# REPORT
StudyTitle: TOXICITY OF DEAIACID ANHYDRIDE REACTION PRODUCT TO SCENEDESMUS SUBSPICATUS IN A 72-HOURALGAL GROWTH INHIBITION TEST Data Requirements ITest Guidelines: OECD No. 201 EU Commission Directive 92/69/EEC, C.3 Study Director: Roger

**B~Uscher** StudyCompletion Date: November 19, 2004 Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen I Switzerland Sponsor: Rockwool InternationalAIS Hovedgaden 584 DK-2640 Hedehusene Denmark Identification: RCC Study Number: Page 1 of59 848634RCC Study Number848634 DENAcid anhydride reaction product 

# GLP CERTIFICATE REPORT

The Swiss GLP Monitoring Authorities
Swiss Federal Officeof Public Health SwissAgencyforthe Environment. Forests and Landscape **~** Swissmedic 

## r . SwissAgencyfor SWlSsrnedlC TherapeuUcProducts 

# Statement of GLP Compliance

It is herebyconfirmed that during the period of thefollowingTest Facilities of November 18 - 22, 2002 

### RCC Ltd 4452 ltingen Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for Therapeutic Products and the Swiss Agency forthe Environment, Forests and Landscape with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities -Toxicology

- Environmental Chemistry and

Pharmanalytics Areas ofexpertise* TOX, ACC, MUT, OTH (Safety Pharmacology) ACC, ECT, ENF, EMN, PCT, RES, OTH (Animal metabolism)
The inspections were performed in agreement with the OECD Guidelines for National GLP Inspections and Audits. Itwas found that the aforementioned test facilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time they were inspected.

Bern, March 2003 Federal Office ofPublic Health The Director

Prof. Th. Zeltner

• TOX=Toxicology:ACC =Analyticaland Clinical Chemislly: ECT=Environmentaltoxicityonaquatic and terresbial organisms ; ENF

### =BehaviourIn water, soliandair. Bioaccumulalion ; EMN=Studieson effecls onmesocosmsand natural ecosystems; MUT

### =Mutagenicity; PCT= Physical-chemical testing ; RES=Residue studies ;OTH "'Other, tobespeclfied. Page2RCC Study Number848634 DEA/Acid anhydride reaction product GOOD LABORATORY PRACTICE REPORT Page 3



# STATEMENT OF COMPLIANCE

RCC Study Number: Test Item: Study Director: StudyTitle: 848634 DENAcid anhydride reaction product Mr. Roger Bi:itscher Toxicity of DENAcid anhydride reaction product to Scenedesmus subspicatus in a 72-hour algal growth inhibition test This study (with the exception of the pre-experiments as mentioned in the report and the analytical standards as mentioned in the analytical phase report) has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February, 2"d, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision ofthe OECD Council [C(97) 186/Final]. There were no circumstances that may have affected the quality or integrityofthe data. Study Director: Mr. Roger Bi:itscher

Date: 

# J/oll'lAMbJJ 

# )'J 2oolf IRCC Study Number848634 DEA/Acid anhydride reaction product 

# SIGNATURES

Study Director:

Management: REPORT

Mr. Roger

**B~Uscher**

Date:

Dr. Uwe Morgenroth Page4



## ;It;

**2oo~** fRCC Study Number848634 DEA/Acid anhydride reaction product 

# QUALITYASSURANCE REPORT Page 5

RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 ltingen 

### I Switzerland STATEMENT
RCC Study Number: Test Item: Study Director: StudyTitle: 848634 DEA/Acid anhydride reaction product Mr. Roger Batscher Toxicity of DEA/Acid anhydride reaction product to Scenedesmus subspicatus in 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 ofthe pre-experiments as mentioned in the report) were periodically inspected. The study plan and this report were audited by the Quality Assurance. The dates are given below.
Dates and Types ofQA Inspections Dates of Reports to the Study Director and to the Management December 12, 2003 Study plan December 12, 2003 July 16, 2004 Process based (analytical work) July 16, 2004 August30, 2004 Report September01, 2004 September 01, 2004 This statement also confirms thatthis final report reflects the raw data.
QualityAssurance: 

# FMrs. Andrea GildelRCC Study Number848634 DEA/Acid anhydride reaction product 

# CONTENTS REPORT Page6

GLP CERTIFICATE.........................................................................................................2 STATEMENT OF COMPLIANCE ....................................................................................3 SIGNATURES .................................................................................................................4 QUALITYASSURANCE ..................................................................................................5 CONTENTS.....................................................................................................................6 CONTENTS CONTINUED (TABLES & FIGURES)..........................................................

### ? PREFACE .......................................................................................................................8 DATA REQUIREMENTS 

### ITEST GUIDELINES...............................................................9 SUMMARY OF STUDY PLAN AMENDMENTS...............................................................9 SUMMARY....................................................................................................................10 1 PURPOSE............................................................................................................11 2 MATERIALSAND METHODS..............................................................................11 2.1 DEFINITIONS .............................................................................................11 2.2 TEST ITEM .................................................................................................12 2.3 ANALYTICAL STANDARD..........................................................................12 2.4 TEST SYSTEM ..........................................................................., ................ 12 2.5 STUDY DESIGN .........................................................................................13 2.5.1 Experimental Conditions................................................................13 2.5.2 Preparation ofTest Solutions and Concentrations
.........................14 2.6 EVALUATIONS ...........................................................................................15 2.6.1 Counting and Examination oftheAlgal Cells .................................15 2.6.2 Determination oftheAlgal Growth Inhibition and the EC Values ...........................................................................................15 2.6.3 WaterQuality Criteria ....................................................................16 2.6.4 Analysis ofthe Test Item Concentrations.......................................16 3 RESULTS AND DISCUSSION .............................................................................18 4 TABLES ...............................................................................................................20 5 FIGURE................................................................................................................23 6 REFERENCES.....................................................................................................24 ATTACHMENT..............................................................................................................25 ANALYTICAL PHASE REPORT (Pages 1to 34)RCC Study Number848634 DENAcid anhydride reaction product REPORT 

# CONTENTS CONTINUED (TABLES 

# &FIGURES) Page 7



### Table 1: Algal cell densities during the test period of72 hours................................20 Table 2: Areas underthe growth curves (AUG) and percentage inhibition ofAUG 

### (lAue) during the test period ...........................................................21 Table 3: Growth rates (r) and percentage inhibition of r 

### (lr) during the test period ................................................................................21 Table 4: Appearance ofthe test medium ................................................................22 Table 5: pH values in the test medium and in the control ........................................22 Table 6: Watertemperature during the test period ..................................................22

Figure 1: Growth curves of Scenedesmus subspicatus, 

### incubated for72 hours
.......23RCC Study Number848634 DEA/Acid anhydride reaction product 

# PREFACE GENERAL StudyTitle:
Sponsor:

Study Monitor:

Test Facility:

Analytical Phase No.:


### RESPONSIBILITIES Study Director: Deputy Study Director: Principal Investigatorfor Analytical Phase: Technical Coordinator: Head of RCC QualityAssurance:


### SCHEDULE Experimental Starting Date: Experimental Completion Date: Study Completion Date:


### ARCHIVING REPORT Page 8

Toxicity of DEA/Acid anhydride reaction product to Scenedesmus subspicatus in a 72-hour algal growth inhibition test Rockwool InternationalA/S Hovedgaden 584 DK-2640 Hedehusene Denmark Mrs. Anne Jensen (until end ofMay 2004) Mrs. Lea Tobiassen, DVM (from June 01, 2004) Danish Toxicology Centre Kogle Aile 2 DK-2970 H0RSHOLM Denmark RCC Ltd Environmental Chemistry & Pharmanalytics Zelgliweg 1 CH-4452 ltingen 

### I Switzerland 848635

Mr. Roger

**B~itscher** Dr. Birgit Seyfried Dr. Tobias Schoop Mrs. Helene Eckenstein Mrs. IrisWuthrich

June 04, 2004 June 21, 2004 November 19, 2004

RCC Ltd, CH-4452 ltingen, 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 withoutthe Sponsor's consent.RCC Study Number848634 DENAcid anhydride reaction product 

# 1 PURPOSE REPORT Page 11



### The purpose of this test was to determine the inhibitory effect of the test item DENAcid anhydride reaction product on the growth ofthe freshwater green algal species Scenedesmus subspicatus. 

### Exponentially growing cultures ofthis algal species were exposed to a single test concentration of nominal 100 mg/L under defined conditions (limit test). The limit test was based on a range-finding test and was performed in compliance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on Scenedesmus subspicatus 

### upto at leastthis concentration. A static, non-renewal exposure system was used. The inhibition ofgrowth in relation to control cultures was determined over a test-period of 72 hours and, thus, over several algal generations. The test method and the test species are recommended by the test guidelines of OECD and EU.



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

NOEC (No Observed £_ffect Concentration): LOEC (1_owest Observed S.ffect Concentration): the number ofcells per ml the increase ofcell density overthe test period the actual number of cells per volume of medium (cells/ml) calculated as the g_rea ynderthe growth _gurve (AUC) the increase ofcell density per unittime the calculated concentration oftest item that results in an x% reduction of biomass (b) relative to the control the calculated concentration oftest item that results in an x% reduction ofgrowth 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 observed relative to the controlRCC Study Number848634 DEA/Acid anhydride reaction product 

## 2.5 STUDY DESIGN 2.5.1 Experimental Conditions REPORT Page 13



### The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified waterto obtain the following final nominal concentrations: Macro-nutrients:

Trace elements: NaHC03 CaCb x 

### 2 H2

### 0 NH4CI MgS04 x 

### 7 HzO MgCI2 x 

### 6 HzO KHzP04 Na2

### EDTA x 

### 2 H2

### 0 FeCb x 

### 6 HzO MnCI2 x 

### 4 HzO H3B03 NazM004 x 

### 2 HzO ZnCI2 

### CoCb x 

### 6 H2

### 0 CuCI2 x 

### 2 HzO 50.0 mg/L 18.0 mg/L 15.0 mg/L 15.0 mg/L 12.0 mg/L

1.6 mg/L 100.0 !Jg/L

80.0 !Jg/L 415.0 !Jg/L 185.0 !Jg/L 7.0 !Jg/L 3.0 !Jg/L 1.5 !Jg/L 0.01 !Jg/L Calculated water hardness ofthe test water: 0.24 mmoi/L (= 

### 24 mg/L as CaC03 ). The test was started (0 hours) by inoculation of 10,000 algal cells per mL test medium. The algal cells were taken from an exponentially growing pre-culture which was set up four days priorto the test underthe same conditions as in the test. One day before the start ofthe test, the pre-culture was diluted threefold to keep the algae in exponential growth. The test design included three replicates of the 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 Erlenmeyerflasks. The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at atemperature between 22 and 23

# oc (Table 6) and continuously illuminated at a measured light intensity of about 8500 Lux (mean value), range: 8010 to 8910 Lux (minimum and maximum value of measurements at nine places distributed overthe 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 assure unmistakable identification. The test duration was 72 hours. For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2003 (72-hour EC50 for the growth rate: 0.69 mg/L, RCC Study 849621) showed that the toxic performance was valid and within the historical range ofthe RCC laboratory (from 1996 to 2003: 72-hour EC50: 0.44- 1.16 mg/L).RCC Study Number848634 DENAcid anhydride reaction product REPORT


### 2.5.2 Preparation ofTest Solutions and Concentrations Page 14



### A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the algae up to and including the nominal test item concentration of 100 mg/L. Thus, a single test item concentration of nominal 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition ofthe test item). The test medium was prepared immediately before the start ofthe test as follows: The amount of 60.3 mg of the test item was mixed into 600 ml test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The actual concentration ofthe test item (i.e. of the different components ofthe test item) in the test medium was analytically determined (see Section 2.6.4). The limit test was based on the results of a range-finding test and on the results of a preexperiment to determine the solubility ofthe test item (without GLP). However, concentrations above nominal 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC.RCC Study Number848634 DENAcid anhydride reaction product 

## 2.6 EVALUATIONS REPORT



### 2.6.1 Counting and Examination ofthe Algal Cells Page 15

Small volumes of the test medium and the control (1.0 ml) were taken out of all test flasks after 24, 48, and 72 hours of 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 leasttwo measurements persample. In addition, after 72 hours exposure, a sample was taken from the control and from the single test concentration. The shape of the algal cells was examined microscopically in these samples.



### 2.6.2 Determination ofthe Algal Growth Inhibition and the EC Values Inhibition ofalgal growth was determined from: a) the area underthe growth curves AUC (= biomass, b) b) the specific growth rates (r) for exponentially growing cultures using the following equations: 

### a) Area underthe growth curve (AUC):

where: 

### No N1 Nz 

### Nn t1 

### tz tn 

# = nominal numberofcells/ml at time t0 (start ofthe test) 

### = mean measured numberofcells/ml at time t1 

### = mean measured number ofcells/ml at time t2 

# = mean measured number ofcells/ml at time tn 

# =time offirst measurement afterstart ofthe test 

### = time ofsecond measurement afterstart ofthe test 

# =time of nth measurement afterstart ofthe test 

### Percentage inhibition ofAUC (lAue):
I - AUCC
- AUCt 1QQOI AUC-

. to AUCC where: AUCc 

### = mean area underthe control growth curve AUCt 

### = mean area underthe growth curve at treatment tRCC Study Number848634 DEA/Acid anhydride reaction product REPORT Page 17



### Duplicate samples per sampling date were taken for each analysis, resulting in a total number ofsix samples per sampling datefrom'the test medium and the control. The samples for analysis by method 1 and 3were analyzed directly afterthe sampling without prior storage. The samples for analysis by method 2were deep-frozen at approximately -20

# oc immediately after sampling and were kept deep-frozen until analysis. The stability of the components to be analyzed by method 2 underthe storage conditions was confirmed in a preexperiment (without GLP). The components of the test item were analyzed in all duplicate test medium samples. From the control samples, one ofthe duplicate samples was analyzed persampling date. The analytical procedures and the results are described in the attached analytical phase report.RCC Study Number848634 DENAcid anhydride reaction product REPORT Page 19



### No remarkable observations were ma9e concerning the appearance ofthe test medium. The test medium appeared to be a clear solution throughoutthe test duration (Table 4). At the start of the test, the pH values in the test medium and the control were 7.7 and 8.1, respectively. Atthe end ofthe test, pH values ofthe test medium and the control were 8.2 and 8.4, respectively {Table 5). The watertemperature ranged from 22 to 23

# oc (Table 6).RCC Study Number848634 DENAcid anhydride reaction product REPORT Page 20 

# 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 of72 hours
Nominal Flask Density ofalgal cells (cell numberx 10,000/ml) after test item No. concentration (mg/L) 24 h 1 3.3 3.1 11.5 Control 2 2.7 2.5 9.5 3 2.9 3.0 11.5 4 2.8 2.7 10.5 5 2.8 2.7 9.5 6 3.5 3.3 9.3 m 2.94 s 0.31 n 6 1 2.9 3.2 11.0 100 2 2.5 2.4 10.5 3 2.8 3.0 11.0 m 2.80 s 0.31 n 3
m: mean value; s: standard deviation; n: numberofflasks Atthe start, 10,000 algal cells/mlwere incubated. 48 h 72 h 10.8 69.7 9.7 49.1 11.4 71.4 10.2 58.6 9.5 49.2 8.7 44.1 10.18 56.99 0.98 11.46

6 6 11.0 64.2 10.1 60.5 11.1 60.2 10.78 61.88 0.42 2.46

3 3 69.8 49.4 71.1 58.0 50.0 43.5

65.2 61.1 60.1RCC Study Number848634 DEA/Acid anhydride reaction product REPORT



### Table 2: Areas underthe growth curves (AUC) and percentage inhibition ofAUC (lAue) 

### during the test period Page 21

Nominal Areas underthe growth curves (AUC) and % inhibition ofAUC test item concentration 0-24 h 0-48h 0-72 h (mg/L) AUC lAue(%) AUC lAue(%) AUC lAue(%) Control 23 0.0 157 0.0 939 0.0

100 22 7.3 161 -2.5 1009 -7.4 AUC x 10,000

- % inhibition: increase in growth relative to that ofcontrol



### Table 3: Growth rates (r) and percentage inhibition of r (lr) 

### during the test period
Nominal Growth rate rand % inhibition of r test item concentration 0-24 h 0-48 h 0-72 h (mg/L) r (1/day) lr (%) r (1/day) lr (%) r (1/day) lr (%) Control 1.07 0.0 1.16 0.0 1.34 0.0

100 1.03 4.6 1.19 -2.6 1.37 -2.4

- % inhibition: increase in growth relative to that ofcontrolRCC Study Number848634 DEA/Acid anhydride reaction product REPORT



### Table 4: Appearance ofthe test

**me,~ium** Abbreviations: 0: no remarkable observations, cleartest medium 1: homogeneous dispersion in thewater, turbidity observable 2: noticeableturbidity caused bythe test item 3: noticeable coloration caused bythe test item 4: inhomogeneous dispersion ofthe test item 5: precipitation ofthe test item 6: test item at the surface 7: test item lying atthe bottom ofthe flask
Nominal test item Exposure time

concentration (mg/L) Oh 24 h 48 h

100 0 0 0



### Table 5: pH values in the test medium and in the control

Nominal test item pH values

concentration (mg/L) Start End Control 8.1 8.4

100 7.7 8.2



### Table 6: Watertemperature during the test period
Temperature (0C} Day 0 (Start) 22 Day 1 23 Day2 22 Day 3 (End) 23 Page22

72h

0RCC Study Number848634 DENAcid anhydride reaction product 

# 5 FIGURE REPORT



### Figure 1: Growth curves of Scenedesmus subspicatus, 

### incubated for 72 hours

100-.-----------------------------------------.

::::J E 

# -- """0

>< !!2 Qj 

## .s. >- 

# - 10 "iii c Q) "0 Qj (.) c m Q) E

1 0 24 48 72
test period (hours)

··•· Control 

### -D- 100 mg/L Page 23RCC Study Number848634 DENAcid anhydride reaction product 

# 6 REFERENCES REPORT

1 AN, S.S., FRIEDL, T., HEGEWALD, E. (1999): Page 24

Phylogenetic Relationships of Scenedesmus and Scenedesmus-like Coccoid Green Algae as Inferred from ITS-2 rONA Sequence Comparisons, Plant Biology 1, 418-428 2 HEGEWALD, E. (2000): New Combinations in the Genus Oesmodesmus (Chlorophytaceae, Scenedesmaceae) Algological Studies 96, 1-18RCC Study Number848634 DENAcid anhydride reaction product REPORT



# ATTACHMENT

ANALYTICAL PHASE REPORT Page 25

# ANALYTICAL PHASE REPORT Analytical Phase to: TOXICITY OF DEAIACID ANHYDRIDE REACTION PRODUCT TO SCENEDESMUS SUBSPICATUS IN A 72-HOUR ALGAL GROWTH INHIBITION TEST Study Director: Mr. Roger Batscher Principal InvestigatorAnalytical Phase: Dr. Tobias Schoop Completion Date oftheAnalytical Phase Report: November 19, 2004
Test Facility: RCC Ltd Environmental Chemistry &Pharmanalytics CH-4452 ltingen/Switzerland Identification: RCC Study Number: 848634 RCC Phase Number: 848635

Page 1to 34RCC Study Number 848634 ANALYTICAL PHASE REPORT DENAcid anhydride reaction product RCC Phase Number848635 GOOD LABORA TORY PRACTICE 

# STATEMENT OF COMPLIANCE

RCC Study No.: Study Director:

Analytical Phase Number: Test Item: Principal Investigatorfor Analytical Phase: Analytical Phase to: 848634 Mr. Roger

**B~itscher** RCC Ltd Environmental Chemistry 

### & Pharmanalytics 848635 DENAcid anhydride reaction product
Dr. Tobias Schaap Toxicity of DENAcid anhydride reaction product to Scenedesmus subspicatus in a 72-hour algal growth inhibition test Page 2

The analytical phase (with the exception of the pre-experiments and the standards used for identification ofthe interesting signals as mentioned in the phase report) has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 261 h, 1997 by decision ofthe OECD Council [C(97)186/Final].
Principal Investigatorfor Analytical Phase: Dr. Tobias Schaap 

# i?;Gn0/

**···················~····**..·················.. Date: 

# ;f/vvA-Gv 

# .-A"1/

**Q_(f!Y~**RCC Study Number848634 ANALYTICAL PHASE REPORT DEA/Acid anhydride reaction product RCC Phase Number 848635 

# PREFACE GENERAL Analytical Phase to:
Sponsor:

Study Monitor:

Test Facility:



## RESPONSIBILITIES Study Director: Principal Investigator for analytical phase: Technical Coordinators: Reporting: Head of RCC QualityAssurance: Toxicity of DEA/Acid anhydride reaction product to Scenedesmus subspicatus in a 72-hour algal growth inhibition test Rockwool International A/S Hovedgaden 584 DK-2640 Hedehusene Denmark Mrs. Anne Jensen (until end of May 2004) Mrs. Lea Tobiassen, DVM (from June 01, 2004) Danish Toxicology Centre Kogle Aile 2 DK-2970 H0RSHOLM Denmark RCC Ltd Environmental Chemistry 

### & Pharmanalytics Zelgliweg 1 CH-4452 ltingen/Switzerland

Mr. Roger Batscher
Dr. Tobias Schoop Mrs. Mandy Broda, Mr. Roman Klaus Dr. Tobias Schoop
Mrs. Iris Wuthrich 

## SCHEDULE OF ANALYTICAL PHASE Experimental starting date: Experimental completion date: Completion date ofthe Analytical Phase Report: June 07, 2004 June 21, 2004 November 19, 2004 Page 5