# REPORT
StudyTitle: READY BIODEGRADABILITYOF DEAIACIDANHYDRIDE REACTION PRODUCT IN A MANOMETRIC RESPIROMETRYTEST Data Requirements ITestGuidelines: OECD No. 301 F EU Commission Directive 92/69 EEC, C.4-D Study Director: Roger

**B~ltscher** StudyCompletion Date: February 17, 2004 Test Facility: RCC Ltd Environmental Chemistry & Pharmanalytics CH-4452 ltingen I Switzerland Sponsor: Rockwool InternationalAIS Hovedgaden 584 DK-2640 Hedehusene Denmark RCC Study Number: 848637 Page 1 of23RCC Study Number848637 DEA/Acid anhydride reaction product 

# GLP CERTIFICATE REPORT

The Swiss GLP Monitoring Authorities
Swiss Federal Officeof PublicHealth SwissAgencyforthe Swissmedlc Environment. Forests / • SwissAgencyfor and Landscape 5WlSSm

## edtC TherapeuticProducts 

# 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 PubJic Health. the Swjss Agency for Therapeutic Products and the Swiss Agency forthe Environment, Forests and Landscape with respectto the compliancewith the Swiss legislation on Good Laboratory Practice. TestFacilities -Toxicology

- Environmental Chemistryand

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 compliancewith the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at thetimetheywere inspected.

Bern, March 2003 Federal Office of Public Health The Director

Prof. Th. Zeltner

•TOX =Toxicology :ACC =Analyticaland Clinical Chemistry: ECT

### = Environmentaltoxlc:ityon aquaticand terreslrlal organisms ; ENF =BehaviourIn water, soil and air. Bioaccumulation ; EMN • Studieson effects on mesocosmsandnatural ecosystems: MUT =Mutagenicity. PCT=Physic:al-chemical testing; RES • Residue stucfJeS ;OTH •

**~·** tobespecified. Page2RCC Study Number848637 DENAcid anhydride reaction product
GOOD LABORATORY PRACTICE REPORT Page 3



# STATEMENT OF COMPLIANCE

RCC Study Number: Test Item: Study Director: Study Title: 848637 DEA/Acid anhydride reaction product Mr. Roger

**B~itscher** Ready Biodegradability of DEA/Acid anhydride reaction product in a Manometric Respirometry Test

This study (with the exception ofthe determination ofthe COD ofthe test item as mentioned in the 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 26th, 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**

Date:RCC Study Number848637 DENAcid anhydride reaction product 

# SIGNATURES

Study Director:

Management: REPORT Page4

Mr. Roger

**B~Uscher**

Date:

Dr. Uwe MorgenrothRCC Study Number848637 DENAcid anhydride reaction product REPORT 

# QUALITYASSURANCE

RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 ltingen I Switzerland
STATEMENT
RCC Study Number: 848637 Test Item: DEA/Acid anhydride reaction product Study Director: Mr. Roger

**B~itscher** Page 5

Study Title: Ready Biodegradability of DEA/Acid anhydride reaction product in a Manometric Respirometry Test The general facilities and activities are inspected periodically and the results are reported to / the responsible person and the management. Study procedures (with the exception of the determination of the COD of the test item as mentioned in the report) were periodically inspected. The study plan and this report were audited by the QualityAssurance. The dates are given below.
Dates and Types ofQA Inspections Dates of Reports to the Study Directorand to the Management July 31, 2003 Study plan July 31, 2003 November27, 2003 Process based November27, 2003 (test item application) January 27, 2004 Report January 27, 2004 This statement also confirms that this final report reflects the raw data.
QualityAssurance: Mrs. Ursula Burri 

# (!.

**lfu_~·** Date:RCC Study Number848637 DEA/Acid anhydride reaction product 

# CONTENTS REPORT Page6

GLP CERTIFICATE.........................................................................................................2 STATEMENTOF COMPLIANCE ....................................................................................3 SIGNATURES.................................................................................................................4 QUALITYASSURANCE..................................................................................................5 CONTENTS.....................................................................................................................6 CONTENTS CONT'D (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..............................................................................12 2.1 TEST ITEM .................................................................................................12 2.2 REFERENCE ITEM.....................................................................................12 2.3 TEST SYSTEM ...........................................................................................13 2.4 TEST PROCEDURES.................................................................................13 2.4.1 TestWater.....................................................................................13 2.4.2 Test Concentrations.......................................................................14 2.4.3 Conditions ofthe Test....................................................................15 2.5 CALCULATIONS.........................................................................................16 2.6 DETERMINATION OFTHE CHEMICAL OXYGEN DEMAND .....................16 3 RESULTS AND DISCUSSION .............................................................................17 3.1 BIODEGRADATION OFTHE TEST ITEM...................................................17 3.2 BIODEGRADATION OF THE REFERENCE ITEM......................................17 3.3 BIODEGRADATION IN THE TOXICITY CONTROL....................... u ...........17 3.4 MEASUREMENTOF pH .............................................................................18 4 TABLES ...............................................................................................................19 5 FIGURES .............................................................................................................22RCC Study Number848637 DEA/Acid anhydride reaction product REPORT 

# CONTENTS CONT'D (TABLES & FIGURES)

Table 1: Cumulative biochemical oxygen demand (BOD in mg 02/L) Page 7

in the testflasks ..........................................................................................19 Table 2: Biodegradation of DEA/Acid anhydride reaction product and the reference item ................................................................................20 Table 3: pH values atthe start and atthe end ofthetest ..........................................21

Figure 1: Cumulative biochemical oxygen demand in thetestflasks during the incubation period ........................................................................22 Figure 2: Biodegradation of DEA/Acid anhydride reaction product and the reference item ................................................................................23RCC Study Number848637 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 PageS

Ready Biodegradability ofDEA/Acid anhydride reaction product in a Manometric RespirometryTest Rockwool International A/S Hovedgaden 584 DK-2640 Hedehusene Denmark Mrs. Anne Jensen Danish Toxicology Centre KogleAIIe 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 Wetzlinge·r Mrs. IrisWUthrich

November 10, 2003 December 11, 2003 February 17, 2004

RCC Ltd, CH-4452 ltingen, Switzerland will retain the study plan, raw data, a sample of the test item and the final report ofthe present studyfor at leastten years. No datawill be discarded withoutthe Sponsor's consent.RCC Study Number848637 DEA/Acid anhydride reaction product REPORT 

# DATA REQUIREMENTS I

# TEST GUIDELINES Page9

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of Chemicals No. 301 F, Ready Biodegradability: Manometric Respirometry Test, 1992. EU Commission Directive 92/69 EEC, C.4-D, Manometric Respirometry Test, 1992.



# SUMMARY OF STUDY PLAN AMENDMENTS

FirstAmendmentto Study Plan Concerning: Alteration: Reason: Preface I Schedule Update ofthe schedule. Rescheduling ofthe study. 2.1 Test Item Update ofexpiration date and A newtest item batch was tested batch number. atthe request ofthe sponsor.RCC Study Number848637 DEA/Acid anhydride reaction product 

# SUMMARY REPORT Page 10

The test item DENAcid anhydride reaction product was investigated for its ready 

### biodegradability in a manometric respirometry test over 28 days based on EU Commission Directive 92/69 EEC, C.4-D (1992) and OECD Guideline for Testing of Chemicals No. 301 F (1992). The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of 1.39 mg 

### 0 2 

### /mg test item. The biochemical oxygen demand (BOD) of DEA/Acid anhydride reaction product in the test media significantly increased from Day 4 until test termination after28 days. At the end ofthe 28-day incubation period, the mean biodegradation of DEA/Acid anhydride reaction product amounted to 53%. Consequently, DEA/Acid anhydride reaction product was found to be partly biodegradable under the conditions of the test within 28 days. However, the pass level for ready biodegradability (biodegradation of at least 60% of the COD in a 1 0-day window within the 28-day period ofthe test) was not reached. In the toxicity control, containing both DEA/Acid anhydride reaction product and the reference item sodium benzoate, no inhibition of the biodegradation of the reference item was determined. Thus, DEA/Acid anhydride reaction product had obviously no inhibitory effect on the activity ofactivated sludge microorganisms. In the procedure controls, the reference item sodium benzoate degraded by an average of 86% until exposure Day 14 and reached an average biodegradation of 92% by the end of the test (Day 28), thus confirming suitability ofthe activated sludge.RCC Study Number848637 DEA/Acid anhydride reaction product 

# 1 PURPOSE REPORT Page 11

The purpose ofthis studywas to determinethe ready biodegradability of DEA/Acid anhydride reaction product in a 28-day biodegradation test by following the BOD (biochemical oxygen demand) ofthe test item underdefined conditions by means ofmanometric methods. This test design was selected according to the guidelines since the test item contains poorly watersoluble components.RCC Study Number848637 DEA/Acid anhydride reaction product 

## 2.3 TEST SYSTEM REPORT Page 13

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, 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 ofwet to dryweight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water (see Section 2.4.1) to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). The diluted activated sludge was used as inoculum to give afinal concentration of30 mg dry material per liter.



## 2.4 TEST PROCEDURES
2.4.1 TestWater The test water was prepared according to testing guidelines. Analytical grade salts were dissolved in purified waterto obtain the following stock solutions: a) ·KH2P04 K2HP04 Na2HP04 x 2H20 NH4CI 8.50 g/L 21.75 g/L 33.40 g/L

0.50 g/L The pH ofthis solution was 7.4. 22.50 g/L 36.40 g/L

0.25 g/L, stabilized with one drop ofconcentrated HCI per liter To obtain the final test water, 10 ml of stock solution a) and 1 ml each of stock solutions b)- d) were combined and made up to 1000 mlwith purified water. The pH ofthe test water was adjusted from 7.8 to 7.4with a diluted hydrochloric acid solution.RCC Study Number848637 DEA/Acid anhydride reaction product 

### 2.4.2 Test Concentrations REPORT Page 14

The amounts oftest item were directlyweighed into the testflasks. No emulsifiers orsolvents were used but ultrasound dispersion was employed forten minutes to obtain a suspension of the test item as homogeneous as possible. For the dosage of the reference item, a stock solution containing 251 mg sodium benzoate per 100 ml oftest water was prepared. From this, aliquots of 10 mlwere added to the corresponding test flasks. To each flask (with the exception of the abiotic control) activated sludge was added (see Section 2.3). Finally, the flasks were made up to avolume of250 mlwith testwater. The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. Concentrations of test and reference item and corresponding oxygen demands in the test flasks:

2 3 Identification Flask Amount oftest item4 No. mg/L COD1 Test item 5 101 140 Test item 6 100 140 Inoculum control 

### 9 

## --- n.a. Inoculum control 10 

## --- n.a. Procedure control 11 

# --- n.a. Procedure control 12 

## --- n.a. Abiotic control3 7 100 138 Toxicity control 

### 8 101 140 Chemical oxygen demand (COD) in mg 0 2/L test solution. Theoretical oxygen demand (ThOD) in mg 0 2/L testsolution Poisoned with mercury dichloride at a concentration of 10 mg/L Amount of reference item4 mg/L ThOD2



## --- n.a.



## --- n.a.



## --- n.a.



## --- n.a. 100 167 100 167



## --- n.a. 100 167

4 The tabulated values represent rounded results obtained by calculation using the exact raw data. n.a.: notapplicableRCC Study Number848637 DEA/Acid anhydride reaction product 

### 2.4.3 Conditions oftheTest REPORT Page 15



### Apparatus: The test flasks (500-ml Erlenmeyer flasks, labeled with all necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT 012 

### (Veith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least each working day. Principle: Electro-chemical analysis process: The biodegradation process consumes the dissolved oxygen in the liquid and generates 

### C02. 

### The 

### C02 

### is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a coppersulfate solution. Test duration: 28 days Light regimen: Darkness 

### Test temperature: 22

# oc. maintained with a built-in thermostat and checked once perweek. pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum (Table 3). At the end of incubation, the pH was measured again in each testflask.RCC Study Number848637 DENAcid anhydride reaction product 

## 2.5 CALCULATIONS REPORT Page 16

Thetabulated values represent rounded results obtained by calculation using the exact raw data. The percentage biodegradation was calculated according to theformula given below:
% biodegradation 

### = BOD (mg0 2 /mg chemical) x 1

### OO% CODand/orThOD(mg 0 2 /mgchemical) where: BOD 

### = Biochemical oxygen demand ofthe test or reference item



### = (mg 0 2uptake/L bytestand/orreferenceitem)- (mg0 2uptake/L by inoculum control) mgtestand/or referenceitemperliter· COD 

### = Chemical oxygen demand ofthe test item (since the ThOD ofthe test item could not be calculated; see Section 2.6) ThOD 

# =Theoretical oxygen demand ofthe reference item Thetheoretical oxygen demand is the total amount ofoxygen required to oxidize a chemical completely. It is calculated from the molecular formula assuming the turnover of H into H20, C into C02, Pinto P20s, Na into Na20, halogens into their corresponding hydrogen halides and N into NH4 and/or N03 according to EU Commission Directive 92/69 EEC C.4-D.



## 2.6 DETERMINATION OF THE CHEMICAL OXYGEN DEMAND Due to the complex composition of the test item, its chemical oxygen demand (COD) was determined according to the EU Commission Directive 92/69/EEC, C.6 following DIN 38414-S9. The determination ofthe CODwas not performed in compliance with the GLP regulations and is, therefore, excluded from the Statement of Compliance

**~in** this report. However, the determination of the COD was performed in compliance with ISO 17025. The results will be archived underthis RCC study number.RCC Study Number848637 DEA/Acid anhydride reaction product REPORT 

# 3 RESULTS AND DISCUSSION 3.1 BIODEGRADATION OF THE TEST ITEM The results are presented in Tables 1and 2, and in Figures 1and 2. Page 17

The percent biodegradation of the test item was calculated based on its chemical oxygen demand (COD) of 1.39 mg 02/mg test item (see Sections 2.4.2, 2.5 and 2.6). The biochemical oxygen demand (BOD) of DEA/Acid anhydride reaction product in the test media significantly increased from Day 4 until testtermination after28 days. Atthe end ofthe 28-day incubation period, the mean biodegradation of DEA/Acid anhydride reaction product amounted to 53%. Consequently, DEA/Acid anhydrk:le reaction product was found to be partly biodegradable under the conditions of the test within 28 days. However, the pass level for ready biodegradability (biodegradation of at least 60% of the COD in a 1 0-day window within the 28-day period of the test) was not reached. The manometric measurement gives no possibilities of identifying which components (monomers, dimers, trimers, polymers) of the test item were degraded during the 28-day incubation period. Abiotic control: No degradation of the test item occurred in the abiotic control under the conditions of the test. 

## 3.2 BIODEGRADATION OF THE REFERENCE ITEM The results are presented in Tables 1and 2, and in Figures 1and 2. The percent biodegradation ofthe reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg 02/mg (ThOD; see Sections 2.4.2 and 2.5). In the procedure controls, the reference item degraded by an average of 86%) by exposure Day 14, thus confirming suitability ofthe activated sludge. At the end ofthe test (Day 28), the reference item degraded by an average of92%. 

## 3.3 BIODEGRADATION IN THE TOXICITY CONTROL The results are presented in Tables 1and 2, and in Figures 1and 2. The percent biodegradation in the toxicity control containing both, the test item and the reference item, was calculated based on the COD of the test item and the ThOD of the reference item (see, Sections 2.4.2, 2.5 and 2.6). In the toxicity control, the biochemical oxygen demand over the 28-day exposure period was similar to the sum ofthe cumulative BOD ofthe test item and ofthe reference item. Within 14 days ofexposure, a biodegradation of65% was observed. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms because

.biodegradation in the toxicity control was >25% within 14 days.RCC Study Number848637 DEA/Acid anhydride reaction product 

## 3.4 MEASUREMENT OF pH The results are presented in Table 3. REPORT Page 18

The pH measured in all flasks atthe start ofthetest was in the range of7.3to 7.4. Atthe end ofexposure (Day 28), the pH values were between 7.2 and 7.9.

### RCC Study Number848637 DEA/Acid anhydride reaction product 

# 4 TABLES REPORT Page 19

The tabulated values represent rounded results obtained by calculation using the exact raw data.

Table 1: Cumulative biochemical oxygen demand {BOD in mg 0 2 /L) in the testflasks

Test item Inoculum Procedure Abiotic Toxicity control control control control Time Flask No. Flask No. Flask No. Flask No. Flask No. (days) 

### 5 6 9 10 11 12 7 8 0 0 0 0 0 0 0 0 0 1 2 1 0 0 4 4 0 10 2 4 3 0 0 91 91 0 99 3 

## -- -- -- -- 

## - -- -- 

## -- 4 15 14 1 0 120 121 0 151 5 19 16 2 0 127 129 0 161 6 31 25 2 1 132 134 0 167 7 34 32 3 1 135 138 0 172 8 37 35 3 2 138 141 0 177 9 39 39 4 2 140 143 0 183 10 41 41 4 2 142 145 0 188 11


## -- -- 

## -- -- 

## -- - 

## -- 

## -- 12 44 44 5 3 144 149 0 196 13 46 46 5 3 145 150 0 200 14 49 48 5 3 146 151 0 205 15 55 51 5 4 147 152 0 211 16 59 57 5 4 148 153 0 215 17 

### - 

## -- -- 

## -- -- 

## - -- 

## -- 18 63 62 6 4 149 155 0 220 19 65 64 6 4 150 156 0 222 20 67 65 6 4 151 156 0 224 21 70 67 6 4 152 157 0 226 22 72 69 6 4 153 158 0 228 23 75 71 6 4 154 159 0 230 24 77 72 6 4 155 159 0 231 25 79 74 6 5 156 160 0 232 26 80 75 6 5 157 160 0 234 27 81 76 6 5 157 160 0 235 28 83 78 7 5 158 161 0 236

No reading takenRCC Study Number848637 DEA/Acid anhydride reaction product REPORT Page 20

Table 2: Biodegradation of DEA/Acid anhydride reaction product and the reference item



### {: Percentage Biodegradation1 

### Testitem Procedure control Time FlaskNo. Flask No. Flask No. Flask No. (days) 5 0 0 1 1 2 3 3


## -- 4 10 5 13 6 21 7 23 8 25 9 26 10 27 11


## -- 12 29 13 30 ;t,,..l:H''':;;';lJi:' ·""""·· ',;'; ::·')'"':!!H::i:: ,, ,,·· ,.,.•:::

**;:'',~~'"':',,.'.::::** 

### \i 5 16 39 17 -- 18 41 19 43 20 44 21 46 22 48 23 50 24 51 25 52 26 53 27 54 28 55 Mean (Day 28) 53 Corrected forthe inoculum controls Notdetermined 6 11 12 0 0 0 1 2 2 2 54 54



## - -- -- 10 71 72 11 75 76 17 78 79 21 79 81 23 81 83 26 82 83 27 83 85



## -- -- 

## -- 29 83 86 30 84 87 '$!, '85 ' 

### 88 33 85 88 38 86 89

-- 

## -- 

## -- 41 86 89 42 86 90 43 87 90 44 88 91 46 88 

### 91 47 89 92 48 89 92 49 90 92 50 90 92 51 90 92 52 91 92 92 Toxicity control Flask No. 8 0 3 32



## -- 49 52 54 55 57 58 60



## -- 62 64 '86 

### 67 68



## -- 70 71 71 72 72 73 73 74 74 75 75RCC Study Number848637 DEA/Acid anhydride reaction product REPORT



### Table 3: pH values at the start and atthe end ofthe test

Flask No. Identification pH Start 

### 5 Test item 

### 7.4 6 Test item 

### 7.4 9 Inoculum control 

### 7.4 10 Inoculum control 

### 7.4 11 Procedure control 

### 7.4 12 Procedure control 

### 7.4 7 Abiotic control 

### 7.3 8 Toxicity control 

### 7.3 Page 21

End 

### 7.2 7.3 7.5 7.5 7.9 7.9 7.3 7.6RCC Study Number848637 DEA/Acid anhydride reaction product 

# 5 FIGURES REPORT Page 22

Figure 1: Cumulative biochemical oxygen demand in the testflasks during the incubation period

:J' "';:! 0 CJ .§. c 0 m 250

200 ·········-····-······-······- ··············-·-·-·--·--···--·-···-·-·-···-··-···

50 -·--- -+-Test item -Testitem .....,._Inoculumcontrol """""'*-- Inoculum control '"""'*-- Procedure control

**~Procedure** control

**~Abiotic** control --Toxicitycontrol

**0~~~~~~~~~~~~~~~~~~~~~~**

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Exposure time (days)RCC Study Number848637 DEA/Acid anhydride reaction product REPORT Page 23
Figure 2: Biodegradation ofDEA/Acid anhydride reaction product and the reference item

100

80

0 2 4 6 8 -+-Testitem -Testitem '"""'*-- Procedure control _...._Procedure control --Toxicitycontrol 10 12 14 16 18 20 22 24 26 28 30 Exposure time (days)