REPORT 

Study Title: 

ACUTE TOXICITY OF 

DEA/ACID ANHYDRIDE REACTION PRODUCT 

TO ZEBRA FISH (BRACHYDAN/0 RER/0) 

IN A 96-HOUR SEMI-STATIC TEST 

Data Requirements I Test Guidelines: 

OECD No. 203 

EU Commission Directive 92/69/EEC, C.1 

Study Director: 

Roger B~Hscher 

Study Completion Date: 

November 19, 2004 

Test Facility: 

RCC Ltd 

Environmental Chemistry & 

Pharmanalytics 

CH-4452 ltingen I Switzerland 

Sponsor: 

Rockwool International AIS 

Hovedgaden 584 

DK-2640 Hedehusene 

Denmark 

Identification: 

RCC Study Number: 848630 

Page 1 of 52 
RCC Study Number 848630 

DENAcid anhydride reaction product 

GLP CERTIFICATE 

REPORT 

The Swiss GLP Monitoring Authorities 

Swiss Federal 

Office of 

Public Health 

Swiss Agency for the 

Environment, Forests 

and Landscape 

./ Swissmedic 

~ • Swiss Agency for SW1ssrned1c TherapeullcProducts 

Statement of GLP Compliance 

It is hereby confirmed that 

during the period of 

the following Test 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 for the 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 of expertise * 

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. It was 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 of Public Health 

The Director 

Prof. Th. Zeltner 

• TOX =Toxicology; ACC =Analytical and Clinical Chemistry; ECT = Environmental toxicity on aquatic and terrestrial 

organisms ; ENF =Behaviour In water, soli and air. Bioaccumulation ; EMN =Studies on effects on mesocosms and natural 

ecosystems; MUT "'Mutagenicity; PCT =Physical-chemical testing; RES"' Residue studies ; OTH =Other, to be specified. 

Page2 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

GOOD LABORATORY PRACTICE 

REPORT 

STATEMENT OF COMPLIANCE 

RCC Study Number: 

Test Item: 

Study Director: 

Study Title: 

848630 

DENAcid anhydride reaction product 

Mr. Roger Bi:itscher 

Acute toxicity of 

DENAcid anhydride reaction product 

to zebra fish (Brachydanio rerio) 

in a 96-hour semi-static test 

Page3 

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 2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of 

Good Laboratory Practice, as revised in 1997 and adopted November 261

h, 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 Bi:itscher 

Date: 
RCC Study Number 848630 

DENAcid anhydride reaction product 

SIGNATURES 

Study Director: 

Management: 

REPORT Page4 

Mr. Roger B~itscher 

Date: 

Dr. Uwe Morgenroth 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

QUALITY ASSURANCE 

REPORT 

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

STATEMENT 

Study Number: 

Test Item: 

Study Director: 

Study Title: 

848630 

DENAcid anhydride reaction product 

Mr. Roger Batscher 

Acute toxicity of 

DENAcid anhydride reaction product 

to zebra fish (Brachydanio rerio) 

in a 96-hour semi-static test 

Page 5 

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. The dates are given below. 

Dates and Types of QA Inspections Dates of Reports 

to the Study Director 

and to the Management 

December 12, 2003 Study plan December 12, 2003 

May 05,2004 Process based May 05,2004 

(application, test system) 

October 8/1 0/11 , 2004 Report October 11, 2004 

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

Quality Assurance: f' Ms. Andrea GOdel 

~~.. . ...... :· .... 0..~ ......................................... . 

Date: N ~9 1 

2GO 4 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

CONTENTS 

REPORT Page6 

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

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

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

QUALITY ASSURANCE· .................................................................................................. 5 

CONTENTS .................................................................................................................... 6 

CONTENTS CONTINUED (TABLES) .............................................................................. 7 

PREFACE ....................................................................................................................... 8 

DATA REQUIREMENTS I TEST GUIDELINES ............................................................... 9 

SUMMARY OF STUDY PLAN AMENDMENTS ............................................................... 9 

SUMMARY .................................................................................................................... 10 

1 PURPOSE ............................................................................................................ 11 

2 MATERIALS AND 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 of Test Solutions and Concentrations ......................... 14 

2.6 EVALUATIONS ........................................................................................... 15 

2.6.1 Determination of the LC50, LCO, LC1 00, NOEC and 

LOEC ............................................................................................. 15 

2.6.2 Water Quality Criteria .................................................................... 15 

2.6.3 Analysis of the Test Item Concentrations ....................................... 15 

3 RESULTS AND DISCUSSION ............................................................................. 16 

4 TABLES ............................................................................................................... 17 

ATTACHMENT .............................................................................................................. 19 

ANALYTICAL PHASE REPORT (Pages 1 to 33) 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

REPORT 

CONTENTS CONTINUED (TABLES) 

Page 7 

Table 1: Mortality and visible abnormalities observed in the test fish ........................ 17 

Table 2: Appearance of the freshly prepared and old test medium ........................... 17 

Table 3: pH-values of the freshly prepared and old test medium 

and in the control. ........................................................................................ 18 

Table 4: Oxygen concentrations (mg/L) of the freshly prepared 

and old test medium and in the control. ....................................................... 18 

Table 5: Temperatures CC) of the freshly prepared and old test medium 

and in the control. ........................................................................................ 18 
RCC Study Number 848630 

DENAcid anhydride reaction product 

PREFACE 

GENERAL 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

Analytical Phase No.: 

RESPONSIBILITIES 

Study Director: 

Deputy Study Director: 

Principal Investigator for 

Analytical Phase: 

Technical Coordinator: 

Head of RCC Quality Assurance: 

SCHEDULE 

Experimental Starting Date: 

Experimental Completion Date: 

Study Completion Date: 

ARCHIVING 

REPORT 

Acute toxicity of 

DEA/Acid anhydride reaction product 

to zebra fish (Brachydanio rerio) 

in a 96-hour semi-static test 

Rockwoollnternational A/S 

Hovedgaden 584 

DK-2640 Hedehusene 

Denmark 

Mrs. Anne Jensen (until end of May 2004) 

Page 8 

Mrs. Lea Tobiassen, DVM (from June 01, 2004) 

Danish Toxicology Centre 

KogleAIIe 2 

DK-2970 H0RSHOLM 

Denmark 

RCC Ltd 

Environmental Chemistry & Pharmanalytics 

Zelgliweg 1 

CH-4452 ltingen I Switzerland 

848631 

Mr. Roger Batscher 

Dr. Armin Peither 

Dr. Tobias Schoop 

Mr. Frederic Levrechon 

Mrs. Iris Wuthrich 

May 31, 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 without the Sponsor's consent. 
RCC Study Number 848630 

DENAcid anhydride reaction product 

REPORT Page 9 

DATA REQUIREMENTS I TEST GUIDELINES 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

OECD Guideline for Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 1992 

EU Commission Directive 92/69/EEC, Part C.1: Acute Toxicity for Fish, 1992 

SUMMARY OF STUDY PLAN AMENDMENTS 

First Amendment to Study Plan 

Concerning: Alteration: Reason: 

Preface I General Change of the Study Monitor Update by the sponsor. 

during the study. 

2.2 Test Item The batch no. 200501.UN2810 Update of the study plan since 

of the test item was additionally the batch no. 200501.UN2810 

used for pre-experiments and was additionally used in the 

analytical purposes. Update of study (for pre-experiments and 

the expiration date (re-analysis analytical purposes). The test 

date). item was re-analyzed by the 

sponsor. 

2.6.3 Analyses of the The analysis of acidic The concentrations of the 

Test Item Concentrations anhydrides, trimellitic and anhydrides in the test item were 

tetrahydrophthalic anhydrides below the limit of quantification 

(method no. 4 according to the of the analytical method. 

study plan) was not performed. Therefore, a quantification of 

the concentrations of the 

anhydrides in the diluted 

aqueous test medium was not 

possible. 

RCC Study Number 848630 

DEA/Acid anhydride reaction product 

1 PURPOSE 

REPORT Page 11 

The purpose of this 96-hour toxicity test was to evaluate the acute toxicity of the test item 

DENAcid anhydride reaction product to fish. Zebra fish (Brachydanio rerio) were exposed in 

a semi-static test to an aqueous test medium containing the test item at a nominal 

concentration of 100 mg/L under defined conditions (limit test). The limit test was based on 

the results of a range-finding test and was performed in compliance with the test guidelines 

to demonstrate that the test item has no toxic effect on fish up to at least this concentration. 

The test fish were observed for mortality and visible abnormalities. 

The test method and the test species are recommended by the test guidelines of OECD and 

EU. 

2 MATERIALS AND METHODS 

2.1 DEFINITIONS 

LCO: The highest test concentration without a significant number 

of dead test organisms. 

LCSO: The median lethal concentration, i.e. the calculated 

concentration of test item which causes death in 50% of the 

test organisms. 

LC1 00: The lowest test concentration at which all test organisms are 

dead. 

NOEC (No Observed 

,!;ffect Concentration): 

LOEC (t:owest Observed 

,!;_ffect Concentration): 

The highest test concentration at which no significant toxic 

effect on the test organisms is observed. 

The lowest test concentration at which a significant toxic 

effect on the test organisms is observed. 

RCC Study Number 848630 

DEA/Acid anhydride reaction product 

2.5 STUDY DESIGN 

2.5.1 Experimental Conditions 

REPORT Page 13 

One glass aquarium with 5 liters test medium was used for each treatment (the single test 

concentration and the control). The test vessels were labeled with the RCC study number 

and all necessary additional information to ensure unmistakable identification. 

At the start of the test, 7 fish were introduced into each aquarium in a random order. The 

loading rate was 0.38 g fish wet weight per liter test medium. The test medium and the 

control were slightly aerated during the test period. The fish were not fed during the test. 

A semi-static test with daily test medium renewal was performed to keep the test item 

concentration in the test medium as constant as possible during the test period of 96 hours. 

During this semi-static test, the test fish were transferred daily into a clean test vessel with 

freshly prepared test medium of the single concentration. 

Test duration: 96 hours 

Water temperature: 21 - 22 oc during the test period (Table 5) 

Light conditions: A 16-hour light to 8-hour dark photoperiod, with a 30-minute ' 

transition period (light intensity during the light period was 

approximately within the range of 50 to 500 Lux). 

Test water: Reconstituted water: analytical grade salts were dissolved in 

deionized water to obtain the following nominal concentrations: 

CaCI2 x 2H20 2.0 mmoi/L (= 294 mg/L) 

MgS04 x 7H20 0.5 mmoi/L (= 123 mg/L) 

NaHC03 0. 75 mmoi/L (= 65 mg/L) 

KCI 0.075 mmoi/L (= 5.8 mg/L) 

Water Hardness 

Alkalinity 

Ratio of Ca : Mg 

Na:K 

= 

= 

2.5 mmoi/L (= 250 mg/L as CaC03) 

0.8 mmoi/L 

4 : 1 (based on molarity) 

1 0 : 1 (based on molarity) 

The test water was aerated prior to the preparation of the test 

medium until oxygen saturation was reached. 
RCC Study Number 848630 

DENAcid anhydride reaction product 

REPORT 

2.5.2 Preparation of Test Solutions and Concentrations 

Page 14 

A limit test was performed in accordance with the guidelines to demonstrate that the test 

item has no toxic effect on the test fish 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 of the test 

item). 

The test medium was prepared before the start of the test and prior to each test medium 

renewal by mixing 501 - 505 mg of the test item into 5000 ml test water using ultrasonic 

treatment for 15 minutes and intense stirring for 25 minutes at room temperature. 

The test medium was freshly prepared just before introduction of the fish (= start of the test) 

and just prior to the test medium renewals. 

The actual concentration of the test item (i.e. of the different components of the test item) in 

the test medium was analytically determined (see Section 2.6.3). 

The limit test was based on the results of a range-finding test and on the results of a preexperiment to determine the solubility of the test item (without GLP). However, 

concentrations above nominal 100 mg/L were not tested according to the guidelines. 


RCC Study Number 848630 

DEA/Acid anhydride reaction product 

4 TABLES 

REPORT 

Table 1: Mortality and visible abnormalities observed in the test fish 

Abbreviations of visible abnormalities for which fish were observed during the study: 

AK: 

AP: 

Strongly extended gills 

Apathy 

OB: 

SA: 

Fish mainly at the water surface 

Mucus secretion 

Page 17 

BA: 

BO: 

Distended abdomen 

Fish mainly at the bottom 

of the aquarium 

Exophthalmus 

Convulsions 

SR: 

SV: 

Fish lying on side or back on the bottom 

Strong ventilation 

TS: Tumbling during swimming 

GA: VF: Changed body color 

KR: 

Number of fish tested at the single concentration and the control: 7 

Nominal Number of affected fish* I number of dead fish 

test item visible abnormalities 

concentration 

Observation time 

(mg/L) 2% hours 24 hours 48 hours 72 hours 96 hours 

Control 010 010 010 010 0/0 

100 010 010 010 010 0/0 

LCSO > 100 mg/L 

* number of dead fish plus number of fish with visible abnormalities 

Table 2: Appearance of the freshly prepared and old test medium 

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: inhomogeneous dispersion of the test item 

5: precipitation of the test item 

6: test item at the surface 

7: test item lying at the bottom of the aquarium 

Nominal 

test item Exposure time 

concentration 

Oh 24 h 48 h 72 h 96 h 

(mg/L) new old new old new old new old 

100 0 0 0 0 0 0 0 0 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

REPORT Page 18 

Table 3: pH-values of the freshly pr~pared and old test medium and in the control 

Nominal 

test item Exposure time 

concentration 

Oh 24 h 48 h 72 h 96 h 

(mg/L) new old new old new old new old 

Control 7.7 7.8 7.8 7.9 7.8 7.8 7.8 7.8 

100 7.6 7.7 7.5 7.6 7.6 7.6 7.5 7.6 

Table 4: Oxygen concentrations (mg/L) of the freshly prepared and old test medium 

and in the control 

Nominal 

test item Exposure time 

concentration 

Oh 24 h 48 h 72 h 96 h 

(mg/L) new old new old new old new old 

Control 8.6 8.4 8.5 8.3 8.5 8.4 8.6 8.5 

100 8.7 8.4 8.4 8.7 8.5 8.5 8.3 8.6 

Table 5: Temperatures CC) of the freshly prepared and old test medium and in the control 

Nominal 

test item Exposure time 

concentration 

Oh 24 h 48 h 72 h 96 h 

(mg/L) new old new old new old new old 

Control 22 22 22 22 22 21 22 21 

100 22 22 22 22 22 21 22 21 
RCC Study Number 848630 

DEA/Acid anhydride reaction product 

REPORT 

ATTACHMENT 

ANALYTICAL PHASE REPORT 

Page 19 
ANALYTICAL PHASE REPORT 

Analytical Phase to: 

ACUTE TOXICITY OF 

DEAIACID ANHYDRIDE REACTION PRODUCT 

TO ZEBRA FISH (BRACHYDAN/0 RER/0) 

IN A 96-HOUR SEMI-STATIC TEST 

Study Director: 

Mr. Roger Batscher 

Principal Investigator Analytical Phase: 

Dr. Tobias Schoop 

Completion Date of the Analytical Phase Report: 

November 19, 2004 

Test Facility: 

RCC ltd 

Environmental Chemistry & Pharmanalytics 

CH-4452 ltingen/Switzerland 

Identification: 

RCC Study Number: 848630 

RCC Phase Number: 848631 

Page 1 to 33 
RCC Study Number 848630 ANALYTICAL PHASE REPORT 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

RCC Study No.: 

Study Director: 

Analytical Phase Number: 

Test Item: 

Principal Investigator for 

Analytical Phase: 

Analytical Phase to: 

848630 

Mr. Roger B~:ltscher 

RCC Ltd 

Environmental Chemistry & Pharmanalytics 

848631 

DENAcid anhydride reaction product 

Dr. Tobias Schoop 

Acute toxicity of 

DENAcid anhydride reaction product 

to zebra fish (Brachydanio rerio) 

in a 96-hour semi-static test 

Page2 

The analytical phase (with the exception of the pre-experiments and the standards used for 

identification of the 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 of Good 

Laboratory Practice, as revised in 1997 and adopted November 261

h, 1997 by decision of the 

OECD Council [C(97)186/Final]. 

Principal Investigator for 

Analytical Phase: Dr. Tobias Schoop 

...... ar0~tP O .. : ... ~v ...... .T} ................... . Date: A / . I Ae;. '"1Cf4t'G !VCJ VC~-.- b :;..y -.1,. L' f 
RCC Study Number 848630 ANALYTICAL PHASE REPORT Page3 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

CONTENTS 

STATEMENT OF COMPLIANCE .................................................................................... 2 

CONTENTS .................................................................................................................... 3 

PREFACE ....................................................................................................................... 5 

GENERAL ....................................................................................................................... 5 

RESPONSIBILITIES ........................................................................................................ 5 

SCHEDULE OF ANALYTICAL PHASE ........................................................................... 5 

1 PURPOSE .............................................................................................................. 6 

2 MATERIALS AND METHODS ................................................................................ 6 

2.1 TEST ITEM ............................................................................................................ 6 

2.2 ANALYTICAL PROCEDURE .................................................................................. 7 

2.2.1 Storage ....................................................................................................... 7 

2.2.2 Reagents and Solvents ............................................................................... 7 

2.2.3 Standard Solutions used for Sample Quantification .................................... 7 

2.2.4 Preparation of Spiked Test Water Samples ................................................ 7 

2.2.5 Analysis of Samples .................................................................................... 8 

2.3 MEASUREMENT CONDITIONS ............................................................................ 8 

2.3.1 Method 1 (diethanolamine) ......................................................................... 8 

2.3.2 Method 2 (dimers, trimers and polymers) .................................................... 9 

2.3.3 Method 3 (tetrahydrophthalic acid and trimellitic acid) ................................. 9 

2.4 EVALUATION OF RESULTS ............................................................................... 10 

2.4.1 DEA/Acid anhydride reaction product on the base of diethanolamine ....... 10 

2.4.2 DEA/Acid anhydride reaction product on the base of dimers, trimers 

and polymers ............................................................................................ 11 

2.4.3 DEA/Acid anhydride reaction product on the base of 

tetrahydrophthalic acid .............................................................................. 12 

2.4.4 DEA/Acid anhydride reaction product on the base of trimellitic acid .......... 13 

3 RESULTS AND DISCUSSION ............................................................................. 14 

3.1 METHOD 1 (DIETHANOLAMINE) ........................................................................ 14 

3.2 METHOD 2 (DIMERS/TRIMERS AND POLYMERS) ............................................ 14 

3.3 METHOD 3 (TETRAHYDROPHTHALIC ACID AND TRIMELLITIC ACID) ............ 15 

4 TABLES ............................................................................................................... 17 

Table 1: Example of calibration data of test item standards for diethanolamine ........ 17 

Table 2: Calibration data of test item standards for dimers, trimers and 

polymers ..................................................................................................... 18 

Table 3: Example of calibration data of test item standards for 

tetrahydrophthalic acid ( cis-4-cyclohexene-1 ,2-dicarbonic acid) .................. 19 

Table 4: Example of calibration data of test item standards for trimellitic acid 

(1 ,2,4-Benzenetricarboxylic acid) ................................................................ 20 

Table 5: Results obtained for the concentrations of the test item in the test 

medium on the base of diethanolamine ....................................................... 21 
RCC Study Number 848630 ANALYTICAL PHASE REPORT 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

Table 6: Results obtained for the concentrations of the test item in the test 

medium on the base of dilners, trimers and polymers ................................. 22 

Table 7: Results obtained for the concentrations of the test item in the test 

medium on the base of tetrahydrophthalic acid (cis-4-cyclohexene-1 ,2-

dicarbonic acid) ........................................................................................... 23 

Table 8: Results obtained for the concentrations of the test item in the test 

medium on the base of trimellitic acid (1 ,2,4-Benzenetricarboxylic 

acid) ............................................................................................................ 24 

5 FIGURES ............................................................................................................. 25 

5.1 Method 1 (diethanolamine) ....................................................................... 25 

Figure 1: HPLC-chromatogram of standard solution (low-level) ..................................... 25 

Figure 2: HPLC-chromatogram of standard solution (high-level) ................................... 25 

Figure 3: HPLC-chromatogram of biological control sample .......................................... 26 

Figure 4: HPLC-chromatogram of spiked test water sample .......................................... 26 

Page4 

Figure 5: HPLC-chromatogram of non-aged treatment sample ..................................... 27 

Figure 6: HPLC-chromatogram of aged treatment sample ............................................ 27 

5.2 Method 2 (Dimers/trimers and Polymers) .................................................. 28 

Figure 7: HPLC-chromatogram of standard solution (low-level) ..................................... 28 

Figure 8: HPLC-chromatogram of standard solution (high-level) ................................... 28 

Figure 9: HPLC-chromatogram of biological control sample .......................................... 29 

Figure 10: HPLC-chromatogram of spiked test water sample ........................................ 29 

Figure 11: HPLC-chromatogram of non-aged treatment sample ................................... 30 

Figure 12: HPLC-chromatogram of aged treatment sample .......................................... 30 

5.3 Method 3 (tetrahydrophthalic acid and trimellitic acid) ............................... 31 

Figure 13: HPLC-chromatogram of standard solution (low/mid-level) ............................ 31 

Figure 14: HPLC-chromatogram of standard solution (high-level) ................................. 31 

Figure 15: HPLC-chromatogram of biological control sample ........................................ 32 

Figure 16: HPLC-chromatogram of spiked test water sample ........................................ 32 

Figure 17: HPLC-chromatogram of non-aged treatment sample ................................... 33 

Figure 18: HPLC-chromatogram of aged treatment sample .......................................... 33 
RCC Study Number 848630 ANALYTICAL PHASE REPORT 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

PREFACE 

GENERAL 

Analytical Phase to: 

Sponsor: 

Study Monitor: 

Test Facility: 

RESPONSIBILITIES 

Study Director: 

Principal Investigator 

for analytical phase: 

Technical Coordinator: 

Reporting: 

Head of RCC 

Quality Assurance: 

Acute toxicity of 

DENAcid anhydride reaction product 

to zebra fish (Brachydanio rerio) 

in a 96-hour semi-static test 

Rockwoollnternational NS 

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 

Dr. Tobias Schoop 

Mrs. Iris Wuthrich 

SCHEDULE OF ANALYTICAL PHASE 

Experimental starting date: 

Experimental completion date: 

Completion date of the Analytical Phase Report: 

June 03, 2004 

June 21, 2004 

November 19, 2004 

Page 5 

RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 7 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

2.2 ANALYTICAL PROCEDURE 

2.2.1 Storage 

Analysis of diethanolamine (method 1) 

The samples were analysed directly after the sampling procedure. 

Analysis of dimers, trimers and polymers (method 2) 

The samples were stored deep-frozen and protected from light until analysis was performed. 

The stability of the frozen samples was tested during non-GLP pre-experiments. 

Analysis of tetrahydrophthalic acid and trimellitic acid (method 3) 

The samples were analysed directly after the sampling procedure. 

2.2.2 Reagents and Solvents 

Acetonitrile (purity> 99.8 %) 

Methanol (purity> 99.8 %) 

Nitric acid (purity 65%) 

Ammoniumdihydrogenphosphate 

(purity> 99 %) 

Purified water for HPLC 

Test water 

J.T.Baker, no. 9017 

J.T.Baker, no. 8402 

J.T.Baker, no. 6080 

Fluka, no. 09717 

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 

Typical example: 

The test item (109.46 mg) was dissolved in methanol and made up to the mark in a 50 ml 

volumetric flask to prepare a stock solution of 2189.2 mg/L. Defined volumes of this stock 

solution were diluted with methanol/water 1 :9 (method 2 & 3) respectively methanol/test water 

1:9 (method 1) to obtain standard solutions of the test item in the range of 10.9 to 219 mg/L. 

The total calibration range covered by these solutions was used for the calibration of all active 

ingredients using the res~ective methods. 

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. 

Typical example: 

The test item (507.2 mg) was dissolved in methanol and made up to the mark in a 50 ml 

volumetric flask to prepare a stock solution of 10144 mg/L. 0.1 ml of this stock solution were 

diluted with test water to obtain spiked samples with a concentration of 101.44 mg/L. These 

solutions were freshly prepared and subjected to the same treatment as a sample without any 

storage. 
RCC Study Number 848630 ANALYTICAL PHASE REPORT PageS 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

In addition, test water without the test item was analyzed (analytical blank). 

2.2.5 Analysis of Samples 

Analysis of diethanolamine (method 1 ), tetrahydrophthalic acid and trimellitic acid 

(method 3) 

The samples were directly analysed without any storage. 900 IJL aliquots of the samples were 

diluted with 100 IJL methanol prior to analysis (dilution factor 1.111). 

Analysis of dimers, trimers and polymers (method 2) 

Treatment samples and control samples were thawed at room temperature for one hour and 

shaken manually to obtain homogeneous sample solutions. 900 !JL aliquots of the samples 

were diluted with 100 !JL methanol prior to analysis (dilution factor 1.111). 

Aliquots of the final solutions were analyzed by the respective methods. 

The results obtained are presented in Table 5 (diethanolamine), Table 6 (dimers, trimers and 

polymers), Table 7 (tetrahydrophthalic acid) and Table 8 (trimellitic acid). 

2.3 MEASUREMENT CONDITIONS 

2.3.1 Method 1 (diethanolamine) 

Auto sampler: 

Pump: 

Detector: 

Column: 

Eluent 

Flow rate: 

Run time: 

Temperature: 

Injection volume: 

Detection: 

Retention time: 

Merck-Hitachi L-7200 

Merck-Hitachi L-6200 

732 IC Detector 

Metrosep Cation 1-2; 125 x 4 mm; 7 1-1m 

2 mM HN03 (w = 65%) /1 L water (milli-Q) 

1 mUminute 

15 minutes 

Room temperature 

electrochemical 

approximately 6 minutes 
RCC Study Number 848630 ANALYTICAL PHASE REPORT 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

2.3.2 Method 2 (dimers, trime~~ and polymers) 

Auto sampler: 

Pump: 

Detector: 

Column oven: 

Column: 

Eluent: 

Flow rate: 

Run time: 

Temperature: 

Injection volume: 

Merck-Hitachi L-7200 

Merck-Hitachi L-7100 

Merck-Hitachi DAD L-7400 

Merck-Hitachi L-7300 

Superdex Peptide 1 0/300 GL 

methanol/ 0.035 M NaCI10/90 (v:v) 

1 mUminute 

25 minutes 

40°C in a thermostated oven 

100 IJL 

Detection wave length: 210 nm 

Retention time: approximately 11-18 minutes 

2.3.3 Method 3 (tetrahydrophthalic acid and trimellitic acid) 

Auto sampler: 

Pump: 

Detector: 

Column: 

Pre-Column: 

Varian 9095 

Varian ProStar 230 

Varian 9050 

Phenomenex Luna C8 ; 150 X 4.6 mm; 3 IJm 

Phenomenex C8 ; 4 X 3 mm 

Page9 

Eluent: 0.1 M NH4HzP04/ acetonitrile 9 + 1, adjusted to pH 2.5 with H3P04 

Flow rate: 0.8 mUminute 

Run time: 30 minutes 

Temperature: Room temperature 

Injection volume: 100 IJL 

Detection wave length: 21 0 nm 

Retention time: -6.5 minutes (Trimellitic acid) 

-25 minutes (Tetrahydrophthalic acid) 
RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 10 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

2.4 EVALUATION OF RESULTS 

2.4.1 DEAl Acid anhydride reaction product on the base of diethanolamine 

Injected samples were quantified by peak areas with reference to the respective calibration 

curve. The latter was obtained by correlation of the 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). The results obtained are presented in Table 1. 

From the calibration curve the concentration x of the test item in an injected sample was 

calculated by the following equation: 

x={f (1) 

where 

y = Peak area of the test item in injected sample [counts] 

x = Concentration of the test item in injected sample [mg/L] 

a, b = constants 

The concentration of the test item in a sample was calculated by equation 2: 

c=x·F (2) 

where 

c = Concentration of the test item in the sample [mg/L] 

x = Amount of the test item in injected sample found by equation 1 [mg/L] 

F = Factor 

The recovery of the test item in a sample was calculated by equation 3: 

R=-c- · 100% 

Cnom 

where 

R = Recovery [%] 

(3) 

c = Concentration of the test item in the sample found by equation 2 [mg/L] 

Cnom = Nominal concentration of the test item in the sample [mg/L] 
RCC Study Number 848630 ANALYTICAL PHASE REPORT Page 11 

DEA/Acid anhydride reaction product 

RCC Phase Number 848631 

2.4.2 DEAl Acid anhydride reaction product on the base of dimers, trimers and 

polymers 

Injected samples were quantified by peak areas with reference to the respective calibration 

curve. The latter was obtained by correlation of the peak area of the standard solutions to 

their corresponding concentration in mg/L. The correlation was performed using a linear 

function given below (equation 4). The results obtained are presented in Table 2. 

From the calibration curve the concentration x of the test item in an injected sample was 

calculated by the following equation: 

y-a X=--

b (4) 

where 

y = Peak area of the test item in injected sample [counts] 

x = Concentration of the test item in injected sample [mg/L] 

a = y-axis intercept 

b = Slope 

The concentration of the test item in a sample was calculated by equation 2: 

c=x·F (5) 

where 

c = Concentration of the test item in the sample [mg/L] 

x = Amount of the test item in injected sample found by equation 4 [mg/L] 

F = Factor 

The recovery of the test item in a sample was calculated by equation 6: 

R=-c- · 100% 

Cnom 

where 

R = Recovery[%] 

(6) 

c = Concentration of the test item in the sample found by equation 5 [mg/L] 

Cnom = Nominal concentration of the test item in the sample [mg/L]