REPORT 

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Assessment of Ready Biodegradability; C02 Evolution Test 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

NBest 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA98052 

UNITED STATES OF AMERICA 

41401177 

28 January 2015 

Page 1 of29 
Harlan Study Number: 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 MATERIALS 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 Preliminary Investigational Work ........................................................... 11 

3.5.2 Experimental Preparation ........................................................................ 11 

3.5.2.1 Test Item .................................................................................... 11 

3.5.2.2 Reference Item ......................................................................... .-12 

3.5.2.3 Toxicity Control ........................................................................ 12 

3.5.3 Preparation of Test System ...................................................................... 12 

3.6 Evaluations ............................................................................................................ 13 

3 .6.1 Observations ............................................................................................ 13 

3.6.2 pH Measurements .................................................................................... 13 

3.6.3 IC Analysis .............................................................................................. 13 

3.6.4 IC/TC Ratio ............................................................................................. 14 

3.7 Data Evaluation ..................................................................................................... 14 

3. 7.1 Calculation of Carbon Content ................................................................ 14 

3.7.2 Percentage Biodegradation ...................................................................... 15 

3.7.3 Total C02 Evolution ................................................................................ 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 .................................................................................................................... 19 
Harlan Study Number: 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 on Each Analysis Occasion .......................................... 20 

Percentage Biodegradation Values ........................................................................ 21 

Total and Inorganic Carbon Values in the Culture Vessels on Day 0 ................... 22 

pH Values of the Test Preparations on Days 0 and 28 .......................................... 23 

Observations on the Test Preparations Throughout the Test Period ..................... 24 

LIST OF FIGURES 

Figure 1 Biodegradation Curves .......................................................................................... 25 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Certificate of Analysis ........................................................................................... 26 

Mineral Medium .................................................................................................... 27 

Dissolved Organic Carbon (DOC) Values from the Preliminary 

Investigational Work ............................................................................................. 28 

Monitoring Authority Statement of GLP Compliance .......................................... 29 
Harlan Study Number: 41401177 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Harlan Study Number: 

Study Title: 

41401177 

D-erythro-Hex-2-enonic acid, .garnrna.-lactone, monosodium salt 

[CAS RN 6381-77-7]: 

Assessment of Ready Biodegradability; C02 Evolution 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 of Directives 2004/9/EC and 2004/1 0/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 of the study or the validity of the data. 

Study Director: NBest 

··~·············· Date: 2 8 JAN 2015 
Harlan Study Number: 41401177 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 41401177 

Study Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7]: 

Assessment of Ready Biodegradability; C02 Evolution Test 

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-related procedures conducted at the test facility were audited and inspected. The details of 

these audits and inspections are given below. 

Reported to the 

Dates and Types of QA Inspections relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection 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 Report Audit N/A 27 November 2014 

This statement confirms that this report reflects the raw data and the procedures followed. 

Quality Assurance: 

2 8 JAN 2015 

Date: 
Harlan Study Number: 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 of Chemicals (1992) No. 301B, "Ready Biodegradability; C02 Evolution 

Test" referenced as Method C.4-C of Commission 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 carbo niL, 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 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 strict terms and conditions ofOECD Guideline No. 301B. 
Harlan Study Number: 41401177 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Deviations from Study Plan 

8 September 20 14 

29 October 2014 

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

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 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: 41401177 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. 

This study was performed based on the U.S. Toxic Substances Control Act (TSCA) Section 4 

Test rule and the regulations set forth under 40 CFR 799.5087. 

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

C02 Evolution Test" 

• Method C.4-C of Commission Regulation (EC) No. 440/2008 

• US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 

(Paragraph (m)) 
Harlan Study Number: 41401177 Report Page 9 

2 TEST AND REFERENCE ITEMS 

2.1 Test Item 

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is 

given in Appendix 1. 

Identification: 

Physical state/ Appearance: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

Intended use/ 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 in the dark 

not supplied 

Information as provided by the Supplier (Sigma-Aldrich). 

Identification: 

Description: 

Batch: 

Purity: 

Expiry Date: 

Storage conditions: 

Sodium benzoate 

white granular solid 

SLBG9515V 

>99.5% 

09 July 2015 

room temperature over silica gel 
Harlan Study Number: 41401177 Report Page 10 

3 MATERIALS AND 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 of the 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. 

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 of the Severn Trent Water Plc sewage treatment plant 

at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. 

3.3 Preparation of Inoculum 

The activated sewage sludge sample was washed twice by settlement and resuspension in 

mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may 

have been present. The washed 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 after rinsing the filter three successive times with 10 mL of deionized reverse osmosis 

water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour 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 prior to drying in an oven 
Harlan Study Number: 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 of test 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 of test item (100 mg) was dissolved in mineral medium and inoculated 

at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. 

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 

per the 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 1 liter to give a 300 mg/L stock solution. 

An aliquot (276 mL) of this 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 carbo niL. The volumetric flask containing the test item was inverted several times to 

ensure homogeneity of the solution. 

A test concentration of 1 0 mg carbon/L was employed in the test following the recommendations 

of the Test Guidelines. 
Harlan Study Number: 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 carbo niL. The volumetric flask containing the reference item 

was inverted several times to ensure homogeneity of the 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 of the test item on the sewage sludge micro-organisms used in the test. 

An aliquot (276 mL) of the test item stock solution was dispersed in inoculated mineral medium 

along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was 

adjusted to 3 liters to give a final concentration of 27.6 mg test item/L plus 17.1 mg sodium 

benzoate/L, equivalent to a total of 20 mg carbo niL. 

3.5.3 Preparation of Test System 

The following test preparations were prepared and inoculated in 5 liter test culture vessels each 

containing 3 liters of solution: 

a) An inoculated control, in duplicate, consisting of inoculated mineral medium. 

b) The procedure control containing the reference item (sodium benzoate), in duplicate, in 

inoculated mineral medium to give a final concentration of 10 mg carbon/L. 

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

of 10 mg carbo niL. 

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

concentration of 20 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, 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 30.0 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 Hach HQ40d 

Flexi handheld meter and the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or 
Harlan Study Number: 41401177 Report Page 13 

sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the 

addition of mineral medium which had been purged 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 of 0.05 M NaOH. The C02 absorbing solutions were prepared using purified de-gassed 

water. 

3.6 Evaluations 

3.6.1 Observations 

The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27. 

3.6.2 pH Measurements 

The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification 

with hydrochloric acid, using a Hach HQ40d Flexi handheld meter and a Hach HQ160 Flexi 

handheld meter. 

3.6.3 IC Analysis 

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 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 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 

of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to 

C02 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by 

reference solutions of sodium 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 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 of the 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 

(Na2C03) in deionized water. Each analysis was carried out in triplicate. 

3. 7 Data Evaluation 

3. 7.1 Calculation of Carbon Content 

The theoretical amount of carbon present in the test item (D-erythro-Hex-2-enonic acid, 

.gamma.-lactone, monosodium salt [CAS RN 6381-77-7] (C6Hs06Na)) was calculated as 

follows: 

No of C atoms x mol wt of C x 100 

mol wt of test item 

6 x 12·

011 x100=36.19% 

199.11 

Thus for a concentration of 10 mg C/L the total organic carbon present was 30 mg C. 

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

was calculated as follows: 

No of C atoms x mol wt of C x 100 

mol wt of sodium benzoate 

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 Study Number: 4140 1177 Report Page 15 

3.7.2 Percentage Biodegradation 

The percentage biodegradation 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 R1 and R2 are meaned for the inoculum control, 

test and reference items before substitution into the following equation: 

01 ThCO ( 01 b" d d . *) mg IC in test flask- mg IC in control flask 100 

;o 2 = ;o 10 egra atwn = x 

mg TOC added as test chemical 

3.7.3 Total C02 Evolution 

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: 

Total C02 evolution (mg C/L) = mg IC in control x 100 x --1-- %C of CO2 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 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 

C02 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window, 

as appropriate, is less than 20%. 

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

exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however 

*The conversion factor for carbon to carbon dioxide is 3.67 
Harlan Study Number: 41401177 Report Page 16 

values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are 

obtained should be critically examined. 

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

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

4.2.1 Validation Criteria 

The total C02 evolution in the inoculum control vessels on Day 28 was 31.88 mg/L and therefore 

satisfied the validation criterion given in the 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% of the 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 off any dissolved C02 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 of samples 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 Study Number: 41401177 Report Page 18 

Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed 

that no significant carry-over of C02 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 strict terms 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 in the test. The slight decrease in biodegradation between days 

14 and 28 was considered to be due to sampling/analytical variation. 

Sodium benzoate attained 83% biodegradation after 14 days and 88% biodegradation after 

28 days thereby confirming the suitability of the inoculum and test conditions. 
Harlan Study Number: 41401177 Report Page 19 

5 CONCLUSION 

The test item attained 70% biodegradation after 28 days and 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 Study Number: 41401177 Report 

TABLES 

Table 1 Inorganic Carbon Values on Each Analysis Occasion 

Day 

0 

2 

6 

8 

10 

14 

21 

28 

29 

R1 -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 

C02 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 

Test Item 

Rt 

Abs2 Abs 1 

1.05 1.05 

- 10.21 

- 28.26 

- 32.11 

- 35.11 

- 37.97 

- 39.21 

- 45.36 

1.39 47.76 
Harlan Study Number: 41401177 Report Page 21 

Table 2 Percentage Biodegradation Values 

% 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 carry-over of C02 detected in Absorber 2 
Harlan Study Number: 4140 1177 Report Page 22 

Table 3 Total and Inorganic Carbon Values in the Culture Vessels on Day 0 

Total Carbon * Inorganic Carbon* 

Test vessel (mg!L) (mg/L) 

IC Content (% of TC) 

Test Item 

10 mg C/L R1 

8.87 -0.01 0 

Test Item 

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 values 
Harlan Study Number: 41401177 Report Page 23 

Table 4 pH Values of the 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 R2 7.8 7.4 7.4 

Procedure Control R1 7.8 7.4 7.5 

Procedure Control R2 7.8 7.4 7.4 

Test Item R1 7.8 7.5 7.4 

Test Item R2 7.8 7.4 7.4 

Toxicity Control 7.8 7.5 7.5 

Replicates 1 and 2 
Harlan Study Number: 4140 1177 Report Page 24 

Table 5 Observations on the Test Preparations Throughout the Test Period 

Observations on Test Preparations 

Test Vessel 

DayO Day6 Day 13 Day20 Day27 

Inoculum R1 Light brown Light brown Light brown Light brown Light brown 

Control dispersion dispersion dispersion dispersion dispersion 

R2 Light brown Light brown Light brown Light brown Light brown 

dispersion dispersion dispersion dispersion dispersion 

Light brown Light brown Light brown Light brown Light brown 

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 

Light brown Light brown 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 

Light brown Light brown Light brown 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 

Light brown Light brown Light brown Light brown Light brown 

R2 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 

Light brown Light brown Light brown Light brown 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 1 and 2 
Harlan Study Number: 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 

e! 50 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 25 
Harlan Study Number: 41401177 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

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 26 
Harlan Study Number: 41401177 

Appendix 2 Mineral Medium 

Solution a 

Solution b 

Solution c 

Solution d 

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) of purified water* was added the following volumes of solutions 

a-d. 

1 0 mL of Solution a 

1 mL of Solution b 

1 mL of Solution c 

1 mL of Solution d 

*Reverse osmosis purified and deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP) 

Page 27 
Harlan Study Number: 41401177 Report 

Appendix 3 Dissolved Organic Carbon (DOC) Values from the Preliminary 

Investigational Work 

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-V csH TOC analyzer. 

DOC Concentration 

Sample mgC/L %of Nominal 

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 of any test item. 

LOQ =Limit of Quantification (determined down to 1.0 mg C/L). 
Harlan Study Number: 41401177 Report 

Appendix 4 Monitoring Authority Statement of GLP Compliance 

Hanan Laboratories Limited 

Shardlow Busin!ll!m$ Park 

London Road 

Shandlow 

TEST 

Environmental 

Environm!ll!nml Toxh::itv 

rn·'!fl!i .. wr••ntl. Ta~~>tini:J 

Page 29