United States Chemical Safety December 2012 

Environmental Protection and Pollution Prevention 

Agency (7510P) 

 

Poly(Hexamethylenebiguanide) 

Registration Review: 

Final Work Plan 

December 2012 

 
Docket Number: EPA-HQ-OPP-2012-0341 

www.regulations.gov 

Poly(Hexamethylenebiguanide) 

Registration Review: 

Final Work Plan 

December 2012 

Case # 3122 

Approved By: 

~NdJw~ p.- Jennifer McLain 

Acting Director, Antimicrobials Division 

Date: 

'" I I 

2 of13 
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Poly(Hexamethylenebiguanide) Hydrochloride Registration Review Team

Human Health & Environmental Effects

William Hazel 

Michelle Centra 

Timothy Dole 

Jonathan Chen 

Srinivas Gowda 

Najm Shamim 

Donna Randall 

Risk Management

Rebecca von dem Hagen 

Lance Wormell 

Office of General Counsel

Philip Ross 

________________________________________ 

Supporting documents for the Poly(Hexamethylenebiguanide) Hydrochloride Case may be found 

in the registration review docket EPA-HQ-OPP-2012-0341 located on the internet at

www.regulations.gov. 
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Introduction

This is the Final Work Plan (FWP) of the Environmental Protection Agency (EPA or “the 

Agency”) for the registration review of Poly(hexamethylenebiguanide) hydrochloride (Case 

3122). The work plan includes the expected registration review timeline. The work plan also 

addresses public comments received concerning the Preliminary Work Plan (PWP) in the 

Summary Document which was posted in the Poly(hexamethylenebiguanide) hydrochloride 

registration review docket, and any other comments concerning the initial docket postings. The 

Summary Document provided information on what EPA knows about the pesticide and what 

additional risk analyses and data or information the Agency believes are needed to make a 

registration review decision. 

The Agency is implementing the registration review program pursuant to Section 3(g) of the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and will review each registered 

pesticide every 15 years to determine whether it continues to meet the FIFRA standard for 

registration. Changes in science, public policy and pesticide use practices will occur over time. 

The registration review program is intended to make sure that, as the ability to assess risk 

evolves and as policies and practices change, all registered pesticides continue to meet that 

statutory standard. The public phase of registration review begins when the initial docket is 

opened for each case. Information on this program is provided at: 

http://www.epa.gov/oppsrrd1/registration_review/. 

The Poly(hexamethylenebiguanide) Case, Case Number 3122, contains one active ingredient, 

Poly(hexamethylenebiguanide) (PC Code 111801) (PHMB). The first product containing 

PHMB was registered in 1982 for use in manufacturing swimming pool sanitizers and 

disinfectants for use in commercial, industrial, and residential settings and to formulate sanitizers 

and disinfectants for use in food handling/storage establishments, premises and equipment, 

including food contact surfaces. Currently, there are 25 products containing PHMB as an active 

ingredient registered with EPA. PHMB is a broad spectrum antimicrobial registered for use as a 

fungicide, algicide, and sanitizer in swimming pools, spas and hot tubs; a materials preservative 

in architectural and industrial coatings, textiles and cellulosic material, glues and adhesives, 

leather and fresh animals skins and hides; a microbicide/microbiostat in industrial processes and 

water systems including cooling towers; and as a hard surface, food and non-food contact, 

disinfectant/sanitizer in a variety of residential and public access premises, commercial, 

institutional, industrial premises and equipment, medical and agricultural premises and 

equipment, and food handling storage establishments and equipment. 

In 2008, the Agency established an exemption from a tolerance in 40 CFR 180.1280 for residues 

of PHMB in or on all food commodities when the residues are the result of the lawful application 

of a food contact surface sanitizer containing PHMB at 550 parts per million (ppm). A 

Reregistration Eligibility Decision (RED) for PHMB was completed in 2004 and the Agency 

intends to issue the post-RED DCI in the near future. The Agency intends to incorporate data 

received in response to the PHMB post-RED DCI into the risk assessments to be performed for 

this registration review. Also, the Agency notes that this registration review will be based on 

updated labels reflecting mitigation measures outlined in Table 13 of the 2004 RED. Labels will 

be updated through the reregistration process. 
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The Poly(hexamethylenebiguanide) Preliminary Work Plan (PWP) was published on July 6, 

2012 and the 60-day comment period ended on September 10, 2012. Documents associated with 

the registration review of PHMB, including the PWP, can be viewed at www.regulations.gov in 

docket EPA-HQ-OPP-2012-0341. Below is a summary of the issues relevant to this registration 

review case. 

Comments Received on the Preliminary Work Plan

In the PWP, the Agency solicited comments on specific topics: environmental justice, water 

body impairment, trade irritants, and structure activity relationships. The Agency did not receive 

any comments specific to these topics. The Agency received five comments during the public 

comment period on the initial docket. The complete Agency responses can be found in 

Responses to the 60-Day Public Comments on the Product Chemistry, Environmental Fate, and 

Ecological Effects Scoping Document in Support of Registration Review of Poly(hexamethylenebiguanide) hydrochloride located in the docket. 

Comments were submitted by the Bay Area Clean Water Agencies (BACWA); the California 

Stormwater Quality Association (CASQA); the Tri-TAC (the Technical Advisory Committee for 

the CASQA, the California Water Environment Association and the League of California Cities); 

and the San Francisco Bay Water Quality Control Board (SFBWQB). The comments that 

resulted in changes to the work plan included the request to require acute and chronic toxicity 

tests for estuarine and marine species as well as data on the impact of PHMB on wastewater 

treatment plants. Specifically, the commenters requested that the Agency require an Activated 

Sludge Respiration Inhibition Study. Commenters also requested clarification on the use 

patterns to be included in the planned risk assessment. The commenters requested that the 

Agency include swimming pool discharge, leaching of paint from exterior surfaces and all 

possible discharges to wastewater treatment plants in the anticipated ecological risk assessment. 

Below is a summary of the changes made since the issuance of the PWP.

Updates Since the PWP Was Issued

The Agency made changes to the anticipated human health and ecological risk assessments as a 

result of the comments received on the PWP and the Focus Meeting held on September 19, 2012. 

Changes from the Preliminary Work Plan to this Final Work Plan include:

 Since the publication of the PWP, it has come to the attention of the Agency that the 

chemical properties of the 20% MUP and 96.3% manufacturing use product (MUP) are 

different. Specifically, as a result of differences in the manufacturing processes, the two 

compounds differ in their polymer chain lengths and molecular weight. At the time of 

the initial registration of the 96.3% TGAI, the Agency was not aware of the potential 

difference in toxicity and the standard Tier I data set was not required. The Agency has 

determined that the toxicity database for the 96.3% manufacturing use product (MUP) is 

notcomplete and the following additional data are anticipated to be required on the 96.3% 

MUP: 

o 870.3100 90-Day Oral Toxicity, 

o 870.3700 Developmental Toxicity, 
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o 870.3250 90-Day Dermal 

o 870.5375 In vitro mammalian cell assay 

o 870.5395 In vivo mammalian cytogenetics 

o 870.5550 Unscheduled DNA Synthesis in Mammalian Cell Cultures 

 The 90-Day Inhalation Toxicity study anticipated to be required on the 20% MUP in the 

PWP is now anticipated to be required on both the 20% MUP and 96.3% MUP. 

 In response to the commenter’s request that the Agency require acute and chronic toxicity 

tests for estuarine and marine species as well as data on the impact of PHMB on 

wastewater treatment plants, the Agency has determined that the following additional 

ecological effects and environmental fate data are necessary: 

o 850.1350 Estuarine/Marine Shrimp Chronic Toxicity 

o 850.1400 Estuarine/Marine Fish Early Life Stage 

o 850.3300 Activated Sludge Respiration Inhibition 

 In response to comments received requesting information on the use patterns to be 

included in the planned risk assessment , the Agency has clarified the use patterns to be 

included in the environmental fate and ecological effects risk assessment. In addition to 

those scenarios listed in the PWP, the Agency will also evaluate risks from swimming 

pool discharges, leaching from use in architectural paints applied to facades and other 

exterior surfaces, and all possible discharges to wastewater treatment plants in the 

ecological risk assessment. 

 Also, please note that on June 27, 2012, EPA announced certain revisions in harmonized 

guideline series 850 - Ecological Effects Tests - including removal of the Tier 

designation on 850.4100 (Seedling Emergence and Seedling Growth), 850.4400 (Aquatic 

Plant Toxicity Test Using Lemna Spp.), 850.4450 (Aquatic Plants Field Study) and 

850.5400 (Algal Toxicity). For more information see "Final Test Guidelines; OCSPP 850 

Series; Notice of Availability" 77 FR 38282, June 27, 2012. 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0154-0028. 

o The Algal Toxicity study (GLN 850.5400) anticipated as required in the PWP and 

FWP will be replaced by the following harmonized guidelines: 850.4500 Algal 

Toxicity and 850.4550 Cyanobacteria (Anabaena flos-aquae) Toxicity 

o The Seedling Emergence and Growth, Tier II study (GLN 850.4225) anticipated 

as required in the PWP and FWP will be replaced by the following harmonized 

guidelines: 850.4100 Seedling Emergence and Seedling Growth 

Additional information on the changes to the ecological and human health risk assessments can 

be found in the Amended Product Chemistry, Environmental Fate, and Ecological Effects 

Scoping Document in Support of Registration Review of Poly(hexamethylene-biguanide) 

hydrochloride and Poly(hexamethylenebiguanide) hydrochloride. Revised Human Health 

Scoping Document for the Registration Review, respectively, at regulations.gov in docket EPAHQ-OPP-2012-0341. 
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This document makes final the work plan for the poly(hexamethylenebiguanide) registration 

review process. 

Anticipated Risk Assessment and Data Needs:

The Agency anticipates the need to conduct a comprehensive ecological risk assessment, 

including an endangered species risk assessment, for all uses of PHMB. The Agency anticipates 

the need to conduct a human health risk assessment. The Agency anticipates needing additional 

data for use in conducting the registration review for PHMB. 

Ecological Risk: 

 Ecological hazards of PHMB were characterized as part of the 2004 RED assessment; 

however, a quantitative risk assessment was not performed. 

 For registration review, the Agency anticipates conducting a comprehensive 

ecological risk assessment, including an endangered species assessment, for use 

patterns resulting in direct discharge of PHMB to water through uses in industrial 

processes and water systems such as cooling towers and paper mill process water as 

well as indirect discharge of PHMB via consumer, institutional and commercial 

product uses, including swimming pools. 

 The Agency has not conducted a risk assessment that supports a complete endangered 

species determination for PHMB. The ecological risk assessment planned during 

registration review will allow the Agency to determine whether PHMB’s use has 'no 

effect' or 'may affect' federally listed threatened or endangered species (listed species) or 

their designated critical habitats. When an assessment concludes that a pesticide’s use 

'may affect' a listed species or its designated critical habitat, the Agency will consult with 

the U.S. Fish and Wildlife Service and/or National Marine Fisheries Services (the 

Services), as appropriate. 

 The Agency anticipates the need to require the following additional ecological data for 

use in the registration review of PHMB. 

850.2100 -- Avian acute oral toxicity test, upland game 

850.1010 -- Aquatic invertebrate acute toxicity, freshwater daphnids 

850.1025 -- Oyster acute toxicity test1

850.1055 -- Bivalve acute toxicity larval (embryo/larval)1

850.1075 -- Fish acute toxicity2

850.1300 -- Daphnid chronic toxicity test 

850.1400 -- Freshwater fish early-life stage toxicity test, using the most acutely 

sensitive fish 

850.1035 -- Estuarine/marine mysid acute toxicity test 

850.1350 -- Estuarine/marine shrimp chronic toxicity 

 

1

 Results from either GLN 850.1025 or 850.1055 are expected to satisfy both anticipated data requirements. 2

 GLN 850.1075 is anticipated to be required for both a freshwater warm water fish and an estuarine/marine fish 

species. 
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850.1400 -- Estuarine/marine fish early life stage 850.4100 -- Seedling 

Emergence and Seedling Growth 

850.4400 -- Aquatic plant toxicology test using Lemna spp. 

850.4500 -- Algal Toxicity 

850.4550 -- Cyanobacteria (Anabeana flos-aquae) Toxicity 

Special Study -- Chronic whole sediment freshwater invertebrate toxicity test, 

amphipod (Hyallela azteca) 

Special Study -- Chronic whole sediment freshwater invertebrate toxicity test, 

midge (Chironomous sp.) 

Special Study -- Chronic whole sediment estuarine/marine invertebrate toxicity 

test, amphipod (Leptochirus plumulosus) 

 The Agency anticipates the need to require the following additional environmental fate 

data for use in the registration review of PHMB. 

835.1230 -- Sediment and soil adsorption/desorption 

850.6800 -- Activated Sludge Respiration Inhibition 

 Data are expected to be required for both the 20% and 96.3% technical grade active 

ingredients (TGAI) because available information is unclear on whether or not PHMB – 

which exists as a mixture of polymer chains of different lengths or sizes and mass – will 

have similar polymer characterization; therefore, ecological hazard across products. If 

information is provided to the Agency establishing that polymer characterization is the 

same for both TGAIs, then data are anticipated to be required on the more toxic TGAI 

only. 

 For more information, please refer to Amended Product Chemistry, Environmental Fate, 

and Ecological Effects Scoping Document in Support of Registration Review of 

Poly(hexamethylene-biguanide) hydrochloride, located in the docket. 

Human Health Risk: 

 The most recent human health risk assessment for PHMB was completed in support of 

the exemption from the requirement of a tolerance established in 2008. 

 The Agency anticipates the need to update or conduct the following human health risk 

assessments based on currently registered use patterns: dietary (food only); residential 

dermal, inhalation and incidental oral including dermal and incidental oral assessments 

for children and toddlers; and occupational dermal and inhalation. In addition, the 

Agency anticipates the need to conduct an aggregate assessment on dietary (food) and 

residential sources of PHMB.

 The Agency does not anticipate the need to conduct a drinking water assessment for 

PHMB because the Agency does not expect PHMB or its degradates to reach or pass 

through drinking water treatment plants. 
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 Past PHMB risk assessments have relied in part on a human study which involved 

intentional exposure to PHMB. In the course of registration review EPA will reconsider 

this study, as well as any other research involving intentional exposure of human subjects 

to PHMB brought to its attention. If EPA decides to rely on any of this study, EPA will 

ensure that all applicable regulatory requirements are met, including but not limited to the 

requirements for EPA ethics review and Human Studies Review Board review of certain 

research involving intentional exposure of human subjects. 

 The Agency anticipates the need to require the following additional human health data 

for use in the registration review of PHMB. 

870.3465 -- 90-day inhalation toxicity (rat) 

870.3100 -- 90-day oral toxicity 

870.3700 -- Developmental toxicity 

870.3250 -- 90-day dermal toxicity 

870.5375 In vitro mammalian cell assay 

870.5395 In vivo mammalian cytogenetics 

870.5550 Unscheduled DNA synthesis in mammalian cell cultures 

 The 90-day inhalation data are expected to be required on both the 20% and 96.3% 

technical grade active ingredients (TGAI) because available information is unclear on 

whether or not PHMB – which exists as a mixture of polymer chains of different lengths 

or sizes and mass – will have similar polymer characterization and; therefore, toxicity 

across products. If the Tier I Toxicity database is completed for the 96.3% TGAI, then 

data are anticipated to be required on the more toxic TGAI only. 

 For more information, please refer to Poly(hexamethylene-biguanide) hydrochloride. 

Revised Human Health Scoping Document for the Registration Review, located in the 

docket. 

Product Chemistry: 

 The Agency does not anticipate the need to require additional product chemistry data for 

use in this registration review at this time. 

 For more information, please refer to Amended Product Chemistry, Environmental Fate, 

and Ecological Effects Scoping Document in Support of Registration Review of 

Poly(hexamethylene-biguanide) hydrochloride, located in the docket. 

Endocrine Disruptor Screening Program

As required by FIFRA and FFDCA, EPA reviews numerous studies to assess potential adverse 

outcomes from exposure to chemicals. Collectively, these studies include acute, subchronic and 

chronic toxicity, including assessments of carcinogenicity, neurotoxicity, developmental, 

reproductive, and general or systemic toxicity. These studies include endpoints which may be 

susceptible to endocrine influence, including effects on endocrine target organ histopathology, 

organ weights, estrus cyclicity, sexual maturation, fertility, pregnancy rates, reproductive loss, 
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and sex ratios in offspring. For ecological hazard assessments, EPA evaluates acute tests and 

chronic studies that assess growth, developmental and reproductive effects in different 

taxonomic groups. As part of the 2008 exemption from the requirements of a tolerance, EPA 

reviewed these data and selected the most sensitive endpoints for PHMB. However, as required 

by FFDCA section 408(p), PHMB is subject to the endocrine screening part of the Endocrine 

Disruptor Screening Program (EDSP). 

EPA has developed the EDSP to determine whether certain substances (including pesticide 

active and other ingredients) may have an effect in humans or wildlife similar to an effect 

produced by a “naturally occurring estrogen, or other such endocrine effects as the Administrator 

may designate.” The EDSP employs a two-tiered approach to making the statutorily required 

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a 

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

systems. Chemicals that go through Tier 1 screening and are found to have the potential to 

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA 

will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 

testing is designed to identify any adverse endocrine-related effects caused by the substance, and 

establish a dose-response relationship between the dose and the E, A, or T effect. 

Under FFDCA section 408(p), the Agency must screen all pesticide chemicals. Between October 

2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, 

which contains 58 pesticide active ingredients and 9 inert ingredients. PHMB is not among the 

group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. 

Accordingly, as part of registration review, EPA will issue future EDSP orders/data call-ins, 

requiring the submission of EDSP screening assays for PHMB. For further information on the 

status of the EDSP, the policies and procedures, the list of 67 chemicals, future lists, the test 

guidelines and the Tier 1 screening battery, please visit our website: http://www.epa.gov/endo/. 

Timeline

EPA has created the following estimated timeline for the completion of the PHMB registration 

review: 

Anticipated Schedule

Activities Estimated Month/Year

Phase 1: Opening the docket 

Open Public Comment Period for Docket June 2012 - Complete

Close Public Comment Period for Docket August 2012 - Complete

Phase 2: Case Development 

Develop Final Work Plan (FWP) December 2012 - 

Complete 

Issue DCI December 2013 

Data Submission December 2015 

Open Public Comment Period for Preliminary Risk Assessments June 2017 

Close Public Comment Period for Preliminary Risk Assessments July 2017 
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Phase 3: Registration Review Decision 

Open Public Comment Period for Proposed Reg. Review Decision December 2017 

Close Public Comment Period for Proposed Reg. Review Decision February 2018 

Final Decision and Begin Post-Decision Follow-up 2018 

Total (years) 6 

Next Steps

A DCI will be developed regarding the data needs listed under the “Risk Assessments and 

Anticipated Data Needs” section of this document. The Agency expects to issue the DCI in 

December 2013. Human health and environmental fate and ecological hazard assessments will 

be conducted for all uses. 

 
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III. Summary of Anticipated Data Requirements – PHMB 

The table below summarizes anticipated data needs for the Poly(Hexamethylenebiguanide) Case 

in Section I of this document (Preliminary Work Plan), Amended Product Chemistry, 

Environmental Fate, and Ecological Effects Scoping Document in Support of Registration 

Review of Poly(hexamethylene-biguanide) hydrochloride, and Poly(hexamethylene-biguanide) 

hydrochloride. Revised Human Health Scoping Document for the Registration Review. 

Table 2. Summary of Anticipated Data Requirements – PHMB 

Guideline 

Number Anticipated Data Requirement Test Material 

Estimated Timeframe 

(Measured in months 

from DCI Receipt) 

870.3465 90-Day Inhalation Toxicity TGAI:20% & 

96.3%3 24 

870.3100 90-day oral toxicity TGAI: 96.3%3 24 

870.3700 Developmental toxicity TGAI: 96.3%3 24 

870.3250 90-day dermal toxicity TGAI: 96.3%3 24 

870.5375 In vitro mammalian cell assay TGAI: 96.3%3 12 

870.5395 In vivo mammalian cytogenetics TGAI: 96.3%3 12 

870.5550 Unscheduled DNA Synthesis in Mammalian 

Cell Cultures TGAI: 96.3%3

 12 

835.1230 Sediment Sorption/Desorption Study TGAI:20% & 

96.3%4 12 

850.33005 Activated Sludge Respiration Inhibition TGAI: 20% & 

96.3%4 12 

850.2100 Avian acute oral toxicity test, upland game or 

waterfowl species 

TGAI: 20% & 

96.3%4 12 

850.1010 Aquatic invertebrate acute toxicity, 

freshwater daphnids 

TGAI: 20% & 

96.3%4 12 

 

3

 Anticipated 90-day inhalation data are expected to be required on both the 20% and 96.3% technical grade active 

ingredients (TGAI) because available information is unclear on whether or not PHMB – which exists as a mixture of 

polymer chains of different lengths or sizes and mass – will have similar polymer characterization and, therefore, 

toxicity across products. If the Tier I Toxicity database is completed for the 96.3% TGAI, then the 90-day inhalation 

study is anticipated to be required on the more toxic TGAI only. 

4

 Anticipated data are expected to be required on both the 20% and 96.3% technical grade active ingredients (TGAI) 

because available information is unclear on whether or not PHMB – which exists as a mixture of polymer chains of 

different lengths or sizes and mass – will have similar polymer characterization and, therefore, ecological hazard 

across products. If information is provided to the Agency proving that polymer characterization is the same for both 

TGAIs, then data are anticipated to be required on the more toxic TGAI only. 

5

 EPA has a published final guideline for this study: http://www.regulations.gov/#!documentDetail;D=EPA-HQOPPT-2009-0154-0021. The Agency anticipates codifying this study in 40 CFR 158(W) in the near future. OECD 

Test Guideline 209 can also be used as guidance for this study, available online at http://www.oecdilibrary.org/content/book/9789264070080-en . 
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850.1025 Oyster acute toxicity test6 TGAI: 20% & 

96.3%4 12 

850.1055 Bivalve acute toxicity larval (embryo/larval)7 TGAI: 20% & 

96.3%4 12 

850.1075 Fish acute toxicity7 TGAI: 20% & 

96.3%4 12 

850.1300 Daphnid chronic toxicity test TGAI: 20% & 

96.3%4 12 

850.1400 Freshwater Fish early-life stage toxicity test, 

using the most acutely sensitive fish 

TGAI: 20% & 

96.3%4 12 

850.1035 Estuarine/marine mysid acute toxicity test TGAI: 20% & 

96.3%4 12 

850.1350 Estuarine/marine shrimp chronic toxicity8 TGAI: 20% & 

96.3%4 24 

850.1400 Estuarine/marine fish early life stage8 TGAI: 20% & 

96.3%4 24 

850.4100 Seedling Emergence and Seedling Growth TGAI: 20% & 

96.3%4 12 

850.4400 Aquatic plant toxicology test using Lemna 

spp. 

TGAI: 20% & 

96.3%4 12 

850.4500 Algal toxicity TGAI: 20% & 

96.3%4 12 

850.4550Error! 

Bookmark not 

defined.

Cyanobacteria (Anabaena flos-aquae) 

toxicity 

TGAI: 20% & 

96.3%4 12 

Special Study 

Chronic Whole Sediment Freshwater 

Invertebrate Toxicity Test with the amphipod 

Hyallela azteca9,10

TGAI: 20% & 

96.3%4 12 

Special Study Chronic Whole Sediment Freshwater TGAI: 20% & 12 

 

6

 Results from either GLN 850.1025 or 850.1055 are expected to satisfy both anticipated data requirements. 7

 GLN 850.1075 is anticipated to be required for both a freshwater warm water fish and an estuarine/marine fish 

species. 

8 For the least acutely sensitive estuarine/marine species a chronic value may potentially be estimated using an 

acute-to-chronic ratio (ACR) approach. If the ACR derived from the estuarine/marine chronic test conducted for the 

more sensitive estuarine/marine organism (based on the acute results) does not differ from ACRs calculated from 

freshwater species, the chronic test for the less sensitive organism may be estimated using existing ACRs and the 

acute value for the less sensitive species. From the Office of Water (OW) guidance ACRs within a factor of ten are 

considered not to differ significantly

9

 EPA has published Agency-wide guidance: Methods for Measuring the Toxicity and Bioaccumulation of 

Sediment-Associated Contaminants with Freshwater Invertebrates, EPA 600/R-99/064 available on-line at 

http://water.epa.gov/polwaste/sediments/cs/freshfact.cfm. 10 The anticipated DCI will provide that a protocol must be submitted for review and approval by the Agency prior 

to initiating this study. 
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Invertebrate Toxicity Test with the midge 

(Chironomous sp.) 9,10

96.3%4

Special Study 

Chronic Whole Sediment Estuarine/Marine 

Invertebrate Toxicity Test with the amphipod 

Leptochirus plumulosus)

 10,11

TGAI: 20% & 

96.3%4 12 

 

11 EPA has published Agency-wide guidance: Method for Assessing the Chronic Toxicity of Marine and Estuarine 

Sediment-associated Contaminants with the Amphipod Leptocheirus plumulosus EPA 600/R-01/020 available online at http://water.epa.gov/polwaste/sediments/cs/leptofact.cfm.