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 C ooperation 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

-4-



### 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

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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 Terminati on 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. Sch aefer, Director of Biodegradation

Molly E. Matthews, Senior Biologist, Biodegradation

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

Aaron Gehris, Biologist, Biodegradation

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TABLE OF CONTENTS

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

Good Laboratory Practice Co mpliance 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 S ubstance............................................................................................................ .... 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

Refere nces ..................................................................................................................... ....................... 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 Concentr ation 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 CO 2 test, inoculated mineral medium was dosed with a known

amount of test substance as the nominal sole source of organic carbon and aerated with CO 2 -free air.

The CO 2 produced from the mineralization of organic carbon within the test chambers was displaced

by the flow of CO 2 -free air and trapped as K 2 CO 3 in KOH trapping solution. The amount of CO 2

produced by the test substance (co rrected for that evolved by the bl ank inoculum) is expressed as a

percentage of the theoretical amount of CO 2 (TCO 2 ) 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 CO 2 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

pH 1 Readily

Biodegradable 2

Alcohols, C18-22, distn. residues

(CAS No. 1160164-88-4) 84.1 1 7.4 1 No

1 Average of three replicates.

2 Meets or exceeds the OECD criteria for ready biodegradability (60% of TCO 2 within a 10-day window of

reaching 10% TCO 2 ).

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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 ultima te biodegradation in the environmen t. 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 additiona l testing may be needed.

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

and was completed on the day the Study Director signe d 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 befo re 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 fi nal report are filed under Project number 527E-128A in

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

Sponsor.

OBJECTIVE

The objective of the study was to measure the amount of carbon dioxide (CO 2 ) produced from

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

the theoretical amount of CO 2 (TCO 2 ) that could have been produced if complete biodegradation of

the test substance occurred.

EXPERIMENTAL DESIGN

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

contained three replicate test chambers. The control group was used to measure the background CO 2

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 sodi um benzoate, a substance

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

were used to evaluate Alcohols, C18-22, dist n. 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 pro cedures outlined in the protocol, “Alcohols,

C18-22, distn. residues (CAS No. 1160164-88-4): R eady 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), a nd Annex to Directive 67/548/EEC. Guideline C.4-C,

Carbon Dioxide (CO 2 ) Evolution (2).

Test Substance

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

International, Ltd. identificati on 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 administ ered 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 measu red 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 Internationa l, 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 administer ed to the reference group test chambers by volumetric addition.

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

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

Name: Sodium benzoate

Manufacturer: Mallinckrodt Bake r, 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 te st 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 moistu re and then through Ascarite to produce CO 2 free air. The

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

approximately 100 mL of 0.5 N KOH to trap the CO 2 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 CO 2 detected in the KOH solution was subtracted from the

CO 2 in the blank control traps to determine the amount of CO 2 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 plat e motors were cycled on and off approximately every

15 minutes to prevent the heating of the stirrer motors.

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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 a nd then aerated at test temperature until use. A

total suspended solids measurement and standard pl ate 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 CO 2 free air for approximately 24 hours at a rate of 50 to

100 mL per minute to purge the systems of CO 2 . After the aeration period, the flow of CO 2 free air

was stopped, and three CO 2 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 nom inal 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 CO 2 -free air through each of the test chambers

at a rate of 50-100 mL per minute. The CO 2 produced from the degradation of organic carbon

sources within the test chamber was trapped as K 2 CO 3 in the KOH solution and the amount of

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

Shimadzu Model TOC-V CSH 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 CO 2 trap nearest the

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

one position closer to the test chamber and a ne w 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 inorga nic carbonate. All chambers were aerated overnight

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

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

Calculations

The results of the inorganic carbon analyses of the CO 2 traps were converted to mg CO 2

produced using the following equation:

mg CO 2 = result (mg C/L) X vol. of KOH (L) X 3.67 mg CO 2 /mg C

The cumulative mg of CO 2 for the test and reference substanc es were corrected for the amount

of CO 2 evolved by the control, using the following equation:

Cumulative mg CO 2 evolved = Σ mg CO 2 test - mean Σ mg CO 2 control

The percentage of theoretical carbon dioxide (%TCO 2 ) evolved was calculated as follows:

% TCO 2 = 

### ) /mg Carbon mg CO

. t) x ( bon in tes (mg of car produced mg CO

2 2



### 67

3 x 100

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RESULTS AND DISCUSSION

Carbon Analysis

The measured total organic carbon (TOC) concen tration of the refere nce substance stock

solution was 394.8mg C/L. The volume of stock so lution 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 perfor med on the inoculum were 7.7 x 10 4 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 CO 2 produced

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

(% TCO 2 ) evolved is presented in tabula r and graphical forms in Table 4 and Figure 1, respectively.

The control chambers evolved an average of 106.2 mg CO 2 over the test period. This value has

been corrected for the amount of CO 2 in the trapping solution sin ce potassium hydroxide solution,

even when freshly prepared, contains carbonates. The amount of CO 2 evolved by the control

chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO 2

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 wh ich an average of 103.8% of theoretical CO 2 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. r esidues (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% TCO 2 was not achieved with 10 days of reaching 10% TCO 2

.

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CONCLUSIONS

Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% TCO 2 within a

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

TCO 2 (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 peri od. Alcohols, C18-22, distn. residues (CAS

No. 1160164-88-4) may not be cons idered readily biodegradable, si nce the pass level of 60% TCO 2

was not achieved with 10 days of reaching 10% TCO 2

.

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REFERENCES

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

CO 2 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 (CO 2 ) Evolution.

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Table 1

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

DOC 1

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 acidifie d with 2 drops of 2 N HCl prior to analysis.Wildlife International, Ltd.Project Number 527E-128A- 18 -Table 2Measured Inorganic Carbon Concentration of Trapping Solutions (mg C/L)*DateDayControlRep. 1ControlRep. 2ControlRep. 3SodiumBenzoateRep. 1SodiumBenzoateRep. 2SodiumBenzoateRep. 3Test SubstanceRep. 1Test SubstanceRep. 2Test SubstanceRep. 3Blank03-Dec-0907-Dec-0910-Dec-0914-Dec-0917-Dec-0921-Dec-0923-Dec-0928-Dec-0930-Dec-0904-Jan-1007-Jan-1011-Jan-1015-Jan-1018-Jan-1021-Jan-1025-Jan-1028-Jan-1031-Jan-1026913162022272934374145485155586120.936.130.336.522.733.018.629.614.623.719.019.023.417.216.114.416.216.520.028.839.434.421.824.517.033.813.120.317.318.018.914.525.214.315.816.021.735.231.835.533.026.417.532.213.827.217.722.417.918.313.923.916.115.992.4169.677.166.533.234.817.632.812.721.714.116.823.216.013.824.316.315.189.3177.184.967.433.234.217.534.114.333.513.920.919.417.018.015.626.117.877.4158.085.377.337.236.619.836.916.826.817.621.021.816.916.016.216.825.618.338.936.949.839.359.922.341.718.537.631.456.847.329.829.834.627.933.318.448.645.258.639.447.227.351.122.748.735.438.737.727.125.224.524.225.918.349.242.353.335.741.624.959.422.052.264.430.639.529.227.227.927.933.16.25.87.210.55.55.84.74.36.15.76.66.36.67.57.87.26.37.5*Values in Table 2 were rounded for presentation purposes.Wildlife International, Ltd.Project Number 527E-128A- 19 -Table 3Cumulative Milligrams of Carbon Dioxide Evolved1,2DateDayControlRep. 1ControlRep. 2ControlRep. 3Sodium3BenzoateRep. 1Sodium3BenzoateRep. 2Sodium3BenzoateRep. 3Test Substance3Rep. 1Test Substance3Rep. 2Test Substance3Rep. 3Control4Rep. 1Control4Rep. 2Control4Rep. 3Blank03-Dec-0907-Dec-0910-Dec-0914-Dec-0917-Dec-0921-Dec-0923-Dec-0928-Dec-0930-Dec-0904-Jan-1007-Jan-1011-Jan-1015-Jan-1018-Jan-1021-Jan-1025-Jan-1028-Jan-1031-Jan-102691316202227293437414548515558617.720.932.045.453.865.972.783.688.997.6104.5111.5120.1126.4132.3137.6143.6149.67.317.932.445.053.062.068.280.685.492.999.2105.8112.8118.1127.3132.6138.4144.28.020.932.645.657.767.473.885.790.7100.7107.2115.4122.0128.7133.8142.6148.4154.326.376.392.1103.5106.2108.7108.7109.0108.6107.8106.4105.3106.4106.2104.5107.0107.1106.725.177.996.6108.3111.0113.3113.2114.0114.2117.8116.2116.6116.4116.5116.4115.7119.4120.020.766.585.4100.7104.9108.1108.9110.7111.8112.9112.8113.2113.8113.9113.0112.5112.8116.3-0.91.12.27.512.424.125.829.431.136.241.154.764.769.573.779.984.390.6-0.94.78.917.322.329.332.939.943.152.358.765.672.075.978.481.084.087.6-0.94.98.014.518.123.125.835.938.949.366.370.377.482.085.389.193.499.65.416.525.034.540.850.855.965.268.374.979.484.190.293.896.999.5103.2106.55.113.525.334.140.146.951.462.264.870.274.178.482.985.591.994.598.0101.15.716.525.534.744.852.357.067.370.178.082.088.092.196.198.3104.4108.0111.12.34.47.110.912.915.116.818.420.622.725.127.429.932.635.538.140.443.21The results of the inorganic carbon analyses of the CO2traps were converted to mg CO2produced using the following equation:mg CO2= cumulative result (mg C/L) Xvol. of KOH (L) X 3.67 mg CO2/mg C2Calculations performed in Excel 2000 full precision mode. Manual calculations may differ.3Corrected for the CO2attributed to the inoculum and the KOH by subtracting the average amount of CO2evolved by the controls.4Corrected for the amount of CO2in the blank to determine the amount of CO2evolved by the inoculum.Wildlife International, Ltd.Project Number 527E-128A- 20 -Table 4Cumulative Percent of Theoretical Carbon Dioxide Evolved1,2DateDayControlRep. 1ControlRep. 2ControlRep. 3SodiumBenzoateRep.1SodiumBenzoateRep.2SodiumBenzoateRep.3TestSubstanceRep. 1TestSubstanceRep. 2TestSubstanceRep. 303-Dec-0907-Dec-0910-Dec-0914-Dec-0917-Dec-0921-Dec-0923-Dec-0928-Dec-0930-Dec-0904-Jan-1007-Jan-1011-Jan-1015-Jan-1018-Jan-1021-Jan-1025-Jan-1028-Jan-1031-Jan-10269131620222729343741454851555861NANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANANA23.969.383.794.096.598.798.799.098.698.096.695.696.796.594.997.297.296.922.870.787.798.4100.8102.9102.8103.6103.7107.0105.6105.9105.7105.8105.7105.0108.4109.018.860.477.591.595.398.298.9100.5101.5102.6102.4102.8103.4103.5102.7102.2102.5105.6-0.91.02.06.811.321.923.426.728.332.937.449.758.763.166.972.676.682.3-0.84.38.015.720.326.729.836.239.247.553.359.665.468.971.273.576.379.5-0.94.47.313.216.521.023.432.635.344.860.263.970.374.577.480.984.890.5Cumulative Average (N=3)Std. Dev.103.86.284.15.71The percentage of TCO2was calculated using the following equation:% TCO2=)/mg Carbonmg CO.t) x (bon in tes(mg of carproducedmg CO26732x 1002Calculations 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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