## 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
CBICControl 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 

### FinalTest Report 

### onthe Biodegradation Study 

### by Univat USA 

### Inc. under the

### TSCA Section 4

### Test Rule on

### the Testing of Cettain 

## lligh Production Volume Chemicals: Second 

### Gtoupof 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) 

### thefinal test 

### reporton 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 aToxic Substances h) ffi efl *T1 t.t f\1

*l F+ -If

### frt -11f-:: -.1 *.

. - ri I ! r-r -:-iU.S. Environmental Protecdon Agency February 9,2075 Page 2 

### Conuol 

## Act (ISCA) Section

## 4 test accordaflce 

### with thistest 

### rule and the 

## I. Genetal 

### Backgtound CONTAINS 

### NO CONFIDENTIAL BUSINESS 

### INFORMATION



### rule.lThe Comparry submits 

### thisfinal test 

### report in regulations set

## foth at 799.5087.


### As a matter

## of backgtound,

### this test 

### rule addresses 

### 19 

### high productionvolume (HP\D chemicals

### that 

### weflt unsponsoted 

### under theAgency'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 

### toxicityto fish Acute 

### toxicity to Daphnia 

### Toxicity to plants (algae) 

### In the

### finalTSCA Section 4

### testrule, EPA specified the use

### ofASTM, 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, theAgency 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 

### notyet begun 

### importing sodium erythorbate 

### with the 

### intentto sell

### it for TSCA purposes 

### atthe 

### 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. 2Id. at 1069. 3With respect to the biodegradation study, the Company requested the use of OECD test guideline 3018 for the biodegtadation test. T T ICONTAINS 

### 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 

### deadlinedue

### to the factthat there has 

### beena 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 hasalso indicated that 

### this same technical issue is 

### being encountered 

### during the 

### paftition coefficient test, 

### though 

### to asomewhat 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 4test 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 advisedthe 

### PCBTF Testing Consortium (ofwhich Univar is a member) 

### in early 2013 

### that robust summaries were 

### not necessary 

### undertheTSCA Section

### 4 

### HPV test rules.

## If EPA decides 

### that 

## it wishesto teceive such 

### robust summaries, 

### Univar 

### will certainly 

### provide them.

aUpon EPA's receipt of this January 27,2A1,4Ietter of intent to test, Univar planned to begin importing this substance for both TSCA purposes andfor continuing exempt FDA pulposes. sIt 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. 6This was also conveyed to the Agency in an electronic mail to N'Ir.John Schaeffer 

### trPA) from Mr. John

### \'. Nfassingale (Greenwich Chemical Consulting) onJanuary 29, 201,5.U.S. Environmental Protection Agency February 9,2015 Page 4



### Univar thanks theAgency 

### 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 usethe 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: iolr n(rDrrr:cci. 

### com

@,r'

### FeGBs"



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OPPT Document Control Office U.5. Environmental Protection Agency EPA East Building Room E6428 1201 Constitution Ave., NW Washlngton, DC 20004 Attn.: TSCA Section 4 (40 CFR 799.5087) ed my of i6 rub.cqhr I Frr! | le for umrpt by FedEx- md thar the rhipoenr i! p.o,p€rly mtkGd. d*E!!!d .d rt I I - | mem ofte rme.



### tr-l ErJ--,tIFtErsf,ffiurE^E,Ij,e

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- I I rb tuspded p.nry orsotery by rca4 bc d'ffi m *plioir !..rl-!.!,i 1 -l l d;qnan^iuBdbrdrdrrho rd-bftdEEll-l.ia.E,q1;a!.driBitu 8nF lmruuu oI xaDilrq I I hfll rcgulrtim., ffi, ffrequMcnb, taGxB mExEu ti.bitity fn ts., ddrgq I ^ _ I tu.s. J20 J8 r kito) tor equiv.t.nr to*l cenoy ff &c d''cr!)*tu I t;DEEr.rrrrrr-u t uErrrr-^r I r.tLrcrrr.rrN^ I Nr^ | qi&d"*b-i+bd-.d. .-ryormsm)un'Im*d 

### '! UREENWICH LHEMTcAL LONSULTING, tNC. I fiH;L*iffiH'Jiiffi:::ilTiiliiiXJ,i.iil or nrE rh.* in effet ', , I

I - | r.r{letur-rqt }ftL,r- d..redvslucftr.eils. I 44(lll lr(.4 Horrr FVAR|I I \IIITF fi.{\ I t"*"yl.whiohevtrissEst€r.Incmrdr,rhemsimu

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## o I LHf;Ef'.it'#Sf,I#ft#' sERvrcEcoN,.DITIoN I I t ANy DArlrAcEs, wrsr.HER DIRECT,IN'DIRECT. NCDENTN,,SPECIAI I I NADANEXCESSoFTHEDECLAmDVilI]EFoR



### tt I I d&FGdbr*rF!-ritu,e I I db.*rqd.!*iiEtrdru 5-r@Fri-- I I w!.&&rtt4@,-,ucbbbr 

## Hi:H#-"H I oPPT Document control

## office I f,r-*Hj;'5i-*.#i*

I U-5. Envllonmental PrOIaCrlOn AtenCV l ri!&r&lfub drd.!. I -' I d@rye{!-L4r6dFGdr 

## -ffi:f. l EPA East

## Building l :iIs,ffi:*i'jI#ffi
incJudhs fa rclrrcd to r I ^ I y,u.rr *mhduiint ticn pordlrg dirpirim, Cutmr



### tt I l rnl a^^!+i+r+i^^ /tva Nrl, I !Et5.!qr!.'ddk,ed{tu ef,EDrdre'r I dLdh,Edr&Llr-b jl|r 

# ffi:ilElwashincton.Dc2oo@lX:'"ffiffi,'F;#

| - | 4-r&riddturt&l&rr li, b.iEr{dLrr I I rr-!ryl6.h4bkk(4ffir sffipsyirSdtrumdl.tin I ^.^- . TrrA ---!:-- ^ l a^ F-n ?^n rn6-l I bnvedallitrfomrtionofmynd*rc 

## ;trffi | Attn.: TSCA Section 4 (ilo CFR

## 799.s087) I tr""-B,L?,H*,ffi.:

tt tt I I r{od***irurltt*& I I !*rld-k..r6.n-r!H



### https://3pl.freedomffship.com/viewlabel.php?airbill:7801 83 

### 147614&,carrier:FedEx 21912015

# horlolr* r*. 

## 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 DE72zGD 

### UK Univar USA 

### Inc. 17425 NE Union 

### Hill Road Redmond 

### wA 98052 

### LNITED STATES OF

### AMERICA

41401177 28 January 2015

Page 

### I of29Harlan 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.....

...... 19Harlan 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 inthe Culture Vessels on Day 0............ .......22 Table

## 4 pH Values of the Test Preparations on Days 0 and

## 28.......... .................23 Table

## 5 Observations on theTest PreparationsThroughout the Test

## Period ......24 

## LIST OF FIGURES


## LIST OFAPPENT}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 MonitoringAuthority Statement of GLP Compliance 

## ........... ........29Harlan 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 asamended 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 anddata generated. There were no circumstances consideredto have affected the integrity of the study orthe

### validity of the data. Study

# Director: N Best



### 2 

### S JAN 2015Harlan 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 andactivities are inspected at least once ayear andthe 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 aregiven below.

Dates and Types 

### ofQA Inspections Reported tothe 

### relevant Study 

### Ilirector andTest 

### 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 thatthis report reflects the raw data and the procedwes followed. 

### eualityAssurance:

# trruil

Date: 

# 2I JAN 20t5Harlan Study Number: 41401177



## SUMMARY Introduction 

## A studywas performed 

### to assess 

### the ready biodegradability 

### of thetest 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 OCSPP835.31l0 (Paragraph (m)). 

### Methods The test 

### item, ata 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 between20 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 strictterms 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 Therewere 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 chargeableto the Sponsor. 

### No data

### will be discarded without contacting the Sponsor to obtain theirwritten 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 

### itemwhen exposed

### to sewage sludge microorganisms under aerobic conditions. The results 

### of the study are

### ofvalue 

### in the assessment 

### of the persistence 

### of thetest item 

### in the aquatic environment. 

### This study was performed basedon the U.S. 

### Toxic Substances Control 

### Act (TSCA) Section 4 Test rule and the regulations setforth under40 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 inthe dark not supplied

Sodium benzoate white granular solid SLBGgs15V >99.sYo 09July 2015 roorntemperature over silica gel Identification: Description: Batch: Purity: Expiry Date: Storage conditions:Harlan Study Number: 41401177 Page l0



## MATERIALSAND 

## METIIODS Definitions and

### Abbreviations 3.1 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-Daywindow 

### 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 isthe sum 

### of the organic carbon 

### in solution and 

### in suspension. 

### This is the organic carbon present 

### in solution 

### or that 

### which passesthrough 

### a 0.45 

### pm

### filter or remains 

### in 

### the supernatant 

### after centrifuging 

### at approximately 4000g

### 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 stageofthe 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 samplewas 

### 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 suspendedsolids 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 wasthen continued 

### for afurther 3 minutes after rinsing the

### filter three successive times 

### with 

### l0 mL of deionized reverse osmosis 

### water. The

### filter paperwasthen dried 

### in an oven atapproximately 105

### 'C for at least 

### I hour and allowed to cool before 

### weighing. This process was repeated 

### until aconstantweight was attained. The suspended solids concenhation was equal 

### to 3.0

## gll prior to use.

* Rinsed three timeswith 20 mL deionized reverse osmosis water prior to drying in an ovenHarlan 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 

### thetest 

### item adsorbed 

### tofilter 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 ashimadzu 

### 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 

### weretaken

### 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 aconcentration 

### of 30 mg suspended solids (ss)/I- prior to adjusting 

### to a

### final volume

### of 

### I liter. 

### Two samples 

### weretaken

### 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 Thetest 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) andthe volume adjusted

### to 1

### liter to give a 300 mg/I- stock solution. 

### An aliquot (276 

### mL) ofthis 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 thetest 

### item was inverted severaltimes to ensure homogeneity of the solution. 

### A test concentration 

### of 

### l0 mg carbon/L was employed in the test

### following the recommendations of theTest 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 stocksolution was added 

### to the testvessel containing inoculated 

### mineral medium and 

### the volume adjusted

## to3 liters

### to give 

### afinaltest concintration 

### of 17

### .I mglL, equivalent 

### to 10 mg 

### carbon/L. The 

### volumetricflask containing the reference item was inverted severaltimes to ensure homogeneity of the solution. 

### 3.5.2.3 Toxicity Control 

### A toxicity control, containingthetest 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 atotal

### 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 

### afinal concentration 

### of 20 mg carbon/L to act as a

### toxicity control (one vessel only). 

### Data 

### fromthe 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 

### testwas carried 

### out 

## in a temperature 

### controlled 

### room flt temperatures between 20 and 22oC, in darkness. Approximately 24 hours 

### prior to addition 

### of thetest and reference itemsthe vessels were 

### filled 

### with 2400 

### mL of mineral medium and 30.0 

### mL of inoculum and aerated

### overnight. On Day 0 thetest and reference items were added and the 

### pHofall vessels measured using a Hach HQ40d Flexi handheld meter andthe pH was adjusted to 

### pH 7.4 

### + 0.2 using diluted hydrochloric acid orHarlan Study Number: 41401177 Page 13

sodium hydroxide solution 

### priorto 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 30to 100 

### ml/min pervessel 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 

### Day28, 1

### mL of concentrated hydrochloric acidwas added 

### to each vessel

### to drive 

### off any inorganic carbonates

### formed. The vessels were resealed, aerated overnight andthe

### final samples takenfrom both absorber vessels on Day 29. The sampleswere analyzed 

### for IC using aTekmar-Dohrmann

### Apollo 9000

### TOC analyzer 

### or a Shimadzu TOC-VcsH TOC 

### analyzer. Samples (300or 50 

### pL) were injected into the 

### IC channel 

### of the

### TOC analyzer. 

### IC analysis occws 

### by means 

### of the conversion

### of an aqueous sampleto CO2 

### by orthophosphoric 

### acid using zero grade 

### air as

### the carrier 

## gas. Calibration was by reference solutions 

### of sodium carbonate OtrazCO3). Eachanalysis wasca:ried out intriplicate.Harlan Study Number:4l4Dll77 Page 14



## 3.6.4 IC/TC Ratio Samples (30 

### mL) were removed 

### from the inoculum control and test item vesselson 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-LcsuTOC 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 asthe 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 intriplicate. 

# 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
6x 

### 12'ol1 x

### loo -36.lg0/o 199.1l Thus for aconcentration 

### 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 molwt

### 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 benzoateHarlan 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 COzevolution 

### in the inoculum control vessels atthe end

### of thetest 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 sametest the reference item yields > 60Yodegradation 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 thetest item to be considered as non-inhibitory. The test is considered 

### valid 

### if the difference 

### ofthe extremes

### of replicatevalues 

### of production

### of COz atthetime the plateau is reached, at the end

### of thetestor atthe end

### ofthe 10-Day window, iffiappropriate, is less than}}o/o. Thetotal COz evolution in the inoculum control vessels atthe endof 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 carbonto carbon dioxide is 3.67Harlan Study Number: 41401177 Report Page 16

values up 

### to 70 

### mg/L areacceptable. Data 

### from studieswhere 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 atthe beginning 

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


## RESULTS 

# 4,1 Preliminary lnvestigational

## Work The results obtained

### from 

### the samples

### taken 

## for DOC analysis 

### fromthe preliminary investigational 

### work indicated 

### that the test 

### item 

### did not adsorb 

### to 

### filter matrices

### or activated sewage sludge (seeAppendix 

### 3). Therefore, 

### for the purpose 

### of the study, the samples taken

### for DOC analysis were 

### filteredto 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 thetest preparations on Days 0 and28 are given 

### in Table

## 4. Observations made on the contents 

### of the test vessels are given 

### in Table 5. 

## 4.2.1 Validation Criteria Thetotal COz evolution inthe inoculum control vessels on Day 28 was 31.88 mg/L andtherefore satisfied thevalidation criterion given intheOECD 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 thevalidation criterion given in the OECD Test Guidelines. 

## 4,2.2 Biodegradation 

### Acidification 

### of thetest 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 presentanddrives 

### 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 istaken as being thefinal 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 absorbervessels on Day 29 confirmed that no significant carry-over 

### of COz intothe 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 thatthetest 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 underthe strictterms 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 1.39Harlan 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 29values corrected to include any carry-over of CO2 detected in Absorber 2Harlan 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 (% ofTC)
Test ltem l0 mg C/L RI 8.87 -0.01 0

Test Item 10 mg C/L R2 8.83 -0.01 0Harlan 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 theTest 

### Prepar*fions on Days 0and 28

Rr

### -

## R, : Replicates 

### I and 2Harlan Study Number: 4l40ll7'1 I I
Report Page24



### Table

## 5 Observations on theTest 

### PreparationsThroughout 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 2Harlan Study Number: 41401177 Report Page 25



## FIGURES
F

### igure 

## I Biodegradation Curues

100

c .o o EI 

### 850 E) o o 

### s Sodium benzoate



### -FTest ltem


### - -a -

### - Toxicity Control

10 Day \\ilndowHarlan Study Number: 41401177 Report Page 26



## APPENDICBS Appendix

## I Certificate ofAnalysis

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## ffi t-rr:lv*ro' 

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### (IttE,Foil Gs&E so92 

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frditt$t s,r -:r,S*4 {4F !1"}.3l;t S rirE{ ' TF;k,re't'r+ecHarlan 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 aShimadzu 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 7oof Nominal Carbon Content mgC/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
100mgil, 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



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