## REPORT D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Assessment ofReady Biodegradability; C02 

## Evolution Test

StudyDirector: TestFacility:

Sponsor:

Harlan StudyNumber: Study Completion Date: NBest 

### HarlanLaboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE722GD 

### UK UnivarUSA Inc. 17425 NEUnion Hill Road Redmond WA98052 UNITED STATES OF AMERICA

41401177 28 January 2015

Page 1of29Harlan StudyNumber: 41401177 Report Page2 

## CONTENTS CONTENTS ....................................................................................................................................2 STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE...................................................

### .4 QUALITY ASSURANCE STATEMENT......................................................................................5 SUMMARY ....................................................................................................................................6 Introduction............................................................................................................................6 Methods...............................................................................................................................6 Results..................................................................................................................................6 GENERAL INFORMATION .........................................................................................................7 Schedule.................................................................................................................................7 Deviations from StudyPlan...................................................................................................7 Archiving ...............................................................................................................................7 1 INTRODUCTIONAND PURPOSE.....................................................................................8 1.1 Guidelines I Regulations.......................................................................................... 8 2 TESTAND REFERENCE ITEMS .......................................................................................9 2.1 TestItem..................................................................................................................9 2.2 Reference Item.........................................................................................................9 3 MATERIALS 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 PreliminaryInvestigational Work ........................................................... 11 3.5.2 Experimental Preparation........................................................................ 11 3.5.2.1 TestItem.................................................................................... 11 3.5.2.2 Reference Item ..........................................................................-12 3.5.2.3 Toxicity Control ........................................................................ 12 3.5.3 Preparation ofTest System...................................................................... 12 3.6 Evaluations ............................................................................................................ 13 3.6.1 Observations............................................................................................ 13 3.6.2 pHMeasurements.................................................................................... 13 3.6.3 IC Analysis .............................................................................................. 13 3.6.4 IC/TC Ratio ............................................................................................. 14 3.7 DataEvaluation ..................................................................................................... 14 3.7.1 Calculation ofCarbon Content................................................................ 14 3.7.2 Percentage Biodegradation...................................................................... 15 3.7.3 Total C02Evolution................................................................................ 15 3.8 Validation Criteria................................................................................................. 15 4 RESULTS ............................................................................................................................ 17 4.1 Preliminary Investigational Work ......................................................................... 17 4.2 Definitive Test....................................................................................................... 17 4.2.1 Validation Criteria................................................................................... 17 4.2.2 Biodegradation ........................................................................................ 17 5 CONCLUSION.................................................................................................................... 19Harlan StudyNumber: 41401177 Report Page 3
TABLES........................................................................................................................................20 FIGURES ......................................................................................................................................25 APPENDICES...............................................................................................................................26 

## LIST OF TABLES Table 1 Table 2 Table 3 Table 4 Table 5 Inorganic Carbon Values onEach Analysis Occasion ..........................................20 Percentage BiodegradationValues........................................................................21 Total and Inorganic Carbon Values in the Culture Vessels on Day 0...................22 pH Values ofthe Test Preparations onDays 0 and 28 ..........................................23 Observations onthe Test Preparations Throughout the Test Period .....................24 

## LIST OF FIGURES Figure 1 BiodegradationCurves ..........................................................................................25 

## LIST OF APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4 Certificate ofAnalysis ...........................................................................................26 Mineral Medium....................................................................................................27 Dissolved Organic Carbon (DOC) Values from the Preliminary Investigational Work .............................................................................................28 Monitoring Authority Statement ofGLP Compliance ..........................................29Harlan StudyNumber: 41401177 Report Page4 

## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: Study Title: 41401177 D-erythro-Hex-2-enonic acid, .garnrna.-lactone, monosodium salt [CAS RN 6381-77-7]: Assessment ofReady Biodegradability; C02Evolution Test This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV /MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affected the integrity ofthe study orthe validity ofthe data. Study Director: NBest



# ··~·············· Date: 

## 28 

## JAN 2015Harlan StudyNumber: 41401177 Report Page 5 

## QUALITYASSURANCE STATEMENT Harlan StudyNumber: 41401177 Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: Assessment ofReady Biodegradability; C02 EvolutionTest

The general facilities and activities are inspected at least once a year and the results are reported to the relevant responsible person and management. Study-relatedprocedures conducted atthe test facility were audited and inspected. The details of these audits and inspections are given below. Reported to the Dates and Types ofQAInspections relevant Study Director and Test Facility Management Date ofInspection Type ofInspection Phase Inspected Report Date 11 April2014 Study Plan N/A 11 April2014 Verification 24 October 2014 Process - based Test Item Preparation 24 October 2014 24 October 2014 Process - based Test System 24 October 2014 Preparation and Exposure 30 October 2014 Process - based Assessment of 30 October 2014 Response 27 November 2014 ReportAudit N/A 27 November 2014
This statement confirms thatthis report reflects the raw data andthe procedures followed. Quality Assurance: 

## 2 8 JAN 2015 Date:Harlan StudyNumber: 41401177 Report Page 6 

## SUMMARY Introduction A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing ofChemicals (1992) No. 301B, "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C ofCommission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 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 temperatures between 20 and 22 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with atoxicity control were used for validationpurposes. 

### Results The test item attained 70% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% 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 ofOECD Guideline No. 301B.Harlan StudyNumber: 41401177 Report 

## GENERAL INFORMATION Schedule Experimental Starting Date: Experimental CompletionDate: 

### Deviations from Study Plan 8 September 2014 29 October2014

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

### Archiving Page 7

Unless instructed otherwise by the Sponsor, the study plan (general studyplan 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 whichinstructions will be sought as to further retention or disposal. Further retention or return ofthe data will be chargeable to the Sponsor. No data will be discarded without contacting the Sponsorto obtaintheir written consent.Harlan StudyNumber: 41401177 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 under aerobic conditions. The results ofthe study are ofvalue in the assessment ofthe persistence ofthe test item in the aquatic environment. This study was performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 Testrule andthe regulations set forth under40 CFR799.5087. 

### 1.1 Guidelines I 

### Regulations This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations: • OECD Guidelines for Testing ofChemicals (1992) No. 301B "Ready Biodegradability; C02 Evolution Test" • Method C.4-C ofCommission Regulation (EC) No. 440/2008 • US EPAFate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m))Harlan StudyNumber: 41401177 Report Page 9 

## 2 TESTAND REFERENCE ITEMS 2.1 Test Item Information as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is given in Appendix 1. Identification: Physical state/ Appearance: Batch: Purity: ExpiryDate: Storage Conditions: Intended use/ Application: 

### 2.2 Reference Item D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] white granular solid 14031314 99.19% 26 March 2016 room temperature inthe dark not supplied

Information as provided bythe Supplier (Sigma-Aldrich). Identification: Description: Batch: Purity: Expiry Date: Storage conditions: Sodiumbenzoate white granular solid SLBG9515V >99.5% 09 July 2015 roomtemperature over silica gelHarlan StudyNumber: 41401177 Report Page 10 

## 3 MATERIALSAND METHODS 3.1 Definitions and Abbreviations Readily biodegradable: 10-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 10-Day windowwithin 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. This is the organic carbon present in solution or that which passes through a 0.45 !liD 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 29 September 2014 from the aeration stage ofthe Severn Trent Water Plc sewage treatmentplant at Loughborough, Leicestershire, UK, whichtreats predominantly domestic sewage. 

### 3.3 Preparation ofInoculum The activated sewage sludge sample was washed twice 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 after rinsing the filter three successive times with 10 mL ofdeionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use. 

### *Rinsed three times with 20 mL deionized reverse osmosis water priorto drying in an ovenHarlan StudyNumber: 41401177 Report Page II 

### 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 Preliminary Investigational Work In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VcsH TOC analyzer (see see Appendix 3). An amount oftest item (100 mg) was dissolved in mineral medium (1 liter) to give a 100 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 f.!m Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further amount oftest item (100 mg) was dissolved in mineral medium and inoculated at a concentration of30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 f.lm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and 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 of30 mg ss/L and then filtering or centrifuging as perthe test item samples. 3.5.2 Experimental Preparation 3.5.2.1 Test Item The test item was dissolved directly in mineral medium. An amount of test item (300 mg) was dissolved in mineral medium with the aid of ultrasonication (5 minutes) and the volume adjusted to 1liter to give a 300 mg/L 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 of27.6 mg/L, equivalent to 10 mg carboniL. The volumetric flask containing the test item was inverted several times to ensure homogeneity ofthe solution. A test concentration of 1 0 mg carbon/L was employed in the test following the recommendations ofthe Test Guidelines.Harlan StudyNumber: 41401177 Report Page 12
3.5.2.2 Reference Item A reference item, sodium benzoate (C6H5C00Na), 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. 3.5.2.3 Toxicity Control A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect ofthe test item onthe sewage sludge micro-organisms used inthe test. An aliquot (276 mL) ofthe test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 27.6 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of20 mg carboniL. 3.5.3 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 medium to give a final concentration of 10 mg carbon/L. 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). Data from the inoculum control and procedure control vessels was shared with similar concurrent studies. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures between 20 and 22

## oc, indarkness. Approximately 24 hours prior to addition ofthe test and reference items the vessels were filled with 2400 mL ofmineral medium and 30.0 mL ofinoculum and aerated overnight. On Day 0 the test and reference items were added and the pH ofall vessels measured using a Hach HQ40d Flexi handheld meter and the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid orHarlan StudyNumber: 41401177 Report Page 13
sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition ofmineral medium which had beenpurged overnight with C02 free air. The test vessels were sealed and C02-free air bubbled through the solution at a rate of 30 to 100 mL!min per vessel and stirred continuously by magnetic stirrer. The C02-free air was produced by

**~assing** compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules. The C02 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of0.05 M NaOH. The C02 absorbing solutions were prepared using purified de-gassed water. 

### 3.6 Evaluations 3.6.1 Observations The appearance ofthe testpreparations was recorded onDays 0, 6, 13, 20 and 27. 3.6.2 pH Measurements The pH ofthe 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 Samples (2 mL) were taken from the first C02 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. All samples were analyzed for IC immediately. 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 IC using a Tekrnar-Dohrrnann Apollo 9000 TOC analyzer or a Shimadzu TOC-VcsH TOC analyzer. Samples (300 or 50 flL) were injected into the IC channel ofthe TOC analyzer. IC analysis occurs by means ofthe conversion of an aqueous sample to C02 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions ofsodium carbonate (Na2C03). Each analysis was carried out intriplicate.Harlan StudyNumber: 41401177 Report Page 14
3.6.4 IC/TC Ratio Samples (30 mL) were removed from the inoculum control and test item vessels on Day 0 and filtered through 0.45 J.Lm 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-LcsH TOC analyzer. Samples (50 or 800 J.LL) were injected into the TC and IC channels ofthe TOC analyzer. TC analysis is carried out at 680 

## oc using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by 2M HCl at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5K04) and sodium carbonate (Na 2C03) in deionized water. Each analysis was carried out intriplicate. 

### 3.7 Data Evaluation 3.7.1 Calculation ofCarbon Content The theoretical amount of carbon present in the test item (D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7] (C6Hs06Na)) was calculated as follows: No ofCatoms x mol wtofC x 100 mol wtoftestitem 6x 12·011 x100=36.19%

199.11 Thus for a concentration of 10 mg C/L the total organic carbonpresent was 30 mg C. The theoretical amount ofcarbon present in the reference item, sodium benzoate (C6HsCOONa) was calculated as follows: No ofCatoms x mol wtofC x 100

mol wtofsodiumbenzoate 7 X 12·011 X 100 = 58.34% 144.11 Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.Harlan StudyNumber: 41401177 Report Page 15
3.7.2 Percentage Biodegradation The percentage biodegradation orpercentage ofTheoretical Amount ofCarbonDioxide (ThC02 ) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values ofReplicates R1 and R2 are meaned for the inoculum control, test and reference items before substitutioninto the following equation:

01 ThCO ( 01 b" d d . *) mgIC intest flask- mgIC incontrolflask 100 ;o 2 

### = ;o 10 egra atwn 

### = x mgTOC added as testchemical 3.7.3 Total C02 Evolution 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 meanedbefore substitutioninto the equation: Total C02 evolution (mg C/L) = mg ICincontrolx 100 x --1

-- %C ofCO 2 test volume

. 100 1 

### = mgiCmcontrolx--x- 27.29 3 

### 3.8 Validation Criteria 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 byDay 14 for the test itemto be considered as non-inhibitory. The test is considered valid ifthe difference ofthe extremes ofreplicate values ofproduction of C02at the time the plateau is reached, at the end ofthe test or at the end ofthe 10-Day window, as appropriate, is less than 20%. The total C02evolution inthe inoculum control vessels atthe end ofthe 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 StudyNumber: 41401177 Report Page 16
values up to 70 mg/L are acceptable. Data from studies where values in excess of70 mg/L are obtained should be critically examined. The IC content ofthe test item suspension in the mineral medium at the beginning ofthe test should be <5% ofthe TC.Harlan StudyNumber: 41401177 Report Page 17 

## 4 RESULTS 4.1 Preliminary Investigational Work The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or activated sewage sludge (see Appendix 3). Therefore, for the purpose ofthe study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss ofany 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 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 onDay 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. 

### 4.2.1 Validation Criteria The total C02evolution inthe inoculum control vessels onDay 28 was 31.88 mg/L and therefore satisfiedthe validation criterion given inthe OECD Test Guidelines. The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% ofthe TC content and hence satisfied the validation criterion given inthe OECD Test Guidelines. 

### 4.2.2 Biodegradation Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives offany dissolved C02present inthe test vessels. Therefore any additional C02detected inthe Day 29 samples originated from dissolved C02thatwas present inthe testvessels onDay 28 and hence the biodegradationvalue calculated from the Day 29 analyses is taken as being the final biodegradationvalue for the testitem. The results ofthe inorganic carbon analysis ofsamples from the first absorber vessels onDay 29 showed an increase in all replicate vessels with the exception ofinoculum control R2 and the toxicity control.Harlan StudyNumber: 41401177 Report Page 18 Inorganic carbon analysis ofthe samples from the second absorber vessels on Day 29 confirmed that no significant carry-over ofC02 into the second absorber vessels occurred. The test item attained 70% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% 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 strictterms and conditions ofOECD GuidelineNo. 301B. The toxicity control attained 75% biodegradation after 14 days and 72% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between days 14 and 28 was consideredto be due to sampling/analytical variation. Sodium benzoate attained 83% biodegradation after 14 days and 88% biodegradation after 28 days thereby confirming the suitability ofthe inoculum and test conditions.Harlan StudyNumber: 41401177 Report Page 19 

## 5 CONCLUSION The test item attained 70% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% 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 strictterms and conditions ofOECD Guideline No. 301B.Harlan Study Number: 41401177 Report TABLES Table 1 Inorganic Carbon Values on Each Analysis Occasion

Day 0 2 6 8 10 14 21 28 29 R 1 -Rz Abs Inoculum Control
Rt Abs 1 Abs2 Abs 1 1.28 1.17 1.17 5.33 - 6.50 10.27 - 9.69 11.47 - 11.35 13.57 - 13.79 16.21 - 16.09 19.27 - 19.04 25.87 - 26.32 26.05 1.16 26.05 Replicates 1 and 2 C0 2 absorber vessels R2 Inorganic Carbon (mg IC) Procedure Control
Rt R2 Abs2 Abs 1 Abs2 Abs 1 Abs2 Abs 1 1.17 1.28 1.17 1.28 1.17 1.28

- 28.77 - 26.80 - 10.67

- 33.79 - 31.60 - 28.49

- 35.32 - 34.97 - 31.07

- 39.33 - 38.07 - 35.23

- 40.80 - 41.25 - 37.29

- 41.69 - 42.25 - 38.76

- 49.95 - 50.06 - 45.25

1.39 53.00 1.51 51.99 1.04 46.20 Page 20

Test Item Toxicity Control

Rt Rz Rt Abs2 Abs 1 Abs2 Abs 1 Abs2

1.05 1.05 1.40 1.05 1.28

- 10.21 - 31.21 -

- 28.26 - 50.63 -

- 32.11 - 55.04 -

- 35.11 - 57.46 -

- 37.97 - 60.97 - I

- 39.21 - 61.63 -

- 45.36 - 70.90 - !

1.39 47.76 1.28 69.22 1.39Harlan StudyNumber: 41401177 Report Page 21


### Table 2 PercentageBiodegradationValues % Biodegradation Day Procedure Control Test Item Toxicity Control

0 0 0 0 2 73 15 42 6 76 61 68 8 79 67 73 10 83 72 73 14 83 72 75 21 76 66 71 28 80 64 75 29* 88 70 72

* Day 29 values correctedto include any carry-over ofC02 detected inAbsorber2Harlan StudyNumber: 41401177 Report Page 22


### Table 3 Total and Inorganic CarbonValues in the CultureVessels on Day 0 Total Carbon* Inorganic Carbon* Testvessel (mg!L) (mg/L) IC Content (% ofTC) Test Item 10 mg C/L R1 8.87 -0.01 0 TestItem 10 mg C/L R2 8.83 -0.01 0

R1 - R2 = Replicates 1 and 2 

### * Corrected for control values. Negative values are due to measured concentrations being less than control valuesHarlan StudyNumber: 41401177 Report Page 23
Table 4 pHValues oftheTestPreparations on Days 0 and 28 pH Test Vessel DayO DayO Day28 Pre-Adjustment Post-Adjustment Inoculum Control R 1 7.8 7.4 7.5 Inoculum Control R2 7.8 7.4 7.4 Procedure Control R 1 7.8 7.4 7.5 Procedure Control R2 7.8 7.4 7.4 Test Item R 1 7.8 7.5 7.4 Test Item R2 7.8 7.4 7.4 Toxicity Control 7.8 7.5 7.5

Replicates 1 and 2Harlan StudyNumber: 41401177 Report Page 24
Table 5 Observations on the Test Preparations Throughout the TestPeriod Observations on Test Preparations Test Vessel DayO Day6 Day 13 Day20 Day27 Inoculum R1 Lightbrown Lightbrown Lightbrown Light brown Light brown Control dispersion dispersion dispersion dispersion dispersion

R2 Lightbrown Light brown Light brown Lightbrown Lightbrown dispersion dispersion dispersion dispersion dispersion Lightbrown Light brown Light brown Lightbrown Lightbrown Procedure dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no

Control R1 undissolved undissolved undissolved undissolved undissolved reference item reference item reference item reference item reference item visible visible visible visible visible Lightbrown Lightbrown Light brown Light brown Light brown dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no R2 undissolved undissolved undissolved undissolved undissolved reference item reference item reference item reference item reference item visible visible visible visible visible Lightbrown Light brown Lightbrown Light brown Light brown Test Item RI dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no undissolved undissolved undissolved undissolved undissolved test item visible test item visible test item visible test item visible test item visible

Lightbrown Light brown Light brown Light brown Light brown R2 dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no undissolved undissolved undissolved undissolved undissolved testitem visible test item visible test item visible test item visible test item visible

Lightbrown Light brown Lightbrown Lightbrown Light brown dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no Toxicity undissolved undissolved undissolved undissolved undissolved Control test or test or test or test or test or reference item reference item reference item reference item reference item visible visible visible visible visible

R1- R2=Replicates 1and 2Harlan StudyNumber: 41401177 Report 

## FIGURES Figure 1 Biodegradation Curves 100
90 - o - Sodium benzoate Test Item

- -1>. -- Toxicity Control
0

# ~

0 80 0 ./-.__ 

# /**~** / /• / 

# - o- 

# - 70 

## • 60
c: 0 1ii "'0 50 e! Cl Q) c <f!. 40
30
20
10
0 0


# ---
•

2 4 6 8 1 0 12 14 16 18 20 22 24 26 28 Time (Days) Page 25Harlan StudyNumber: 41401177 Report 

## APPENDICES Appendix 1 Certificate ofAnalysis

GS&c5>PO# L.ot:rl!ifumbe:n

**Produl!tiot~** ,IJate: Rll-ovaluat!cm Data: Souroe Cod:11: I"Htory; Product Cod:e: **GS~l<E** S.OS:¢ 14031314 M11r 26,2014 MaL 2$, 2016 010·DSI.01

**~g** Plllrd"!tl, Duing, .f!SrtQXI, etma 6112511 {21> kgG

**~~artqn)** Page 26Harlan StudyNumber: 41401177 Appendix 2 Mineral Medium

Solution a

Solutionb Solution c Solutiond KH2P04 K2HP04 Na2HP04.2H20 NH4Cl

CaCb MgS04.7H20 FeCh.6H20 Report

8.50 g/L 21.75 g/L 33.40 g/L 0.50 g/L pH= 7.4 27.50 g/L 22.50 g/L 0.25 g/L To 1 liter (final volume) ofpurified water* was added the following volumes ofsolutions a-d. 1 0 mL ofSolution a 1 mL ofSolutionb 1 mL ofSolutionc 1 mL ofSolutiond

*Reverse osmosis purified anddeionized water (Elga Optima 15+ orElga Purelab Option R-15 BP) Page 27Harlan StudyNumber: 41401177 Report Appendix 3 Dissolved Organic Carbon (DOC) Values from the Preliminary InvestigationalWork Page 28



### In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge the following samples were analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VcsH TOC analyzer. DOC Concentration Sample mgC/L %ofNominal Carbon Content mgC/L Corrected for Appropriate Control Mineral medium <LOQ 

### - 

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

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

## - - 100 mg/L Untreated 32.18 32.18 89 100 mg/L Filtered .32.51 32.51 90 100 mg/L, inoculated at 30 mg ss/L, Filtered 31.97 31.97 88 100 mg/L, inoculated at 30 mg ss/L, 31.89 31.89 88 Centrifuged 

### 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 ofany test item.

LOQ =LimitofQuantification (determined down to 1.0 mg C/L).Harlan StudyNumber: 41401177 Report Appendix 4 Monitoring Authority Statement ofGLP Compliance

Hanan Laboratories Limited Shardlow Busin!ll!m$ Park London Road Shandlow TEST Environmental Environm!ll!nml Toxh::itv rn·'!fl!i..wr••ntl.

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