( AN ACTIVATED SLUDGE, 

RESPIRATION INHIBITION TEST

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

DOW STUDY NUMBER: 

Organisation for Economic Cooperation and Development

OECD Guideline 209

Council of the European Communities Directive 88/302/EEC

Annex V, Guideline C.11

STUDY INITIATION DATE: 14 May 2012

STUDY COMPLETION DATE: 23 August 2012

SUBMITTED TO:

The Dow Chemical Company

Midland, Michigan 48640

Wildlife International, Ltd. 

8598 Commerce Drive

Easton, Maryland 21601 

1 (410) 822-8600

Page 1 of 35

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Wildlife International, Ltd. 

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Project Number 103E-l28 

Dow Study Number 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: The Dow Chemical Company 

TITLE: ( An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

DOW STUDY NUMBER: 

STUDY COMPLETION: 23 August 2012 

This study was conducted in 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 (11 NohSan, Notification No. 6283, Agricultural 

Production Bureau, I October 1999), with the following exceptions: 

The characterization of the reference substance and stability of the test and reference substances 

under conditions of storage at the test site were not determined in accordance with Good Laboratory 

Practice Standards. 

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

determined. 

Periodic analyses of municipal water (NANOpnre® source) for potential contaminants were not 

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

certified laboratory and standard U.S. EPA analytical methods. 

STUDY DIRECTOR: 

Wildlife International, Ltd. 

- 2 -

Project Number 103E-l28 

Dow Study Number 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: The Dow Chemical Company 

TITLE: ( An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

DOW STUDY NUMBER: 

STUDY COMPLETION: 23 August 2012 

This study was conducted in 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 (11 NohSan, Notification No. 6283, Agricultural 

Production Bureau, I October 1999), with the following exceptions: 

The characterization of the reference substance and stability of the test and reference substances 

under conditions of storage at the test site were not determined in accordance with Good Laboratory 

Practice Standards. 

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

determined. 

Periodic analyses of municipal water (NANOpnre® source) for potential contaminants were not 

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

certified laboratory and standard U.S. EPA analytical methods. 

STUDY DIRECTOR: 

Wildlife International, Ltd. 

- 2 -

Project Number 103E-l28 

Dow Study Number 

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: The Dow Chemical Company 

TITLE: ( An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

DOW STUDY NUMBER: 

STUDY COMPLETION: 23 August 2012 

This study was conducted in 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 (11 NohSan, Notification No. 6283, Agricultural 

Production Bureau, I October 1999), with the following exceptions: 

The characterization of the reference substance and stability of the test and reference substances 

under conditions of storage at the test site were not determined in accordance with Good Laboratory 

Practice Standards. 

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

determined. 

Periodic analyses of municipal water (NANOpnre® source) for potential contaminants were not 

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

certified laboratory and standard U.S. EPA analytical methods. 

STUDY DIRECTOR: 
- 

Wildlife International, Ltd. 

- 3 -

Project Number I 03E-128 

Dow Study Number 

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 (ENV /MC/CHEM (98) 17); and Japan MAFF (1 I NohSan, Notification No. 6283, Agricultural 

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

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

ACTIVITY: DATE CONDUCTED: 

Protocol 24 February 2012 

Test Vessel Preparation 15 May 2012 

Data and Draft Report 17-22 May 2012 

Final Report 23 August 2012 

All inspections were study-based unless otherwise noted. 

Quality Assurance Representative 

Wildlife International, Ltd. 

DATE REPORTED TO: 

STUDY DIRECTOR: MANAGEMENT: 

24 February 2012 15 May 2012 

15 May 2012 16 May 2012 

22 May 2012 29 May 2012 

23 August 2012 23 August 2012 

DATE 

Wildlife International, Ltd. 

- 3 -

",",.,.- --- -,. .-~. -." -,.-. 

Project Number I 03E-128 

Dow Study Number 

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 (ENV /MC/CHEM (98) 17); and Japan MAFF (1 I NohSan, Notification No. 6283, Agricultural 

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

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

ACTIVITY: DATE CONDUCTED: 

Protocol 24 February 2012 

Test Vessel Preparation 15 May 2012 

Data and Draft Report 17-22 May 2012 

Final Report 23 August 2012 

All inspections were study-based unless otherwise noted. 

Quality Assurance Representative 

Wildlife International, Ltd. 

DATE REPORTED TO: 

STUDY DIRECTOR: MANAGEMENT: 

24 February 2012 15 May 2012 

15 May 2012 16 May 2012 

22 May 2012 29 May 2012 

23 August 2012 23 August 2012 

DATE 

':;~." 

Wildlife International, Ltd. 

- 3 -

",",.,.- --- -,. .-~. -." -,.-. 

Project Number I 03E-128 

Dow Study Number 

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 (ENV /MC/CHEM (98) 17); and Japan MAFF (1 I NohSan, Notification No. 6283, Agricultural 

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

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

ACTIVITY: DATE CONDUCTED: 

Protocol 24 February 2012 

Test Vessel Preparation 15 May 2012 

Data and Draft Report 17-22 May 2012 

Final Report 23 August 2012 

All inspections were study-based unless otherwise noted. 

Quality Assurance Representative 

Wildlife International, Ltd. 

DATE REPORTED TO: 

STUDY DIRECTOR: MANAGEMENT: 

24 February 2012 15 May 2012 

15 May 2012 16 May 2012 

22 May 2012 29 May 2012 

23 August 2012 23 August 2012 

DATE 

':;~." 
- 

Wildlife International, Ltd. 

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REPORT APPROVAL 

SPONSOR: The Dow Chemical Company 

Project Number 

Dow Study Number 

TITLE: (Catechol Dibeuzoate, An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

STUDY DIRECTOR: 

Biologist, Biodegradatio 

MANAGEMENT: 

Director of Biodegradation 

23 A'?J',A 20(2 

DATE 

Wildlife International, Ltd. 

- 4-

REPORT APPROVAL 

SPONSOR: The Dow Chemical Company 

Project Number 

Dow Study Number 

TITLE: (Catechol Dibeuzoate, An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

STUDY DIRECTOR: 

Biologist, Biodegradatio 

MANAGEMENT: 

Director of Biodegradation 

23 A'?J',A 20(2 

DATE 

Wildlife International, Ltd. 

- 4-

REPORT APPROVAL 

SPONSOR: The Dow Chemical Company 

Project Number 

Dow Study Number 

TITLE: (Catechol Dibeuzoate, An Activated Sludge, Respiration 

Inhibition Test 

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 

STUDY DIRECTOR: 

Biologist, Biodegradatio 

MANAGEMENT: 

Director of Biodegradation 

23 A'?J',A 20(2 

DATE 
Wildlife International, Ltd. 

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Project Number 

Dow Study Number 

STUDY INFORMATION

Study Initiation Date: 14 May 2012

Experimental Start Date (OECD): 14 May 2012

Experimental Start Date (EPA): 15 May 2012

Experimental Termination Date: 15 May 2012

Study Completion Date: 23 August 2012

Study Director:

Biologist, Biodegradation

Wildlife International, Ltd.

Sponsor: The Dow Chemical Company

 Midland, Michigan 48640

 

Sponsor’s Representative: 

Study Personnel:

 

 

 

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Project Number 

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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............................................................................................................................................... 10

Objective................................................................................................................................................... 10

Experimental Design................................................................................................................................. 10

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

Test Substance.............................................................................................................................. 11

Reference Substance .................................................................................................................... 11

Reference Stock Solution Preparation.......................................................................................... 12

Test System.................................................................................................................................. 12

Test Conditions and Apparatus.................................................................................................... 12

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

Procedure...................................................................................................................................... 13

Sample Analysis........................................................................................................................... 13

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

Quality Control............................................................................................................................. 14

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

Conclusion ................................................................................................................................................ 15

References................................................................................................................................................. 16

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TABLE OF CONTENTS

- Continued -

TABLE

Table 1. Respiration Rates and Percent Inhibitions................................................................................ 17

FIGURES

Figure 1. Percent Inhibition Versus Concentration for Reference Substance 3,5-Dichlorophenol.......... 18

Figure 2. Percent Inhibition Versus Concentration for 

.................................................................................................... 18

APPENDICES

Appendix I. Measured Dissolved Oxygen (DO) Concentrations (mg O2/L)...................................... 19

Appendix II. Protocol........................................................................................................................... 20

Appendix III. Certificate of Analysis for ........... 32

Appendix IV. Certificate of Analysis for 3,5-Dichlorophenol .............................................................. 34

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SUMMARY

The effect of the test su bstance, o n activated 

sludge m icroorganisms maintained i n an aer obic en vironment was as sessed b y t he Activated Sl udge 

Respiration I nhibition T est (OECD Guideline 209 ( 1)). T he t est co ntained co ntrol, r eference and 

treatment groups. T he control replicates were used to determine the background respiration rate of the 

sludge an d wer e n ot d osed wi th t he t est o r r eference su bstance. T he r eference g roup was d osed wi th 

3,5-dichlorophenol, a kno wn i nhibitor of r espiration, a t c oncentrations of 3, 15 a nd 50 mg/L. T he 

treatment group was dosed with at concentrations of 

10, 1 00, a nd 10 00 m g/L. An ab iotic control was d osed wi th t he t est su bstance at a co ncentration o f 

1000 mg/L to discriminate between abiotic uptake by the test substance and microbial respiration. After 

an exposure period of three hours, the respiration rates of the test solutions were determined using a YSI 

Model 50B-115 Dissolved Oxygen Meter. 

The r espiration r ates i n t he t wo c ontrols w ere 31. 7 a nd 46. 8 mg O 2/L/hr. B oth c ontrols had 

respiration rates above 29.8 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures 

with 1. 49 g dry w eight of s uspended s olids pe r l iter. T he c oefficient of va riation of t he t wo c ontrol 

respiration rates was 27.2%, and was within the 30% 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 22.0 mg/L, with 95 percent confidence limits of 15 mg/L and 50 mg/L. The EC50 for the 

reference substance was within the 2 to 25 mg/L range considered acceptable for the test. 

An inhibitory dose response effect was not observed for the treatment group. The EC50 value for 

is greater than 1000 mg/L, the highest concentration 

tested. The abiotic tr eatment mixture dosed w ith 1000 mg/L of 

 had a r espiration rate of –1.2 mg O2/L/hr showing there was no chemical uptake of 

oxygen by the test substance. The following is a summary of the results:

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Nominal Respiration Percent

Test Concentration Rate Respiration

Substance mg/L mg O2/L/hr1 Inhibition1

Control 1 0 31.7 NA

Control 2 0 46.8 NA

3,5-dichlorophenol 3 30.0 23.6%

3,5-dichlorophenol 15 24.4 37.8%

3,5-dichlorophenol 50 9.8 75.1%

10 34.2 12.8%

100 45.0 -14.6%

1000 43.6 -11.1%

- Abiotic Treatment 1000 -1.2 NA

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 p urpose o f t his t est was t o p rovide a scr eening m ethod t o i dentify substances t hat m ay 

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

concentrations for use in biodegradability tests.

This study was co nducted by Wildlife International, Ltd. for The Dow Chemical Company at the 

Wildlife I nternational, Ltd . b iodegradation f acility in Ea ston, M aryland. The t est was p erformed o n 

15 May 20 12. A co py o f the raw d ata generated at Wildlife International, Ltd. an d a co py of the final 

report are filed under Project Number in the archives located at the Wildlife International, Ltd. 

site. The original raw data and final report will be archived at Wildlife International, Ltd. prior to transfer 

to The Dow Chemical Company.

OBJECTIVE

The o bjective o f t his st udy was t o ass ess t he ef fects o f t he t est su bstance, 

( on activated sludge microorganisms by determining the respiration rate, and 

to calculate an EC50 value, if possible.

EXPERIMENTAL DESIGN

The t est co ntained co ntrol, r eference, a nd t reatment g roups. T he co ntrol r eplicates w ere u sed t o 

determine t he b ackground r espiration r ate of t he s ludge a nd w ere not e xposed t o t he t est or r eference 

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, 100, 

and 1000 mg/L. The test substance was also tested at a nominal concentration of 1000 mg/L in an abiotic 

test mixture.

MATERIALS AND METHODS

This s tudy was c onducted a ccording t o t he pr ocedures out lined i n t he pr otocol, “

 ( An Activated Sludge, Respiration Inhibition Test,” (Appendix II). 

The protocol was b ased on the procedures specified in the OECD Guideline for Testing of Chemicals,

Method 209 (1) and t he Council of t he E uropean Communities Directive 88/ 302/EEC, Guideline C.11 

(2). 

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Test Substance

The t est su bstance was r eceived f rom Do w o n 1 3 F ebruary 2 012 an d was assi gned W ildlife

International, L td. i dentification num ber 1040 4. T he f ollowing i s a de scription of t he t est s ubstance 

(Appendix III) used in this study:

Name: 

Lot Number: 

Chemical Abstract Number: 

Recertification Date: 1 February 2014

Purity %: 

Physical Description: Solid

Handling Precautions: Standard laboratory precautions

Storage Conditions: Ambient, protected from light

The test substance was administered by direct weight addition for the 10, 100, and 1000 mg/L test 

concentrations. The 10 mg/L test treatment was dosed with 0.0050 g of the test substance. The 100 mg/L 

test treatment was d osed with 0.0500 g of the test substance. The 1000 mg/L test treatment was d osed 

with 0.5001 g of the test substance. The 1000 mg/L abiotic test treatment was dosed with 0.5000 g of the 

test substance. This method was deemed appropriate based on the limited solubility of the test substance 

in water.

Reference Substance

The r eference su bstance was r eceived f rom Sigma-Aldrich C hemical C ompany o n 7 September 

2010 and w as a ssigned Wild life I nternational, Ltd . id entification n umber 9698 . Th e f ollowing is a 

description of the reference substance (Appendix IV) used in this study:

Name: 3,5-dichlorophenol

Lot Number: MKBC3078

Chemical Abstract Number: 591-35-5 

Expiration Date: 24 January 2015

Physical Description: Solid

Purity (%): 99.7%

Handling Precautions: Standard laboratory precautions

Storage Conditions: Ambient

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Reference Stock Solution Preparation

A s tock solution ( 500 m g/L) of t he r eference s ubstance, 3, 5-dichlorophenol, w as pr epared on 

11 April 2012 by dissolving 500.2 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, 8.9 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 volume of 1L with NANOpure water and mixed to ensure homogeneity. The pH 

of the solution measured 8.0. The reference stock solution had an expiration date of 11 October 2012 and 

was stored ambient and protected from light. The reference substance solution was ad ministered to the 

reference gr oup b y v olumetric addition. V olumetric a ddition i s t he most a ppropriate r oute of 

administration of aqueous solutions.

Test System

The b iological t est sy stem was a co nsortium of microorganisms common t o t he activated sludge 

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

aerobic an d f acultative aer obic b acteria. T he t est s ystem w as ch osen b ecause i t i s r epresentative of a 

treatment process that may receive the test substance.

Test Conditions and Apparatus

Control, r eference, an d t reatment t est mixtures wer e i ncubated at 2 0 ± 2 oC an d aer ated f or t hree 

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

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

oxygen d emand ( BOD) b ottles t o co nduct d issolved o xygen ( DO) measurements. T est mixtures wer e 

identified b y p roject n umber, t est su bstance i dentification, t est co ncentration, an d bottle n umber. T est 

temperatures were measured with min/max thermometer each working day.

Test Inoculum

Activated sludge collected from the Cambridge Wastewater Treatment Facility, Cambridge, 

Maryland on 14 M ay 20 12 w as ut ilized t o pr epare t he i noculum f or t he test. T he C ambridge f acility 

receives wastes from predominately domestic sources. T he sludge was sieved using a 2 mm screen and 

allowed to settle for approximately 30 minutes. Th e supernatant above the settled solids was removed 

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

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solids i n t he s ettled s ludge w ere adjusted t o a no minal c oncentration of a pproximately 400 0 mg/L by 

dilution with municipal water. Fifty milliliters 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 ove rnight. T he pH a nd t otal s uspended solids co ncentration o f t he act ivated sl udge wer e 

determined on the day of use in the study.

Procedure

Test mixtures wer e p repared at 1 5-minute in tervals s tarting w ith th e f irst c ontrol. Th e c ontrol 

contained 16.0 mL of synthetic sewage, 200 m L of inoculum, and enough municipal water to bring the 

total vol ume up t o 5 00 mL. T he mixture w as pr omptly a erated a t a r ate s ufficient t o p rovide a erobic 

conditions a nd ke ep t he s olids i n s uspension. S ubsequent m ixtures c ontained 16 .0 m L of s ynthetic 

sewage, 200 m L of i noculum, t he ap propriate a mount o f test su bstance o r r eference su bstance st ock 

solution, a nd e nough m unicipal w ater t o br ing t he t otal vol ume up t o 500 mL. A n a biotic mixture 

contained 16 .0 m L of s ynthetic s ewage, t he a ppropriate a mount of t est s ubstance, a nd 484 m L of 

municipal wa ter. F inally, a se cond co ntrol was p repared i n t he same manner as t he first control. Al l 

mixtures were aerated for three hours. 

Sample Analysis

Three hours after the start of aeration, the contents of the first control vessel were transferred to a 

BOD bottle. Dissolved oxygen concentrations were measured every 10 seconds over a 10 minute period 

or until dissolved oxygen measurements dropped below 1.0 mg/L using a YSI Model 50B-115 Dissolved 

Oxygen M eter. T he r espiration r ate i n su bsequent v essels was d etermined in an i dentical manner a t 

15 minute i ntervals. T he contact t ime f or each co ncentration o f r eference o r t est su bstance wi th t he 

activated sludge was three hours.

Calculations

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

was c alculated u sing DO v alues b etween ap proximately 6 .5 mg O 2/L a nd 2. 5 m g O 2/L, o r o ver 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:

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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

RCRC

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 pr ogram w as de signed t o c alculate t he E C50 value a nd t he 95% c onfidence i nterval b y pr obit 

analysis, the moving average, or binomial probability with nonlinear interpolation (4). Confidence limits 

(95%) f or t he E C50 a nd t he E C50 v alue i tself ( when a pplicable) w ere de termined us ing bi nomial 

probability with nonlinear interpolation only.

Quality Control

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

- the two control respiration rates should not be less than 20 mg oxygen per one gram

 of activated sludge in an hour;

- the two control oxygen uptake rates have a coefficient of variation of 30% or less;

- the EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L.

RESULTS AND DISCUSSION

Temperatures measured t hroughout t he pe riod of s ludge m aintenance a nd dur ing t he t est r anged 

from 19.8 – 20.5°C. The initial total suspended solids (TSS) concentration of the activated sludge after 

removal of the supernatant layer measured 16,883 mg/L. The measured TSS concentration of the sludge 

on the day of testing was 3727 mg/L. The measured pH of the sludge on the day of testing was 7.6.

A pre-test pH evaluation of the test substance treated test mixtures was performed on 14 May 2012. 

A control was prepared with 3.2 mL of synthetic sewage, 40 mL of sludge inoculum, and 56.8 mL of 

municipal w ater. Test m ixtures were p repared w ith 3. 2 m L of s ynthetic s ewage, 40 mL of s ludge 

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inoculum, the appropriate amount of test substance, and the appropriate volume (56.8 mL) of municipal 

water to bring the total volume to 100 mL. Each mixture was stirred with a magnetic stir bar and the pH

was measured. The following is a summary of the pH evaluation:

Test Substance

Concentration Measured

(mg/L) pH

pH mixture 1 0 7.4

pH mixture 2 10 7.4

pH mixture 3 100 7.4

pH mixture 4 1000 7.4

Respiration rates and percent inhibitions are presented in Table 1. A graph of percent inhibition 

versus concentration for the reference substance, 3, 5-dichlorophenol is presented in Figure 1. A graph of 

percent inhibition versus concentration for the test substance 

is presented in Figure 2. The measured dissolved oxygen concentrations are presented in Appendix 

I. The respiration rates observed in the two controls were 31.7 and 46.8 mg O2/L/hr. Both controls had 

respiration rates above 29.8 mg O2/L/hr, which is the low limit based on the validity criteria for mixtures 

with 1. 49 g dry w eight of s uspended s olids pe r l iter. T he c oefficient of va riation of t he t wo c ontrol 

respiration rates was 27.2%, and was within the 30% limit established for the test. The EC50 value for the 

reference substance was 22.0 mg/L with a 95% confidence interval of 15 to 50 mg/L, and was within the 

2 t o 25 mg/L r ange c onsidered accep table f or t he t est. C onfidence l imits ( 95%) f or t he E C50 an d t he 

EC50 value itself were calculated using binomial probability with nonlinear interpolation. The observed 

percent inhibitions for ranged from –14.6 to 12.8%. 

The ab iotic t reatment mixture d osed wi th 1 000 m g/L o f 

had a respiration rate of –1.2 mg O2/L/hr showing there was no chemical uptake of oxygen by the 

test substance.

CONCLUSION

An inhibitory dose response effect upon respiration was not observed for the treatment group at any 

of the tested concentrations. The EC50 value for the test substance is greater than 1000 mg/L, the highest 

concentration tested.

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REFERENCES 

1. Organisation for Economic Cooperation and Development. 2010. Activated Sludge, Respiration 

Inhibition T est ( Carbon and A mmonium O xidation). O ECD Guideline f or T esting o f C hemicals, 

Guideline 209. Adopted 22 July 2010.

2. Annex to Commission Directive 88/302/EEC. 1992. Activated Sludge Respiration Inhibition Test. 

Official Journal of the European Communities No. L133. Guideline C.11. 

3. Stephan, C .E. 1978. U .S. EPA , En vironmental R esearch La boratory, D uluth, M innesota. Personal 

Communication.

4. Stephan, C .E. 1977. “ Methods f or C alculating a n LC 50,” Aquatic T oxicology and H azard

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 O2/L/hr1 Inhibition1

Control 1 0 31.7 NA

Control 2 0 46.8 NA

3,5-dichlorophenol 3 30.0 23.6%

3,5-dichlorophenol 15 24.4 37.8%

3,5-dichlorophenol 50 9.8 75.1%

10 34.2 12.8%

100 45.0 -14.6%

1000 43.6 -11.1%

- Abiotic Treatment 1000 -1.2 NA

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 Concentration for Reference Substance 3,5-Dichlorophenol

Percent Inhibition vs. Concentration for 3,5-

dichlorophenol

0.00

20.00

40.00

60.00

80.00

1 10 100

Concentration (mg/L)

% Inhibition

Series1

Figure 2. Percent Inhibition Versus Concentration for 

Percent Inhibition vs. Concentration for 

-20.00

-10.00

0.00

10.00

20.00

30.00

40.00

50.00

10 100 1000

Concentration (mg/L)

% Inhibition

Series1

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Wildlife International, Ltd. 

- 19 -

 

Project Number 

Dow Study Number 

Appendix I

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

3,5-dichlorophenol

Time Control Control 3 15 50 10 100 1000 Abiotic

(sec) 1 2 mg/L mg/L mg/L mg/L mg/L mg/L 1000 mg/L

10 *8.1 8.2 8.2 *8.3 *8.8 8.1 7.8 8.0 *9.2

20 7.7 7.3 7.6 7.8 8.6 7.5 7.1 7.2 9.3

30 7.5 7.0 7.3 7.6 8.6 7.3 6.7 6.9 9.3

40 7.4 6.8 7.1 7.5 8.5 7.0 *6.5 6.7 9.3

50 7.3 6.6 6.9 7.4 8.4 6.9 6.3 *6.5 9.3

60 7.1 *6.4 6.8 7.3 8.4 6.7 6.1 6.3 9.3

70 7.0 6.2 6.6 7.2 8.4 6.6 5.9 6.1 9.4

80 6.9 6.0 *6.5 7.1 8.3 *6.4 5.7 5.9 9.4

90 6.8 5.9 6.4 7.0 8.3 6.3 5.6 5.8 9.4

100 6.7 5.7 6.3 6.9 8.3 6.2 5.5 5.7 9.4

110 6.7 5.6 6.2 6.8 8.3 6.1 5.3 5.5 9.4

120 6.6 5.5 6.1 6.8 8.2 6.0 5.2 5.4 9.4

130 6.5 5.3 6.0 6.7 8.2 5.9 5.0 5.3 9.4

140 6.4 5.2 5.9 6.6 8.2 5.7 4.9 5.1 9.4

150 6.3 5.0 5.8 6.6 8.2 5.7 4.8 5.0 9.4

160 6.2 4.9 5.7 6.5 8.2 5.5 4.7 4.9 9.4

170 6.2 4.8 5.6 6.4 8.1 5.4 4.6 4.8 9.4

180 6.1 4.7 5.5 6.4 8.1 5.4 4.4 4.7 9.4

190 6.0 4.5 5.4 6.3 8.1 5.3 4.3 4.6 9.4

200 5.9 4.4 5.4 6.2 8.1 5.2 4.2 4.5 9.4

210 5.8 4.3 5.3 6.2 8.1 5.1 4.1 4.3 9.4

220 5.8 4.2 5.2 6.1 8.0 5.0 4.0 4.2 9.4

230 5.7 4.0 5.1 6.0 8.0 4.9 3.9 4.1 9.4

240 5.6 3.9 5.0 6.0 8.0 4.8 3.8 4.0 9.4

250 5.5 3.8 5.0 5.9 8.0 4.7 3.7 3.9 9.4

260 5.4 3.7 4.9 5.8 7.9 4.6 3.6 3.8 9.4

270 5.4 3.6 4.8 5.8 7.9 4.5 3.5 3.7 9.4

280 5.3 3.4 4.7 5.7 7.9 4.4 3.3 3.6 9.4

290 5.2 3.3 4.6 5.7 7.9 4.3 3.2 3.5 9.4

300 5.1 3.2 4.5 5.6 7.8 4.2 3.1 3.4 9.4

310 5.1 3.1 4.5 5.6 7.8 4.1 3.0 3.3 9.4

320 5.0 3.0 4.4 5.5 7.8 4.1 2.9 3.2 9.4

330 4.9 2.9 4.3 5.4 7.8 4.0 2.8 3.0 9.4

340 4.8 2.7 4.2 5.4 7.8 3.9 2.7 2.9 9.4

350 4.8 2.6 4.2 5.3 7.7 3.8 2.6 2.8 9.4

360 4.7 *2.5 4.1 5.3 7.7 3.7 *2.5 2.7 9.4

370 4.6 2.4 4.0 5.2 7.7 3.6 2.4 2.6 9.4

380 4.5 2.3 3.9 5.2 7.7 3.5 2.3 *2.5 9.4

390 4.5 2.1 3.8 5.1 7.7 3.4 2.2 2.4 9.4

400 4.4 2.0 3.7 5.1 7.6 3.3 2.1 2.3 9.4

410 4.3 1.9 3.7 5.0 7.6 3.3 2.0 2.2 9.4

420 4.2 1.8 3.6 5.0 7.6 3.2 1.9 2.1 9.4

430 4.2 1.7 3.5 4.9 7.6 3.1 1.8 2.0 9.4

440 4.1 1.6 3.4 4.9 7.5 3.0 1.7 1.9 9.4

450 4.0 1.4 3.4 4.8 7.5 2.9 1.6 1.8 9.4

460 3.9 1.3 3.3 4.8 7.5 2.8 1.5 1.7 9.4

470 3.9 1.2 3.2 4.7 7.5 2.7 1.3 1.6 9.4

480 3.8 1.1 3.1 4.7 7.5 2.6 1.2 1.5 9.4

490 3.7 1.0 3.0 4.7 7.4 *2.5 1.1 1.4 9.4

500 3.6 0.9 3.0 4.6 7.4 2.4 1.0 1.2 9.4

510 3.6 1.1 2.9 4.6 7.4 2.4 0.9 1.1 9.4

520 3.5 1.0 2.8 4.5 7.4 2.3 1.0 9.4

530 3.4 0.9 2.7 4.5 7.3 2.2 0.9 9.4

540 3.3 2.6 4.5 7.3 2.1 9.4

550 3.3 2.6 4.4 7.3 2.0 9.4

560 3.2 *2.5 4.4 7.3 1.9 9.4

570 3.1 2.4 4.4 7.3 1.8 9.4

580 3.0 2.3 4.3 7.2 1.7 9.4

590 3.0 2.2 4.3 7.2 1.6 9.4

600 *2.9 2.2 *4.3 *7.2 1.6 *9.4

*Dissolved oxygen values used to calculate respiration rates

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Wildlife International, Ltd. 

- 20 -

 

Project Number 

Dow Study Number 

Appendix II

Protocol

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Project Number 

Dow Study Number 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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- 
Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Wildlife International, Ltd. 

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Dow Study Number 

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Wildlife International, Ltd. 

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Dow Study Number 

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Wildlife International, Ltd. 

- 32 -

 

Project Number 

Dow Study Number 

Appendix III

Certificate of Analysis for 

- 
Wildlife International, Ltd. 

- 33 -

 

Project Number 

Dow Study Number 

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Wildlife International, Ltd. 

- 34 -

 

Project Number 

Dow Study Number 

Appendix IV

Certificate of Analysis for 3,5-Dichlorophenol

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Wildlife International, Ltd. 

- 35 -

 

Project Number 

Dow Study Number 

-