GnEENWTcH CHEMTcAL CoNsuLTtNc, Ir'{c.

3300 PGA BouLEVARD . Surrn 635

Pnrnr Bp,ncn Ganogxs, FL 33410

Tr.lEpHonn 561.578.8 1 10

Fer:srurlu 551.578.8120

CBIC Control Number

February 9, 2015 363866

CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

Bv CDX and Federal Exuress

OPPT Docurnent Conttol Offrce

U.S. Environmental Protection Agency

EPA East Burtding

Room 86428

1,201, Constitution Ave., I.ISfl

Washington, DC 20004

Attn.: TSCA Section 4

Re: Submission of the Final Test Report on the Biodegradation Study by Univat

USA Inc. under the TSCA Section 4 Test Rule on the Testing of Cettain lligh

Production Volume Chemicals: Second Gtoup of Chemicals

D-erythro-Flex-2-enonic acid, .gamma.-lactone, moilosodium salt

cAs RN 6381-77-7

(Sodium erythofiate)

Docket Identification No. EPA-HQ-OPPT-2007-0531; 40 CFR 799.5087

Deat Sir/Madam:

On behalf of its client, Univar USA Inc. (Univar or the Company), Greenwich Chemical

Consulting, Inc. (Greenwich Chernical Consulfud submits to the U.S. Envitonmental

Protection Agency FPA or the Agency) the final test report on the biodegtadation study

conducted ofl the chemical substance, D-erythto-hex-2-enonic acid, .gamma.-lactone,

monosodiutn salt ICAS RNI 6381,-77-71 which is currently subject to a Toxic Substances

h) ffi

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U.S. Environmental Protecdon Agency

February 9,2075

Page 2

Conuol Act (ISCA) Section 4 test

accordaflce with this test rule and the

I. Genetal Backgtound

CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

rule.l The Comparry submits this final test report in

regulations set foth at 799.5087.

As a matter of backgtound, this test rule addresses 19 high production volume (HP\D

chemicals that weflt unsponsoted under the Agency's voluntary HPV Chemical Challenge

Program. One of the 19 chemicals identified in the nrle is D-erythro-hex-2-enonic acid,

.gafirma.-lactone, monosodium salt which is identified herein as sodiwm trythorbate. The rule

tequires manufactutets and importers of these HPV chemicals, including sodium erythorbate,

to conduct testing to obtain screening level data for human health effects, environmental

effects, chemical fate, and physical/ chemical properties.

The Agency has issued this test rule under its TSCA Section a(a)(t)@) authority based on its

determination that HPV compounds, including sodium erythorbate, are "produced in

substantial quantities; thete is or may be substantial human exposure to them; existing data ate

insufficient to determine or predict their health and environmental effects; and testing is

necessary to develop such data."z The frnal rule idennfies the following studies on sodium

erythorbate:

n-Octanol/Watet Partition Coefficient

Ready Biodegradation

Aquatic Toxicity Testing:

Acute toxicity to fish

Acute toxicity to Daphnia

Toxicity to plants (algae)

In the finalTSCA Section 4 testrule, EPA specified the use of ASTM, ISO, andf or CFR test

guidelines fot this testing. In its letter dated April 75, 2014, Univat requested that the

corresponding OECD test guidelines be used in the conduct of these studies. In its May 19,

201,4 response letter, the Agency granted this request.3

Hatlan Laboratodes Ltd. (Hadan Laboratories) was selected to conduct all of these tests. AIso,

Gteenwich Chernical Consulting is serving as the study monitor for this testing.

Because Univar had not yet begun importing sodium erythorbate with the intent to sell it for

TSCA purposes at the time of the rule's effective date, the specific dates, as set forth in the

fural rule, for the submission of the letter o[intent and test reports \Mere not applicable. Univar

rTanuarv 7-201,1:76 Fed. Res. 10(r7.

2 Id. at 1069.

3 With respect to the biodegradation study, the Company requested the use of OECD test guideline 3018 for the

biodegtadation test.

T

T

I
CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

U.S. Environmental Protection Agency

February 9,2015

Page 3

subsequently submitted its letter of intent to test onJanuary 27,201,4.4 Based on this, EPA

stated in its response lettet dated February 1,L,201,4 that study plans would be due April 15,

2014 and final reports would be due February 11,,2015.s

II. Recent Extension Request

In its tecent lettet dated January 27, 201,5, Univar requested from EPA an extension to the

February 11,201.5 deadline due to the fact that there has been a signifrcant technical issue

encountered dudng the development of the analytical method which is to be used in support

of the tlrree ecotoxicity studies. The labotatory has also indicated that this same technical issue

is being encountered during the paftition coefficient test, though to a somewhat lesset degree.

Therefore, Univat requested an extension through Octobet 31,,201,5 for the submission of the

frnal test teports. However, this extension need not apply to the biodegradation test which

was timely completed and is being submitted herein ahead of the February 71,,201,5 deadline.6

III. Final Test Report List

Consistent with the final TSCA Section 4 test rule, the following final test report, datedJarilara1

28,2015, is included herein:

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-

7]: Assessment of Ready Biodegradability; COz Evolution Test

IV. Submission of Robust Summaties through HPVIS

In the final test rule, EPA requests, but does not require, that a robust sumrnary also be

included for each test identified in the rule consistent with the Agency's Draft Gwidance on

Deueloping Robust Summaries. The Agency has signaled to other testing consortia that the

submission of these robust summaries is unnecessary. For example, EPA advised the PCBTF

Testing Consortium (of which Univar is a member) in early 2013 that robust summaries were

not necessary under the TSCA Section 4 HPV test rules. If EPA decides that it wishes to

teceive such robust summaries, Univar will certainly provide them.

a Upon EPA's receipt of this January 27,2A1,4Ietter of intent to test, Univar planned to begin importing this

substance for both TSCA purposes and for continuing exempt FDA pulposes.

s It is noted that since the time this test rule was issued through today, no other companies have come forward

indicating their intent to test sodium erythorbate under this rule.

6 This was also conveyed to the Agency in an electronic mail to N'Ir. John Schaeffer trPA) from Mr. John \'.

Nfassingale (Greenwich Chemical Consulting) on January 29, 201,5.
U.S. Environmental Protection Agency

February 9,2015

Page 4

Univar thanks the Agency for

and determinatioris coflcerning

Sincerely,

John V. Massingale

Principal

Ms. K. Gilje - Univar

Ms. S. Lilley - Univar

Mt.J. Schaeffer - EPA

CONTAINS NO CONFIDENTIAL

BUSINESS INFORMATION

its consideration in this matter. For all questions, corrrrneflts,

this matter, please use the contact information given below.

Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, F'L 33410

Telephone (561) 578-8110

Facsirnilc (561) 578-81 20

E -mail: @,r' i olr n(rDrrr:cci. com
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Origin lD:PBIA

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33(1t} PGA BLVD

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PALM BEACH GARDENS, FL 33410

SHIP To: (202) 564-8S30

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I)-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium

salt [CAS RN 6381-77-7lz

Assessment of Ready Biodegradabilityi COz Evolution Test

Study I)irector:

Test Facility:

Sponsor:

Harlan Study Number:

Study Completion Date:

N Best

Harlan Laboratories Ltd.

Shardlow Business Park

Shardlow

Derbyshire

DE72 zGD

UK

Univar USA Inc.

17425 NE Union Hill Road

Redmond

wA 98052

LNITED STATES OF AMERICA

41401177

28 January 2015

Page I of29
Harlan Study Number: 4140fi77

CONTENTS

CONTENTS .......... .,...............2

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE .......4

QUALITY ASSURANCE STATEMENT....... ...................5

suMMARy ........ ...................6 Introduction......... ..........6 Methods. ...............6 Results. ................6

GENERAL TNFORMATION .,,,,7 Schedule ..,.......7

Deviations from Study Plan... ...........7

Archiving ............ ...........7 I INTRODUCTTON AND PURPOSE............ ...........,.8 l.l Guidelines / Regulations......... ....,.8 2 TEST AND REFERENCE ITEMS ............ ....,..........9 2.1 Test Item .............9 2.2 Reference Item.... ...........9 3 MATERIALS AND METHODS.......... .... IO 3.1 Definitions and Abbreviations ,........ l0 3.2 Test System.,.......... ...... 10 3.3 Preparation of Inoculum ............... 10 3.4 Mineral Medium .............. 1l

3.5 Procedure ...... 1l

3.5.1 PreliminaryInvestigationalS/ork .............. 11

3.5.2 Experimental Preparation .,............. l l

3.5.2.1 Test ltem....... ...........,..... 11

3.5.2.2 Reference Item ..............12

3.5.2.3 Toxicity Control ............ 12

3.5.3 Preparation of Test System.......... ...............12

3.6 Evaluations.......... ........ 13

3.6.1 Observations........ ......... 13

3.6.2 pH Measurements ......... 13

3.6.3 IC Analysis.......... .............. 13

3.6.4 IC/TC Ratio .................. 14

3.7 DataEvaluation............ .............. 14

3.7.1 Calculation of Carbon Content., ................. 14

3.7.2 Percentage Biodegradation .......... 15

3.7.3 Total COz Evolution ..... 15

3.8 Validation Criteria ....,.. 15

4 RESULTS.....,...... ....... 17

4.L Preliminary Investigational Work ............. 17

4.2 Definitive Test .................. 17

4.2.1 Validation Criteria ......., l7

4.2.2 Biodegradation ...-.-.--...- 17

5 CONCLUSTON..... ...... 19
Harlan Study Number: 4l4OIl77

TABLES ,...............20 FIGURES............ .........25 APPENDICES...... ................26

LIST OF'TABLES

Table 1 Inorganic Carbon Values on Each Analysis Occasion ............20

Table 2 Percentage Biodegradation Values ....,.......21

Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0............ .......22

Table 4 pH Values of the Test Preparations on Days 0 and 28.......... .................23

Table 5 Observations on the Test Preparations Throughout the Test Period ......24

LIST OF FIGURES

LIST OF APPENT}ICES

Appendix I Cer.tificate of Ana1ysis............ ....26

Appendix 2 Mineral Medium .......,..27

Appendix 3 Dissolved Organic Carbon (DOC) Values from the Preliminary

Investigational Work ..................28

Appendix 4 Monitoring Authority Statement of GLP Compliance ........... ........29
Harlan Study Number: 41401177 Repoft Page 4

STUDY I}IRECTOR STATEMENT OF GLP COMPLIANCE

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshirc,DB7z zGD, UK

Harlan Study Number: 41401177

Study Title: D-erythro-Hex-2-enonic acid, .gffirma.-lactone, monosodium salt

lcAS RN 6381-77-71:

Assessment of Ready Biodegradability; COz Evolution Test

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory

Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994)). These Regulations are

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice

(revised 1997 , ENV/h{C/CHEM(98) 17); and are in accordance with, and implement, the

requirements of Directives 2004/9lEC and 2004110/EC.

These principles are compatible with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Commumty, the United States (EPA and FDA),

and Japan (MHLW, MAFF and METI).

This report fully and accuately reflects the procedures used and data generated. There were no

circumstances considered to have affected the integrity of the study or the validity of the data.

Study Director: N Best

2 S JAN 2015
Harlan Study Number: 41401177 Report Page 5

QUALITY ASSURANCE ST?TTEMENT

Harlan Study Number: 41401177

Study Title: D-erythro-Hex-2-enonic acid, .gailrma.-lactone, monosodium salt

lcAS RN 6381-77-7):

Assessment of Ready Biodegradability; COz Evolution Test

The general facilities and activities are inspected at least once a year and the results are reported

to the relevant responsible person and management.

Study-related procedures conducted at the test facility were audited and inspected. The details of

these audits and inspections are given below.

Dates and Types of QA Inspections

Reported to the

relevant Study

Ilirector and Test

Facility Management

Date of Inspection Type of Inspection Phase Inspected Report Date

1l April 2014

24 October 2014

24 October 2014

30 October 2014

27 November 2014

Study Plan

Verification

Process - based

Process - based

Process - based

Report Audit

N/A

Test Item Preparation

Test System

Preparation and

Exposure

Assessment of

Response

N/A

l l April 2014

24 October 2014

24 October 2014

30 October 2014

27 November 2014

This statement confirms that this report reflects the raw data and the procedwes followed.

eualityAssurance: trruil

Date:

2I JAN 20t5
Harlan Study Number: 41401177

SUMMARY

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic

aqueous medium. The method followed was designed to be compatible with the OECD

Guidelines for Testing of Chemicals (1992) No. 3018, "Ready Biodegradability; COz Evolution

Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA

Fate, Transport, ffid Transformation Test Guidelines OCSPP 835.31l0 (Paragraph (m)).

Methods

The test item, at a concentration of l0 mg carbon/L, was exposed to activated sewage sludge

micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures

between 20 and 22 "C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide

produced. Control solutions with inoculum and the reference item, sodium benzoate, together

with a toxicity control were used for validation purposes.

Results

The test item attained 70% biodegradation after 28 days and satisfied the lO-Day window

validation criterion, whereby 60% biodegradation must be attained within l0 days of the

biodegradation exceeding L0%. The test item can therefore be considered to be readily

biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Harlan Study Number: 41401177

GENERAL INFORMATION

Schedule

Experimental Starting Date:

Experimental Completion Date :

8 September 2014

29 October 2Al4

Ileviations from Study Plan

There were no deviations (unplanned changes) from the study plan.

Archiving

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific

supplement), all raw data (paper and electronic) and the final report will be retained in the Harlan

Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as

to further retention or disposal. Further retention or return of the data will be chargeable to the

Sponsor.

No data will be discarded without contacting the Sponsor to obtain their written consent.
Harlan Study Number: 414011,77

I INTRODUCTIONAND PURPOSE

This report contains a description of the methods used and results obtained during a study to

investigate the ready biodegradability of the test item when exposed to sewage sludge microorganisms under aerobic conditions.

The results of the study are of value in the assessment of the persistence of the test item in the

aquatic environment.

This study was performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4

Test rule and the regulations set forth under 40 CFR 799.5087.

1.1 Guidelines / Regulations

This study was designed to be compatible with the procedures indicated by the following

internationally accepted guidelines and recommendations :

. OECD Guidelines for Testing of Chemicals (1992) No. 3018 "Ready Biodegradability;

COz Evolution Test"

. Method C.4-C of Commission Regulation (EC) No. 440i2008

. US EPA Fate, Transport, ffid Transformation Test Guidelines OCSPP 835.3110

(Paragraph (r"))
Harlan Study Number: 41401177

2 TEST ANI} REFERENCE ITEMS

2.1 Test Item

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is

given in Appendix 1.

Identification:

Physical state/Appearance :

Batch:

Purity:

Expiry Date:

Storage Conditions:

Intended use/Applic ation :

2.2 Reference ltem

Information as provided by the Supplier (Sigma-Aldrich).

D -erythro - Hex-2 - enonic acid, . gamma. -lactone,

monosodium salt [CAS RN 6381-77-7)

white granular solid

1403 I 3 l4

99.19 %

26 March 2016

room temperature in the dark

not supplied

Sodium benzoate

white granular solid

SLBGgs15V

>99.sYo

09 July 2015

roorn temperature over silica gel

Identification:

Description:

Batch:

Purity:

Expiry Date:

Storage conditions:
Harlan Study Number: 41401177 Page l0

MATERIALS AND METIIODS

3.1 Definitions and Abbreviations

Readily biode gradable :

10-Day window:

Inorganic carbon (IC):

Total carbon (TC):

Total organic carbon (TOC):

Dissolved organic carbon (DOC):

Test items may be considered to be readily

biodegradable if > 60% biodegradation is attained in a

1O-Day window within the 28 days of the test.

The l0 days immediately following the attainment of

10% biodegradation.

Carbon derived from inorganic sources e.g. carbon

dioxide, carbonic acid, and carbonate.

This is the sum of the organic carbon and the

inorganic carbon present in a sample.

This is the sum of the organic carbon in solution and

in suspension.

This is the organic carbon present in solution or that

which passes through a 0.45 pm filter or remains in

the supernatant after centrifuging at approximately

4000 g for 15 minutes.

3.2 Test System

A mixed population of activated sewage sludge micro-organisms was obtained on

29 September 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant

at Loughhorough, Leicestershire, UK, which treats predominantly domestic sewage.

3.3 Preparation of fnoculum

The activated sewage sludge sample was washed twice by settlement and resuspension in

mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may

have been present. The washed sample was then maintained on continuous aeration in the

laboratory at a temperature of approximately 2l oC and used on the day of collection.

Determination of the suspended solids level of the activated sewage sludge was carried out by

filtering a sample (100 mL) of the washed activated sewage sludge by suction ttrough preweighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further

3 minutes after rinsing the filter three successive times with l0 mL of deionized reverse osmosis

water. The filter paper was then dried in an oven at approximately 105 'C for at least I hour and

allowed to cool before weighing. This process was repeated until a constant weight was attained.

The suspended solids concenhation was equal to 3.0 gll prior to use.

* Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven
Harlan Study Number: 4l4}ll77 Report Page n

3.4 Mineral Medium

The mineral medium used in this study (see Appendix 2) was that recommended in the

OECD Guidelines.

3.5 Procedure

3.5.1 PreliminaryInvestigationalWork

In order to investigate whether the test item adsorbed to filter matrices and/or the activated

sewage sludge, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of

the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge

solids.

Thus the following work was conducted and samples analyzed for Dissolved Organic Carbon

(Doc) using a shimadzu Toc-vg5H ToC analyzer (see see Appendix 3).

An amount of test item (100 mg) was dissolved in mineral medium (1 titer) to give a 100 mg/L

stock solution. Two samples were taken for DOC analysis; one untreated and one filtered

through a 0.45 pm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the

filter). A further amount of test item (100 mg) was dissolved in mineral medium and inoculated

at a concentration of 30 mg suspended solids (ss)/I- prior to adjusting to a final volume of I liter.

Two samples were taken for DOC analysis; one after filtration through a 0.45 pm Gelman

AcroCap filter (discarding the initial 5 mL to pre-condition the filter) *d the other after

centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral

medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as

per the test item samples.

3.5.2 Experimental Preparation

3.5.2.1 Test Item

The test item was dissolved directly in mineral mediurn.

An amount of test item (300 mg) was dissolved in mineral medium with the aid of

ultrasonication (5 minutes) and the volume adjusted to 1 liter to give a 300 mg/I- stock solution.

An aliquot (276 mL) of this stock solution was dispersed in inoculated mineral medium and the

volume adjusted to 3 liters to give a final concentration of 27.6mglL, equivalent

to 10 mg carhon/L. The volumetric flask containing the test item was inverted several times to

ensure homogeneity of the solution.

A test concentration of l0 mg carbon/L was employed in the test following the recommendations

of the Test Guidelines.
Harlan Study Number: 41401177 Page 12

3.5.2.2 Reference Item

A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control

vessels. An initial stock solution of 1000 mglL wim prepared by dissolving the reference item

directly in mineral medium with the aid of ultrasonication for approximately 5 minutes. An

aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated

mineral medium and the volume adjusted to 3 liters to give a final test concintration of

17 .I mglL, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item

was inverted several times to ensure homogeneity of the solution.

3.5.2.3 Toxicity Control

A toxicity control, containing the test item and sodium benzoate, wffi prepared in order to assess

any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An aliquot(276 mL) of the test item stock solution was dispersed in inoculated mineral medium

along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was

adjusted to 3 liters to give a final concentration of 27.6 mg test item/L plus 17.1 mg sodium

benzoate/L, equivalent to a total of 20 mg carbon/L.

3.5.3 Preparation of Test System

The following test preparations were prepared and inoculated in 5 liter test culture vessels each

containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.

b) The procedure control containing the reference item (sodium benzoate), in duplicate, in

inoculated mineral medium to give a final concentration of l0 mg carbon/L.

c) The test item, in duplicate, in inoculated mineral medium to give a final concentration

of 10 mg carbon/L.

d) The test item plus the reference item in inoculated mineral medium to give a final

concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Data from the inoculum control and procedure control vessels was shared with similar

concurrent studies.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 rng

suspended solids (ss)/L. The test was carried out in a temperature controlled room flt

temperatures between 20 and 22oC, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled

with 2400 mL of mineral medium and 30.0 mL of inoculum and aerated overnight. On Day 0

the test and reference items were added and the pH of all vessels measured using a Hach HQ40d

Flexi handheld meter and the pH was adjusted to pH 7.4 + 0.2 using diluted hydrochloric acid or
Harlan Study Number: 41401177 Page 13

sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the

addition of mineral medium which had been purged overnight with COz free air.

The test vessels were sealed and C0z-free air bubbled through the solution at a rate of 30 to

100 ml/min per vessel and stirred continuously by magnetic stirrer.

The Coz-free air was produced by passing compressed air through a glass column containing

self-indicating soda lime (Carbosorb@) granules.

The COz produced by degradation was collected in two 500 mL Dreschel bottles containing

350 mL of 0.05 M NaOH. The COz absorbing solutions were prepared using purified de-gassed

water.

3.6 Evaluations

3.6.1 Observations

The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and??.

3.6.2 pH Measurements

The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification

with hydrochloric acid, using a Hach HQ40d Flexi handheld meter and a Hach HQ160 Flexi

handheld meter.

3.6.3 IC Analysis

Sarnples (2 mL) were taken from the first COz absorber vessels on Days 0,2,6,8, 10, 14,21,28

and 29. The second absorber vessels were sampled on Days 0 and 29.

AII samples were analyzed for IC immediately.

On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any

inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples

taken from both absorber vessels on Day 29.

The samples were analyzed for IC using a Tekmar-Dohrmann Apollo 9000 TOC analyzer or a

Shimadzu TOC-VcsH TOC analyzer. Samples (300 or 50 pL) were injected into the IC channel

of the TOC analyzer. IC analysis occws by means of the conversion of an aqueous sample to

CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by

reference solutions of sodium carbonate OtrazCO3). Each analysis was ca:ried out in triplicate.
Harlan Study Number: 4l4Dll77 Page 14

3.6.4 IC/TC Ratio

Samples (30 mL) were removed from the inoculum control and test item vessels on Day 0 and

filtered through 0.45 pm Gelman AcroCap filters (first approximate 5 mL discarded in order to

pre-condition the filter) prior to DOC analysis.

The samples were analyzed for IC and TC using a Shimadzu TOC-Lcsu TOC analyzer. Samples

(50 or 800 pL) were injected into the TC and IC channels of the TOC analyzer. TC analysis is

carried out at 680 'C using a platinum based catalyst and zero grade air as the carrier gas. IC

analysis involves conversion by 2M HCI at ambient temperature. Calibration was performed

using reference solutions of potassium hydrogen phthalate (CaHsKOo) *d sodium carbonate

(NazCO:) in deionized water. Each analysis was ca:ried out in triplicate.

3.1 Data Evaluation

3.7.1 Calculation of Carbon Content

The theoretical amount of carbon present in the test item (D-erythro-Hex-2-enonic acid,

.gtrrnma.-lactone, monosodium salt ICAS RN 6381-77-7] (CrftONa)) was calculated as

follows:

No of C atoms x mol vrt of C

x 100

mol wt of test item

6 x 12'ol1 

x loo -36.lg0/o 199.1l

Thus for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference item, sodium benzoate (CeHsCOONa)

was calculated as follows:

No of C atoms x mol wt of C

x 100

mol wt of sodium benzoate

7 x12'o11x1oo 

=58.34Yo

144.1 I

Thus for a l0 mg CiL test concentration the total organic carbon

was 30 mg C.

present for sodium benzoate
Harlan Study Number: 41401177 Report Page 15

3.1.2 PercentageBiodegradation

The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2)

produced is calculated by substituting the inorganic carbon values, given in Table 1, into the

following equation. The values of Replicates Rr and R2 are meaned for the inoculum control,

test and reference items before substitution into the following equation:

% ThCO z(= Yobiodegradation*) = x 100

3.7.3 Total COz Evolution

The total COz evolution in the inoculum control vessels at the end of the test is calculated from

the equation below. The inorganic carbon values for Replicates R1 and R.2 on Day 28 are

meaned before substitution into the equation:

Total CO2 evolution (mg C/L) : mg IC in control * 

= 

, 

= 

1?0= 

%C of CO, = test volume

= ms IC in control " #h. i

3.8 Validation Criteria

The results of the degradation test are considered valid if in the same test the reference item

yields > 60Yo degradation by Day 14.

The test item may be considered to be readily biodegradable if > 60% degradation is attained

within 28 days. This level of degradation must be reached within 10 days of biodegradation

exceeding 1,0%.

The toxicity control (test item and sodium benzoate) should attain > 25yo degradation by Day 14

for the test item to be considered as non-inhibitory.

The test is considered valid if the difference of the extremes of replicate values of production of

COz at the time the plateau is reached, at the end of the test or at the end of the 10-Day window,

iffi appropriate, is less than}}o/o.

The total COz evolution in the inoculum control vessels at the end of the test should not normally

exceed 40 mg/L medium (: 120 mg/3 liters, corresponding to 33 mg C per flask), however

* The conversion factor for carbon to carbon dioxide is 3.67
Harlan Study Number: 41401177 Report Page 16

values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are

obtained should be critically examined.

The IC content of the test item suspension in the mineral medium at the beginning of the test

should be <5% of the TC.
Harlan Study Number: 41401177 Report Page 17

RESULTS

4,1 Preliminary lnvestigational Work

The results obtained from the samples taken for DOC analysis from the preliminary

investigational work indicated that the test item did not adsorb to filter matrices or activated

sewage sludge (see Appendix 3). Therefore, for the purpose of the study, the samples taken for

DOC analysis were filtered to remove the suspended solids present without the loss of any test

item.

4.2 Definitive Test

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum

control vessels at each analysis occasion are given in Table L Percentage biodegradation values

of the test and reference items and the toxicity control are given in Table 2 and the

biodegradation curves are presented in Figure l. Total and Inorganic Carbon values in the

culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0

and 28 are given in Table 4. Observations made on the contents of the test vessels are given in

Table 5.

4.2.1 Validation Criteria

The total COz evolution in the inoculum control vessels on Day 28 was 31.88 mg/L and therefore

satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test

(see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given

in the OECD Test Guidelines.

4,2.2 Biodegradation

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was

conducted according to the methods specified in the Test Guidelines. This acidification

effectively kills the micro-organisms present and drives offany dissolved COz present in the test

vessels. Therefore any additional COz detected in the Day 29 samples originated from dissolved

COz that was present in the test vessels on Day 28 and hence the biodegradation value calculated

from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29

showed an increase in all replicate vessels with the exception of inoculum control Rz and the

toxicity control.
Harlan Study Number: 41401177 Report Page 18

Inorganic carbon analysis of the sarnples from the second absorber vessels on Day 29 confirmed

that no significant carry-over of COz into the second absorber vessels occurred.

The test item attained 70% biodegradation after 28 days and satisfied the l0-Day window

validation criterion, whereby 600/o biodegradation must be attained within 10 days of the

biodegradation exceeding 10%. The test item can therefore be considered to be readily

biodegradable under the strict terms and conditions of OECD Guideline No. 3018.

The toxicity control attained 75% biodegradation after 14 days and 72% biodegradation after

28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage

treatment micro-organisms used in the test. The slight decrease in biodegradation between days

14 and 28 was considered to be due to sampling/analytical variation.

Sodium benzoate attained 83% biodegradation after 14 days and 88% biodegradation after

28 days thereby confirming the suitability of the inoculum and test conditions.
Harlan Study Number: 41401177 Report Page 19

5 CONCLUSION

The test item attained 70% biodegradation after 28 days and satislied the 10-Day window

validation criterion, whereby 60% biodegradation must be attained within l0 days of the

biodegradation exceeding 10%. The test item can therefore be considered to be readily

biodegradable under the strict terms and conditions of OECD Guideline No. 3018.
Harlan Study Number: 41401177 Report Page 20

TABLES

Table I Inorganic Carbon Values on Each Analysis Occasion

Rr - R+ : Replicates I and 2

Abs = COz absorber vessels

Day

Inorganic Carbon (mg IC)

Inoculum Control Procedure Control Test ltem Toxicity Control

RI R, RI R, R1 Rr Rr

Abs I Abs 2 Abs I Abs 2 Abs 1 Abs 2 Abs I Ahs 2 Abs I Abs 2 Abs I Abs 2 Abs I Abs 2

0 1.28 l.l7 t.t7 1.17 1.28 t.t7 r.28 t.t7 1.28 1.05 1.05 1.40 1.05 1.28

2 5.33 6.50 28.77 26.80 10.67 t0.21 3t.21

6 10.27 9.69 33.79 31.60 28.49 28.26 s0.63

I 11.47 I 1.35 1s.32 34.97 31.07 32.1 1 55.04

l0 13.57 13.79 39.33 38.07 35.23 35.1 1 57.46

t4 16.21 r6.09 40.80 4t.25 37.29 37.97 60.97

21 19.27 19.04 41.69 42.25 38.76 39.21 61.63

28 25.87 26.32 49.95 s0.06 45.25 45.36 70.90

29 26.05 L16 26.05 1.39 53.00 I.51 51.99 1.04 46.20 1.39 47.76 1.28 69.22 1.39
Harlan Snudy Number: 414011,77 Page 2l

Tahle 2 Percentage Biodegradation Values

Day

7o Biodegradation

Procedure Control Test ltem Toxicity Control

0 0 0 0

2 73 15 42

6 76 6l 68

I 79 67 73

10 83 72 73

t4 83 72 75

2t 76 66 7l

28 80 64 75

29* 88 70 72

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2
Harlan Study Numher: 41401177 Report Page22

Table 3 Total and Inorganic Carhon Yalues in the Culfure Vessels on Day 0

Rr - Rz : Replicates I and 2

+ Corrected for conrot values. Negative values are due to measured concentrations being less than control values

Test vessel

Total Carbon*

(ms/L)

Inorganic Carbon*

(mg/L) IC Content (% of TC)

Test ltem

l0 mg C/L RI

8.87 -0.01 0

Test Item

10 mg C/L R2

8.83 -0.01 0
Harlan Study Number: 41401177 Page23

Test Vessel

pH

Day 0

Pre-Adjustment

Day 0

Post-Adjustment Day 28

lnoculum Conffol R1 7.8 7.4 7.5

lnoculum Control R2 7.8 7.4 7.4

Procedure Control R1 7.8 7.4 7.5

Procedure Control R2 7.8 7.4 7.4

Test Item R1 7.8 7.5 7.4

Test ltem R2 7.8 7.4 7.4

Toxicity Control 7.8 7.5 7.5

Table 4 pH Values of the Test Prepar*fions on Days 0 and 28

Rr - R, : Replicates I and 2
Harlan Study Number: 4l40ll7'1

I

I

Report Page24

Table 5 Observations on the Test Preparations Throughout the Test Period

Test Yessel

Observations on Test Preparations

Ilay 0 Day 6 Day 13 Day 20 Day 27

Inoculum

Control K1

R2

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Light brown

dispersion

Procedure

Control R1

R2

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Light brown

dispersion, no

undissolved

reference item

visible

Test Item R1

&

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

rurdissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Light brown

dispersion, no

undissolved

test item visible

Toxicity

Conhol

Light brown

dispersion, no

undissolved

test or

reference item

visible

Light brown

dispersion, no

undissolved

test or

reference item

visible

Light brown

dispersion, no

undissolved

test or

reference item

visible

Light brown

dispersion, no

undissolved

test or

reference item

visible

Light brown

dispersion, no

undissolved

test or

reference item

visible

Rr - Rz : Replicates I and 2
Harlan Study Number: 41401177 Report Page 25

FIGURES

F igure I Biodegradation Curues

100

c

.o

o

EI850

E) o

o

s

Sodium benzoate

-FTest ltem

- -a - - Toxicity Control

10 Day \\ilndow
Harlan Study Number: 41401177 Report Page 26

APPENDICBS

Appendix I Certificate of Analysis

Jfsr+mr*,,-"." .*pptErafis?

Hftr{+-lll}E*tiorr

s!il*f

Hsr.ny'fitct$li 1ra FUI

tnsfiih'su$rqt FCG)"

"Lrf,S"

Lrr*E an drylrig

ffi t-rr:lv*ro'

Sodlum EryCl+erh ,

r-ffC Grndt l(oshrr

(IttE,Foil Gs&E so92 ldJtfurntrn 148t314 Frcdtrctlpn.trril: lillr 20- ?Ot4 ft*rlxrlultlon Ehtr: [arr.8*. efiE

Eauror *odrl 01O{lSI-gI Froiloryr ffitg Prdm, Drl*rg. Jirrgrr,Cl*r

F'0drrct Gcdrr 6ea$l t (26 lrgil crrbn)

It't niffi. ir$r,

;1li1".rr'rn'rirur:

l.!.!:j#! r):i*-r- r

Ii. tI

q i;1J

*+r.'f+rl,=:

s6 51}',

Ox*t*tr ?JE lrilr; iErti

ssocilrc to.Hqtgn tgbj* - - 

.ih*j I ,vE 0l

Gsrdir*rt Ttrh rnffi drdin,Pflrdudlggrdfidofl il$druEilt|.

Atora.m#st rErfilffiu{*r,a:r*riltrr',firqirt,*#d}tf+t*,e*iiltfif tit.F.t6iitri*l*ryr 

. 

€A

ster*d:.hr; yr**{144l

Tr*i dffirrlf{ }rD lrii iltfi#rn

UdF *;r

J.r"{ss,tt{Etlr*rr: tr* atll

f rditt$t s,r -:r,S*4 {4F !1"}.3l;t S

r irE{ '

TF;k,re't'r+ec
Harlan Study Nurnber: 4I4AU77 Report Page27

Appendix 2

Solution a

Solution b

Solution c

Solution d

Mineral Medium

KH2POa

KzHPO+

NazHPO+.ZHzO

NH+Ct

CaC12

MgSOa.TH2O

FeCh.6HzO

8.50 s/L

21.75 stL

33.40 s/I,

0.50 g/L

pH : 7.4

27.s0 slL

22.50 glL

0.25 g/L

To I liter (final volume) of purified water* was added the following volumes of solutions

a-d.

10 mL of Solution a

I mL of Solution b

1 mL of Solution c

I mL of Solution d

* Reverse osmosis purified and deionized water (Elga Optima l5+ or EIga Purelab Option R-15 BP)
Harlan Study Numher: 41401177 Reporl Page 28

Appendix 3 Ilissolved Organic Carbon (DOC) Values from the Preliminary

Investigational Work

In order to investigate whether the test item adsorbed to filter matrices and/or the activated

sewage sludge the following samples were analyzed for Dissolved Organic Carbon (DOC) using

a Shimadzu TOC-Vcss TOC analyzer.

These results indicated that the test item did not adsorb to filter matrices or activated sewage

sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were

filtered to remove the suspended solids present without causing a loss of any test item.

Sample

DOC Concentration

7o of Nominal

Carbon Content

mg C/L

mg C/L

Corrected for

Appropriate

Control

Mineral medium <LOQ

Control, inoculated at 30 mg ss/L, Filtered <LOQ

Control, inoculated at 30 mg ss/L, Centrifuged <LOQ

100 mgil, Untreated 32.1 8 32. t8 89

100 mg/L Filtered .32.51 32.51 90

100 mg/L, inoculated at 30 mg ssll, Filtered 31.97 3t.97 88

100 mg/L, inoculated at 30 mg ss/L,

Centrifuged 3 1.89 31.89 88

LOQ: Limit of Quantification (determined down to 1.0 mg C/L).
Harlan Study Number: 41401177 Report

d

Page 29

Appendix 4 Monitoring Authority Statement of GLP Compliance

{Dfr Department \- - of Heatth

THE T}EPARTHEHT OF HEALTfI OF THE GOVERHilJIHHT

oF THE UHTTG.fi KINGD$I'I

SGfi N tABOEfrfrOftY PRAIETICE

STATETITENT OF

IH ITSGORDAHCE TYITH

TEST FAC'LITY

Harlrn t afromtorirs Linnftnd

Sherdlow Bt*lrror Frrk

London Ft$fid

$ha,rdlou

Osrbltlhlrs

T}E?T TGN

GOTTPLIAHGE

BI#IHCTIVE EOO*'SIEG

TEST TYPE(S)

Ardytlee l/GIIn lcd Ch*nist4r

Env*rorrmentfil F*ts

Envilronrn*:rtal Toxlnitlr

Fhyr.fihem" Tt*tlng

Itlut*gnnleity

Toxlcaiegy

D*"TE fiF IHSPEGT*OH tg to t4 H,*rch ffit{

An inspsction fior *ompt*anr.e with th€ Principles of Good Lsboratot y Frac,lce was

carriad out at the ahotte test facility as psrt of the UK Good Lebo'rstory Freetice

Com plianue Monitoring trrCIg ra rnrn€.

Thin etatarytsfit confirmc that- un the date *f iseue, thCI UK Good Laborato,ry

Frsctlcs lutonitoring Authority rrrere satiEfied thst the flbsvo trst tauility was

oparating in oornpliance with the OEOD Principlae nf {isod Labortaiary Fractice.

This statement constitutes a Go#d I'absratory Practim lnstrument {as defined in the

UK GCICId l-ehwatrry Proc,tlce R*gulatione 1S99)"

vw-.;*-S*ffi-*k*

a* /* l,,t

Sr" Andrerv J. Br*y

Hmsd, Ut( GLF tllenltorJns *uthority