United States Chemical Safety December 2012 Environmental Protection and Pollution Prevention Agency (7510P)



# Poly(Hexamethylenebiguanide) Registration Review: Final Work Plan December 2012Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov



## Poly(Hexamethylenebiguanide) Registration Review: Final Work Plan December 2012

Case # 3122

Approved By :

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Jennifer McLain Acting Director, Antimicrobials Division

Date:

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2 of13Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

3 of 14 Poly(Hexamethylenebiguanide) Hydroc hloride 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(Hexamethyleneb iguanide) Hydrochloride Case may be found in the registration review docket EPA-HQ-OPP-2012-0341 located on the internet at www.regulations.gov

.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

4 of 14 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 concerni ng 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 wh at EPA knows about the pesticide and what additional risk analyses and da ta or information the Agency believes are needed to make a registration review decision.

The Agency is implementing the registration revi ew 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 th e FIFRA standard for registration. Changes in science, public policy an d pesticide use practices will occur over time. The registration review program is intended to ma ke sure that, as the ability to assess risk evolves and as policies and practi ces change, all registered pestic ides continue to meet that statutory standard. The public pha se 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, an d residential settings and to formulate sanitizers and disinfectants for use in food handling/stor age establishments, premises and equipment, including food contact surfaces. Currently, there are 25 products containing PHMB as an active ingredient registered wi th EPA. PHMB is a broad spectrum an timicrobial registered for use as a fungicide, algicide, and sa nitizer in swimming pools, spas an d 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 micr obicide/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 reside ntial and public access premises, commercial, institutional, industrial premises and equipm ent, 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 commod ities when the residues are the re sult of the lawful application of a food contact surface sani tizer 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 fu ture. 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 outlin ed in Table 13 of the 2004 RED. Labels will be updated through the reregistration process.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

5 of 14 The Poly(hexamethylenebiguanide) Preliminary Work Plan (PWP) was published on July 6, 2012 and the 60-day comment period ended on Se ptember 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 re levant to this registration review case.

Comments Received on the Preliminary Work Plan

In the PWP, the Agency solicited comments on sp ecific topics: environmental justice, water body impairment, trade irritants, and structure act ivity 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 th e Product Chemistry, Environmental Fate, and Ecological Effects Scoping Docume nt in Support of Registration Re view of Poly(hexamethylenebiguanide) hydrochloride located in the docket.

Comments were submitted by the Bay Area Clea n Water Agencies (BACWA); the California Stormwater Quality Association (CASQA); the Tri-TAC (the Technical Advisory Committee for the CASQA, the California Water Environment Asso ciation and the League of California Cities); and the San Francisco Bay Water Quality C ontrol Board (SFBWQB). The comments that resulted in changes to the work plan included th e 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 as sessment. The commenters requested that the Agency include swimming pool discharge, leachi ng 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 differe nces in the manufacturing processes, the two compounds differ in their polymer chain length s and molecular weight. At the time of the initial registration of the 96.3% TGAI, th e 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 fo r 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,Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

6 of 14 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 anticipat ed 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 th e 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 ha s determined that the following additional ecological effects and environmen tal 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 Re spiration Inhibition

 In response to comments received requesti ng 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 Agen cy will also evaluate risks from swimming pool discharges, leaching from use in archit ectural 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 an nounced 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 informa tion see "Final Test Guidelines; OCSPP 850 Series; Notice of Availability" 77 FR 38282, June 27, 2012. http://www.regulations.gov/#!docum entDetail;D=EPA-HQ-OPPT-2009-0154-0028

.

o The Algal Toxicity study (GLN 850.5400) an ticipated 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 Emer gence and Seedling Growth

Additional information on the changes to the ecol ogical and human health risk assessments can be found in the Amended Product Chemistry, Environm ental Fate, and Ecological Effects Scoping Document in Support of Registrati on Review of Poly(hexamethylene-biguanide) hydrochloride and Poly(hexamethylenebiguanide) hydrochloride

. Revised Human Health Scoping Document for the Registration Review , respectively, at regula tions.gov in docket EPAHQ-OPP-2012-0341.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

7 of 14 This document makes final the work plan for the poly(hexamethyleneb iguanide) 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 hea lth risk assessment. The Agency anticipates needing additional data for use in conducting the registration review for PHMB.

Ecological Risk:

 Ecological hazards of PHMB were characte rized as part of the 2004 RED assessment; however, a quantitative risk as sessment 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 PH MB to water through uses in industrial processes and water systems such as cooling towers and paper mill process water as well as indirect discharg e of PHMB via consumer, institutional and commercial product uses, including swimming pools.

 The Agency has not conducted a risk asse ssment that supports a complete endangered species determination for PHMB. The eco logical risk assessment planned during registration review will allow the Agency to determine whether PHMB’s use has 'no effect' or 'may affect' federall y listed threatened or endange red 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 cr itical 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 toxi city test, upland game 850.1010 -- Aquatic invertebrate acute toxicity, freshwater daphnids 850.1025 -- Oyster acute toxicity test 1 850.1055 -- Bivalve acute toxicity larval (embryo/larval) 1 850.1075 -- Fish acute toxicity 2 850.1300 -- 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 sh rimp chronic toxicity

1 Results from either GLN 850.1025 or 850.1055 are exp ected 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.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

8 of 14 850.1400 -- Estuarine/marine fish ea rly 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 fr eshwater invertebrate toxicity test, amphipod ( Hyallela azteca ) Special Study -- Chronic whole sediment fr eshwater invertebrate toxicity test, midge ( Chironomous sp.) Special Study -- Chronic whole sediment es tuarine/marine invertebrate toxicity test, amphipod ( Leptochirus plumulosus )

 The Agency anticipates the need to require the following additional environmental fate data for use in the regi stration review of PHMB.

835.1230 -- Sediment and soil adsorption/desorption 850.6800 -- Activated Sludge Respiration Inhibition

 Data are expected to be required for bot h the 20% and 96.3% t echnical grade active ingredients (TGAI) because available informa tion 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; ther efore, ecological hazard across products. If information is provided to the Agency establishing that polymer characterization is the same for both TGAIs, then data are anticipat ed 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 assessm ent 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 followi ng human health risk assessments based on currently registered us e patterns: dietary (f ood only); residential dermal, inhalation and incidental oral includ ing 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 dri nking water assessment for PHMB because the Agency does not expect PH MB or its degradates to reach or pass through drinking water treatment plants.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

9 of 14  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 invo lving intentional exposure of human subjects to PHMB brought to its attention. If EPA d ecides to rely on any of this study, EPA will ensure that all applicable re gulatory requirements are met, including but not limited to the requirements for EPA ethics re view 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 registra tion 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) b ecause available information is unclear on whether or not PHMB – which exists as a mixt ure 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 da tabase is completed for the 96.3% TGAI, then data are anticipated to be requi red on the more toxic TGAI only.

 For more information, please refer to Poly(hexamethylene-biguanide) hydrochloride. Revised Human Health Scoping Docu ment 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 num erous studies to asse ss potential adverse outcomes from exposure to chemicals. Collectiv ely, these studies includ e acute, subchronic and chronic toxicity, including asse ssments of carcinogenicity, ne urotoxicity, developmental, reproductive, and general or systemic toxicity. These studies include e ndpoints which may be susceptible to endocrine influe nce, including effects on endocri ne target organ histopathology, organ weights, estrus cyclicity, sexual maturatio n, fertility, pregnancy rates, reproductive loss,Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

10 of 14 and sex ratios in offspring. For ecological hazard assessments, EPA evaluates acute tests and chronic studies that assess gr owth, developmental and reprod uctive effects in different taxonomic groups. As part of the 2008 exemption from the requirements of a tolerance, EPA reviewed these data and select ed the most sensitive endpoints for PHMB. However, as required by FFDCA section 408(p), PHMB is subject to the endocrine scr eening part of the Endocrine Disruptor Screening Program (EDSP).

EPA has developed the EDSP to determine whet her 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 ot her such endocrine effect s as the Administrator may designate.” The EDSP employs a two-tiered approach to making th e statutorily required determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where EPA will determine which, if any, of th e Tier 2 tests are necessary base d on the available data. Tier 2 testing is designed to identify a ny adverse endocrine-related eff ects caused by the substance, and establish a dose-response relationship between the dose and the E, A, or T effect.

Under FFDCA section 408(p), the Agency must scre en all pesticide chemicals. Between October 2009 and February 2010, EPA issued test orders/dat a call-ins for the firs t group of 67 chemicals, which contains 58 pesticide activ e ingredients and 9 inert ingred ients. PHMB is not among the group of 58 pesticide active ingredients on the in itial 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 assa ys 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 scr eening battery, please visit our website: http://www.epa.gov/endo/.

Timeline

EPA has created the following estimated timelin e 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 Pre liminary Risk Assessments July 2017Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

11 of 14 Phase 3: Registration Review Decision Open Public Comment Period for Propos ed 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 fa te and ecological hazard assessments will be conducted for all uses.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

12 of 14 III. Summary of Anticipated Data Requirements – PHMB

The table below summarizes anticipated data needs for the Poly(Hexamethylenebiguanide) Case in Section I of this documen t (Preliminary Work Plan), Amended Product Chemistry, Environmental Fate, and Ecological Effects Sc oping Document in Support of Registration Review of Poly(hexamethylene-biguanide) hydrochloride , and Poly(hexamethylene-biguanide) hydrochloride. Revised Human Health Scop ing 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

850.3300 5 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 – w ill 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 gr ade 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 pol ymer characterization and, th erefore, ecological hazard across products. If information is provided to the Agency proving that polymer characteri zation 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 gu idance for this study, available online at http://www.oecdilibrary.org/content/book/9789264070080-en

.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

13 of 14 850.1025 Oyster acute toxicity test 6 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 toxicity 7 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 toxicity 8 TGAI: 20% & 96.3% 4 24

850.1400 Estuarine/marine fish early life stage 8 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

850.4550 Error! 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 azteca 9,10 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 exp ected 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 gu idance: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater In vertebrates, 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.Docket Number: EPA-HQ-OPP-2012-0341 www.regulations.gov

14 of 14 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 As sessing 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

.