Assessment of Ready Biodegradebilityi COz Evolutro" ,J

har{sff >.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 of27
Harlan Study Number: 41207010

CONTENTS

Report Page 2

CONTENTS 2

STUDY DIRECTOR STATEME. IT OF GLP COMPLIANCE 4

QUALITY ASSURANCE STATEMENT.. 5

SUMMARY 6

Introduction 6

Methods 6

Results 6

GENERAL INFORMATION 7

Schedule 7

Deviations from Study Plan 7

Archiving 7

1 INTRODUCTION AND PURPOSE 8

1.1 Guidelines / Regulations 8

2 TEST AND REFERENCE ITEMS 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 of Inoculum 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 of Data 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 25
Harlan Study Number: 41207010

LIST OF TABLES

Report Page 3

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 of the Test Preparations on Days 0 and 28 22

Observations on the Test Preparations Throughout the Test Period 23

LIST OF FIGURES

Figure I Biodegradation Curves 24

LIST OF APPENDICES

Appendix 1

Appendix 2

Appendix 3

Mineral Medium 25

Pre-Study Solubility Work 26

Monitoring Authority Statement ofGLP Compliance 27
Harftn 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:

Study Title:

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

item formul*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 gencral facitities and sctil'ities are inspccted at least once a year 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

S9 April 2013

i0 April 2013

?a April 2013

?6 Jdy 2013

$ndy Plan

Verificaticn

Prwes * bascd

Process - based

Proccss - based

Proess$ - based

Report Audit

NIA

Tes ltem Prcparalion

Test System

Preparation

Exposure

Asscssment of

Rcspoffie

N/A

3l Decernber 20t2

l0 April 2013

09 April 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 ffifi
Harlan 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 of Chemicals (1992) No. 301B, "Ready Biodegradability; CO, Evolution

Test" referenced as Methnd C.4-C of Commission 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 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 80% degradation after 28 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 item can therefore be considered to be readily biodegradable under the

strict terms and conditions ofOECD Guideline No. 3018.
Harlan Study Number: 412070 I0

GENERAL INFORMATION

Schedule

Report Page 7

Experimental Starting Date:

Experimental Completion Date:

Deviations from Study Plan

l1lune 2013

10 luly 2013

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: 41207010 Report Page 8

1 INTRODUCTION AND 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.

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 of Chemicals (1992) No. 301 B "Ready Biodegradability;

C02 Evolution Test"

• Method C.4-C ofCommission Regulation (EC) No. 44012008

• US EPA Fate, Transpon, and Transfonoation Test 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 temperattrre
Harlan 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 carbon present in a sample.

This is the sum of the organic carbon in solution and

In suspension.

Dissolved organic carbon is the organic carbon present

in solution or that which passes through a 0.45 ~m

filter or remains in the supernatant after centrifuging

at approximately 4000 g for 15 minutes.

A mixed population of activated sewage sludge micro-organisms was obtained on 10 June 2013

from the aeration stage of the Severn Trent Water PIc sewage treatment plant at Loughborough,

Leicestershire, UK, which treats 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 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 21°C 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 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 successive times with 10 mL of deionized 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 concentration was equal to 4.3 gIL prior to use.

• Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven
Harlan Study Number: 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

suitable method of preparation (see Appendix 2).

3.5.2 Experimental Preparation

For the purpose ofthe test, the test item was dispersed directly in mineral medium.

An amount of test item (42.9 mg) was dispersed in approximately 400 mL of mineral medium

with the aid of high 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

of 14.3 mgIL, equivalent to 10 mg carbonIL.

A test concentration of 10 mg carbonIL was employed in the test following the recommendations

of the Test Guidelines.

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the

homogeneity, concentration or stability of the test item formulation. This is an exception with

regard to GLP and has been reflected in the GLP compliance statement.

3.5.3 Reference Item

For the purposes of the test, a reference item, sodium benzoate (C6HsCOONa), 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) 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

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 Sludy Number: 412070I0

3.5.4 Toxicity Control

Report Page 12

For the purposes ofthe test, a toxicity 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 of test item (42.9 mg) was dispersed in approximately 400 mL of mineral medium

with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in

inoculated mineral medium. An aliquot (51.4 mL) of the 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 itemIL plus 17.1 mg sodium benzoate/L, equivalent to a total of20 mg carbonIL.

3.5.5 Preparation of Test 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 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 10 mg carbonIL.

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

of 10 mg carboniL.

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

concentration of20 mg carbon/L to act as a toxicity 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 of the test and reference items the vessels were filled

with 2400 mL of mineral medium and 20.9 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 WTW

pHlOxi 3401 pH and dissolved oxygen meter. If necessary 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 of mineral medium which had been purged

overnight with CO2 free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to

100 mUmin per vessel and stirred continuously by magnetic stirrer.

The COrfree air was produced by 'passing compressed air through a glass column containing

self-indicating soda lime (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 of 0.05 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 absorber vessels were all sampled on Days 0 and 29.

All the samples were analyzed for CO2 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 CO:~ using a Tekmar-Dohnnann Apollo 9000 TOC analyzer.

Samples (300 ~L) were injected into the IC (Inorganic Carbon) cbannel of tbe TOC analyzer.

Inorganic carbon analysis occurs 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 ofsodium carbonate (Na2C03). Each analysis was carried out in triplicate.

3.5.6.2 Inorganic CarbonITotal Carbon Analysis

Samples (30 roL) were removed from the test item vessels on Day 0 prior to the addition of the

test item in order to 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 order to pre-condition the filter) prior to DOC analysis.

ICITC analysis of the test item dispersions after dosing was not possible due to the insoluble

nature ofthe test 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

of the 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 carried out in triplicate.
Harlan Study Number: 41207010

3.5.6.3 pH Measurements

Report Page 14

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

with hydrochloric acid, using a WTW pHlOxi 3401 pH and dissolved oxygen meter.

3.5.7 Evaluation of Data

3.5.7.1 Determination of Carbon Content

The test item contains 70% carbon (data supplied by the Sponsor) and so for a concentration of

10 mg C/L (a total of 42.9 mg of test item in 3 liters) the total organic carbon present was

30 mg C.

The theoretical amolUlt of carbon present in the reference item, sodium benzoate (C6HsCOONa)

was calculated as follows:

No of C atoms x mol wtofC x 100

mol wt of sodium benzoate

7 x 12.0 I I x I00 58.34%

144.11

Thus for a 10 mg C/L test concentration (a total of 51.4 mg of sodium benzoate in 3 liters) the

total organic carbon present for sodium benzoate was 30 mg C.

3.5.7.2 Percentage Degradation

The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThC02)

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

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

test and reference items before substitution into the following equation:

fig IC in test flask - mg 1C in control flask %ThC02(~%degradatjon')= TOe dd d h' I xlOO

mg a e as test c emlca

The total C02 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 R2 on Day 28 are

meaned before substitution into the equation:

• The conversion factor for carbon to carbon dioxide is 3.67
Harlan 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

%Cof CO2 test volume

. 100 1 =: mgIC In control x-- x27.29 3

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

yields 60% 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 10%.

The toxicity control (test item and sodium benzoate) should attain 25% 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

CO2 at the time the plateau is reached, at the end ofthe test or at the end of the lO-Day window,

as appropriate, is less than 20%.

The total C02 evolution in the inoculum control vessels at the end ofthe test should not normally

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 of 70 mgfL are

obtained should be critically examined.

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

should be <5% ofthe TC.
Harlan Study Number: 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 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.

The total C02 evolution in the inoculum control vessels on Day 28 was 30.72 mgIL and therefore

satisfied the validation criterion given in the 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 of 5% of the 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 of the test for the replicate

vessels was <20% and hence satisfied the validation criterion given in the OECD Test

Guidelines.

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 off any dissolved CO2 present in the test

vessels. Therefore any additional C02 detected in the Day 29 samples originated from dissolved

C02 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 ofsamples from the first absorber vessels on Day 29

showed an increase in all replicate vessels, with the exception of procedure control R] and R2•

This decrease was considered to be due to sampling/analytical variation. Inorganic carbon

analysis ofthe samples from the second absorber vessels on Day 29 confirmed that no significant

carry-over of CO2 into the second absorber vessels occurred.

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 biodegradable under the

strict temlS and conditions of OECD Guideline No. 301 B.
Harlan Study Number: 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 in the test.

Sodium benzoate attained 107% degradation after 14 days and 116% degradation after 28 days

thereby confirming the suitability of the inoculum and test conditions. Degradation values in

excess of 100% were considered to be due 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 biodegradable under the

strict tenns 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) Benzoate Toxicity

(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 - 11.0 I - 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 - CO2 absorber vessels
Harlan Study Number: 41207010 Report Page 20

Table2 Percentage Biodegradation Values

% Ingradation -/0 Degradation

Day Sodium Benzoate

-I_ Degradation Test Item plus Sodium

Test Item 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

- Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2
Harlan Study Number: 41207010 Report Page 21

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

Test Item

10.36" 1.21 12

10 mg C/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 item
Harlan Study Number: 41207010 Report Page 22

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

pH pH

Test Vessel prior to after pH on Day 28 adjustment adjustment

on Day 0 on Day 0

Inoculum Control R1 7.8 7.2 7.3

Inoculum Control Rz 7.7 7.3 7.3

Procedure Control

7.8 7.5 7.3 10 mg CIL R1

Procedure Control

7.7 7.4 7.2 10 mg CIL R2

Test Item

7.7 7.4 7.2 10 mg CIL Rj

Tesl Item

7.7 7.3 7.1 10 mg CIL Rz

Toxicity Control

7.7 7.3 7.2 20 mg ell

Rl - R2 "" Replicates I and 2
Harlan Study Number: 41207010 Report Page 23

Table 5 Observations on the Test Preparations Throughout the Test Period

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 Pale bro....n Pale brown Pale brown 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

Test Item 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 test item

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

or reference or reference or reference or reference test or

reference item item visible item visible item visible item visible visible

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

FIGURES

Figure 1 Biodegradation Curves

I • Sodium benzoate

0 TeSl11em

- - " - - To)(icity Control

./

I 0

0

- - - .. - -

V0-

0

,./0

0/

/ . / 0

/

f /

!

10 Day window

)

o

o 2 4 6 8 10 12 14 16 18 20 22 24 26 28

nne (Days)

80

40

60

20

100

120

'40
Harlan Study Number: 41207010

APPENDICES

Appendix 1 Mineral Medium

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 I liter (final volume) of purified water· was added the following volumes ofsolutions

a-d.

10 mL of Solution a

I roL of Solution b

1 mL of Solution c

1 rnL of Solution d

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

Appendix 2 Pre-Study Solubility Work

Report Page 26

The following pre-study solubility/dispersibility work was perfonned in order to determine the

most suitable method of preparation

(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 on the surface.

(ii) High Shear Mixing: An amount of test 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 a cloudy 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

biodegradation test.

(iii) Ultrasonication: An amount of test item (50 mg) was dispersed in approximately

400 mL of mineral media with the aid of ultrasonication for 15 minutes. The volume

was then adjusted to a final volume of 3 liters. This fonned a hazy dispersion with

particles of test item visible.

(iv) High Shear Mixing: An amount of test item (50 mg) was dispersed in approximately

400 mL of mineral media with the aid ofligh shear mixing (approximately 7500 rpm,

15 minutes). The volume was then adjusted to a final volume of31iters. This formed

a hazy dispersion with no particles oftest item visible.

(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 of mineral media. This formed a

clear colorless media column with filter paper and test item adhered.

(vi) Additton onto an Inert Support: An amount of test item (50 rng) was weighed on a

glass slide. The glass slide was added to 3 liters ofmineral media. This fanned a clear

colorless media column with a glass slide with a globule 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 of test item was dispersed in approximately 400 mL mineral

media with the aid of high shear mixing (approximately 7500 rpm for 15 minutes), prior to

addition to the test vessel.
Harlan Study Number: 412070 I0 Report Page 27

Appendix 3 Monitoring Authority Statement of GLP Compliance

,oH) Department

'C of Health

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)

Analytlca IIClinical

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 a Good Laboratory Practice Instrument (as defined in the

UK Good laboratory Practice Regulatior.s 1999).

~~

30{" I ,z..

Dr. Andrew J. Gray •

Head, UK GLP Monitoring Authority •