# REPORT
StudyTitle: TOXICITYOF DEAIACID ANHYDRIDE REACTION PRODUCT TO ACTIVATED SLUDGE IN A RESPIRATION INHIBITION TEST Data Requirements ITestGuidelines: OECD No. 209 EU Commission Directive 88/302/EEC, C.11 Study Director: Roger

**B~Uscher** StudyCompletion Date: June 07, 2004 Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen/Switzerland Sponsor: RockwoollnternationalA/S Hovedgaden 584 DK-2640 Hedehusene Denmark RCC Study Number: 848636 Page 1 of20RCC Study Number848636 DENAcid anhydride reaction product 

# GLP CERTIFICATE REPORT

The Swiss GLP Monitoring Authorities Swiss Federal Office of PublicHealth SwissAgencyforthe Environment, Forests and Landscape **~** Swissmedic ' • Swiss Agencyfor SWlSSm

### ed1C Therapeutic Products 

# Statement of GLP Compliance

Itis 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 Agencyforthe Environment, Forests and Landscape with respect to the compliance with the Swiss legislation on Good Laboratory Practice. Test Facilities

- Toxicology -Environmental Chemistryand

Pharmanalytics Areas ofexpertise* 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. Itwas found thatthe aforementioned testfacilities were operating in compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the time theywere inspected.

Bern, March 2003 Federal Office of Public Health The Director

Prof.Th. Zeltner

• TOX =Toxicology :ACC =Analyticaland ainicalChemistry : ECT=Environmental toxicityon aquaticand terrestrial organisms : ENF=BehaviourIn water, soil and air. Bloaccumulation ; EMN =Studieson effects on mesocosmsand natural ecosystems: MUT:Mutagenicity; PCT:Physical-chemicaltesting; RES • Residue studies;OTH•

**~·to**bespecified. Page2RCC Study Number848636 DEA/Acid anhydride reaction product 

### GOOD LABORATORY PRACTICE REPORT Page 3



# STATEMENT OF COMPLIANCE

RCC Study Number: Test Item: Study Director: StudyTitle: 848636 DEA/Acid anhydride reaction product Mr. Roger

**B~:itscher** Toxicity ofDEA/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 November261 h, 1997 by decision ofthe OECD Council [C(97) 186/Final]. There were no circumstances that may have affected the quality or integrity ofthe data.
Study Director: Mr. Roger

**B~itscher**



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Date: 

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# SIGNATURES

Study Director:

Management: REPORT Page4

Mr. Roger

**B~itscher**

Date:

Dr. Uwe Morgenroth



# ............L
............................................. Date:RCC Study Number848636 DEA/Acid anhydride reaction product REPORT Page 5 

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

### I Switzerland STATEMENT RCC Study Number: Test Item: Study Director: StudyTitle: 848636 DEA/Acid anhydride reaction product Mr. Roger

**B~Uscher** Toxicity ofDEA/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 bythe QualityAssurance. The dates are given below:
Dates and Types ofQA Inspections Dates ofReports to the Study Directorand tothe Management October 03, 2003 Study plan October03, 2003 November25, 2003 Test system November25, 2003 January 16+21, 2004 Report January21, 2004 This statement also confirms thatthis final report reflects the rawdata. QualityAssurance:



# ~Mrs. Margot Richter-AuerRCC Study Number848636 DENAcid anhydride reaction product 

# CONTENTS REPORT Page6

GLP CERTIFICATE.........................................................................................................2 STATEMENT OF COMPLIANCE ....................................................................................3 SIGNATURES .................................................................................................................4 QUALITYASSURANCE ..................................................................................................5 CONTENTS.....................................................................................................................6 CONTENTS CONTINUED- TABLES & FIGURES..........................................................7 PREFACE .......................................................................................................................8 DATA REQUIREMENTS ITEST 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 ofthe Respiration Rates..........................................16 2.6.2 Determination ofthe Inhibition Rates .............................................16 2.6.3 WaterQuality Criteria ....................................................................16 3 RESULTS AND DISCUSSION .............................................................................17 3.1 TOXICITY OF DEA/ACIDANHYDRIDE REACTION PRODUCT
.................17 3.2 TOXICITY OF 3,5-DICHLOROPHENOL (POSITIVE CONTROL)
................17 4 TABLES ...............................................................................................................18 5 FIGURE................................................................................................................19 6 REFERENCES.....................................................................................................20RCC Study Number848636 DEA/Acid anhydride reaction product REPORT 

# CONTENTS CONTINUED- TABLES 

# &FIGURES

Table 1: Influence ofDEA/Acid anhydride reaction product Page 7

on oxygen consumption ofactivated sludge ................................................18 Table 2: Influence of3,5-dichlorophenol on oxygen consumption ofactivated sludge ......................................................................................18

Figure 1: Influence ofDEA/Acid anhydride reaction product and 3,5-dichlorophenol on the respiration rate ofactivated sludge .....................19RCC Study Number848636 DEA/Acid anhydride reaction product 

# PREFACE
GENERAL StudyTitle: Sponsor:
Study Monitor:

Test Facility:



### RESPONSIBILITIES Study Director: Deputy Study Director: 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 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. RenateWetzlinger Mrs. Iris Wuthrich

November24, 2003 November26, 2003 June 07, 2004

RCC Ltd, CH-4452 ltingen, Switzerland will retain the study plan, raw data, a sample ofthe test item and the final report ofthe present studyfor at leastten years. No data will be discarded withoutthe Sponsor's consent.RCC Study Number848636 DEA/Acid anhydride reaction product REPORT Page 9 

# DATA REQUIREMENTS 

# ITEST 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

FirstAmendmentto Study Plan Concerning: Alteration: Reason: 2.5.1 Dosage and The test media were stirred for The test media were clearafter Concentrations one hour instead of24 hours as the stirring time ofone hour. stated in the study plan.RCC Study Number848636 DENAcid anhydride reaction product 

# SUMMARY REPORT Page 10

The inhibitory effect ofthe 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 forTesting ofChemicals, 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 nottested. 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 ofthe 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 ofactivated sludge after the incubation period ofthree hours. The 3-hour EC20, ECSO, and EC80 could not be calculated, butwere clearly higherthan 1000 mg/L.RCC Study Number848636 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 ofthe test item was expressed as a percentage ofthe mean respiration rate oftwo 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 oftest item at which the respiration rate is inhibited by x% compared to the respiration rate ofthe controlRCC Study Number848636 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, thereafterdried and the ratio ofwetto dryweightwas 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 oftwo 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 ofthe activated sludge was measured again in the inoculum used in the test. The pH ofthe activated sludge inoculum was 6.6. * Syntheticwastewater: 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 afinal volume of 1 liter with deionized waterRCC Study Number848636 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 testflask. Then, 284 mL oftap waterwas 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 ofsynthetic wastewater and 200 mL ofthe 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 ofthe 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 afterthe 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 ofincipient 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 ofthis 3,5-dichlorophenol stock solution were mixed with syntheticwastewater and tap water in the respective testflasks to obtain the desired concentrations (see scheme below). Composition ofthe test media:

Flask Identification Synthetic Amount Aliquot Activated Tap Final

No. waste- oftest ofstock 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 amountswere directlyweighed onto glass slides and transferred directly into the testflasks ** Stock solution of3,5-dichlorophenol (500 mg/L)RCC Study Number848636 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 ofthe reference item, then to the test solutions ofthe 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 aflow ofapproximately one liter per minute. The concentration ofdissolved 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 atthe start and at the end ofthe incubation period.RCC Study Number848636 DEA/Acid anhydride reaction product 

## 2.6 EVALUATIONS REPORT



### 2.6.1 Measurement ofthe 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-1minute-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 ofat least 10 minutes.


### 2.6.2 Determination ofthe Inhibition Rates The inhibitory effect ofthe test item or ofthe 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 atthe start and at the end ofthe test series (flasks 1and 1 0). (1 - 2Rs 

# J x 1 00% =percent inhibition Rc1 

### +Rc2 where Rs 

# = oxygen consumption rate (in mg 

### 0 2 L-1minute-1 ) attested 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 ofatoxic 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 ofthe test item.


### 2.6.3 WaterQuality 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 ofthe 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 Number848636 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 1and 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 ofactivated 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 higherthan 1000 mg/L. The oxygen consumption rates ofthe two controls (run at the start or at the end ofthe 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 ofthe 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 of5-30 mg/L, confirming the suitability ofthe activated sludge used.RCC Study Number848636 DEA/Acid anhydride reaction product 

# 4 TABLES REPORT Page 18

Table 1: Influence ofDEA/Acid anhydride reaction product on oxygen consumption ofactivated sludge
Flask Test chemical Nominal Oxygen Inhibition pH values Oxygen concentration consumption concentration oftest 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 ofthe 3-hour incubation period -%inhibition: increased oxygen consumption rate relative to that ofcontrol

Table 2: Influence of3,5-dichlorophenol on oxygen consumption ofactivated sludge

Flask Testchemical Nominal Oxygen Inhibition pH values Oxygen concentration consumption concentration oftest 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-dichlorophenol 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 ofthe 3-hour incubation periodRCC Study Number848636 DENAcid anhydride reaction product 

# 5 FIGURE REPORT

Figure 1: Influence ofDEA/Acid anhydride reaction product and 3,5-dichlorophenol on the respiration rate ofactivated sludge

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

Testconcentration (mg/L) Page 19RCC Study Number848636 DENAcid anhydride reaction product 

# 6 REFERENCES
1) FINNEY, D.J. (1971): ProbitAnalysis, REPORT

3rd Edition, Cambridge University Press, London 2) DAVIES, R.G. (1971): Computer Programming in Quantitative Biology, Academic Press, London Page 20