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 C ooperation 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

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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

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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.



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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

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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

Refere nces..................................................................................................................... ............................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 Ox ygen (DO) Concentrations (mg O 2 /L) ......................................17
Appendix II. Protocol and Amendments.......................................................................................... ....18
Appendix III. Certifi cate 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 environmen t was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The t est contained control, reference and treatment groups. The control replicates were used to determ ine the background respiration rate of the sludge and
were not dosed with the test or reference su bstance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respirati on, 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 me asured using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rates in the two controls were 26.2 and 29.3 mg O 2 /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 fo r the reference substance was within the 5 to 30 mg/L range considered acceptable for the test. An in hibitory 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/hr 1 Inhibition 1
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 perform ed using MS Excel 2000 in full precision mode . Manual calculations may differ slightly. N A - Not Applicable

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INTRODUCTION The purpose of this test was to provide a scr eening method to identify substances that may adversely affect aerobic microbial treatment plants a nd to indicate suitable non-inhibitory test substance concentrations for use in biodegradability tests.

This study was conducted by Wildlife Internationa l, 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 unde r Project Number 527E-127 in the archives located on the Wildlife International, Ltd. site.

OBJECTIVE The objective of this study was to assess the effect s 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 treat ment 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 w as dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The test substa nce was tested at nominal concentrations of 10, 30, 100, 300 and 1000 mg/L.

MATERIALS AND METHODS This study was conducted according to the proce dures 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 sp ecified 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 fo llowing 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. identif ication 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 a dding 20 mL of NANOpure ® water. The NANOpure ® water is not known to contain any contamin ants that are reasonably expected to be present at levels capable of interfering with the study. While stirring, 8.6 mL of 1N H 2 SO 4 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. Volumetr ic 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 sy stem 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 fl asks 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 identif ication, test concentration, and bottle number.

Test Inoculum Activated sludge collected from the Denton W astewater 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 siev ed using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above th e 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 Prot ocol) 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 interv als 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. Sub sequent 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 to tal 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 O 2 /L/hour. The rate was calculated using DO values between approximately 6.5 mg O 2 /L and 2.5 mg O 2 /L, or over a 10 minute period if the DO did not reach approximately 2.5 mg O 2 /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 -

2 1 s 

### RC RC 2R

+ 

### ) X 100

where:

R s = oxygen consumption rate at a given concentration of the test substance RC 1 = oxygen consumption rate, Control 1 RC 2 = oxygen consumption rate, Control 2
When the dose response pattern (percent inhibition ve rsus 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 va lue 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 de termined 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) c oncentration 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 presen ted in Table 1. The resp iration rates observed in the two controls were 26.2 and 29.3 mg O 2 /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 nonlin ear 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 substa nce at the concentrations evaluated in this study were not observed, and did not exhibit a concentr ation dependent dose response pattern. The EC50 value for the test substance is therefore greater th an 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 Chemi cals, 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 Co mmunities 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/hr 1 Inhibition 1
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 perform ed using MS Excel 2000 in full precision mode . Manual calculations may differ slightly. N A - Not Applicable

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Figure 1. Percent Inhibition Versus Log Concentration

Percent Inhibition Versus Log Concentration

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-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 O 2 /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