REPORT 

Study Title: 

READY BIODEGRADABILITY OF 

DEAIACID ANHYDRIDE REACTION PRODUCT 

IN A MANOMETRIC RESPIROMETRY TEST 

Data Requirements I Test Guidelines: 

OECD No. 301 F 

EU Commission Directive 92/69 EEC, C.4-D 

Study Director: 

Roger B~ltscher 

Study Completion Date: 

February 17, 2004 

Test Facility: 

RCC Ltd 

Environmental Chemistry & 

Pharmanalytics 

CH-4452 ltingen I Switzerland 

Sponsor: 

Rockwool International AIS 

Hovedgaden 584 

DK-2640 Hedehusene 

Denmark 

RCC Study Number: 

848637 

Page 1 of 23 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

GLP CERTIFICATE 

REPORT 

The Swiss GLP Monitoring Authorities 

Swiss Federal 

Office of 

Public Health 

Swiss Agency for the Swissmedlc 

Environment. Forests / • Swiss Agency for 

and Landscape 5 Wl S Sm edtC 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 lti ngen 

Switzerland 

were inspected by the Federal Office of PubJic Health. the Swjss 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 toxlc:ity on aquatic and terreslrlal organisms ; ENF = Behaviour In water, soil and air. Bioaccumulation ; EMN • Studies on effects on mesocosms and natural ecosystems: MUT =Mutagenicity. PCT = Physic:al-chemical testing; RES • Residue stucfJeS ; OTH • ~· to be specified. 

Page2 
RCC Study Number 848637 

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 of the determination of the COD of the 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 of Good Laboratory Practice, as revised in 1997 and adopted November 

26th, 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 

Date: 
RCC Study Number 848637 

DENAcid anhydride reaction product 

SIGNATURES 

Study Director: 

Management: 

REPORT Page4 

Mr. Roger B~Uscher 

Date: 

Dr. Uwe Morgenroth 
RCC Study Number 848637 

DENAcid anhydride reaction product 

REPORT 

QUALITY ASSURANCE 

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 Quality Assurance. The dates are given below. 

Dates and Types of QA Inspections Dates of Reports to the Study 

Director and to the Management 

July 31, 2003 Study plan July 31, 2003 

November 27, 2003 Process based November 27, 2003 

(test item application) 

January 27, 2004 Report January 27, 2004 

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

Quality Assurance: Mrs. Ursula Burri 

(!. lfu_~· 

Date: 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

CONTENTS 

REPORT Page6 

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

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

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

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

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

CONTENTS CONT'D (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 .............................................................................. 12 

2.1 TEST ITEM ................................................................................................. 12 

2.2 REFERENCE ITEM ..................................................................................... 12 

2.3 TEST SYSTEM ........................................................................................... 13 

2.4 TEST PROCEDURES ................................................................................. 13 

2.4.1 Test Water ..................................................................................... 13 

2.4.2 Test Concentrations ....................................................................... 14 

2.4.3 Conditions of the Test .................................................................... 15 

2.5 CALCULATIONS ......................................................................................... 16 

2.6 DETERMINATION OF THE CHEMICAL OXYGEN DEMAND ..................... 16 

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

3.1 BIODEGRADATION OF THE TEST ITEM ................................................... 17 

3.2 BIODEGRADATION OF THE REFERENCE ITEM ...................................... 17 

3.3 BIODEGRADATION IN THE TOXICITY CONTROL ....................... u ........... 17 

3.4 MEASUREMENT OF pH ............................................................................. 18 

4 TABLES ............................................................................................................... 19 

5 FIGURES ............................................................................................................. 22 
RCC Study Number 848637 

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 test flasks .......................................................................................... 19 

Table 2: Biodegradation of DEA/Acid anhydride reaction product 

and the reference item ................................................................................ 20 

Table 3: pH values at the start and at the end of the test .......................................... 21 

Figure 1: Cumulative biochemical oxygen demand in the test flasks 

during the incubation period ........................................................................ 22 

Figure 2: Biodegradation of DEA/Acid anhydride reaction product 

and the reference item ................................................................................ 23 
RCC Study Number 848637 

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 PageS 

Ready Biodegradability of DEA/Acid anhydride 

reaction product in a Manometric Respirometry Test 

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. Iris WUthrich 

November 1 0, 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 of the present study for at least ten years. 

No data will be discarded without the Sponsor's consent. 
RCC Study Number 848637 

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 

First Amendment to Study Plan 

Concerning: Alteration: Reason: 

Preface I Schedule Update of the schedule. Rescheduling of the study. 

2.1 Test Item Update of expiration date and A new test item batch was tested 

batch number. at the request of the sponsor. 
RCC Study Number 848637 

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 after 28 days. At the end of the 

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 of the 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 of activated 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 of the activated sludge. 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

1 PURPOSE 

REPORT Page 11 

The purpose of this study was to determine the ready biodegradability of DEA/Acid anhydride 

reaction product in a 28-day biodegradation test by following the BOD (biochemical oxygen 

demand) of the test item under defined conditions by means of manometric methods. 

This test design was selected according to the guidelines since the test item contains poorly 

water soluble components. 

RCC Study Number 848637 

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 of wet to dry weight 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 (±1 0%) 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 a final concentration of 30 mg dry material per 

liter. 

2.4 TEST PROCEDURES 

2.4.1 Test Water 

The test water was prepared according to testing guidelines. Analytical grade salts were 

dissolved in purified water to 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 of this solution was 7 .4. 

22.50 g/L 

36.40 g/L 

0.25 g/L, stabilized with one drop of concentrated 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 ml with purified water. The pH of the test water 

was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution. 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

2.4.2 Test Concentrations 

REPORT Page 14 

The amounts of test item were directly weighed into the test flasks. No emulsifiers or solvents 

were used but ultrasound dispersion was employed for ten 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 of test water was prepared. From 

this, aliquots of 10 ml were 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 a volume of 250 ml with test water. 

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 of test 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 test solution 

Poisoned with mercury dichloride at a concentration of 1 0 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.: not applicable 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

2.4.3 Conditions of the Test 

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 copper sulfate solution. 

Test duration: 28 days 

Light regimen: Darkness 

Test temperature: 22 oc. maintained with a built-in thermostat and checked once per week. 

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 test flask. 
RCC Study Number 848637 

DENAcid anhydride reaction product 

2.5 CALCULATIONS 

REPORT Page 16 

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

data. 

The percentage biodegradation was calculated according to the formula given below: 

% biodegradation = 

BOD (mg 0 2/mg chemical) x 1 OO% 

COD and/or ThOD (mg 0 2/mg chemical) 

where: 

BOD = Biochemical oxygen demand of the test or reference item 

= (mg 0 2 uptake/L by test and/or reference item)- (mg 0 2 uptake/L by inoculum control) 

mg test and/or reference item per liter· 

COD = Chemical oxygen demand of the test item (since the ThOD of the test item could 

not be calculated; see Section 2.6) 

ThOD = Theoretical oxygen demand of the reference item 

The theoretical oxygen demand is the total amount of oxygen 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 of the COD was 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 under this RCC study number. 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

REPORT 

3 RESULTS AND DISCUSSION 

3.1 BIODEGRADATION OF THE TEST ITEM 

The results are presented in Tables 1 and 2, and in Figures 1 and 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 test termination after 28 days. At the end of the 

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 1 and 2, and in Figures 1 and 2. 

The percent biodegradation of the 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 of the activated sludge. At the end of the test (Day 28), the 

reference item degraded by an average of 92%. 

3.3 BIODEGRADATION IN THE TOXICITY CONTROL 

The results are presented in Tables 1 and 2, and in Figures 1 and 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 of the cumulative BOD of the test item and of the reference item. Within 

14 days of exposure, a biodegradation of 65% 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 Number 848637 

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 at the start of the test was in the range of 7.3 to 7.4. At the end 

of exposure (Day 28), the pH values were between 7.2 and 7.9. 
RCC Study Number 848637 

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 test flasks 

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 taken 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

REPORT Page 20 

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

{: 

Percentage Biodegradation 1 

Test item Procedure control 

Time Flask No. 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 for the inoculum controls 

Not determined 

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 

75 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

REPORT 

Table 3: pH values at the start and at the end of the 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 

7.6 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

5 FIGURES 

REPORT Page 22 

Figure 1: Cumulative biochemical oxygen demand in the test flasks during the incubation 

period 

:J' 

"';:! 

0 

CJ 

.§. 

c 

0 

m 

250 

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

50 -·---

-+-Test item 

-Test item 

.....,._Inoculum control 

"""""'*-- Inoculum control 

'"""'*-- Procedure control 

~Procedure control 

~Abiotic control 

--Toxicity control 

0~~~~~~~~~~~~~~~~~~~~~~ 

0 2 4 6 8 1 0 12 14 16 18 20 22 24 26 28 30 

Exposure time (days) 
RCC Study Number 848637 

DEA/Acid anhydride reaction product 

REPORT Page 23 

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

100 

80 

0 2 4 6 8 

-+-Test item 

-Test item 

'"""'*-- Procedure control 

_...._Procedure control 

--Toxicity control 

10 12 14 16 18 20 22 24 26 28 30 

Exposure time (days)