P. 1 

oP10T-26o5-ooj,3 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

rhqw. .32Sa'D.~' 

WASHINGTON, D.C. 20460 R fF C F '-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 and Testing Branch 

TO: Mike Mattheisen, Chief 

Chemical Information and Testing Branch 

r 

J 

I received the attached review of a biodegradation study of 1-chlorododecane ~'. 

from Bob 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, EPA should 

try to obtain information on "ready" biodegradation (by the closed bottle method) and not 

"inherent" biodegradation . Although Bob recommended the OECD test guideline 301D, 

OPPT cannot require testing by OECD guidelines or the 800 series 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) 

4 0 1 0 0 U 0 0 0 1 4 

CONTAINS No CBI 

Internet Address (URL) e http :llwww.epa .gov 

Recycled/Recyclable e Printed with Vegetable Oil Based Inks on 100% Postconsumer, Process Chlorine Free Recycled Paper 
p. 2 

Review of 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 of the followin 

Applicable fNAI or Cannot Determine fC] and add comment below, as needed 

[1p] 1 . The final study report indicates evidence of gross, unauthorized or undocumented deviation from the EPA test 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 from the "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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) 

lSa q4tse9 ( ~~ - (v~.c,~X.4_ ,`~.~` 

5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. 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 from that 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 from temperature, 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 of the checkboxes 1 through 13 : A 

REVIEW OF TSCA SECTION 4 FINAL STUDY REPORT March 2006 Version 
] 11 . The final study report inadequately describes the dosage information. 

12 . The final study report does not accurately reflect the individual animal data (or that of other 

discrete observations pertinent to the type of study conducted), 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 . Conclusions 

Place a check mark beside the summary statement s) that apply to this review and add comments below as 

appropriate , 

Part A. To be filled out bV 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 

reasons in notes below) . 

Data Reliability 4. The study results are precise and accurate . 

5. Appropriate methods were used to determine 

statistical significance . 

6 . If repeated, 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). [ ] 

9. The data are not useful for any purpose (describe in notes below) . 

s 

omments (conti on separate page, if neede~d) : 

~ 

. .. 

. 

A I ' 

if I 

e ranch/Div s~on/Office: T,~Q/~ `` 

. 

Part B. To be filled out by CITB PM 

Tiered Testing Decision 

(when applicable) 

Study Acceptability 

_ . _ kt, (W V D I-- 

[l~ 

. 

1 . The tiered testing trigger was 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 TSCA SECTION 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 that when filing the sec 4 study report I should have suggested running a different test, not 

the Closed Bottle test (OECD 301 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 ISO test. Let me know if you need more information. 

OPPTS835.3140.pdF 

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 F 1720 Developed by Subcommittee : E47.04 jBook of Standards 

Volume: 11 .06 

Buy Standard (PDF) more info 6 pages $38.00 

Buy Standard (Print) more info 6 pages $38.00 

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redline? 12 pages $45.60 

Historical (view previous versions of ~ ASTM License ~ Shipping & 

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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 acclimatization to occur. It may 

therefore--be assumed that a chemical yielding a-positive-result in this-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: oT CO2 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 of a 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 of the speed, economy, and space efficiency of the sealed vessel test . 

The sealed vessel test is ideal for the comparative testing of groups of chemicals and for 

generating structure-activity data bases also because of its speed, economy, and space efficiency . 

1 . Scope 

1 .1 This test method covers procedures for determining the ready, ultimate, aerobic 

biodegradability of organic chemicals by monitoring CO, production in sealed vessels 

containing the test compound and a dilute sewage inoculum. Because of the stringency of the 

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 from the sealed vessel procedures of Birch (1), Struijs (2), 

Boatman (3), and Peterson (4), which were developed as simpler, more economical alternatives 

to the CO, production techniques reported by Gledhill (5) and Sturm (6), the Sturm report being 

the basis of the Modified Sturm Test of the Organization for Economic Cooperation and 
Development (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 of at least 25 ppm of carbon, 

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 of carbon. 

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 of the test chemical with a dilute inoculum of microbes 

from domestic 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 on just the liquid phase after the addition of alkali, or on just the headspace 

following acidification. The determinations are made using commercial carbon analyzers based 

on the IR detection of CO2. The determination of CO, production provides unequivocal proof of 

biodegradation, barring the unlikely event of abiotic production of CO Z from the 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 of dissolved organic carbon (DOC) 

in the liquid phase. 

1 .6 The simplicity of the 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 of the test may be reduced by 

substituting an acclimated inoculum in order to provide a measure of inherent biodegradability . 

1 .8 Materials that are toxic to the microbial inoculum at 10 ppm of carbon 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 of the safety concerns, if any, associated with 

its use. It is the responsibility of the user of this 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 ; C02 production ; Organic chemicals; Sealed vessel ; Ultimate 

analysis; ICS Number Code 13 .080.30 (Biological properties of soil) 

DOI: 10.1520/E 1720-01 R08 

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 

Exposure Assessment BranchUS EPA, Office of Pollution Prevention and Toxics 

1200 Pennsylvania Ave, NW (7406M) 

Washington, DC 20460 

202-564-8533 (P) 

202-564-8892 (F) 

[EPI Suite] http://www.epa.gov/opptintrlexposure/pubslepisuite .htm 

[FatePointer] httpa/esc .syrres .com/fatepointer/search .asp 
United 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 ublica -t; jons - 0 f-the Natiotia r Tec -h-ni- ical Infor - --mifon i---S-e-- rvice -(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 from source 

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 from http ://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 from the six test methods described in OECD Test Guideline 301 A to 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 from the gaseous 
phase, as in classical WarburgBarcroft respirometer flasks . 

(2) However, IC has been shown to accumulate in the medium during the 

application of the modified Sturm test (see paragraph (o)(9) of this guideline) . A 

concentration of IC as high as 8 mg/L was found during the degradation of 20 mg 

C/L of aniline . Thus, the collection of COZ in the alkaline traps did not give a true 

reflection of the amount of C02 produced 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 

biodegraded will not be met for some substances that would be so classified using 

DOC removal. 

(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 Sturm methodology (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) of this guideline) . Boatman et al . (see paragraph (o)(12) of this guideline) 

reviewed the earlier methods and adopted an enclosed headspace system in 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 DIC in the liquid phase was 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 medium that 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, 

DIC in 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 (see 
paragraph (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 Sturm test . 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) of this 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 : A classification of chemicals for which there is 

unequivocal evidence of biodegradation (primary or ultimate) in any test of 

biodegradability . 

Lag Phase: The time from the 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 ; these 
tests 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 of carbon dioxide 

calculated to be produced from the 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 of degradation achieved when 

-- the -test- -substance-- is -totally- utilized- --by -micro=organisms-resulting- in-the- --

production of carbon dioxide, water, mineral salts and new microbial cellular 

constituents (biomass). 

10-d window: The 10 days immediately following the attainment of 10% 

biodegradation . 

(e) Principle of the test . (1) The test substance, normally at 20 mg 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 

from sewage treatment effluent obtained from a 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 Test 
Method and Standard No. 7916). 

(3) D 1293-84: Standard Test Methods for pH of 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-19 on Water. 

(7) D4839-88: Standard Test Method for Total 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 from its chemical structure or 

by measurement, so that the percentage degradation may be calculated. For 

volatile test substances, a measured or calculated Henry's law constant is helpful 

for determining a suitable headspace-to-liquid volume ratio. Information on the 

toxicity of the test substance to microorganisms such as from an 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 law constant 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% ThIC within 14 days. 

(j) Reproducibility. (1) In the ISO ring test of the method (see paragraph 

(o)(16) of this guideline), the results in Table 1 were obtained using the 

recommended conditions, including 20 mg test substance as C/L. 

Table 1 . Summary of results from ISO ring test 

Test -Mean-Percent-age of variation 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 low with 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% of test 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 of variation would result when lower 

concentrations of test 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 parag ra ph 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 2 were obtained . 
Table 2. Summary of results from EU 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 reference cited in paragraph (o)(23).of this 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 of effluent. 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 serum bottles 

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) of this 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-ovet the-range-of- ---

0 to 1 mg C (a suitable instrument is an Ionics model 1555b TOC analyzer with 

Horiba model PIR2000 NDIR COz detector, or equivalent). 

(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-cm coarse 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) A supply 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 of f 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. Use analytical grade reagents throughout. 

- ---- ----- -- --- -- ---- (3) Water. (i) Distilled or-de--nized w- 

-- --- --ater----shoul-----db-- -- 

used-- 

- -dco -nt -- --ioning .ai- <1 mg/L as total organic carbon . This represents <5% of the initial organic carbon 

content introduced by the recommended dose of the 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 medium are 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 ammonium chloride (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) Potassium dihydrogen phosphate (KHZPO4) . . . . . . . . . . . . . .  . .  , . .  . .  g,50g Dipotassium hydrogen phosphate (KZHP04) . . . . . . . . . . . . . . . . , . . , .  21 .75g Disodium hydrogen phosphate dehydrate (Na2HP04.2Hz0) . . . . . . . . . . . . . . . . . . . 33 .40g Ammonium chloride (NH4C1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.50g Dissolve in water and make up to I liter . The pH of this solution should be 

7.4 (f 0.2) . If this is not the case, then prepare a new solution. 

(B) Calcium chloride dihydrate . . . . . . . . . . . . . . . . ., . . , ., (CaC12.2H20) 36 .40g Dissolve in water and make up to 1 liter . 

(C) Magnesium sulfate heptahydrate . . . . . . . . . . . . . . . . . . . . . . , . . (MgS04.7H20) 22.50g 
Dissolve 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 

of solutions (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 of dissolved 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) Sodium hydroxide solution 7M. Dissolve 280 g of sodium hydroxide 

(NaOH) in 1 liter of water (see paragraph (1)(3) of this guideline) . Determine the 

concentration of DIC of this 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 of the validity criterion in paragraph (n)(5)(i)(B) of this guideline. Prepare a 

fresh solution if the concentration of DIC is 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 medium to 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 sodium acetate, dextrose (glucose), and sodium benzoate may 

be too biodegradable to be useful for this purpose. 

(iii) For test compounds with very low or negligible water solubility, it 

may be desirable to employ reference compounds of similar solubility; for 

example, sodium stearate . 

(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 inoculum at 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 show that 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 medium containing both test substance 

11 
and 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) of this 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 30 mg/L may be 

used but may significantly increase COZ production of the blanks (see paragraph 

(o)(19) of this 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 inoculum for 1 liter of test medium. 

(iii) Inoculum may 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 from the 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 mm mesh sieve) and the sludge should be kept aerobic 

until it is used. 

12 
(ii) Alternatively, after removal of any 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 from a high 

rate treatment plant, or is thought to 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 

from excess substrate or inhibitors . 

(iv) After complete resuspension is achieved, or with untreated sludge, 

withdraw a 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 

medium speed. Settle the blended sludge for 30 minutes or longer if required and 

decant liquid for use as inoculum at the rate of about 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 medium when the test is 

started (see paragraph (o)(13) of this guideline) . 

(12) Secondary sewage effluent . (i) Alternatively, the inoculum can be 

derived from the 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 sodium hydroxide (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) Surface waters . A sample 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 inoculum to be used in each test vessel should meet the criteria given 

in paragraph (1)(10)(ii) of this guideline. 

(14) Soils. A sample is taken of an appropriate soil, collected to a depth of 

up to 20 cm below the soil surface. Stones, plant remains and invertebrates should 

be removed from the 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 . If the sample is transported in a loosely tied black polythene bag, it can 

be stored at 2 to 4°C in the bag for 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 window may 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 of the test, to enable 95% confidence intervals to be calculated for the mean 

percentage biodegradation value. 

(2) Inoculated medium. Prepare immediately before use sufficient 

inoculated medium by 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 medium and 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) of this 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 10 to 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) of this guideline) according to the nature of 

the substance, either before or after addition of the 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 from the volume injected and the density of the 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 allow for 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 from the test vessel . The test vessel and its contents 

are then discarded . 

15 
(viii) The sealed 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°C f 1°C. 

(4) Sampling. The pattern of sampling will depend on the lag period and 

rate of biodegradation of the 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) if a complete degradation curve is required. The requisite number 

of replicate vessels are taken from the shaker, representing FT, FB and Fc and, if 

used, FI and Fs vessels (see paragraph (m)(3) of this guideline) . The test normally 

runs for 28d. If the biodegradation curve indicates that a plateau has been attained 

before 28d, the test may be concluded earlier than 28d. Take samples from the 

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) of this guideline. 

Since the methods can give slightly different results, only one should be used in a 

given test run. 

(ii) Method-A (see paragraph (m)(6) of this 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 if a 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 sodium hydroxide 

solution (see paragraph (1)(6) of this 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 septum of each 

vessel that is to be sampled, in order to lower the pH of the medium to <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 from the 

shaker, and an aliquot (e.g . 1 ml) of gas is withdrawn from the headspace of each 

16 
vessel 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 for the test system at 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 of the headspace 

volume to the liquid volume is the same as in the tests (e .g . 1 :2) . Acidify and 

equilibrate as described in-paragraph (m)(6)(i) of this guideLne, and- measure the 

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 review the 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 from separate (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) of this 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 septum of each 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. If absolute blank IC values are needed at all sampling 

occasions, IC determinations of the NaOH solution will be needed each time it is 

used. The vessels are removed from the 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 system at least once by using IC standards, adding alkali and 

17 
equilibrating, 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 of that produced in 

the blank controls equals the TOC added to each test vessel at the start of the test, 

-- -that i 

ThIC = TOC. 

The total mass (mg) of inorganic carbon (TIC) in each vessel is : 

Equation [1] 

where : 

TIC = (mg inorganic C in the liquid + mg inorganic C in the headspace) 

= (VL X CL) + (VH x CH) 

VL = volume of liquid in the vessel (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 TIC for the two analytical methods used for measuring IC in 

this test are described in paragraphs (n)(2) and (n)(3) of this 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 

18 
included, inhibition monitoring control (FI) vessels, from the respective amounts 

of TIC produced up to each sampling time. 

(ii) If there has been a significant increase in the TIC content 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 %D in 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, from Equation [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 window and 

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 of the 

last day of the 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 from only the reference component of the mixture. If, at day 28, 

19 
Figure 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 DFC is the %D in vessels FC containing 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 inoculum and 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 of TIC is observed, abiotic degradation 

processes may have occurred. 

(5) Validity of results. (i) A test 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 of TIC 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 

inoculum from another 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) of this guideline should be followed . 

(iii) If the test substance does not reach 60% ThIC and was 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 

20 
100 ml effluent/L) or inocula from other sources, especially if degradation was in 

the range 20 to 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) If the pass value of 60% ThIC is not attained, determine the pH value 

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 OECD guidelines 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) of test substance . 

(ii) Test conditions: 

(A) Reference to this test guideline ; 

(B) Description of the test system (e .g . volume of the vessel, head spaceto-liquid ratio, method of stirring, 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 of solvents ; 

(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) of this 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) Any other 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 Sturm test 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 . Cunningham and D.A. Ziegler (1986) . A method 

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 

23 
products : 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 ASTM Standards, 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 4 April 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 inoculum in 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 PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

RECD VI" 

19 1 7` 

MAR 0 2 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) which you submitted in response to the TSCA section 4 test 

rule entitled "Testing of Certain High Production Volume Chemicals" (71 FR 13708, 

March 16, 2006). As stated in my July 13, 2009, letter, the wrong guideline (ASTM E 

1279) was followed in conducting the test for biodegradation instead of the guidelines 

specified by the test rule (ASTM 1625 or ISO 9888). Nevertheless, EPA scientists 

reviewed the submitted study to see if it 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 of one of the specified guidelines 

would have provided. I am, therefore, requesting that Dyno Nobel arrange to have the 

study repeated according to one of the specified guidelines, ASTM 1625 or ISO 9888 . 

By so doing, you will avoid being in violation of the test rule as stated in 40 CFR 790.59 

(attached). The scientific review also provides additional advice on how to properly 

conduct the repeat study. If you 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 Paper 
p. 35 

C1TB Project Manager: 

Chemical Name: 

CAS Registry Number : 

Report Title: 

Test Sponsor: 

Testing Lab: 

Test Standard: 

Review of TSCA Section 4 Final Studv Report 

Catherine Roman 

Pentaerythritol tetranitrate (PETN); EPA-HQ-OPPT-2005-0033-0294 .2 

78-11-5 

"Biodegradation Potential of PETN" 

DyN- A/0 b`/ 

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 EPA test 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 from the "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 

not signed. 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 from that 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 from temperature, 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 SECTION 4 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 (or that of other 

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 summary statement(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 . If repeated, 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 not useful for any 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 a period of time on the order of days 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, ASTM 1279- 89 is not an acceptable 

s bsti te 

The test report reveals significant departure from the 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. 

From the USACHPPM test reports for biodegradation and water solubility, it appears that 

achievement of the expected concentration of test substance in the amended media required 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 from scratch and run another test for biodegradability, but 

only after properly researching and then achieving understanding of the behavior of this test substance with 

respect to preparing stock (dosing) solutions, dosing flasks, abiotic losses if any, and possible interactions 

with sterilants like formalin if they are to be used in the actual biodegradation test (note-autoclaving 

REVIEW OF TSCA SECTION 4 FINAL STUDY REPORT 

March 2006 Version 
p. 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 of any testing is to find a 

way to reliably dose the test system quickly to a desired concentration, so that the investigator knows 

what's in the test systems from zero time . 

Reviewer Name/Title/Branch/Division/Office : R. Boethling, EAB/EETD/OPPT 

Reviewer Signature: signed-my word is my bond 

Part B. To be filled 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 SECTION 4 FINAL STUDY REPORT 

March 2006 Version 

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2) Tie health effects, e)Evironr ent~ 1 (l 

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