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# oP10T-26o5-ooj,3 UNITED STATES ENVIRONMENTAL PROTECTIONAGENCY 

# rhqw . .32Sa'D .~' WASHINGTON, D.C. 20460 RfF CF '-r, - 10 r~ .'-; 

## MAR 2 2 2010

MEMORANDUM 

## 28

OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES

SUBJECT: Biodegradation Study of 1-Chlorododecane (CAS No. 112-52-7) per

HPV-1 Test Rule Found Unacceptable FROM : Catherine Roman, Project Manager C . 1~e-lr-ez. , Chemical Information andTesting Branch TO : Mike Mattheisen, Chief

Chemical Information and Testing Branch r J I received the attached review ofa biodegradation study of 1-chlorododecane ~' . fromBob Boethling of EETD . Bob found that although the study was properly conducted and a test guideline specified by the test rule was used, the study did not provide the needed data because 1-chlorododecane is a volatile chemical and the specified guideline was not appropriate. Also, per Bob's recommendation, EPAshould try to obtain information on "ready" biodegradation (by the closed bottle method) and not "inherent" biodegradation. Although Bob recommended the OECDtest guideline 301D, OPPT cannot require testing by OECDguidelines or the 800series guidelines, and there is no guideline in the CFR . If and when ready biodegradation information is needed in the future for 1-chlorododecane, an appropriate guideline will be selected at that time. Attachment (1) . cc: tierry Brown (7405M)/ Docket No. EPA-HQ-OPPT-2005-0033 Bob Boethling (7406M)

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# CONTAINS NoCBI

Internet Address (URL) e http:llwww .epa.gov Recycled/Recyclable e Printed with VegetableOil Based Inks on 100%Postconsumer, Process Chlorine Free Recycled Paperp. 2 Reviewof TSCA Section 4 Final Study Report CITB Project Manager: X_6~~ Chemical Name: / CAS Registry Number : Report Title: Test Sponsor: Testing Lab: Test Standard: Date Study Received by EPA : Date Study Due to EPA : Special Instructions (e.g., regarding approved protocol modifications, etc.) : Part I Test Report and Study Conduct Enter one ofthe followin Applicable fNAI or Cannot Determine fC] and add comment below, as needed [1p] 1 . The final studyreport indicates evidence ofgross, unauthorized or undocumented deviation from the EPAtest standard requirements, including "shall," "must," or "will" violations. List significant test standard deviations and "shall," "must," or "will" violations in comments below . t] 2. The final study report indicates evidence of deviations fromthe "should" statements in the study protocol that may have a bearing on the study conclusions. List these in comments below . 3 . Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented. ' ' [~ 4 . There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately -followed

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5 . The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. There is evidence that QAprocedures may be inadequate. 6 . There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed. 7. The test, control, or reference substances are different fromthat specified in the section 4 test rule or Enforceable Consent Agreement (ECA) . 8 . Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations fromtemperature, lighting, dosing) are apparent. 9 . The final study report inadequately describes the study method . [D] 10. The final study report inadequately describes the test system . codes into each ofthe checkboxes 1 through 13: A

REVIEW

OF TSCA SECTION4 FINAL STUDY REPORT March 2006 Version] 11 . The final study report inadequately describes the dosage information. 12. The final study report does notaccurately reflect the individual animal data (or that of other discrete observations pertinent to the type of studyconducted), or individual data were not provided. rAl 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used. ' Part II . Concl usions Place a check mark beside the summary statement s) that apply to this reviewand add comm ents below

as appropriate , Part A . To be filled out bV Reviewer Study Conduct 1 . The studywas well-conducted. 2 . The studywas generally well-conducted, with some minor deficiencies (list in notes below) . 3 . The studyconduct was significantly deficient or questionable, (give reasons in notes below) . Data Reliability 4. The study results are precise and accurate. 5. Appropriate methods were used to determine statistical significance. 6. Ifrepeated, the test results would be expected to be the same. Data Utility 7 . The data are useful for risk assessment forthe endpoint tested. [ ] 8 . The data are useful for some other purpose (e.g., dose-setting) . [ ] 9 . The data are not useful for any purpose (describe in notes below) . s omments (conti on separate page, ifneede~d) : ~ . ..

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. Part B . To be filled out by CITB PM Tiered Testing Decision (when applicable) Study Acceptability 

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1 . The tiered testing triggerwas met, additional testing is indicated. 2 . The trigger for additional (tiered) testing was not met. 3. The study is acceptable . 4 . The study may not be acceptable (provide rationale in memo to CITB chief) . 

# 10

REVIEW

OF TSCASECTION 4 FINAL STUDY REPORT March 2006 Version

### Biodegradation study on 1-chlorododecane (112-52-7) Bob Boethling to; Catherine Roman 03/24/2010 11 :53 AM Cc: Mike Mattheisen I realize now

thatwhen filing the sec 4 study report I should have suggested running a different test, not the Closed Bottle test (OECD301 D) . I tend to forget the "Sealed Vessel C02 test" but it was designed and approved for, among other things, volatile chemicals. Best of all, not only is it an OPPTS 835 guideline but also an approved ASTM and ISOtest. Let me know if you need more information. OPPTS835.3140

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# ASTM E1720- 01(2008) ASTNLE172Q- D1(2048) Standard-Test M-ethod-for- - -- - -- Determining Ready, Ultimate, Biodegradability of Organic Chemicals in a Sealed Vessel C02 Production Test Active Standard ASTM F1720Developed by Subcommittee: E47.04jBook of Standards Volume: 11 .06

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# ASTM E1724 Significance and Use As a ready biodegradability test, when using non-adapted inoculum, the sealed vessel method provides only a limited opportunity for biodegradation and acclimatizationto occur. It may therefore--be assumed that a chemicalyielding a-positive-resultinthis-stringent test-will_ __ _ biodegrade rapidly and ultimately in the environment. Generally, no further biodegradability testing would be required for a chemical that passes this test unequivocally. 

### The sealed vessel test isapplicable to the testiiig'of~volatile'test cheiriical's because the biodegradative formatiori :oTCO2 occurs in a closed system . The sealed vessel test is also appropriate for testing sparingly soluble chemicals and for chemicals that bind to inoculum, since biodegradability is based on the analysis ofa soluble formation product rather than on the disappearance of the sparingly soluble substrate. Ample replicate sampling for rate determination or statistical evaluation, or both, is feasible because ofthe speed, economy, and space efficiency ofthe sealed vessel test. Thesealed vessel test is ideal for the comparative testing ofgroups ofchemicals and for generating structure-activity data bases also because ofits speed, economy, and space efficiency. 1 . Scope 1 .1 This test method covers procedures for determining the ready, ultimate, aerobic biodegradability oforganic chemicals by monitoring CO, production in sealed vessels containing the test compound and a dilute sewage inoculum . Because ofthe stringency ofthe test conditions, it can be assumed that a chemical that is 60 % or better biodegraded in this test method will biodegrade in most aerobic environmental compartments. 1 .2 This test method is derived fromthe sealed vessel procedures of Birch (1), Struijs (2), Boatman (3), and Peterson (4), whichwere developed as simpler, more economical alternatives to the CO, production techniques reported by Gledhill (5) and Sturm(6), the Sturmreport being the basis ofthe Modified Sturm Test ofthe Organization for Economic Cooperation andDevelopment (OECD) (7) .

1 .3 The procedures are applicable to pure materials, including sparingly solubles, which can be dissolved or dispersed homogeneously in aqueous stock solutions ofat least 25 ppm ofcarbon, or which can be introduced reproducibly to test bottles as pure test material in 1 to 2-mg portions. The test chemical should be nontoxic to sewage microorganisms at 10 ppm ofcarbon. The test may be applied to volatile materials with Henry's Law Constants of up to approximately 10-z atm/m3/mole. The testing of mixtures, extracts, or fully formulated products can lead to serious problems in data interpretation. 1 .4 The procedures involve incubation ofthe test chemical with a dilute inoculumof microbes fromdomestic wastewater secondary sewage treatment effluent in small, sealed vessels for up to 28 days. Biodegradability is determined by monitoring CO, production as dissolved inorganic carbon (DIC) in the liquid phase, and as gaseous CO Z in the head space. Alternatively, analysis can be performed onjust the liquid phase after the addition ofalkali, or onjust the headspace following acidification. The determinations are made using commercial carbon analyzers based on the IRdetection of CO2 . The determination of CO, production provides unequivocal proofof biodegradation, barring the unlikely event ofabiotic production of CO Z fromthe test material . 1 .5 For water-soluble materials that do not adsorb to glass or biological solids, biodegradation may be confirmed further by monitoring the disappearance ofdissolved organic carbon (DOC) in the liquid phase. 1 .6 Thesimplicity ofthe sealed vessel method permits ample replicate sampling for rate determination or statistical evaluation, or both. 1 .7 For a chemical that fails the test as written, the stringency ofthe test may be reduced by substituting an acclimated inoculumin order to provide a measure of inherent biodegradability. 1 .8 Materials that are toxic to the microbial inoculumat 10 ppm ofcarbon may not be amenable to testing by this test method, or they may require special method modification such as reducing the test concentration if instrumental sensitivity permits. For some cationics, complexing the test material with a nondegradable anionic may reduce toxicity. 1 .9 The values stated in SI units are to be regarded as the standard. 1 .10 This standard does not purport to address all ofthe safety concerns, if any, associated with its use. It is the responsibility ofthe user ofthis standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific precautionary statements, see Section 6 .Index Terms

aerobic; biodegradation; CO, production; ready; sealed vessel ; Aerobic/anaerobic biodegradation; Biodegradation; C02production; Organic chemicals; Sealed vessel ; Ultimate analysis; ICS Number Code 13.080.30(Biological properties of soil) DOI : 10.1520/E1720-01R08 llllllllllllllllllllllllllll ISO 14593 (1999) . Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in an aerobic medium-method by analysis of inorganic carbon in sealed vessels (C02 headspace test) International Organization for Standardization, Geneva. Robert Boethling, PhD ExposureAssessment BranchUS EPA, Office of Pollution Prevention and Toxics 1200 Pennsylvania Ave, NW (7406M) Washington, DC20460 202-564-8533 (P) 202-564-8892 (F) [EPI Suite] http://www

.epa.gov/opptintrlexposure/pubslepisuite .htm [FatePointer] httpa/esc.syrres .com/fatepointer/search.aspUnited States Prevention, Pesticides EPA 712-C-08-001 Environmental Protection And Toxic Substances October 2008 Agency (7101) 

# ~- .EPA Fate, Transport and Transformation Test Guidelines OPPTS 835. 3140 Ready Biodegradability -C02 in Sealed Vessels (Headspace Test)

### INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data to meet the data requirements of the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a) . OPPTS developed this guideline through a process of harmonization of the testing guidance and requirements that existed for the Office of Pollution Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in 

## p -t; 0 r -h-ni- - --mifon rvice ublica jons- f-the Natiotia Tec icalInfor i---S-e-- -(NTISYand-in-the--- guidelines published by the Organization for Economic Cooperation and Development (OECD) . For additional information about OPPTS harmonized guidelines and to access this and other guidelines, please go to http://www .epa .gov/oppts and select "Test Methods &Guidelines" on the left side menu.OPPTS 835.3140 : Ready biodegradability-C02 in sealed vessels (Headspace Test) (a) Scope--{1) Applicability . This guideline is intended for use in testing pursuant to the Toxic Substances Control Act (TSCA) (15) U.S.C. 2601) . (2) Background. This OPPTS test guideline is based on the OECD Guideline for the Testing of Chemicals no. 310, Ready Biodegradability - COZ in Sealed Vessels (Headspace Test) (adopted 23 March 2006), available fromsource OECD at http://masetto.sourceoecd .or;g and U.S. EPA Fate, Transport and Transformation Test Guidelines no. 835.3120, Sealed-Vessel COz Production Test (Jan. 1998), available fromhttp ://www .epa.gov/opptsfrs/home/guidelin.htm . (b) Purpose

. This guideline is a screening method for the evaluation of ready biodegradability of chemical substances and provides similar information to that fromthe six test methods described in OECDTest Guideline 301 Ato F (see paragraph (o)(1) of this guideline) . Therefore, a chemical substance that shows positive results in this guideline can be considered readily biodegradable and consequently rapidly degradable in the environment. (c) General considerations. (1) The well-established OECD carbon dioxide (COz) method (i .e. 30113) (see paragraph (o)(1) of this guideline), based on Sturm's original test (see paragraph (o)(2) of this guideline) for assessing biodegradability of organic chemicals, by the measurement of the COZ produced by microbial action, has normally been the first choice for testing poorly soluble chemicals and those that strongly adsorb. It is also chosen for soluble (but not volatile) chemicals, since the evolution of COZ is considered by many to be the only unequivocal proof of microbial activity. Removal of DOC can be effected by physical or chemical processes - adsorption, volatilization, precipitation, and hydrolysis - as well as by microbial action, and many nonbiological reactions consume oxygen. However, rarely is COZ produced from organic chemicals abiotically. In the original and modified Sturm test, COZ is removed from the liquid phase to the absorbing vessels by sparging (i .e. bubbling air treated to remove COz through the liquid medium), while in the version of Larson (see paragraphs (o)(3) and (o)(4) of this guideline), COZ is transferred from the reaction vessel to the absorbers by passing C02-free air through the headspace and, additionally, by shaking the test vessel continuously. Only in the Larson modification is the reaction vessel shaken; stirring is specified only for insoluble substances in the ISO version (see paragraph (o)(5) of this guideline) and in the original US version (see paragraph (o)(6) of this guideline), both of which specify sparging rather than headspace replacement . In another official US EPA method (see paragraph (o)(7)) of this guideline based on Gledhill's method (see paragraph (o)(8) of this guideline), the shaken reaction vessel is closed to the atmosphere and C02 produced is collected in an internal alkaline trap directly fromthe gaseousphase, as in classical WarburgBarcroft respirometer flasks. (2) However, IC has been shown to accumulate in the mediumduring the application ofthe modified Sturmtest (see paragraph (o)(9) ofthis guideline) . A concentration of IC as high as 8 mg/L was found during the degradation of 20 mg C/L ofaniline. Thus, the collection of COZ in the alkaline traps did not give a true reflection ofthe amount of C02produced microbiologically at intermediate times during the degradation. As a result, the specification that >60%of ThCOz should be collected within a 10-d window for a test substance to be classified as readily biodegradedwill not be met for some substances that would be so classified using DOCremoval . (3) When the percentage degradation is a lower value than expected, IC -has-pcrssibly-accumulated--in-the-test-solution:-Then,th-e- -degradability -may- be assessed with the other OECD ready biodegradability tests. (4) Other drawbacks of the Sturmmethodology (cumbersome test flasks; time-consuming; more prone to experimental error; applicable to volatile substances) had earlier prompted a search for a sealed vessel technique, other than Gledhill's, rather than one based on gas flow-through (see paragraphs (o)(10) and (o)(11) ofthis guideline) . Boatman et al . (see paragraph (o)(12) ofthis guideline) reviewed the earlier methods and adopted an enclosed headspace systemin which the COz was released into the headspace at the end of incubation by acidifying the medium . COZ was measured by gas chromatography (GC)/IC analysis in automatically taken samples of the headspace, but DICin the liquid phasewas not taken into account. Also, the vessels used were very small (20 ml), containing only 10 ml of medium, which caused problems e.g. when adding the necessarily very small amounts of insoluble test substances. Another shortcoming is that due to the amount of inoculated test medium, there may in some cases be insufficient or no microorganisms present in the inoculated mediumthat are competent to degrade a given test substance. (5) These difficulties have been overcome as a results of the independent studies of Struijs and Stoltenkamp (see paragraph (o)(13)) and of Birch and Fletcher (see paragraph (o)(14) of this guideline) . In the former study COZ was measured in the headspace after acidification and equilibration, while in the latter, DICin both the gaseous and liquid phases was measured, without treatment; over 90% of the IC formed was present in the liquid phase. Both methods have advantages over the Sturm test in that the test system is more compact and manageable, volatile chemicals can be tested, and the possibility of delay in measuring COz produced is avoided. (6) The two approaches were combined in the ISO Headspace COZ Standard (see paragraph (o)(15) of this guideline), which was ring-tested (seeparagraph (o)(16) of this guideline) and it is this standard that forms the basis of the present guideline. Two methods of measuring C02 have been recommended; namely, COZ in headspace after acidification, and IC in the liquid phase after the addition of excess alkali . The latter method was introduced during the CONCAWE ring test of this headspace method modified to measure inherent biodegradability (see paragraph (n)(17) of this guideline) . The changes made in the 1992 revision of OECD Guidelines for Ready Biodegradability have been incorporated into this guideline (see paragraph (o)(11) of this guideline), so that the conditions (medium, duration, etc.) are otherwise the same as those in the revised Sturmtest. Birch and Fletcher (see paragraph (o)(14) of this guideline) obtained very similar results with this headspace test as were obtained with the same chemicals in the 1988 OECD ring test of the revised ready biodegradability test methods (see paragraph (o)(18) ofthis guideline) . (d) Definitions. DIC : Dissolved inorganic carbon DOC : Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0.45 micrometer filter or remains in the supernatant after centrifuging at approx. 4000 g (about 40.000 m sec-z) for 15 min. IC

: Inorganic carbon Inherent Biodegradability: Aclassification ofchemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any test of biodegradability. Lag Phase: The time fromthe start of a test until acclimatization and/or adaptation of the degrading microorganisms is achieved and the biodegradation degree of a chemical substance or organic matter has increased to a detectable level (e.g. 10%of the maximum theoretical biodegradation, or lower, dependent on the accuracy of the measuring technique) . Mineralization: Mineralization is the complete degradation of an organic compound to COz and H20 under aerobic conditions, and CH4, COZ and Hz0 under anaerobic conditions. Plateau phase: Plateau phase is the phase in which the maximal degradation has been reached and the biodegradation curve has leveled out. Readily Biodegradable: An arbitrary classification of chemicals which have passed certain specified screening tests for ultimate biodegradability ; thesetests are so stringent that it is assumed that such substances will rapidly and completely biodegrade in aquatic environments under aerobic conditions. ThC0z: Theoretical carbon dioxide (mg) is the quantity ofcarbon dioxide calculated to be produced fromthe known or measured carbon content of the test substance when fully mineralized; also expressed as mg carbon dioxide evolved per mg test substance. ThIC : Theoretical inorganic carbon TIC : Total inorganic carbon Ultimate Aerobic Biodegradation: The level ofdegradation achieved when --the -test- -substance-- is -totally- utilized- --by -micro=organisms-resulting- in-the-

-- production of carbon dioxide, water, mineral salts and newmicrobial cellular constituents (biomass) . 10-d window

: The 10 days immediately following the attainment of 10% biodegradation. (e) Principle ofthe test. (1) The test substance, normally at 20mg C/L, as the sole source of carbon and energy, is incubated in a buffer-mineral salts medium that has been inoculated with a mixed population of microorganisms fromsewage treatment effluent obtained froma local domestic sewage treatment plant. The test is performed using sealed bottles with a headspace of air, which provides a reservoir of oxygen for aerobic biodegradation. The COZ evolution resulting from the ultimate aerobic biodegradation of the test substance is determined by measuring the IC produced in the test bottles in excess of that produced in blank vessels containing inoculated medium only. The extent of biodegradation is expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of test substance (as organic carbon) added initially. (2) The DOC removal and/or the extent of primary biodegradation of the test substance can also be measured for water soluble materials that do not adsorb to glass or biological solids. (f) Applicable ASTM standards. Refer to the documents referenced in paragraph (o)(19) of this guideline for the standards in paragraphs (f)(1) through (f)(8) of this guideline: (1) D1129-90

: Standard Terminology Relating to Water. (2) D1193-91 : Standard Specifications for Reagent Water (Federal TestMethod and Standard No. 7916) . (3) D1293-84: Standard Test Methods for pHof Water. (4) D2579-85: Standard Test Method for Total and Organic Carbon in Water. (5) D2777: Standard Practice for Determination of Precision and Bias of Applicable Methods of Committee D-19 on Water. (6) D4375-90: Standard Terminology for Basic Statistics in Committee D-19on Water. (7) D4839-88

: Standard Test Method forTotal Organic-Carbon in Water by Ultraviolet, or Persulfate Oxidation or Both, and Infrared Detection. (8) E178-80

: Standard Practice for Dealing with Outlying Observations. (g) Information on the test substance. The organic carbon content (% w/w) of the test substance should be known, either fromits chemical structure or by measurement, so that the percentage degradation may be calculated. For volatile test substances, a measured or calculated Henry's lawconstant is helpful for determining a suitable headspace-to-liquid volume ratio. Information on the toxicity of the test substance to microorganisms such as froman Activated Sludge Respiration Inhibition test (OECD 209; see paragraph (o)(20) of this guideline), is useful in selecting an appropriate test concentration and for interpreting results showing poor biodegradability. It is also recommended to include the inhibition control unless it is known that the test substance is not inhibitory to microbial activities (see paragraph (1)(9) of this guideline) . (h) Applicability of the method. The test is applicable to insoluble as well as water-soluble test substances, but good dispersion of the substance should be ensured. Using the recommended headspace-to-liquid volume ratio of 1 :2, volatile substances with a Henry's lawconstant of up to 50 Pa.m3.mol"1 can be tested, as the proportion of test substance in the headspace will not exceed 1 % (see paragraph (o)(13) of this guideline) . A smaller headspace volume may be used when testing substances that are more volatile, but their bioavailability may be limiting especially if they are poorly soluble in water. However, users should ensure that the headspace-to-liquid volume ratio and the test substance concentration are such that sufficient oxygen is available to allow complete aerobic biodegradation to occur (e.g. avoid using a high test substance concentration and a small headspace volume)

. Guidance on this can be found in references cited in paragraphs (o)(13) and (o)(21) of this guideline.(i) Reference substances. In order to check the test procedure, a reference substance of known biodegradability should be tested in parallel . For this purpose, aniline, sodium benzoate or ethylene glycol may be used when testing watersoluble test substances, and 1-octanol for poorly soluble test substances (see paragraph (o)(13) of this guideline) . Biodegradation of these substances must reach >60%

ThICwithin 14days. (j) Reproducibility. (1) In the ISOring test ofthe method (see paragraph (o)(16) of this guideline), the results in Table 1 were obtained using the recommended conditions, including 20mg test substance as C/L.

Table 1 . Summary of results fromISOring test Test -Mean-Percent-age ofvariation Number of Substance Biod(gg jation N Laboratories Aniline 90 16 17 1-Octanol 85 12 I 14 Within-test variability (replicability), using aniline, was lowwith coefficients of variability not greater than 5%in nearly all test runs. In the two cases in which the replicability was worse, the greater variability was probably due to high IC production in the blanks. Replicability was worse with 1-octanol, but still less than 10%

for 79% oftest runs. This greater within-test variability may have been due to dosing errors, as a small volume (3 to 4 pl) of 1-octanol had to be injected into sealed test bottles. Higher coefficients ofvariation would result when lower concentrations oftest substance are used, especially at concentrations lower than 10 mg ClL. This could be partially overcome by reducing the concentration of total inorganic carbon (TIC) in the inoculum . see paragraph o 16 of this guideline)

(2) In an EU ring-test (see paragraph (o)(22) of this guideline) of five surfactants added at 10 mg C/L, the results in Table 2were obtained.Table 2 . Summary of results fromEU ring test

Mean Percentage Coefficient of Test Substance :1 Biodegradation variation Number of (28d) (%) laboratories etrapropylenebenzenesulphonat 17 45 10 Di-isooctylsulfosuccinate 72 22 9 Hexadecyltrimethylammonium --- -- --- --

c-hToricTe---- -- ---- 75--- - - --- - 13-- - -- Isononylphenolethoxylate)

(NPE9) 41 32 10 Cocoamidopropyl 60 23 11 dimethylhydroxysulfobetaine SiOz was added to neutralize toxicity . The results show that generally, the variability was higher for the less well-degraded surfactants. Within-test variability was less than 15%for over 90% of cases, the highest reaching 30-40% . NOTE: Most surfactants are not single molecular species but are mixtures of isomers, homologues, etc. which degrade after different characteristic lag periods and at different kinetic rates, resulting in blurred, extenuated curves, so that the 60%pass value may not be reached within the 10-d window, even though each individual molecular species would reach >60% within 10 days if tested alone. This may be observed with other complex mixtures as well . See the referencecited in paragraph (o)(23) .ofthis guideline. (k) Safety precautions. (1) This method involves the use of nonchlorinated sewage treatment plant effluent. Consequently, individuals performing this test may be exposed to microorganisms that are dangerous to human health. Disposable latex gloves and laboratory eyewear with splash guards should be worn during procedures involving the handling ofeffluent. When large volumes of effluent are being handled, for example during filtering and sparging operations, a dust/mist respirator and laboratory footwear should also be worn. (2) Individuals who work with sewage microorganisms may want to keep current with immunizations for polio, typhoid, hepatitis B, and tetanus.

see ara ra h o 22 of this uideline

(3) Test media should be treated with 5 percent chlorine bleach prior to disposal .(1) Description of the method--(1) Apparatus . (i) Glass serumbottles sealed with butyl rubber stoppers and crimp-on aluminum seals. The recommended size is `125 ml' which have a total volume of approximately 160 ml (In this case the volume of each bottle should be known to be 160 ± 1 ml) . A smaller size of vessel may be used when the results fulfill the conditions described in paragraph (n)(5) ofthis guideline) ; (ii) Carbon analyzer or other instrument (e.g. gas chromatograph) for measuring inorganic carbon. For analysis of carbon in the test medium (liquid phase), the analyzer should be capable of measuring DIC and DOC in aqueous. media over the range of 0 to 20 mg C/L (a suitable instrument is an Ol Corporation model 700 TOC analyzer, or equivalent) . For analysis of COZ in

--- headspacagas,-th"nalyzer-shoul-d-be-capafrle-of measuring-C-Oz-ovetthe-range-of- 0to 1 mg C(a suitable instrument is an Ionics model 1555b TOC analyzer with Horiba model PIR2000 NDIRCOz detector, orequivalent) . (iii) Syringes- A gas-tight, 1,000 mL cemented-needle syringe with 22° beveled, bent point, for piercing butyl rubber or neoprene septa and injecting gasphase samples into the gas-phase analyzer (for example, Hamilton no. 1001 with no. 81317 tip, or equivalent) . A spring-loaded syringe with square end, for injecting liquid samples into Ionics-type analyzers (or equivalent), if used. (iv) Filter apparatus - Filter flask, 2- or 3-L capacity, 20-cm Buchner funnel, 18.5-cmcoarse filter paper (Whatman no. 41, or equivalent), and vacuum source, for filtering sewage treatment effluent inoculum . (v) Orbital shaker in a temperature-controlled environment; (vi) Asupply of CO2-free air - this can be prepared by passing air through soda lime granules or by using an 80% NZ / 20%

OZ gas mixture (optional) ; (vii) Organic carbon analyze (optional) . (viii) pH meter (ix) Flasks/bottles - Three 100-mL volumetric flasks and one 1,000-mL volumetric flask for preparing mineral nutrient medium stock solutions. One 2,000-mL volumetric flask for each test compound, for preparing test compound stock solutions. For water-soluble test compounds that do not precipitate in the presence of the mineral nutrient test medium, more concentrated stock solutions may be used. In this case smaller volumetric flasks are suitable. Glass bottles or flasks, 6-L capacity, for preparation of mineral nutrient medium .(x) Magnetic stirrers for media and sample preparation. (xi) Automated pipetting device for delivering variable volumes of liquid up to 100 mL with an accuracy off 1 percent (for example, EM Science Optifix, or equivalent)

. (xii) Large laboratory oven, for drying glassware after cleaning. (xiii) Ultrasonic processor (sonicator) , for dispersing sparingly soluble test compounds, if needed. (2) Reagents. Useanalytical grade reagents throughout. - ---- ----- -- --- -- ---- (3) Water. (i) Distilled or-de--nized w- -- --- --ater----shoul-----db-- -- used-- --dco -nt -- .ai- --ioning <1 mg/L as total organic carbon. This represents <5%of the initial organic carbon content introduced by the recommended dose ofthe test substance. (ii) Deionized or distilled water should be free of calcium and toxic substances, especially metals such as copper. It may be desirable to saturate the water with oxygen before initiating the experiment by sparging for 20 min. with clean, filtered compressed air. (4) Preparation of mineral nutrient medium-(i) Stock solutions. The stock solutions and the mineral salts mediumare similar to those in ISO 14593 (see paragraph (o)(15) of this guideline) and OECD 301 ready biodegradability tests (see paragraph (o)(1) of this guideline) . The use of a higher concentration of ammoniumchloride (2.0 g/L instead of 0.5 g/L) should only be necessary in very exceptional cases, e.g. when the test substance concentration is >40 mg C/L. Stock solutions should be stored under refrigeration and disposed of after six months, or earlier if there is evidence of precipitation or microbial growth. Prepare the stock solutions in paragraphs (1)(4)(i)(A) through (1)(4)(i)(D) of this guideline: (A) Potassiumdihydrogen phosphate (KHZPO4) . . . . . .. . . . . .. . . . , . . . . g,50g Dipotassiumhydrogen phosphate (KZHP04) . . . .. . . . . .. . . . . . , . . , . 21 .75g Disodiumhydrogen phosphate dehydrate (Na2HP04.2Hz0) . . .. . . . . . . . . .. . . . . . 33.40g Ammoniumchloride (NH4C1) . . . .. . . . . . . .. . . . . . . . .. . . . . . . .. . . . . .. .. .. . . . . . .. . . . . . . . . .. . . . . . . .. . . . 0.50g Dissolve in water and make up to I liter. The pHof this solution should be 7.4 (f 0 .2) . If this is not the case, then prepare a new solution. (B) Calciumchloride dihydrate . . . .. . . . . . .. . . . . ., . . , ., (CaC12.2H20) 36.40g Dissolve in water and make up to 1 liter. (C) Magnesium

sulfate heptahydrate. . . .. .. . . . . . . .. . . .. . . . ,.. (MgS04.7H20) 22.50gDissolve in water and make up to 1 liter. (D) Iron (III) chloride hexahydrate. . ... . .. . . .. . . .. . .. . . . .. .. . . . .. .. . . .. .. .. . . . (FeC13.6Hz0) 0 .25g Dissolve in water and make up to 1 liter and add one drop of concentrated hydrolchloric acid. (ii) Preparation of medium . Mix 10 ml of solution (A) with approximately 800 ml water (see paragraph (1)(3) of this guideline), then add 1 ml ofsolutions (B), (C) and (D) and add water to 1 liter. (5) Other reagents. (i) Concentrated orthophosphoric acid (H3P04) (>85%mass per volume) .

- ---- Oi)-C02=free--campressed-air-or-nitrogeir-for-sparging--the-inacuhurr- to---- - -- - - ensure that it is free ofdissolved CO . The delivery line should be equipped with a large gas diffusing stone (Fisher no. 11-139A or equivalent) for maximum sparging efficiency. (iii) Calibration gas for headspace analysis should be of certified standard grade and contain no more than approximately 0 .25 percent COZ by volume, with the balance being nitrogen. (6) Sodiumhydroxide solution 7M . Dissolve 280g ofsodiumhydroxide (NaOH) in 1 liter of water (see paragraph (1)(3) of this guideline) . Determine the concentration of DICofthis solution and consider this value when calculating the test result (see paragraphs (m)(7)(i) and (n)(3) of this guideline), especially in the light ofthe validity criterion in paragraph (n)(5)(i)(B) ofthis guideline. Prepare a fresh solution ifthe concentration of DICis too high. (7) Test substance. (i) Prepare a stock solution of a sufficiently watersoluble test substance in water (see paragraph (1)(3) of this guideline) or in the test medium(see paragraph (1)(4) of this guideline) at a concentration preferably 100- fold greater than the final concentration to be used in the test. It may be necessary to adjust the pH of the stock solution to 7.2 f 0.2 with HCl or NaOH . The stock solution should be added to the mineral mediumto give a final organic carbon concentration of between 2 and 40 mg C/L, preferably 20 mg/C/L. If concentrations lower than these are used, the precision obtained may be impaired. Soluble and insoluble liquid substances may be added to the vessels directly using high precision syringes. Test compounds known to be toxic to sludge microorganisms at 10 mg C/L may be tested at lower initial concentrations down to a minimum level of 2 to 5 mg C/L, the actual value depending on the sensitivity of the carbon analyzer. Poorly soluble and insoluble test substances may require special treatment (see paragraph (o)(24) of this guideline) . The choices are: 10(A) Direct addition of known weighed amounts ; (B) Ultrasonic dispersion before addition; (C) Dispersion with the aid of emulsifying agents to be required to establish whether they have any inhibitory or stimulatory effects on microbial activity before addition; (D) Adsorption of liquid test substances, or a solution in a suitable volatile solvent, on to an inert medium or support (e.g. glass fiber filter), followed by evaporation of the solvent, if used, and direct addition of known amounts; (E) Addition of known volume of a solution of the test substance in an easily volatile solvent to an empty test vessel, followed by evaporation of the solvent . (ii) Any solvents or dispersing agents should be tested for any stimulatory or inhibitory effect on microbial activity (see paragraph (m)(3)(ii)(B) of this guideline)

. (8) Reference substance. (i) Prepare a stock solution of the (soluble) reference substance in water (see paragraph (l)(3) of this guideline) at a concentration preferably 100-fold greater than the final concentration to be used (20 mg C/L) in the test. (ii) Suitable substances are aniline (freshly distilled) and phthalic or trimellitic acid, but sodiumacetate, dextrose (glucose), and sodiumbenzoate may be too biodegradable to be useful for this purpose. (iii) For test compounds with very lowor negligible water solubility, it may be desirable to employ reference compounds of similar solubility; for example, sodiumstearate. (9) Inhibition check. Test compounds frequently show no significant degradation under the conditions used in ready biodegradation tests. One possible cause is that the test substance is inhibitory to the inoculumat the concentration at which it is applied. An inhibition check may be included in the test design to facilitate identification (in retrospect) of inhibition as a possible cause or contributory factor. Alternatively, the inhibition check may rule out such interferences and showthat zero or slight degradation is attributable solely to nonsusceptibility to microbial attack under the conditions of the test. In order to obtain information on the toxicity of the test substance to (aerobic) microorganisms, prepare a solution in the test mediumcontaining both test substance 11and reference substance, each at the same concentration as when present

individually. (10) Inoculum

. (i) The inoculum may be derived from a variety of sources: activated sludge; sewage effluent (non-chlorinated) ; surface waters and soils; or a mixture of these (see paragraph (o)(1) of this guideline) . The biodegradative activity of the source should be checked by using a reference substance. Whatever the source, microorganisms previously exposed to the test substance should not be used if the procedure is to be used as a test for ready biodegradability. See also the precautions in paragraph (k) . (ii) Based on experience, the optimal volume for the inoculum is that which: (A) Is sufficient to give adequate biodegradative activity; (B) Degrades the reference substance by the stipulated percentage (see paragraph (n)(5)(i) ofthis guideline) ; (C) Gives 102 to 105 colony-forming units per milliliter in the final mixture; (D) Normally gives a concentration of 4 mg/L suspended solids in the final mixture when activated sludge is used. Concentrations up to 30mg/L may be used but may significantly increase COZ production of the blanks (see paragraph (o)(19) ofthis guideline) ; (E) Contributes less than 10% of the initial concentration of organic carbon introduced by the test substance; (F) Is generally 1-10 ml of inoculumfor 1 liter oftest medium . (iii) Inoculummay be preconditioned to the experimental conditions, in order to reduce the blank COZ evolution. Preconditioning consists of aerating with moist C02-free air for up to 5-7 days at the test temperature, after diluting in test medium, prior to starting the test. (11) Activated sludge. (i) Activated sludge should be freshly collected on the day of the experiment fromthe aeration tank of a sewage treatment plant treating predominantly domestic sewage, or laboratory-scale unit treating predominantly domestic sewa~e. If necessary, coarse particles should be removed by sieving (e.g. using a 1 mmmesh sieve) and the sludge should be kept aerobic until it is used. 12(ii) Alternatively, after removal ofany coarse particles, settle or centrifuge (e.g. 1100 x g for 10 minutes) . Discard the supernatant liquid. The sludge may be washed in the mineral solution. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g suspended solids/L. Thereafter aerate until it is used. (iii) Sludge should be taken from a properly working conventional (i .e. activated sludge process) treatment plant. If sludge has to be taken froma high rate treatment plant, or is thoughtto contain inhibitors, it should be washed. Settle or centrifuge the resuspended sludge after thorough mixing, discard the supernatant liquid, and again suspend the washed sludge in a further volume of mineral medium

. Repeat this procedure until the sludge is considered to be free fromexcess substrate or inhibitors. (iv) After complete resuspension is achieved, or with untreated sludge, withdrawa sample just before use for the determination of the dry weight of the suspended solids. (v) Another alternative is to homogenize activated sludge (3-5 g suspended solids/L) . Treat the sludge in a Waring blender for 2 minutes at mediumspeed. Settle the blended sludge for 30 minutes or longer if required and decant liquid for use as inoculumat the rate ofabout 10 mg/L of mineral medium . (vi) Still further reduction of the blank COz evolution can be achieved by aerating the sludge overnight with C02-free air. Use 4 mg/L activated sludge solids as the concentration of the inoculum in the test mediumwhen the test is started (see paragraph (o)(13) ofthis guideline) . (12) Secondary sewage effluent . (i) Alternatively, the inoculumcan be derived fromthe secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. The undiluted effluent should contain approximately 1 x 106 microorganisms per liter. Maintain the sample under aerobic conditions and use on the day of collection, or precondition if necessary. The effluent should be filtered through a coarse filter to remove gross particulate matter and the pH is measured. (ii) To reduce its IC content, the filtrate should be sparged with C02-free air for 1 h while maintaining the pH at 6.5 using orthophosphoric acid. The pH is restored to its original value with sodiumhydroxide (see paragraph (1)(6) of this guideline) and after settling for about 1 h a suitable volume of the supernatant is taken for inoculation. This sparging procedure reduces the IC content of the inoculum

. For example, when the maximum recommended volume of filtered sparged effluent (100 ml) per liter was used as inoculum, the amount of IC present in blank control vessels was in the range 0.4 to 1 .3 mg/L (see paragraph 13(o)(14) of this guideline), representing 2-6.5%of test substance C at 20 mg C/L and 4-13%

at 10 mg C/L. (13) Surfacewaters. Asample is taken of an appropriate surface water. It should be kept under aerobic conditions and used on the day of collection. The sample should be concentrated, if necessary, by filtration or centrifugation. The volume of inoculumto be used in each test vessel should meet the criteria given in paragraph (1)(10)(ii) ofthis guideline. (14) Soils. Asample is taken ofan appropriate soil, collected to a depth of up to 20cm

below the soil surface. Stones, plant remains and invertebrates should be removed fromthe sample of soil before it is sieved through a 2 mm mesh (if the sample is too wet to sieve immediately, then partially air dry to facilitate sieving) : It--should-be-kept--under- aerobiE-c-onditions-and-used--on--the- day -ofcollection. Ifthe sample is transported in a loosely tied black polythene bag, it can be stored at 2 to 4°C in the bagfor up to one month. (m) Test procedure-(1) Number of test vessels. (i) The number of vessels needed for a test will depend on the frequency of analysis and the test duration. (ii) It is recommended that triplicate vessels be analyzed after a sufficient number of time intervals that the 10-d windowmay be identified. In addition, at least five test vessels from the test substance, reference substance and blank control series (see paragraph (m)(3)(ii) of this guideline should analyzed at the end ofthe test, to enable 95%confidence intervals to be calculated for the mean percentage biodegradation value. (2) Inoculated medium . Prepare immediately before use sufficient inoculated mediumby adding, for example, 2 ml suitably treated activated sludge at 2000 mg/L to 1 liter of mineral nutrient medium (paragraph (1)(4) of this guideline) . When secondary sewage effluent is to be used, add up to 100 ml effluent to 900 ml mineral salts mediumand dilute to 1 liter with medium . (3) Preparation of vessels. (i) Aliquots of inoculated medium are dispensed into replicate vessels to give a headspace to liquid ratio of 1 :2 (e.g. add 107 ml to 160 ml-capacity bottles) . Other ratios may be used, but see the warning given in paragraph (h) . When using either type of inoculum, care should be taken to ensure that the inoculated medium is adequately mixed to ensure that it is uniformly distributed to the test vessels. (ii) Sets of vessels are prepared as listed in paragraphs (m)(3)(ii)(A) through (m)(3)(ii)(E) of this guideline: 14(A) Test vessels (denoted FT) containing the test substance; (B) Blank controls (denoted FB) containing only the test medium plus inoculum ; any chemicals, solvents, agents or glass fiber filters used to introduce the test substance into the test vessels should also be added; (C) Vessels (denoted FC) for checking the procedure using the reference substance; (D) If needed, vessels (denoted F,) for checking a possible inhibitory effect of the test substance containing both the test substance and reference substance at the same concentrations as in bottles FT and Fc, respectively (see paragraph (1)(9) ofthis guideline) ; (E) Vessels (denoted Fs) for checking possible abiotic degradation, set up as for vessels FT but including 50 mg/L HgC12, or sterilized by some other means (e.g. autoclaving) . (iii) Water-soluble test substances and reference substances are added as aqueous stock solutions to give a starting concentration of 10to 20 mg as C/L. (iv) Insoluble test and reference substances may be added to vessels in a variety of ways (see paragraph (1)(7) ofthis guideline) according to the nature of the substance, either before or after addition ofthe inoculated medium, depending on the method of treatment of the substance. If one of the procedures given in paragraph (1)(7) of this guideline is used, then the blank vessels FB should be treated the same as test vessels FT but excluding the test or reference substance. (v) Volatile test substances should be injected into sealed vessels (see paragraph (m)(3)(vii) of this guideline) using a microsyringe. The dose is calculated fromthe volume injected and the density ofthe substance. (vi) Water should be added to vessels, where necessary, to give the same liquid volume in each vessel . The headspace-to-liquid ratio (usually 1 :2) and concentration of the test substance should be such that sufficient oxygen is available in the headspace to allowfor complete biodegradation. (vii) All vessels are then sealed, for example with butyl rubber septa and aluminum caps. Volatile test substances should be added at this stage. If the decrease in DOC concentration of the test solution is to be monitored and for time zero analyses to be performed for initial IC concentration or other determinants, remove an appropriate sample fromthe test vessel . The test vessel and its contents are then discarded. 15(viii) Thesealed vessels are placed on a rotary shaker, with a shaking rate

sufficient to keep the contents well mixed and in suspension (e.g. 150 to 200 rpm), and incubated in the dark at 20°Cf 1°C . (4) Sampling. The pattern of sampling will depend on the lag period and rate of biodegradation ofthe test substance. Vessels are sacrificed for analysis on the day of sampling, which should be at least weekly, or more frequently (e.g. twice per week) ifa complete degradation curve is required. The requisite number of replicate vessels are taken fromthe shaker, representing FT, FB and Fc and, if used, FI and Fs vessels (see paragraph (m)(3) ofthis guideline) . The test normally runs for 28d. Ifthe biodegradation curve indicates that a plateau has been attained before 28d, the test may be concluded earlier than 28d. Take samples fromthe five vessels reserved for the 28th day of the test for analysis, and use the results to -calculate~-the- confidence--limits -or coeffrcient-of-~variatiun-of--pereentagebiodegradation. Vessels representing the checks for inhibition (FI) and for abiotic degradation (Fs) need not be sampled as frequently as the other vessels; sampling at day 1 and day 28 is sufficient. (5) Inorganic carbon (IC) analysis. (i) COZ production in the vessels is determined by measuring the increase in the concentration of IC during the test. There are two recommended methods available for measuring the amount of IC produced in the test, described in paragraphs (m)(6) and (m)(7) ofthis guideline. Since the methods can give slightly different results, only oneshould be used in a given test run. (ii) Method-A (see paragraph (m)(6) ofthis guideline) is recommended if the medium is likely to contain remnants of, for example, a glass-fiber paper and/or insoluble test substance. This analysis can be performed using a gas chromatograph ifa carbon analyzer is not available. It is important that the vessels should be at or close to the test temperature when the headspace gas is analyzed. Method-B (see paragraph (m)(7) of this guideline) can be easier for laboratories using carbon analyzers to measure IC. It is important that the sodiumhydroxide solution (see paragraph (1)(6) ofthis guideline) used to convert COZ to carbonate is either freshly prepared or its IC content is known, so that this can be taken into account when calculating the test results (see paragraph (n)(5)(i)(B) of this guideline. (6) Method-A: acidification to pH<3. (i) Before each batch of analyses, the IC analyzer is calibrated using an appropriate IC standard (e.g. 1% w/w COZ in NZ) . Concentrated orthophosphoric acid is injected through the septumof each vessel that is to be sampled, in order to lower the pH of the mediumto <3 (e.g. add 1 ml to 107 ml test medium) . The vessels are placed back on the shaker. After shaking for one hour at the test temperature, the vessels are removed fromthe shaker, and an aliquot (e.g. 1 ml) of gas is withdrawn fromthe headspace of each 16vessel and injected into the IC analyzer. The measured IC concentrations are recorded as mg C/L. (ii) The principle of this method is that after acidification to pH <3 and equilibration at 20°C, the equilibrium constant for the distribution of COZ between the liquid and gaseous phases in the test vessels is 1 .0 when measured as a concentration (see paragraph (o)(13) of this guideline) . This should be demonstrated forthe test systemat least once as outlined in this paragraph: Set up vessels containing 5 and 10 mg/L as IC using a solution of anhydrous sodium carbonate (NazC03) in C02-free water, prepared by acidifying water to pH 6 .5 with concentrated orthophosphoric acid, sparging overnight with C02-free air, and raising the pH to neutrality with alkali . Ensure that the ratio ofthe headspace volume to the liquid volume is the same as in the tests (e.g. 1 :2) . Acidify and equilibrate asdescribed in-paragraph (m)(6)(i) of this guideLne, and- measurethe IC concentrations of both the headspace and liquid phases. Check that the two concentrations are the same within experimental error. If they are not, the operator should reviewthe procedures. This check on the distribution of IC between liquid and gaseous phases need not be made every time the test is performed. (iii) If DOC removal is to be measured (water-soluble test substances only), samples should be taken of the liquid phase fromseparate (non-acidified) vessels, and the samples membrane-filtered and injected into the DOC analyzer. These bottles can be used for other analyses as necessary, such as to measure primary biodegradation. (7) Method-B: conversion of COZ to carbonate . (i) Before each batch of analyses, the IC analyzer is calibrated using an appropriate standard - for example, a solution of sodium bicarbonate (NaHC03) in C02-free water (see paragraph (m)(6)(ii) ofthis guideline) in the range 0 to 20 mg/L as IC . Sodium hydroxide solution (7M ; see paragraph (1)(6) of this guideline) (e.g. 1 ml to 107 ml medium) is injected through the septumofeach vessel sampled and the vessels are shaken for 1 h at the test temperature. Use the same NaOH solution on all vessels sacrificed on a particular day, but not necessarily on all sampling occasions throughout a test. Ifabsolute blank ICvalues are needed at all sampling occasions, IC determinations of the NaOH solution will be needed each time it is used. The vessels are removed fromthe shaker and allowed to settle. Suitable volumes (e.g. 50 to 1000 pl) of the liquid phase in each vessel are withdrawn by syringe. The samples are injected into the IC analyzer and the concentrations of IC are recorded. It should be ensured that the analyzer used is equipped properly to deal with the alkaline samples produced in this method. (ii) The principle of this method is that after the addition of alkali and shaking, the concentration of IC in the headspace is negligible. This should be checked for the test systemat least once by using IC standards, adding alkali and 17equilibrating, and measuring the concentration of IC in both the headspace and liquid phases. The concentration in the headspace should approach zero. This check need not be made every time the test is performed. (iii) If DOC removal is to be measured (water-soluble test substances only), samples should be taken of the liquid phase from separate vessels (containing no added alkali), and the samples membrane filtered and injected into the DOC analyzer. These vessels can be used for other analyses as necessary, such as to measure primary biodegradation. (n) Data and reporting - (1) Calculations. (i) Assuming 100% mineralization of the test substance to C02, the ThIC in excess ofthat produced in the blank controls equals the TOCadded to each test vessel at the start ofthe test, -- -thati ThIC =TOC . The total mass (mg) of inorganic carbon (TIC) in each vessel is: Equation [1] where : TIC= (mg inorganic Cin the liquid + mg inorganic Cin the headspace)

= (VL X CL) + (VH x CH) VL =volume of liquid in thevessel (liter); CL =concentration of IC in the liquid (mg/L as inorganic carbon) ; VH=volume of the headspace (liter) ; CH =concentration of IC in the headspace (mg/L as inorganic carbon) . The calculations of TICfor the two analytical methods used for measuring IC in this test are described in paragraphs (n)(2) and (n)(3) ofthis guideline. Percentage biodegradation (%D) in each case is given by: Equation [2] %D = (TICt - TICn) x 100 TOC where : TICt = mg TIC in test vessel at time t;

TICb = mean mg TIC in blank vessels at time t; TOC = mg TOC added initially to the test vessel . The percentage biodegradation is calculated for the test (FT), reference (Fc) and, if 18included, inhibition monitoring control (FI) vessels, fromthe respective amounts ofTICproduced up to each sampling time. (ii) Ifthere has been a significant increase in the TICcontent of the sterile controls (Fs) over the test period, then it may be concluded that abiotic degradation of the test substance has occurred and this should be taken into account in the calculation of %Din Equation [2] . (2) Acidification to pH<3. Since acidification to pH <3 and equilibration (Method-A) results in the equalization of the concentration of TIC in the liquid and gaseous phases, only the concentration of IC in the gas phase needs to be measured. Thus, fromEquation [I] TIC = (VL + VH) x CH = VB x CH, where VB = volume of the test vessel . (3) Conversion of COZ to carbonate. In this method (Method-B) calculations are performed as in Equation [1], but the negligible amount of IC in the gaseous phase is ignored, that is VH x CH =0, and TIC =VL x CL. (4) Expression of results. (i) Compile a table of %D for each test (FT), reference (Fc) and, if included, inhibition control vessel (FI) for each day samples. Record the amount of TIC in the blanks (vessels FB) and sterile controls (vessels Fs) . (ii) A biodegradation curve is obtained by plotting %D against time of incubation and if possible, the lag phase, biodegradation phase, 10-d windowand plateau phase, (phase in which the maximal degradation has been reached and the biodegradation curve has leveled out), are indicated. If comparable results are obtained for parallel test vessels FT (<20%difference), a mean curve is plotted (see Fig.l); if not, curves are plotted for each vessel . The mean value of the %D in the plateau phase is determined or the highest value is assessed (e.g. when the curve decreases in the plateau phase), but it is important to assess that in the latter case the value is not an outlier. Indicate this maximum level of biodegradation as degree of biodegradation of the test substance in the test report. If the number of test vessels was insufficient to indicate a plateau phase, the measured data ofthe last day ofthe test are used to calculate a mean value. This last value, the mean of five replicates, serves to indicate the precision with which %D was determined. Also report the value obtained at the end of the 10-d window . (iii) In the same way, plot curves for the reference substance, (vessels FC) the sterile control (vessels Fs) and the inhibition control (vessels FI). (iv) Calculate %D for the FI vessels, based on the theoretical IC yield anticipated fromonly the reference component of the mixture. If, at day 28, 19Figure 1. Biodegradation of 1-octanol in the headspace COZ test a _o

0

0 m 5 10 16 20 25 30 Test time (days) [(DFC - DFI)/DFC] x 100 > 25%, where DFCis the %D in vessels FCcontaining the reference substance and DFI is the %D in Vessels Fl, it may be assumed that the test substance inhibited the activity of the inoculum, and this may account for low values of DFT obtained under the conditions of the test. In this case the test could be repeated using a lower test substance concentration and preferably reducing the DIC in the inoculumand TIC formed in the blank controls, since the lower test substance concentration will otherwise reduce the precision of the method. Alternatively, another inoculum may be used. If in vessels Fs (abiotic check) a significant increase (>10%) in the amount ofTIC is observed, abiotic degradation processes may have occurred. (5) Validity of results. (i) Atest is considered valid if: (A) The mean %D in vessels Fc containing the reference substance is >60%by the 14th day of incubation; and (B) The mean amount ofTIC present in the blank controls FB at the end of the test is <3 mgC/L. (ii) If these limits are not met, the test should be repeated with an inoculumfromanother source and/or the procedures used should be reviewed. For example, if high blank IC production is a problem the procedure given in paragraphs (1)(11) and (1)(12) ofthis guideline should be followed. (iii) If the test substance does not reach 60% ThIC andwas shown not to be inhibitory (paragraph (n)(4)(iv) of this guideline), the test could be repeated with increased concentration of inoculum (up to 30 mg/L activated sludge and 20100 ml effluent/L) or inocula fromother sources, especially if degradation was in the range 20to 60%

. (6) Interpretation of results. (i) Biodegradation >60%ThIC within the 10-d window in this test demonstrates that the test substance is readily biodegradable under aerobic conditions. (ii) Ifthe pass value of 60% ThIC is not attained, determine the pHvalue in media in vessels that have not been made acid or alkaline; a value of less than 6 .5 could indicate that nitrification occurred. In such a case, repeat the test with a buffer solution of higher concentration. (7) Quality assurance. To assure the integrity of data developed using this method and to comply with current regulatory requirements, a quality assurance program meeting EPA, FDA (United States Food and Drug Administration), or OECDguidelines should be followed. (8) Test Report. The test report should include the information in paragraphs (n)(8)(i) through (n)(8)(iii) of this guideline: (i) Test substance: (A) Common name, chemical name, CAS number, structural formula and relevant physical-chemical properties ; (B) Purity (and known impurities) oftest substance. (ii) Test conditions: (A) Reference to this test guideline; (B) Description of the test system(e.g. volume ofthe vessel, head spaceto-liquid ratio, method ofstirring, etc) ; (C) Application of test substance and reference substance to test system : test concentrations used in the test and amount of carbon dosed into each test vessel, any use ofsolvents; (D) Details of the inoculum, including any pretreatment and preconditioning; (E) Incubation temperature; (F) Validation of the principle of IC analysis; 21(G) Main characteristics of the IC analyzer employed (and any other analytical methods used) ; (H) Number of replicates. (iii) Results: (A) Raw data and calculated values of %D in tabular form(see paragraph (n)(4)(i) ofthis guideline. (B) The plots of %D against time for the test and reference substances, including identification of the lag phase, degradation phase, and 10-d window ---(see-paragraph~nX4)(ii)-of-this guidetirro) ;-_--

(C) Percentage removal at plateau, at the end of test, and after the 10-d window ; (D) Reasons for any rejection of the test results; (E) Anyother facts that are relevant to the procedure followed; (F) Discussion of results. (o) References. (1) OECD (1992) . Ready biodegradability, no. 301, adopted 17 July 1992. Organization for Economic Cooperation and Development, Paris. (2) Sturm, R

.N. (1973) . Biodegradability of nonionic surfactants: screening test for predicting rate and ultimate biodegradation. J. A . Oil Chem

. Soc. 50, 159-167. (3) Larson, R

.J. (1979) . Estimation of biodegradation potential of xenobiotic organic chemicals. Appl . Environ. Microbiol . 38, 1153-1161 . (4) Larson, R

.J, M .A. Hansmann, and E.A. Bookland (1996) . Carbon dioxide recovery in ready biodegradability tests: mass transfer and kinetic constants. Chemosphere 33, 1195-1210 . (5) ISO 9439 (1999) . Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test. International Organization for Standardization, Geneva. 22(6) US EPA (1996) . Carbon dioxide evolution test, Fate, Transport and Transformation Test Guidelines 835.3110 . U.S. Environmental Protection Agency, Washington, DC. (7) US EPA (1996) . Aerobic aquatic biodegradation, Fate, Transport and Transformation Test Guidelines 835. 3100 . U.S. Environmental Protection Agency, Washington, DC . - (8) Gledhill, W

.E. (1975) . Screening test for assessment of biodegradability: linear alkyl benzene sulfonate. Appl . Microbiol . 30, 922-929 . (9) Weytjens, D

., I . Van Ginneken and H .A. Painter (1994) . The recovery of carbon dioxide in the Sturmtest for ready biodegradability. Chemosphere 28, 801-812. - (10) Ennis, D

.M . and A. Kramer (1975) . A rapid microtechnique for testing biodegradability of nylons and polyamides. J . Food Sci . 40, 181-185. (11) Ennis, D.M

., A. Kramer, C.W

. Jameson, P.H. Mazzocchi and W .J. Bailey (1978) . Structural factors influencing the biodegradation of imides. Appl . Environ. Microbiol . 35, 51-53. (12) Boatman, R

.J., S.L. Cunninghamand D .A. Ziegler (1986) . Amethod for measuring the biodegradation of organic chemicals. Environ. Toxicol . Chem . 5,233-243. (13) Struijs, J. and J. Stoltenkamp (1990) . Head space determination of evolved carbon dioxide in a biodegradability screening test. Ecotox. Environ. Safe. 19, 204-211 . (14) Birch, R

.R. and R

.J. Fletcher (1991) . The application of dissolved inorganic carbon measurements to the study of aerobic biodegradability. Chemosphere 23, 507-524 . (15) ISO 14593 (1999) . Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in an aerobic medium-method by analysis of inorganic carbon in sealed vessels (COZ headspace test) International Organization for Standardization, Geneva. (16) Battersby, N

.S. (1997) . The ISO headspace COZ biodegradation test. Chemosphere, 34, 1813-1822 . (17) Battersby N

.S., D. Ciccognani, M .R. Evans, D

. King, H

.A. Painter, D .R. Peterson, and M

. Starkey (1999) . An inherent biodegradability test for oil

23products: description and results of an international ring test. Chemosphere 38, 3219-3235. (18) OECD (1988) . OECD Ring-test of methods for determining ready biodegradability: Chairman's report (M . Hashimoto; MITI) and final report (M . Kitano and M

. Takatsuki ; CITI) . Organization for Economic Cooperation and Development, Paris. (19) ASTM(1993) . Annual Book of ASTMStandards, Volumes 11 .01 and 11 .02 on Water and Environmental Technology, and Volume 14.02 on General Methods and Instrumentation. American Society for Testing and Materials, Philadelphia, PA .

- ---- -(20) OEC-D-(1'984) :-A-etivattd-sludge,--respira{ion-inhibitian-test, no.--~09~--- - ---- -- adopted 4April 1984. Organization for Economic Cooperation and Development, Paris. (21) Struijs J., M .J. Stoltenkamp-Wouterse and A .L .M . Dekkers (1995) . A rationale for the appropriate amount of inoculumin ready biodegradability tests. Biodegradation 6, 319-327. (22) WRC (1999) . Ring-test of the ISO headspace COZ method: application to surfactants: surfactant ring test-1, Report EU4697. Water Research Centre, May 1999, Medmenham, UK. (23) Richterich, K

. and J. Steber (2001) . The time-window - an inadequate criterion for the ready biodegradability assessment of technical surfactants. Chemosphere 44, 1649-1654. (24) ISO 10634 (1996) . Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium . International Organization for Standardization, Geneva.p. 34 J~~~ED STqr~s a'~ A '? UNITED STATES ENVIRONMENTAL PROTECTIONAGENCY WASHINGTON, D.C. 20460 RECDVI"



## 19 1 7` 

## MAR 02 2010 " ~ r' r 

## ;1L: 28

OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES Mr. Neal Olsen, Manager, Environmental Affairs Dyno Nobel, Inc. 2650 Decker Lake Blvd., Suite 300 Salt Lake City, Utah 84119 Dear Mr . Olsen, This letter concerns the biodegradation study on pentaerythritol tetranitrate (PETN) (CAS No. 78-11-5) whichyou submitted in response to the TSCA section 4test rule entitled "Testing of Certain High Production Volume Chemicals" (71 FR 13708, March 16, 2006) . As stated in my July 13, 2009, letter, the wrongguideline (ASTM E 1279) was followed in conducting the test for biodegradation instead ofthe guidelines specified by the test rule (ASTM 1625 or ISO9888) . Nevertheless, EPAscientists reviewed the submitted study to see ifit provided the information the Agency was seeking when it required the test. The attached scientific review indicates the study did not provide the needed information on inherent biodegradability that the use ofone ofthe specified guidelines would have provided. I am, therefore, requesting that Dyno Nobel arrange to have the study repeated according to one ofthe specified guidelines, ASTM 1625 or ISO9888. By so doing, youwill avoid being in violation ofthe test rule as stated in 40 CFR 790 .59 (attached) . The scientific reviewalso provides additional advice on how to properly conduct the repeat study. Ifyou have any questions about the scientist's advice, please contact Catherine Roman, the project manager, (roman.catherine(a~,epa.gov, 202-564- 8172) .

Attachments (2) 11is,1'Jirector al Control Division

cc: Gerry Brown (7405M)/ Docket No. EPA-HQ-OPPT-2005-0033 Bob Boethling (7406M) Catherine Roman (7405M)

Internet Address (URL) * http://www .epa.gov Recycled/Recyclable o Printed with Vegetable Oil Based Inks on 100%Postconsumer, Process Chlorine Free Recycled Paperp. 35

C1TB Project Manager: Chemical Name: CAS Registry Number : Report Title: Test Sponsor: Testing Lab: Test Standard: ReviewofTSCA Section 4 Final Studv Report Catherine Roman Pentaerythritol tetranitrate (PETN) ; EPA-HQ-OPPT-2005-0033-0294.2 78-11-5 "Biodegradation Potential ofPETN" 

## DyN- A/0b`/ US Army Center for Health Promotion and Preventive Medicine ASTM E1279-89 Date Study Received by EPA : Date Study Due to EPA : Special Instructions (e.g., regarding approved protocol modifications, etc.): Part I . Test Report and Study Conduct Enter one of the following codes into each of the checkboxes 1 throuah 13: A Applicable [NA], or Cannot Determine [C]and add comment below, as needed [c] [c] [na] [c] [c] [c] [c) [na] 1 . The final study report indicates evidence of gross, unauthorized or undocumented deviation from

the EPAtest standard requirements, including "shall," "must," or "will" violations. List significant test standard deviations and "shall," "must," or "will" violations in comments below . 2 . The final study report indicates evidence of deviations fromthe "should" statements in the study protocol that may have a bearing on the study conclusions. List these in comments below . 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not followed or are inadequately documented. 4 . There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed . 5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or notsigned. There is evidence that QA procedures may be inadequate. 6 . There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed. 7. The test, control, or reference substances are different fromthat specified in the section 4test rule or Enforceable Consent Agreement (ECA) . 8 . Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e .g., deviations fromtemperature, lighting, dosing) are apparent . [ a ] 9 . The final study report inadequately describes the study method. [ a ] 10. The final study report inadequately describes the test system . REVIEW

OF TSCA SECTION4 FINAL STUDY REPORT March 2006 Version[ d ] 11 . The final study report inadequately describes the dosage information. [ na ] 12. The final study report does not accurately reflect the individual animal data (orthat ofother discrete observations pertinent to the type of study conducted), or individual data were not provided. [ d ] 13. The final study report inadequately describes the calculations, interpolations, data transformations, statistical analyses, or modeling parameters used. Part lI . Conclusions Place a check mark beside the summarystatement(s) that apply to this review and add comments below as appropriate Part A . To be filled out by Reviewer Study Conduct 1 . The study was well-conducted. [ ] 2 . The study was generally well-conducted, with some minor [ ] deficiencies (list in notes below) . 3. The study conduct was significantly deficient or questionable, (give [ x ] reasons in notes below) . Data Reliability 4 . The study results are precise and accurate. [ ] 5. Appropriate methods were used to determine [ ] statistical significance. 6. Ifrepeated, the test results would be expected to be the [ ] same. Data Utility 7. The data are useful for risk assessment for the endpoint tested. [ ] 8 . The data are useful for some other purpose (e.g., dose-setting) . [x ] 9 . The data are notuseful forany purpose (describe in notes below) . [ ] Reviewer Comments (continue on separate page, if needed) : On its face, it does appear that the basic conclusion on biodegradability is correct; namely, that PETN biodegraded in one test over aperiod oftime on the order ofdays to weeks. However, the biodegradation study was poorly conceived and conducted . ISO 9888 and ASTM

E1625 are the Zahn-Wellens (Z-W) test and Semi-Continuous Activated Sludge (SCAS) test, respectively. Their objective is to determine ultimate inherent biodegradability. ASTM 1279-89 measures parent chemical loss of identity in natural water samples, which is completely different. Because the test rule objective was aligned with the former, A STM 1279- 89 is not an acceptable s bsti te

The test report reveals significant departure fromthe ASTM standard. For example, in the standard, the "control" consists of site water that is sterilized and amended with test substance; but in the submitted USACHPPM study, the "control" consists of distilled water amended with sterilant and test substance. This could impact interpretation of results because site water should contain many organic substances not present in purified (distilled) water. Interactions among test substance, sterilant and other substances are possible.

Fromthe USACHPPM test reports for biodegradation and water solubility, it appears that achievementofthe expected concentration of test substance in the amended mediarequired many days, and this was not anticipated. This finding complicates interpretation of the test results. Moreover, an unexplained and disturbing finding was that no test substance at all was identified in amended media in two tests that were run prior to the December experiment, which in the end yielded the only presentable data.

The correct response is to start over fromscratch and run another test for biodegradability, but only after properly researching andthen achieving understanding of the behavior ofthis test substance with respect to preparing stock (dosing) solutions, dosing flasks, abiotic losses if any, and possible interactions with sterilants like formalin if they areto be used in the actual biodegradation test (note-autoclaving REVIEW

OF TSCASECTION4 FINAL STUDY REPORT March 2006 Versionp. 37 might be preferable) . The "other" biodegradation test could be one for inherent biodegradability, if that is consistent with the assessment objectives under the test rule. An important aspect ofany testing is to find a way to reliably dose the test systemquickly to a desired concentration, so that the investigator knows what's in the test systems fromzero time. Reviewer Name/Title/Branch/Division/Office: R . Boethling, EAB/EETD/OPPT Reviewer Signature: signed-my word is my bond Part B . To befilled out bv C1TB PM Tiered Testing Decision 1 . The tiered testing trigger was met, additional testing is indicated. (when applicable) 2 . The trigger for additional (tiered) testing was not met. Study Acceptability 3. The study is acceptable . 4. The study may not be acceptable (provide rationale in memo to CITB chief) .

REVIEW

OF TSCA SECTION4 FINAL STUDY REPORT March 2006 Version Il~t~ [l ` 

### [l V C

379 .59 an ; ot 1 e or( nt ng o e ru e . successfu .ly completed

the test standarl speci 50 1 d 206,7, Ma;r 17, 19F5. Rede:ignated 'R 9713, iune 30, 1986. <.nd amen led at 5 657 Sep . 3o, 1987; 59 FR 36314, 3ept . 1, 0F ~ 3446 i, July 3, 1995] ;79 .59 Failure to comply with a -ule. teed ,51 FR )89; est Peisons who notified EPA of t .eii nt nt t > conduct a test re luired n a tes rul( in part 799 of this c hapter i .nc wh fail to conduct the test in ac( Ird an( -with the test standard: and sc "ed ule adopted in the test ~ule, o ats mo lifie( _ in accordance with §7! 1.55, wi i be i n violation of the rule. ( ) An y person who fails c r refus col )ply with ),ny aspect of this pa a i :st rile under part 799 cf this ( te" is i-i violatiorL of section 15 o Ac .. EPA will treat viola' ions o G( )d L.Lboratory Practice ;,tandai int icatiA in §792.17 of this c hapter s tii t or)aPth c : th(° s a: S4 opa t D--Implementation, Er~ folcement and- Modificl : tior of Consent Agreements

ouxc s: 51 FR 2371 .5, June 3), 1986, nle ~ ot erwi: e noted. §' ; )0 .60 Contents of cor sent ,+ ;re (- o )d I 1 ,t ,on ( 1) 1 n ~s I n 1/0' (3) . p )ns. IIAi 110) e ;till e it : h 3 pn e 3u) ~1 :t : ;e ner !I nec, 11 ; ~i rer ;( ns: ~l .e i ti on t' Eis ~ e tpo' v hic q zirl s :at( . ecpo S ~an.~ a bov r ren (1:'. 1 ;PA I ule! i ndc ,gre a) Standard proaisions. All cc ise:( ; 1 est al reerr ents will contain the foll wi) :j ; p- )visi )ns: l ~ ;roc all! , ) ~ ;oni 1) hlentif,catior, of the chem tc be t ;sted. lui r 2) Tie health effects, e)Evironr ent~ 1 (l e 'ects and/cr other chara~terist~ .s fc r Mr( w)ich ~esting will be required . tgr' l .~sses o eac 1 'eq 3) The names and addr n ?,nuf tcturer and/or processor w] ) wi 1 -rib i s ;n tl e agreement . Ac~, A) The name and addree3 of thl m~, -- rS( u actu rer, processor or other nt: ( y tiol v . lo h is agreed ;,o act as the pr (cil) .1 (1 t st sl onsor. oni :~ (5) The technical or com)~ ~rci d me: ade, level of pu~~ity or )ther ( iar.a ;- abl t rist:cs of the test sukstance s) ;r agr

,e(s) . val r ixtu )f a r (6) tandards for the de velop)^ ~nt st d Eta. the t !(7) E, requirement that 1esting ;ill )e or c )ndu-1i in accordance with tl ~ El A pr~= me] its.

.f~ ) CFR Ch. I (7- -0S Edition) atory -~rac' .ice 'FR pa-~t 792,) . Iles with reas rbmitt :-ng i nte reports to EP: Iirement that it submit , :. s :)rdana. wit

l §' itement that ' ducted pur :ua: nent will be in accordarce ;cified in sE;cti hat the discl( by such testin :ction l4(b) of t luirement that 1/or processor: ;reement will ;ation require 1) of the Act , t er if they expo

, -3 substance o 1 ; submission i )der the agr( I that any otb ~ intends to e), c mixture is : d export notifi 5) A st :ct, J-. ac ndi : i~ /or s ud; ~- i ~ 11: dnct ;d v eeme nt n ~ rity an( ~ion 1 c t : . 6)' . A st,,ter- oi a u -~= 'I ,utes "fi Ez E n ~ 1 m ~`a) `ac "ai tl .1 1 SE si p~ t] es .of 5 ti S

. ' 7) .~:Ey ~

: t.~ t ties Lgr( e e , "pu:^1 ose ; ; l i )) Co ~titer ts ) : ~, ' r t h o ; d o t: P f( n P li n `a g w g a 0 tent if n z,~ elo pi Lien sent ag:e_ c ,'-TSCA 5 ts,79I, 4' 7, i 3 ar Ecoi .orr, c E I cul D rit (C~EC:)) tl ticid ; a3:~ u r c . !ol :ne red blTl-e 6 ) .'. .ni ca tfon Ser ,i, 1 ~ ~ n",hE e-tes ~ m

;tt i ~I .cr : ug dat[cn o' : ( . .~ : , In, nt h :,-~in .tia, l ;~ L 1 =~ c:t Lb] de1ires ~s ( ~ till- I L''st s; ;,m,rnu 'a( c , t : . : - : o-es iO ;Jrter s : r . . : .tv th t-: a .teri<. r (1 dog dific .Ltio i : ~ e P) rielii es . ' : . .ti ve )l :ngi(s )I I . : 1~~1 :5 V ipted onl y ' a -u lgi A,_t;hey ~ ril l ~ p :( i .,t table ar d c :mioE.l s(b: r _ :ture the_a;re( nt t St, cterr e, i c ' < < I ~r eetne: ~-,r -.,ai ilana,ior J~~ .- ree :n, . ' .,~ _ime) rirnarize ;1 ueFcr : .aestin; ( c r ~i ." . ~~ ;~_s 6E.1 it v: E v . i ~ t `ie 's~=us,, a] d _ ,, . i r~,vte t'-exp lair 1 ol tI to ~e c : ))( c c ": :i 1 -'ie ra i ~ .the- spe :il a l e(:te( iuinent v il : e a :1 m tl , : 1L-Rl1GI .i .^l ; I ~~'' i l'(_"-desi Zls, a r ti ;' the nt~ 0!1 E

:'i, L no, ng, r, tler (a : l r tu__ ~ : hY )(1)(E',) of t F . Ef ironrier 'o r 7=n (C ,P) regu-

,he principal Lid " plan to )0. 2. ze results of t t ) the connn )unced to vit i the prom :(d) of the ;u) e of data w11 be govie . ~ct . th manufacs gning the co nply with -lei ts of secid part 707 of k

: t c r intend to r,ixture for ,-: f , ata is re- ',. ;m:nt and a, ; ; :r )erson who )or - such sub- t, zb; ~ct to the ;~, at on require- ; :-, ~quirernent the ~~, i 1 the event : ;- g : iulgates a sign fic Lnt new use I? 1 icable to the te: t chemical i ;! g ;ction 5(a) :2), t-,e consent',, : o : t will have t e >tatus of a ;~, a for pur .pos s of section~~k E c ~ and manufa ;tu ~ers andlor' .' r' c s signing the .gr tement will~ ;, ; c vith the data 3ut mission re-,,, ; t' ; ts imposed by th~ t provision.,~;

, ;tatement that ea ;h manufac ;'; ;, a ; id/or processc ~ igning the,;,, e )t agrees that ria ation of its,~~ s~ ents will con: ~it ite a "Pro-' ; ; h ,ct" under ; ;cc .or 15(1) of the~, ;, c will trigger a 1 1 rovisions of;; : plicable to a ~ iol ction of sech, ;; c a statement thc ~, n the even~ ;,; f~ nore provi=for ; c f the agree,t! d ( : determined 1 ) 1 e unenforce! E S a court, the r m~ inder of the; ; c: nt would not b pi ,sumed to bn

, 9 EPA will the ) e ther initiate,,! a 41 ; iking proceedi ;g n' publish 0 ;1tAL REGIS''ER thf Administra' : ison for not i iti tting such a ; F ng" FR 2.^,715, J i ,G i :nand 36314, Sel : ; . :