REPORT

Study Title:

TOXICITY OF TKA 40278 (CGI 827)

TO ACTIVATED SLUDGE

IN A RESPIRATION INHIBITION TEST

Data Requirements / Test Guidelines:

OECD No. 209

EU Commission Directive 87/302/EEC, C. 11

Study Director:

Roger B~tscher

~letlon Date:

January 29,2003

RCC Ltd

Environmental Chemistry &

Pharmanalytics Division

CH-4452 Itingen/Switzerland

S_gonsor:

Ciba Specialty Chemicals Inc,

CH-4002 Basel / Switzerland

RCC Study Numn,ber:

845447

Page 1 of 21

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REPORT Page2

GLP CERTIFICATE

The Swiss GLP Monitoring Authorities

Statement of GLP Compliance

It is hereby confirmed that

dudng the period of

the following Test Facilities of

August 15- 17, 2000

August 28 - 29,2001 and

April 15,2OO2

RCC Ltd

4452 Itingen

Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for the

Environment, Forests and Landscape and the Intercantonal Office for the Control of

Medicines with respect to the compliance with the Swiss legislation on Good Laboratory

Practice,

Test Facilities areas of expertise*

- Toxicology Division TOX, ACC, MUT

. Environmental Chemistry and

Pharmanalytics Division

Microbiological Diagnostics by

Btotechnology & Animal Breeding Division

ACC, ECT, ENF, EMN,

PCT,RES,OTH (Animal

metabolism)

OTH (Microbiology)

The inspection was performed in agreement with the C~ECD Guidelines for National GLP

Inspections and Audits. it was found that the aforementioned test facilities were operating in

compliance w~th the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at

the time they were inspected.

Federal Offce of Public Health

The Director

Bern, May 2002 Prof, "Tn. Zeltner

 ̄ TOX = Toxicology ; ACC = An =l-~¢al =nd C~ntcal Chemist,/: ECT = Env~ronn~enlal toxicity on aqua~ and ter~estr~[

ecosys e~ts: ML~ = Mut~geni~ity; PCT = phys ~.a~.chem~l testing ; RES = Res~lue studle= ; OTH = Other, to be tpec~d.

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REPORT Page3

GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE

RCC Study Number:

Testltem:

Study Director:

StudyTitle:

845447

TKA 40278 (CGI 827)

Mr. Roger B&tscher

Toxicity of TKA 40278 (CGI 827)

to activated sludge in a respiration inhibition test

This study (with the exception of the pre-experiments as mentioned in the report) has been

performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice,

adopted February 2’~, 2000 [RS 813.016.5], This Ordinance is based on the OECD

Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26t~, 1997

by decision of the OECD Council [C(97) 186/Final],

The purity of the test item is not yet available and is therefore excluded from the Statement of

Compliance.

There were no circumstances that may have affected the quality or integrity of the data.

Study Director: Mr. Roger B~tscher

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RCC Study Number 845447

TKA 40278 (CGI 827)

SIGNATURES

REPORT Page 4

Study Director: Mr. Roger B~.tscher

Management: /Jr Dr. Uwe Morgenroth

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TKA 40278 (CGI 827)

REPORT Page 5

QUALITY ASSURANCE UNIT

RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland

STATEMENT

RCC Study Number:

Test Item:

Study Director:

Study Title:

845447

TKA 40278 (CGI 827)

Mr. Roger B~tscher

Toxicity of TKA 40278 (CGI 827)

to activated sludge in a respiration inhibition test

The general facilities and activities are inspected periodically and the results are reported to

the responsible person and the management.

Study procedures (with the exception of the pre-experiments as mentioned in the report)

were periodically inspected. The study plan and this report were audited by the Quality

Assurance Unit. The dates are given below:

Dates and Types of QAU Inspections Dates of Reports to the Study

Director and to the Management

November 11, 2002 Study plan November 11, 2002

November 14, 2002 Process based November 15, 2002

January 27, 2003 Final report January 27, 2003

This statement also confirms that this final report reflects the raw data.

Quality Assurance: Mrs. Ch ’tine Schlumpf

Date: /’ J~:20 2~ZO~

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REPORT Page 6

CERTIFICATION OF GLP AND

VERIFICATION OF THE REPORT

The Statement of Compliance with Good Laboratory Practice found in this report, and signed

by the Study Director is truthful and accurate, and this report as provided by the testing

facility is complete and unaltered.

Forthe Sponsor:

Date:

645
RCC Study Number 845447

TKA 40278 (CGI 827)

REPORT Page 7

CONTENTS

GLP CERTIFICATE ......................................................................................................... 2

STATEMENT OF COMPLIANCE .................................................................................... 3

SIGNATURES ................................................................................................................. 4

QUALITY ASSURANCE UNIT ......................................................................................... 5

CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT ............................... 6

CONTENTS ..................................................................................................................... 7

CONTENTS CONTINUED - TABLES & FIGURES .......................................................... 8

PREFACE ....................................................................................................................... 9

DATA REQUIREMENTS / TEST GUIDELINES ............................................................. 10

SUMMARY OF STUDY PLAN AMENDMENTS ............................................................. 10

SUMMARY .................................................................................................................... 11

1 PURPOSE ............................................................................................................ 12

2 MATERIALS AND METHODS .............................................................................. 12

2.1 DEFINITIONS ............................................................................................. 12

2.2 TEST ITEM ................................................................................................. 13

2.3 REFERENCE ITEM ..................................................................................... 13

2.4 TEST SYSTEM ........................................................................................... 14

2.5 STUDY DESIGN ......................................................................................... 15

2.5.1 Dosage and Concentrations .......................................................... 15

2.5.2 Experimental Conditions ................................................................ 16

2.6 EVALUATIONS ........................................................................................... 17

2.6.1 Measurement of the Respiration Rates .......................................... 17

2.6.2 Determination of the Inhibition Rates ............................................. 17

2.6.3 Water Quality Criteria .................................................................... 17

3 RESULTS AND DISCUSSION ............................................................................. 18

3.1 TOXICITY OF TKA 40278 (CGI 827) ........................................................... 18

3.2 TOXICITY OF 3,5-DICHLOROPHENOL (POSITIVE CONTROL) ................ 18

4 TABLES ............................................................................................................... 19

5 FIGURE ................................................................................................................ 20

6 REFERENCES ..................................................................................................... 21

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REPORT Page 8

CONTENTS CONTINUED -TABLES & FIGURES

Table 1:

Table 2:

Influence of TKA 40278 (CGI 827) on oxygen consumption

of activated sludge ...................................................................................... 19

Influence of 3,5-dichtorophenol on oxygen consumption

of activated sludge ...................................................................................... 19

Figure 1: Influence of TKA 40278 (CGI 827) and 3,5-dichlorophenol

on the respiration rate of activated sludge ................................................... 20

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REPORT Page 9

PREFACE

GENERAL

Study Title:

Sponsor:

Monitoring Scientist:

Test Facility:

Toxicity of TKA 40278 (CGI 827)

to activated sludge in a respiration inhibition test

Ciba Specialty Chemicals Inc.

Segment Plastic Additives

P.O. Box, R-1045.1.36

CH-4002 Basel / Switzerland

Dr. Moung Sook Lee

RCC Ltd

Environmental Chemistry & Pharmanatytics Division

Zelgliweg 1

CH-4452 Itingen / Swffzerland

RESPONSIBILITIES

Study Director:

Deputy Study Director:

Technical Coordinator:

Head of RCC Quality Assurance:

Mr. Roger B~tscher

Dr. Ulrich Memmert

Mrs. GSraldine Rappine

Mrs. Iris WOthrich

SCHEDULE

Experimental Starting Date:

Experimental Completion Date:

Study Completion Date:

November25,2002

November28,2002

January29, 2003

ARCHIVING

RCC Ltd, CH-4452 Itingen, Switzerland will retain the study plan, raw data, a sample of the

test item and the final report of the present study for at least ten years.

No data will be discarded without the Sponsor’s consent.

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RCC Study Number 845447 REPORT

TKA 40278 (CGI 827)

DATA REQUIREMENTS / TEST GUIDELINES

Page 10

This study followed the procedures indicated by the following internationally accepted

guidelines and recommendations:

OECD Guideline for Testing of Chemicals, No. 209, Activated Sludge, Respiration Inhibition

Test, 1984.

EU Commission Directive 87/302]EEC, Part C.11, Activated Sludge Respiration Inhibition

Test, 1988.

SUMMARY OF STUDY PLAN AMENDMENTS

There were no amendments to the study plan.

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REPORT Page 11

SUMMARY

The inhibitory effect of the test item TKA 40278 (CGI 827) on the respiration rate of aerobic

wastewater microorganisms of activated sludge was investigated in a 3-hour respiration

inhibition test according to the EU Commission Directive 87/302/EEC, and the OECD

Guideline for Testing of Chemicals, No. 209.

The following nominal concentrations of TKA 40278 (CGI 827) were tested: 6.3, 12.5, 25, 50,

and 100 mg/L. Concentrations above 100 mg/L were not tested.

In addition, two controls and three different concentrations of the reference item

3,5-dichlorcphenol (5, 16 and 50 rag/L) were tested in parallel. The results of these

treatments confirmed the suitability of the activated sludge and the method used.

Up to and including the concentration of 100 mg/L, the test item TKA 40278 (CGI 827) had

no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the

incubation period of three hours. The saturation concentration (water solubility limit of

TKA 40278 (CGI 827) under the present test conditions) was obviously reached and no

significant inhibition was observed after three hours contact time.

Thus, the 3-hour NOEC, EC20, and EC50 are clearly higher than the water solubility limit of

TKA 40278 (CGI 827) under the present test conditions, or higher than 100 mg/L.

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REPORT Page12

1 PURPOSE

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item

TKA 40278 (CGI 827) on activated sludge microorganisms. Aerobic activated sludge was

incubated for three hours in the presence of various concentrations of the test item under

defined conditions. The respiration rates (oxygen consumption) were measured. The

inhibitory effect of the test item was expressed as a percentage of the mean respiration rate

of two controls.

The test method of application and the test system are recommended by the test guidelines.

2 MATERIALS AND METHODS

2.1 DEFINITIONS

Respiration rate: the oxygen consumption of wastewater microorganisms in aerobic

activated sludge, expressed as mg O2 per liter per minute

ECx: the calculated concentration of test item at which the respiration rate

is inhibited by x% compared to the respiration rate of the control

NOEC: the highest test concentration at which no significant toxic effect is

observed (_<15% inhibition)

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TKA 40278 (CGI 827)

2,2 TEST ITEM

The test item and the following information concerning the test item were provided by the

sponsor:

Identity:

Product name:

Batch no.:

Expiration date:

Purity:

Solubility in water:

Stability in water:

Aggregate state/physical form

at room temperature:

Color:

Storage conditions:

TKA 40278

CGI 827

KL 01/02

August 30, 2004

To be determined

(excluded from the statement of compliance)

Not indicated by the sponsor

Stable

Solid / powder

Slightly yellow

At room temperature at about 20 °C, in the dark

since the test item is light sensitive.

2.3 REFERENCE ITEM

The reference item and the following information concerning the reference item were

provided by Aldrich Chemie:

Identity:

Product no.:

Lot no.:

Purity:

Expiration date:

Storage conditions:

3,5-dichlorophenol

D7060-0

02611ES

99.1% (GLC)

July 17, 2005

In tightly closed original container, at room

temperature at about 20 °C

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TKA 40278 (CGI 827)

2.4 TEST SYSTEM

The study was performed with aerobic activated sludge from a wastewater treatment plant

(ARA Ergolz II, FOllinsdorf, Switzerland) treating predominantly domestic wastewater. The

sludge was washed once twice with tap water by centrifugation and the supernatant liquid

phase was decanted. A homogenized aliquot of the final sludge suspension was weighed,

thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a

concentration equivalent to 3 g dry material per liter. During holding of three days prior to

use, the sludge was fed daily with 50 mL synthetic wastewater* per liter and was kept at

room temperature under continuous aeration until use. Immediately before use, the dry

weight of the activated sludge was measured again in the inoculum used in the test. The pH

of the activated sludge inoculum was adjusted from 8.3 to 7.1 with a diluted sulfuric acid

solution.

* Synthetic wastewater:

16 gpeptone

11 g meat extract

3 g urea

0.7 g NaCI

0.4 g CaCI2  ̄ 2H20

0.2 g MgSO,=  ̄ 7H20

2.8 g K2HPO4

filled up to a final volume of 1 liter

with deionized water

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TKA 40278 (CGI 827)

2.5 STUDY DESIGN

2.5.1 Dosage and Concentrations

The nominal test item concentrations tested were 6.3, 12.5, 25, 50, and 100mg/L.

Concentrations above 100 mg test item per liter were not tested.

According to the results of a pre-test (without GLP), no stock solution could be prepared due

to the low water solubility of TKA 40278 (CGI 827). Therefore, test item amounts of 3.1, 6.5,

12.0, 24.2, 48.4 mg were directly weighed (by means of an analytical balance) into the test

flasks. Then, 284 mL of tap water was added to each flask. The test item was mixed into the

tap water by ultrasonic treatment over 15 minutes and intense stirring over 24 hours at room

temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as

homogeneously as possible. Then, 16 mL of synthetic wastewater and 200 mL of the

activated sludge inoculum were added (see scheme below). The test solutions were

prepared as far as possible in the dark since the test item is light sensitive.

Additionally, two controls (tap water, synthetic wastewater, and activated sludge inoculum,

without test item) and the reference item 3,5-dichlorcphenol (positive control) at nominal

concentrations of 5, 16, and 50 mg/L were tested in parallel under otherwise identical test

conditions.

For test media containing the reference item, a stock solution of 3,5-dichlorophenol was

prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL

1 M NaOH and diluted to about 30 mL with purified water. Excess of NaOH was neutralized

with 0.5 mot,’L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made

up to one liter with purified water. The pH was determined to be 7.5. The final concentration

of 3,5-diohlorophenol amounted to 500 mg/L. Aliquots of this 3,5-dichlorophenol stock

solution were mixed with synthetic wastewater and tap water in the respective test flasks to

obtain the desired concentrations.

Composition of test media:

Flask Identification Synthetic Amount Aliquot Activated Tap Final

No. waste- of test of stock sludge water nominal

water item solution conc.

(mL) (mg)* (mL)** (mE) (mE) (mg/L)

1 Inoculum control 16 200 284

2 3,5-dichlorophenol 16 5 2O0 279 5

3 3,5-dichlorophenol 16 16 200 268 16

4 3,5-dichlorophenol 16 5O 200 234 50

5 Test item 16 3.1 20O 284 6.3

6 Test item 16 6.5 200 284 12.5

7 Test item 16 12.0 200 284 25

8 Test item 18 24.2 2OO 284 50

9 Test item 16 48.4 2O0 284 100

10 Inoculum control 16 2OO 284

Amounts were directly weighed into the designated test flasks

** Stock solution of 3,5-dichlorophenol

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TKA 40278 (CG1827)

2.5.2 Experimental Conditions

The test was performed in 1000-mL brown glass flasks. Brown glass flasks were used for the

test since the test item is light sensitive. The test flasks were labeled with the necessary

information to ensure unmistakable identification.

At the start of the test, 200 mL activated sludge inoculum (see Section 2.4) with a sludge

concentration of 3.4 g]L dry weight (corresponding to about 1.3 g dry material per liter test

medium) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but

convenient interval) first to a control, then to the test solutions of the reference item, then to

the test solutions of the test item, and finally to the second control.

During the incubation period of exactly 3 hours, all test media and the controts were

continuously aerated with compressed air at a flow of approximately one liter per minute. The

concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period,

and just before the measurements of the respiration rates the dissolved oxygen

concentrations were at least 8.7 mg/L (Tables 1 and 2). The temperature in the test media,

measured in one control, was 19 °C at the start and at the end of the incubation period.

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TKA 40278 (CG1827)

2.6 EVALUATIONS

2.6.1 Measurement of the Respiration Rates

For measurement of the respiration rate, a well-mixed sample of each test medium was

poured into a BOD-flask after exactly three hours Incubation time, and was not further

aerated. Then, the dissolved oxygen concentration was measured with an oxygen electrode

(VVTW OXI 539 meter, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim,

Germany), and was continuously recorded for about ten to fifteen minutes. During

measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen

consumption rate (in mg O2 L"1 minute"~) was determined from the linear part of the

respiration curve in the range of 6.5-2.5 mg O2/L. In case of very rapid oxygen consumption,

the range used was below the limits indicated above but always within the linear part of the

respiration curve. In case of low oxygen consumption, the rate was determined over a period

of at least 10 minutes.

2,6.2 Determination of the lnhibition Rates

The inhibitory effect of the test item or of the reference item on the respiration rate (oxygen

consumption per minute) is expressed as a percentage of the mean respiration rate of the

two controls measured at the start and at the end of the test series (flasks 1 and 10).

1 2Rs x 100% = percent inhibition

Rcl + Rc2

where

Rs = oxygen consumption rate (in mg 02 L"~ minute"1) at tested concentration

Re1 = oxygen consumption rate in control flask 1

Rc~ = oxygen consumption rate in control flask i0

The 3-hour EC50-, EC20-, and EC80-values for the test item and their 95%-confidence limits

could not be calculated due to the absence of a toxic effect.

The 3-hour EC50-value of the reference item 3,5-dichlorophenol and its 95%-confidence

limits were calculated by Probit Analysis (Ref. 1,2).

All test results were based on the nominal test item concentrations.

2.6.3 Water Quality Criteria

The pH-values and the dissolved oxygen concentrations were determined in all test media

and the controls at the start and at the end of the 3-hour incubation period. The water

temperature was measured in one control at the start and end of the incubation period, and

was continuously monitored in all test media and the controls during measurement of the

respiration rate. Before the addition of activated sludge, the appearance of the test media

was recorded.

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TKA 40278 (CGI 827)

3 RESULTS AND DISCUSSION

3.1 TOXICITY OF TKA 40278 (CGI 827)

The influence of TKA 40278 (CGI 827) on the respiration rate of activated sludge is

presented in Table 1 and Figure 1.

Down to the test concentration of 25 mg/L, a part of the test item was suspended in the test

media. Thus, these test concentrations were clearly above the water solubility limit of

TKA 40278 (CGI 827) under the present test conditions. The appearance of the test solutions

of the two lowest test concentrations of 6.3 and 12.5 mg/L could not be assessed since

brown glass flasks were used for the test (because of the light sensitivity of the test item).

Up to and including the concentration of 100 mg/L, the test item TKA 40278 (CGI 827) had

no significant inhibitory effect (<15%) on the respiration rate of activated sludge affer the

incubation period of 3 hours (Table 1 and Figure 1).

Concentrations above 100 mg]L were not tested. The saturation concentration (water

solubility limit of TKA 40278 (CGI 827) under the present test condition) was obviously

reached and no significant inhibition was observed after three hours contact time.

Thus, the 3-hour NOEC, EC20, and EC50 are clearly higher than the water solubility limit of

TKA 40278 (CGI 827) under the present test conditions, or higher than 100 mg]L.

The oxygen consumption rates of the two controls (run at the start or at the end of the test)

differed only by 3% (guideline-recommended maximum variation: 15%).

3.2 TOXICITY OF 3,5-DICHLOROPHENOL (POSITIVE CONTROL)

The results are presented in Table 2 and Figure 1.

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to

be 14 mg/L with 95% confidence limits from 7 to 27 mg/L. The 3-hour EC50 is within the

guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge

used.

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TKA 40278 (CGl 827)

4 TABLES

Table 1: Influence of TKA 40278 (CGI 827) on oxygen consumption of activated sludge

Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen

concentration consumption concentration

of test rate (rag O2/L)

chemical

(rag/L) (mg O~/L min"1) (%) start* end* start* end*

1 Control 0 1.386 7.6 8.2 8.6 9.5

10 Control 0 1.427 7,6 8.2 8.7 8.7

Mean 1.406

Deviation(%) 3.0

5 TKA 40278 (CGI 827) 6.3 1.416 -0.7 7.6 8.1 8.5 8.9

6 TKA 40278 (CGI 827) 12.5 1.300 7.6 7.6 8.2 8.5 8.8

7 TKA 40278 (CGI 827) 25 1.390 1.2 7,6 8.1 8.5 9,1

8 TKA 40278 (CGI 827) 5O 1.311 6.7 7,6 8.2 8.9 8.7

9 I TKA 40278 (CGI 827) 100 1.450 -3.1 7.5 8.0 8.8 9.5

Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to that of control

Table 2: Influence of 3,5-dichlorophenol on oxygen consumption of activated sludge

Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen

concentration consumption concentration

of test rate (rag O~/L)

chemical

(mg/L) (rag O~/L min"1} start* end* start* end*

1 Control 0 1.386 7.6 8.2 8.6 9.5

10 Control 0 1.427 7.6 8.2 8.7 8.7

Mean 1.406

Deviation (%) 3.0

2 3,5-dichlorophenol 5 1,209 14.0 7.5 8.3 8.8 8.7

3 3,5-dichlorophenol 16 0.664 52.8 7.5 8.2 8.4 9.3

4 3,5-dichlomphenol 50 0.119 91.5 7.5 8.3 8.5 8.8

Start and end of the 3-hour incubation period

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TKA 40278 (OG1827)

5 FIGURE

Figure 1: Influence of TKA 40278 (CGI 827) and 3,5-dichlorophenol on the respiration rate

of activated sludge

100

8O

-2O

P

.-. o... 3,5-dlchlorophenol

~ -t’KA 40278 (CG1827)

1000

Test concentration (rag/L)

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TKA 40278 (CGI 827)

6 REFERENCES

1)

2)

FINNEY, D.J. (1971):

Probit Analysis,

3rd Edition, Cambridge University Press, London

DAVIES, R.G. (1971):

Computer Programming in Quantitative Biology,

Academic Press, London

660