## REPORT 

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

## Evolution Test

StudyDirector: TestFacility:

Sponsor:

HarlanStudyNumber: Study Completion Date: NBest 

### Harlan Laboratories Ltd. Shard!owBusiness Park Shardlow Derbyshire DE722GD 

### UK UnivarUSA Inc. 17425 NE UnionHill Road Redmond WA 98052 UNITED STATES OF

### AMERICA

41401177 28 January 2015

Page 1 of29Harlan StudyNumber: 41401177 Report Page2


## CONTENTS CONTENTS ....................................................................................................................................2 STUDY DIRECTOR STATEMENT 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 I 

### Regulations
.......................................................................................... 8 2 TEST AND REFERENCE ITEMS .......................................................................................9 2.1 Test Item ..................................................................................................................9 2.2 Reference Item.........................................................................................................9 3 MATERIALSANDMETHODS......................................................................................... lO 3.1 Definitions and Abbreviations
............................................................................... 10 3.2 Test System............................................................................................................ 10 3.3 Preparation ofInoculum
........................................................................................ 10 3.4 Mineral Medium
.................................................................................................... 11 3.5 Procedure
............................................................................................................... II 3.5.1 Preliminary Investigational Work
........................................................... 11 3.5.2 Experimental Preparation
........................................................................ !! 

### 3.5.2.1 Testltem
.................................................................................... ll 3.5.2.2 Reference Item .......................................................................... 12 3.5.2.3 Toxicity Control
........................................................................ l2 3.5.3 Preparation ofTest System...................................................................... l2 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 Data Evaluation
..................................................................................................... 14 3.7.1 Calculation ofCarbon Content................................................................ l4 3.7.2 Percentage Biodegradation
...................................................................... l5 3.7.3 Total C02 

### Evolution
................................................................................ l5 3.8 Validation Criteria
................................................................................................. 15 4 RESULTS ............................................................................................................................ !? 

### 4.1 Preliminary Investigational Work
......................................................................... 17 4.2 Definitive Test ....................................................................................................... 17 4.2.1 Validation Criteria
................................................................................... 17 4.2.2 Biodegradation
........................................................................................ 17 5 CONCLUSION.................................................................................................................... l9Harlan Study Number: 4I40ll77 Report Page3

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 andInorganic Carbon Values inthe Culture Vessels onDay 0 ...................22 pHValues ofthe Test Preparations onDays 0 and 28 ..........................................23 Observations onthe Test Preparations Throughoutthe Test Period
.....................24 

## LIST OFFIGURES Figure I Biodegradation Curves ........................:.................................................................25 

## LIST OFAPPENDICES Appendix I Appendix 2 Appendix 3 Appendix4 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 HarlanLaboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: Study Title: 41401177 D-erythro-Hex-2-enonic acid, .ganuna.-lactone, monosodium salt [CAS RN 6381-77-7]: Assessment ofReady Biodegradability; C02EvolutionTest 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 andMETI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affected the integrity ofthe study or the validity ofthe data. StudyDirector: NBest



## ..

# ~............. Date: 

## 2 BJAN 2015Harlan StudyNumber: 41401177 Report Page 5 

## QUALITYASSURANCE STATEMENT Harlan StudyNumber: 41401177 StudyTitle: 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 relevantresponsible person and management. Study-related procedures conducted at the test facility were audited and inspected. The details of these audits andinspections are given below. Reported to the Dates and Types ofQA Inspections relevantStudy Director and Test Facility Management Date ofinspection Type ofinspection Phase Inspected ReportDate 11 April 20 14 Study 

### Plan N/A 11 April2014 Verification 24 October 2014 Process

- based Test ItemPreparation 24 October 2014 24 October 2014 Process

- based Test System 24 October 2014 Preparation and Exposure 30 October2014 Process

- based Assessment of 30 October 2014 Response 27 November 2014 Report Audit N/A 27 November 2014

1bisstatement confirms thatthis report reflects the raw data and theprocedures followed. Quality Assurance:


## 2 8 JAN 2015 Date:Harlan StudyNumber: 41401177 Report Page6


## 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 carbon!L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 20 and 22 

# oc for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. 

### Results The test item attained 70% biodegradation after 28 days and satisfied the 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 StudyNumber: 4140 1177 Report


## GENERAL INFORMATION Schedule Experimental StartingDate: Experimental Completion Date: 

### Deviations from Study Piau 8 September 2014 29 October 2014

There were no deviations (unplanned changes) from the studyplan. 

### Archiving Page 7

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 whichinstructions willbe 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 obtain their writtenconsent.Harlan StudyNumber: 41401177 Report Page 8


## 1 INTRODUCTIONAND PURPOSE This report contains a description of the methods used and results obtained during a study to investigate the ready biodegradability ofthe test item when exposed to sewage sludge microorganisms uuder 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 and the regulations set forth uuder 40 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 EvolutionTest" • 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 Page9


## 2 TESTAND REFERENCE ITEMS 2.1 TestItem Infmmation as provided by the Sponsor. A Certificate ofAnalysis supplied by the Sponsor is giveninAppendix 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: ExpityDate: Storage conditions: Sodiumbenzoate white granular solid SLBG9515V >99.5% 09 July 2015 room temperature over silica gelHarlan Study Number: 4l40ll77 Report Page lO 

## 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 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. This is the organic carbon present in solution or that which passes through a 0.45 flm 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 Pic sewagetreatment plant at Loughborough, Leicestershire, UK, which treats 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 I hour and allowed to cool before weighing. This process was repeated until a constantweight was attained. The suspended solids concentration was equal to 3.0 giL prior to use. 

### *Rinsed three times with 20 rnL deionized reverse osmosis water prior to 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 reconunended 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 removethe sewage sludge solids. Thns 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 (!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 11m 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 vohune of !liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 11m 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 testitem 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 !literto 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 carbon/L. The volumetric flask containing the test item was inverted several times to ensure homogeneity ofthe solution. A test concentration ofI0 mgcarbon/L was employed inthe test following the reconunendations ofthe Test Guidelines.Harlan StudyNumber: 41401177 Report Page 12



### 3.5.2.2 Reference Item A reference item, sodium benzoate (C6H5

### 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) 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 carbon/L. 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 ofthetestitem on the sewage sludge micro-organisms used in the 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 carbon/L. 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, induplicate, consisting ofinoculated mineral medium. b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculatedmineral 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 carbon/L. 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 "C, 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 Study Number: 41401177 Report Page 13

sodium hydroxide solutionprior to the volume in all the vessels being adjusted to 3 liters by the addition ofmineralmedium whichhad beenpurged overnight with C02free air. The test vessels were sealed and CO,-free air bubbled through the solution at a rate of 30 to I00 mL!min pervessel and stirred continuouslybymagnetic stirrer. The C02-free air was produced by J?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 ofthetestpreparations was recorded on Days 0, 6, 13, 20 and 27. 

### 3.6.2 pHMeasurements 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 C02absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled onDays 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 off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples takenfrom both absorber vessels on Day29. The samples were analyzed for IC using a Tekmar-Dohrmann Apollo 9000 TOC analyzer or a Shimadzu TOC-VcsH TOC analyzer. Samples (300 or 50 IlL) 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 in triplicate.Harlan Study Number: 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**~m** 

### Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) priorto DOC analysis. The samples were analyzed for lC and TC using a Shimadzu TOC-LcsH TOC analyzer. Samples (50 or 800**~L)** 

### 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 (C8

### H5

### K04) 

### and sodium carbonate (Na2C03) 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, .gannna.-lactone, monosodium salt [CAS RN 6381-77-7] (C6 H8

### 06Na)) was calculated as follows: No ofCatoms xmol wt 

### ofC xlOO mol wt

### oftest item 6 X 

### 12.011 X 

### 100 = 36.19%

199.11 Thus for a concentration of 10 mg C/Lthe total organic carbonpresentwas 30 mg C. The theoretical amount ofcarbon present in the reference item, sodium benzoate (C6HsCOONa) was calculated as follows: No ofCatomsxmol wt 

### ofCx 100

mol wt

### ofsodiumbenzoate 7 x 12

·011 x100=58.34%

144.11 Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was30mgC.Harlan StudyNumber: 4l40ll77 Report Page !5

3.7.2 Percentage Biodegradation Thepercentage biodegradation orpercentage ofTheoretical Amount ofCarbon Dioxide (ThC02 ) produced is calculated by substituting the inorganic carbon values, given in Table 

### I, into the following equation. The values ofReplicates Rt and R2 are meaned for the inoculum control, test andreference items before substitution into the following equation:
o/ ThCO ( o/ b' d d t' *) mgICintestflask- mgICin controlflask lOO ro 2 =**;~o** 10 egra a wn = x mgTOC added as test chemical 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 substitution into the equation: Total C02evolution (mg C/L) Ic . I 100 I = mg m contro x x ---'=--- %C ofCO2 testvolume

. 100 1 = mgiCmcontrolx--x- 27.29 3 

### 3.8 Validation Criteria The results of the degradation test are considered valid ifin the same test the reference item yields 2 60% degradation by Day 14. The test item may be considered to be readily biodegradable if 2 60% degradation is attained within 28 days. This level of degradation must be reached within I0 days of biodegradation exceeding 10%. The toxicity control (test item and sodium benzoate) should attain 2 25% degradationby Day 14 for the test item to 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%. Thetotal C02 evolution in the inoculum control vessels atthe end ofthe test should notnormally exceed 40 mg/L medium 

### (= 120 mg/3 liters, corresponding to 33 mg C per flask), however

*The conversion factor for carbon to carbon dioxide is 3.67Harlan Study Number: 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 shouldbe <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 of any test item. 

### 4.2 Definitive Test Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 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 inTable 3. The pHvalues ofthe test preparations onDays 0 and 28 are given in Table 4. Observations made onthe contents ofthe test vessels are given in Table 5. 

### 4.2.1 Validation Criteria Thetotal C02evolution 

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

### in the OECD Test Guidelines. 

### 4.2.2 Biodegradation Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives offany dissolved C02 present inthe test vessels. Therefore any additional C02detected in the Day 29 samples originated from dissolved C02thatwas present in the test vessels onDay 28 and hence the biodegradationvalue calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. 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 of inoculum 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 thatno significantcarry-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 Guideline No. 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 inthe test. The slight decrease inbiodegradation 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 testconditions.Harlan StudyNumber: 41401177 Report Page 19


## 5 CONCLUSION The test item attained 70% biodegradation after 28 days and satisfied the I0-Day window validation criterion, whereby 60% biodegradation must be attained within I0 days of the biodegradation exceeding I0%. 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 Page 20 

## TABLES Table 1 

### Inorganic CarbonValues onEachAnalysis Occasion Inorganic Carbon (mg IC) Inoculum Control Procedure Control Test Item Toxicity Control Day Rt R, Rt R, Rt R, Rt Abs 1 Abs2 Abs1 Abs2 Abs 1 Abs2 Abs1 Abs2 Abs 1 Abs2 Abs1 Abs2 Abs 1 Abs2 0 1.28 1.17 1.17 1.17 1.28 1.17 1.28 !.17 1.28 1.05 1.05 1.40 1.05 1.28 2 5.33 

### - 6.50 

### - 28.77 

## - 26.80 

## - 10.67 

### - 10.21 

## - 31.21 

## - 6 10.27 

### - 9.69 

### - 33.79 

### - 31.60 - 28.49 

## - 28.26
- 50.63 

## - 8 11.47 

### - 11.35 

## - 35.32 - 34.97 

### - 31.07 

### - 32.11 - 55.04 

### - 10 13.57 

### - 13.79 

### - 39.33 

### - 38.07 

### - 35.23 - 35.11 

## - 57.46 

### - 14 16.21 

### - 16.09 

## - 40.80 

## - 41.25 

## - 37.29 

## - 37.97
- 60.97 

## - 21 19.27 

## - 19.04 

## - 41.69 

### - 42.25 

## - 38.76 

## - 39.21 

### - 61.63 

### - 28 25.87 

### - 26.32 

### - 49.95 

### - 50.06 

### - 45.25 - 45.36 

## - 70.90 

### - 29 26.05 1.16 26.05 1.39 53.00 1.51 51.99 1.04 46.20 1.39 47.76 1.28 69.22 1.39

-------.L
........... ... 

### ' '

. --·----

R1 - Rz = Replicates 1 and 2 Abs = C02 absorber vesselsHarlan StudyNumber: 41401177 Report Page 21



### Table 2 Percentage Biodegradation Valnes % 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 corrected to include any canyMover ofC02 detected inAbsorber 2Harlan StudyNumber: 41401177 Report Page22



### Table 3 Total andInorganic CarbonValues in the CultureVessels on Day 0 Total Carbon

# . Inorganic Carbon* Testvessel IC Content(% ofTC) (mg!L) (mg/L) TestItem IOmgC/L Rt 8.87 -0.01 0
TestItem !Omg C/LR 2 8.83 -0.01 0

R 1 - R2 = Replicates I and 2 

### * Corrected for control values. Negative values are due to measured concentrations being less than control valuesHarlan Study Number: 4!40!!77 Report Page23



### Table 4 pHValues ofthe Test Preparations on Days 0 and 28 pH Test Vessel DayO DayO Day28 Pre-Adjustment Post-Adjustment Inoculum Control R1 7.8 7.4 7.5 Inoculum Control Rz 7.8 7.4 7.4 Procedure ControlR 1 7.8 7.4 7.5 Procedure Control R2 7.8 7.4 7.4 TestItem Rt 7.8 7.5 7.4 Test ItemRz 7.8 7.4 7.4 Toxicity Control 7.8 7.5 7.5

Replicates l and 2Harlan Study Number: 41401177 Report Page 24



### Table 5 Observations on the TestPreparations Throughoutthe TestPeriod Observations on TestPreparations Test Vessel DayO Day6 Day13 Day20 Day27
Inoculum R, Lightbrown Lightbrown Lightbrown Lightbrown Lightbrown Control dispersion dispersion dispersion dispersion dispersion

R, Light brown Lightbrown Lightbrown Lightbrown Light brown dispersion dispersion dispersion dispersion dispersion Light brown Lightbrown Lightbrowa Lightbrown Light 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 Light brown Lightbrown Light brown Lightbrown Light brown dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no R, undissolved undissolved undissolved undissolved undissolved reference item reference item reference item reference item reference item visible visible visible visible visible Light brown Light brown Light brown Lightbrown Lightbrown Test Item R, dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no undissolved undissolved undissolved undissolved undissolved test item visible test item visible test item visible testitem visible test item visible

Lightbrown Lightbrown Light brown Lightbrown Lightbrown R, 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 Lightbrown Light brown Lightbrown Lightbrown dispersion, no dispersion, no dispersion, no dispersion, no dispersion, no Toxicity lUldissolved 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** I and 2Harlan StudyNarnber: 41401177 Report Page 25


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

- -tr. -- Toxicity Control

0 80

# /~ 0 0 

# - - 0 

# -- • • 

## • • 0 / 0 70 

### • / 

## • • 60**~ ·~**  

### "" 50**~** 

## "' m 0 

# "' 40

30

20 

### • 10 E------10 Day Window
0 2 4 6 6 10 12 14 16 18 20 22 24 26 28 Time (Days)Harlan StudyNumber: 41401177 Report


## APPENDICES Appendix 1 Certificate ofAnalysis



### $odium Eryth0rfbate FCC,GtRdc,**K05-h~i G~&,E·Pp#** **LoU~urt'lben** ProduQtlon--Oilttt: Re-ov•tuatlon Dater: Source.Code: Fac.lory: Product Codl':

### {~**~l~u~lili~-** ldtJIJtlflt>allon**~iisAY,** , . Holff>/y· Mli_t'I!Jl'l (.a'S fib) (n_.,l;tl)'curro_.lt FCC)· Liintt· GlS&E 5092 14031314**M~r.** 26, 2014 Mer. 2(1:,.2016 01i)..DSI·D1 QetXIng·Perohn,**Pe~<lng, Jieng~ti,**  China 692611 (25 kge e&rh;m)**~!l)fi;:'--ril•ri'lil:.:** D,:l'f'b ;•\a<. '**P;'js:~"<.-\lJ"i.\** L(l!lt; on r:ltyhf{) 0:11DlOtJl1 Spo_1ll!Jc**~i:!!.'!.I.!~J~l~{~~~--- --~-**  ..**:-~~2-~-!'>t_B~I?~** Conc!ualon:**Thlam&\~tlal rh$8ts•Ptod'uet$ped.~u_on** **~~fremitnW.** 0.17 Cq_nlr.-rm-**-~t-** ____
Above·maturial

### manuriactur8df,&·,fttste~:-~y,ji~Jl~}(J;ti-ei'~~-f{~~hri·S~{~_J\ilsb~~-~~-,i:·¢~.'fLj~:~t~~-l:\b1Y~!-=_usA . '-" ,,--- -- ""'1·:- ' --' ,_ • ";, Page 26Harlan Study Number: 41401177


### Appendix 2 Mineral Medium

Solution a

Solution b Solution c Solution d KH2P04 K2HP04 Na2HP04.2H20 NH4Cl

CaC12 MgS04.7H20 FeCb.6H20 Report

8.50 g/L 21.75 giL 33.40 giL 0.50 giL pH= 7.4 27.50 g/L 22.50 g/L 0.25 g/L To !liter(final volume) ofpurified water* was added the following volumes ofsolutions a-d. 10 mL ofSolution a 1 mL ofSolutionb 1 mL ofSolution c I mL ofSolution d

*Reverse osmosis purified and deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP) Page27Harlan StudyNumber: 4140 1177 Report
Appendix 3 Dissolved OrganicCarbon (DOC) Values from thePreliminary 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 ShimadzuTOC-VcsH TOC analyzer.
DOC Concentration Sample mg C/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 

### - - I00 mg!L Untreated 32.18 32.18 89 I00 mg/LFiltered .32.51 32.51 90 I00 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 tbat the test item did not adsorb to filter matrices or activated sewage sludge. Therefore, for the purpose of the study, tbe samples taken for DOC analysis were filtered to remove the suspended solids presentwithout causing a loss ofany test item.

LOQ**~Limit** ofQuantification (determined down to 1.0 mgC/L).Harlan Study Number: 4 l401177 Report


### Appendix 4 MonitoringAuthority StatementofGLP Compliance 

# i ,;;;, Department

### ~- · ofHealth
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC TEST FACILITY

Harlan Laborato·ries Limited Shardlow Business Park London Road Shardlow Dorbyshlre DE72 2GD

DATE OF INSPECTION TEST TYPE(S) Analytical/Clinical Chemistry Environmental Fate Environmental Toxicity Phys.Chem. Testing Mutagenlcity

Toxtcology

12 to 14 March 2014 An Inspection for compliance with the Principles of Good Laboratory Practlce was carried out at the above test facility as part of the UK Good Labnratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of Issue, the UK Good Laboratory Practice Monitoring Authority ware satisfied that the above test facUity was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory Pr;ictlce Instrument (as defined in the UK Good Laboratory Piactice Regulations 1999).

Dr. Andrew .J. Gray Hand, UK GLP Mo-nitoring Authority Page 29