Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): AN ACTIVATED SLUDGE, 

RESPIRATION INHIBITION TEST 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527E-127 

Organisation for Economic Cooperation and Development 

OECD Guideline 209 

Council of European Communities Directive 92/69/EEC 

Annex V, Guideline C.11 

AUTHORS: 

Edward C. Schaefer 

Brandt C. Twilley 

STUDY INITIATION DATE: November 23, 2009 

STUDY COMPLETION DATE: January 7, 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 

1 (410) 822-8600 

Page 1 of 32
Wildlife International, Ltd. Project Number j27~-127 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4): An Activated Sludge, Respiration 

lnhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 527E-127 

STUDY COMPLETION: January 7; 2010 

This shldy was conducted in compliance with Good Laboratory Practice Standards as published by 

the US. Environmental Protection Agency in 40 CFR Part 792; OECD Principles of Good Laboratory 

Practice (ENVIMCICHEM (98) 17); and Japan MAFF (1 1 NohSan, Notification No. 6283; Agricultural 

Production Bureau, 1 October 1999), with the following exccptious: 

Characterization of the test and reference substances and their stabilities undcr conditions of 

storage at the test site were not determined in accordance with Good Laboratory Practice Standards. 

The concentration. stablllty and homogeneity of the reference substance m the carrler were not 

determined 

Periodic analyses of municipal water (NA~Opure~ source) for potential contaminants were not 

performed according to Good Laboratory Practice Standards; however, they were performed using a 

certified laborato~ and standard US. EPA analytical methods. 

STUDY DIRECTOR: 

Brandt C. Twilley 
Wildlife httemational, Ltd. Project Number 527~-127 

QUALITY ASSURANCE STATEMENT 

This study was examined for compliance with Good Laboratory Practice Standards as published by 

the U.S. Environmental Protection Agency in 40 CFR Part 792; OECD Principles of Good Laboratory 

Practice (ENVIMCICHEM (98) 17) and Japan MAFF (1 1 NohSau, Notification No. 6283, Agricultural 

Production Bureau; 1 October 1999). The dates of all inspections and audits and the dates that any 

findings were reported to the Study Director and Laboratory Management wcre as follows: 

DATE REPORTED TO: 

ACTIVITY: DATE CONDUCTED: STUDY DIRECTOR: 

Protocol October 14,2009 October 14, 2009 

Test Substance Preparation November 25,2009 Novcntber 25,2009 

Dissolved Oxygen November 25,2009 Novanber 25,2009 

Measurements 

Data and Draft Report December 12,2009 December 14,2009 

Final Rcporl January 7; 201 0 January 7,2010 

MANAGEMENT: 

November 24,2009 

Novcmber 30: 2009 

November 30; 2009 

December 15,2009 

January 7; 2010 

All inspections were study-based unless otherwise noted. 

07 3kd Wo 

DATE 

Wildllfe International, Ltd. 
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REPORT APPROVAL 

SPONSOR: Sasol Germany GmbH 

TITLE: Alcohols, CIS-22, distn. residues (CAS No. 1160164-88-4): An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 52E-127 

STUDY DIRECTOR: 

Biologist, ~iodegadationl 

MANAGEMENT: 

?LJ 0. 

Edward C. ~cxaefcr 
Wildlife International, Ltd. Project Number 527E-127

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STUDY INFORMATION 

Study Initiation Date: November 23, 2009 

Experimental Start Date (OECD): November 24, 2009 

Experimental Start Date (EPA): November 25, 2009 

Experimental Termination Date: November 25, 2009 

Study Completion Date: January 7, 2010 

Study Director: Brandt C. Twilley 

 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. 

Study Personnel: Edward C. Schaefer, Director of Biodegradation 

 Brandt C. Twilley, B.S., Biologist, Biodegradation 

 
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TABLE OF CONTENTS

Title 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................................................................................................................................9 

 Test Substance................................................................................................................................9 

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

 Reference Stock Solution Preparation..........................................................................................10 

 Test System ..................................................................................................................................11 

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

 Test Inoculum...............................................................................................................................11 

 Procedure......................................................................................................................................11 

 Sample Analysis...........................................................................................................................12 

 Calculations..................................................................................................................................12 

 Quality Control.............................................................................................................................13 

Results and Discussion .............................................................................................................................13 

Conclusion ................................................................................................................................................13 

References.................................................................................................................................................14 
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TABLE OF CONTENTS

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TABLE

Table 1. Respiration Rates and Percent Inhibitions................................................................................15 

FIGURE 

Figure 1. Percent Inhibition Versus Log Concentration ..........................................................................16 

APPENDICES

Appendix I. Measured Dissolved Oxygen (DO) Concentrations (mg O2/L) ......................................17 

Appendix II. Protocol and Amendments..............................................................................................18 

Appendix III. Certificate of Analysis ....................................................................................................32 
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SUMMARY 

 The effect of Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) on activated sludge 

microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration 

Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment 

groups. The control replicates were used to determine the background respiration rate of the sludge and 

were not dosed with the test or reference substance. The reference group was dosed with 

3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The 

treatment group was dosed with Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) at 

concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three hours, the 

respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved Oxygen 

Meter. The respiration rates in the two controls were 26.2 and 29.3 mg O2/L/hr. The difference between 

the two control respiration rates was approximately 10.6 - 11.8%, and was within the 15% difference limit 

established for the test. The validity of the test was further supported by the results from the 

3,5-dichlorophenol reference group, which resulted in an EC50 value of 19.5 mg/L, with 95 percent 

confidence limits of 3 mg/L and 50 mg/L. The EC50 for the reference substance was within the 5 to 30 

mg/L range considered acceptable for the test. An inhibitory dose response effect was not observed for 

the treatment group. Therefore, the EC50 value for Alcohols, C18-22, distn. residues (CAS No. 

1160164-88-4) is greater than 1000 mg/L, the highest concentration tested. The following is a summary 

of the results: 

 Nominal Respiration Percent 

Test Concentration Rate Respiration 

Substance mg/L mg/L/hr1 Inhibition1

Control 1 0 26.2 NA 

Control 2 0 29.3 NA 

3,5-dichlorophenol 3 29.9 -7.7% 

3,5-dichlorophenol 15 16.5 40.5% 

3,5-dichlorophenol 50 5.5 80.2% 

Alcohols, C18-22, distn. residues 10 28.1 -1.3% 

Alcohols, C18-22, distn. residues 30 28.7 -3.4% 

Alcohols, C18-22, distn. residues 100 28.7 -3.4% 

Alcohols, C18-22, distn. residues 300 29.3 -5.6% 

Alcohols, C18-22, distn. residues 1000 29.3 -5.6% 

1

Calculations performed using MS Excel 2000 in full precision mode. Manual calculations may differ slightly. 

NA - Not Applicable 
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INTRODUCTION 

 The purpose of this test was to provide a screening method to identify substances that may 

adversely affect aerobic microbial treatment plants and to indicate suitable non-inhibitory test substance 

concentrations for use in biodegradability tests. 

 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 the final report are filed under Project Number 527E-127 in the archives located on 

the Wildlife International, Ltd. site. 

OBJECTIVE 

 The objective of this study was to assess the effects of the test substance, Alcohols, C18-22, distn. 

residues (CAS No. 1160164-88-4), on activated sludge microorganisms by determining the respiration 

rate, and to calculate an EC50 value, if possible. 

EXPERIMENTAL DESIGN 

 The test contained control, reference, and treatment groups. The control replicates were used to 

determine the background respiration rate of the sludge and were not exposed to the test or reference 

substances. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at 

concentrations of 3, 15 and 50 mg/L. The test substance was tested at nominal concentrations of 10, 30, 

100, 300 and 1000 mg/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): An Activated Sludge, Respiration Inhibition Test,” (Appendix 

II). The protocol was based on the procedures specified in the OECD Guideline for Testing of 

Chemicals, Method 209 (1) and the Council of the European Communities Directive 92/69/EEC, Annex 

V, Guideline C.11 (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: 
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 Name: Alcohols, C18-22, distn. residues (CAS No. 1160164-88-4) 

 Lot: 03585/MA 

 Chemical Abstract Number: 1160164-88-4 

 Date Prepared: August 25, 2009 

 Expiration Date: August 2013 

 Physical Description: Solid 

 Purity (%): See Certificate of Analysis (Appendix III) 

 Storage Conditions: Ambient, protected from light 

 The test substance was dosed by direct weight addition. This method was deemed appropriate based 

on the relative insolubility of the test substance in water. 

Reference Substance 

 The reference substance was received from Sigma-Aldrich Chemical Company on August 18, 2005 

and was assigned Wildlife International, Ltd. identification number 7320. The following is a description 

of the reference substance used in this study: 

 Name: 3,5-dichlorophenol 

 Lot Number: 15809KI 

 Chemical Abstract Number: 591-35-5 

 Expiration Date: 8/2010 

 Physical Description: Solid 

 Purity (%): 97 

 Handling Precautions: Standard laboratory precautions 

 Storage Conditions: Ambient 

Reference Stock Solution Preparation 

 A stock solution of the reference substance, 3,5-dichlorophenol, was prepared by dissolving 

500.0 mg of the reference substance in 10 mL of 1N NaOH and then adding 20 mL of NANOpure® water. 

The NANOpure® water is not known to contain any contaminants that are reasonably expected to be 

present at levels capable of interfering with the study. While stirring, 8.6 mL of 1N H2SO4 was added to 

reach the point of incipient precipitation. The solution of 3,5-dichlorophenol was then diluted to a final 
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volume of 1L with NANOpure® water. The reference substance solution was administered to the 

reference group by volumetric addition. Volumetric addition is the most appropriate route of 

administration of aqueous solutions. 

Test System 

 The biological test system was a consortium of microorganisms common to the activated sludge 

treatment process. The organisms responsible for the decomposition of organic materials are principally 

aerobic and facultative aerobic bacteria. The test system was chosen because it is representative of a 

treatment process that may receive the test substance. 

Test Conditions and Apparatus 

 Control, reference, and treatment test mixtures were incubated at 20 ± 2oC and aerated for three 

hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures 

were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical 

oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements. Test mixtures were 

identified by project number, test substance identification, test concentration, and bottle number. 

Test Inoculum 

 Activated sludge collected from the Denton Wastewater Treatment Facility, Denton, Maryland on 

November 24, 2009 was utilized as the inoculum for the test. The Denton facility receives wastes from 

predominately domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for 

approximately 30 minutes. The supernatant above the settled solids was removed and the total suspended 

solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled 

sludge were adjusted to a nominal concentration of approximately 4000 mg/L by dilution with municipal 

water. 50 mL of synthetic sewage (Appendix I of Protocol) was added to each liter of adjusted sludge. 

The sludge was maintained at a temperature of 20 ± 2°C and continuously aerated overnight. The pH and 

total suspended solids concentration of the activated sludge were determined on the day of use in the 

study. 

Procedure 

 Test mixtures were prepared at 15 minute intervals starting with the first control. The control 

contained 16.0 mL of synthetic sewage, 200 mL of inoculum, and enough municipal water to bring the 
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total volume up to 500 mL. The mixture was promptly aerated at a rate sufficient to provide aerobic 

conditions and keep the solids in suspension. Subsequent mixtures contained 16.0 mL of synthetic 

sewage, 200 mL of inoculum, the appropriate amount of test substance, or reference substance stock 

solution, and enough municipal water to bring the total volume up to 500 mL. Finally, a second control 

was prepared. All mixtures were aerated for three hours. 

Sample Analysis 

 The contents of the first control vessel were transferred to a BOD bottle and dissolved oxygen 

concentrations were measured every 10 seconds over a 10 minute period using a YSI Model 50B-115 

Dissolved Oxygen Meter. The respiration rate in subsequent vessels was determined in an identical 

manner at 15 minute intervals. The contact time for each concentration of reference or test substance with 

the activated sludge was three hours. 

Calculations 

 A respiration rate was calculated for each test mixture and expressed in mg O2/L/hour. The rate 

was calculated using DO values between approximately 6.5 mg O2/L and 2.5 mg O2/L, or over a 

10 minute period if the DO did not reach approximately 2.5 mg O2/L. The respiration rate was calculated 

using the following equation: 

Respiration Rate = [(initial DO – final DO)/(final time – initial time)] x 3600 s/hr

 Percent inhibition was calculated using the following equation: 

Percent Inhibition = ( 1 -

1 2

s

RC RC

2R

+

 ) X 100

where: 

Rs = oxygen consumption rate at a given concentration of the test substance 

RC1 = oxygen consumption rate, Control 1 

RC2 = oxygen consumption rate, Control 2 

 When the dose response pattern (percent inhibition versus test substance concentration) allowed for 

the calculation of an EC50 value, the data were analyzed using the computer program of C.E. Stephan (3). 

The program was designed to calculate the EC50 value and the 95% confidence interval by probit 
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analysis, the moving average, or binomial probability with nonlinear interpolation (4,5,6). Confidence 

limits (95%) for the EC50 and the EC50 value itself (when applicable) were determined using binomial 

probability with nonlinear interpolation only. 

Quality Control 

 The test is considered valid only if the following criteria were met: 

- the two control respiration rates were within 15% of each other; 

- the EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 5 to 

30 mg/L. 

RESULTS AND DISCUSSION 

 Temperatures measured throughout the period of sludge maintenance and during the test ranged 

from 19.4 – 20.7°C. The initial total suspended solids (TSS) concentration of the activated sludge after 

removal of the supernatant layer measured 11,300 mg/L. The measured TSS concentration of the sludge 

on the day of testing was 4113 mg/L. The measured initial pH of the sludge on the day of testing was 8.0. 

 Respiration rates and percent inhibitions are presented in Table 1. The respiration rates observed in 

the two controls were 26.2 and 29.3 mg O2/L/hr, or a difference of approximately 10.6 - 11.8%. The 

EC50 value for the reference substance was 19.5 mg/L and was within the 5 to 30 mg/L range considered 

acceptable for the test. Confidence limits (95%) for the EC50 and the EC50 value itself, were calculated 

using binomial probability with nonlinear interpolation. The observed percent inhibitions for Alcohols, 

C18-22, distn. residues (CAS No. 1160164-88-4) ranged from –5.6 to –1.3%. 

CONCLUSION 

 Inhibitory effects upon respiration by the test substance at the concentrations evaluated in this study 

were not observed, and did not exhibit a concentration dependent dose response pattern. The EC50 value 

for the test substance is therefore greater than 1000 mg/L, the highest concentration tested. 
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REFERENCES 

1. Organisation for Economic Cooperation and Development. 1984. Activated Sludge Respiration 

Inhibition Test. OECD Guideline for Testing of Chemicals, Guideline 209. Adopted 4 April 1984 

2. Annex to Commission Directive 92/69/EEC. 1992. Activated Sludge Respiration Inhibition Test. 

Official Journal of the European Communities No. L383. Guideline C.11. 

3. Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal 

Communication. 

4. Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. 

5. Thompson, W.R. 1947. Biological Reviews. Vol. II, No. 2. Pp. 115-145. 

6. Stephan, C.E. 1977. “Methods for Calculating an LC50,” Aquatic Toxicology and Hazard

Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 
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Table 1 

Respiration Rates and Percent Inhibitions 

 Nominal Respiration Percent 

Test Concentration Rate Respiration 

Substance mg/L mg/L/hr1 Inhibition1

Control 1 0 26.2 NA 

Control 2 0 29.3 NA 

3,5-dichlorophenol 3 29.9 -7.7% 

3,5-dichlorophenol 15 16.5 40.5% 

3,5-dichlorophenol 50 5.5 80.2% 

Alcohols, C18-22, distn. residues 10 28.1 -1.3% 

Alcohols, C18-22, distn. residues 30 28.7 -3.4% 

Alcohols, C18-22, distn. residues 100 28.7 -3.4% 

Alcohols, C18-22, distn. residues 300 29.3 -5.6% 

Alcohols, C18-22, distn. residues 1000 29.3 -5.6% 

1

Calculations performed using MS Excel 2000 in full precision mode. Manual calculations may differ slightly. 

NA - Not Applicable 
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Figure 1. Percent Inhibition Versus Log Concentration 

Percent Inhibition Versus Log Concentration

-15

-10

-5

0

5

10

1 10 100 1000

Log Concentration (mg/L)

% Inhibition 
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Appendix I 

Measured Dissolved Oxygen (DO) Concentrations (mg O2/L) 

Reference Alcohols C18-22, distn. residues (CAS No. 1160164-88-4)

Time Control Control 3 15 50 10 30 100 300 1000

(sec) 1 2 mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L

10 7.8* 7.9* 7.9* 8.5* 8.7* 8.0* 8.0* 8.0* 8.0* 7.6*

20 7.4 7.4 7.4 8.2 8.7 7.5 7.4 7.5 7.4 7.1

30 7.2 7.2 7.2 8.1 8.6 7.4 7.2 7.4 7.3 6.9

40 7.1 7.1 7.1 8.0 8.6 7.3 7.1 7.3 7.2 6.8

50 7.1 7.0 7.0 8.0 8.6 7.2 7.1 7.2 7.1 6.7

60 7.0 7.0 6.9 7.9 8.6 7.1 7.0 7.1 7.0 6.7

70 6.9 6.9 6.8 7.9 8.6 7.1 6.9 7.0 6.9 6.6

80 6.8 6.8 6.8 7.8 8.6 7.0 6.8 7.0 6.9 6.5

90 6.8 6.7 6.7 7.8 8.6 6.9 6.8 6.9 6.8 6.4

100 6.7 6.7 6.6 7.7 8.5 6.9 6.7 6.8 6.7 6.3

110 6.6 6.6 6.5 7.7 8.5 6.8 6.6 6.8 6.6 6.3

120 6.6 6.5 6.5 7.7 8.5 6.7 6.6 6.7 6.6 6.2

130 6.5 6.5 6.4 7.6 8.5 6.6 6.5 6.6 6.5 6.1

140 6.4 6.4 6.3 7.6 8.5 6.6 6.4 6.6 6.4 6.1

150 6.4 6.3 6.3 7.5 8.5 6.5 6.4 6.5 6.4 6.0

160 6.3 6.2 6.2 7.5 8.5 6.4 6.3 6.4 6.3 5.9

170 6.2 6.2 6.1 7.5 8.5 6.4 6.2 6.3 6.2 5.8

180 6.2 6.1 6.0 7.4 8.4 6.3 6.2 6.3 6.1 5.8

190 6.1 6.0 6.0 7.4 8.4 6.2 6.1 6.2 6.1 5.7

200 6.0 5.9 5.9 7.3 8.4 6.2 6.1 6.1 6.0 5.6

210 6.0 5.9 5.8 7.3 8.4 6.1 6.0 6.1 5.9 5.6

220 5.9 5.8 5.7 7.3 8.4 6.0 5.9 6.0 5.9 5.5

230 5.8 5.7 5.7 7.2 8.4 6.0 5.8 5.9 5.8 5.4

240 5.8 5.7 5.6 7.2 8.4 5.9 5.8 5.9 5.7 5.3

250 5.7 5.6 5.5 7.1 8.3 5.8 5.7 5.8 5.7 5.3

260 5.7 5.5 5.5 7.1 8.3 5.8 5.6 5.7 5.6 5.2

270 5.6 5.4 5.4 7.1 8.3 5.7 5.6 5.6 5.5 5.1

280 5.5 5.4 5.3 7.0 8.3 5.6 5.5 5.6 5.4 5.1

290 5.5 5.3 5.2 7.0 8.3 5.6 5.4 5.5 5.4 5.0

300 5.4 5.2 5.2 6.9 8.3 5.5 5.4 5.4 5.3 4.9

310 5.3 5.2 5.1 6.9 8.3 5.4 5.3 5.4 5.2 4.8

320 5.3 5.1 5.0 6.9 8.2 5.4 5.2 5.3 5.2 4.8

330 5.2 5.0 4.9 6.8 8.2 5.3 5.2 5.2 5.1 4.7

340 5.1 4.9 4.9 6.8 8.2 5.2 5.1 5.2 5.0 4.6

350 5.1 4.9 4.8 6.7 8.2 5.1 5.0 5.1 5.0 4.6

360 5.0 4.8 4.7 6.7 8.2 5.1 5.0 5.0 4.9 4.5

370 4.9 4.7 4.7 6.7 8.2 5.0 4.9 5.0 4.8 4.4

380 4.9 4.7 4.6 6.6 8.2 4.9 4.8 4.9 4.7 4.4

390 4.8 4.6 4.5 6.6 8.1 4.9 4.8 4.8 4.7 4.3

400 4.8 4.5 4.4 6.6 8.1 4.8 4.7 4.7 4.6 4.2

410 4.7 4.4 4.4 6.5 8.1 4.7 4.6 4.7 4.5 4.1

420 4.6 4.4 4.3 6.5 8.1 4.7 4.6 4.6 4.5 4.1

430 4.6 4.3 4.2 6.4 8.1 4.6 4.5 4.5 4.4 4.0

440 4.5 4.2 4.2 6.4 8.1 4.5 4.4 4.5 4.3 3.9

450 4.4 4.2 4.1 6.4 8.0 4.5 4.4 4.4 4.3 3.9

460 4.4 4.1 4.0 6.3 8.0 4.4 4.3 4.3 4.2 3.8

470 4.3 4.0 3.9 6.3 8.0 4.3 4.2 4.2 4.1 3.7

480 4.2 3.9 3.9 6.2 8.0 4.2 4.1 4.2 4.0 3.6

490 4.2 3.9 3.8 6.2 8.0 4.2 4.1 4.1 4.0 3.6

500 4.1 3.8 3.7 6.2 8.0 4.1 4.0 4.0 3.9 3.5

510 4.1 3.7 3.6 6.1 8.0 4.0 3.9 4.0 3.8 3.4

520 4.0 3.7 3.6 6.1 7.9 4.0 3.9 3.9 3.8 3.4

530 3.9 3.6 3.5 6.0 7.9 3.9 3.8 3.8 3.7 3.3

540 3.9 3.5 3.4 6.0 7.9 3.8 3.7 3.8 3.6 3.2

550 3.8 3.4 3.4 6.0 7.9 3.8 3.7 3.7 3.5 3.2

560 3.7 3.4 3.3 5.9 7.9 3.7 3.6 3.6 3.5 3.1

570 3.7 3.3 3.2 5.9 7.9 3.6 3.5 3.5 3.4 3.0

580 3.6 3.2 3.1 5.8 7.9 3.6 3.5 3.5 3.3 2.9

590 3.5 3.2 3.1 5.8 7.8 3.5 3.4 3.4 3.3 2.9

600 3.5* 3.1* 3.0* 5.8* 7.8* 3.4* 3.3* 3.3* 3.2* 2.8*

* Numbers indicate oxygen concentrations used to calculate respiration rates
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Appendix II 

Protocol and Amendments 
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Appendix III 

Certificate of Analysis