## 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 StudyDirector: Roger B~tscher ~letlonDate: January 29,2003

RCCLtd Environmental Chemistry & Pharmanalytics Division CH-4452 Itingen/Switzerland S_gonsor: Ciba Specialty Chemicals Inc, CH-4002 Basel / Switzerland RCC StudyNumn,ber: 845447 Page 1 of 21



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## 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. FederalOffce 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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### 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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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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## 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:



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## 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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## 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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## 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.



### 648RCC 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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## 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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## 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 SyntheticAmountAliquot 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 Testitem 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"1minute"~)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 InhibitionpH 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 InhibitionpH 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