 

between federal agencies. 

Once completed, a public notice of the availability of the document for public review must be 

issued. As a general rule for the NPS the document must undergo a 30 day public review and 

comment period. This process is to be guided by agency specific mandates and guidelines. 

Based on the information gained and developed from the review, the park will decide if an EIS is 

necessary or to issue a FONSI. If the latter, then the FONSI must specify the reasons why the 

action will not have a significant impact on the human or natural environment and state why the 

EIS is not necessary. This document is recommended for approval by the Park Superintendent 

and forwarded to the regional director for final approval and signature. This process is to be 

guided by agency specific mandates and guidelines. 

Endangered Species Protection Act 

Federal regulations implementing Section 7 of the Endangered Species Act, 16 U.S.C. Section 

1536(a) (2), require 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 (hereafter referred to as the 

“Services”), if they are proposing an "action" that may affect listed species or their designated 

habitat. Each Federal agency is required under the Act to ensure 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. EPA, a Federal agency, 

authorizes through product labeling the use of pesticides such as antimycin A. Pesticide users, in 

turn, must comply with product labeling that EPA approves under the Federal Insecticide, 

Fungicide and Rodenticide Act, 7 U.S.C. Section 136-136y. , et seq, for the purpose of 

preventing jeopardy to listed species or adverse modification of their critical habitat. FWS 

defines adverse modification to designated critical habitat as “a direct or indirect alteration that 

appreciably diminishes the value of critical habitat for both the survival and recovery of a listed 

species” (50 CFR 402.02). 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" (50 C.F.R. § 402.02). 

To facilitate compliance with the requirements of the Endangered Species Act subsection (a)(2), 

the Environmental Protection Agency (hereafter referred to as the EPA or “the Agency”) Office 

of Pesticide Programs has established methodologies for evaluating whether a proposed 

registration action may directly or indirectly affect listed species or their designated critical 

habitat (U.S. EPA 2004). Based on the Agency’s screening-level risk assessment, levels of 
10

concern (LOCs) are exceeded for direct or indirect effects for listed or candidate aquatic species 

that co-occur in the area of the proposed antimycin use or areas downstream that could be 

contaminated if the proper neutralization procedures are not used (U. S. EPA 2006). If 

determined that listed or candidate species are present in the proposed treatment area, further 

biological assessment must be undertaken. The extent to which listed species may be at risk 

from the proposed action determines the need for development of a more comprehensive 

consultation package as required by the Endangered Species Act (ESA). 

The process for determining risk to endangered species is discussed in the document entitled 

“Overview of the Ecological Risk Assessment Process in the Office of Pesticide Programs.”1 If 

the assumptions associated with the screening-level action area result in risk quotients (RQ or 

ratio of exposure to toxicity) that are below the Agency’s level of concern (LOC) for risk to 

Federally-listed endangered and/or threatened (“listed”) species, a “no effect” determination 

conclusion is made with respect to listed species in the project area. EPA’s ‘no effect’ 

determinations also takes into account the potential indirect effects to listed species by also 

evaluating the RQs for species that listed species may depend upon for survival. 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 

should 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. 

 1 http://www.epa.gov/oppfead1/endanger/consultation/ecorisk-overview.pdf
11

Figure 2. A conceptual model outlining the interaction processes of working with the Services 

under the Endangered Species Act (ESA). 

Once the action area is defined, the U.S. Fish and Wildlife Service and/or the National Marine 

Fishery Service, hereafter referred to as “the Services,” will provide action agencies such as the 

National Park Service and the EPA with a list of designated critical habitat and listed species that 

occur in the action area and the action agency determines that the action “may affect” listed 

species. If designated critical habitat or listed species co-occur or overlap with the action area, 

then the Agencies will engage in consultation with the Services. The consultation package will 

include: use site information, listed species biological characteristics (direct effects analysis), 

biological/ecological information for indirect and critical habitat effects. Additional details on 

information to be provided to the Services as required by the ESA are presented in (AOP 3). 

The action agency is responsible for determining whether or not the proposed action “may 

affect” designated critical habitat or protected species. This determination must be submitted to 

the appropriate Service for concurrence. 

Biological Assessment 

Listed Species/Critical Habitat 

Present 

No Effect May Affect 

Not Likely to 

Adversely Affect 

Likely to Adversely 

Affect 

Informal Consultation 

End Informal 

Consultation 

Formal Consultation 

Biological Opinion 

USFWS Concurrence USFWS No 

Concurrence 
12

No Effect Determination 

If the action agency determines that the proposed action has “no effect” on designated critical 

habitat or listed species it is not required to consult with the Services on the action. 

 

Not Likely to Adversely Affect Determination

If the action agency determines that the proposed action “may affect” designated critical habitat 

or protected species, there are two possible determinations. If the determination is that the 

proposed action is “not likely to adversely affect” and the Services provide written concurrence, 

the action agency has completed its consultation obligation under the ESA. 

Likely to Adversely Affect Determination 

If the determination is that the proposed action is “likely to adversely affect,” (LAA) or the 

Services “non-concur” with the action agency’s “not likely to adversely affect determination” 

(NLAA) the action agency must enter “formal consultation” with the appropriate Service. 

In response to a LAA determination where the Service provides a nonjeopardy opinion, it may 

issue reasonable and prudent measures (RPMs) when incidental take is anticipated that are 

necessary or appropriate to minimize the impacts incidental of any such incidental take. 

In response to a LAA determination where the Service provides a jeopardy opinion, the Services 

will provide (1) reasonable and prudent alternatives (RPAs) to the action agency’s proposed 

action in its Biological Opinion, (2) issue an incidental take statement together with reasonable 

and prudent measures that are necessary or appropriate to minimize take, or (3) determine that 

there are no RPA(s) nor can it issue an incidental take statement 

Overview of Project Planning and Implementation 

This section provides an overview of the qualifications for the project manager(s), required 

training for project personnel, planning, steps and documentation required to conduct a safe, 

effective, and lawful antimycin A application. Detailed information and instructions for training 

and documentation in compliance with federal law and this manual are presented in the 

referenced Administrative Operating Procedures (AOP) and Technical Operating Procedures 

(TOP). 

Training

Detailed information on training is presented in AOP 5 and 6. Because antimycin A is a 

restricted use pesticide, it may only be applied by or under the direct supervision of applicators 

that have passed the required state pesticide applicator certification course. Additionally, a 

project manager is required to participate in all aspects of a minimum of two antimycin projects 

prior to supervising an antimycin A application. Formal training from the Rotenone and 

Antimycin Use in Fishery Management course taught at the National Conservation Training 

Center (http://training.fws.gov/, can be substituted for participation in one on-site application. 

Adherence to these actions will insure that professional and public credibility are maintained 

during native fish restoration projects using antimycin. 
13

Project Manager Qualifications 

The project manager must have demonstrated experience in the following areas: 

a. Certified Pesticide Applicator: the project manager must be certified by the state 

in which the restricted use pesticide will be applied 

b. Water chemistry: pH, conductivity, dissolved oxygen, and temperature 

c. Determination of on-site toxicity 

d. Determination of water travel time using a tracer dye 

e. Discharge measurements 

f. Project Implementation: The project manager must have experience in all aspects 

of an antimycin project. This includes the use and operation of all equipment used 

in applications (drip stations, handling antimycin, and washing cans, using all 

safety equipment and keeping project records.) and the application of potassium 

permanganate for neutralization of antimycin A. Experience can come from 

participation in a minimum of two antimycin A projects under the supervision of 

an experienced project manager. 

g. The Project Manager is responsible for keeping all records pertaining to the 

project as specified in Administrative Operating Procedure (AOP) 8. The Project 

Manager should also oversee the preparation of the final report. 

Project Personnel Qualifications 

The project manager(s) must document that all project personnel have attended the Safety 

Meeting and have received the following training: 

a. Safety: each team member must be trained on how to safely handle antimycin A. 

This training will stress compliance with label directions 

b. Personal Protective Equipment: each team member must be trained on how to 

properly use the personal protective equipment required for handling and/or 

applying antimycin A. 

Reporting 

For each antimycin A project, the project manager will prepare a preliminary project plan to 

document the purpose and rational for the project. Additionally once completed the project 

manager will complete a final report that will include documentation of project planning, 

treatment records, protocol deviations and project evaluation. These documents are to be filed in 

the project manager’s office and a copy of the final report should be filed in the agency’s central 

office. The actual amount of antimycin A and potassium permanganate applied should also be 

recorded on a pesticide use log and submitted annually per each agency's recording requirements. 

NPS and USFWS require annual reporting of pesticide use through the agency's Pesticide Use 

Proposal System 

Preliminary Project Plan 

Detailed information on preparing the Preliminary Project Plan is presented in AOP 1. As part 

of the Preliminary Planning stage, the project manager will prepare a “Preliminary Project Plan.” 

The primary objective of this document is to communicate the purpose, rationale, and a general 

overview of the intended application. The document will consist of the following draft sections: 

Background: This section will include the objectives, rationale, fish species management 

strategy, legal authority, and project team. 
14

Treatment Overview: This section will include the application dates, treatment area, project area, 

and equipment needs. 

Regulatory Compliance: This section will include relevant information on NEPA and ESA. 

Final Project Plan 

Detailed information on preparing the Final Project Plan is presented in AOP 1. After the 

Preliminary Project Plan has been reviewed by management, other relevant federal and state 

agencies, and the public, the project manager will revise and finalize the document as the “Final 

Project Plan.” The main objectives of the Final Project Plan are to provide the project manager 

with detailed instructions for conducting the application and to serve as a record of the project 

which can be used to gauge the success of the treatment and improve best practices for future 

projects. The document will consist of the following finalized sections: 

Background: This section will include the objectives, rationale, fish species management 

strategy, legal authority, and project team. 

Treatment Overview: This section will include the application dates, treatment area, project 

area, treatment area characterization, pre-treatment bio-monitoring, and equipment needs. 

Regulatory Compliance: This section will include relevant information on NEPA, and ESA. 

Appendices: This section will include the NEPA compliance records, final environmental 

assessment decision, and official ESA correspondence. 

Treatment Records 

Detailed information on documenting treatment records is presented in AOP 8. Treatment 

records include relevant water body and application data collected during the treatment. The 

project manager will maintain original data sheets for the treatment and copies of treatment 

records (including all data collected prior to, during and after the treatment). For NPS units it is 

recommended that treatment records will be retained for a minimum of five years and that 

guidance provided in Directors Order 12 for administrative records be followed. Guidance for 

other agencies may be different and projects on their lands should follow their policies. 

Protocol Deviations 

Detailed information on documenting protocol deviations is presented in AOP 11; an example 

Protocol Deviation Form is presented in Appendix B.10. Throughout the project, the project 

manager will document any deviation from the standard operating procedures identified in this 

manual.

Final Report

Detailed information on preparing the Final Report is presented in AOP 10. At the completion 

of the antimycin A project, the project manager will prepare a “Final Report.” The primary 

objective of the Final Report is to document the intended and actual outcomes of the antimycin A 

application. 
15

Overview of Antimycin A Application 

This section provides an overview of the activities to be completed before, during, and after an 

antimycin A treatment to ensure a safe, effective, and lawful application. Detailed information 

and instructions for applying antimycin A in compliance with federal law and this manual are 

presented in the referenced AOP’s and TOP’s. 

Before Application 

Prior to applying antimycin A, the project manager and appropriate team members will conduct 

and/or prepare for the following activities: planning and public involvement, defining the 

treatment area and project areas, characterization of the physical treatment area, pre-treatment 

bio-monitoring, public notification and closures, safety training and use of personal protective 

equipment, and regulatory compliance. 

Planning and Public Involvement

Planning identifies the steps, timing of planning, and final decision making process regarding an 

antimycin A application. Public (stakeholder) involvement informs parties interested in the 

proposed treatment of any potential direct impacts [on the public] such as area closures or 

drinking water restrictions (AOP 1 or 2). Through public involvement, potential issues/concerns 

are identified and resolved and are later discussed in the Final Project Plan. The general steps for 

planning and public involvement can be divided into Preliminary Planning, Intermediate 

Planning, and Final Planning (Figure 3). Ideally, stakeholder involvement occurs concurrently 

during each stage of the project, with most of the stakeholder involvement occurring during the 

Intermediate Planning stage. 

Figure 3. Example of a typical antimycin A project implementation planning and application 

chronology beginning with project conception through implementation. 

Preliminary Planning Stage: Preliminary Planning typically begins between one and two years 

before treatment and initiates the formal documentation process for the project. As part of the 

Preliminary Planning stage, the project manager will prepare a draft Preliminary Project Plan. 

The draft Preliminary Project Plan will be distributed within the lead agency for appropriate 

internal review and approval. After comments are incorporated and the document is approved, 

the revised Preliminary Project Plan is typically distributed to other appropriate federal and state 

Preliminary Planning Intermediate Planning Final Planning

 18-24 months 12-18 months 6 -12 months 

Project 

Implementation

Begin NEPA 

Compliance 

Draft 

FONSI 

Public 

Outreach & 

Review 

Draft EA 

Brief 

Partner 

Agencies 

Macroinvertebrate 

and Fish 

Monitoring 

Post Area 

Closures 

Verify 

Barrier 

Approved 

FONSI 

Public 

Notification 

Safety 

Training 

Regulatory 

Compliance 

USFWS 

Consultation 

Preliminary 

Project Plan 

Final Project 

Plan 

Treatment Area 

Characterization 

Public 

Education 
16

agencies and non-governmental organizations for review. After additional comments are 

incorporated, the revised draft Preliminary Project Plan is typically distributed to other 

appropriate federal and state agencies and non-governmental organizations for review. After 

additional comments are incorporated and the revised document is approved, it is considered the 

final Preliminary Project Plan. 

Although the exact timing is left to the discretion of the project manager, NEPA compliance, and 

if applicable endangered species consultation with FWS and/or the NMFS, is typically initiated 

during the Preliminary Planning Stage. 

Intermediate Planning Stage: Once the Preliminary Project Plan is completed and approved, the 

document will be distributed for public review thus beginning the Intermediate Planning stage. 

The Intermediate Planning stage allows for appropriate revision of the Preliminary Project Plan 

to address issues regarding new information on the affected environment, regulatory compliance, 

and response to agency and public concerns. During this stage, project managers are encouraged 

to conduct outreach (e.g. stakeholder meetings) as appropriate to inform and educate 

stakeholders about the upcoming application. 

Upon completion of the Preliminary and Intermediate Planning steps, the Preliminary Project 

Plan will be revised and finalized as the “Final Project Plan.” The Final Project Plan will be 

used to conduct the application and, eventually, will form a large portion of the Final Report. In 

addition, the Final Project Plan will include documentation demonstrating compliance with 

NEPA and ESA. 

 

Final Planning Stage: Once the Final Project Plan is approved and all necessary regulatory 

requirements/permitting are complete, Final Planning begins. During the Final Planning phase, 

the project manager will make final preparations to conduct the application such as procuring 

necessary supplies and equipment and briefing team members on the details of the project. 

Defining the Treatment Area and Project Area 

As part of the Preliminary Project Plan, the project manager will clearly define the treatment and 

project areas. Clear definition of these areas facilitates regulatory compliance (e.g. NEPA, ESA, 

etc) and prompts useful stakeholder input. The “treatment area” comprises the water body or 

bodies, or portions thereof, into which antimycin A is applied. In streams, a typical “treatment 

area” would begin with the upper treatment stations and end just downstream or beyond the 

deactivation site. In ponds or lakes, the “treatment area” would consist of the lake or pond, all 

tributary inlets, and possibly the lake outlet. Clear definition of the treatment area is required to 

comply with NEPA and ESA, and also provides stakeholders with an understanding of the exact 

location of areas potentially affected by the treatment. Proper identification will include a 

written description as well as supporting maps and other appropriate documentation. In streams, 

proper identification will include the downstream physical barrier (e.g. waterfall, cascade or dry 

channel) that will prevent upstream migration of non-native fish and downstream movement of 

the piscicide (TOP 1). 

The “project area” includes the location(s) where antimycin A will be mixed and measured for 

use, the treatment area, the areas closed to public access during the project, the location(s) of the 
17

deactivation site(s), and the location of the barrier that will prevent invasive species from reentering the treatment area. Proper identification will include a written description as well as 

supporting maps and other documentation. 

Treatment Area Characterization 

Characterizing the treatment area provides the project manager with detailed water body and 

water quality data needed to complete the Final Project Plan. Characterization includes 

determining target species distribution, delineating the treatment area, water quality parameters 

(e.g., pH, hardness, temperature, dissolved oxygen), identifying discharge sites (e.g. where 

treated water will or may outflow to or join other water bodies) and, for flowing water bodies, 

measuring mean stream gradients, approximate flow times and discharge for each treatment 

section. 

Determining Target Species Distribution: The distribution of target fish within the proposed 

project area will be mapped and the treatment area defined prior to the completion of the Final 

Project Plan (TOP 1). For stream applications, defining the fish distribution includes the 

elevation of the upper distribution limit for every tributary (TOP 1). If native fish exist in 

headwater areas, the distribution work must identify sympatric zones and the treatment area 

defined based on this information. 

Fish distribution information may be collected in a variety of ways, but electrofishing gear is 

commonly used as this technique efficiently captures fish in small high gradient streams (TOP 

1). It is important that only staff experienced in the use of backpack electrofishing gear and fish 

identification verify all distribution information such that no areas are inadvertently over looked 

or specimens misidentified. All distribution information will be recorded and important 

locations noted on either topographical maps or entered into a Geographic Information System 

(GIS) database so that the project area and treatment area can be clearly defined later in the 

Preliminary and Final Project Plan. 

Delineating the Treatment Area: Proper delineation and marking of key locations throughout the 

treatment area facilitates site navigation and also provides reference points useful in record 

keeping and for determining application sites. In some instances, it may be desirable to collect 

gradient and habitat data for 100m stream sections (TOP 1). If the determination is made to 

collect these data, it is recommended that sequentially numbered tags (aluminum and/or plastic 

flagging tape) be placed every 100m (or other appropriate interval) where practical. For flowing 

water bodies, begin at the lower barrier (typically just upstream of the deactivation site) and 

continue upstream to the uppermost end of the treatment area. For some applications, 

determination of elevation of each measured interval is helpful. For example, site markers for 

the barrier site at 457m elevation and the 100m site at 472m elevation would read “0m, 457m” 

and “100m, 472m”, respectively. Once completed, the vertical drop between each 100m site, 

total length of treatment area, and the total vertical elevation drop from the top to the bottom of 

the treatment area can be determined. A study area map depicting these data is useful for project 

planning and crew instruction during daily treatment (TOP 1). 

For lakes or small impoundments begin site measurements at a marked point and continue 

around the perimeter of the treatment area to determine the surface area. Currently, a hand held 
18

GPS unit is probably the best way to delineate the size and area of the lake. The location of all 

inlet streams, springs and wetlands that need to be treated should also be marked on the project 

map. Additionally, a depth profile needs to be completed for the water body. For preliminary 

planning purposes, this can be extrapolated from a USGS 1:24000 TOP maps. But this 

information needs to be verified (ground-truthed) on site prior to the completion of the final 

project plan. 

Discharge Sites: Discharge measurements can be in ft3

/sec or m3/sec. Based on field surveys of 

the project area, the project manager or an experienced team member will identify and 

appropriately mark suitable stream discharge sites or these sites may be arbitrarily selected daily 

for the collection of these data. Discharge measurements will be taken daily during the treatment 

to ensure proper daily treatment concentrations (TOP 5). 

Areas recommended for the collection of discharge data are: 

• just upstream of the confluence of each tributary stream within the treatment area 

with the main stream; 

• just after the confluence of each tributary within the treatment area and the main 

stream; 

• immediately upstream of the detoxification site; and 

• immediately downstream of the detoxification site. 

Discharge measurements can generally be conducted several months prior to actual treatment if 

flows are similar to those expected during Antimycin A application and during normal flow 

conditions (i.e. not during or directly after large rain events) for estimating the quantity of 

antimycin A needed and for project planning purposes only. The day prior to treatment, stream 

discharge for the main stream and any tributary stream to be treated will be measured at predetermined measurement locations to determine the amount of antimycin A needed to achieve an 

effective concentration in the stream. Stream discharge sites and measurements will be noted on 

the study stream map (TOP 1). All discharge sites will be clearly marked with flagging and 

denoted on the site map so that discharge measurements can be repeated and compared 

throughout the treatment period. 

Water Travel Times: For flowing water bodies, the determination of approximate water travel 

times from the upper end of the treatment area to all tributaries and to the downstream end of the 

project area using fluorescene or rhodamine dye is recommended (TOP 6). Water travel times 

should also be determined for all tributaries that will be treated. If the project area is sub-divided 

into sections or reaches, this information should be collected for each reach or section. These 

data provide project managers with information that aids daily planning and helps determine 

travel time of antimycin between dispensing stations and for the total project area. This process 

also aids in the identification of potential problem areas (e.g. backwater eddies, springs, seeps, or 

other hydrological anomalies where the dye does not mix well). Data may be added to the study 

area map for easy reference (TOP 1). 

Dye studies can generally be completed prior to actual treatment during normal flow conditions 

and can be completed by one or more teams working simultaneously at different sites. However, 

travel time measurements also should be made just prior to application at several locations within 

treatment area to ensure previously collected data still accurately reflect water travel times. 
19

Stream Gradients: For flowing water bodies, mean stream gradient should be calculated for each 

100m section within the treatment area. Mean stream gradients help the project manager identify 

potential trouble spots or areas. Work in Great Smoky Mountains National Park has 

demonstrated that if the stream gradient exceeds 8%, the length of stream that can be effectively 

treated by one dispensing unit decreases (TOP 1). This information helps the project manager 

determine the proper location of dispensing units so an effective concentration of antimycin is 

maintained throughout the stream segment being treated. 

Pre- and Post-Treatment Bio-Monitoring 

Pre-treatment fish population biomonitoring is recommended to establish carrying capacity prior 

to treatment. Recommended sample locations are upstream of the treatment area, within the 

treatment area and downstream of the treatment area in the deactivation area. Post-treatment 

biomonitoring is recommended in these same locations to determine if the treatment was 

successful, to evaluate effects of deactivation on fish populations and to determine when 

reintroduced native fish species have recovered (TOP 4). 

While not required, pre- and post-application bio-monitoring is recommended for aquatic 

macroinvertebrates upstream of, within and downstream of the treatment area and deactivation 

area (TOP 4). Aquatic macroinvertebrate monitoring will provide pre-treatment data that can be 

compared to post-treatment data to assess short and long-term impacts as well as recovery from 

the antimycin and neutralization with potassium permanganate. However, if data from several 

projects in different geographical areas provides conclusive evidence that these communities 

recover from the treatment, then this monitoring should be considered on a project by project 

basis. If a project is to be conducted in an area for which no information exist, aquatic 

macroinvertebrate monitoring is highly recommended. 

Although there are no specific guidelines for determining the number of locations that aquatic 

macroinvertebrates should be sampled, at least two locations within the treatment area and one 

downstream of the neutralization site should be sampled both prior to treatment (optimally 

within a month) and after treatment (optimally within a month). At each monitoring site, a 

minimum of three follow-up collections should be made (winter, spring and summer/fall) to 

evaluate recovery. The area of the country in which the project occurs will dictate the sampling 

method used (e.g. Surber or Hess samplers are commonly used quantitative collection methods). 

The Rapid Bioassessment method is not as quantitative as the other two methods (e.g. number of 

individuals per unit area) but is a recognized as a semi- quantitative sampling method and is used 

throughout the U.S. 

Pre-treatment fish sampling provides managers with a realistic, measurable population density by 

which population recovery of native species can be measured. In some projects in the Southeast, 

four100-m sites for fish sampling (i.e. two in the treatment area and one immediately 

downstream of the deactivation site, and one control) have been used to collect these data. 

Three-pass depletion or mark-recapture techniques that are normally used to quantify fish 

abundance in wadeable streams. Other accepted methods can also be used. Pre-treatment 

abundance data are the benchmark used to evaluate recovery of the desired native species during 

post treatment evaluations. Species abundance data from the neutralization site and 

control/reference site can be compared pre- and post-treatment to evaluate the impacts of 
20

potassium permanganate on fishes downstream of the treatment area. Post-treatment collections 

are used to assess establishment of the desired species and to evaluate the rate of recovery. 

Public Notification and Closures 

Public access to the treatment area is prohibited while antimycin A is being applied (i.e. the dates 

specified in the Final Project Plan) and during any additional times determined to be necessary 

by the project manager (AOP 4). The project manager will take the necessary steps to inform the 

public of the planned closures and to prevent access to the treatment area. 

The project manager may inform the public through general press release, public meetings, 

newspaper, radio, and/or television. At least 3 - 7 days prior to the project start date, notice of 

closure and dates of closure will be posted on appropriate road crossings or access ways to the 

treatment area. The information presented to the public and on posted notices will include, at a 

minimum, the following: 

• a description and map of the area closed to public entry 

• the time period during which the closure will occur 

• a summary of the objectives and rationale of the closure 

• a contact name, telephone number, and email address for additional information. 

All steps taken to notify the public and prevent access to the treatment area must be documented 

in the Final Report. 

Safety Training and Use of Personal Protective Equipment 

Safety training, including the use of personal protective equipment (PPE), is required prior to 

any application of antimycin A (AOP 5). Additional pertinent safety information on required 

PPE is also presented on the Fintrol® product label. Prior to applying antimycin A, the project 

manger or their designee will lead a detailed training/briefing on how to safely handle antimycin 

A and how to properly use the necessary PPE that is required to be worn when handling and 

applying antimycin A. In addition, the project manager or their designee will brief team 

members on the legal requirements of the label under FIFRA and any applicable nuances of that 

particular application related to FIFRA, CWA, NEPA, ESA, and any other applicable 

regulations. 

Participation in this meeting is required for all individuals who will be present in the project area 

during the application of antimycin A. The project manager will document the presence of all 

individuals using a sign-in sheet (Appendix B.6) that will be included in the Final Report. 

Regulatory Compliance 

Prior to and during treatment, the project manager will ensure appropriate compliance with 

FIFRA, CWA, NEPA, ESA, state, agency specific and other applicable regulations. All

antimycin A projects on Federal lands must include the proper NEPA compliance documentation 

prior to treatment (AOP 3). 
21

During the Application Dates 

During the application dates, the project team will apply and deactivate antimycin A under the 

supervision of the project manager (See Overview of Project Planning and Implementation). 

This will include the appropriate storage, transportation, decontamination (e.g. proper cleansing 

of equipment), and spill containment (AOP 7); pre-treatment site preparation and setup (TOP 1-

8); pre-treatment bioassay (TOP 9); applying antimycin A (TOP 13, 14); minimize or eliminate 

personal exposure (AOP 5); and deactivating with potassium permanganate (TOP 16). 

In addition, at the beginning and conclusion of each work day, the project manager will assemble 

all team members. During these meetings, the project manager will provide the team with an 

update on project status, daily roles (duty roster)/responsibilities emphasize safety and other 

appropriate matters. As appropriate, team members will inform the project manager and other 

team members of any pertinent information from his/her duty station. 

Storage, Transportation, and Spill Containment of Antimycin A and Equipment 

Proper handling of antimycin A is essential to conducting a safe, effective, and lawful 

application. All project staff should be briefed and clearly understand the details on storage, 

transportation, decontamination, and spill containment prior to treatment (AOP 7). 

Storage: A unit of antimycin A is packaged in two glass bottles (antimycin A and dilutent), 

padded with thick paper sheeting, and housed in a 3.75L metal pail. Prior to use, antimycin A 

will be stored in the pail provided and according the instructions on the EPA-approved product 

label. 

Transportation: Antimycin A will be transported from the storage area to the project area in 

appropriate containers that minimize or eliminate breakage. To transport from the project area to 

the individual application sites, antimycin A and dilutent bottles, with original padding in place, 

will be removed from the pails, mixed and the appropriate amount for each treatment station to 

achieve the desired concentration will be measured and placed in drip-proof, glass or plastic (e.g.

Nalgene®) specimen containers. The date, station number and amount of antimycin in each bottle 

needs to be recorded on each bottle (duct tape and permanent markers has been used effectively 

for this purpose). These plastic containers will be placed in sealed plastic bags and placed in 

padded backpacks or padded, plastic panniers and transported to the individual antimycin station 

for that day’s treatment. 

Spill Containment: In the event of an antimycin A spill on land or in water, the spill will be 

contained as described below. The project manager will document all spills as protocol 

deviations (AOP 11; TOP 18). 

To contain spills on land, stop the spill at its source; dike solution in pools; absorb with clay, 

soil, or other noncombustible, absorbent material; and deactivate materials resulting from an 

antimycin spill and containment with an oxidizing agent such as potassium permanganate. 

To contain spills in water, first determine whether or not the location of the spill is designated for 

immediate treatment. An accidental spill of antimycin A into waters not designated for 

immediate treatment requires the initiation of neutralization procedures using potassium 
22

permanganate as described in TOP 16. The project manager will initiate actions to deactivate the 

chemical to minimize further contamination. 

An accidental spill of antimycin A into waters designated for immediate treatment requires the 

project manager to note the time of the spill and the amount that was spilled on the Antimycin 

Application SOP Deviation Form (Appendix B.10). Additionally, the project manager and 

project personnel should keep detailed notes on the effects on fish in live cages (bioassay) and 

conduct on site monitoring to determine if material from the spill travels further downstream 

than antimycin applied from a dispensing unit. 

Pre-Treatment Bioassay 

The Fintrol® label identifies the lethal treatment concentrations required to kill many fish species 

(Appendix E, Table C.1); however, lethal concentrations for other species and/or varying 

environmental conditions (e.g. pH, alkalinity, temperature, stream gradient) may require 

adjustments in the concentration to be used. Because of these variations, it is highly 

recommended that the project manager conduct a bioassay to determine the proper target 

concentration of antimycin A (TOP 9). These on-site bioassays are conducted to ensure that 

antimycin A is applied in amounts lethal to target organisms while minimizing adverse effects on 

non-target organisms and the environment. 

The project manager should record this information in the final report. Based upon the results of 

this bioassay and experience from previous projects, the concentration identified should be 

applicable to similar streams throughout the region with similar water chemistry. 

Bioassays are used to ascertain the concentrations of antimycin and potassium permanganate 

necessary for a successful treatment and deactivation, including: 1) the treatment concentration 

in μg/L (parts per billion; ppb) of antimycin and treatment duration (hours) needed to kill 100% 

of the target species; 2) the concentration of potassium permanganate in mg/L (parts per million; 

ppm), applied to water to deactivate antimycin and reduce antimycin A non-target mortality to 

0%, and 3) the mortality rate to target and non-target species associated with various 

concentrations of potassium permanganate. 

The concept of “exposure” is an important principal for project managers to understand and 

utilize. “Exposure” is defined as the concentration of antimycin A (ppb) multiplied by the 

amount of time (hours) the organism was exposed to that particular concentration. For example, 

a typical treatment for rainbow trout may be 8ppb for 8 hours, or 64 exposures. In some cases, 

the LD100 concentration observed during field bioassays for a particular species (e.g. 50ppb) over 

8 hours (i.e. 400 exposures) may be higher than the antimycin A label permits (25ppb) 

(Appendix E). In order to treat the target species without violating the product label, the project 

manager could plan a treatment of 25ppb for 8 hours (200 exposures each) for day one and 

repeat the treatment on day two to achieve project objectives. Using this technique, the project 

manager can meet restoration goals while limiting impacts on non-target specimens due to higher 

concentrations. 

Application concentration and exposure time varies by species, region, and habitats. In order to 

determine the proper application concentration to achieve a 100% kill for a given species, the 
23

project manager may: 1) use the recommended treatment concentrations from the antimycin A 

label (Appendix E, 2) conduct laboratory bioassays, or 3) on-site bioassays to determine 

appropriate concentration for specific project objectives (TOP 9). Because of the many chemical 

and physical differences between different regions of the nation, a laboratory or on-site bioassay 

is recommended for initial projects to determine appropriate concentrations for the complete 

removal of non-native fish. If the decision is to conduct a laboratory bioassay, it is 

recommended that water from the body of water to be treated be brought to the laboratory for the 

bioassay and that lethality tests are conducted at temperatures that are similar to what may occur 

under field conditions. 

 

Once the bioassay is completed, it is imperative that the appropriate distance between antimycin 

stations (dispensing stations) be determined for stream projects (TOP 12). This is best 

accomplished on day one of the initial treatment, in a region or area. 

The person operating the station(s) is also responsible for observing the fish in each live cage 

(bioassay) located just upstream of each application station. Observations should occur at one to 

two hour intervals after treatment begins and information on fish appearance and condition must 

be recorded on the antimycin dosage and stream temperature data sheet (Appendix B.7). If fish 

die during the day, dead fish are to be removed from live cages and the number and time at 

which the fish were removed recorded. Prior to leaving the station for the day, record the 

appearance of surviving fish should be recorded on the appropriate data sheet. 

In a lake or pond project, live cars with the target species should be randomly distributed 

throughout the water body as described in TOP 14. 

Pre-Treatment Site Preparation and Setup 

It is recommended that pre-treatment site preparation and setup proceed in the following order: 

1. Set one to two antimycin stations in the upstream reach of the project area. 

2. Place holding cages with fish at 100 m intervals for 1 km downstream of the 

stations. 

3. Determining the appropriate spacing for antimycin A dispensing stations based on 

the distance 100% kill was achieved in 24 hours. 

4. Set additional antimycin A stations at the appropriate distance in remainder of 

treatment reach. 

5. Set up and test the potassium permanganate deactivation equipment 

(neutralization site) at a minimum of two to three days prior to antimycin arrival 

at the station. 

Detoxification Methods: 

Potassium Permanganate Station: Based on water travel times, the potassium permanganate 

station should be setup and calibrated two to three days prior to the anticipated arrival of the 

antimycin. Additionally, this will ensure that, in the event of an antimycin A spill in the 

downstream portion of the treatment area that the antimycin can be deactivated prior to 

movement of the chemical beyond the treatment area (TOP 16). 
24

The detoxification site should be located within 100m, or as close as possible, to the barrier 

identified in the Final Project Plan. The detoxification site should have a level area of ground 

large enough to hold the deactivation dispensers. Generally, the dispensing system depends on 

gravity to deliver the liquified potassium permanganate from the barrels to the dispensing units, 

therefore, the barrels must have sufficient vertical drop to ensure continuous and constant flow. 

Experience from previous projects has shown that a drop of approximately eight feet is sufficient 

to meet this need. Additionally, the site must be accessible for staff and equipment; and have 

appropriate morphology to safely allow proper installation and hourly calibration of the 

equipment. Other methods that have been used successfully can also be used. 

Application Methods: Antimycin A is applied to water by a drip-feed device (as part of a drip 

station), backpack sprayer, boat bailer, or sprayer. Drip stations (Figure 4) are typically used in 

streams and rivers inaccessible to boats. 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 Fintrol® 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, lakes or small reservoirs. Application to these 

deeper water bodies may require the use of a pump mechanism to ensure adequate mixing 

throughout the water column where antimycin A is dispensed through a perforated hose 

stretching the depth of the water column or is delivered through the propeller wash (Wormell 

2005). 

Antimycin A Station: Antimycin dispensing stations should be set at pre-determined locations 

following the results of earlier field trials (TOP 12). Locate a fairly level area next to the water 

body for the location of the dispensing unit. Dispensing containers should be leveled on the 

bank at least 15 to 45 mm (6-18 inches) above the water surface (TOP 13). Depending on type 

of dispensing apparatus, it may be placed directly over the water or on shore. Once the 

dispensing container is in place, it should be tested to ensure that it is functioning properly. For 

best results, lift the bucket (filled with a couple gallons of water) for ten seconds to remove air 

from the hose (Figure 4). Moore et al. (2005) provide additional information on this type of unit. 

Steffereud and Propst (1996) provide information on an alternative dispensing unit they have 

used. 
25

Figure 4. Antimycin A drip station in operation during a small stream treatment. The apparatus 

includes a dispensing bucket (19L or 5-gal) containing formulated product (water, antimycin A, 

and the surfactant nonoxyl-9). The dispensing bucket is calibrated to deliver 3.8L or 1-gal per 

hour. 

Applying Antimycin A 

Antimycin A must be handled in accordance with EPA-approved product labeling and OSHA 

standards (AOP 3). Antimycin A may be applied using drip stations (of various designs), boat 

bailer, and, when necessary, a backpack or hand held sprayer. 

 

Antimycin Station Operation: The project manager will authorize preparation or “loading” of 

that day’s antimycin A treatment stations. For flowing water bodies, begin with the upper 

treatment stations and work downward. Where possible, the upper treatment stations should be 

set at least 50m linear distance above the upper distribution of target species to allow sufficient 

mixing of the antimycin A with water prior to reaching the beginning of the target area. A 

similar proven approach from another region may also be used. The method should be 

documented in the agencies procedures antimycin treatments and in the final report. 

Once all of the day’s treatment stations are loaded (TOP 13), the project manager will authorize 

treatment initiation. For NPS projects, treatment begins at the upper station(s) and proceeds 

downstream and/or to lower elevations. When treatment is initiated at the upstream most station, 

a fluorescene or rhodoamine dye is added to the stream as an aid to tracking the progress of the 

antimycin flume. The arrival of the dye plume at downstream stations lets these operators know 

when their antimycin station should be “turned on. A similar proven approach from another 

region may also be used and should be documented in the final report. 

Backpack Sprayer Operation: A backpack sprayer is used to treat backwater areas not readily 

exposed to antimycin A through natural water circulation (TOP 15). These areas, including 

pools, springs, and seeps, were initially identified when determining water travel times through 

the use of a dye. Supplementing drip stations with a backpack sprayer prevents ensures complete 
26

elimination of unwanted species. Sprayers capable of holding11 to 15L (3 to 4 gallons) are 

preferred over larger models (>15 liters) because the weight is difficult to carry safely throughout 

the day over difficult terrain. 

Typically, the sprayer applies two applications. Once the fluorescence dye is applied and 

treatment begins, the sprayer mixes half the antimycin assigned to them along with enough dye 

(fluorescene or rhodoamine) to keep track of where they have sprayed. The sprayer slowly 

follows the dye downstream spraying areas where the dye is not mixing. The sprayer also sprays 

a short distance up (50 – 200 m) the downstream segment of any small tributaries normally 

fishless that are within the treatment zone that might provide some refuge for fish. It is 

important to note that the concentration of antimycin in the sprayer is usually relatively high 

(>50ppb), therefore areas only need be sprayed for several seconds to be effective. Once all 

appropriate areas have been sprayed using the backpack sprayer, a second spraying is normally 

conducted approximately 4 hours later. As a general rule the operator works from the bottom of 

the treatment area to the top for the second application. Once complete, wash and clean all 

equipment as discussed in TOP 13 and 15. 

Exposure Mitigation 

A successful antimycin A application results in no exposure to handlers (i.e. antimycin A mixers, 

loaders, and applicators) or the public. To ensure adequate protection of handlers, all persons 

handling antimycin A wear personal protective equipment (PPE) as required by the EPAapproved product label including, at a minimum, long-sleeved shirt, long pants, chemical 

resistant gloves, and goggles (AOP 5). 

To ensure adequate protection of the public, the following protective measures will be put into 

place: 

• The antimycin A and potassium permanganate product labels will be followed 

• The treatment area will be clearly marked and identified with posted signs 

• Public access to the project area will be prohibited during treatment and for at least two 

days after the last treatment day for streams and two weeks for lakes or small 

impoundments 

• The public will not be permitted to enter (e.g. swim, bathe) or fish in treated waters 

during treatment 

• Dead fish will not be consumed 

• Treated water will not be ingested 

• Out-flow from the treatment area will be deactivated with potassium permanganate any 

time treated water will leave the defined project area or if the terminus of the project area 

is within one kilometer of public access that is not in the defined project area. 

Deactivating with Potassium Permanganate 

Potassium permanganate is a very strong oxidizing agent capable of inflicting serious burns and 

must be handled in accordance with product labeling and OSHA/other standards. Care must be 

taken to minimize exposure to the skin, eyes, and lungs. If skin/eye contact occurs, rinse the 

affected area with cold fresh water for at least 5 minutes and seek immediate medical attention. 
27

The method described below is one that has proven effective during projects in National park 

units. A similar proven method from another region may also be used provided the techniques 

are documented in agency approved plans and in the final report. 

The current label indicates that 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. The following discussion is intended to provide guidance for the types of 

situations in which a project manager may decide not to deactivate with potassium 

permanganate. In remote areas where the risk of human contact is negligible and there is at least 

a one to one dilution from another tributary just downstream of the treatment area the project 

manager may decide not to deactivate. In stream reaches downstream of a water fall or steep 

canyon/gorge that exceed the vertical elevation drop known to naturally deactivate antimycin A, 

the project manager may decide not to deactivate with potassium permanganate provided access 

to the area is closed and there is no chance of human exposure . However, to ensure public 

safety, neutralization is mandatory any time treated water will leave the defined project area. If 

the terminus of the project is within one kilometer of public access that is not in the defined 

project area, neutralization is mandatory. 

Use the potassium permanganate application chart (TOP 16) to determine how much KMnO4

must be applied per hour, based on stream discharge (ft3/sec) at the neutralization station. The 

stream discharge at the neutralization station must be measured daily to determine the correct 

potassium permanganate application rate for each day of the treatment (TOP 5). Normally, a 

potassium permanganate concentration of 1 mg/L is necessary to accommodate background 

potassium demand, 1 mg/L (ppm) to neutralize antimycin A, and 1 mg/L residual to ensure 

complete neutralization, for a total concentration of 3 mg/L. However, some stream systems 

may have high organic loads that demand more KMnO4. If a project area is thought to have a 

high organic demand, pre-treatment bioassays will be necessary to determine the appropriate 

amount of KMnO4 required to neutralize the antimycin. 

Approximately 1- 4 hours prior to the anticipated start time of the deactivation station, measure 

out the appropriate amount of potassium permanganate and add it to one drum/barrel. Stir the 

solution thoroughly (avoid splashing) using a boat paddle or other appropriate method and wait 

for the arrival of the dye to begin the application of potassium permanganate. 

Prior to deactivation, calibrate all drip boxes to ensure proper application rates when applied 

(TOP 16). It is important to re-calibrate the boxes every hour to ensure they are working 

properly and have not gotten clogged. Although potassium permanganate is relatively soluble in 

water (6.4 g/mL at 20oC), experience in GRSM projects has shown that adding more than 100g 

KMnO4 per 3.8 liters of water results in excessive clogging of the drip box drain ports at normal 

operating temperatures of 7 - 10°C. Therefore, for 114L (30 gallon) drums, you may use 57L 

(15 gallons) per hour up to 100g and then switch to 114L (30 gal) per hour delivery when 

application exceeds 100g per hour. If the drip boxes or tanks begin to precipitate potassium 

permanganate, flush the tanks and lines with clean water prior to refilling the tanks for the next 

round. 
28

When the dye is observed approximately 100 – 200 m upstream of the deactivation station 

attendants must carefully stir the potassium permanganate solution again to ensure all potassium 

permanganate is in solution. Once the dye reaches the detoxification station, begin detoxification 

by opening the valve of one barrel. Record the date and time deactivation begins on the 

Detoxification Station Datasheet (Appendix B.8) including any other pertinent information or 

observations. Use a graduated cylinder to calibrate the discharge of potassium permanganate 

solution from the dispensing boxes to ensure the proper application rate. If necessary, adjust the 

valve on the drip box to get the appropriate discharge rate. 

Once the station is operating, stir the solution occasionally to prevent the potassium 

permanganate from settling and check the discharge rate every hour to ensure proper the correct 

amount of potassium permanganate is being applied. Previously inscribed graduations on the 

side of the stock containers (barrels) are also a reliable way to ensure the station is running at the 

desired application rate. Have the secondary barrel filled, mixed, and ready to use to ensure 

there is no gap in the potassium permanganate application process. During stirring, refilling, and 

when the barrel is empty, the operator should visually inspect the container for any potassium 

permanganate precipitate. Report any precipitate to the site supervisor so he/she may determine 

whether or not it is necessary to flush each off-line system thoroughly before refilling for the 

next run. These actions are recorded on the detoxification station datasheet and included in the 

Final Report. 

Note that the purpose of the water in the barrels is to act as a delivery mechanism for the 

KMnO4. Once the barrels are set to deliver a fixed volume of water per hour, you can add the 

respective amount of KMnO4 for the stream discharge measured. A two barrel (container) 

system is an efficient means of delivering the KMnO4 because it provides a backup tank in case 

of part failure, and an uninterrupted flow can be maintained throughout the deactivation process. 

The deactivation station attendant will run the station for a previously determined time (typically 

eight hours) and record the start/stop times of operation. Note any important safety, logistical, or 

functional problems on the datasheet and report to the project manager or site supervisor. 

Although not required, most systems perform better when they are flushed with water at the 

conclusion of each day of use. 

At the conclusion of the final potassium permanganate application and prior to exiting the project 

area, the project team will gather and remove all items brought into the project area. The project 

manager will perform a final walk-through of the project area to ensure no chemicals or 

equipment is left behind. At a later date, in accordance with the timing designated in the Final 

Project Plan, the project manager will have any postings or notices removed. 

After Application 

Upon completion of the application, and in accordance with the Final Project Plan the project 

manager will make arrangements to retreat or restock the treatment area and will prepare the 

Final Report. 
29

Re-Treatment 

The goal of every project is to achieve complete removal of the target organisms with one 

treatment. However, experience has shown this goal may not be achieved for various reasons. 

When additional antimycin A applications are necessary, all aforementioned procedures should 

be followed, including the annual application guideline limits detailed in TOP 13.

Restocking 

Although the timing of stocking desired fish species is at discretion of project manager, it should 

not occur until there is a sufficient forage base in stream to support the reintroduced fish. 

Post-Treatment Bio-Monitoring 

Pre- and post-treatment bio-monitoring of fish species is required to properly identify the 

treatment area and gauge the application’s success (TOP 4). Pre- and post-treatment biomonitoring of aquatic macroinvertebrates is optional, but strongly recommended as these data 

can be used to evaluate the extent to which non-target species are affected by antimycin A and 

for determining whether a sufficient forage base exists to support stocking.

.

Final Report 

At the completion of the antimycin A project, the project manager will prepare a Final Report. 

In the Final Report, the project manager will document the intended and actual outcomes of the 

antimycin A application (AOP 10). 
30

Administrative Operating Procedures 

AOP 1 Preliminary and Final Project Plan 

Applicability 

A Preliminary Project Plan and Final Project Plan will be developed for each antimycin A 

treatment. 

Purpose

To provide standardized documents for the Preliminary Project Plan and the Final Project Plan 

prior to initiation of an antimycin A application. 

Procedure 

As part of the Preliminary and Intermediate Planning stages, respectively, the project manager 

will prepare a “Preliminary Project Plan” and “Final Project Plan.” These documents 

communicate the purpose, rationale, and overview of the intended application to promote 

internal and external feedback. The outlines for completing the required Preliminary and Final 

Project Plan are presented herein. 

Preliminary Project Plan 

The Preliminary Project Plan provides an overview of the intended application to promote 

feedback prior to finalizing the plan. The Preliminary Project Plan will consist of the following 

sections: 

Background: 

Objectives: Explain the overall purpose and objectives of the project. 

Rationale: Explain the rationale for the project. 

Fish Species Management Strategy: explain the overall fish species management strategy and 

how it relates to the objectives and rationale of the project. 

Legal Authority: Explain the legal authority for conducting the application including citations of 

applicable statutes. 

Project Team: Identify the project manager and key team members along with corresponding 

roles, responsibilities, and a summary relevant experience. 

Treatment Overview:

Application Dates: List the projected start and end date of the project. 

Treatment Area: Clearly identify the water bodies or portions thereof to which antimycin A will 

be applied beginning with the highest altitude treatment stations (i.e.., the “upper” treatment 

stations) and ending just beyond the deactivation site. 

Project Area: Clearly identify the location(s) where antimycin A will be formulated (i.e., mixed 

and measured for use), the areas potentially affected by the treatment (i.e., where antimycin A 

and potassium permanganate will be measurably present in the environment), the location(s) of 

the deactivation site(s), the location of the barrier that will prevent invasive species from reentering the treatment area, and potential public access ways. 

Equipment Needs: Describe the equipment that will be used to conduct the application. 
31

Final Project Plan: The Final Project Plan provides the project manager with detailed 

instructions for conducting the application and to serve as a record of the intended project that 

can be used to gauge the success of the treatment and potentially derive best practices for future 

projects. 

Regulatory Compliance: 

FIFRA: Describe the steps that will be taken during the application to comply with the 

requirements of FIFRA (e.g. Federal and all appropriate state labels are strictly adhered to). 

CWA: Describe the steps that will be taken during the application to comply with the 

requirements of CWA. The project manager must ensure compliance with all federal and state 

requirements with CWA. 

NEPA: Describe the steps that will be taken during the application to comply with the 

requirements of NEPA 

ESA: Describe the steps that will be taken during the application to comply with the 

requirements of ESA 

Appendices: 

Final Environmental Assessment (if applicable) 

Final Environmental Impact Statement (if applicable) 

Official ESA Correspondences

Table 1. Necessary components of the Preliminary and Final Project Plans. 

 Preliminary 

Project Plan 

Final 

Project Plan 

Background X X 

 Objectives X X 

 Rationale X X 

 Fish Species Management Strategy X X 

 Legal Authority X X 

 Project Team X X 

Treatment Overview X X 

 Application Dates X X 

 Treatment Area X X 

 Project Area X X 

 Pre-Treatment Bio-Monitoring X 

 Equipment Needs X X 

Regulatory Compliance 

 FIFRA X X 

 CWA X X 

 NEPA X X 

 ESA X X 

Appendices X 

 Final EA or EIS X 

 Official ESA Correspondence X 

 FONSI X 
32

AOP 2 Planning and Public Involvement

Applicability 

This procedure defines the planning and public involvement steps necessary to develop and 

implement an antimycin A treatment. 

Purpose

The purpose of this procedure is to ensure that an antimycin A treatment is fully planned and 

public concerns addressed prior to initiation. Actions identified in this procedure are consistent 

with the requirements of National Environmental Policy Act for federal, state and local agencies 

and tribes. Adequate planning ensures that environmental, regulatory, and public concerns and 

information needs are addressed and incorporated into antimycin A treatment design. 

Procedure 

Adequate planning and public involvement steps are defined below to address the application of 

antimycin A for control of target fish species in flowing and static water. Planning identifies the 

steps, timing of planning, and final decision process regarding antimycin A treatment. Public 

Involvement educates interested parties on the proposed treatment and any potential direct 

impacts such as area closures or drinking water restrictions. Through public involvement, 

potential issues/concerns are identified and resolved to create a more meaningful and complete 

Final Project Plan. Ideally, public involvement occurs concurrently during each stage of the 

project, with most of the public involvement occurring during the Intermediate Planning stage. 

Planning 

The general steps for planning and public involvement can be divided into Preliminary Planning, 

Intermediate Planning, and Final Planning. 

Preliminary Planning: Preliminary Planning begins the formal documentation process for the 

project. As part of the Preliminary Planning stage, the project manager will prepare a draft 

Preliminary Project Plan. The draft Preliminary Project Plan will be distributed to management 

for appropriate internal agency review and approval. After comments are incorporated and 

management approves the document, the revised Preliminary Project Plan is typically distributed 

to other appropriate Federal and state agencies for review and comments. After agency 

comments are incorporated and management approves the revised document, the document is 

considered the final Preliminary Project Plan. 

Although the exact timing is left to the discretion of the project manager, NEPA documentation, 

and, if necessary, material for endangered species consultation with FWS are typically initiated 

and prepared during the Preliminary Planning Stage. The steps involved in this process are: 

1. Define the project proposal, including relationship to identified fisheries management 

strategies and description of the treatment area (later used to define action area). 

2. Preparation of the Preliminary Project Plan 

3. Identification of all applicable laws and regulations. 

4. Receive and incorporate agency review and feedback. 

5. Public scoping to identify issues and concerns. 
33

6. Presentation of preliminary application plan, including all pre- and post- application 

activities. 

Intermediate Planning: Intermediate planning serves as a response to the results of Preliminary 

Planning and finalizes project design for implementation. This planning stage allows for 

revision, as appropriate, of the original project design to address issues regarding new 

information on the affected environment, regulatory compliance, and response to agency and 

public concerns. Components of intermediate planning are: 

• Finalization of project design and application plan. 

• Completion of environmental analysis (e.g. air and water quality, biological resources, 

recreational uses, economics, benefits and costs). 

• Development of public involvement plan. 

• Define pre- and post- application activities, including resource protection and monitoring. 

Final Planning: Once the Final Project Plan is approved by management and all necessary 

regulatory requirements/permitting are complete, Final Planning can begin. During Final 

Planning, the project manager will make final preparations to conduct the application such as 

procuring necessary supplies and equipment and briefing team members on the details of the 

project. The Project Managers Planning and Implementation Check list is found in Appendix 

B.3. 

Public Involvement: Public involvement provides for a process to identify significant public 

issues/concerns and allow input in final project design and implementation. Included in public 

involvement is the development of a Public Involvement Plan, with primary components 

identified below: 

• Identification of interest groups. 

• Definition of process for public information and education and input into final project 

design. 

• Identification of methods to notify public and obtain public comment. 

• Public notification of application. 

• Post-application public information. 
34

AOP 3 Environmental Compliance 

Applicability 

Compliance with all environmental laws and regulations required for safe and legal application 

of antimycin A. 

Purpose 

The purpose of this procedure is to ensure that relevant environmental compliance measures are 

met prior to the initiation of an antimycin A treatment. This includes a variety of laws and 

regulations administered by federal and state agencies, local government, and municipalities and 

tribes. Satisfaction of requirements in relevant sections of the laws and regulations are necessary 

to safely and legally apply antimycin A. 

Procedure 

During initial planning of an antimycin A treatment project, applicable environmental laws and 

regulations are identified. Federal laws and regulations are contained within Federal Insecticide, 

Fungicide, and Rodenticide Act (FIFRA), Clean Water Act (CWA), National Environmental 

Policy Act (NEPA), and Endangered Species Act (ESA). Applicable State laws vary from state 

to state. Laws and regulations by local government entities also vary for location to location. 

Processes for adherence to laws and regulations for State and local governance will vary on a 

project by project basis. 

Federal Laws and Regulations 

FIFRA 

• Basis for piscicide-use regulations. 

• Requires registration of pesticide with EPA. 

• Prohibits use of registered pesticide “in a manner inconsistent with its labeling.” 

• Provides for certification of pesticide applicators.

CWA 

• NPDES permit not required for pesticides applied directly to water to control pests in the 

water. 

• Application is consistent with relevant requirements of FIFRA. 

NEPA: Any antimycin A project occurring on federal lands or otherwise having a federal nexus 

requires adherence to NEPA. This includes the preparation of an Environmental Assessment 

(EA) or Environmental Impact Statement (EIS). For the NPS, detailed information on the 

preparation of an EA or EIS can be found in Director’s Order #12 and Handbook. An equivalent 

manual will guide the preparation of these documents for other federal agencies. 

Basic steps for this process include the following: 

• Define project proposal and conduct internal agency scoping. 

• Identify potential environmental impacts. 

• Consider alternatives (action, no action). 

• Determine “significance” of impact(s). 
35

• Provide for public review and comment on the proposal. 

• Decision notice is completed and provided to agencies and public. 

ESA 

• EPA is the federal action agency responsible for labeling and use of pesticides. For this 

reason, all users of antimycin A must act in compliance with the Endangered Species Act. 

• Section 7 consultations are required to ensure actions of federal agencies do not 

jeopardize existence of listed species or adversely modify critical habitat. 

• The Federal action agency develops and provides to FWS an environmental assessment 

that describes the antimycin A application and assessment of impacts. 

• If action is likely to “adversely affect,” formal consultation is required. 

• Biological Opinion is issued by FWS to action agency and renders a decision of 

“jeopardy” or “non-jeopardy”. 

Endangered Species Act: In compliance with Section 7 of the Endangered Species Act2

, each 

Federal agency shall, in consultation with and with the assistance of the Secretary of the Interior 

(hereafter referred to as “the Secretary”), ensure that any action authorized, funded, or carried 

out by such agency (hereinafter in this section referred to as an "agency action") is not likely to 

jeopardize the continued existence of any endangered species or threatened3 species or result in 

the destruction or adverse modification of habitat of such species which is determined by the 

Secretary, after consultation as appropriate with affected States, to be critical4, unless such 

agency has been granted an exemption for such action by the Committee pursuant to Section 7 

subsection (h) of the Endangered Species Act. In fulfilling the requirements of this paragraph 

each agency shall use the best scientific and commercial data available. 

Species Location Information: Section 7(a) (4) requires Federal agencies to confer with the U. S. 

Fish and Wildlife Service and/or the National Marine Fisheries Service (hereafter referred to as 

the “Services”) on any agency action which is likely to jeopardize the continued existence of any 

species proposed for listing or result in the adverse modification of critical habitat proposed to be 

designated. A conference may involve informal discussions between the Services, the action 

agency, and the applicant. Following informal conference, the Services issue a conference report 

containing recommendations for reducing adverse effects. These recommendations are 

discretionary, because an agency is not prohibited from jeopardizing the continued existence of a 

proposed species or from adversely modifying proposed critical habitat. However, as soon as a 

listing action is finalized, the prohibition against jeopardy or adverse modification applies, 

regardless of the stage of the action. 

Defining the Action Area: Actions necessary to define the project area are described in TOP 1. 

The documentation used by a Federal action agency to initiate consultation should contain a 

 2 http://www.fws.gov/endangered/esa.html 3

 The term "threatened species" means any species which is likely to become an endangered species within the foreseeable future 

throughout all or a significant portion of its range. 

4 The term "critical habitat" for a threatened or endangered species means- (i) the specific areas within the geographical area occupied by the 

species, at the time it is listed in accordance with the provisions of section 4 of the Endangered Species Act, on which are found those physical or 

biological features (I) essential to the conservation of the species and (II) which may require special management considerations or protection; 

and (ii) specific areas outside the geographical area occupied by the species at the time it is listed in accordance with the provisions of section 4 

of this Act, upon a determination by the Secretary that such areas are essential for the conservation of the species.
36

description of the action area5 as defined in the Services regulations and explained in the 

Services' consultation handbook. 

Issues Related to Incidental Take: When the Services determine that a proposed action may 

jeopardize the continued existence of a listed species in the wild or result in adverse modification 

to designated critical habitat, the Services, with the assistance of the Federal agency and/or 

applicant, develop Reasonable and Prudent Alternatives (RPA) that may be undertaken to avoid 

the likelihood of jeopardy or adverse modification. While these RPAs must avoid jeopardy or 

adverse modification, they may result in adverse effects to or take6

 of listed species. If take will 

occur from the implementation of an RPA, an incidental take statement must be developed to 

exempt such take from section 9 prohibitions.7

 

An incidental take statement identifies the level of take that is anticipated from implementation 

of a project as proposed. However, a statement also contains reasonable and prudent measures 

and terms and conditions that are nondiscretionary actions designed to minimize the effects of 

the take, and that must be implemented in order for such take to be exempt from the section 9 

prohibitions. 

State Laws and Regulations 

State Pesticide Applicator Training and Certification: FIFRA provides for state certification of 

pesticide applicators. State requirements are variable, but include requirements for initial 

applicator certification and renewal processes. 

Other State Requirements: Satisfaction of other State laws and regulations may include 

additional technical and public review and associated compliance measures, above and beyond 

federal requirements. 

Local Government Laws and Regulations

Local laws and regulations may be administered by a variety of agencies, commissions, and/or 

boards and in a variety of formats. Preliminary planning steps will include identification of these 

laws and regulations and requirements for implementation of antimycin A application. 

 

5 The action area is defined by regulation as all areas to be affected directly or indirectly by the Federal action and not merely the immediate area 

involved in the action (50 CFR §402.02). This analysis is not limited to the "footprint" of the action nor is it limited by the Federal agency's 

authority. Rather, it is a biological determination of the reach of the proposed action on listed species. Subsequent analyses of the environmental 

baseline, effects of the action, and levels of incidental take are based upon the action area.. Taken from 

http://www.fws.gov/endangered/consultations/sec7_faq.html#4

6

 The term "take" means to harass, harm, pursue, hunt, shoot, wound, kill, trap, capture, or collect, or to attempt to engage in any such conduct. 

Taken from http://www.fws.gov/endangered/consultations/sec7_faq.html#4 

7

 For additional information see pages 4-41 through 4-48 of the Section 7 Consultation Handbook. 
37

AOP 4: Public Notification and Closures

Applicability 

This procedure is applicable to all antimycin A application projects. 

Purpose 

The purpose of this administrative protocol is to provide guidance for public notification prior to 

applications of antimycin A and for notification of closure of the action area to public entry 

during treatment. 

Procedure 

Policy on Pre-Treatment Media Notification (applicable to federal, state, local, and 

tribal agencies) 

General press release: General press releases are sent to media outlets (i.e. newspapers and radio 

and television stations) within the watershed and adjoining areas a minimum of one week in 

advance of treatment. Press releases are provided to the media for voluntary publication or for 

broadcast to the public. This should include a map of the project area and the length of the 

period of closure to the public. 

Policy on Closure and Posting of Treatment Sites as Closed to Public Entry

The project area will be closed to public entry and used during the application. This closure will 

minimize if not eliminate exposure of the public to antimycin. 

 

Potential posting locations adjacent to the proposed project area: 

Designated public access sites 

• Public fishing sites 

• Public parks 

• Boat launch sites 

• Public trail access 

Non-designated sites routinely used by the public 

• Road crossings 

• Known trails crossing or terminating on public lands 

Commercial sites 

• Public businesses in immediate treatment area 

Government agency offices near the treatment area 

All treatment area access points 

Time period: Access sites are posted the evening before treatment is initiated. Postings are 

removed the day after completion of the treatment for streams and one week after treatment for 

lakes or ponds. 

Accepted posting materials: 

Description of area closed to public entry

Press releases 

Laminated treatment notice and project area map and information sheet (Appendix B.4) 
38

AOP 5 Safety Training and Use of Personal Protective 

Equipment

Applicability 

These procedures address the training and use of Personal Protective Equipment (PPE) to ensure 

applicator safety. 

Purpose 

The purpose of this training is to ensure that personnel are provided with and have knowledge of 

and experience with safe handling and use of antimycin A during applications, including use of 

personal protective equipment. Conformance to the training standards and operating procedures 

outlined in this document guarantees that personnel conduct operations in safe and legal 

methods. 

Procedure 

Training with regard to Standard Operating Procedure and General Safety 

• Ensure personnel are familiar with facilities and Station Safety Plan. 

• Ensure personnel are familiar with areas of the facility requiring either safety glasses or 

chemical splash goggles for entry. 

• Personnel must be familiar with safety procedures of all chemicals used during treatment. 

• Antimycin A can be used only by persons who are properly trained. 

• Chemicals are stored in a secured area. 

• Ensure personnel have received all relevant safety training for implementation of an 

antimycin A treatment (e.g., electrofishing procedures and safety, ATV usage, boat safety 

and operations). 

Training related to Field Operations (where applicable)

• Ensure personnel are familiar with health precautions in the use of antimycin A. 

• Personnel are trained about use of PPE. 

• Personnel are trained in pesticide spill procedures.

• Ensure personnel are familiarized with all safety and hazard considerations for an 

individual treatment (e.g., MSDS, First Aid awareness).
39

AOP 6 Quality Assurance (QA) Measures Utilized in 

Antimycin A Application 

Applicability 

These QA measures apply to procedures used in the chemical control of target fish species for a 

variety of management goals. 

Purpose 

Conformance to the training standards and operating procedures outlined in this document 

guarantees that personnel conduct operations by similar, proven, and accepted methods 

regardless of project location. The QA measures described will ensure that data generated by 

workers are accurate and reliable. 

Procedure 

This synopsis outlines the steps taken to assure and to document those day-to-day functions in 

the application of antimycin A for control of target fish species are conducted properly. The 

primary areas addressed in this document include training in standard procedures used in field 

work during antimycin A application treatments. The Training section outlines training offered 

to workers. The Documentation section describes applicable record keeping procedures. 

Training 

The following training is offered to antimycin A applicators. Only training which supports 

working skills necessary to successfully complete an antimycin A treatment are listed. Other 

forms of training which support skills not directly related to antimycin A control are not 

included. 

• Toxicity testing 

• Dye application 

• Discharge measurement 

• Certification of pesticide applicators 

• Antimycin A application 

• Potassium permanganate application 

Training Session Content 

Certification of pesticide applicators: Certified pesticide applicator(s) associated with a 

treatment project must have passed a current state certification examination prior to the initiation 

of a project. A minimum of one certified applicator must be present on site during all phases of a 

treatment project. 

Toxicity testing: Senior staff participates in and supervise bioassay procedures to determine 

toxicity to target species prior to project implementation. Additional personnel receive limited 

introduction. Flow-through test procedures are demonstrated and explained. Data records are 

extensive, so attention is paid to completing all forms accurately. 

Dye application: A training session for dye application to determine water travel time between 

drip stations and/or known points is presented to field personnel. Application of dye is described 

for the purposes of determining water travel time within the project area. 
40

Discharge measurement: Personnel responsible for conducting measurements of stream 

discharge will be proficient in the use of the flow meter to be used and in the collection of 

discharge data. During the treatment, personnel who have no experience in the collection of 

discharge data will be teamed with experienced personnel and trained in the proper use of the 

meter and the collection and recording of discharge data. Additional instruction on conducting 

function checks on the meter, completing calculations of stream discharge, and selecting 

locations to conduct discharge measurements will also be given to inexperienced personnel. 

Antimycin A application: Personnel who apply antimycin A are instructed in the operation of all 

equipment used in applications. An experienced operator demonstrates the equipment and 

procedures used for applications to trainees at a drip station location. Trainees are instructed in 

setting up drip stations, handling antimycin A, keeping records, washing cans, glass or plastic 

containers, and using all safety equipment. After instruction, the operator trainees are required to 

demonstrate their abilities to properly operate an antimycin A drip station during an application. 

Experienced applicators are stationed at antimycin stations adjacent to trainees and they or the 

project manager visits each trainee periodically each day of treatment. 

Potassium permanganate application: Prior to the operation of the potassium permanganate 

(KMnO4) station, an experienced operator demonstrates the equipment and procedures used for 

potassium permanganate applications to trainees at the deactivation site. Trainees are instructed 

in setting up the deactivation station, weighing and mixing KMnO4, keeping records, washing 

containers, proper and secure storage of KMnO4 and using all safety equipment. After 

instruction, the operator trainees are required to demonstrate their abilities to conduct potassium 

permanganate applications. A minimum of one experienced applicator must be at the site at all 

times during the operation of the KMnO4 station 

Documentation 

Training 

A record of training is maintained for each person participating in antimycin A applications. 

This record, kept by the Project Manager for each project, lists training provided to all antimycin 

A application personnel. 

Data Collection and Reporting 

All data collected during field operations is recorded on standardized data forms. Specific data 

collection forms include Discharge Measurement (Appendix B.5), Antimycin A Daily 

Application Form (Appendix B.9), Potassium Permanganate Daily Application Form, 

,(Appendix B.10,), and Daily Antimycin and Potassium Permanganate application Summary 

Sheet (Appendix B.11). In addition, a series of forms are used to record data from on-site 

toxicity tests. The forms are organized to allow the data to be easily transferred to computer data 

files. Transcription of the data is conducted as time allows, if possible, on the day of data 

collection. Forms are stamped "ENTERED" to prevent duplicate entries into data files. Original 

data forms are retained after data entry. 

Data are recorded in ink; however, pencil is acceptable if data forms are used in wet conditions. 

Entry errors are crossed out with a single pen stroke, corrected, and initialed. 
41

AOP 7 Storage, Transportation, and Spill Containment of 

Antimycin A during a Treatment Project 

Applicability 

Procedures apply to all activities related to the storage, transportation, and use of antimycin A 

during a treatment project. 

Purpose 

The purpose of this procedure is to provide instruction for the safe handling and storage of 

antimycin A in the field. 

Procedure

General Storage 

• Antimycin A stockpiles are secured in locked buildings. 

• Antimycin A is stored in locked, covered vehicles in treatment areas having vehicle 

access. 

• Antimycin A is stored in secured areas in treatment areas lacking vehicle access. 

 

Specific Storage 

A unit of antimycin A is packaged as discreet components of active ingredient and diluent in two 

glass bottles, padded with thick paper sheeting and held in metal, 3.75L pails. Inventory logs are 

maintained to record the use of antimycin A. 

In the field, antimycin A bottles, will be transported to the base camp or base of operations in the 

original 3.75L pails, or other appropriate containers (e.g., hard panniers). At this point the 

project supervisor may elect to mix the antimycin A needed for the next day’s treatment or the 

bottles can be removed from the pails with original padding in place and placed in plastic bags, 

and transported to drip station sites in padded backpacks or padded, plastic panniers.. If the first 

option is selected, a mixing station must be set up with absorbent pads to absorb any material 

that may be spilled. If the project supervisor decides to mix the antimycin A on site then the 

chemical must be mixed over the pads used to wrap the bottles and mixing must take place at 

least 10 feet from the stream. 

Transportation 

Antimycin A is transported from the storage facility to the treatment site in a variety of methods 

ranging from motor vehicles, pack mules, watercraft or backpacks. Each mode of transportation 

is supplied with chemically absorbent materials (spill kit) and personnel are supplied with a twoway radio to allow rapid communication if problems arise. Passengers and foodstuffs are not 

allowed in compartments used to transport antimycin A. 

Proper precautions are taken to evenly distribute and secure loads. Containers are secured to 

prevent shifting or tipping and are protected from the weather regardless of the mode of 

transportation. After application, empty containers are returned to the storage facility for 

disposal. 
42

Container Disposal 

Empty metal containers and padding are disposed of in an approved municipal landfill or offered 

for recycling. 

Spills 

Spills on land: In the event of a spill during storage or transport or at an application site it is of 

greatest importance that the spill is stopped at its source, the spilled material is contained and the 

Project Leader is notified. Shovels and other hand tools are used for immediate containment or 

channelization of the spilled antimycin A into a containment area. The following actions are 

taken, as necessary, to contain and clean up a spill on the ground: 

1. Stop the spill at its source 

2. Dike solution in pools 

3. Absorb with clay, soil, or noncombustible, absorbent material 

4. Deactivate materials resulting from an antimycin A spill to the extent possible. 

Spills into water: If antimycin A is spilled near or into a waterway, containment is initiated to 

prevent or minimize movement into the waterway. If an antimycin A spill occurs into a stream 

not scheduled for immediate antimycin A treatment, a deactivation station should be deployed 

using potassium permanganate according to neutralization techniques described in TOP 16. 

Detoxification records must be completed for this incident. Accidental spills of antimycin A into 

a stream during treatment operations may occur during a period when the treatment project is 

already underway. In such an instance, monitoring is extended to ensure that the area of impact 

of the plume does not exceed previous projections. 
43

AOP 8 Procedure for Management of Project Records 

Applicability 

Procedures apply to all records of treatments using antimycin A. 

Purpose 

To ensure continuity in the management and archiving of all project records regardless of 

location and to assure compliance with environmental regulations. 

Procedure 

Management of Routine Treatment Records 

Hard copies of treatment data: Treatment data are given to the project manager daily and stored 

in camp until he can return them to his office for storage. Original data are stored in a series of 

folders organized by stream which contain all data collected during each stream treatment (i.e. 

water chemistry, discharge measurements, antimycin A application records, etc.) and summaries 

of data sets collected daily during the treatment (i.e. Daily Antimycin and Potassium 

Permanganate Application Summary Sheet (Appendix B.11). The treatment supervisor is 

responsible for maintaining all treatment folders. Duplicates are made as required. 

Computer records: Data are transferred to the station system at the end of the field season. 

Electronic treatment records are backed up and stored in a secure space. Data are retained in a 

secure space for the length of time required by NPS data management requirements or agency 

specific requirements. 
44

AOP 9 Contingency Management for Environmental Changes 

Applicability 

Procedures apply to environmental contingencies that may arise during antimycin A application. 

Purpose

To provide for standardized responses to changing environmental conditions that occur just prior 

to or after initiation of antimycin A application. 

Procedure 

Changes in environmental conditions may occur just prior to or after initiation of an antimycin A 

application. Changing environmental conditions primarily relate to climatic changes that can 

decrease water temperature, alter quantity of water to be treated within the project area, and/or 

hinder personnel performance due to inclement weather. A process for identifying and 

responding to environmental factors that may disrupt safe application or alter the effectiveness of 

antimycin A application are defined herein. 

Pre-Application Planning 

Scheduling of application activities will consider local weather patterns. Pre-application review 

of weather will include both long- and short-term forecast to determine likelihood of consistent 

environmental conditions during planned application duration. Reduced water temperature may 

increase the exposure time necessary to effectively remove target fishes and may also increase 

the distance at which antimycin A remains toxic. Change in stream flow may result from rain 

and associated runoff into the project area, thereby requiring additional measurement of flow to 

ensure effective application according to label directions. 

Response to Changing Environmental Conditions during Application 

• A significant decline in air temperature may result in a decrease in water temperature. 

Water temperature declines will slow fish metabolic rates and may increase the length of 

time before antimycin A effects are observed. 

• Increases in stream flow will require recalculation of the amount of antimycin A needed 

to achieve the appropriate treatment concentration. 

• Inclement weather may detrimentally affect personnel performance in field operations. 

Treatment Termination and/or Postponement 

• Postponement prior to initiation of application will occur if inclement weather results in 

prolonged unstable environmental conditions.

• Termination after initiation of application will occur if inclement weather results in 

unstable environmental conditions.

• Rescheduling a treatment will occur if prolonged instability in environmental conditions 

is predicted for the duration of planned treatment.
45

AOP 10 Final Report 

Applicability 

The development of a Final Report for each antimycin A treatment project is highly 

recommended as it provides a concise administrative history of the project. 

Purpose 

To provide for a standardized document for the Final Report at the conclusion of an antimycin A 

project. This report is normally completed once follow-up monitoring indicates that all nonnative fish and project objectives have been completed (about one year after treatment). 

Procedure

At the completion of the antimycin A project, the project manager will prepare a “Final Report.” 

The primary objective of the Final Report is to document the intended and actual outcomes of the 

antimycin A application. The outline for completing the required Final Report is presented 

herein. 

Final Report 

Background 

Objectives: Explain the overall purpose and objectives of the project. 

Rationale: Explain the rationale for the project. 

Fish Species Management Strategy: Explain the overall fish species management strategy and 

how it relates to the objectives and rationale of the project. 

Legal Authority: explain the legal authority for conducting the application including citations of 

applicable statutes. 

Project Team: identify the project manager and key team members along with corresponding 

roles, responsibilities, and a summary relevant experience. 

Treatment Overview 

Application Dates: List the start and end date of the project. 

Treatment Area: Clearly identify the water bodies or portions thereof to which antimycin A was 

intended to be and, if different, actually applied. 

Project Area: Clearly identify the intended and, if different, actual location(s) where antimycin A 

was formulated (i.e., mixed and measured for use), the areas affected by the treatment (i.e., 

where antimycin A and potassium permanganate was measurably present in the environment), 

the location(s) of the deactivation site(s), the location of the barrier that will prevent invasive 

species from re-entering the treatment area, and potential public access ways. 

Treatment Area Characterization: Summarize the results of the gradient and discharge analysis. 

Pre-Treatment Bio-Monitoring: Summarize the results of the pre-treatment bio-monitoring and 

how they were used to define the treatment area. 

Equipment Used: Describe the equipment used to conduct the application. 

Regulatory Compliance 

FIFRA: Describe the steps taken to comply with the requirements of FIFRA. 

CWA: Describe the steps taken to comply with the requirements of CWA. 
46

NEPA: Summarize the steps taken to comply with the requirements of NEPA. 

ESA: Summarize the steps taken to comply with the requirements of ESA and append the 

Services’ final determination and, if necessary, how reasonable and prudent 

measures/alternatives were implemented to the Final Project Plan. 

Public Involvement

Public Concerns: Describe substantive public concerns. 

Response: Describe project manager’s response to each public concern. 

Results and Discussion

Treatment Results: Describe the extent to which the application accomplished the objectives. 

Bioassay Results: Summarize the results of any bioassays conducted prior to, during, and after 

the application. 

Bio-Monitoring Results: Summarize the results of the pre- and post-application bio-monitoring. 

Discussion: Describe any conclusions that can be drawn from the treatment including 

effectiveness of the treatment, best practices for future applications, and any conclusions that can 

be drawn regarding non-target species recovery. 

Appendixes

Final Project Plan 

“Safety and Personal Protective Equipment” meeting sign-in sheet 

Protocol Deviation Forms 

Upon completion, the project manager will distribute and properly file the Final Report along 

with other project documentation in accordance with applicable record keeping policies. 
47

AOP 11 Protocol Deviations 

Applicability 

A Protocol Deviation Form will be completed for each deviation from an AOP/TOP/SOP for 

each antimycin A treatment. 

Purpose 

To provide for a standardized document for tracking deviations from the administrative, 

technical, or other standard operating procedures in this document. 

Procedure 

Throughout the project, the project manager will document on the Protocol Deviation Form 

(Appendix B.10) any deviation from the standard operating procedures identified in this manual. 

Extenuating circumstances that may permit the project manager to act in this manner include 

those that require deviating from standard operating procedures to accomplish any of the 

following: 

• protect worker/bystander safety; 

• prevent release of antimycin A beyond the treatment area; 

• prevent violation of Federal or other regulation(s); 

For each deviation, the project manager will document the following information: 

• the TOP, AOP, and/or SOP from which the deviation occurred; 

• a description of the cause and nature of deviation event; 

• the date and time the deviation began; 

• the date and time the deviation ended or was corrected; 

• the date and time the deviation was identified; 

• the location of the deviation within the treatment area; 

• a description of the action taken to correct the deviation; and 

• signature of the Project Manager on the deviation form. 

It is a violation of Federal law to apply antimycin A in a manner inconsistent with its labeling. A 

protocol deviation that violates the antimycin A product label, while properly documented, does 

not absolve the violator(s) from potential administrative or legal action under FIFRA. 
48

Figure 5. A cascade barrier to the upstream movement of salmonids in Great 

Smoky Mountains National Park. 

Lessons Learned! 

It is important that only 

qualified staff 

experienced in the use of 

backpack electrofishing 

gear verify all 

distribution information 

so that no areas are 

inadvertently missed. 

One untreated area can 

compromise the entire 

project! 

Technical Operating Procedures 

TOP 1 Determination and Characterization of Treatment Area 

for Streams 

Applicability 

Procedure applies to the determination of the area to be treated during the project and collection 

of physical stream attributes to assist with defining and characterizing the treatment area. 

Principle

Prior to an antimycin treatment, it is necessary to determine the distribution of target fish species 

within the watershed to ensure success of the project. Identification and location of selected 

physical stream attributes will help locate potential trouble spots and assist with project planning. 

Equipment Required 

• 7.5’ USGS topographic maps of project area 

• GPS unit 

• Measuring tape or hip chain 

• Surveyor flagging 

• Aluminum tree tags 

• Electrofishing gear, minnow traps, seines, or entanglement nets 

• Dip nets 

Procedures 

Upstream Limits 

Upper target fish distributions should be verified early in 

the initial planning process and re-checked within a few 

weeks of the treatment date. Backpack electrofishing units 

are the method of choice to determine upstream distribution 

as they offer the greatest opportunity for capture in small 

high gradient streams. However, other appropriate 

sampling techniques may also be used. The main stem of 

the stream and all tributaries must be surveyed. 

Experienced personnel should conduct pre-treatment 

surveys. Once upper distribution limits are found, GPS 

coordinates and elevation should be recorded and the site 

marked (e.g. flagging or rock cairn) for future reference. In 

most cases, upper treatment stations are 50-200m upstream 

of the upper known distribution of target species to ensure 

target species are eliminated. 
49

Lessons Learned! 

Many times, physical 

validation of barriers is 

unnecessary given a large 

waterfall or cascade >5m 

vertical (Figure 5), a thermal 

barrier, or when streamflow 

becomes subterranean. In 

other cases, plunge pools, 

side channels, stair-step 

pools, and other complex 

habitat make visual barrier 

validation difficult. In such 

cases, barrier validation is 

necessary using the “mark-nmove” technique (TOP 3).

Downstream Limits 

In all proposed treatment streams, a downstream physical barrier (e.g. waterfall, cascade, 

constructed barrier, dry stream reach, or thermal barrier) to upstream movement of target fishes 

should be located (Figure 5). 

Data Requirements 

The GPS coordinates of all distributional information and location of downstream physical 

barrier should be placed on topographical maps and a Geographic Information System (GIS) so 

that the project area can be clearly defined (Figure 6). 

Figure 6. An example of a study area map depicting distribution of native and non-native fish 

species prior to removal of non-native fish. 

Tagging 100m Sites Throughout Treatment Area 

Once the target species distribution is determined, sequentially 

numbered markers (aluminum or plastic flagging tape) should be placed 

on trees or on stakes along the stream bank every 100m (or other predetermined frequency), starting at the downstream physical barrier and 

continuing upstream to the upper limits of target species distribution. 

Relevant information (e.g. tag tree species, side of stream, elevation) 

should be recorded for each marker. Once markers are placed along 

entire treatment reach, the vertical drop between each marker location, 

total length of treatment reach, and total vertical elevation drop from top 

to bottom of the entire treatment reach can be determined. A schematic 

Stream Segment

Non-native Rainbow Trout

Southern Appalachian Brook Trout

Fish Barrier (W=Waterfall)

Legend

 W

2,000'

 W

3,000'
50

depicting all these data (Figure 7) is useful for project planning and crew instruction during 

antimycin application. 

The stream schematic is also helpful in determining amount of antimycin needed and length of 

stream that can be feasibly treated each day. In addition, markers provide excellent points of 

reference for workers throughout the treatment and elevation changes between markers are 

useful for determining daily treatment station placement (TOP 11). 

Measurement of Mean Stream Gradient (%) For Each Site 

Mean stream width (m) and gradient (%) should be calculated for each section within the 

treatment area. The first stream width is collected at the bottom end of the site followed by 

additional wetted stream widths every 10m upstream to the upper end of the site. Mean stream 

width (m) is generated by averaging all the wetted stream widths (m) for a given site. Stream 

gradient (%) is generated using a clinometer to measure the gradient between each 10m wetted 

stream width, beginning at the bottom of the site and proceeding upstream to the top of the site. 

Mean site gradient (%) is then calculated by calculating the mean for all gradient measurements 

within each site. Once stream gradients are calculated, they can be graphed to identify potential 

trouble spots (Figure 4). Mean stream gradients >8%, which typically indicate areas where the 

antimycin degrades rapidly (a function of increased oxidation associated with increased 

turbulence) and dispensing station intervals may need to be less than where gradient is less steep. 

Other similar techniques may also be used to determine stream gradient for the project area. 
51

Site # Elevation Flow Time Discharge

Mean % 

Gradient

30 2326 7 1.2 3.5

29 2313 7 1.2 3 Trib.1

27 2303 7 2.0 3.1 Trib. 2

26 2293 6 3.1

25 2290 7 2.9

24 2285 7 2.7

23 2280 8 3.3

22 2277 7 6

20 2257 10 3.5

19 2218 7 2.9

18 2211 7 2.7 2.6

17 2198 4 4.9

16B 2192 6 6.2

16 2178 6 5.4

15 2170 7 5.6 9.5

14 2153 10 4.9 Trib. 3

13 2144 10 4.5 Trib.4

12 2122 11 5.1

11 2102 9 6.1

10 2096 9 2.8

9 2086 9 2.9

8 2069 4 2.6

7 2063 4 2

6 2062 8 7.3 2.4

5 2050 6 1.9 Burrels Ford Road

4 2033 9 3.1 Legend

3 2030 5 2.6

2 2028 7 2 Road/Bridge

1 1995 8 8.9 7.3

212 Detox Station

Waterfall

Waterfall/Barrier

Discharge Station

Small tributary

Burrels Ford Road

Detox

  ‐  15 gal of KMnO4 /water applied per hour

    ‐  Need 946 ml/min, OR Waterfall

    ‐  Need 237 ml/15sec

Detox

example:

Detox Station cfs = 3.0 cfs King Creek

‐  KMnO4 for 4ppm for 1 hr = 1224g/hr

  ‐  Mix 2,448 g in each 30‐gal drum

  ‐  Apply at 15gal/hr

Chattooga River

1 ppm 2 ppm 4 ppm

Time 8 ppb Antimycin Calculation Time g/hr g/hr g/hr

8 hr 32.5 1.  Calculate Ac ft of water = 1 hr 51 102 204

8 hr 64.9 CFS x 449(GPM/CFS) x 60(min/hr) x 8 (total hr) 1 hr 102 204 408

8 hr 97.4 1 hr 153 306 612

8 hr 129.9 1 hr 204 408 816

8 hr 162.4 Total Ac ft x 12.3 = mls required for 1 ppb 1 hr 255 510 1,020

8 hr 194.8 1 hr 306 612 1,224

8 hr 227.3 Total Ac ft x 49.2 = mls required for 4 ppb 1 hr 357 714 1,428

8 hr 259.8 1 hr 408 816 1,632

8 hr 292.2 Total Ac ft x 98.4 = mls required for 8 ppb 1 hr 459 918 1,835

8 hr 324.7 1 hr 510 1,020 2,039

8 hr 357.2 1 hr 561 1,122 2,243

8 hr 389.7 1 hr 612 1,224 2,448

8 hr 422.1 1 hr 663 1,326 2,652

8 hr 455.6 1 hr 714 1,428 2,856

Note: 212 minutes or 3' 32"

small falls and some pools

waterfall in section

Burrels Ford Road

Extra Tag 16

site length 120m & a waterfall(~11m)

Tag 21 skipped

Trib.3 to the right

Trib. 4 at last 10m segment

Notes:

**Marked as site 30

Trib. 1 enters - marked as site 29

Trib. 2

6.5 6.5

7.0 7.0

49.2 ml/ac ft = 4 ppb

98.4 ml/ac ft = 8 ppb

5.5 5.5

6.0 12.3 ml/ac ft = 1 ppb 6.0

Also:

4.5 4.5

5.0 5.0

3.5 3.5

4.0 4.0

2.5 2.5

3.0 3.0

1.5 325,800 (gal/Ac ft) 1.5

2.0 2.0

0.5 0.5

1.0 1.0

Antimycin Use (mls concentrate for 8 hours) KMnO4 Use

Stream CFS Stream CFS

King Creek Brook Trout Restoration Project

 August 15-20, 2005

Sites start downstream 

(working from waterfall above 

detox station (site 1) upstream 

to shoals above the second 

fork in the creek (site 30)).

King Creek Gradient Profiles

June 21-22, 2005

June 27-28, 2005

 

Figure 7. An example of a project area map for a stream slated for treatment. The map includes 

stream discharge, mean stream gradient, elevation, key projection locations and application rate 

tables. 
52

Figure 8. Mean stream gradient profile of Sams Creek, Great Smoky Mountains National Park. 

Bars represent standard error (SE). Note the red line at roughly 8% represents the line above 

which identifies sites in which the effective vertical travel distance of antimycin declined. Site 

42 is the upper most treatment site (3,460 ft.) and site 0 is located at the lower barrier (2,180 ft.).

0

2

4

6

8

10

12

14

16

18

42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2

Site Number

% Gradient
53

TOP 2 Determination and Characterization of Treatment Area 

for a Lake 

Applicability 

Procedure applies to the determination of the lake area and volume to be treated if one exists in 

the treatment area. 

Principle 

Prior to treatment with antimycin, it is necessary to determine the surface area, volume (acrefeet) inflows and outflow of the lake or pond to be treated. 

Specific information to be obtained prior to treatment should include: 

• Total surface area and volume of the lake 

• Maximum depth, thermal stratification and temperature/oxygen/pH profile 

• Identification of all inlets and outlets 

• Inlet and outlet discharge, temperature and pH 

• Distribution of fish within the inlets 

• Distribution of fish within the lake (fish below thermocline) 

• Presence of sensitive species 

• Location of a permanent fish barrier below the lake 

• Species of fish present, and spawning/fry emergence 

Equipment Required 

• 7.5’ USGS Topographic map of the project area 

• GPS unit 

• raft and boat motor 

• depth finder 

• water quality kit (pH, oxygen, temperature) 

• gill nets, or other appropriate entanglement gear 

• field book 

Procedures 

For initial planning purposes, the area and volume of the lake may be extrapolated from a USGS 

map prior to going to the site. However, to accurately determine the amount of antimycin 

required for the lake treatment, it is necessary to accurately determine the total volume (in acrefeet of water) of the lake/impoundment prior to the treatment. It is recommended that site a 

visits be made to the project area at the same time of the year that the project is proposed, in 

order to obtain as much information on water volume/inflows and outflows, water chemistry and 

fish distribution. On the year the project is to be conducted, a site visit should be completed just 

prior to application, in order to make appropriate adjustments for current conditions. 
54

TOP 3 Validation of A Potential Fish Barrier 

Applicability 

Procedures apply to determining if a natural feature (i.e. waterfall or cascade) is a barrier to 

upstream movement of undesirable fish. This TOP applies only to potential barriers for which 

their ability to preclude upstream movement of undesirable fish is questionable. 

Principle 

To determine if a questionable natural or constructed feature (i.e. waterfall or cascade) is a 

barrier to upstream movement of undesirable fish. 

Equipment Required 

• Backpack electrofishing units 

• Safety equipment for electrofishing 

• Data sheets and/or field data logger 

• Water quality instruments 

• Tape measure or hip chain 

• Scissors for fin clips 

• Measuring boards, scales and data boards 

• Holding cages 

• Fish anesthetic 

• USGS topographical maps 

Procedures 

Experience in GRSM has shown that the best method for determining if a presumed barrier 

actually blocks movement of undesirable fish is to conduct a ‘mark-and-move’ study. Data from 

previous ‘mark-and-move’ studies has shown displaced fish will return to their home range if 

possible. Target fish are collected upstream of the presumed barrier and transported below the 

feature. If marked fish are later found above the feature, it is not a barrier to fish movement. 

Identify and map the location of the feature on USGS topographic map (7.5’ map is optimal). 

The UTM or Lat-Long location of the feature should be recorded. Establish one to two 100-m 

sites downstream of the obstruction and two to three 100-m sites upstream of the obstruction. 

Conduct three-pass electrofishing sampling in each site. The length and weight of each fish 

collected should be recorded on field data sheets or entered into a data logger. Each fish 

collected should receive a fin clip (i.e. adipose for salmonids) that identifies the fish as being 

collected upstream or downstream of the feature. If the study is conducted over >6 months or on 

species without an adipose fin, PIT or VIE tags should be used. Release all marked fish 

collected in upstream sites in the downstream sites. Between 6 months and 1 year later, return to 

the site and sample all previously sampled locations. Record length, weights and mark of all fish 

collected at each site. From these data, determine if fish collected in upstream sites returned 

upstream to the area from which they were originally collected. If marked fish are collected 

upstream of the obstruction, it is not a barrier to upstream movement and the stream segment 

should not be considered for restoration unless the feature can be rendered impassable. 
55

TOP 4 Pre- and Post-Treatment Aquatic Macroinvertebrate 

and Fish Monitoring 

Applicability 

Procedure applies to conducting aquatic macroinvertebrate and fish sampling prior to and 

immediately after treatment to evaluate impacts of antimycin application to non-target organisms 

within and downstream of treatment area. Should data from current and future projects 

demonstrate only short term impacts and complete recovery within months, implementing this 

TOP should be considered on a case by case basis. 

Principle 

Prior to and immediately following antimycin application, aquatic macroinvertebrate and fish 

collections are conducted to evaluate the effects of antimycin and potassium permanganate 

within and downstream of the treatment reach. These data are also useful for public and 

professional project evaluation, identifying potential resource impacts, and adjusting protocols 

for future projects. 

Equipment

Aquatic Macroinvertebrates 

• Surber or Hess aquatic macroinvertebrate sampler for quantitative sampling (or similar 

sampler) 

• Kick nets and seines for Rapid Bio-assessments 

• Specimen containers (ziplocks, whirl paks, or nalgene jars) 

• Collection labels 

• Sorting tray 

• Forceps 

• Preservative (alcohol) 

Fish 

• Backpack electrofishing gear 

• Waders and electrical gloves 

• Dip nets 

• Buckets 

• Block Nets 

• Measuring tape or hip chain 

• Measuring board 

• Scales 

PROCEDURES 

Aquatic Macroinvertebrates 

Prior to treatment, aquatic macroinvertebrate and fish assemblages should be sampled within the 

treatment area as well as downstream of the antimycin deactivation dispensing station. Pretreatment aquatic macroinvertebrate sampling provides data that can be compared to posttreatment data to characterize short and long-term impacts as well as recovery (Figure 5). Pretreatment fish sampling provides a realistic, measurable population density goal for the posttreatment population. Pre- and post-treatment fish sampling downstream of the deactivation site 

also provides data to assess impacts of potassium permanganate. 
56

Figure 9. —Dr. David Etnier sorting a 

pre-treatment aquatic macroinvertebrate collection from Sams 

Creek, Great Smoky Mountains 

National Park. 

Etnier (2005) recommended a minimum of six 

macroinvertebrate sampling sites within the treatment area: 

two in treatment area, two in the deactivation reach, and two 

in control (reference) sites outside the treatment and 

deactivation area. Each site is sampled bi-annually in the 

spring (May) and fall (September) within one month of 

planned treatment. Spring samples afford the majority of the 

invertebrate fauna while the fall sample will capture potential 

short-term treatment impacts. Each site should be sampled 

within 1 month post-treatment and quarterly thereafter for 

one to two years depending on project objectives. A similar 

approach may also be used for different regions of the 

country, but the project manager should decide which method 

will best meet project objectives during the initial project 

planning phase. 

PROCEDURES 

Fish 

A minimum of four 100m fish sampling sites should be established prior to treatment: two in the 

treatment area, one in the deactivation reach, and one in a control area. Three-pass depletion or 

mark-recapture techniques should be used to quantify fish abundance at all sites. Data from pretreatment surveys can be used as benchmarks to evaluate recovery of the desired species. 

Species abundance data from the deactivation and control sites can be compared to that from preand post-treatment sites within project area to characterize impacts of potassium permanganate in 

non-target areas. Post-treatment surveys should continue until pre-determined population goals 

are met. 
57

TOP 5 Conducting Stream Discharge Measurements 

Applicability 

Procedure applies to determination of stream discharge (Q) prior to application of antimycin or 

potassium permanganate to streams (Murphy and Willis 1996). 

Principle

Prior to an antimycin treatment it is necessary to measure stream discharge (ft3/sec or m3/sec) at 

various sites throughout the project area to calculate application rates of antimycin and 

potassium permanganate. At a minimum, discharge data must be collected at the site(s) where 

treatment is initiated, immediately downstream of each tributary stream that has surface water, 

and at the deactivation site. Discharge measurements should be made at the mouth of all 

tributary streams that are treated. If a rain event occurs during a treatment, discharge must be 

measured before resuming antimycin application and potassium permanganate dispensing. 

Equipment Required 

• Marsh-McBirney® , Pigmy® flow meter, or equivalent meter 

• Topset rod with vernier scale 

• Measuring tape 

• Data forms (e.g. Appendix B.3.) 

Potential Interferences 

Ideally, the discharge measurement site should be located where stream bottom contour is 

smooth and relatively solid (not necessarily flat), there is perceptible flow from bank to bank, 

and no instream obstructions. Conditions detrimental to accurate discharge measurement include 

obstructions such as logs and boulders, aquatic vegetation, undercut banks, shallow water, riffle 

areas, dead water areas, eddies, soft substrate, bridges and culverts, multiple stream channels. 

 

Safety 

Discharge measurement in deep or swift water may be hazardous. An uneven bottom, soft, loose 

or slippery substrate, turbidity, and obstructions may increase the danger. Select areas where the 

stream bed is firm and provides good footing. Avoid potentially dangerous situations such as 

deep pools or waterfalls immediately downstream of the gauging site. 

Procedures 

General Principles 

Stream discharge (Q) may be defined as the volume of water moving through a cross section of 

stream per unit of time and is expressed as cubic feet per second (cfs) or cubic meters per second 

(cms). The procedure described here is the velocity-area technique whereby the computed 

discharge of a stream is the product of the cross-sectional area and the average velocity. 

The cross section of the stream at the measurement site is divided into sufficient sections so that 

the depth measurements will provide an accurate profile of the bed, and the velocity 

measurements are made sufficiently close to obtain an accurate representation of velocities in 

each of the sections. The width of individual sections will largely depend on the overall width of 

the stream, the unevenness of the stream bed and the variation of velocities across the channel. It 
58

Figure 10. — Measurement of stream 

discharge using the cross sectional 

area method. Note personnel 

standing downstream of the meter. 

is important to space the sections more closely where the depths and velocities are more variable 

in order to define accurately the discharge for any given section. 

In most streams the ideal discharge site cannot be found. In this situation it is recommended that 

three discharge measurements be taken in a 20-50 m section of stream. If the three 

measurements are similar, average the values to determine the discharge for that site. If one 

value is significantly different from the other two, take a fourth measurement, determine if it is 

close to the two closest measurements. If so, determine the use theses values to determine the 

discharge for the site. 

The number of cells will vary dependent on stream width. For streams with a width of ≤ 1m, a 

minimum of five cells is recommended. For streams with width >1m, 10 cells are recommended. 

No more than 10% of the discharge should be in one cell. 

Velocity is determined for each cell. Velocity should be measured at 0.6 the depth from the 

surface. In larger streams where depths >1m, velocity is measured at 0.2 and 0.8 the depth from 

the surface. This is based on both mathematical theory and on observations from numerous 

vertical velocity curves. The mean of the two velocities is used for that cell. The standard 

equation to calculate stream discharge is: 

 Q = Σ (V · D · W) where, 

 Q = stream discharge (ft3/sec or 

m3/sec) 

 V = mean velocity of cell (ft/sec or 

m/sec) 

 D = depth of cell (ft or m) 

 W = width of cell (ft or m) 

Site Selection Criteria 

The discharge measurement site should be in a safe 

location, with a firm bottom and depth of less than 1 

meter. The measurement cross section is perpendicular to 

the general direction of flow and stream bed cross section 

is as uniform as possible and free from instream flow 

obstructions. 

Conducting the Measurement 

Stream discharge is measured by using a flow meter 

attached to a topset rod that has depth measurement 

capability. To begin a measurement, a tape measure 

(English or metric) is placed across the stream (Figure 

10). Anchor one end at the initial point and proceed 

across the stream at right angles to the direction of the 

current. 
59

While wading across the stream, an overall impression of the depths and velocities can be 

obtained. This is a good time to look for rocks and debris which can be removed from the stream 

bed to improve accuracy of measurements. On smaller streams it may be possible to construct 

small dikes to cut off sections of shallow flows and dead water. After any modifications to 

obtain more accurate flow estimates, be certain to allow sufficient time for conditions to stabilize 

before proceeding with measurement. 

Based on wetted perimeter width, the cross-section is typically divided into 10 equidistant cells 

(see above). Measure begins on left bank (looking upstream). The first measurement is made 

one-half cell width. The second measurement is made 1 cell-width from the first (i.e. middle of 

second cell) and so on across the stream’s wetted width. At each cell’s center point, measure 

velocity, depth, and visually characterize substrate (e.g. sand, gravel, or cobble). Depths are 

measured by reading the level of the water surface on the rod when the base plate rests on the 

stream bed. The current meter is set to the correct depth to obtain and record velocity. This 

procedure is repeated until the stream is traversed and all cells measured. The total discharge for 

the site is the sum of discharges of each individual cell. 

The position of the operator with respect to the current meter is important when making a 

discharge determination by wading. The operator should stand to the side and downstream of the 

meter to avoid influencing the measurement of velocity. Studies indicate that the position that 

has minimal effect on the operation of the current meter is when the operator stands facing either 

shore and is no less than 0.4 m (1.25 ft) downstream and to the side of the current meter. 

Data Requirements 

A depth measurement is required for each side (or one in center) of a cell and a velocity 

measurement is taken at the center of each cell. The stream discharge data sheet (Appendix B.3) 

is an example that can be used to record discharge data. 
60

Figure 11. Fluorescene dye 

traveling downstream during 

a flow study to establish 

project timelines..

TOP 6 Determination of Stream Water Travel Time Using 

Fluorescent Dyes (Fluorescene & Rhodamine) 

Applicability 

This procedure determines time required for antimycin to travel from 

one point to the next in treatment. 

Principle 

Water travel time determines the length of time required for antimycin 

to travel from the upstream most antimycin station to the deactivation 

site each day. Travel times are also useful in developing time lines to 

bring treatment in a tributary stream and the main stream at the same 

time. 

Equipment Required 

• Goggles 

• Rubber Gloves 

• Rhodamine® WT (20% solution) or Uranine (fluorescene) 

• Graduated cylinder 

• Transport containers with Zip-loc bag 

• Watch or stopwatch 

• Flagging/tags 

• Measuring tape or hip chain 

 

Potential Interferences

There can be significant loss of Rhodamine® WT to adsorption on clay. 

Safety

No special safety precautions. 

Procedures

Determine the section of stream for which water travel times are needed and proceed to the 

upstream end of the segment. Water travel time should be calculated using previously marked 

stream segments (see TOP 1). Record elevation and segment number where dye travel time 

estimation will begin. Measure 100ml of dye into a graduated cylinder and pour dye into the 

stream at the site marker. Record the time the dye was introduced into the stream. Follow the 

leading edge of the dye downstream recording the time the dye first appears at each tag or 

flagging at every 100m increment (Figure 11) or at known points. In addition, observers should 

make detailed notes of any abnormal flow areas along the stream corridor (i.e. side channels, 

meadows, beaver complexes, or anywhere the dye does not appear to mix with stream water). 

Knowledge of the location of these areas is critical to project success and this information is 

critical for project planning. 

Over time, the dye plume will begin to dissipate and become difficult to see. If it becomes 

difficult to determine when the dye plume is approaching, you may refresh the plume by adding 

an additional 100ml of dye and noting the time and location it was added. Continue following 
61

the dye plume downstream recording arrival times at each 100m tag downstream to the 

deactivation site. Once completed, travel times for each 100m segment can be added to study 

area maps to assist with project planning (TOP 1, Figure 7). In some cases, it may be desirable 

to collect water travel time from the deactivation site to other areas of interest downstream, such 

as stream confluences, hatcheries, public areas, or other significant landmarks. 

If the stream reach to be treated is longer than 3 to 5 km, water travel time may be calculated in a 

subset of measured segments. The gradient profile prepared in TOP 1 should be used to 

determine how many discrete water travel time measurements are necessary to obtain a good 

estimate of water travel time through the entire treatment reach. 
62

TOP 7 Closure of Treatment Area and Public Notification of 

Closure 

Applicability 

Closure is required to preclude non-project personnel from entering the project area. 

Principle 

Exclusion of non-project personnel from project area eliminates potential for disturbance of 

project equipment and precludes public contact with the piscicide or the potential consumption 

of treated fish or water during project implementation. 

Authorities 

A. Reference will be made to appropriate agency authority for administrative closure of 

the action area. For example, the authority used for the National Park Service, is Title 

36, Code of Federal Regulations, Section 1.5 (Appendix B.4.). 

B. Action areas within private property must be posted to ensure adequate public 

notification. 

Procedures

The first step is to advise the public of the planned closure 2 or 3 weeks prior to the treatment. 

The next step is actual closure of the project area to the public during project implementation. A 

map delineating the project area, and all access locations to be closed during the project, will be 

prepared for federal and state sponsored projects. This map will become part of public notice 

documents. Signs advising the public of project area closures should be posted at all project 

access points. Access points include, but are not limited to, roads, trails or boat ramps. These 

locations should be marked on the project area map. Signs will advise the public of closure, 

duration of closure, and purpose of closure (Appendix B.2). It is recommended that signs 

advising the public of the upcoming area closure are posted at least 14 days prior to project 

implementation. Project areas located on private property must be posted to no trespassing to 

ensure that non-project personnel do not enter the project area. 

The land management agency/organization responsible for conducting the project will post the 

closure schedule and duration. Closures should cite relevant agency responsibilities and 

mandates (e.g. the authority used for the National Park Service, is Title 36, Code of Federal 

Regulations, Section 1.5) (Appendix B.2). 

 

Public service announcements (except for private lands) should be provided to local media (print 

and broadcast) outlets in vicinity of project at least 14 days prior to project implementation. 

Statements should include description of project area, purpose of project, duration of closure, and 

agencies involved in the project. 

Upon completion of the project, all signs for stream applications will be removed within 2 days 

and within 7 days for lake or impoundment projects. 
63

Figure 12. A pretreatment antimycin 

A application safety meeting to discuss 

project supervision, safety, technique, 

assignments, and timelines. 

TOP 8 Conducting a Pre-Treatment Safety Meeting 

Applicability 

Procedure applies to conducting a safety meeting for all project staff prior to treatment. 

Principle 

Prior to an antimycin treatment, it is necessary to conduct a safety meeting to ensure that 

personnel are provided with and have knowledge of and experience with safe handling and use 

of antimycin A during applications, including use of personal protective equipment. 

Conformance to the training standards and operating procedures outlined in this document 

ensures that personnel conduct operations in safe and legal methods. This meeting will identify 

the project leaders, review safety concerns, identify who issues daily work assignments, and 

communicate other project topics. 

 

Procedures

Prior to treatment, the project manager(s) should have a 

meeting with all project staff to review safety and to 

provide an overview of the project. This meeting covers 

several key issues and ensures that everyone understands 

key public and personnel safety concerns, project leaders 

and contacts, work schedules and assignments, and other 

important housekeeping items. All safety and project 

overview attendees should sign a sign-up sheet or safety 

meeting checklist that can be found in Appendix B.6. 

Personnel from non-governmental organizations (NGO’s) 

must be signed up as volunteers if they participate in any 

phase of the project. 

Typical antimycin projects involve personnel from several 

different agencies and pre-approved personnel from NGO’s (Figure 12). Begin each meeting 

with introductions of project staff and prior antimycin experience. Those with prior working 

knowledge of the treatment process are very useful in assisting others during treatment. After 

introductions, identify key project leaders, their roles and responsibilities during the project. 

If there is a chance members of public may wander into the project area, appropriate procedures 

to escort them from the project area should be discussed. Specific personnel to deal with such 

situations should be identified. Other assignments that should be discussed include identifying 

project leaders, safety officer, antimycin station staff, sprayer operators, deactivation station 

staff, sentinel fish personnel, biotic and abiotic data collection personnel, and other personnel. 

Work schedules should be reviewed daily to be sure all staff fully understands their assignments. 

Personnel safety gear for all staff should be identified and its use reviewed. Personnel safety 

gear will vary by project assignment. 

Communication methods and protocols, both among project personnel and with off-site agency 

personnel should be reviewed to ensure all personnel know equipment operation and protocols. 
64

Personal and project first aid stations should be identified and discussed to ensure all project 

personnel are familiar with procedures in case of accident or injury. Material Safety Data Sheets 

(MSDS) forms and other relevant safety informational materials on treatment compounds should 

be made available to all project personnel and be available [on site] throughout the project. 

The project manager/safety officer, or other appropriate personnel, should review all safety 

issues or concerns that might be specific to project area. For example, if pack animals are used 

to transport gear, personnel should be advised of correct behavior around pack animals. Or, if 

venomous snakes are in project area, personnel should be advised on avoidance and 

identification methods.
65

TOP 9 Setup of a Bioassay to Determine a Target Antimycin 

A Concentration 

Applicability 

Procedure applies to the determination of the lethal concentration (ppb) of antimycin needed to 

eliminate all target fish in a 24-hour period. This procedure also applies to the determination of 

the effective concentration (ppm) of potassium permanganate needed to effectively deactivate 

antimycin based upon the water chemistry of the project area. 

Principle

Application of any pesticide requires a thorough knowledge of the correct application rates in 

order to effectively treat the target organism (s) and eliminate waste. A bioassay (laboratory or 

field) is a tool used to define the lethal dosage (LD100) of a piscicide in the environment where 

the project will be conducted. The Fintrol® label clearly identifies the recommended lethal 

concentrations of antimycin A required to kill select fish species, however, lethal concentrations 

for many other species are not provided. Additionally, varying environmental conditions (e.g.

pH, alkalinity, temperature, stream gradient) can and do affect antimycin efficacy in a stream or 

lake. A laboratory bioassay using water from the project area or a bioassay conducted on-site 

will ensure the correct concentration of antimycin is applied and that impacts to non-target 

organisms will be minimized. 

Typical bioassays seek to address three key uncertainties: 1) the concentration in micrograms per 

liter (μg/L) or parts per billion (ppb) of antimycin and exposure duration needed to kill 100% of 

the target species in a 24 hour period; 2) the concentration of potassium permanganate in 

milligrams per liter mg/L or parts per million (ppm), applied to water to deactivate antimycin and 

minimize or eliminate non-target mortality downstream of the project area and 3) the mortality 

rates of target species exposed to the desired treatment concentration of potassium permanganate 

downstream of the project area. 

Procedures

There are two methods used to determine the working concentration of antimycin needed for a 

given project: on-site bioassays and/or laboratory bioassays. On-site bioassays are preferred 

because stream water chemistry will be identical to that which will be encountered during actual 

treatment thus eliminating concerns that different environmental conditions may yield different 

results. If laboratory bioassays are used, investigators should use water from the project area if 

possible or attempt to mimic the water quality conditions of the target stream. Any project 

planned for a geographic area where antimycin has not been previously applied must complete a 

field or laboratory bioassay prior to project implementation. Once completed, the results should 

be applicable to similar habitat in the study area and bioassays do not have to be repeated. There 

are several methods for conducting on-site bioassays; methodologies for two streamside toxicity 

tests are described herein. The methodology and reporting needs for a laboratory trial are 

identical. 
66

Figure 13. Setup of streamside 

bioassay tubs and buckets used 

to determine the target 

concentration of antimycin (ppb) 

and potassium permanganate 

(ppm)

Find a suitable location along the stream which will allow 

enough space for 8-10 wash tubs or other suitable 

containers. This number of tubs will provide enough space 

for three replicates of each target concentration. Fill each 

wash tub with water (the water in the wash tubs will serve 

as a water bath for the test buckets during the bioassay). 

Within each wash tub, place three 5-gallon buckets filled 

with water (18.9L), place an aerator hose and air-stone in 

each bucket. Place ten target species in each bucket (a 

suitable surrogate species may be used). The larger-sized 

fish found in the population are preferred as these fish tend 

to be the least sensitive to the treatment. If possible, the 

test fish should be obtained from the area to be treated, but 

may be obtained from other sources if necessary. Cover 

each bucket with mesh netting or some other breathable 

material to reduce stress on the test specimens. 

Three experiments should be performed to evaluate toxicity 

of antimycin and potassium permanganate to target species. Test # 1 examines the toxicity of 

different concentrations of antimycin (ppb), test # 2 examines the concentration of potassium 

permanganate (ppm) needed to deactivate a given concentration of antimycin, and test # 3 

examines the toxicity of potassium permanganate prior to deactivation. A control tub (with 3 

buckets, each stocked with 10 test animals) should be maintained. Be sure to properly dispose of 

all test fish upon study completion (TOP 10). An example of the Sams Creek (GRSM) bioassay 

can be found in Appendix D. 

Antimycin Toxicity 

Prepare the stock solution #1 by mixing 5ml of antimycin concentrate (20% active ingredient) 

with 5ml dilutent. The final solution is 10ml at 10% active ingredients or 0.1 ml/ml. Next, mix 

stock solution #2 by mixing 1ml of stock # 1 in 1 liter stream water. The final solution is 

0.0001ml/ml or 100 ppm vol/vol. Note: Stock # 2 is stable for only about 8 hours and must be 

prepared fresh thereafter. 

For concentrations of 2, 4, and 8 ppb, mix and stir in 0.4, 0.8, and 1.6ml of stock solution #2 in 

each respective set of buckets. Be sure each bucket is appropriately marked and add enough 

stream water to fill the bucket to the 20 L. One wash tub will serve as a control and receive no 

antimycin stock solution. Once the appropriate amount of antimycin is added to each bucket, 

fish should be monitored with observations noted hourly over an eight hour period. Fish that 

survive the exposure period are placed in holding cages directly in the stream and total percent 

mortality is recorded at 24 and 48-hours after the initial 8-hour exposure period. Record and 

summarize results in a summary bioassay table (Table 2) to determine the proper concentration 

of antimycin and potassium permanganate. 
67

Amount of Potassium Permanganate Needed to Deactivate Antimycin 

The bucket setup for test #2 is set up the same as test #1, except three concentrations of 

potassium permanganate are used in addition to the antimycin. Begin by determining the 

effective concentration from test #1 (e.g. 8ppb) and adding the appropriate amount of antimycin 

(stock solution #2) in each bucket (e.g. 1.6ml) to get the effective concentration. Be sure to have 

one set of buckets as a control. Next, prepare the potassium permanganate stock solution by 

adding 1g potassium permanganate to 100 ml of stream water. The resulting stock solution 

equals 10g/L potassium permanganate. For 1, 2, 3, and 4 ppm of potassium permanganate, add 

2, 4, 6, or 8ml of stock solution to the appropriate buckets. Be sure each bucket is appropriately 

marked and add enough water to fill the bucket for a total of 20L. Once the appropriate amount 

of, potassium permanganate has been added to each antimycin bucket, mix thoroughly and then 

let the mixture set for 30 minutes before adding fish. After the fish have been added to the 

buckets, mortality and behavioral observations are to be recorded hourly for eight hours. Fish 

that survive the exposure period should be placed in holding cages directly in the stream. 

Holding cages are checked at 24 and 48-hr intervals and number of dead fish recorded. Record 

and summarize results in a summary bioassay table (Table 2) to determine the proper 

concentration of antimycin and potassium permanganate. 

Potassium Permanganate Toxicity 

The bucket setup for test #3 is set up the same as tests #1 and #2, except only various 

concentrations of potassium permanganate are used. Be sure to have one set of buckets as a 

control. Next, prepare the potassium permanganate stock solution by adding 1g potassium 

permanganate to 100 ml of stream water. The resulting stock solution equals 10g/L potassium 

permanganate. For 1, 2, 3, and 4 ppm of potassium permanganate, add 2, 4, 6, or 8ml of stock 

solution to the appropriate buckets. Be sure each bucket is appropriately marked and add enough 

water to fill the bucket for a total of 20L. Once the potassium permanganate and fish have been 

added to the buckets, monitor each bucket of fish and record mortality and behavioral 

observations hourly over an eight hour period. Fish that survive the exposure period should be 

placed in holding cages directly in the stream and percent mortality recorded at 24 and 48-hours 

after the initial 8-hour exposure period. Record and summarize results in a summary bioassay 

table (Table 2 to determine the proper concentration of antimycin and potassium permanganate. 

Table 2. Results of a field bioassay conducted on rainbow trout in a GRSM stream. 

 % Mortality Through Time (hours) % Mortality in 

holding cages 

Antimycin 

concentration 1 2 3 4 5 6 7 8 24 48 

1 ppb 0 0 0 0 0 0 0 0 0 0 

2 ppb 0 0 0 0 0 0 0 10 10 20 

4 ppb 0 0 0 10 20 20 30 40 40 70 

8 ppb 20 20 40 40 50 60 60 80 100 100 

0 ppb 

(Control) 

0 0 0 0 0 0 0 0 0 0 

Alternatively, on-site bioassays may be conducted within the stream. In-stream bioassays should 

be conducted only if the test site is a sufficient distance from downstream terminus of project 
68

that no active antimycin will be carried downstream of project terminus. TOP 9 provides details 

on instream bioassays. 

In addition to determining if label recommended concentration is appropriate for a project, instream bioassays provide information that may be used to determine optimal dispensing station 

placement, and thus more efficient and safe application of antimycin. For example, if all fish in 

a holding cage 200-m downstream of the dispensing station die within 48 hrs and a fish survives 

in the holding cage 300-m downstream of the station, dispensing stations should be placed at 

200-m intervals.
69

TOP 10 Collection, Maintenance, and Disposal of Sentinel 

Fish 

Applicability 

Procedure applies to the collection, handling, and disposal of sentinel (bioassay) fish to monitor 

lethality of antimycin applied to stream or lake. 

Principle

Sentinel fish are used to ensure that antimycin stations are placed at appropriate distance to 

maintain an effective concentration of antimycin to achieve complete elimination of fish between 

stations. All sentinel fish should be collected, handled, and disposed of properly in order to 

eliminate unwanted attraction of scavengers. 

Sample Collection, Transportation, and Maintenance

Bioassay fish are either collected from treatment area or may be transported in from sources 

external to treatment area. Species of fish used as sentinels should be species targeted for 

removal, but congeners may be used. 

Equipment Required 

• Fish collecting gear (e.g. electrofisher, seine, minnow trap, and trammel nets) 

• Live cars 

• Dip nets 

• Fish transport equipment 

• Fish transport chemicals (to minimize stress) 

 

Safety 

Established safety standards for each type of collecting gear used should be followed. 

Procedures 

Collection 

Sentinel fish may be collected using a variety of sampling methods (i.e. backpack electrofishing, 

seines, trap nets, etc.) but gear type should be appropriate for habitat. In stream systems, sentinel 

fish collection is typically accomplished using backpack electrofishing gear. As fish are 

collected, they are temporarily placed in buckets and then transported to and held in holding 

cages located outside the project area until needed. Test animals may also be held in coolers 

filled with water from the project stream. If coolers are used, they should be properly shaded 

and adequately aerated to reduce stress. Portable backpack storage tanks with aerators are ideal 

for transporting fish to and from collection, storage, and deployment sites (Figure 14). 

Maintenance 

Once the desired number of fish is collected, they may be transported to the holding site and held 

in large holding cages until ready for deployment. Holding sites must be located outside of the 

treatment area to avoid impacting or killing sentinel fish. For lakes and some stream systems, 

holding areas may be neighboring streams or lakes which already contain the target species. DO 

NOT use fishless or any other system where the target species is not already present to avoid 
70

Figure 14. A backpack fish transporter and 

aerator used to transport sentinel fish to and 

from holding cages and the treatment area. 

accidental introduction. It is best to avoid holding target fish more than 7 days prior to 

deployment in order to reduce stress and handling mortality. 

All sentinel fish should be collected and transported 

in a manner that minimizes stress. Possible stress 

factors include temperature, dissolved oxygen, and 

handling/transport. To limit temperature stress, 

avoid changes in water temperature during transport 

or holding of more than 3o

C over a 24-h period. To 

minimize critical drops in dissolved oxygen, change 

or aerate transport water often and do not place too 

many fish in transport container. Maintain 

dissolved oxygen concentration at greater than 5 

mg/L or 60% of saturation. Limit transport stress by 

avoiding overcrowding fish in tanks, limiting 

transfers from device to device (e.g. buckets, trucks, 

and tanks), or by adding stress reducing 

compounds. Examples of stress reducing 

compounds include Stresscoat®, buffered 

tricainemethylsulfonate, and salt. Each of these 

reduces “hauling stress” and enhances survival of 

sentinel fish. All test animals should be transferred 

to stream water as soon as possible to maximize 

acclimation time. Test animals should be in stream 

a minimum of 24 hours before exposure. 

Disposal 

Sentinel fish should be buried off channel. 
71

TOP 11 Sentinel and Wild Fish Observations for Daily 

Treatment Evaluation 

Applicability 

This procedure will determine where to physically place holding cages with sentinel fish during 

treatment and provides the project manager with a tool to evaluate the success of daily 

treatments. This information coupled with observations of wild fish in the stream will help 

ensure a thorough treatment of the entire target area for the day and project. 

Principle 

Currently, on-site procedures and instruments to detect antimycin in the waters of the project 

area are available but are not practical for real time monitoring of treatment concentrations. 

Therefore sentinel fish in holding cages (bioassays) and wild fish in the stream are used as bioindicators to evaluate and monitor the efficacy of the treatment. As the time of exposure 

duration for antimycin increases, sentinel and wild fish undergo predictable physiological and 

morphological changes which trained observers can recognize and use to evaluate the 

progression of each days treatment. 

Procedures

Prior to treatment, holding cages should be placed 5-10m upstream of each drip station in 

operation for that day’s treatment. Additionally, holding cages should be placed at a 

predetermined interval (i.e. 100 or 200m) for an additional 1,000m downstream of the 

downstream lower most downstream treatment station each day of the project. Following this 

procedure will allow project managers to determine how far downstream of the daily treatment 

area antimycin eliminated fish and will help determine where antimycin stations need to set for 

subsequent days. In addition, one holding cage should be placed 5-50m above and one holding 

cage 100-300m below the deactivation site prior to treatment. 

Ten fish should be placed in each holding cage, preferably using larger individuals from the 

target population or another source that is close to the project area. The date and time fish are 

placed in the holding cage must be recorded on flagging tape and attached to the cage. Youngof-year (YOY) fish may be used but are not preferred as they are more susceptible to the 

antimycin than adult fish. However, in situations where sub-adults or adults may not be 

available nor can be feasibly transported into the project area, YOY fish can be used. A daily log 

indicating holding cage placement should be maintained. This information is useful to project 

managers if they need to quickly locate project personnel incase of an accident or spill and will 

help ensure all project equipment is removed during project cleanup. 

During treatment, drip station operators should check the holding cages hourly and record all 

observations on the antimycin A daily application form (Appendix B.9.). All dead fish should be 

removed from the cage, properly disposed of (TOP 10), and the date and time noted on the 

corresponding datasheet. Sentinel fish should remain in the cage until they have fully expired so 

that project managers can evaluate the progress of each day’s treatment and the entire project. 

When all fish have expired and the project manager is confident that all non-native fish have 

been eliminated from upstream reaches, remove the cage from the stream. If subsequent 
72

treatments are planned, move the cages to the appropriate location downstream for the next day’s 

treatment. 
73

TOP 12 Determining Distance between Antimycin Dispensing 

Stations 

Applicability 

This procedure ensures that antimycin stations are placed at the appropriate locations to ensure a 

uniform concentration of the piscicide throughout the treatment area for the day. This procedure, 

in conjunction with the placement of sentinel (bioassay) fish, provides the project manager with 

daily on-site bioassays for the stream, lake or impoundment being treated. This procedure 

applies to streams being treated in new areas or regions where no data are available. 

Principle 

Antimycin dispensing stations placed at appropriate locations along the treatment reach ensure 

that each segment receives the appropriate dosage of antimycin per hour for the prescribed 

amount of time. The location of each station is determined by the physical characteristics of the 

area being treated and information obtained from the previous days treatment. 

Equipment Required 

• Goggles 

• Gloves 

• Antimycin dispensing buckets w/ accessories 

• Antimycin A (formulated end product) 

• Graduated cylinders 

• Stopwatches 

• Exposure containers 

• Measuring tape 

• Flagging 

Safety

Standard field safety procedures are followed. No special safety procedures are required. 

Procedures 

Determining Vertical and/or Linear Distance between Drip Station(s) 

Select a location within the treatment area which can be used to determine the effective distance 

between antimycin dispensing stations. One of two locations is recommended. The first location 

is just above the upper end of the target species distribution. This area is recommended as it will 

determine the effective lethal distance and serve as the initial phase of the project. A second 

option is to select a reach with higher stream gradients, given that the effective distance of 

antimycin is typically lower in higher gradient areas. Selection of a reach ≥1 km upstream of the 

project terminus will enable natural deactivation of antimycin, precluding the need for artificial 

deactivation. 

Once the location is selected, place holding cages with sentinel fish (TOP 10) at 100m intervals 

for 1km downstream of the station. Adhere to the procedures outlined for station operation in 

TOP 12 and begin treatment. The station operator and project managers must keep accurate 

records on fish condition in each holding cage (TOP 10) at one hour intervals. 
74

The target distance between stations (linear or vertical) is determined by the distance from the 

antimycin dispensing station to the last holding cage in which 100% mortality occurs in 24 

hours. This distance will be used to guide the placement of stations for the remainder of the 

project. Keep in mind that the distance between stations may vary because of the effects of 

stream gradient, the amount of organic material in the stream, water chemistry or other factors. 

Daily observations of sentinel fish in holding cages will help the project managers refine the 

placement of antimycin stations for each day’s treatment. 
75

Figure 15. A Operational 

antimycin dispensing station on 

Sams Creek, GRSM during 

treatment in 2000. 

TOP 13 Operation of a Dispensing Station to Apply Antimycin 

to Streams 

Applicability 

This procedure ensures that dispensing stations are properly set-up and operated to ensure a 

uniform concentration of antimycin throughout the treatment area for the day and to ensure 

necessary data are recorded. 

Principle 

A properly set dispensing station will deliver the appropriate amount of antimycin for a specified 

time period. 

Safety 

Antimycin A is a restricted use pesticide. The pesticide label (Appendix E) and Material Safety 

Data Sheets (MSDS) (Appendix F) safety precautions must be followed. 

Potential Interferences 

Several potential interferences have been identified that reduce or eliminate the effectiveness of 

antimycin A on target fish. These interferences include, but are not limited to, temperature, pH 

(>8.0), alkalinity, elevated organic load, (e.g. deciduous leaves), and high stream gradient (i.e.

>8%). (See pertinent literature in Appendix A for additional information.) 

Equipment Required 

There are several methods for dispensing antimycin at a constant rate to the stream. The 

particular method selected by an applicator will be based upon on-site conditions, applicator 

experience and preferences, available equipment, and transport considerations. In addition, to 

the specific dispenser detailed below, information on other dispensing methods is detailed in 

literature referenced in Appendix A. 

• Drip bucket (18.9L) 

• Hose (6’) 

• Drip pan 

• Hammer (3 lb) 

• Rebar – 3/8”x2’ (2) 

• Leveling blocks 1”x1”x1’(2) 

• Pitcher (2.5L) 

• Torpedo level 

• Socket driver or screwdriver 

• Mesh filter 

• Funnel 

• Paper clip 

Procedures

Antimycin is best applied to the stream from a container (5-gallon 

or 18.5L containers are frequently used) and delivery device (e.g.

Farnum float pan or Mariotte bottle principle) that dispenses 

toxicant at a constant rate (Figure 15). Other devices that provide 
76

Figure 16. An operational 

antimycin drip station on Bear 

Creek, GRSM during the 2003 

treatment. 

a constant flow are also appropriate. According to the label, the maximum single treatment 

concentration is 25 μg a.i./L. The maximum number of treatments per year is three (3) per 

project area. The minimum reapplication treatment interval is one (1) day. The maximum 

annual treatment concentration is 50 μg a.i./L per project area. The maximum daily treatment 

duration is 8 hours. 

 

Setup 

Begin by verifying that all the necessary equipment listed in the equipment list above or for the 

particular dispensing apparatus chosen are available. After each dispensing location is identified, 

find the best spot to set the antimycin dispensing bucket. An ideal location is a level stream bank 

located 1-2m above a shallow mainstream pool, run or riffle (Figure 16). In most cases, a fairly 

level stream bank is not available. In such instances, use the leveling blocks and any other 

available items to level the bucket on a rock, bank, or other surface (Figure 16). Be sure the 

bucket is level and can support 18kg (40 lb) without tipping over. Once the bucket is level, 

attach the hose to the base of the bucket (outlet) and place the bucket lid on so no debris enters 

the bucket. 

Identify a suitable location just in the stream thalweg just below the drip bucket to set the drip 

pan (Figure 16). Suitable locations are those which are of shallow to moderate depth (2-20cm) 

with substrate preferably composed of medium to small gravel or smaller. Hammer both pieces 

of rebar into the substrate within hose length of the drip bucket. Mount the drip pan to the rebar 

and attach the hose. If necessary, prop the hose up with branches or other available material so 

there are no low spots for air to settle into before the water reaches the drip pan. 

Sometimes when the bucket is filled with water, air can become trapped in the hose. To remove 

air from the hose, raise the bucket 0.5-1.5m off the ground and open the valve on the bucket to 

allow the water to flow through the hose and purge all the air from the line. Once all the air is 

purged, close the valve on the bucket and set it back on the blocks. Check to be sure the drip pan 

and bucket are level and the station is ready for operation. 

Placement of Upper Antimycin Dispensing Station(s)

Antimycin dispensing station placement should begin just 

upstream of the upper most end of the target fish distribution. 

In most cases, placement of the first station should be 100-

200m upstream of the upper fish distribution location to ensure 

the antimycin is completely mixed in the water column when it 

reaches the target area. When treating tributary streams follow 

the same procedure. In this situation, use dye travel times to 

determine when to begin treatment on each tributary so that 

treated water from each stream segment will “merge” at their 

confluences at about the same time. The practice of merging 

treated segments is important in order to maintain exposure 

levels throughout the treatment area and reduce the opportunity 

for lapses or gaps which may offer temporary sanctuaries for 

the target species. 
77

Treatment 

Roughly 15 minutes before starting the treatment, prepare the drip bucket by filling it half full of 

water. The container should be filled with filtered (e.g. using a sieve or cheese cloth) water from 

the stream. Using the graduated cylinder, measure out half of the prescribed antimycin and 

nonoxyl-9 to the bucket and mix thoroughly with a stirring stick or clean twig. Fill the graduated 

cylinder with stream water three times and empty the rinseate into the bucket. Place the “dirty” 

graduated cylinder in the stream to rinse and secure it with a stone or small twig. After adding 

half the antimycin, add the remainder of the water to fill the bucket to the level needed for four 

hours (e.g. for 4 hours at a rate of 4L/hr, fill to 16L) and mix the solution again. The mixture is 

now ready (charged) for deployment. 

The amount of antimycin needed for each dispensing unit is calculated by the following 

formulas: 

 (3) CFS x 449 (GMP/CFS) x 60(min/hr) x 4 or 8 (Total hours) 

 325,800 (gallons/acre foot) 

 (4) Acre Feet of Water x 98.4 mls = mls required for 8 ppb 

Approximately 100ml of a tracer dye should be added to the stream when treatment is initiated 

each day at the first charged station. Treatment will either begin at a specified time (uppermost 

dispenser) or when the fluorescene dye reaches the next downstream drip station indicating 

treated water is present. To begin treatment, simply open the valve on the bucket and the 

antimycin should travel through the hose to the float pan and enter the stream. If there is no 

stream of antimycin entering the stream, check the drip hole in the drip pan with the paper clip 

and also check the hose for air. Once filled with treatment solution (charged), each container 

should be checked at least every 30 minutes to ensure the proper dispensing rate of diluted 

antimycin (i.e. L/hr or gal/hr). Each container must be under the direct control of an applicator, 

with one applicator monitoring no more than two antimycin stations. Containers should be 

charged sequentially in a downstream direction. If the unit is running fast or slow, adjust the 

float inside the drip pan up or down to either speed up or slow down the application rate. Once 

adjusted, tighten the set screw on the float to ensure a consistent application rate. Using the 

antimycin application data sheet (Appendix B.7), record bucket stage, water temperature, and 

sentinel fish observations hourly for the duration of the treatment. Additionally, note the time 

and amount of antimycin and water that is added to the bucket during the treatment. 

Approximately three hours after a dispensing station was turned on, one gallon of the mixture 

should remain in the bucket. At this point, pour the remaining antimycin into the bucket. Rinse 

the antimycin bottle three times with stream water, emptying the contents into the bucket. The 

bottle and graduated cylinder can then be placed in the stream under a stone or limb for 

additional rinsing or placed in the original Zip-loc bag. Enough water most be added to the 

bucket to bring the level up to 20L (about 16L). Mix the solution and place the lid back on top 

of the bucket to keep debris out. At the end of the day, place the goggle and gloves in the Ziploc bag and return all equipment to the designated cleaning area. Once the treatment is complete, 

rinse the bucket and hose system in the stream. Collect all the soiled safety gear, graduated 

cylinder, and antimycin bottle and safely transport them to the washing area. 
78

Evaluate the status of sentinel fish in the holding cages downstream and upstream of each 

antimycin dispensing station hourly during application and at the 24 hour milestone. Record the 

physical condition, appearance, and mortality for the fish in each cage on the antimycin station 

datasheet. The placement of the following days dispensing stations will be based on vertical 

and/or linear distance between the drip station and the live car in which all fish have died 24 

hours after initiation of dispensing. Using this vertical and/or linear distance (m) (TOP 12) and 

the study area gradient/elevation map (Figure7), place each drip station no more than the 

determined distance below the drip station immediately above it. The total target treatment 

segment (km) and/or manpower will determine the number of stations set each day. Caution 

should be used not to set more stations than personnel can manage. Typically, one person can 

easily manage two stations along a stream segment given no terrain limitations. The vertical 

and/or linear distance between each additional downstream drip station should be similar to the 

effective distance determined during the “on-site bioassay”. 
79

TOP 14 Application of Antimycin to a Lake 

Applicability 

This procedure ensures that antimycin is properly dispensed and a uniform concentration 

throughout the treatment area for the day and to ensure necessary data is recorded. 

Principle 

Fish do not detect and avoid antimycin, which provides the applicator an advantage in removing 

fish from standing waters. However, it is important to use proper dispensing techniques to 

ensure an even distribution antimycin. In most cases, the injection of antimycin into an outboard 

motor prop wash through a Venturi pump or boat bailer system will deliver the appropriate 

amount of antimycin evenly throughout the lake surface. Aerial application of antimycin is not 

recommended, except for limited spraying of shallow surfaces around the lake perimeter. 

Alpine lake treatments have been successful in removing trout with as little as 2 ug/l (25 

mls/acre-foot), based upon total volume of lake and applied to the surface of the lake. However, 

application rates for alpine lakes with low inflow rates and low turn-over rates (inflows equal 

total lake volume in 30 to 60 days), have ranged from 2-5 ug/l, with 4-5 ug/l used for most alpine 

lake applications. Treatment rates need to be as high as 8 ug/l in lakes and ponds with pH 

nearing 8.0, and high turn-over rates (inflows equal total lake volume in one to seven days). 

Independent of the application rate, planning and application needs to focus on obtaining an even 

distribution of the chemical throughout at least the surface of the lake, and means of treating the 

lower water column, if it is demonstrated that fish are concentrated throughout all depths. A 

battery operated bilge pump can be used to dispense antimycin into deeper areas of the lake. 

Selection of the date of treatment should be based upon the spawning of the target species. 

Overall application should occur prior to spawning, or long enough after spawning to allow fish 

to develop to at least the swim-up stage. 

Safety

Antimycin A is a restricted use pesticide. The pesticide label (Appendix E) and Material Safety 

Data Sheets (MSDS) (Appendix F) safety instructions must be followed. 

Potential Interferences

Several potential interferences have been identified that reduce or eliminate the effectiveness of 

antimycin A on target fish. These interferences include, but are not limited to, pH (>8.0), 

alkalinity, elevated organic load, deciduous leaves, and algal blooms. 

Equipment Required

Lake treatments will use the stream treating apparatus and kits to treat the inlet and 

outlets, and sprayer kits to spray the edge of the lake, bogs, shallow areas and seeps. In 

addition to the equipment previously listed, the following equipment is needed for 

back-country lake renovations projects: 

• Good raft, with floor, oars, oar locks and motor mount 

• Outboard motor (>4 hp) gas and oil 

• Holding tank for mixing water and chemical, with lid to reduce spill and fumes 
80

• Venturi pump/battery bilge pump system 

• Buckets 

• Boat/motor tools, including starting fluid, and raft repair kit 

• Latex or Nitrile gloves 

• Goggles 

• Life jackets 

• Dip nets 

• Rain jackets 

• Dip nets 

• Sand bags (for blocking outlet flows) 

 

Miscellaneous Equipment 

In addition to the above equipment, the following equipment should be available: 

• Dye 

• Water bags (drinking water) 

• Dip nets 

• Shovel 

• Flagging 

• Block Nets 

• Rope 

• Extra drill bits (52-38) 

• First Aid kit 

• Radio 

• Measuring board and weighing scales (measuring fish) 

• Length frequency sheets 

• Scales 

• Live cages (net bags) 

• pH meter 

• Extra thermometers 

• Extra bottle for antimycin split-out 

• Marking pens 

• Gill Net and setting rope 

Procedures

Boat and Motor 

The use of a raft/zodiac with a minimum of a 4 hp outboard motor is recommended for lake 

applications. Although projects have been completed with the use of electric motors, the use of a 

4-5 hp gas motor for lakes in the 2-10 ha range, helps ensure mixing in a variety of conditions 

(high winds/no wind). Planning lake operations without the use of a gas motor should be 

considered only in rare circumstances. 

Mixing and Application. 

Antimycin should be diluted with lake water to at least 1/50 within an on-board holding tank, 

with the hold tank to include a lid to limit spills and fumes. The antimycin/water mixture should 

then be injected into the prop wash of the outboard motor, using a venture or battery-powered 

bilge pump to ensure effective mixing of antimycin across the surface of the lake. It is 
81

recommended that antimycin be applied around the perimeter of the lake, and across the lake in 

multiple grid patterns. A minimum of 0.5 hours per hectare should be spent applying diluted 

antimycin to help assure an even distribution. Approximately 9-10% of the total antimycin for 

the lake treatment should be sprayed around the edge of the lake to ensure a lethal concentration 

around the shoreline, using multiple applications (3 passes of 3%) to ensure thorough mixing. If 

it has been determined that fish occupy all depths of the lake, the bilge pump can be used to 

apply antimycin into the lower sections of the lake. In order to check the effectiveness of the 

prop wash injection system and shoreline spraying, the use of dye mixed with the antimycin 

mixture will help in adjusting the motor and highlight areas not sprayed in previous passes. 

Inlet Streams 

It is extremely important to survey and document the distribution of fish in the inlets to the lake 

system, using the procedures outlined in TOP 1. It is important to remember that some small 

inlet streams with limited flow may not contain adult fish, but fry may be present. 

Generally, the procedures for treating small inlet streams that enter the lake must follow the 

procedures outlined in TOP 2. However, lakes with inlet flows colder than the surface 

temperature may attract fish to greater depths than found on the average throughout the lake. To 

address the attraction of lake fish to inlet streams, consideration should be given to treating inlet 

streams at about 4 ug/l for up to 24 hours to ensure complete removal of target species. 

Outlet Stream 

The procedures for treating the outlet stream must follow the procedures described in TOP 12. 

Monitoring 

Live cages with fish from the lake or nearby stream should be placed at various locations and 

depths to aid monitoring the progress of the project and the response of the target species as 

described in TOP 11. 

Deactivation of Antimycin 

If a barrier exists downstream of the lake and serves as the terminus of the project, deactivation 

procedures must follow those outlined in TOP 16. If the project area continues downstream for 

several kilometers from the lake, then the project should follow the procedures outline in AOP 9: 

Preliminary and Final Project Plan. 

In some reservoirs, and even some natural lakes, it may be possible to block the outlet flow of 

the lake using existing valves or sandbags, thus allowing photo-oxidation and hydrolysis to 

neutralize the antimycin. This approach must be identified in the Final Project Plan. Project 

managers must have the necessary detoxification equipment on site in case the temporary dam 

fails or weather events increase flows. In this event, detoxification must follow the procedures in 

TOP 16. In addition, applicators must determine if antimycin is detoxified prior to releasing 

water from reservoir systems. 
82

TOP 15 Use of a Backpack or Hand-Held Sprayer to Apply 

Antimycin A 

Applicability 

This procedure will outline the procedures used to safely apply antimycin with a backpack or 

hand-held sprayer. 

Principle

Any areas identified during the dye travel studies where the dye does not mix well are areas that 

must be treated with a backpack sprayer. Typical areas treated with a backpack sprayer include 

backwater pools, seeps, side channels, beaver complexes, bogs, wet meadows, and springs along 

the stream corridor. 

 

Equipment Required 

• Goggles 

• Rubber gloves 

• Waders 

• Backpack sprayer (3 gal) with coarse nozzle 

• Graduated cylinder 

• Pitcher 

• Antimycin 

• Fluorescene or rhodamine dye (100ml) 

 

Safety

Goggles, gloves, and waders are required when dispensing antimycin using a sprayer. A long 

sleeved shirt or Tyvek® poncho is recommended to keep any spillage off the operator (Figure 

13). To eliminate inhalation of vaporized antimycin, only sprayers with a coarse nozzle are 

approved for applying antimycin with a backpack sprayer. An 11 to 15L (3 to 4 gallon) 

backpack sprayer will weigh 11 to 15kg (24 to 33lb). Crew members assigned the duty of 

backpack sprayer operation should be in good physical condition and able to carry moderate 

loads (e.g. 10 to 20kg) without difficulty. 

Procedures

Backwaters, seeps, springs and any other areas along the stream corridor where dye is not mixing 

should be treated with a backpack sprayer. Backpack sprayers must not be charged with more 

than 10% vol/vol solution of antimycin, and the total amount of antimycin used in backpack 

sprayers must not exceed 10% of total amount used daily in all dispensing stations. 

Prior to daily treatment, determine the length of treatment reach and total amount of antimycin to 

be used for that day. The backpack sprayer applicator will spray the entire treatment area and 

will be assigned ≤10% of the total daily amount of antimycin (ml), which is determined by the 

project leader, and 50ml of dye. For ease of application, the sprayer typically begins at the upper 

end of the daily treatment zone and follows the initial dye release downstream in order to 
83

Figure 17.A backpack sprayer 

applies antimycin to back water 

eddies during an antimycin project. 

Note the operator is wearing all 

required safety gear for safe 

application of antimycin A with a 

sprayer. 

identify areas where mixing is not occurring. The sprayer will make two trips through the daily 

treatment zone dispensing 1⁄2 the total allotment of antimycin and dye during each trip. 

Immediately after the initial release of antimycin and dye at the 

upper end of the daily treatment area, the sprayer will measure out 

half of the allotted antimycin and dye and place it into the sprayer 

tank. Fill the remainder of the tank with stream water and mix 

gently with a stirring stick. Securely replace the cap, being sure not 

to cross-thread the cap, and the tank is ready for application. After 

donning goggles, gloves, and waders, and backpack sprayer, the 

applicator begins treatment. To properly treat, follow the dye 

released by the upper drip station downstream, spraying any area 

where dye is not readily observed in the stream, such as backwater 

pools and side channels. The dye in the sprayer will assist the 

sprayer in assuring all areas are treated. Note problem areas and 

record location for second sprayer application. Apply moderate 

amounts of antimycin to all untreated areas keeping in mind the 

prescribed amount is needed to treat the entire treatment reach. 

Once the sprayer reaches the end of the daily treatment reach, 

return to the top of the daily treatment reach to refill and make the 

second pass through the same section. Once the second spraying 

pass is complete, return the sprayer to the cleaning area, rinse 

sprayer with clean water from an untreated tributary or other 

source, and hang the sprayer to air dry. Be sure to pressurize the 

sprayer and run clean water through the nozzle to prevent future 

clogging. 
84

Figure 18. The potassium permanganate 

deactivation station used to neutralize 

antimycin during a native fish restoration 

project in GRSM. 

Lessons Learned! 

Once a deactivation site is identified, 

clearly mark drum and drip box setup 

areas with flagging so setup crews 

know where to place equipment when 

they return. 

TOP 16 Deactivation of Antimycin A in a Stream 

Applicability 

Procedure ensures that the deactivation station is located at the downstream terminus of the 

treatment reach and that active antimycin does not impact areas outside the project area. 

Principle

Prior to treatment, the deactivation station is set up, calibrated, and prepared for operation to 

preclude any possibility of antimycin impacting areas downstream of the treatment area. 

Equipment Required

The particular apparatus for dispensing potassium permanganate will vary, depending on 

location condition, accessibility, applicator experience and preference. The following equipment 

list is for one commonly used dispenser: 

• Hanging Scale 

• 5-gal bucket 

• Small boat paddle 

• Holding drums 

• CDS drip boxes 

• Garden hose 

• Hose fittings 

• Graduated cylinder (1 litre) 

• Dust mask 

• Goggles 

• Chemical resistant gloves 

• Long sleeve shirts 

• Waders 

• Shoes/boots and socks 

• Potassium permanganate 

Procedures

Locate Suitable Deactivation Setup Area 

Once the barrier and study area have been clearly identified, it 

is important to survey the area to locate a suitable location for 

the deactivation site. The deactivation site does not have to be 

located directly below the barrier, but should be located within 

100m of the barrier to avoid non-target impacts and should 

have some of the following characteristics (Figure 18 & 19): 

1) level area large enough to hold potassium 

permanganate dispensing units (typically 5 to 55 gallon containers) 

2) level area with at least 8 feet vertical difference from stream 

3) fairly accessible for staff from trail or road to transport equipment in/out 

4) stream morphology such that you can safely work in the stream to install and calibrate 

equipment on an hourly basis 
85

Figure 20. An example of the drip box 

system used to deliver potassium 

permanganate to a stream to neutralize 

antimycin A. 

Figure 19. A Potassium permanganate 

deactivation station configuration used 

to detoxify antimycin during native fish 

restoration project in Crater Lake NP. 

Note large drums used for larger stream 

volume (~15cfs). 

Setup of deactivation drums 

Setup storage tanks or drums on previously selected level 

streamside surface. Be sure the surface is located ≥ 1 foot 

vertically above the drip boxes to ensure adequate flow to 

the drip boxes. If needed, level tanks using boards, rocks, 

or other means, being sure drums are stabile and will 

accommodate weight of the water without tipping over. 

Safety note: Water weighs ~1 kg per L, care should be 

exercised when using drums of ≥ 113.55L (i.e. ≥ 113.6 kg). 

`Next, setup saw horses in the stream paralleling the direction 

of flow. Using wire or rope, secure planks (e.g. 1”x 8”) 

about 25 cm apart across the top of saw horses. Secure drip 

boxes to planks and ensure entire structure is level (Figure 

19). Once tanks and drip boxes are level, attach and secure 

hoses from tanks to drip boxes. If there is considerable 

distance between the tanks and drips boxes, a support system 

for the hose may be needed. 

Test the fire pump and hose that will be used to fill the 

drums. Place a small intake hose in the stream and begin 

filling drums, being sure to use a filter on the intake to 

reduce uptake of debris. Be sure system works properly as 

the tanks will need to be filled many times during the 

operation of the deactivation station. Once the tanks are 

filled, open valves on drip boxes to test system for stability, 

leaks, and ensure no air is trapped in the hoses. 

It is important to understand that the purpose of the water 

in the tanks is to act as a delivery mechanism for the 

potassium permanganate. Once the tanks are set to deliver 

a fixed volume of water per hour, you can add the amount 

of potassium permanganate required to deactivate 

antimycin for the stream discharge measured. Two tanks 

are an efficient means of delivering the potassium 

permanganate because: 1) a backup tank is available if primary tank malfunctions, and 2) an 

uninterrupted flow is maintained because the second tank can operate while the first tank is 

refilled (Figure 19). 

To calibrate the drip boxes, open the valve on one tank and use a large graduated cylinder to 

measure the amount of water exiting the drip box over a 15 second period. For 56.775L (15 

gallons) an hour, 946ml per minute, or 237ml per 15 seconds is needed. If 2 drip boxes are 

working from 1 tank, these rates are halved (Figure 20). For example, for 56.775L an hour from 

two drip boxes, about 119ml per 15 seconds must be dispensed from each drip box. If the drip 

rates are off, turn the valves accordingly to adjust the flow rates. Drip boxes should be recalibrated every hour to ensure proper functioning. 
86

Data Requirements: 

Stream discharge at the site must be determined daily prior to the initiation of deactivation. The 

hourly amount (g) of potassium permanganate applied (ppm) during deactivation is based upon 

stream discharge (Table 3). For any given discharge, the weight of potassium permanganate 

added to each barrel is calculated and recorded on the potassium permanganate data sheet 

(Appendix B.8). 

Table 3. — Antimycin and potassium permanganate application rate table based upon stream 

discharge (ft3/sec) and desired application concentration. 

Antimycin Potassium Permanganate

Stream

CFS Time 8 ppb Time

1 ppm

g/hr

2 ppm

g/hr

4 ppm

g/hr

0.5 8 hr 32.5 1 hr 51 102 204

1.0 8 hr 64.9 1 hr 102 204 408

1.5 8 hr 97.4 1 hr 153 306 612

2.0 8 hr 129.9 1 hr 204 408 816

2.5 8 hr 162.4 1 hr 255 510 1,020

3.0 8 hr 194.8 1 hr 306 612 1,224

3.5 8 hr 227.3 1 hr 357 714 1,428

4.0 8 hr 259.8 1 hr 408 816 1,632

4.5 8 hr 292.2 1 hr 459 918 1,835

5.0 8 hr 324.7 1 hr 510 1,020 2,040

5.5 8 hr 357.2 1 hr 561 1122 2,243

6.0 8 hr 389.7 1 hr 612 1224 2,447

6.5 8 hr 422.1 1 hr 663 1326 2,650

7.0 8 hr 455.6 1 hr 714 1428 2,854

7.5 8 hr 487.9 1 hr 765 1530 3,058

8.0 8 hr 520.5 1 hr 816 1,632 3,262

8.5 8 hr 553.2 1 hr 867 1734 3,465

9.0 8 hr 585.9 1 hr 918 1836 3,669

9.5 8 hr 618.6 1 hr 969 1938 3,873

10.0 8 hr 651.2 1 hr 1020 2040 4,077

Cleaning and Maintenance of Deactivation Equipment 

Deactivation operators should pay close attention to limit the amount of potassium permanganate 

sediment build up in hoses and drips boxes. Adding more than 26g potassium permanganate per 

1L of water will cause excessive sedimentation and clogging in the drip boxes. Therefore, for 

113.55L (30 gallon) drums, deliver 56.775L (15 gallons) per hour up to 26g/L and then switch to 

113.55L (30 gallons) per hour delivery when application exceeds 26g/L. Prior to deactivation 

each day, or if the drip boxes or tanks begin to collect potassium permanganate sediment, flush 

the tanks and hoses with clean water prior to refilling the tanks for the next round. 
87

TOP 17 Equipment and Safety Gear Cleaning 

Applicability 

Procedure ensures that all equipment and safety gear used daily during antimycin A treatment is 

properly cleaned and sanitized or disposed of. 

Principle 

To ensure project personnel are not exposed to contaminated equipment during treatment. 

Equipment 

• Alconox detergent 

• Sponges 

• Graduated cylinder brushes 

• Chemical resistant gloves 

• Buckets or tubs dedicated to washing equipment in the field 

• Clothes line and clothes pins (if necessary) 

Procedures

All bottles, gloves, goggles, and graduated cylinders used during antimycin A treatment or 

deactivation must be returned to a designated washing area to be cleaned daily. Designated 

washing areas may be a nearby wet laboratory or for remote projects, all antimycin mixing and 

glassware washing must be done in the field. 

Fill two buckets (or wash tubs) with water adding Alconox to one of the buckets. Pre-rinse all 

equipment and safety gear with water and then place them in the detergent bucket. Wash all 

equipment and safety gear with either a sponge or brush and place in the rinse bucket. Allow all 

equipment and safety gear to fully dry on a drying rack or clothesline prior to re-use. Dispose of 

rinsates on the forest soil at least 100m from any water body or campsite. 

Disposal

Original shipment bottles must be completely emptied. Prior to disposal or re-use, these bottles 

must be triple-rinsed with an oxidizing detergent (e.g Alconox®). If bottles will not be re-used 

for future applications, they should be disposed of in an approved sanitary landfill or offered for 

recycling. 
88

TOP 18 Documenting Field Deviations from the Protocols 

Applicability 

Procedures apply to all actions taken preparing for and conducting an antimycin A application as 

defined in this document. 

Principle 

All deviations from the protocols including technical operating procedures (TOP’s) and 

administrative operating procedures (AOP’s) are recorded to ensure that noncompliance is 

identified, corrected (where possible), and explained. 

Equipment Required 

• Pen or pencil 

• Protocol Deviation Form (Appendix B.12.) 

Procedures

If at any time during the project, a deviation from the any of the stated AOP’s or TOP’s occurs, 

the project manager will record such using the protocol deviation form (B. 10). Identify the 

TOP, AOP, or SOP from which the deviation occurred and record it on the protocols deviation 

form (Appendix B.12.). Respond to the deviation appropriately and expeditiously to minimize 

continued/future deviations and potential impacts of the action. Report the deviations in the 

Final Report on the Application of Antimycin A (see AOP 11). 

Steps to Document the Deviation 

1. Indicate the TOP, AOP, or protocol(s) from which the deviation occurred. 

2. Indicate the date and time the deviation was identified. 

3. Indicate the date and time the deviation began. 

4. Indicate the date and time the deviation ended or was corrected. 

5. Indicate the location of the deviation within the treatment area. 

6. Provide a description of the cause and nature of deviation event. 

7. Provide a description of the action taken to correct the deviation. 

8. Obtain the signature of the Project Manager on the deviation form. 

9. Summarize the deviations and the corrective actions.

10. Identify lessons learned or best practices that could potentially prevent future deviations.
89

Literature Cited 

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Label. 

Berger, B.L., R.E. Lennon, and J.W. Hogan. 1975. Laboratory studies on antimycin A as a fish 

toxicant. U.S. Bureau of Sport Fisheries and Wildlife, Investigations in Fish Control

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Bettermann, A.D., J.M. Lazorchak and J.C. Dorofi. 1996. Profile of toxic response to sediments 

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Etnier, D. A. and C.D. Hulsey. 2005. Effects of antimycin A treatment on benthic 

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

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90

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Tiffan, K.F. and E.P. Bergersen. 1996. Performance of antimycin in high-gradient streams. North 

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http://www.epa.gov/oppfead1/endanger/consultation/ecorisk-overview.pdf 

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91

Appendix A. Additional literature concerning the use, chemistry, detoxification, toxicity, and 

other data concerning antimycin A and potassium permanganate. 

Alward, W.D. 1971. Proud lake to Milford Millpond: Fish Eradication Project. Michigan 

Department of Natural Resources, Michigan, December 1971. 8 pp. 

Avault, J.W., Jr. 1968. Rid trash fish from catfish ponds: Louisiana Conservationist 20(7-8):8-9. 

Avault, J.W., Jr. 1972. Watch those wild fish; they rob you of profits. Fish Farming Industries 

3(3): 24-26. 

Avault, J.W., Jr., and G.C. Radonski. 1968. Use of antimycin as a fish toxicant with emphasis on 

removing trash fish from catfish ponds. Proceedings of the Southeastern Association of 

Game and Fish Commissioners 21(1967):472-475. 

Avery, E.L. 1974. Experimental reclamation of trout streams through chemical treatment in 

Westfield Creek. Wisconsin Department of Natural Resources Report F-083-R. 24pp. 

Baker, G.M., N.W. Darby and T.B. Williams. 2004. Balancing Bonneville Cutthroat Trout with 

non-native salmonids in Great Basin National Park. S.E. Moore,Carline, R.F.and Dillion, 

J., eds. Working together to ensure the future of wildtrout; proceedings of Wild Trout 

VIII symposium – 30th anniversary; 2004 September 20 – 22; Yellowstone National Park, 

Wyoming. 398 pages. 

Berger, B.L. 1964a. Trials of antimycin in ponds at the National Fish Hatchery, Berlin, New 

Hampshire, September 1964. U.S. Fish and Wildlife Service, Fish Control Laboratory, La 

Crosse, Wisconsin, typewritten, December 1964. 4 pp. 

Berger, B.L. 1964a. Trials of antimycin in ponds at the National Fish Hatchery, Cape 

Vincent, New York. U.S. Fish and Wildlife Service, Fish Control Laboratory, La Crosse, 

Wisconsin, typewritten, October 1964. 3 pp. 

Berger, B.L. 1965a.An application of FINTROL-5 (antimycin) to Veterans Memorial Park Pond, 

West Salem, Wisconsin. U.S. Fish and Wildlife Service, Fish Control Laboratory, La 

Crosse, Wisconsin, typewritten, November12, 1965. 11 pp. 

Berger, B.L. 1965b. Field testing of antimycin at Stuttgart, Arkansas. U.S. Fish and Wildlife 

Service, Fish Control Laboratory, La Crosse, Wisconsin, typewritten, May 5, 1965. 21 

pp. 

Berger, B.L. 1965c. Rough fish removal with antimycin in Coe and Sidie Hollow Creeks, 

Vernon County, Wisconsin on August 13, 1965. U.S. Fish and Wildlife Service, Fish 

Control Laboratory, La Crosse, Wisconsin, typewritten, 4 pp. 
92

Berger, B.L. 1966a. Reclamation of Lake Creek with FINTROL-5 at the National Fish Hatchery, 

Saratoga, Wyoming. U.S. Fish and Wildlife Service, Fish Control Laboratory, La Crosse, 

Wisconsin, typewritten, January 13, 1966. 13 pp. 

Berger, B.L. 1966b. Field trials of Fintrol-5 at Stuttgart, Arkansas, in December 1965. U.S. Fish 

and Wildlife Service, Fish Control Laboratory, La Crosse, Wisconsin, typewritten, March 

7, 1966. 20 pp. 

Berger, B.L. 1966c. Antimycin (Fintrol) as a fish toxicant. Proceedings of the Southeastern 

Association of Game and Fish Commissioners 19(1965):300-301. 

Berger, B.L. 1971. Fish toxicant compositions and method of using them. U.S. Patent 3,608,072. 

Filed March 21, 1969, patented September 21, 1971. 1pp. 

Berger, B.L. and J.W. Hogan. 1966. Intensive screening: laboratory trials with antimycin, pages 

75-76. In Progress in Sport Fishery Research, 1965. U.S. Bureau of Sport Fisheries and 

Wildlife, Resource Publication No. 17. 

Berger, B.L. and R.E. Lennon. 1967. A test of antimycin A, a fish toxicant, in Beauty Lake, 

Quebec. U.S. Fish and Wildlife Service, Fish Control Laboratory, La Crosse, Wisconsin, 

typewritten, February 1967. 20 pp. 

Berger, B.L., P.A. Gilderhus, and R.E. Lennon. 1967. Attempted reclamation of Whitewater 

Lake, Valentine National Wildlife Refuge, Nebraska. U.S. Fish and Wildlife Service, 

Fish Control Laboratory, La Crosse, Wisconsin, mimeo, October 251967. 12 pp. 

Brown, R.T. 1973. Toxicity of antimycin and rotenone to crawfish, Procambarus spp., and the 

possible use of antimycin as a fish poison in fish ponds. M.S. Thesis, Louisiana State 

University, Baton Rouge, August 1973. 49 pp. 

Brynildson, C. 1970. Selective chemical fish eradication of Mill Creek, Richland County. 

Wisconsin Department of Natural Resources, Division of Fish, Game and Enforcement, 

Bureau of Fish Management, Management Report No. 32:1-12. 

Burress, R.M. 1966. Populations of channel catfish in three antimycin-treated ponds in 

Georgia. U.S. Fish and Wildlife Service, Southeastern Fish Control Laboratory, Warm 

Springs, Georgia, typewritten, November 1966. 18pp. 

Burress, R. M. 1968a. Antimycin for controlling sunfish populations in ponds. Farm Pond 

Harvest 2(1):11, 12, 22. 

Burress, R. M. 1968b. Field trials of antimycin for selective control of gizzard shad in ponds 

near Atlanta, Georgia. U. S. Fish and Wildlife Service, Southeastern Fish Control 

Laboratory, Warm Springs, Georgia, typewritten, September 1968. 10 p. 
93

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United States Department of the Interior. 1975. Water Measurement Manual. A Bureau of 

Reclamation Water Resources Technical Publication. United States Government Printing 

Office. 

van Tamelen, E. E., J. P. Dickie, M. E. Loomans, R. S. Dewey, and F. M. Strong. 1961. The 

chemistry of antimycin A. X. Structure of the antimycins. Journal of the American 

Chemical Society, 83:1639-1646. 

Vezina, C. 1967. Antimycin A, a teleocial antibiotic. Antimicrobial Agents and Chemotherapy1966:757-766. 

Vezina, C. 1971. Antibiotics for nonhuman uses. Pure and Applied Chemistry 28(4):681-698. 

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. Fish and Wildlife Service, 

Investigations in Fish Control No. 2 and Circular No. 186:1-18. 
105

Walker, C.A. 2003. Effects of Antimycin treatment on benthic macroinvertebrates in Sams Creek 

and Starkey Creek, Great Smoky Mountains National Park, Blount/Sevier counties, 

Tennessee. Masters Thesis. University of Tennessee. Knoxville, Tennessee. 175 pages. 

Wisconsin Alumni Research Foundation. 1964. Antimycin poisoned fish feeding study– Rat. 

Wisconsin Alumni Research Foundation, Madison, Wisconsin. 2 p. 

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. Wisconsin Alumni Research Foundation, Madison, Wisconsin, June 10, 

1965. 36 p. 

Wilson, J.F. Jr. Time-of-Travel Measurements and Other Applications of Dye Tracing. 

International Association of Science and Hydrology. Publication No. 76. 1968. 

Wormell, L. 2005. Use Closure Memo. Special Review and Reregistration Division Memo 

dated October 26, 2005. USEPA 2006. Environmental Fate and Ecological Risk 

Assessment of Antimycin. Office of Pesticide Programs 

Wright, G., Dixon, and Thompson. 1933. Fauna of the national parks. NPS Fauna No. 1. 

Annual Report to the National Park Service. 
106

Appendix B.1. Integrated Pest Management Legislation and Policy 

Title 7 USC 136r-1 Federal Fungicide Insecticide and Rodenticide

Acthttp://www.chemalliance.org/Handbook/background/back-fifra.asp

 (http://uscode.house.gov/usc.htm) SEC. 303. Integrated Pest Management states: “The Secretary of 

Agriculture, in cooperation with the Administrator, shall implement research, demonstration, and 

education programs to support adoption of Integrated Pest Management. Integrated Pest Management is a 

sustainable approach to managing pests by combining biological, cultural, physical, and chemical tools in 

a way that minimizes economic, health, and environmental risks. The Secretary of Agriculture and the 

Administrator shall make information on Integrated Pest Management widely available to pesticide users, 

including Federal agencies. Federal agencies shall use Integrated Pest Management techniques in 

carrying out pest management activities and shall promote Integrated Pest Management through 

procurement and regulatory policies and other activities”. 

Department of Interior Manual, Sec.517 

Integrated Pest Management Policy: Including the Use of Pesticides and Biological Control 

Agents. 

1.1 Purpose - The purpose of this document is to incorporate Integrated Pest Management (IPM) 

in all Department pest management activities. As defined in 7USC136r-1, “Integrated Pest 

Management is a sustainable approach to managing pests by combining biological, cultural, 

physical, and chemical tools in a way that minimizes economic, health, and environmental 

risks.” 

1.2 Scope - This chapter applies to all Department and Bureau activities involving planning, 

procurement, prevention, design, detection, control, and management of native and nonnative 

pest species on DOI lands and properties. 

Public Contracts and Property Management, Facility Management, 2001 Code of Federal 

Regulations (C.F.R.) Title 41, Volume 2, 102.74.35, directs executive agencies to provide IPM 

services. 

Federal Register, July 19, 2000, Standard Concession Contract Language directs concession 

managers to use an integrated pest management program to manage weeds, harmful insects, rats, 

mice and other pests on Concession Facilities and that weed and pest management activities shall 

be in accordance with Applicable Laws and guidelines established by the Director.” 

National Park Service Management Policies 2006 

4.4.5.2 Integrated Pest Management Program 

The Service conducts an integrated pest management (IPM) program to reduce risks to the 

public, park resources, and the environment from pests and pest- related management strategies. 

IPM is a decision- making process that coordinates knowledge of pest biology, the environment, 

and available technology to prevent unacceptable levels of pest damage, by cost- effective 

means, while posing the least possible risk to people, resources, and the environment. 
107

Appendix B.2. 11 Step Process to Developing and Implementing an Integrated Pest 

 Management Strategy 

1. Describe your site management objectives and establish short and long term priorities. 

2. Build consensus with stakeholders-occupants, decision makers and technical experts 

(ongoing). 

3. Document decisions and maintain records. 

4. Know your resource (site description and ecology). 

5. Know your pest. Identify potential pest species, understand their biology, and conditions 

conducive to support the pest(s) (air, water, food, shelter, temperature, and light). 

6. Monitor pests, pathways, and human and environmental factors, including population levels 

and phenological data. 

7. Establish “action thresholds,” the point at which no additional damage or pest presence can 

be tolerated. 

8. Review available tools and best management practices. Develop a management strategy 

specific to your site and the identified pest(s). Tools can include: 1) no action, 2) physical, 3) 

mechanical, 4) cultural, 5) biological, and 6) chemical management strategies. 

9. Define responsibilities and implement the lowest risk, most effective pest management 

strategy, in accordance with applicable laws, regulations, and policies. 

10. Evaluate results; determine if objectives have been achieved; modify strategy if necessary 

(adaptive management). 

11. Education and outreach. Continue the learning cycle, return to Step 1. 
108

Appendix B.3. Example of an antimycin project managers planning and implementation 

checklist. The checklist provides a general outline of the key tasks and considerations of the 

project manager for any antimycin project. 

____ Validate and Geo-Reference Barrier 

____ Work through all NEPA requirements prior to project planning and implementation 

____ Acquire proper permits from state water quality enforcement division (e.g. NPDES, 404, 

etc.) 

____ Acquire Pesticide Applicator Permit from appropriate state (FIFRA requirement) 

____ Meet with local resource groups to educate and solicit volunteer assistance where 

necessary 

____ Determine species distribution in all waters above barrier (flag upper ends) 

____ Determine if any special fishing opportunities will be offered prior to treatment 

____ Purchase & build appropriate supplies & equipment for treatment 

____ Test all equipment to be sure it works and application rates are set accordingly 

____ Measure Out & tag treatment area 

____ Determine & mark elevations for each site 

____ Establish fish monitoring Sites 

____ Establish discharge sites throughout treatment area 

____ Construct site map of treatment area including site numbers, tributaries, elevations, dye 

times, discharge sites, fish sites, & landmarks 

____ Print signs and other education material to support closure of treatment area 

____ Meet with in-house and neighboring law enforcement officials to inform and request any 

support for project 

____ Set location and take hose measurements for detox station 

____ Determine how to transport all gear into treatment & detox area 

____ Record preliminary round of discharge and dye time data 

____ Implement OPEN FISHING portion of project (optional) 

____ Conduct pre-treatment fish monitoring 

____ Post "CLOSED TO PUBLIC" signs just prior to project start-up 

____ Redo upper stream discharges at upper end of treatment area to prepare for first day
109

Appendix B.4. Example of a National Park Service close order sign including federal authorities 

and Superintendents signature under which such closures are authorized. 

NOTICE 

TRAIL CLOSURE 

The following area is closed to public entry from 8:00 PM September 11, 2005 

until 8:30 PM September 16, 2005. 

The Middle Prong Trail will be closed from the junction with Panther Creek 

Trail up to its junction with the Lynn Camp Prong Trail and this trail will be 

closed to it’s junction with the Mirey Ridge Trail. The Greenbrier Ridge 

Trail will be closed from its junction with the Lynn Camp Prong Trail to the 

Appalachian Trail from 6:00 PM September 11, 2005 until 8:30 PM 

September 16, 2005.

Please refer to the project map on the board that shows these closures. 

 

During this time, a native brook trout restoration project will be conducted in 

a 1.5 mile segment of Indian Flats Prong. 

This closure is necessary “for the maintenance of public health and safety... 

and aid to scientific research ” [36 CFR 1.5. (a) (1)]. 

By Order of the Superintendent, per authority vested in Title 36, Code of 

Federal Regulations, Section 1.5 (a) (1) 

________________________ Date ______________ 

Dale Ditmanson 

Superintendent 
110

Appendix B.5. Example of Great Smoky Mountains National Park stream discharge sheet. 

Stream: Date:

Watershed: Station:

Cell Width: (circle one) Fixed _______________ ft Variable _______________ ft

DISTANCE ON DEPTH VELOCITY CELL VOLUME

TAPE (feet) (feet) (feet/second) (cubic ft/sec)

grsmflow.xls DISCHARGE (cfs) =

Stream Discharge Datasheet

19

20

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18
111

Appendix B.6. An example of a pre-treatment safety meeting checklist and personnel signup 

datasheet.. 

Safety Meeting Checklist & Sign-Up Sheet

Stream Name: _____________________________________ Date: _________________

Introduction & Project Command/Control

Introduction of Team & Affiliation 1

Project Coordinators and responsibilities 2

3

Antimycin Properties 4

Properties of Antimycin 5

PPE (Use, technique, cleaning procedures) 6

MSDS location 7

Exposure to eyes or skin 8

9

Antimycin Station Use & Safety 10

Safety 11

Station Setup 12

Common Problems & Fixes 13

Data Recording 14

End of Day & Cleanup 15

16

Potassium Permanganate Station Use & Safety 17

Properties of KMnO4 18

PPE (Use, technique, cleaning procedures) 19

MSDS location 20

Exposure to eyes or skin 21

22

Potassium Permanganate Station Use & Safety 23

Safety 24

Station Setup 25

Common Problems & Fixes 26

Data Recording 27

End of Day & Cleanup 28

29

30

31

32

33

34

35

36

Attendee Agency

Antimycin A1

C28H40N2O9

O

O O CH3

O

O

H3C

OH O

N

H

O

H

O
112

Appendix B.7. Antimycin station equipment checklist and vendor contact information. 

Antimycin Station Equipment Checklist

Quantity Item # Vendor 1 Cost per Unit

4 13058 1 $13.50

4 25730 1 $25.10

8 3 or 6

4 3 or 6

4 SZ-01-2946 4 $1.99/ea

4 3 or 6

2 3 or 6

80 feet 3 or 6

12 3 or 6

24 3 or 6

12 3 or 6

8 3 or 6

8 30775 2 $18.95

8 3 or 6

1 3 or 6

1 95925 1 or 8 $11.50

2 3 or 6

2 11566-2 2 or 7 $2.40/ea

2 105393L 2 or 7 $9.80/100

8 3 or 6

36 Misc.

24 3 or 6

8 3 or 6

12 02-543-36D 8 $81.36 per 12

8 47312 1 or 8 $4.70/ea

8 47313 1 or 8 $5.90/ea

24/case 13091 1 or 5 $90.40

12/case 28794 1 or 5 $80.60

96 3 or 6

1-gal 8

12 8

1 8

12 88333 1 or 5 $39.20

12 3 or 6

Extras Misc.

* January 2004 Costs

1 Vendor Information

Index

1

2

3

4

5

6

7

Male PVC Top Cap w/ Threaded Cap (1-1/4" Used to hold funnel and fill bucke

www.cabelas.com

https://www1.fishersci.com/index.jsp

Keeps waterer level

Phone Number Web Site

(800) 419-9524

(800) 356-0783

www.northernsafety.com

www.lowes.com

Notes

Keeps level

www.valleyvet.com

www.labsafety.com

Northern Safety Supply, Inc.

(800) 237-4445

(800) 631-1246

Lowe's Home Improvement

Cabela's

Fisher Scientific

Reinforced hose is best

Tightens rebar and hose clamps

Keeps debris out of waterer

Vendor Name

Valley Vet

Lab Safety Supply

All bucket water should be filtered

Plastic Graduated Pitcher (2-l)

Torpedo Level

Paper Clips

Rebar (1/2" x 24")

Funnel (6")

Garden Hose Male Thread

Farnam Floats Waterer for Dogs

Hose couplings

Hose Clamps (3/4-1")

Screwdriver or socket

Plastic 5-gal Bucket

Mesh Filter or Red Devil Nylon Bag Straine

Goggles or Safety Glasses

Gloves

Wood or Plexiglass Lid for Waterer

90o

 Through Hull Fitting (3/4")

Rubber Gasket (1" hold, 2-1/4" wide, 1/8")

Two-Way Valve

Mesh Reinforced Garden Hose (6')

Wood Blocks (2"x2"x1')

Local Hardware Store

Item

Plastic 10-gal Bucket

Wooden Dowels (0.5"diam x 12" length) Stirs water/antimycin in bucket

Used to unclog waterer outflow

5lb Hammer

Graduated Cylinders (100ml)

Amber Widemouth Bottles (250ml) Widemouths easier to use & clean

Misc. Garden Hose Gaskets, Fittings, Screen Always Handy

Handheld Thermometers

Nalgene Bottles (250ml) Transport dye for trials & stations

Bic Lighters To warm NoNox-9

Solo Sprayer (2.5 gal) Smaller tank easier on slopes

Graduated Cylinders (250ml)

Flourescent Dye

Amber Widemouth Bottles (500ml) Widemouths easier to use & clean

Ziploc Bags (1-gal) For Gloves/Goggles & Bottles
113

Appendix B.8.. Potassium permanganate equipment checklist and vendor contact information 

Potassium Permanganate (KMnO4) Station Equipment Checklist

Quantity Item # Vendor 1 Cost per Unit

200 lb CAIROX FF 1 $1.98/lb

2 3972 2 $66.60/ea

4 - 3 or 6

1 - Misc.

4 SZ-01-2946 4 $2.99/ea

4 - 3 or 6

2 - 3 or 6

80 feet - 3 or 6

12 - 3 or 6

24 - 3 or 6

"T" Hose Fitting (1-1/4") 2 - 3 or 6

1 - 3 or 6

1 - 3 or 6

4 - 5 $68/ea

2 - 3 or 6

2 - 3 or 6

30 ft - 3 or 6

1 - 3 or 6

2 - 3 or 6

2 11566-2 2 or 7 $2.40/ea

2 105393L 2 or 7 $9.80/100

1 38024 2 or 7 $26.30/10

1 47321 1 or 8 $13.30

1 47322 1 or 8 $16.60

1-8

1 - Misc.

1 93016 8 $39.90/ea

1 77008 2 $18.20/ea

1-8

1 - Misc.

100 ft - Misc.

as needed - Misc.

1-gal - Misc.

1 - Misc.

* January 2004 Costs

1 Vendor Information

Index

1

2

3

4

5

6

7

8

Item

KMnO4 Powder (97%)

30 Gallon Drums

Graduated Cylinders (1l)

Mesh Reinforced Hose (1-1/4 inch)

Hose couplings (1-1/4 inch)

Hose Clamps (1 to 1-1/2 inch)

Screwdriver or socket

Small Boat or Kayak Paddle

90o

 Through Hull Fitting (1-1/8")

Rubber Gasket

Two-Way Valve

A BIG HELP if available!

Optional - but VERY HELPFUL!

Wood Blocks (4"x4"x2')

Utility Knife

Pesola Hanging Scale (2.5kg x 20g)

Plastic Scoop (200ml)

Dykes

Mesh Filter

Goggles or Safety Glasses

Watch (w/second hand)

Gloves

CDS Float Box (2-gal)

Saw Horses

CARUS Chemical Company

 Fire Pump Couplings (Pump to Hose)

Electic Drill w/socket bits

Pressure Treated Boards (1"x6"x8')

Malleable Wire

Hose for Small Gas Fire Pump (1-inch)

Waders (size to fit operator)

Lab Safety Supply

Plastic 5-gal Bucket

Phone Number

Use two at once & two for backup

Vendor Name

Small Gas Fire Pump

Particulate Mask Filter

Graduated Cylinders (500ml)

Gas for Fire Pump 50:1)

Reinforced hose works better

Northern Safety Supply, Inc. (800) 631-1246

Forestry Suppliers

Lowe's Home Improvement

Cabela's

Schall Chemical Company

Local Hardware Store

(800) 647-5368

Web Site

(815) 223-1500

(800) 356-0783

www.caruschem.com

www.labsafety.com

(800) 237-4445

(719) 852-5938

www.lowes.com

Notes

Transport in smaller buckets 

www.forestry-suppliers.com

www.cabelas.com

www.northernsafety.com
114

Appendix B.9. An example of an Antimycin A daily application form. 

ANTIMYCIN DOSAGES AND TEMPERATURE FORM

Stream Name:

Operator: Date:

Station #:

Flow (cfs):

Concentration:

Duration:

ml Antimycin/hr:

Time

Water 

Temp

Antimy.

added

* Please bring your goggles, gloves, bottles, and this datasheet out with you at the 

end of the day!

Comments or Observations

Time

Starting:

Time

Ending:

! ! PLEASE RECORD Time, Water Temp., Comments/Observations, and Fish Status hourly below ! !

Fish Status
115

Appendix B.10. – An example of a potassium permanganate daily application form.

KMnO4 APPLICATION AND TEMPERATURE FORM

Stream Name:

Operator(s): Date:

Stream Flow (cfs):

g KMnO4 per hr:

ppm KMnO4:

Duration:

Time

Water 

Temp

KMnO4 

added

* Please bring this datasheet out with you at the end of the day!

Comments or Observations

Time

Starting:

Time

Ending:

! ! PLEASE RECORD Time, Water Temp., Comments/Observations, and Fish Status hourly below ! !

Fish Status

Remember : Adjust flow rates so NOT to adding more than 100g KMnO4 per gal.

Stir mix every 30 minutes to prevent settling in drums and hoses.
116

Appendix B.11. An example of a antimycin and potassium permanganate application summary sheet. 

Station ppb

Flow

(cfs)

TOTAL 

Hours

TOTAL

cfs

Antimycin 

(ml) F Elevation E

Range 

Temp (oC)

Time 

Treated pH Staff Equipme

Spray

TOTAL

KMnO4

Notes:

Antimycin and KMnO4 Application Table
117

Appendix B.12. 

PROTOCOL DEVIATION FORM 

Treatment/Project Name: 

Description and Cause of Deviation: 

SOP(s) Violated: AOP 

TOP 

Event Timing and Location 

Start Date/Time of Deviation (e.g., 01/01/2007, 3:30 PM): 

End Date/Time of Deviation: 

Identification Date/Time of Deviation: 

Location of Deviation (include site number or short description): 

Reason(s) for Deviation (check all that apply): 

 To protect worker/bystander safety 

 To prevent release of antimycin A beyond the treatment area 

 To prevent violation of Federal or other regulation(s) 

 To maintain efficacy of treatment 

 Other (describe): 

Action(s) Taken to Minimize Continued/Future Deviations and Potential Impacts 

Project Manager Signature Signature Date 

Project Manager Printed Name 
118

Appendix C. A summary of case history of antimycin and potassium permanganate dosage data 

of various fish species from throughout the country. 

Bioassay Case Study 1: Sams Creek, Great Smoky Mountains National Park (GRSM), 

Tennessee. October 1998, pH 6.6-6.7, conductivity 15Ms/cm, temperature 14-15.5oC – 

Bioassays conducted in GRSM indicated 8ppb antimycin applied for 8 hours resulted in a 100% 

mortality rate of rainbow trout after 24 hours. Using 3ppm potassium permanganate and 8ppb 

antimycin, rainbow trout mortality was 0% after 48 hours. 

Bioassay Case Study 2: Snake Creek, Great Basin National Park (GRBA), Nevada. July 

2002, pH 7.8-7.9, conductivity 187 , temperature 7.8-7.9oC – Bioassays conducted in GRBA 

indicated 2,4, and 8ppb antimycin applied for 8 hours resulted in a 100% mortality rate of 

rainbow trout after 24 hours. Using 4ppm potassium permanganate and 8ppb antimycin, 

rainbow trout mortality was 30% after 24 and 48 hours. 

Table C.1. List of antimycin treatment concentration and exposure period estimated to result in 

100% mortality of various fish species across the United States. Note, exposure period reflects 

the time period when antimycin treatment area was maintained at the treatment concentration. 

Family Target Species Scientific Name 

Water 

Temp. 

(

o

C) 

pH Exposure 

Time (hrs) 

LD100

(ppb) Citation 

Atherinidae Brook Stickleback Labidesthes 

sicculus 

5 7.5 8 7.5 Gilderhus et 

al.1969 

Catastomidae White Sucker Catastomus 

commersoni 

5 7.5 8 7.5 Gilderhus et 

al.1969 

 Northern 

Hogsucker 

Hypentelium 

nigricans 

10-18 8.3 15 5 Gilderhus et 

al.1969 

Centrarchidae Bluegill Lepomis 

macrochirus 

20 8.8 10 10 Gilderhus et 

al.1969 

 Largemouth Bass Micropterus 

salmoides 

20 8.8 10 10 Gilderhus et 

al.1969 

 Black Crappie Pomoxis 

nigromaculatus 

20-22 8.4 8 7.5 Gilderhus et 

al.1969 

Cottidae Mottled Sculpin Cottus bairdi 10-18 8.3 15 5 Gilderhus et 

al.1969 

Cyprinidae Central 

Mudminnow 

Umbra limi 20-22 8.4 8 7.5 Gilderhus et 

al.1969 

 Central Stoneroller Campostoma 

anomalum 

10-18 8.3 15 5 Gilderhus et 

al.1969 

 Common Carp Cyprinous carpio 25 8.6 10 10 Gilderhus et 

al.1969 

 Fathead Minnow Pimphales 

promelas 

20 8.8 10 10 Gilderhus et 

al.1969 

 Blacknose Dace Rhinichthys 

atratulus 

10-18 8.3 15 5 Gilderhus et 

al.1969 

 Longnose Dace Rhinichthys 

cataractae 

10-18 8.3 15 5 Gilderhus et 

al.1969 

 Creek Chub Semotilis 

atromaculatus 

10-18 8.3 15 5 Gilderhus et 

al.1969 

Percidae Fantail Darter Etheostoma 

flabbellare 

20 8.8 10 10 Gilderhus et 

al.1969 

 Johnny Darter Etheostoma 5 7.5 8 7.5 Gilderhus et 
119

nigrum al.1969 

Petromyzontidae American Brook 

Lamprey 

Lampetra 

appendix 

5 7.5 8 7.5 Gilderhus et 

al.1969 

Poecilidae Western 

Mosquitofish 

Gambusia affinis 28.3 6.9 48 0.6 Lennon and 

Berger 1970 

Salmonidae Rainbow Trout Oncorhynchus 

mykiss 

14.0 6.6 8 8 Moore and Kulp 

2005 

 Brook Trout Salvelinus 

fontinalis 

7.9 7.8 8 8 Moore and Kulp 

2005 

 Brook Trout Salvelinus 

fontinalis

9-16 7.4 10 8 Gresswell 1991 

 Brook Trout Salvelinus 

fontinalis

5-8 6.5-

7.0 

10 6 Rinne et al. 1981 

 Brook Trout Salvelinus 

fontinalis

12 7.5 5 7 Stevens and 

Rosenlund 1986 
120

Appendix D. The antimycin toxicity report for Sams Creek, Great Smoky Mountains National 

Park in 1998. 

Sams Creek Toxicity Test Report 

Great Smoky Mountains National Park 

October 26, 1998 

Procedure

Toxicity tests were performed streamside, next Sams Creek, using water directly from the 

stream. Tests were conducted from October 19 - 22, 1998. Fish were collected and held 

approximately 15 hours prior to tests. Ten rainbow trout (Oncorhynchus mykiss) were exposed 

to concentrations of antimycin and KMnO4 for eight hours and observed throughout this period. 

Fish that survived the exposure period were placed in holding cages directly in the stream and 

percent mortality was recorded at 24 and 48-hour intervals after initial 8-hour exposure period. 

Three experiments were performed to evaluate toxicity of antimycin and KMnO4 to rainbow 

trout. Test # 1 examines the toxicity of antimycin, test # 2 indicates the amount of KMnO4

needed to detoxify antimycin, and test # 3 examines the toxicity of KMnO4. 

I. Antimycin solutions. 

A. Stock # 1. Mix 5 mls of antimycin concentrate (20% active ingredients) with 5 mls 

dilutant. Final solution is 10 mls at 10% active ingredients or 0.1 mls/ml.

B. Stock # 2. Mix 1 ml Stock # 1 in 1 liter Sams Creek water. Final solution is 0.0001-

mls/ml or 100 ppm vol/vol. Note Stock # 2 is stable for only about 8 hours and must be 

prepared fresh thereafter.

C. Exposure Concentrations. For antimycin exposure concentrations tested the 

following amounts of Stock # 2 was added to five-gallon buckets to make 20 liters

Exposure Concentration Stock # 2 added 

2 ppb 0.4 ml 

4 ppb 0.8 ml 

8 ppb 1.6 ml 

II. KMnO4 solutions. 

D. Stock Solution. Add 1 gram KMnO4 to 100 mls water. The resulting stock solution 

equals 10g/l KMnO4.

E. Exposure Concentrations. The following amounts of KMnO4 were added to fivegallon buckets to make 20 liters. 

Exposure Concentration KMnO4 stock solution added for 20 liters. 

1 ppm 2 ml 

2 ppm 4 ml 

3 ppm 6 ml 

4 ppm 8 ml 

III. Test Fish. 

Test fish were adult rainbow trout from Sams Creek. Mean length was 161.8 millimeters and 

mean weight was 41.6 grams. Ten fish were added to each 20-liter test solution. There was no 

significant difference between mean total length within each group of fish tested (P>0.05). 
121

IV. Dilution Water. 

Dilution water was Sams Creek water which ranged in pH from 6.7-6.6 and was unaffected by 

the additions of antimycin and KMnO4. Temperatures ranged from 14.5-15° C and were 

maintained by a water bath of ambient temperatures from Sams Creek. 

A. Test Results Test # 1. Toxicity of antimycin in Sams Creek water. 

% Mortality Through Time (hours) 

% Mortality in 

holding cages 

Antimycin 

concentration 1 2 3 4 5 6 7 8 24 48 

0 ppb (Control) 0 0 0 0 0 0 0 0 0 0 

2 ppb 0 0 0 0 0 0 0 10 100 100 

4 ppb 0 0 0 0 0 10 10 20 100 100 

8 ppb 0 10 10 20 20 30 60 90 100 100 

B. Test # 2. KMnO4 demand in Sams Creek water. 

 

% Mortality Through Time (hours) 

% Mortality in 

holding cages 

Antimycin 

concentration 

KMnO4

concentration 1 2 3 4 5 6 7 8 24 48 

8 ppb 1 ppm 0 0 0 0 0 0 0 0 10 10 

8 ppb 2 ppm 0 0 0 0 0 0 0 0 0 0 

8 ppb 3 ppm 0 0 0 0 0 10 10 10 10 10 

8 ppb 4 ppm 30 50 50 50 50 50 50 50 50 50 

0 ppb 0 ppm (Control #1) 0 0 0 0 0 0 0 0 0 0 

8 ppb 0 ppm (Control #2) 0 0 0 0 30 80 100 100 100 100 

C. Test # 3. KMnO4 toxicity in Sams Creek water. 

% Mortality Through Time (hours) 

% Mortality in 

holding cages 

KMnO4

concentration 1 2 3 4 5 6 7 8 24 48 

1 ppm 0 0 0 0 0 0 0 0 0 30 

2 ppm 0 0 0 0 0 0 0 0 0 0 

3 ppm 0 0 0 0 0 0 0 0 0 0 

4 ppm 20 20 20 20 20 20 20 20 30 40 

0 ppm (Control) 0 0 0 0 0 0 0 0 0 0 

VI. Discussion. 

At 24 hours 100% mortality was reached in all fish exposed to concentrations of antimycin. In 

Test # 1, 90% mortality was the maximum within the 8-hour exposure period at 8-ppb antimycin. 

These results conflict with control # 2 (8-ppb antimycin: 0-ppm KMnO4) in Test # 2 where 100 

% mortality was reached within the 8 hour exposure period. 

Two-ppm was the only concentration of KMnO4, used to detoxify antimycin in Test # 2, to 

generate 0% mortality. One and 3-ppm KMnO4, used to detoxify antimycin, produced only 10% 

mortality. This occurred after the initial exposure period in 1-ppm KMnO4. Four-ppm KMnO4
122

had 40% mortality. This shows that 4-ppm KMnO4, initially, has some sort of toxicity. The 

same concentration caused initial mortality in Test # 3 also. 

Two and three-ppm KMnO4 produced 0% mortality in Test # 3. No concentrations of KMnO4

caused 100% mortality while 4-ppm KMnO4 caused 40% mortality. However, the highest 

mortality beyond 4-ppm KMnO4 was 1-ppm at 30% mortality. 
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Appendix E. A copy of the Fintrol® (antimycin) product label 
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Appendix F. The Material Safety Data Sheet (MSDS) for Fintrol® (antimycin). 
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The U.S. Department of the Interior (DOI) is the nation's principal conservation agency, charged with the mission "to protect and 

provide access to our Nation's natural and cultural heritage and honor our trust responsibilities to Indian tribes and our 

commitments to island communities." More specifically, Interior protects America’s treasures for future generations, provides 

access to our nation’s natural and cultural heritage, offers recreation opportunities, honors its trust responsibilities to American 

Indians and Alaska Natives and its responsibilities to island communities, conducts scientific research, provides wise stewardship 

of energy and mineral resources, fosters sound use of land and water resources, and conserves and protects fish and wildlife. The 

work that we do affects the lives of millions of people; from the family taking a vacation in one of our national parks to the 

children studying in one of our Indian schools. 

NPS D-1966, January 2008 
132

National Resource Program Center 

Water Resources Division

1201 Oakridge Drive, Suite 250 

Fort Collins, CO 80525 

www.nature.nps.gov

EXPERIENCE YOUR AMERICA TM

National Park Service 

U.S. Department of the Interior 

Natural Resource Program Center 
133