Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): READY BIODEGRADABILITY BY 

THE CARBON DIOXIDE EVOLUTION TEST METHOD 

WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 527E-128A 

Organisation for Economic Cooperation and Development 

OECD Guideline 301B 

and 

 Council of the European Communities Directive 67/548/EEC 

Annex V, Guideline C.4-C 

AUTHORS: 

Edward C. Schaefer 

Molly E. Matthews 

STUDY INITIATION DATE: October 09, 2009 

STUDY COMPLETION DATE: March 18, 2010 

Submitted to: 

Sasol Germany GmbH 

Building 1033/PB 02 

Paul-Baumann-Str. 1 

D-45764 Marl 

Germany 

Wildlife International, Ltd.

8598 Commerce Drive 

Easton, Maryland 21601 

(410) 822-8600 

Page 1 of 34 
wildlife International, Ltd. Project Number 527E-128A 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): Ready Biodegradability by 

the Carbon Dioxide Evolution Test Method 

WILDLIFE INTERNATIONAL, LTD PROJECT NO 527E-128A 

STUDY COMPLETION March 18,2010 

This study was conducted in compliance with Good Laboratory Practice Standards as 

published by the US. Environmental Protection Agency, 40 CFR Part 792, 17 August 1989, and 

OECD Principles of Good Laboratop Practice (ENVIMCICHEM (98) 17); with the following 

exceptions: 

The test and reference substances were not characterized in accordance with Good Laboratop 

Practice Standards; nor were their stabilities under storage conditions at the test site determined in 

accordance with Good Laboratory Practice Standards. 

The concentration, stability and homogeneity of the reference substance in thc camer were not 

determined. 

Periodic analyses of municipal water (NANO~U~~@ source) for potential contaminants were not 

performed according to Good Laboratory Practice Standards, but were performed using a certified 

laboratory and standard U.S. EPA analytical methods. 

The carbon analysis on the test substance was not performed in accordance with Good 

Laboratory Practice Standards. 

STUDY DIRECTOR: 
Wildlife International, Ltd. Project Number 527E-128A 

QUALITY ASSURANCE STATEMENT 

This study was examined for colnpliance with Good Laboratory Practice Standards as 

published by the US. Environmental Protection Agency, 40 CFR Part 792: 17 August 1989, and 

OECD Principles of Good Laboratory Practice (ENVIMCICHEM (98) 17). The datcs of all 

inspections and audits and the dates that any findings were reported to the Study Director and 

Laboratory Management were as follows: 

ACTIVITY: 

Protocol 

Initial Trial 527E-128: 

Test Substance Administration 

Sample Collection 

Test Termination 

Definitive Trial 527E-128A: 

Test Substance Administration 

Samplc Collection 

Data and Draft Report 

Final Report 

DATE CONDUCTED: 

October 13.2009 

October 20.2009 

Norember 09,2009 

Novcmbor 17; 2009 

December 01 : 2009 

Januaq I 1 2010 

Februq 22 March 01 

2010 

March 18: 20 0 

All Inspections were stud) based unless othemlsc noted 

4Lk!!,& 

M~chael D Greene 

Quality Assurance Representative 

DATE REPORTED TO: 

STUDY DIRECTOR: MANAGEMENT: 

October 13,2009 October 22,2009 

October 20,2009 November 06; 2009 

November 09; 2009 November 16,2009 

November 17,2009 November 20,2009 

Dccembcr 01.2009 December 03,2009 

Januzry 11:2010 Januar). 12,2010 

March 02,201 0 March 03,2010 

March 18,2010 March 18: 201 0 
Wildlife International, Ltd Project Number 527E-128A 

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REPORT APPROVAL 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols: C18-22, dism. residues (CAS No. 1160164-88-4): Ready Biodegradability by 

the Carbon Dioxide Evolution Test Method 

WILDLIFE INTERNATIONAL, LTD PROJECTNUMBER: 527E-128A 

STUDY DIRECTOR: 

L. 

Senior Biologist Biodegradahon 

MANAGEMENT: 

Edward C. Schaefer 1 

D~rector of Biodegradation 

DATE 
Wildlife International, Ltd. Project Number 527E-128A 

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STUDY INFORMATION 

Study Initiation Date: October 09, 2009 

Experimental Start Date (OECD): October 16, 2009 

Experimental Start Date (EPA): December 01, 2009 

Experimental Termination Date: February 01, 2010 

Study Completion Date: March 18, 2010 

Study Director: Molly E. Matthews 

 Senior Biologist, Biodegradation. 

 Wildlife International, Ltd. 

Sponsor: Sasol Germany GmbH 

 Building 1033/PB 02 

 Paul-Baumann-Str 1 

 D-45764 Marl 

 Germany 

Sponsor’s Representative: Ricky Stackhouse, Ph.D 

 Sasol North America, Inc. 

 Research and Development 

 2201 Old Spanish Trail 

 Westlake, LA 70669 

Study Personnel: Edward C. Schaefer, Director of Biodegradation 

Molly E. Matthews, Senior Biologist, Biodegradation 

Alexander A. Ponizovsky, Ph.D., Scientist, Biodegradation 

Aaron Gehris, Biologist, Biodegradation 

 
Wildlife International, Ltd. Project Number 527E-128A 

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TABLE OF CONTENTS 

Title/Cover Page..................................................................................................................................... 1 

Good Laboratory Practice Compliance Statement ................................................................................. 2

Quality Assurance Statement ................................................................................................................. 3 

Report Approval ..................................................................................................................................... 4 

Study Information................................................................................................................................... 5 

Table of Contents ................................................................................................................................... 6 

Summary ................................................................................................................................................ 8 

Introduction ............................................................................................................................................ 9 

Objective ................................................................................................................................................ 9 

Experimental Design .............................................................................................................................. 9 

Materials and Methods ......................................................................................................................... 10 

Test Substance ......................................................................................................................... 10 

Reference Substance................................................................................................................ 10 

Test Medium............................................................................................................................ 11 

Test Apparatus and Conditions................................................................................................ 11

Test Inoculum .......................................................................................................................... 12 

Preparation of Test Chambers ................................................................................................. 12

Biodegradation Test Initiation ................................................................................................. 12 

Sample Collection and Analysis.............................................................................................. 13 

Test Termination...................................................................................................................... 13 

Calculations ............................................................................................................................. 13 

Results and Discussion......................................................................................................................... 14 

Carbon Analysis....................................................................................................................... 14 

Observations and Measurements ............................................................................................. 14 

Conclusions .......................................................................................................................................... 15 

References ............................................................................................................................................ 16 
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TABLE OF CONTENTS 

- Continued -

TABLES 

Table 1. Dissolved Organic Carbon (DOC), and pH of Test Solutions at Test Termination............. 17 

Table 2. Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L) ..................... 18 

Table 3. Cumulative Milligrams of Carbon Dioxide Evolved ........................................................... 19 

Table 4. Cumulative Percent of Theoretical Carbon Dioxide Evolved.............................................. 20 

FIGURES 

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved ........................................ 21 

APPENDIX 

Appendix I. Protocol and Deviation............................................................................................... 22 
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SUMMARY 

The ready biodegradability of Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) was 

determined by the Carbon Dioxide Evolution Test Method (OECD Guideline 301B). Tests of ready 

biodegradability are stringent tests that provide limited opportunity for acclimation and 

biodegradation to occur. In the CO2 test, inoculated mineral medium was dosed with a known 

amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air. 

The CO2 produced from the mineralization of organic carbon within the test chambers was displaced 

by the flow of CO2-free air and trapped as K2CO3 in KOH trapping solution. The amount of CO2

produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a 

percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete 

biodegradation of the test substance occurred. The test contained a blank control group, a reference 

group and a treatment group. Each group contained three replicate test chambers. The blank control 

was used to measure the background CO2 production of the inoculum and was not dosed with a 

carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be 

biodegradable, at a nominal concentration of 10 mg C/L. The treatment group test chambers were 

used to evaluate Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) at a nominal 

concentration of 10 mg C/L. The results indicated that the activated sludge inoculum was active, 

degrading the reference substance 103.8%. The average cumulative percent biodegradation for 

Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) was 84.1%.

Test Substance 

Average 

Cumulative 

Percent 

Biodegradation 

Average 

Final 

pH1

Readily 

Biodegradable2

Alcohols, C18-22, distn. residues 

(CAS No. 1160164-88-4) 

84.11 7.41 No 

1 Average of three replicates. 2 Meets or exceeds the OECD criteria for ready biodegradability (60% of TCO2 within a 10-day window of 

reaching 10% TCO2). 
Wildlife International, Ltd. Project Number 527E-128A 

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INTRODUCTION 

 Tests of ready biodegradability, by definition, provide limited opportunity for acclimation and 

biodegradation to occur. A positive result in a test of ready biodegradability is an indication that the test 

substance will undergo rapid and ultimate biodegradation in the environment. A negative result in a test 

of ready biodegradability does not necessarily mean that the test substance will not be biodegraded 

under relevant environmental conditions but that additional testing may be needed. 

 The study was initiated on October 09, 2009, the day the Study Director signed the protocol, 

and was completed on the day the Study Director signed the final report. The experimental start date 

for the initial trial, 527E-128 was October 16, 2009 and the experimental termination date was 

November 18, 2009. The experimental start date for the definitive trial was November 25, 2009 and 

the experimental termination date was February 01, 2010. The definitive trial, 527E-128A was 

conducted because the initial trial was terminated before the optional extension listed in the protocol 

could be initiated.

This study was conducted by Wildlife International, Ltd. for Sasol Germany GmbH at the 

Wildlife International, Ltd. biodegradation facility in Easton, Maryland. Original raw data generated 

by Wildlife International, Ltd. and a copy of final report are filed under Project number 527E-128A in 

the archives located on the Wildlife International, Ltd. site. The original final report was sent to the 

Sponsor. 

OBJECTIVE 

The objective of the study was to measure the amount of carbon dioxide (CO2) produced from 

the biodegradation of a nonvolatile organic test substance. This value is expressed as a percentage of 

the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation of 

the test substance occurred. 

EXPERIMENTAL DESIGN 

The test contained a blank control group, a reference group and a treatment group. Each group 

contained three replicate test chambers. The control group was used to measure the background CO2

production of the inoculum and was not dosed with a carbon source. The reference group chambers 

were used to check the viability of the inoculum and were dosed with sodium benzoate, a substance 
Wildlife International, Ltd. Project Number 527E-128A 

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known to be biodegradable, at a nominal concentration of 10 mg C/L. The treatment group chambers 

were used to evaluate Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) at a nominal 

concentration of 10 mg C/L. 

MATERIALS AND METHODS 

This study was conducted according to the procedures outlined in the protocol, “Alcohols, 

C18-22, distn. residues (CAS No. 1160164-88-4): Ready Biodegradability by the Carbon Dioxide 

Evolution Test Method”. The protocol was based on the procedures specified in the OECD Guideline 

for Testing of Chemicals, Method 301B (1), and Annex to Directive 67/548/EEC. Guideline C.4-C, 

Carbon Dioxide (CO2) Evolution (2). 

Test Substance 

 The test substance was received from Sasol on September 11, 2009 and was assigned Wildlife 

International, Ltd. identification number 9187. The following is a description of the test substance 

used in this study: 

 Name: Alcohols, C18-22, distn. residues 

 Lot Number: 03585/MA 

 CAS Number: 1160164-88-4 

 Physical Description: Solid 

 Purity: 100% 

 Expiration Date: August, 2013 

 Storage Conditions: Ambient Temperature 

 Carbon Content: 81.65% . 

The test substance was administered to the treatment group test chambers by direct weight 

addition. Direct weight addition of a test substance that is relatively insoluble in water is the most 

appropriate route of administration. The amounts of test substance used to dose the treatment group 

test chambers were calculated based on the measured carbon content. The carbon content of the test 

substance was determined by a non-GLP elemental analysis performed by Quantitative Technologies, 

Inc., Whitehouse, New Jersey. 

Reference Substance 

The reference substance was manufactured by Mallinckrodt Chemicals, was received on 

October 30, 2006 and was assigned Wildlife International, Ltd. Identification number 7787. A stock 

solution of the reference substance was prepared in NANO®pure water at a carbon concentration of 
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approximately 400 mg C/L. The stock solution was analyzed for total organic carbon (TOC). The 

reference substance was administered to the reference group test chambers by volumetric addition. 

Dosing amounts were based on the measured carbon content of the reference substance solution. 

Following is a description of the reference substance used in this study: 

 Name: Sodium benzoate 

Manufacturer: Mallinckrodt Baker, Inc. Phillipsburg, New Jersey 

Lot Number: C24593 

Physical Description: Solid 

Chemical Abstract Number: 532-32-1 

Expiration Date: October 30, 2011 

Purity: 99.2% 

Storage Conditions: Ambient 

Test Medium 

The test medium was a modified biochemical oxygen demand (BOD) test dilution water and 

was prepared using high quality water as described in the Protocol (Appendix I). All chemicals and 

reagents used in the preparation of the test medium were reagent grade or better. 

Test Apparatus and Conditions 

The test chambers were amber 4-liter bottles. The air entering the chambers was passed 

through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air. The 

air exiting the test chambers was passed through a series of three gas washing bottles, each containing 

approximately 100 mL of 0.5 N KOH to trap the CO2 that had evolved within the chamber. An 

additional set of gas washing bottles that were not connected to a chamber were maintained 

concurrently with the traps connected to the chambers. These bottles contained approximately 

100 mL of 0.5 N KOH and the amount of CO2 detected in the KOH solution was subtracted from the 

CO2 in the blank control traps to determine the amount of CO2 produced by the inoculum in the blank 

control. The test was conducted at 20±2oC, except on January 19, 20 and 29, 2010 when the 

measured temperature range was 20.4-22.5oC. Test chambers were identified by project number, test 

substance ID, test concentration and vessel number. Magnetic stir bars and stir plates were used to 

mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 

15 minutes to prevent the heating of the stirrer motors. 
Wildlife International, Ltd. Project Number 527E-128A 

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Test Inoculum 

Activated sludge was collected from the Cambridge Wastewater Treatment Facility, 

Cambridge, Maryland on November 25, 2009. The Cambridge facility treats predominantly 

residential wastes. The sludge was sieved using a 2-mm screen, adjusted to approximately 1,000 mg 

total suspended solids (TSS)/L with mineral media and then aerated at test temperature until use. A 

total suspended solids measurement and standard plate count were performed on the inoculum on the 

day of test chamber preparation. Plates were incubated at 20±2oC for approximately 48 hours. 

Preparation of Test Chambers 

The following were added to each chamber: 

1) 2470 mL of NANO®pure water 

2) 3 mL calcium chloride solution (2.75%) 

3 mL of ferric chloride solution (0.025%) 

3 mL of magnesium sulfate solution (2.25%) 

30 mL of phosphate buffer (pH 7.4) 

3) A volume of the activated sludge inoculum to achieve a final TSS concentration of 

approximately 30 mg/L 

All chambers were aerated with CO2 free air for approximately 24 hours at a rate of 50 to 

100 mL per minute to purge the systems of CO2. After the aeration period, the flow of CO2 free air 

was stopped, and three CO2 traps, each containing approximately 100 mL of 0.5 N KOH, were 

connected to the exit air lines of each chamber. Sufficient volumes of reference substance stock 

solution to achieve a nominal concentration of 10 mg C/L were added to the appropriate chambers. 

Sufficient amounts of test substance to achieve a nominal concentration of 10 mg C/L were added to 

the appropriate chambers. The final volume within all chambers was brought up to 3000 mL by the 

addition of NANO®pure water. 

Biodegradation Test Initiation 

The biodegradation test was started by bubbling CO2-free air through each of the test chambers 

at a rate of 50-100 mL per minute. The CO2 produced from the degradation of organic carbon 

sources within the test chamber was trapped as K2CO3 in the KOH solution and the amount of 
Wildlife International, Ltd. Project Number 527E-128A 

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inorganic carbon in the trapping solution was measured at various intervals during the study, using a 

Shimadzu Model TOC-VCSH carbon analyzer. 

Sample Collection and Analysis 

 On days 2, 6, 9, 13, 16, 20, 22, 27, 29, 34, 37, 41, 45, 48, 51, 55 and 58 the CO2 trap nearest the 

test chamber was removed and analyzed for inorganic carbon. The two remaining traps were placed 

one position closer to the test chamber and a new trap was placed on the end of the series. 

Test Termination 

 On the 60th day of the test, an aliquot of the contents of each test chamber was removed and 

the pH determined. The contents of all chambers were then acidified by the addition of 3 mL of 

concentrated hydrochloric acid to drive off inorganic carbonate. All chambers were aerated overnight 

and then a sample from each test chamber was removed for dissolved organic carbon (DOC) analysis 

and the trapping solutions closest to the test chambers were removed for inorganic carbon analysis. 

Calculations 

The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2

produced using the following equation: 

mg CO2 = result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C 

The cumulative mg of CO2 for the test and reference substances were corrected for the amount 

of CO2 evolved by the control, using the following equation: 

Cumulative mg CO2 evolved = Σ mg CO2 test - mean Σ mg CO2 control 

The percentage of theoretical carbon dioxide (%TCO2) evolved was calculated as follows: 

% TCO2 = (mg of carbon in test) x ( . mg CO /mg Carbon)

mg CO produced

2

2

3 67

x 100 
Wildlife International, Ltd. Project Number 527E-128A 

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RESULTS AND DISCUSSION 

Carbon Analysis 

The measured total organic carbon (TOC) concentration of the reference substance stock 

solution was 394.8mg C/L. The volume of stock solution used to dose the reference chambers was 

adjusted based on the measured TOC values so that approximately 10 mg C/L was delivered. 

Observations and Measurements 

The temperature range recorded during the test was 19.4-22.5oC. The results of the standard 

plate count and TSS measurement performed on the inoculum were 7.7 x 104

 CFU/mL and 920 mg/L, 

respectively. 

 The measured dissolved organic carbon (DOC) from Day 61 and pH values of the test chamber 

contents on Day 60 are presented in Table 1. The measured concentrations of inorganic carbon in the 

carbon dioxide trapping solutions are presented in Table 2. The cumulative amounts of CO2 produced 

over the test period are presented in Table 3. The cumulative percent of theoretical carbon dioxide 

(% TCO2) evolved is presented in tabular and graphical forms in Table 4 and Figure 1, respectively. 

The control chambers evolved an average of 106.2 mg CO2 over the test period. This value has 

been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, 

even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control 

chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2

evolution tests (2). 

 The viability of the inoculum and validity of the test were supported by the results of the 

reference substance, sodium benzoate, from which an average of 103.8% of theoretical CO2 was 

evolved. An average percent biodegradation of greater than 60% was achieved by Day 6, thereby 

fulfilling the criteria for a valid test by reaching the pass level by Day 14 (1). The final mean percent 

biodegradation for Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) was 84.1%. Alcohols, 

C18-22, distn. residues (CAS No. 1160164-88-4) may be not considered readily biodegradable, since 

the pass level of 60% TCO2 was not achieved with 10 days of reaching 10% TCO2. 

 
Wildlife International, Ltd. Project Number 527E-128A 

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CONCLUSIONS 

 Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% TCO2 within a 

28-day test period (1,2). In addition, the pass level must be reached within 10 days of achieving 10% 

TCO2 (1,2). The final mean percent biodegradation for Alcohols, C18-22, distn. residues (CAS 

No. 1160164-88-4) was 84.1% over a 60-day test period. Alcohols, C18-22, distn. residues (CAS 

No. 1160164-88-4) may not be considered readily biodegradable, since the pass level of 60% TCO2

was not achieved with 10 days of reaching 10% TCO2. 
Wildlife International, Ltd. Project Number 527E-128A 

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REFERENCES 

1. Organisation for Economic Cooperation and Development. 1992. Ready Biodegradability: 

CO2 Evolution (Modified Sturm Test). OECD Guideline 301B. 

2. Council of the European Communities. Directive 67/548/EEC. Annex V. Guideline C.4-C, 

 Carbon Dioxide (CO2) Evolution. 

 
Wildlife International, Ltd. Project Number 527E-128A 

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Table 1 

Dissolved Organic Carbon (DOC) and pH of Test Solutions at Test Termination 

 DOC1 

Test Chamber (Nominal Concentration) (mg C/L) pH 

 

Control Rep. 1 <1.0 7.4 

Control Rep. 2 <1.0 7.4 

Control Rep. 3 <1.0 7.4 

 

Sodium Benzoate Rep. 1 (10 mg C/L) <1.0 7.5 

Sodium Benzoate Rep. 2 (10 mg C/L) <1.0 7.5 

Sodium Benzoate Rep. 3 (10 mg C/L) <1.0 7.5 

 

Test Substance Rep. 1 (10 mg C/L) <1.0 7.4 

Test Substance Rep. 2 (10 mg C/L) <1.0 7.4 

Test Substance Rep. 3 (10 mg C/L) <1.0 7.4 

 

 

1

 Samples were filtered (0.45 μm), and acidified with 2 drops of 2 N HCl prior to analysis. 

 
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Table 2 

Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L)*

Date Day Control 

Rep. 1 

Control 

Rep. 2 

Control 

Rep. 3 

Sodium 

Benzoate 

Rep. 1 

Sodium 

Benzoate 

Rep. 2 

Sodium 

Benzoate 

Rep. 3 

Test Substance 

Rep. 1 

Test Substance 

Rep. 2 

03-Dec-09 07-Dec-09 10-Dec-09 14-Dec-09 17-Dec-09 21-Dec-09 23-Dec-09 28-Dec-09 30-Dec-09 

04-Jan-10 07-Jan-10 11-Jan-10 15-Jan-10 18-Jan-10 21-Jan-10 25-Jan-10 28-Jan-10 31-Jan-10 

2 6 9 

13 16 20 22 27 29 34 37 41 45 48 51 55 58 61 

20.9 36.1 30.3 36.5 22.7 33.0 18.6 29.6 14.6 23.7 19.0 19.0 23.4 17.2 16.1 14.4 16.2 16.5 

20.0 28.8 39.4 34.4 21.8 24.5 17.0 33.8 13.1 20.3 17.3 18.0 18.9 14.5 25.2 14.3 15.8 16.0 

21.7 35.2 31.8 35.5 33.0 26.4 17.5 32.2 13.8 27.2 17.7 22.4 17.9 18.3 13.9 23.9 16.1 15.9 

92.4 

169.6 

77.1 66.5 33.2 34.8 17.6 32.8 12.7 21.7 14.1 16.8 23.2 16.0 13.8 24.3 16.3 15.1 

89.3 

177.1 

84.9 67.4 33.2 34.2 17.5 34.1 14.3 33.5 13.9 20.9 19.4 17.0 18.0 15.6 26.1 17.8 

77.4 

158.0 

85.3 77.3 37.2 36.6 19.8 36.9 16.8 26.8 17.6 21.0 21.8 16.9 16.0 16.2 16.8 25.6 

18.3 38.9 36.9 49.8 39.3 59.9 22.3 41.7 18.5 37.6 31.4 56.8 47.3 29.8 29.8 34.6 27.9 33.3 

18.4 48.6 45.2 58.6 39.4 47.2 27.3 51.1 22.7 48.7 35.4 38.7 37.7 27.1 25.2 24.5 24.2 25.9 

*

Values in Table 2 were rounded for presentation purposes. 
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Table 3 

Cumulative Milligrams of Carbon Dioxide Evolved1,2

Date Day Control 

Rep. 1 

Control 

Rep. 2 

Control 

Rep. 3 

Sodium3

Benzoate

Rep. 1 

Sodium3

Benzoate 

Rep. 2 

Sodium3

Benzoate 

Rep. 3 

Test Substance 3

Rep. 1 

Test Substance 3

Rep. 2 

Test Substance 3

Rep. 3 

Control

Rep. 1 

03-Dec-09 07-Dec-09 10-Dec-09 14-Dec-09 17-Dec-09 21-Dec-09 23-Dec-09 28-Dec-09 30-Dec-09 

04-Jan-10 07-Jan-10 11-Jan-10 15-Jan-10 18-Jan-10 21-Jan-10 25-Jan-10 28-Jan-10 31-Jan-10 

2 6 9 

13 16 20 22 27 29 34 37 41 45 48 51 55 58 61 

7.7 

20.9 32.0 45.4 53.8 65.9 72.7 83.6 88.9 97.6 

104.5 111.5 120.1 126.4 132.3 137.6 143.6 149.6 

7.3 

17.9 32.4 45.0 53.0 62.0 68.2 80.6 85.4 92.9 99.2 

105.8 112.8 118.1 127.3 132.6 138.4 144.2 

8.0 

20.9 32.6 45.6 57.7 67.4 73.8 85.7 90.7 

100.7 107.2 115.4 122.0 128.7 133.8 142.6 148.4 154.3 

26.3 76.3 92.1 

103.5 106.2 108.7 108.7 109.0 108.6 107.8 106.4 105.3 106.4 106.2 104.5 107.0 107.1 106.7 

25.1 77.9 96.6 

108.3 111.0 113.3 113.2 114.0 114.2 117.8 116.2 116.6 116.4 

116.5 116.4 115.7 119.4 120.0 

20.7 66.5 85.4 

100.7 104.9 108.1 108.9 110.7 111.8 112.9 112.8 113.2 113.8 113.9 113.0 112.5 112.8 116.3 

-0.9 

1.1 2.2 7.5 

12.4 24.1 25.8 29.4 31.1 36.2 41.1 54.7 64.7 69.5 73.7 79.9 84.3 90.6 

-0.9 

4.7 8.9 

17.3 22.3 29.3 32.9 39.9 43.1 52.3 58.7 65.6 72.0 75.9 78.4 81.0 84.0 87.6 

-0.9 

4.9 8.0 

14.5 18.1 23.1 25.8 35.9 38.9 49.3 66.3 70.3 77.4 82.0 85.3 89.1 93.4 99.6 

5.4 

16.5 25.0 34.5 40.8 50.8 55.9 65.2 68.3 74.9 79.4 84.1 90.2 93.8 96.9 99.5 

103.2 106.5 

1The results of the inorganic carbon analyses of the CO2 traps were converted to mg CO2 produced using the following equation: 

 mg CO2 = cumulative result (mg C/L) X vol. of KOH (L) X 3.67 mg CO2/mg C 

2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ. 3Corrected for the CO2 attributed to the inoculum and the KOH by subtracting the average amount of CO2 evolved by the controls. 4Corrected for the amount of CO2 in the blank to determine the amount of CO2 evolved by the inoculum. 
Wildlife International, Ltd. Proje

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Table 4 

Cumulative Percent of Theoretical Carbon Dioxide Evolved1,2

Date Day Control 

Rep. 1 

Control 

Rep. 2 

Control 

Rep. 3 

Sodium 

Benzoate 

Rep.1 

Sodium 

Benzoate 

Rep.2 

Sodium 

Benzoate 

Rep.3 

Test 

Substance 

Rep. 1 

Tes

Substa

Rep

03-Dec-09 07-Dec-09 10-Dec-09 14-Dec-09 17-Dec-09 21-Dec-09 23-Dec-09 28-Dec-09 30-Dec-09 

04-Jan-10 07-Jan-10 11-Jan-10 15-Jan-10 18-Jan-10 21-Jan-10 25-Jan-10 28-Jan-10 31-Jan-10 

2 6 9 

13 16 20 22 27 29 34 37 41 45 48 51 55 58 61 

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 

23.9 69.3 83.7 94.0 96.5 98.7 98.7 99.0 98.6 98.0 96.6 95.6 96.7 96.5 94.9 97.2 97.2 96.9 

22.8 70.7 87.7 98.4 

100.8 102.9 102.8 103.6 103.7 107.0 105.6 105.9 105.7 

105.8 105.7 105.0 108.4 109.0 

18.8 60.4 77.5 91.5 95.3 98.2 98.9 

100.5 101.5 102.6 102.4 102.8 103.4 103.5 102.7 102.2 102.5 105.6 

-0.9 

1.0 2.0 6.8 

11.3 21.9 23.4 26.7 28.3 32.9 37.4 49.7 58.7 63.1 66.9 72.6 76.6 82.3 

-0.

4.3 8.0 

15.20.26.29.36.39.47.53.59.65.68.71.73.76.79.

Cumulative Average (N=3) Std. Dev. 

 103.8  6.2

 84.1  5.7

1The percentage of TCO2 was calculated using the following equation: 

% TCO2 = 

(mg of carbon in test) x ( . mg CO /mg Carbon)

mg CO produced

2 3 67

2 x 100 

2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ. 

NA – Not Applicable. 
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0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

110.0

120.0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62

Time (days)

Cumulative Percent of Theoretical CO2 Production 

Reference Rep. 1 Reference Rep. 2 Reference Rep. 3

Treatment Rep. 1 Treatment Rep. 2 Treatment Rep. 3

Figure 1. Cumulative Percentage of Theoretical Carbon Dioxide Evolved 
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APPENDIX I 

Protocol and Deviation 
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