REPORT 

Study Title: 

TOXICITY OF 

DEAIACID ANHYDRIDE REACTION PRODUCT 

TO ACTIVATED SLUDGE 

IN A RESPIRATION INHIBITION TEST 

Data Requirements I Test Guidelines: 

OECD No. 209 

EU Commission Directive 88/302/EEC, C.11 

Study Director: 

Roger B~Uscher 

Study Completion Date: 

June 07, 2004 

Test Facility: 

RCC Ltd 

Environmental Chemistry & 

Pharmanalytics 

CH-4452 ltingen/Switzerland 

Sponsor: 

Rockwoollnternational A/S 

Hovedgaden 584 

DK-2640 Hedehusene 

Denmark 

RCC Study Number: 

848636 

Page 1 of 20 
RCC Study Number 848636 

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 SWl S Sm ed1 C Therapeutic Products 

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,EC~ENF,EMN,PC~ 

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 ainical Chemistry : ECT = Environmental toxicity on aquatic and terrestrial 

organisms : ENF = Behaviour In water, soil and air. Bloaccumulation ; EMN =Studies on effects on mesocosms and natural 

ecosystems: MUT: Mutagenicity; PCT: Physical-chemical testing; RES • Residue studies; OTH • ~·to be specified. 

Page2 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

GOOD LABORATORY PRACTICE 

REPORT Page 3 

STATEMENT OF COMPLIANCE 

RCC Study Number: 

Test Item: 

Study Director: 

Study Title: 

848636 

DEA/Acid anhydride reaction product 

Mr. Roger B~:itscher 

Toxicity of DEA/Acid anhydride reaction product 

to activated sludge in a respiration inhibition test 

This study 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 B~itscher 

fZ.~ .. £ .................. 

Date: 7uAu_ ()':2. 2tt:J· "- (/ -- -- r' or 
RCC Study Number 848636 

DENAcid anhydride reaction product 

SIGNATURES 

Study Director: 

Management: 

REPORT Page4 

Mr. Roger B~itscher 

Date: 

Dr. Uwe Morgenroth 

............ L ............................................. 

Date: 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

REPORT Page 5 

QUALITY ASSURANCE 

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

STATEMENT 

RCC Study Number: 

Test Item: 

Study Director: 

Study Title: 

848636 

DEA/Acid anhydride reaction product 

Mr. Roger B~Uscher 

Toxicity of DEA/Acid anhydride reaction product 

to activated sludge in a respiration inhibition test 

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

the responsible person and the management. 

Study procedures 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 

October 03, 2003 Study plan October 03, 2003 

November 25, 2003 Test system November 25, 2003 

January 16+21, 2004 Report January 21, 2004 

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

Quality Assurance: ~Mrs. Margot Richter-Auer 
RCC Study Number 848636 

DENAcid anhydride reaction product 

CONTENTS 

REPORT Page6 

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

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

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

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

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

CONTENTS CONTINUED- TABLES & FIGURES .......................................................... 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 REFERENCE ITEM ..................................................................................... 12 

2.4 TEST SYSTEM ........................................................................................... 13 

2.5 STUDY DESIGN ......................................................................................... 14 

2.5.1 Dosage and Concentrations .......................................................... 14 

2.5.2 Experimental Conditions ................................................................ 15 

2.6 EVALUATIONS ........................................................................................... 16 

2.6.1 Measurement of the Respiration Rates .......................................... 16 

2.6.2 Determination of the Inhibition Rates ............................................. 16 

2.6.3 Water Quality Criteria .................................................................... 16 

3 RESULTS AND DISCUSSION ............................................................................. 17 

3.1 TOXICITY OF DEA/ACID ANHYDRIDE REACTION PRODUCT ................. 17 

3.2 TOXICITY OF 3,5-DICHLOROPHENOL (POSITIVE CONTROL) ................ 17 

4 TABLES ............................................................................................................... 18 

5 FIGURE ................................................................................................................ 19 

6 REFERENCES ..................................................................................................... 20 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

REPORT 

CONTENTS CONTINUED- TABLES & FIGURES 

Table 1: Influence of DEA/Acid anhydride reaction product 

Page 7 

on oxygen consumption of activated sludge ................................................ 18 

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

of activated sludge ...................................................................................... 18 

Figure 1: Influence of DEA/Acid anhydride reaction product and 

3,5-dichlorophenol on the respiration rate of activated sludge ..................... 19 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

PREFACE 

GENERAL 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

RESPONSIBILITIES 

Study Director: 

Deputy Study Director: 

Technical Coordinator: 

Head of RCC Quality Assurance: 

SCHEDULE 

Experimental Starting Date: 

Experimental Completion Date: 

Study Completion Date: 

ARCHIVING 

REPORT Page 8 

Toxicity of DEA/Acid anhydride reaction product 

to activated sludge in a respiration inhibition test 

Rockwool International A/S 

Hovedgaden 584 

DK-2640 Hedehusene 

Denmark 

Mrs. Anne Jensen 

Danish Toxicology Centre 

Kogle Aile 2 

DK-2970 H0RSHOLM 

Denmark 

RCC Ltd 

Environmental Chemistry & Pharmanalytics 

Zelgliweg 1 

CH-4452 ltingen I Switzerland 

Mr. Roger B~itscher 

Dr. Birgit Seyfried 

Mrs. Renate Wetzlinger 

Mrs. Iris Wuthrich 

November 24, 2003 

November 26, 2003 

June 07, 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 848636 

DEA/Acid 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. 209, Activated Sludge, Respiration Inhibition 

Test, 1984. 

EU Commission Directive 88/302/EEC, Part C.11, Activated Sludge Respiration Inhibition 

Test, 1988. 

SUMMARY OF STUDY PLAN AMENDMENTS 

First Amendment to Study Plan 

Concerning: Alteration: Reason: 

2.5.1 Dosage and The test media were stirred for The test media were clear after 

Concentrations one hour instead of 24 hours as the stirring time of one hour. 

stated in the study plan. 
RCC Study Number 848636 

DENAcid anhydride reaction product 

SUMMARY 

REPORT Page 10 

The inhibitory effect of the test item DEA/Acid anhydride reaction product on the respiration 

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

respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, 

and the OECD Guideline for Testing of Chemicals, No. 209. 

The following nominal concentrations of DEA/Acid anhydride reaction product were tested: 

10, 32, 100, 320 and 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested. 

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

3,5-dichlorophenol {5, 16 and 50 mg/L) were tested in parallel. The results of these 

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

Up to and including the concentration of 1000 mg/L, the test item DEA/Acid anhydride 

reaction product had no significant inhibitory effect (<15%) on the respiration rate of activated 

sludge after the incubation period of three hours. The 3-hour EC20, ECSO, and EC80 could 

not be calculated, but were clearly higher than 1000 mg/L. 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

1 PURPOSE 

REPORT Page 11 

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

DEA/Acid anhydride reaction product on activated sludge microorganisms. Aerobic activated 

sludge was incubated for three hours in the presence of various concentrations of the test 

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

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

rate of two controls. 

The test method and the test system are recommended by the OECD guideline No. 209 and 

the EU Commission Directive 88/302/EEC, C.11. 

2 MATERIALS AND METHODS 

2.1 DEFINITIONS 

Respiration rate: 

ECx: 

the oxygen consumption of wastewater microorganisms in aerobic 

activated sludge, expressed as mg 0 2 per liter per minute 

the calculated concentration of test item at which the respiration rate 

is inhibited by x% compared to the respiration rate of the control 
RCC Study Number 848636 

DENAcid anhydride reaction product 

2.4 TEST SYSTEM 

REPORT Page 13 

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

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

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

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

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

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

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

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

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

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

of the activated sludge inoculum was 6.6. 

* Synthetic wastewater: 

16 g peptone 

11 g meat extract 

3 g urea 

0.7 g NaCI 

0.4 g CaCI2 · 2H20 

0.2 g MgS04 · ?H20 

2.8 g K2HP04 

filled up to a final volume of 1 liter 

with deionized water 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

2.5 STUDY DESIGN 

2.5.1 Dosage and Concentrations 

REPORT Page 14 

The final nominal concentrations tested were 10, 32, 100, 320, and 1000 mg/L. Test item 

amounts of 4.9, 16.1, 49.6, 159.4, and 500.0 mg were weighed onto glass slides and 

transferred into the respective test flask. Then, 284 mL of tap water was added to each flask. 

The test item was mixed into the tap water using ultrasonic treatment for 5 minutes and 

intense stirring for one hour at room temperature. After the stirring period of one hour, the 

test media appeared to be clear solutions. Then, 16 mL of synthetic wastewater and 200 mL 

of the activated sludge inoculum were added (see scheme below). 

In accordance with the test guidelines, one replicate was tested for each test item 

concentration. Additionally, two controls (tap water, synthetic wastewater and activated 

sludge inoculum, without test item) and the reference item 3,5-dichlorophenol (positive 

control) at nominal concentrations of 5, 16, and 50 mg/L were tested in parallel under 

otherwise identical conditions of the test. 

The chemical oxygen consumption of the test item was investigated in RCC Study 

no. 848637 (28-day ready biodegradability test). Over 28 days, no oxygen consumption was 

determined in an abiotic control containing the test item at a nominal concentration of 

100 mg/L (without activated sludge). Therefore, no abiotic control was included to measure 

the chemical oxygen consumption after the 3-hour incubation period. 

For the dosage of the reference item, a stock solution of 3,5-dichlorophenol was prepared 

according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 M 

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

0.5 moi/L H2S04 to the point of incipient precipitation. Thereafter, the mixture was made up to 

one liter with purified water. The pH was determined to be 7.6. The final concentration of 

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

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

desired concentrations (see scheme below). 

Composition of the test media: 

Flask Identification Synthetic Amount Aliquot Activated Tap Final 

No. waste- of test of stock sludge water nominal 

water item solution cone. 

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

1 Inoculum control 16 --- --- 200 284 ---

2 3,5-dichlorophenol 16 --- 5 200 279 5 

3 3,5-dichlorophenol 16 --- 16 200 268 16 

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

5 Test item 16 4.9 --- 200 284 10 

6 Test item 16 16.1 --- 200 284 32 

7 Test item 16 49.6 --- 200 284 100 

8 Test item 16 159.4 --- 200 284 320 

9 Test item 16 500.0 --- 200 284 1000 

10 Inoculum control 16 --- --- 200 284 ---

* Test item amounts were directly weighed onto glass slides and transferred directly into the test flasks 

** Stock solution of 3,5-dichlorophenol (500 mg/L) 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

2.5.2 Experimental Conditions 

REPORT Page 15 

The test was performed in 1 000-ml glass flasks. The test flasks were labeled with the 

necessary information to ensure unmistakable identification. 

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

concentration of 3.0 g/L dry weight (corresponding to about 1.2 g dry material per liter test 

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

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

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

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

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

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

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

concentrations were at least 8.0 mg/L (Tables 1 and 2). The temperature in the test media, 

measured in one control, was 20 oc at the start and at the end of the incubation period. 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

2.6 EVALUATIONS 

REPORT 

2.6.1 Measurement of the Respiration Rates 

Page 16 

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

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

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

(WTW TriOxmatic® 300 and oxygen meter WTW Oxi 539, Wissenschaftlich-Technische 

Werkstaetten WTW, Weilheim/Germany), and was continuously recorded for about ten to 

fifteen minutes. During measurement, the samples were continuously stirred on a magnetic 

stirrer. The oxygen consumption rate (in mg 0 2 L-1 minute-1

) was determined from the linear 

part of the respiration curve in the range of 6.5-2.5 mg 0 2/L. In case of very rapid oxygen 

consumption, the range used was below the limits indicated above but always within the 

linear part of the respiration curve. In case of low oxygen consumption, the rate was 

determined over a period of at least 10 minutes. 

2.6.2 Determination of the Inhibition Rates 

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

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

two controls measured at the start and at the end of the test series (flasks 1 and 1 0). 

(

1 - 2

Rs J x 1 00% =percent inhibition 

Rc1 + Rc2 

where 

Rs = oxygen consumption rate (in mg 0 2 L-1 minute-1

) at tested concentration 

Rc1 = oxygen consumption rate in control flask 1 

Rc2 = oxygen consumption rate in control flask 1 0 

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

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

The 3-hour EC50-value of the reference item 3,5-dichlorophenol was calculated by Probit 

Analysis (Ref. 1, 2). The 95%-confidence limits could not be calculated. 

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

2.6.3 Water Quality Criteria 

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

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

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

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

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

was recorded. 
RCC Study Number 848636 

DENAcid anhydride reaction product 

REPORT 

3 RESULTS AND DISCUSSION 

Page 17 

3.1 TOXICITY OF DEAIACID ANHYDRIDE REACTION PRODUCT 

The influence of DEA/Acid anhydride reaction product on the respiration rate of activated 

sludge is presented in Table 1 and Figure 1. 

Up to and including the concentration of 1000 mg/L, the test item DEA/Acid anhydride 

reaction product had no significant inhibitory effect (<15%) on the respiration rate of activated 

sludge after the incubation period of three hours (Table 1 and Figure 1). No concentration 

effect relationship was determined. Thus, the 3-hour EC20, EC50, and EC80 could not be 

calculated, but were clearly higher than 1000 mg/L. 

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

differed by 3.4% (guideline-recommended maximum variation: 15%). 

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

The results are presented in Table 2 and Figure 1. 

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

be 11 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the 

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

used. 
RCC Study Number 848636 

DEA/Acid anhydride reaction product 

4 TABLES 

REPORT Page 18 

Table 1: Influence of DEA/Acid anhydride reaction product on oxygen consumption 

of activated sludge 

Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen 

concentration consumption concentration 

of test rate (mg Oil) 

chemical 

(mg/l) (mg Oil min-1

) (%) start* end* start* end* 

1 Control 0 0.991 7.0 8.0 8.8 8.2 

10 Control 0 0.957 7.0 8.3 8.6 8.0 

Mean 0.974 

Deviation (%) 3.4 

5 Test item 10 1.105 -13.5 7.0 8.3 8.3 8.2 

6 Test item 32 0.997 -2.3 7.0 8.3 8.8 8.5 

7 Test item 100 0.945 3.0 7.0 8.2 8.2 8.5 

8 Test item 320 1.010 -3.7 7.0 8.3 8.2 8.4 

9 Test item 1000 0.944 3.1 7.0 8.0 8.7 8.2 

* Start and end of the 3-hour incubation period 

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

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

Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen 

concentration consumption concentration 

of test rate (mg Oil) 

chemical 

(mg/l) (mg Oil min-1

) (%) start* end* start* end* 

1 Control 0 0.991 7.0 8.0 8.8 8.2 

10 Control 0 0.957 7.0 8.3 8.6 8.0 

Mean 0.974 

Deviation (%) 3.4 

2 3,5-d ichlorophenol 5 0.761 21.8 7.0 8.1 8.5 8.2 

3 3,5-dichlorophenol 16 0.321 67.1 7.0 8.2 8.3 8.2 

4 3,5-dichlorophenol 50 0.082 91.6 7.0 8.1 8.1 8.1 

* Start and end of the 3-hour incubation period 
RCC Study Number 848636 

DENAcid anhydride reaction product 

5 FIGURE 

REPORT 

Figure 1: Influence of DEA/Acid anhydride reaction product and 3,5-dichlorophenol 

on the respiration rate of activated sludge 

100 

80 

.$ 60 l! 

s::::: 

0 

:;:; 

l! 

·c. 

U) 

e 40 

-

0 

s::::: 

0 

:;:; 

;e 

J: 

..E 20 

0 ~ 

-20 

c5 

. 

p·· 

.... o 

. 

--- .o--- 3,5-dichlorophenol 

-Test item 

Test concentration (mg/L) 

Page 19 
RCC Study Number 848636 

DENAcid anhydride reaction product 

6 REFERENCES 

1) FINNEY, D.J. (1971): 

Probit Analysis, 

REPORT 

3rd Edition, Cambridge University Press, London 

2) DAVIES, R.G. (1971): 

Computer Programming in Quantitative Biology, 

Academic Press, London 

Page 20