## REPORT

TOXICITY OF TKA 40278 (CGI 827) TO SCENEDESMUS SUBSP/CATUS IN A 72-HOUR ALGAL GROWTH INHIBITION TEST
DataReauiremente /Test Guidelines: OECD No. 201 EU Commission Directive 92/69/EEC, C.3 Study Director: Roger B~tscher Stud Corn letion Date: April 16, 2003 TestFacility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 Itingen / Switzerland
Ciba Specialty Chemicals Inc. CH-4002 Basel / Switzerland RCC Study Number: 845445 Page 1 of 40



### 576RCC Bludy Number 845445 REPORT Page 2

TKA 40278 (CGI 827) 

## GLP CERTIFICATE
The Swiss GLP Monitoring Authorities



# Statement of GLP Compliance
It is hereby confirmed that during the periodof
the following Test Facilities of August15- 17,2000 August28-29,2001 and Apd115,2002 RCC Ltd 4452 Itingen Switzerland were inspected by the FederalOffice of Publia Health, the Swiss Agency for the Environment, Forests and Landscape and the Intercantonal Office for the Controlof Medicines with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities - Toxicology Division

- Environmental Chemistry and Pharmanalytics Division

- Microbiological Diagnostics by Biotechnology & Animal Breeding Division areas of oxpert|se* TOX, ACC, MUT ACC, ECT, ENF, EMN, PCT, RES,OTH(Animal metabolism) OTH(Microbiology) The inspection was performed in agreement with the OECD Guidelines for National GLP Inspections and Audits. It was found that the aforementioned test facilities wore operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time they were inspected.

Bern, May2002 FederalOffice of PublicHealth TheDirector

Prof. Th. Zeltner  ̄ TOX = Toxicology ; ACC = Ana~yllcal and C~nlca[ Chemist P/; ECT = EnvJronrnenla] t oY, Jc;ly on aquett¢ and tetresttlal



### 577RCC Study Number 845445 REPORT Page3

TKA 40278 (CGt 827)
GOOD LABORATORY PRACTICE 

## STATEMENT OF COMPLIANCE
RCC Study Number: 845445 Testltem: TKA 40278 (CGI 827) Study Director: Mr. Roger B~itscher Study Title: Toxicity of TKA 40278 (CGI 827) toScenedesmus subspicatusin a 72-hour algal growth 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 2n~, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26~, 1997 by decision of the OECD Council [C(97) 186/Final]. There were no circumstances that may have affected the quality or integrity of the data.
Study Director: Mr. Roger B~itscher



## Date:

578RCCStudy Number 845445 REPORT Page 4

TKA40278 (CG1827) 

## SIGNATURES

Study Director: Mr. Roger B~tscher

Date: A)0,’I"[ ,’~’, 2ooO~

Management: Dr. Uwe Morgenroth

Date:



### 579RCC Study Numbar 845445 REPORT Page 5

TKA 40278 (¢GI 827) 

## QUALITY ASSURANCE UNIT
RCC Ltd, Environmental Chemistry & Pharmanalytics Division, CH-4452 Itingen / Switzerland

STATEMENT
RCC Study Number: Test Item: Study Director: Study Title: 845445 TKA 40278 (CGI 827) Mr. Roger B~itscher Toxicity of TKA 40278 (CGI 827) toScenedesmus subspicatusin a 72-hour algal growth 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-experimants 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 07, 2002 Study Plan November 07, 2002 December 17-19, 2002 Application December 19, 2002 March 13, 2003 Final report March 13, 2003 This statement also confirms that this final report reflects the raw data.
Quality Assurance: Mr. Henry Schneylin



### 58ORCC Study Number 845445 REPORT Page 6

TKA 40278 (OGI 827) 

## 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 test facility is complete and unaltered.

For the Sponsor:

Date:



### 581Page7

RCC Study Number 845445 REPORT TKA 40278 (CGI 827) 

## 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) .......................................................... PREFACE

....................................................................................................................... 9 DATA REQUIREMENTS / TEST GUIDELINES ............................................................. 10 SUMMARY OF STUDY PLAN AMENDMENTS ............................................................. 10 SUMMARY

.................................................................................................................... 11 1 PURPOSE ............................................................................................................ 1.~ 2 MATERIALS AND METHODS .............................................................................. 2.1 DEFINITIONS ............................................................................................. 12 2.2 TESTITEM ................................................................................................. 13 2.3 ANALYTICAL STANDARD

.......................................................................... 13 2.4 TEST SYSTEM ........................................................................................... 13 2.5 STUDY DESIGN ......................................................................................... 14 2.5.1 Experimental Conditions................................................................ !4~ 2.5.2 Dosage and Concentrations .......................................................... , 2.6 EVALUAT ONS ........................................................................................... 11~ 2.6.1 Counting and Examination of the Algal Cells ................................. Determination of the Algal Growth Inhibition and the EC 2.6.2 Va,ues ........................................................................................... 1176 2.6.3 Water Quality Criteria .................................................................... 2.6.4 Analysis of the Test Item Concentrations ....................................... 17 3 RESULTS AND DISCUSSION ............................................................................. 19 4 TABLES ............................................................................................................... 21 5 FIGURE ......................................................... " ...................................................... 26 6 REFERENCES ..................................................................................................... 27
ATTACHMENT ........................................................................ ’" 28 ANALYTICAL REPORT (Pages A1 to A12)



### 582RCC Study Number 845445 REPORT Page 8

TKA 40278 (CG1827) 

## CONTENTS CONTINUED (TABLES & FIGURES)

Table 1: Table 2:
Table 3: Table 4:
Table 5: Table 6: Table 7: Table 8: Algal cell densities during the test period of 72 hours

.................................. 21 Areas under the growth curves (AUC) and percentage inhibition 22 of AUC (IAuc) during the test period ......................................................... Growth rates r and percentage inhibition of r (I,) during the test period ........ 22 Results of a Dunnett-test with the biomass (b; areas under 23 the growth curves) ................................................................................ Results of a Dunnett-test with the growth rates r ......................................... 23 Appearance of the test media

...................................................................... 24 pH values in the test media and in the control ............................................. 24 Water temperature during the test period .................................................... 25

Figure 1: Growth curves of Scenedesmus subspicatus,incubated for 72 hours

at different concentrations of the test item ..... 26



### 583RCC Study Number 845445 REPORT Page 9

TKA 40278 (CG1827) 

## PREFACE
GENERAL Study Title: Sponsor:

Monitoring Scientist: Test Facility:

Analytical Identification No.: Toxicity of TKA 40278 (CGI 827) toScenedesmus subspicatusin a 72-hour algal growth 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 & Pharmanalytics Zelgliweg1 CH-4452 Itingen / Switzerland 845446


### RESPONSIBILITIES Study Director: Deputy Study Director: Responsible for Analytic.s: Technical Coordinator: Head of RCC Quality Assurance: Mr, Roger B~tscher Dr. Ulrich Memmert Mr. Tobias Schoop Mrs. Geraldine Rappine Mrs. Iris W{Jthrich


### SCHEDULE Experimental Starting Date1: Experimental Completion Date: Study Completion Date: November 29, 2002 January 14, 2003 April 16, 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.

Start of the first test. The test had to be repeated since the validity criteria were not met.



### 584RCC StudyNumber 845445 REPORT Page 10

TKA 40278 (CG1827) 

## DATA REQUIREMENTS / TEST GUIDELINES

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of Chemicals, No. 201, Alga, Growth Inhibition Test, 1984. EU Commission Directive 92/69/EEC, C.3, Algal Inhibition Test, 1992.



## SUMMARY OF STUDY PLAN ’AMENDMENTS
First Amendment to Study Plan Concerning: Alteration: Reason: 2.2 Test Item Indication of purity An analytical certificate was issued by the sponsor



### 585RCC Study Number 845445 REPORT Page 11

TKA 40278 (CG1827) 

## SUMMARY
The influence of the test item "I’KA 40278 (CGI 827) on the growth of the green algal species Scenedesmus subspicatusCHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD Guideline No. 201, 1984. Since the test item contains two different isomers, five WAFs (water accommodated fractions) with loading rates of 1.0, 3.2, 10, 32, and 100 mg/L were prepared prior to the start of the test. Individual dispersions of the test item in test water with loading rates mentioned above were continuously stirred at room temperature in the dark over 96 hours to dissolve a maximal amount of the test item in test water. No auxiliary solvent or emulsifier was used. The dispersions were filtered through membrane filters (pore size 0.45 ~m) just before the start of the test. Additionally, a control was tested in parallel. The measured test item concentration in the analyzed test media samples (loading rates 1.0, 3.2, 10, 32, and 100 mg/L) amounted to 0.95, 2.9, 5.0, 4.4, and 6.0 mg/L at the start of the test (sum of both isomers). In the WAFs with loading rates of 10 to 100 mg/L, approximately the same test item concentration of about 4 to 6 mg/L was measured. Thus, the saturation concentration of the test item in test water was obviously reached. In the test media, incubated under the test conditions during the test period (but without algae), the measured concentrations of TKA 40278 (CGI 827) decreased to values below the limit of quantification of the analytical method of 0.23 mg/L after 72 hours. This decrease of the test item concentrations was considered to be due to photolytical degradation of the test item due to the intensive irradiation. The mean measured test item concentrations (calculated as the average over all measurements par test concentration) were 0.53 mglL (loading ratel.0 mg/L), 1.5 mg/L (loading rate 3.2 mg/L), 2.6 mg/L (loading rate 10 rag/L), 2.3 mg/L (loading rate 32 rag/L), and 3.0 mg/L (loading rate 100 mg/L). All biological results are related to the loading rates of the WAEs and to the mean measured test item concentrations (sum of both isomers). The 72-hour NOEC (highest concentration tested without toxic effects after the exposure period of 72 hours) was determined to be at the loading rate of 1.0 mg/L (mean measured 0.53 rag/L) since at this loading rate, the mean growth rate of Scenedesmus subspicatuswas only 5% lower than in the control. This inhibition was considered to be within the normal range of biological variations and was not considered to be a toxic effect caused by the test item. The 72-hour LOEC (lowest concentration tested with toxic effects) was determined to be at the loading rate of 3.2 mg/L (mean measured 1.5 mg/L) since the growth was statistically significantly reduced at this loading rate and above. The EC50-values for the algal biomass (b) and the growth rate (r) after 72 hours test duration could not be determined since the inhibition of growth was at the loading rates of 3.2 to 100 mg/L below 50%. At the loading rates of 10 to 100 mg/L, the inhibition of growth rate was about 10 to 13% and the inhibition of biomass amounted to about 30 to 38%. No concentration-response relationship was determined. This corresponds to the analytical results which indicate~’that the saturation concentration of the test item in test water was reached at these loading rates and resulted in less than 50% inhibition of growth rate and biomass.



### 586RCC Study Number 845445 REPORT Page 12

TKA 40278 (CG1827) 

## 1 PURPOSE
The purpose of this test was to determine the inhibitory effect of the test item TKA 40278 (CGI 827) on the growth of the freshwater green algal species Scenedesmus subspicatus. Exponentially growing cultures of this green algal species were exposed to water accommodated fractions (WAFs) with different loading rates of the test item. A static, non-renewal exposure system was used. The inhibition of growth in relation to control cultures was determined over a test-period of 72 hours and, thus, over several algal generations. The method of test item application and the test system are recommended by the test guidelines.



## 2 MATERIALS AND METHODS
2.1 DEFINITIONS Cell density: Growth: Biomass (b): Growth rate (r): EbCx:

E~Cx: NOEC (No Observed Effect Concentration):
LOEC ~owest Observed Effect Concentration): the number of cells per mL the increase of cell density over the test period the actual number of cells per volume of medium (cells/mL) calculated as the area under the growth curve (AUC) the Increase of cell density per unit time the calculated concentration of test item that results in an x% reduction of biomass b relative to the control the calculated concentration of test item that results in an x% reduction of growth rate r relative to the control the highest test concentration at which no significant inhibition of growth is observed relative to the control the lowest test concentration at which a significant inhibition of growth is observedrelativeto the control



### 587RCC Study Number 845445 REPORT Page 13 TKA 40278 (CG1827) 

### 2.2 TESTITEM 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 94.1% (sum ofp,p- and m,p-isomer) Solubility in test water was investigated within this study. Stable (stability in test water under test conditions was investigated within this study) Solid / powder Slightly yellow At room temperature at about 20 °C, in the dark since the test item is light sensitive.



### 2.3 ANALYTICAL STANDARD The test item was used as analytical standard.



### 2.4 TEST SYSTEM The test organism used for the study was Scenedesmus subspicatusCHODAT, Strain No. 86.81 SAG, supplied by the Sammlung von Algenkulturen GSttingen (SAG, Experimentelle Phykologie und Sammlung yon Algenkulturen, Albrecht-von-Haller-lnstitut for Pflanzenwissenschaften, Universit~it GSttingen, D-37073 GSttingen, Germany). The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.



### 588RCC Study Number 845445 REPORT Page 14 TKA 40278 (CGI 827) 

### 2.5 STUDY DESIGN
2.5.1 Experimental Conditions The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations: Macro-nutrients: NaHCO3 CaCl2 x 2 H20 NH4Cl MgSO4 x 7 H20 MgCI~ x 6 H~O KH2PO4 Trace elements: 50.0 mg/L 18.0 mg/L 15.0 mg/L 15.0 mg/L 12.0 mg/L 1.6 mg/L Na~EDTA x 2 H20 100.0 p.g/L FeCI3 x 6 H~O 80.0 p,g/L MnCl~ x 4 H20 415.0 ~g/L H3BO~ 185.0 p.g/L Na~MoO,~ x 2 H~O 7.0 ~g/L ZnCl~ 3.0 p,g/L CoCI~ x 6 H~O 1.5 I~g/L CuCt2 x 2 H~O 0.01 p.g/L Calculated water hardness of the test water: 0.24 mmol/L (= 24 

### mg/L) as CaCO3 The test was started (0 hours) by inoculation of 10,000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test under the same conditions as in the test, One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth. The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 22 °C (Table 8), and continuously illuminated at the measured light intensity of about 8500 Lux (mean value), range: 7890 to 9150 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), Installed above the test flasks. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification. The test duration was 72 hours. For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control at least once per year to demonstrate satisfactory test conditions.



### 589RCC Study Number 845445 REPORT Page 15 TKA 40278 (CG1827) 

### 2.5.2 Dosage and Concentrations In order to assess the toxicity of the test item TKA 40278 (CGI 827) containing two different isomers on algae, five WAFs (Water Accommodated Fractions) were prepared according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (Ref 1) as described below: Prior to the start of the test, five individual mixtures with loading rates of 1.0, 3.2, 10, 32, and 100 mg/L were prepared. The dispersions were prepared by mixing 3.0, 9.6, 30.2, 96.2, and 300.6 mg of the test item into 3000 mL test water, respectively, by ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was used. The dispersions were stirred on a magnetic stirrer at room temperature in the dark for 96 hours to dissolve a maximal amount of the test item in test water. The long stirring period of 96 hours was chosen based on the results of a pre-test (without GLP) which showed that the test concentration of dissolved test item steadily increased during the stirring period of 96 hours. After the stirring period of 96 hours, the dispersions were filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 p.m) just before the start of the test. The filtrates of the dispersions with dissolved test item were tested on algae as WAFs. Additionally, a control was tested in parallel. The test media were freshly prepared just before inoculation of the algae. The actual concentrations of the test item in the test media were analytically determined (see Section 2.6.4).The loading rates were based on the results of a range-finding test and on the results of preexperiments to the solubility of the test item (without GLP). However, loading rates above 100 mg/L were not tested according to the guidelines. The enlarged spacing factor of 3.2 between the loading rates was chosen since according to the results of the range-finding test, a large concentration range had to be tested in the definitive test.



### 59ORCC Study Number 845445 TKA 40278 (CGI 827) REPORT Page 16



### 2.6 EVALUATIONS 2.6.1 Counting and Examination of the Algal Cells Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48, and 72 hours exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter ®, Model ZM), with at least two measurements per sample. In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (loading rate 32 mg/L). The shape of the algal cells was microscopically examined.
2.6.2 Determination of the Algal Growth Inhibition and the EC Values Inhibition of algal growth was determined from: a) the area under the growth curves AUC (= biomass, b) b) the specific growth rates (r) for exponentially growing cultures using the following equations: a) Area under the growth curve (AUC): AUC=(N1-No).tl + (NI +N2)-2No (t2_t~)+...+ (Nn-1 + Nn)-2No

2 2 2 where:No = nominal number of cells/mL at time to (start of the test) N1 = mean measured number of cells/mL at time t~ N2 = mean measured number of cells/mL at time t2 Nn = mean measured number of cells/mL at time tn t~ = time of first measurement after start of the test t~ = time of second measurement after start of the test tn = time of n~measurementafterstart of the test (tn - tn-l)

Percentage inhibition of AUC (IAuc): AUCc - AUC~ IAUc= 100%

AUCo where: AUCc = mean area under the control growth curve AUC~ = mean area under the growth curve at test concentration i



### 591ROCStudy Number845445 REPORT Page 17 TKA 40278 (CGI 827) 

## b) Growth rate (r):

r = InNn"InN° tn Percentage inhibition of growth rate (Ir): 

### I~=rc-rl .100% ro where: rc = mean growth rate of the control r~ = mean growth rate of test concentration i AUC and r were calculated for each test flask. Then the mean area and mean growth rate were determined as the arithmetic mean value over all test flasks per treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data. The EC10 and ECS0 values for the growth rate (the respective concentrations of the test item corresponding to 10% and 50% inhibition of growth rate, respectively, compared to the control) could not be calculated due to the low inhibitory effect of the test item. Moreover, no clear loading rate - effect relationship was determined. For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences compared to the control values by a Dunnett-test (Ref. 2, 3).

2.6.3 Water Quality Criteria The pH was measured and recorded in each test concentration and the control at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was recorded daily. 

### 2,6.4 Analysis of the Test Item Concentrations For determination of the actual test item concentrations, the following samples were taken: Just before the start of the test:
After 72 hours: (stability samples) -duplicate samples from each test medium (without algae)

-duplicate samples from the control (without algae)

-duplicate samples from each test medium (without algae)

-duplicate samples from the control (without algae)



### 592RCC Study Number 845445 REPORT Page 18

TKA 40278 (CGI 827)
For the 72-hour stability samples, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability (without GLP) the test item is sufficiently stable in the test water under the storage conditions, The concentrations of the test item TKA 40278 (CGI 827) were analyzed in all test medium samples from both sampling times (0 and 72 hours). From the control samples, only one of the duplicate samples was analyzed from each of both sampling times. The analytical procedure and the results are described in the attached analytical report.



### 593RCC Study Number 845445 REPORT Page 19 TKA 40278 (CGI 827) 

## 3 RESULTS AND DISCUSSION

The measured test item concentration in the analyzed test media samples (loading rates 1.0, 3.2, 10, 32, and 100 mg/L) amounted to 0.95, 2.9, 5.0, 4.4, and 6.0 mg/L at the start of the test (see analytical results and Table 2 in the attached analytical report). In the WAFs with loading rates of 10 to 100 mg/L, approximately the same test item concentration of about 4 to 6 mg/L was measured. Thus, the saturation concentration of the test item in test water was obviously reached. in the test media, incubated under the test conditions during the test period (but without algae), the measured concentrations of TKA 40278 (CGI 827) decreased to values below the limit of quantification (LOQ) of the analytical method of 0.23 mg/L aftero72 hours. This decrease of the test item concentrations was considered to be due to photoiyticat degradation of the test item due to the intensive irradiation. The mean measured test item concentrations (calculated as the average over all measurements per test concentration) were: Loading rate (mg/L) 1.0 0.53 3.2 1.5 10 2.6 32 2.3 100 3.0

Mean measured test item concentration* (mg/L) (% of initially measured)

56 52 51 51 51

*the value was obtained by taking half of LOQ of 0.23 mg/L into calculation The influence of the test item TKA 40278 (CGI 827) on the growth of Scenedesmus subspicatusis shown in Tables 1-5 and Figure 1. The 72-hour NOEC (highest concentration tested without toxic effects after the exposure period of 72 hours) was determined to be at the loading rate of 1.0 mg/L (mean measured 0.53 rag/L) since at this loading rate, the mean growth rate of Scenedesmus subspicatuswas only 5% lower than in the control (Table 3). This inhibition was considered to be within the normal range of biological variations and was not considered to be a toxic effect caused by the test item. The 72-hour LOEC (lowest concentration tested with toxic effects) was determined to be at the loading rate of 3.2 mg/L (mean measured 1.5 mg/L) since the growth was statistically significantly reduced at this loading rate and above (results of Dunnett-tests, one-sided, c~ = 0.05, see Tables 4 and 5). The EC50-values for the algal biomass (b) and the growth rate (r) after 72 hours test duration could not be determined since the inhibition of growth was at the loading rates of 3.2 to 100 mg/L below 50%. At the loading rates of 10 to 100 mg/L, the inhibition of growth rate was about 10 to 13% and the inhibition of biomass amounted to about 30 to 38% (Tables 2 and 3). No concentration-response relationship was determined. This corresponds to the analytical results which indicate that the saturation concentration of the test item in test water



### 594RCC Study Number 845445 REPORT Page 20 TKA 40278 (CG1827) was reached at these loading rates and resulted in less than 50% inhibition of growth rate and biomass. The microscopic examination of the algal cells after 72 hours test period showed no difference between the algae growing in the WAF with the loading rate of 32 mg/L and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at up to this test concentration were obviously not affected. In the control, the cell density has increased from nominal N = 1 x 104 cells/mL at the start of the test (0 hours) to N = 64 x 104 cells/mL (mean value) after 72 hours (Table 1). Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled. No remarkable observations were made concerning the appearance of test media (Table 6). The test media of all test concentrations remained clear throughout the entire test period, At the start of the test, the pH value of control and test media was 7,9. At the end of the test, pH values between 7,9 and 8.1 were measured (Table 7). Thewater temperature was constantly 22 °C during the test period (Table 8).



### 595RCC Study Number 845445 REPORT Page 21 TKA 40278 (CG1827) 

## 4 TABLES The tabulated values represent rounded results obtained by calculation using the exact raw data. Table 1: Algal cell densities during the test period of 72 hours Loading rate (mg/L) Control

1.0

3.2

10

32

100 Mean

measured concentration (mg/L) Flask Density of algal

NO. 24 h 2.0 2.3 2.3 2.4 2,4 2.6 2.2 2.4 2.3 2.1 2.6 2.3 .:ells (ceil number x

48 h 10,000/mL) after

72 h 1 2 3 4 5 6 10.5 10.2 10.5 9.8 8.8 8.5 8.9 8.6 9.3 8.9 8.6 8.3 68.3 70.4

71.8 69,9 54.8 53.7 69.1 72.1 51.6 52.4 64,6 66.9

0.53

1.5

2.6

2.3

3.0 m 2.33

s 0.14 n 6 1 2.2 2.2 2 2.2 2.1 3 2.5 2.4 m 2.27 s 0.16 n 3 1 2.8 2.7 2 2.9 2.8 3 2.8 2.9 m 2.82 s 0.06 n 3 1 3.3 2.9 2 3.0 3.1 3 2.6 2.6 m 2.92 s 0.28 n 3 1 2.4 2,3 2 2.8 3.0 3 2.8 2.5 m 2.63 s 0.28 n 3 1 3.1 3.2 2 2.7 2.7 3 2.8 3.0 m 2.92 s 0.23 n 3 8.8 9.7 8.9

9.4 8.8 9,1

7.9 8.1 8.6 8.25 0.22 3 8.1 8.2 7.8 8.00 0.28 3 5.4 4.7 3.4 4.45 0.98 3 9.24 0.81 6 8.4 10.1 8.6 9.08 0.71 3 9.8 8.8 8.9 9.13 0.42 3 8.3 8.2 8.4

8.4 7.9 7.6

5.1 4.8 3.3 63.80

8.50 6 50.7 48.2 55.2 55.6 49.2 48.8 51.28 3.57 3 40.0 41.1 47.8 47.8 45.1 45.8 44.60 3.70 3 36.0. 36.9 38.3 38.6 37.3 37.4 37.42 1.00 3 38.4 37.7 43.3 42.1 42.4 42.4 41.05 2.60 3 43.0 42.9 43.5 41.6 39.7 38.1 41.47 2.23 3 m: arithmetic mean; s: standard deviation; n: number of replicates At the start, 10,000 algal cells/mL were inoculated.



### 596RCC Study Number 845445 REPORT Page 22

TKA 40278 (CGI 827)

Table 2: Areas under the growth curves (AUC) and percentage inhibition ofAUC (IAuc)

during the test period
Loading Mean Areas under the growth curves (AUC) and % inhibition of AUC rate measured concentration 0~4h 0~8h 0~2h (mg/L) (rag/L) AUC 

### t^ua (%) AUC 

### l~ua (%) AUC 

### IAuc (%) Control 16 0.0 131 0.0 983 0.0 1.0 0.53 15 4.4 127 2.5 828 15.8 3.2 1.5 22 -37.1 141 -8.0 762 22.5 10 2.6 23 -44.7 133 -1.8 657 33.2 32 2.3 20 -23.3 123 5.7 688 30.0 100 3.0 23 -44.7 87 33.1 614 37.5 AUC x 10,000 - % inhibition: increase in growth relative to that of control

Table 3: Growth rates r and percentage inhibition of r (It) during the test period
Loading Mean Growth rate r and % inhibition of r rate measured

concentration 0-24 h 0~8h 0~2h (mg/L) (rag/L) r (l/day) 

### I,- (%) r (l/day) r (l/day) 

### (%) Control 0.84 0.0 1.11 0.0 1.38 0.0 1.0 0.53 0.82 3.0 1.10 0.7 1.31 5.1 3.2 1.5 1.04 -22.9 1.11 0.4 1.27 8.5 10 2.6 1.07 -26.7 1.05 5.0 1.21 12.7 32 2.3 0.96 -14.5 1.04 6.4 1.24 10.5 100 3.0 1.07 -26.9 0.74 33.6 1.24 10.2 - % inhibition: increase in growth relative to that of control



### 597RCC Study Number 845445 REPORT Page 23 TKA 40278 (CGI 827)
Table 4: Results of a Dunnett-test with the biomass (b; areas under the growth curves)
Loading Mean Calculated t-value rate measured concentration (rag/L) (rag/L) 0-24 h 0-48 h 0-72 h 1.0 0.53 0.43 0.58 3.04 * 3.2 1.5 -3.61 -1.83 4.32 * 10 2.6 -4.34 -0.40 6.37 * 32 2.3 -2.26 1.31 5.77 * 100 3.0 -4,34 7.56 * 7,20 * Tabulated t-value 2.44 2.44 2,44 Degrees of freedom 15 15 15 * mean value significantly different from the control (~ = 0.05, one-sided smaller)

Table 5: Results of a Dunnett-test with the growth rates r
Loading Mean Calculated t-value rate measured concentration (mg/L) (mg/L) 0-24 h 0-48 h 0-72 h 1.0 0.53 0.49 0.22 3.15" 3.2 1.5 -3,72 0.12 5,23 * 10 2.6 -4.33 1.48 7.81 * 32 2~3 -2.35 1,89 6.45 * 100 3.0 -4.35 9.96 * 6.30 * Tabulated t-value 2.44 2.44 Degrees of freedom 15 15 15 * mean value significantly different from the control (~ = 0.05. one-sided smaller)



### 598RCCStudy Number 845445 TKA 40278 (CGI 827) REPORT Page 24

Table 6: Appearance of the test media

Abbreviations: 0: no remarkable observations, clear test medium 1: homogeneous dispersion in the water, turbidity observable 2: noticeable turbidity caused by the test item 3: noticeable coloration caused by the test item 4: non-homogeneous dispersion of the test item 5: precipitation of the test ~tem 6: test item at the surface 7: test item lying at the bottom of the flask Loading rate Mean measured Exposure time

concent~tion (mg/L) (mg/L) 

### Oh t 24h I 48h 72h 1.0 0.53 0 0 0 0 3.2 1.5 0 0 0 0 10 2.6 0 0 0 0 32 2.3 0 0 0 0 100 3.0 0 0 0 0

Table 7: pH values in the test media and in the control

Loading rate Mean pHvalues

measured (rag/L) concentration Start End

(rag/L) Contml 7.9 8.0 1.0 0.53 7.9 7.9 3.2 1.5 7.9 8.0 10 2.6 7.9 8.0 32 2.3 7.9 8.1 100 3.0 7.9 8.0



### 599RCC Study Number 845445 REPORT Page 25 TKA 40278 (CG1827)
Table 8: Water temperature during the test period
Temperature (°C) Day0 (Star~) 22 Day 1 22 Day2 22 Day3 (End) 22



### 600RCC Study Number 845445 REPORT Page 26 TKA 40278 (CGI 827) 

## 5 FIGURE
Figure 1: Growth curves of Scenedesmus subspicatus,incubated for 72 hours

at different concentrations of the test item

lOO

E

~10



### 1 I I 0 24 48 72

test period (hours)
"’O" Control ~ 10 mg/L (2.6 rag/L) "-O- 1.0 mg/L (0.53 mg/L) ~ 32 mg/L (2.3 mg/L) --~’-- 3.2 mg/L (1.5 mg/L) --~ - 100 mg/L (3.0 mg/L)
Concentration given as loading rates, .mean measured concentrations in parentheses



### 601RCC Study Number 845445 REPORT Page 27 TKA 40278 (CGI 827) 

## 6 REFERENCES

1
2

3 OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control, Journal of the American Statistical Association 50, 1096-1121 DUNNETT, C.W. (1964): New tables for multiple comparisons with a control, Biometrics 20, 482-491



### 602RCC Study Number 845445 REPORT Page28 TKA 40278 (CGI 827)



## ATTACHMENT

ANALYTICAL REPORT

603

## ANALYTICAL REPORT

Study Title: TOXICITY OF TKA 40278 (CGI 827) TO SCENEDESMUS SUBSPICATUS IN A 72-HOUR ALGAL GROWTH INHIBITION TEST Subtitle of Analytical Report: DETERMINATION OF THE CONCENTRATIONS OF THE TEST ITEM IN TEST MEDIUM
Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics Division CH-4452ItingenlSwitzerland RCC Study Number: 845445 Analytical Identification Number: 845446

Page A1 to A12



### 604RCC Study Number 845445 ANALYTICAL REPORT Page A2. TKA 40278 (CGt 827) Analytical ld. No, 845446 

## CONTENTS

CONTENTS ................................................................................................................. A2 PREFACE

.................................................................................................................... A3 GENERAL

.................................................................................................................... A3 RESPONSIBILITIES ..................................................................................................... A3 SCHEDULE .................................................................................................................. A3 1 PURPOSE ........................................................................................................... A4 2 MATERIALS AND METHODS ............................................................................. A4 2.1 2.2

2.3 2.4 TESTITEM ................................................................................................ A4 ANALYTICAL PROCEDURE ...................................................................... A4 2.2.1 Storage ........................................................................................... A4 2.2.2 Reagents and Solvents ................................................................... A4 2.2.3 Standard Solutions used for Sample Quantification ........................ A4 2.2.4 Preparation of Spiked Test Water Samples .................................... A5 2.2.5 Analysis of Samples ........................................................................ A5 HPLC CONDITIONS .................................................................................. A5 EVALUATION OF RESULTS ..................................................................... A6 3 RESULTS AND DISCUSSION ............................................................................ A7 4 TABLES .............................................................................................................. A8 Table 1: Calibration data of test item-standards ....................................................... A8 Table 2: Results obtained for the concentrations of the test item in

test medium ............................................................................................... A9 5 FIGURES .......................................................................................................... A10 Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: HPLC-chromatogram of standard solution (mid-level) .............................. A10 HPLC-chromatogram of standard solution (high-level) ............................. A10 HPLC-chromatogram of biological control sample .................................... A11 HPLC-chromatogram of spiked test water sample ................................... A11 HPLC-chromatogram of non-aged treatment sample ............................... A12 HPLC-chromatogram of aged treatment sample ...................................... A12



### 605RCC Study Number 845445 ANALYFICAL REPORT Page A3 TKA 40278 (CG1827) Analytical Id. No. 845446 , - 

## PREFACE
GENERAL Study Title:
Subtitle: Sponsor:

Monitoring Scientist: Test Facility:

Analytical Id. No.: Toxicity of TKA 40278 (CGI 827) toScenedesmus subspicatusin a 72-hour algal growth inhibition test Determination of the concentrations of the test item in test medium 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 & Pharmanalytics Division Zelgliweg 1 CH-4452 Itingen/Switzerland 845446


### RESPONSIBILITIES Responsible for Anatytics: Mr. Tobias Schoop

Technical Coordinators: Reporting:


### SCHEDULE Experimental starting date: Experimental completion date: January 07, 2003 January 14, 2003



### 606RCC Study Number 845445 ANALY-t’ICAL REPORT Page A4 TKA 40278 (CGI 827) Analytica~ Id. No. 845446 

## 1 PURPOSE In this analytical report the results obtained for the concentrations of TKA 40278 (CGI 827) in test medium are described. The quantification of the test item was performed by HPLC analysis with UVNIS-detection. The test item consisted of two different isomers. However, the two isomers were not separated by the HPLC method.


## 2 MATERIALS AND METHODS
2.1 TEST ITEM The test item as described in the biological part of this study was also used for analytical purposes. 

### 2.2 ANALYTICAL PROCEDURE 2.2.1 Storage The samples were stored deep-frozen and protected from light until analysis was performed.


### 2.2.2 Reagents and Solvents Acetonitrile Tetrahydrofurane Purified water for HPLC Test water Baker, no. 9017 Baker, no. 9441 in-house prepared by a water purification system (Millipore) as described in the biological part of this study


### 2.2.3 Standard Solutions used for Sample Quantification The test item (23.08 rag) was dissolved in acetonitrile and made up to the mark in a 100 mL volumetric flask to prepare a stock solution of 23t mg/L. Defined volumes of this stock solution were diluted with a mixture of acetonitrile/test water (1:1; v:v) to obtain standard solutions in the range of 0.115 to 69.2 mg/L of the test item. These solutions were used to calibrate the HPLC-system.



### 607RCC Study Number 845445 ANALYTICAL REPORT Page A5 TKA 40278 (CGI 827) Analytical Id. No. 845446 

### 2.2.4 Preparation of Spiked Test Water Samples To demonstrate the validity of the method, untreated test water was spiked with the test item. The test item (105.10 mg) was dissolved in acetonitdie and made up to the mark in a 100 mL volumetric flask to prepare a stock solution of 1051 mg/L. This stock solution (10 mL) was diluted to 100 mL with test water to obtain a solution with a concentration of 105 mg/L of the test item. Defined volumes of this solution or of the stock solution were diluted with test water to obtain spiked test water samples of the test item with concentrations of 1.05 and 105 mg/L. These solutions were subjected to the same treatment as a sample. In addition, test water without the test item was analysed (analytical blank). 2,2.5 Analysis of Samples The samples from the biological test were thawed at room temperature for 4 hours and shaken mechanically to obtain homogeneous sample solutions. The samples (100 mL) were diluted to 200 mL using acetonitrile. Aliquots of the samples were analysed by HPLC with UV/VIS-detection. For results obtained see Table 2, 

### 2.3 HPLC CONDITIONS Auto sampler: Pump: Detector; Column: Eluent A: Eluent B: Eluent C: Gradient: Merck-Hitachi L-7200

Merck-Hitachi L-7100 Merck-Hitachi DAD-L 7450 Discovery; 150 x 4.6 mm; 5 pm purified water acetonitrile tetrahydrofurane minutes 0 3 10 15 15.1 2O % eluent A

70 7O 0 0 7O 7O % eiuent B

15 15 85 85 15 15 % eluent C

15 15 15 15 15 15 Flow rate: 1 mlJmin Temperature: Injection volume: Detection wave length: Retention time: room temperature

10pL 260 nm approx. 8.9 min



### 608RCC Study Number 845445 ANALYTICAL REPORT Page A6 TKA 40278 (CG1827) Analytical Id. No. 845446 

### 2.4 EVALUATION OF RESULTS Injected samples were quantified by peak areas with reference to the respective calibration curve. The latter was obtained by correlation of peak area of the standard solutions to their corresponding concentration in mg/L. The correlation was performed using a potential function given below (equation 1). For results obtained see Table 1. From this curve the concentration x of the test item in an injected sample was calculated by the following equation: y=a. x~ (1)
where Peak area of test item in injected sample [counts] Concentration of test item in injected sample [mg/L] y-axis intercept Slope 

### The concentration of the test item in a sample was calculated by equation 2: c =x  ̄ F (2) where C = X = F = Concentration of test item in sample [mg/L] Amount of test item in injected sample found by equation 1 [mg/L] Factor (= Dilution Factor) The recovery of the test item in a sample was calculated by equation 3: R= c~ . 100% (3) O,rlom where R = C = C~rn = Recovery [%] Concentration of test item in sample found by equation 2 [mg/L] Nominal concentration of test item in sample [mglL]



### 609RCC Study Number 845445 ANALYTICAL REPORT Page A7 TKA 40278 (CG1827) Analytical td. No. 845446 

## 3 RESULTS AND DISCUSSION
The test item TKA 40278 (CGI 827) consisted of two different isomers. However, the two isomers were not separated by the HPLC method, The results obtained for the concentrations of TKA 40278 (CGI 827) in test medium are presented in Table 2. For the preparation of the treatment samples, water accommodated fractions (WAFs) with different loading rates of the test item were prepared (see biological part of this study). The calibration data for test item-standards are given in Table 1, The R2fit was 0,9997 (optimum 1.0000). This reflects the linearity of the HPLC-syatem within the calibration range of 0.115 - 69.2 mg/L of the test item. Typical HPLC chromatograms are shown in the attached Figures 1 to 6. The routine limit of quantification (LOQ) for the test item in test medium was derived from the lowest standard solution, which fits into the calibration curve: the value is 0,115 mg/L. With a factor of 2 from sample dilution, a LOQ of approximately 0.23 mg/L of the test item results. "]he biological control samples and an anal~,sed analytical blank (test water) did not affect the HPLC-chromatogram at the retention time of the test item. Concurrent with the sample analysis, recoveries of spiked test water samples in the relevant concentrations (1.05 and 105 mg/L of the test item) were performed in duplicate. The average concentrations were found to be 103 % and 104 % of the spiked values, with an overall mean of 103 % (n = 4). Therefore, no correction for possible losses during the analytical procedure is necessary. The average concentrations found in the non-aged treatment samples were 0.947 mg/L (loading rate 1.0 mg/L), 2.90 mg/L (loading rate 3.2 rag/L), 4.99 mg/L (loading rate 10 mg/L), 4.39 mg/L (loading rate 32 rag/L) and 5.97 mg/L (loading rate 100 rag/L). The values in the aged samples were below the LOQ of 0.23 mg/L of the test item. The found concentrations of the test item in the non-aged treatment samples can be reasonable explained by the low solubility of the test item. The losses in the aged samples can be due to photolysis of the test item. The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data,



### 610RCC Study Number 845445 ANALYTICAL REPORT Page A8 TKA 40278 (CGI 827) Analytical Id. No. 845446 

## 4 TABLES Table 1: Calibration data of test item-standards

Standard Peak area Deviation from

measured calculated value [mg/L] [counts] 

### [%] 0.115 58OO 5.1 0.462 21680 

### 1.1 1.15 49620 -5.8 4.62 197500 -3.5 11.5 491340 -2.1 23.1 992920 0.5 69.2 3045730 5.2

3500000 3000000 2500000- 2000000 1500000- 1000000- 500000- y = 45737x°.9791

~

10 20 30 40 50 60 70 80

where y = Peak area of test item in injected solution [counts] x = Concentration of test item in injected solution [mg/L]



### 611RCC Study Number 845445 ANALYTICAL REPORT Page A9 TKA 40278 (CGI 827) Analytical [d. No, 845446 Table 2: Results obtained for the concentrations of the test item in test medium

Nominal concentration of Sampling Age of RCC sample TKA 40278 (CG1827) date sample code [mg/L] [day] [hoursl Treatment samples Loading rata 1.0 0

0 3 3 0

0 72 72 A-32

A-33 A..44 A-45 Loading rate 3.2 o

o 3 3 0 A-34

0 A-35 72 A-46 72 A-47 Loading rate 10 0

0 3 3 0 A-36

0 A-37 72 A-48 72 A-49 Loading rate 32 0

0 3 3 0 A-38

0 A-39 72 A-50 72 A-51 Loading rate 100 0

0 3 3 0 A.40

0 A-41 72 A-52 72 A-53 Biological control samples Loading rate 0 ~ Spikedtest watersamples 1.05 0 TKA 40278 (CG1827) measured ling/L] [% of [mg/L] [% of

0.876 n.a. 1,018 n.a. 0.947 n.a.

mean : 0.531"* n.a. 2.939 n.a. 2.868 n.a. 2,904 n.a.

5,02 n.a, 4.96 n.a. 4.99 n.a,

4.39 n.a. 4.39 n.a. 4.39 n.a.

6.05 n.a. 5.90 n.a. 5.97 n.a.

,~Z3 0 AZ4 105 0 AZ1 o AZ2 1.09 103

1.07 102 1.08 103 109 104 110 105 110 104 mean: 103 Analytical blank

0 I I0 l AZ5 I n.d. I n.a. n.a. I n.a" no test item detected not applicable the value was below the LOQ of 0.23 mg/L of the test item (see Results and Discussion) the value was obtained by taking half of LOQ into calculation



### 612RCC Study Number 845445 ANALYTICAL REPORT Page A10 TKA 40278 (CGI 827) Analytical Id. No. 845446 

## 5 FIGURES

0,015 0,010 0,005

-0,005

Figure 1: 2 4 6 8 10 12 14 Retention Ti~e (min) HPLC-chromatogram of standard solution (mid-level) Concentration: 0.462 mg/L of the test item 16 18 20

0,2 0,0

Figure 2: 2 4 6 8 10 12 14 16 18 20 Retention T~ (rain) HPLC-chromatogram of standard solution (high-level) Concentration: 69.2 mg/L of the test item



### 613RCC Study Number 845445 ANALYTICAL REPORT Page A11 TKA 40278 (CGI 827) Analytical Id. No. 845446

0,010
0,005
0,000

-0~005
-0,010

Figure 3: 0 4 6 8 10 12 14 16 18 20 Retention Time (m/n) HPLC-chromatogram of biological control sample Sample Id. No.: A-30 Sampling day 0; age of sample: 0 hours

0,015

-0,005

Figure4: Retention Time (m/n)

HPLC-chromatogram of spiked test water sample Sample Id. No.: AZ4 (spiked with 1.05 mg/L of the test item; nominal concentration in injected sample: 0.526 mg/L) Recovery: 102 % of the nominal concentration



### 614RCC Study Number 845445 ANALYTICAL REPORT Page A12

TKA 40278 (CG1827) Analytical Id. No. 845446

0,005
O,000
-0,005

Figure 5: 4 6 8 I0 12 14 16 IB 20 Retention Ti~e (rain) HPLC-chromatogram of non-aged treatment sample Sample Id. No.: A-33 Sampling day 0; age of sample: 0 hours (nominal concentration: 1.0 mg/L of the test item; nominal concentration in injected sample: 0.5 mg/L) Measured concentration: 1.02 mg/L

0,005

0,000

-0,005

Figure 6: 6 8 I0 12 Retention Time HPLC-chromatogram of aged treatment sample Sample Id. No.: A-52 Sampling day 3; age of sample: 72 hours (nominal concentration: 100 mg/L of the test item; nominal concentration in injected sample: 50 mg/L) Value in diluted, injected sample: < LOQ of 0.23 mg/L of the test item 20



### 615