### ( AN ACTIVATED SLUDGE, RES PIRATION INHIBITION TEST

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

DOW STUDY NUMBER:

Organisation for Economic Cooperation and Development OECD Guideline 2 09

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

Mid land, Michigan 48640



# Wildlife International, Ltd.

8598 Commerce Drive Easton, Maryland 21601 1 (410) 822

- 8600

Page 1 of 35

# Wildlife International, Ltd.

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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.

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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 (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.

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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.

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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

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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 Repres entative:

Study Personnel:

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TABLE OF CONTENTS

Title Page

................................................................................................ .................................................... 1

Good Laboratory Practice Com pliance 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 Referenc e 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 O 2 

### /L)

...................................... 19

Appendix II. Protocol

........................................................................................................................... 20

Appendix III. Certificate of Analysis for

........... 32

Appendix IV. Cer tificate 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

Res piration 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 O 2 

### /L/hr, which is the low limit based on the validit y 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 support ed 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 O 2 

### /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 Respiratio n Percent
Test Concentration Rate Respiration
Substance mg/L mg O 2 /L/hr 1 Inhibition 1

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. Manu al 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 a ckground 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 test ed 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 ot ocol, “ ( 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 we ight 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

wi th 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 umbe r 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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Refer ence 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 N ANOpure  

### 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 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 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 nsort ium 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 maint ain 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 roje ct 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, Cambrid ge, 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

d ilution 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 O 2 

### /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 O 2 

### /L. The respiration rate was calculated

u sing 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 -

2 1 s 

### RCRC 2R

+ 

### ) X 100

where:

R s 

### = oxy gen 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 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 varia tion 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 t otal 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 te sting 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 repare d 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 fo r 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 O 2 

### /L/hr. Both controls had respiration rates above 29.8 mg O 2 

### /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 onside red 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 O 2 

### /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 Econ omic 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. Activat ed 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 Concentra tion Rate Respiration
Substance mg/L mg O 2 /L/hr 1 Inhibition 1

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 sl ightly.
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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Appendix I Measured Dissolved Oxygen (DO) Concentrations (mg O 2 

### /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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Appendix II

Protocol

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Appendix III

Certificate of Analysis for

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Appendix IV

C ertificate of Analysis for 3,5

- Dichlorophenol

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