### Assessment 

### of Ready 

### Biodegradebilityi COz 

### Evolutro" 

# ,J 

# har{sff & gt.r3 

### REFORT

Study Dire*tor: Test Fecilit-v:

$ptnsor:



### H*rl*n Study Numhcr: $fudy Completion Drtet C Bayliss Ifarlan Lrbomtories Ltd. Shardlglnr Busiress Park Shardlow Derbyshire DE?? ?GI} UK

{tx0?ot0
01 O€tober

### f0l3

Page

### I of27Harlan StudyNumber: 41207010


## CONTENTS Report Page 2

CONTENTS 2 STUDY DIRECTOR STATEME. IT OF GLP COMPLIANCE 4 QUALITYASSURANCE STATEMENT.. 5 SUMMARY 6 Introduction 6 Methods 6 Results 6 GENERAL INFORMATION 7 Schedule 7 Deviations 

### from Study Plan 7 Archiving 7 1 INTRODUCTIONAND PURPOSE 8 1.1 Guidelines / Regulations 8 2 TEST AND REFERENCEITEMS 9 2.1 Test Item 9 2.2 Reference Item 9 3 MATERlALS AND METHODS 10 3.1 Definitions and Abbreviations 10 3.2 Test System 10 3.3 Preparation ofInoculum 10 3.4 Mineral Medium 11 3.5 Procedure 11 3.5.1 Pre-Study Solubility Work 1I 3.5.2 Experimental Preparation II 3.5.3 Reference Item I I 3.5.4 Toxicity Control 12 3.5.5 Preparation afTest System 12 3.5.6 Sampling and Analysis 13 3.5.6.1 CO, Analysis 13 3.5.6.2 Inorganic Carbonffotal Carbon Analysis 13 3.5.6.3 pH Measurements 14 3.5.7 Evaluat.ion ofData 14 3.5.7.1 Detennination ofCarbon Content 14 3.5.7.2 Percentage Degradation 14 3.5.7.3 Validation Criteria 15 4 RESULTS 16 4.1 Definitive Test 16 5 CO CLUSION 18 TABLES 19 FIGURES 24 APPENDICES 25Harlan StudyNumber: 41207010


## LIST OFTABLES Report Page3

Table 1 Table 2 Table 3 Table 4 Table 5 Inorganic Carbon Values on Each Analysis Occasion 19 Percentage Biodegradation Values 20 Total and Inorgar:tic Carbon Values in the Culture Vessels on Day 0 21 pH Values ofthe Test Preparations on Days 0 and 28 22 Observations on the TestPreparations Throughoutthe Test Period 23 

## LIST OFFIGURES Figure I Biodegradation Curves 24 

## LISTOFAPPENDICES Appendix 1 Appendix 2 Appendix 3 Mineral Medium 25 Pre-Study Solubility Work 26 Monitoring Authority StatementofGLP Compliance 27Harftn Sh* Numbei: 41207010 Rryort



### STTIDY 

### I}IRECTOR STATEilTENT OF 

### GLP 

### COMPLIAI{CE Hadan rabonfories Ltrl., shaadlow Business Park, shtrdlow, Dabyshiru, DE?z 2GD, 

### uK Page d

Harlan Study Number: StudyTitle: 41207010 .-trr .'-.-. -. r ,t tia 7 Assessmc*r rlr rrv.--, -.*rdtgradability; COz Evolution Test

### - Wi& thc exeptioa nobd bclo*'this study nas pwformed in compliancc with UK GLP stardards (Selredulc 1, Cood Laboralory Prrctice Regulations teOe 1St t9994106 as amcnded by 

### SI2m#099f0. 

## lbs Rcgulatiotrs are in accordancc qd& GLP stardtrds publislwd as OECD ftinciples on Good taborafory Prasticc (rcvised tgg?, ENV/T\{CICHEM(9S)I?); aod arc in accordancc with, 

### ad implemcnt, the requiranarts of Dircctivcs 200419,€C and 2004/l01EC. 

### Th.T principlcs 

### art corrpatible E'ith Oood Laboratory Practice rcgulations specified by rcgutatory arnhorities throrryhotn

### fu Europcar Comnunity, th€

### Unitd St"lcs {EPAtnd 

### fO*j, md Japan (MHLW, MAFF and METD. No analysis trqs crricd out fo

### essrine thc hornogtrity, conccnration or sability of thc rcst itemformul*tion" This elccsion is considercd nor to "ffe"t thc purpose or integrity oitn" "nray. This rcport fully and accurd€ly rcflocts

### tk plocedures uscd and data generatd. Ttrcrc were no circtlmstancca consi&rcdto have afrccted the integrity of thc study orthc validityof rhc data Study ilircctor: C Saytis$Hgrlsa $udy' Numbs: .*t2{}?0I0 Repart Page 5



### QUALITY ASSURANCE 

### STATEMENT 

## lt#ffi:ttrtt N'mber:

# [ ^r-*: ofReady Biodesradability; co2 Evorr*ian

# r*f

The gencralfacitities and sctil'ities are inspccted at least once ayear and the results *re reportcd to rhe relcrrmt rcsponsiblc person and uranagement $tudy-rela:cd proccdurus'condrrted at thc tcst facility wcre audited aad inspmted. 

### Tb dctsils 

### of thcse audits md inspcctions 

### re given below.

Ilat*s rnd Typw sf QA Inspcctions 

## _*-. Repcrt*.d to the rcl*r,ant ,"$fud1- 

### Ilircttsr rrd T*tt Fecilitv Mrnrgement Date of Inspection Typc of Inspectioa Phase Insprcted Report Date 3t 

### ksmber?012 

### I* April 2013 S9April 2013 i0 April 2013 ?aApril 2013 ?6 Jdy 2013 $ndy Plan Verificaticn Prwes * bascd Process 

### - based Proccss 

### - based Proess$

### - based ReportAudit NIA

Tes ltem Prcparalion Test System Preparation Exposure Asscssment 

### of RcspoffieN/A 

### 3l Decernber 20t2 l0 April 2013 09April 2013 

# l

I I fl I 

### l l0 April 2013

I i 24 April 201

## 3

I 

# LuJuryzor3 |

This stdcment mnfrms drar this report reflects the raw dara urd the procedures followed. Qudiry Assurarrce:


### L-glRr.ez
t.attaatrartatrrl/7 

### {t 

## I $cr

## ffifiHarlan Study Number: 41207010


## SUMMARY Introduction Report Page 6



### 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 GECD Guidelines for Testing ofChemicals (1992) No. 301B, "Ready Biodegradability; CO, Evolution Test" referenced as Methnd C.4-C ofCommission Regulation (EC) No. 44012008 and US EPA Fate, Transport, and Transfonnation Test Guidelines OPPTS 835.3110 (Paragraph (m)). 

### Methods The test item, at a concentration of 10 mg CarbonIL, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 "C for 28 days. 

### The degradation ofthe test item was assessed by the determination ofcarbon 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 80% degradation after28 days and satisfied the lO-Day window validation criterion, whereby 60% degradation must be 

### attained within 10 days of the degradation exceeding 10%. The test itemcan therefore be 

### considered to 

### be readily biodegradable under the strictterms and conditions ofOECD GuidelineNo. 3018.HarlanStudy Number: 412070I0


## GENERALINFORMATION Schedule Report Page7

Experimental Starting Date: Experimental CompletionDate:


### Deviations from Study Plan l1lune 2013 10 luly 2013

There were no deviations (unplannedchanges) from the studyplan. 

### Archiving Unless instructed otherwise by the Sponsor, the study plan(general study plan and study specific supplement), all raw data (paperand electronic) and the final reportwill beretained inthe 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 ofthe data will be chargeable to the Sponsor. No datawill be discarded withoutcontacting the Sponsorto obtain theirwritten consent.Harlan StudyNumber: 41207010 Report Page 8


## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the ready biodegradability ofthe test item when exposed to sewage sludge microorganisms underaerobic conditions. The results ofthe study are ofvalue in the assessment ofthe persistence ofthe test item in the aquatic environment.


### 1.1 

### Guidelines/ Regulations This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recOlnmendations:

• OECD Guidelines for Testing ofChemicals (1992) No. 301 B "Ready Biodegradability; C02 Evolution Test"
• Method C.4-C ofCommission Regulation (EC) No. 44012008
• US EPA Fate, Transpon, andTransfonoationTest Guidelines OPPTS 835.3110 (Paragraph (m))Harlan S$dy Nunrber: 4120701S Repon Pag* 

### I



# 2 TESTAND REFERENCE 

### ITEMS 

# 2.I Tcst 

### ltem Infanrration as provided by the Sponsor. Identifieafion: Description: Batch: Prnity: Expiry Date: Storage Canditions: amber eolored liquid not supplied 19.570 ffIt supplied roorn tenperature in the dark 

# 2.2 Ref*renee 

### ltem Infonnatian as provided by thc

## Supplier.

, 

## Identification: $odiurn krrzoate 

## trssffiption: whitc solid 

## Batch: MKBH11$9V 

## Furity: SV/o Expiry

## Date: 26 June 2013 $torage

## c*nditions: roonr temperattrreHarlan Study Number: 41207010 Report Page 10


## 3 MATERIALS AND METHODS 3.1 Definitions and Abbreviations Readily biodegradable:

1O-Day window: Inorganic carbon (IC): Total carbon (TC): Total organic carbon (TOC): Dissolved organic carbon (DOC):



### 3.2 Test System Test items may 

### be considered to be readily biodegradable if 60% biodegradation is attained in a IO-Day window within the 28 days ofthe test. The 10 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 carbonpresent in a sample. This is the sum ofthe organic carbon in solution and In suspension. Dissolved organic carbon is the organic carbonpresent in solution or that which passes through a 0.45**~m** filter or remains in the supernatant after centrifuging at approximately 4000 gfor 15 minutes.

A mixed population ofactivated sewage sludge micro-organisms was obtained on 10 June 2013 from the aeration stage ofthe Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, whichtreats predominantly domestic sewage. 

### 3.3 Preparation oflnocuJum The activated sewage sludge sample was washed two times by settlement and resuspension in mineral medium to remove any excessive amounts ofdissolved 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 21°C and used on the day of collection. Determination ofthe suspended solids level ofthe activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through preweighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes afte-r rinsing the filter three successivetimes with 10mL ofdeionized reverse osmosis water. The filter paper was tben 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 concentrationwas equal to 4.3 gIL priorto use.

• Rinsed three times with 20 mL deionized reverse osmosis waterpriorto drying in an ovenHarlan StudyNumber: 41207010



### 3.4 Mineral Medium Repon Page 11



### The mineral medium used in this study (see Appendix 1) was that recommended m the DEeD 

### Guidelines. 

### 3.5 Procedure 3.5.1 Pre-Study Solubility Work Information provided by the Sponsor indicated that the test item was partly soluble in water. Therefore pre-study solubility/dispersibility work was performed in order to determine the most suitablemethod ofpreparation (see Appendix 2).
3.5.2 Experimental Preparation 

### Forthe purpose ofthe test, the test itemwas dispersed directly in mineral medium. 

### An amount oftest item (42.9 mg) was dispersed in approximately400 mL ofmineral medium with the aid ofhigh shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of14.3 mgIL, equivalentto 10 mg carbonIL. A test concentrationof 10 mg carbonILwas employed inthe test following the recommendations ofthe Test Guidelines. As it was not a requirement ofthe Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability ofthe test item formulation. This is an exception with regard to GLP andhas been reflected inthe GLP compliance statement.
3.5.3 Reference Item 

### For the purposes ofthe test, a reference item, sodium benzoate (C6

### Hs COONa), was used to prepare the procedure control vessels, An 

### initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly 

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

### aliquot (51.4 mL) 

### ofthis stocksolution was added to the testvessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbonIL. The volumetric flask containing the reference item was inverted several times to ensure homogeneity ofthe solution,Harlan SludyNumber: 412070I0



### 3.5.4 Toxicity Control Report Page 12

Forthe purposes ofthe test, atoxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge microorganisms used in the test. An amount oftest item (42.9 mg) was dispersed in approximately 400 mL ofmineral medium with the aid ofhigh shear mixing (approximately 

### 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) ofthe sodium benzoate stock solution was also added to the test vessel and the volwne adjusted to 3 liters to give a final concentration of l4.3 mg test itemILplus 17.1 mg sodiumbenzoate/L, equivalentto atotal of20 mg carbonIL.
3.5.5 Preparation ofTest System The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters ofsolution: a) 

### An inoculated control, in duplicate, consisting ofinoculated mineral medium. b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral mediumto give a final concentration of10 mg carbonIL. c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of10 mg carboniL. d) The test item plus the reference item in inoculated mineral medium to give a final concentration of20 mg carbon/Lto act as atoxicity control (one vessel only). Each test vessel was inoculated with the prepared inoculum at a final concentration of 

### 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at approximately 21 °C, in darkness. Approximately 24 hours prior to addition ofthe test and reference items the vessels were filled with 2400 mL ofmineral medium and 20.9 mL ofinoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pHlOxi 3401 pH and dissolved oxygen meter. Ifnecessary the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodiwn hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by tbe addition ofmineral medium which had been purged overnightwith CO2 free air. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of30 to 100 mUmin per vessel and stirredcontinuously by magnetic stirrer. The COrfree air was produced by 'passing compressed air through a glass column containing self-indicating sodalime (Carbosorbj granules.Harlan Study Number: 41207010 Repon Page 13

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de·gassed water.
3.5.6 Sampling and Analysis 3.5.6.1 CO, Analysis Samples (2 mL) were taken from tbe first CO, absorber vessels on Days 0, 2, 6, 8, 10, 14, 21,28 and 29. The second absorbervessels were all sampledonDays 0 and 29. All the samples were analyzed for CO2immediately. On Day 28, 1 mL ofconcentrated hydrochloric acid was added to each vessel to drive offany 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**CO:~** using a Tekmar-Dohnnann Apollo 9000 TOC analyzer. Samples (300**~L)** were injected into the IC (Inorganic Carbon) cbannel oftbe TOC analyzer. Inorganic carbon analysis occurs by means ofthe conversion ofan aqueous sample to CO2by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions ofsodium carbonate (Na2C03). Each analysis was carriedout intriplicate. 3.5.6.2 Inorganic CarbonITotal CarbonAnalysis Samples (30 roL) were removed from the test item vessels on Day 0 prior to the addition ofthe test item in orderto calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45**~m** Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45**~m** Gelman AcroCap filters (first approximate 5 mL discarded in orderto pre-condition the filter) priorto DOC analysis. ICITC analysis ofthe test item dispersions after dosing was not possible due to the insoluble nature ofthetest item in water. The samples were analyzed for IC and TC using a Shimadzu**TOC~VCPH** TOC Analyzer. Samples (50**~L)** were injected into the Total Carbon (TC) and Inorganic Carbon (lC) cbannels ofthe TOC analyzer. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (CsHsK04) and sodium carbonate (NalC03 ) in deionized water. Each analysis was carriedout intriplicate.Harlan StudyNumber: 41207010

3.5.6.3 pH Measurements Report Page 14

The pH ofthe test preparations was determined on Day

# aand on Day 28 prior to acidification with hydrochloric acid, using a

### WTWpHlOxi 

### 3401 pH and dissolved oxygen meter.


### 3.5.7 Evaluation of

### Data 3.5.7.1 DeterminationofCarbon Content The test item contains 70% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg 

### C/L (a total of42.9 mg of test item in 3 liters) the total organic carbon present was 30 mg C. The theoretical amolUlt ofcarbon present in the reference item, sodium benzoate (C6HsCOONa) was calculated as follows: NoofCatoms x mol wtofC x 100

mol wtofsodiumbenzoate 

### 7 

### x 12.0I I

### xI00 58.34% 144.11 Thus for a 10 mg C/L test concentration (a total of51.4 mg ofsodium benzoate in 3 liters) the total organic carbon presentfor sodiumbenzoate was 30 mg C.
3.5.7.2 Percentage Degradation The percentage degradation or percentage ofTheoretical Amount ofCarbon Dioxide (ThC02) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values ofReplicates Rl and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation: fig ICin test flask

-mg 1Cincontrolflask**%ThC02(~%degradatjon')=** TOe dd d h' I 

### xlOO mg a e as testc emlca The total C02 evolution in the inoculum control vessels at the end ofthe test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:

• Theconversion factor for carbon to carbon dioxide is 3.67Harlan Study Number: 4 1207010

Total CO2 evolution (mg CIL)



### 3.5.7.3 Validation Criteria Report Page 15

IC . I 100

__,-:-_ = 

### mg mcontro x x %CofCO2 

### testvolume



### . 100 1 =: 

### mgIC In 

### control x-- x

### 27.29 3

The results ofthe degradation test are considered valid ifin the same test the reference item yields 60% degradationby Day 14. The test item may be considered to be readily biodegradable if 60% degradation is attained within 28 days. This level ofdegradation must be reached within 10 days ofbiodegradation exceeding 10%. The toxicity control (test item and sodium benzoate) should attain 25% degradation by Day 14 for the test item to beconsideredas non-inhibitory. The test is considered valid ifthe difference ofthe extremes ofreplicate values ofproduction of CO2 

### at the time the plateau is reached, at the end ofthe test or at the end ofthe lO-Day window, as appropriate, is less than 20%. Thetotal C02 evolution inthe inoculum control vessels at the end ofthe test should notnormally exceed 40 mg/L medium (= 

### 120 rng/3 liters, corresponding to 33 mg C per flask), however values up to 70 mgIL are acceptable. Data from studies where values in 

### excess of70 mgfL are obtained shouldbecriticallyexamined. The IC content ofthe test item suspension in the mineral medium at the begirming ofthe test should be <5% ofthe TC.Harlan StudyNumber: 41207010


## 4 RESULTS 4.1 Definitive Test Report Page 16



### Inorganic carbon values for the test item. procedure comrol. toxIcity control and inoculum control vessels at each analysis occasion are given in Table 1. 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 1. 

### Total and Inorganic Carbon values in the culture vessels on Day 

### 0 are given in Table 3. The pH values ofthe test preparations on Days 

### 0 and 

### 28 are given in Table 4. Observations made on the contents ofthe test vessels are given in 

### Table 5. Thetotal C02 evolution in the inoculumcontrol vessels on Day 

### 28 was 

### 30.72 mgIL and therefore satisfied the validationcriterion given inthe OECD Test Guidelines. The IC content of the test item suspension in the mineral mediwn at the start of the test (see Table 3) exceeded the recommended level of5% ofthe TC content given in the OECD Test Guidelines. This was considered to be due to the low TC concentration in the mineral medium and hence the IC contribution was relatively high. This was considered to have had no adverse effect on the study given that the CO2 

### evolution in the control vessels satisfied the validation criterion given in the OECD Test Guidelines. The difference between the values for CO2 

### production at the end ofthe test for the replicate vessels was 

### <20% and hence satisfied the validation criterion given in the OECD Test Guidelines. Acidification ofthe 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 effectivelykills the micro-organisms present and drives offany dissolved CO2

### present in the test vessels. Therefore any additional C02 detected inthe Day 

### 29 samples originated from dissolved C02 that was present in the test vessels on Day 

### 28 and hencethe biodegradationvalue calculated from the Day 

### 29 analyses is taken as beingthe final biodegradationvalue for the testitem. The results ofthe inorganic carbon analysis ofsamples from the first absorber vessels on Day 

### 29 showed an increase in all replicate vessels, with the exception ofprocedure control R] and R2• 

### This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis ofthe samples from the second absorbervessels onDay 

### 29 confirmedthatno significant carry-overofCO2

### into the second absorbervessels occurred. Thetest item attained 

### 80% degradation after

### 28 days and satisfied the IO·Day window validation criterion. whereby 

### 60% degradation must be 

### attained within 

### 10 days of the degradation exceeding 

### 10%. The test item can therefore be considered to be readily biodegradable under the stricttemlS and conditionsofOECD Guideline No. 

### 301B.Harlan StudyNumber: 41207010 Report Page 17



### The toxicity control attained 66% degradation after 14 days and 71% 

### degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used inthe test. Sodium benzoate attained 107% degradation after 14 days and 116% degradation after 28 days thereby confirming the suitability ofthe inoculum and test conditions. Degradation values in excess of100% wereconsideredto bedue to sampling/analytical variation.Harlan Study Number: 41207010


## 5 CONCLUSION Report Page 18

The test item attained 80% degradation after 28 days and satisfied the IO-Day window validation criterion. whereby 60% degradation must 

### be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradableunderthe stricttenns and conditions ofOECD Guideline No. 301B.Harlan Study Number: 41207010 

# TABLES Report Page 19



### Table 1 Inorganic Carbon Values on Each Analysis Occasion
Test Hem Sodium Benzoate plus Sodium Inoculum Control (mg IC) Procedure Control Test Item (mg IC) BenzoateToxicity (mg IC) Control Day (rnA IC) 

### R, R, R, R, R, R, R, Ahsi Abs2 Abs I Abs2 Abs 1 Abs2 Abs 1 Abs 2 Abs 1 Abs 2 Abs I Abs2 Abs t Abs2 0 2.22 2.10 2.10 1.75 2.10 1.87 2.10 1.98 1.75 2.10 1.75 2.10 1.98 2.22 2 8.35 

# - 8.24 

# - 1t.25 

# - 11.25 

# - 10.09 

# - 9.74 - 25.40 

# - 6 9.46 

# - 26.64 

# - 40.94 

# - 13.15 

# - 34.25 

# - 31.72 

### - 50.86 

# - 8 7.68 - 30.39 

# - 44.72 

# - 11.0I 

# - 37.15 

# - 33.60 

### - 54.24 

# - 10 19.26 

# - 20.75 

# - 47.31 

# - 45.03 

# - 41.49 

# - 35.34 - 58.48 

# - 14 24.59 

# - 24.14 

# - 57.01 

# - 55.99 

# - 47.49 

# - 42.05 - 64.03 

# - 21 25.46 

# - 27.60 

# - 61.18 

# - 60.28 

# - 53.18 

# - 47.43 - 69.29 

# - 28 26.32 

### - 23.97 

# - 62.39 

# - 63.73 

# - 51.07 - 49.62 - 68.54 

### - 29 26.72 3.48 30.39 4.06 60.12 7.31 60.79 5.80 54.1 t 2.78 50.77 3.13 70.90 4.29

R1 - Rz = Replicates I and 2 Abs

- CO2absorber vesselsHarlan StudyNumber: 41207010 Report Page 20



### Table2 Percentage Biodegradation Values

% Ingradation -/0 Degradation Day Sodium Benzoate 

### -I_ Degradation Test Item plus Sodium TestItem Benzoate Toxicity Procedure Control Control 0 0 0 0 2 10 5 29 6 30 50 55 8 29 54 59 10 87 61 64 14 107 68 66 21 114 79 71 28 126 84 72 29' 116 80 71

- Day29 values corrected to include any carry-over ofCO2detected in Absorber 2Harlan Study Number: 41207010 Report Page21

Table3 

### Total and Inorganic Carbon Values in tbe Culture Vessels on Day 0
Test vessel Tolal Carbon* Inorganic Carbon* IC Content (*I. ofTC) (mglL) (mglL) Test Item 10.70" 0.73 7 10 mg C/L R1 TestItem 10.36" 1.21 12 10 mgC/L R2

R1- R2 ::II Replicates I and 2
• Corrected for control values.

.. TOlal carbon value given is the sum oflhe TC value oblained from analysis and !he nominal TC contribution of the test itemHarlan StudyNumber: 41207010 Report Page22



### Table4 pHValues ofthe TestPreparations on Days0and 28 pH pH Test Vessel priorto after pHon Day28 adjustment adjustment

on Day 0 on Day 0 Inoculum Control R1 7.8 7.2 7.3 Inoculum Control R z 7.7 7.3 7.3 Procedure Control 7.8 7.5 7.3 10mg CIL R 1 Procedure Control 7.7 7.4 7.2 10 mg CIL R 2 TestItem 7.7 7.4 7.2 10 mg CIL R j Tesl Item 7.7 7.3 7.1 10mg CIL R z Toxicity Control 7.7 7.3 7.2 20 mg

## ell

R

l - R 2 "" Replicates Iand2Harlan Study Number: 41207010 Report Page 23



### Table 5 Observations on the TestPreparations Throughoutthe TestPeriod
Observations on Test Preparations Test Vessel Day 0 Day 6 Day 13 Day 20 Day 28
Inoculum 

### R, Pale brown Pale brown Pale brown Pale bro'A'll Pale brown Control dispersion dispersion dispersion dispersion dispersion


### R, Pale brown Pale brown Pale brown Pale brown Pale brown dispersion dispersion dispersion dispersion dispersion
Pale brown Palebro
....n Pale brown Palebrown Pale brown Procedure dispersion no dispersion no dispersion no dispersion no dispersion no

Control 

### R, undissolved undissolved undissolved undissolved undissolved reference item reference item reference item reference item reference item visible visible visible visible visible Pale bro"'ll Pale brO\'1l Pale brown Pale bro'All Pale brown dispersion no dispersion no dispersion no dispersion no dispersion no 

### R, undissolved undissolved undissolved undissolved undissolved reference ilem reference item rderence item reference item reference item visible visible visible visible visible Pale brown Pale brown Pale brov.n Pale brown Pale brown dispersion no dispersion no dispersion no dispersion no dispersion no TestItem 

### R, undissolved test undissolved test undissolved tesl undissolved test undissolved item visible item visible item visible item visible test item visible Pale brown Pale brown Pale brown Pale bro'A'll Pale brown dispersion no dispersion no dispersion no dispersion no dispersion no 

### R, undissolved test undissolved test undissolved test undissolved test undissolved item visible item visible item visible item visible testitem visible Pale brown Pale brown Pale brown Pale brown Pale brown dispersion no dispersion no dispersion no dispersion no dispersion no Toxicity undissolved test undissolved test undissolved test undissolved test undissolved Control orreference orreference orreference orreference test or reference item item visible item visible item visible item visible visible

R1 - R2 "" Replicates 1 and 2Harlan StudyNumber: 41207010 Report Page 24


## FIGURES Figure 1 Biodegradation Curves 

# I
• Sodium benzoate 0 TeSl11em



## - 

# - " - - T o)(icity Control 

# ./ 

# I
0 0



# - - - 

# .. - - 

# V 0- 0 ,./0 0/ 

### / 

# . / 0 

# / 

# f / !

10 Daywindow 

### )



### o o 2 4 6 8 10 12 14 16 18 20 22 24 26 28 nne (Days) 80

40 60

20 100 120 '40Harlan StudyNumber: 41207010


## APPENDICES Appendix 1 MineralMedium Report Page 25



### Solution a

Solution b Solution 

### c Solution d 

### KH,PO, K,HPO, Na,HPO,.2H,O NH,Cl

CaCh MgSO,.7H,O FeCl,.6H,O 8.50 giL 21.75 gIL 33.40 

### gIL 0.50 

### gIL

27.50 giL 22.50

### gIL 0.25 gIL 

### To Iliter(final volume) ofpurified water· was addedthe following volumes ofsolutions a-d. 

### 10 mL ofSolution a IroLofSolution b 1mL ofSolutionc 1 rnL ofSolution d

• Reverse osmosis purified and deionized water (Elga Optima 15+or Elga Purelab Option R-15 BP)Harlan StudyNumber: 41207010


### Appendix 2 Pre-Study SolubilityWork Report Page 26



### The following pre-study solubility/dispersibility work was perfonned in order to determine the most suitable method ofpreparation (i) Ultrasonication: An amount of test item (500 mg) was dispersed in I liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 30 minutes. This formed a hazy dispersion with few particles oftest item floating onthe surface. (ii) 

### High Shear Mixing: An 

### amount oftest item (250 mg) was dispersed in SOD 

### mL of deionized reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. This formed acloudy dispersion with foam on the surface. This work confinned that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradationtest. 

### (iii) Ultrasonication: 

### An amount oftest item (50 mg) was dispersed in approximately 400 mL ofmineral mediawith the aid ofultrasonication for 15 minutes. The volume was then adjusted to a final volume of3 liters. This fonned a hazy dispersion with particles oftest item visible. (iv) High ShearMixing: An 

### amount oftest item (50 mg) was dispersed in approximately 400 mL ofmineral media with the aidofligh shearmixing (approximately 7500rpm, 15 minutes). Thevolumewas then adjusted to afinal volume of31iters. This formed ahazy dispersion withno particles oftestitemvisible. (v) Adsorption onto an Inert Support: An 

### amount oftest item (50 mg) was weighed on a filter paper. The filter paper was added to 3 liters ofmineral media. This formed a clear colorless mediacolumn with filter paperand test item adhered. (vi) Additton onto an Inert Support: An amount oftest item (50 rng) was weighed on a glass slide. The glass slide was addedto 3 liters ofmineral media. This fanned aclear colorless mediacolumnwith aglass slidewith aglobule oftest item adhered. From the pre-study solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion 

### was found to be obtained when an aliquot oftest item was dispersed in approximately 400 mL mineral media with the aid ofhigh shear mixing (approximately 7500 rpm for 15 minutes), prior to additionto the testvessel.Harlan StudyNumber: 412070I0 Report Page 27


### Appendix 3 Monitoring Authority StatementofGLP Compliance



# ,oH) Department 

# 'C ofHealth

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004l9/EC
TEST FACIUTY Harlan Laboratories ltd Shardlow Business Park london Road Shardlow Derby OE72 ZCD

DATE OF INSPECTION

10 July 2012 TEST TYPE(S) AnalytlcaIIClinical Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys!Chem. Tests Toxicology

An inspection for compliance with the Principles 01 Good Laboratory Practice was carried out at the above test facility as pari of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that. on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance wllh the OECD Principles of Good Laboratory Practice. This statement conslilules aGood Laboratory Practice Instrument (as defined in the UKGood laboratory Practice Regulatior.s 1999).

# ~~ 30{" 

## I ,z.. Dr. AndrewJ. Gray • Head, UK GLP Monitoring Authority •