p.1 

CBIC Control Number 

HUNTSMA N 356399 

•n-ti n • CTI^IJ^IM RECEIVED 

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20I3OCT 18 AHIhi»7 

October 11, 2013 

Document Control Office (DCO) (7407M) ed&P«ceSse< i , n 

Office of Pollution Prevention and Toxics (OPPT) _ A

 s " e uy sHM* 0

*

1 

US EPA V C 

1200 Pennsylvania Ave., NW 

Washington, DC 20460-0001 

Attention: Mr. John Schaeffer 

TSCA Section 4 

40 CFR 799.5089 

Docket ID Number EPA-HQ-OPPT-2009-0112 

RE: Testing of Certain High Production Volume Chemicals; Third Group of Chemicals 

2-Oxiranemethanamine, N-[4-(2-oxiranylmethoxy)phenylJ-N-(2-oxiranylmethyl)- (CAS No. 

5026-74-4) 

Dear Mr. Schaeffer: 

Enclosed please find the final reports for the following studies on 2-Oxiranemethanamine, N-[4-(2-

oxiranylmethoxy)phenylj~N-(2-oxiranylmethyl)- {CAS No. 5026-74-4): 

Determination of Physico-Chemical Properties 

Ready Biodegradability (OECD 301 B) 

Fresh Water Algal Growth Inhibition Test (OECD 201) 

Daphnia magna, Reproduction Test (OECD 211) 

96-hour Acute Toxicity Study in Carp (OECD 203) 

Pseudomonas Cell Multiplication Inhibition Test 

Huntsman previously committed to performing both a Reproduction/Developmental Toxicity Screening 

Test and a Repeated Dose 28-Day Oral Toxicity Study in rodents, however, because preliminary results 

from the Repeated Dose 28-Day Oral Toxicity Study in rodents on a surrogate, (m-(2,3-epoxypropoxy)-NN-bis(2,3-epoxypropyl)aniline, CAS # 71604-74-5, resulted in a No Observed Adverse Effect Level 

(NOAEL) of 50 mg/kg/day based on atrophy of the female reproductive organs observed at 150 

mg/kg/day, Huntsman determined the Re production/Develop mental Toxicity Screening Test was not 

needed. The data generated from the 28-day study is being used as read across for the test rule 

substance, 5026-74-4. The final report will be sent once received. Enclosed is a letter sent to the TSCA 

8(e) Coordinator for your review. 

Please do not hesitate to contact me at the number listed below if additional information is required. 

Sincerely, 

Lynne Oreschnigg 

Regulatory Compliance Specialist 

Huntsman Corporation 

Phone: 281-719-3021 

Fax: 281-719-3050 

lynne_oreschnigg@huntsman.com 

HUNTSMAN CORPORATION 

10003 Woodloch Forest Drive, The Woodlands, Texas 77380, USA 

Tel.:+1-281-719-6000 Fax .:+1-281-719-6416 www.huntsman.com 
HUIMTSMAIVI 

Enriching lives through innovation 

April 30, 2013 

Via Overnight Courier 

TSCA Confidential Business Information Center (7407M) 

EPA East - Room 6428 

US Environmental Protection Agency 

1201 Constitution Ave, NW 

Washington, DC 20004-3302 

Attention: TSCA Section 8(e) Coordinator 

Draft Final Report of a Repeated Dose 28 Day Oral Toxicity Study with m-(2,3-

epoxypropoxy)-N,N-bis(2,3-epoxypropyl) aniline, CAS# 71604-74-5. 

Dear Sir or Madam: 

The European office of Huntsman Advanced Materials (Huntsman) has received a draft final 

report from a Repeated Dose 28 Day Oral Toxicity Study with m-(2,3-epoxypropoxy)-N,Nbis(2,3-epoxypropyi)aniline) CAS# 71604-74-5. This study was conducted using a standard 

OECD 407 protocol by WiL Research Europe B.V. in the Netherlands. The results of this 

repeated dose study have established a No Adverse Effect Level (NOAEL) at 50 mg/kg/day, 

based on observed atrophy of female reproductive organs at the next highest dose level, 150 

mg/kg/day, 

Huntsman is submitting this information pursuant to Section 8(e) of the Toxic Substances 

Control Act (TSCA). Huntsman has not made a determination as to .whether a significant risk of 

injury to human health or the environment is actually presented by these findings. 

Study Design: 

The test substance was administered, by gavage, to groups of 5 male and 5 female Wistar rats 

over 28 consecutive days. Dose levels used in this study, formulated in propylene glycol, were 

0, 50, 150, and 450 mg/kg/day, with the untreated control group receiving only propylene glycol. 

The following study parameters were evaluated: daily clinical signs; functional observation 

battery at study week 4, body weights and food consumption determined weekly; clinical 

pathology and macroscopic evaluation at study termination, and organ weights and 

histopathology evaluations on specified tissues. 

Study Findings: 

• No mortality occurred during this study. However, a poor health status was noted for all 

animals at the highest dose level of 450 mg/kg/day. Although the functional observation 

battery (FOB) revealed no effects on hearing, pupil reflex, righting reflex and grip 

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strength, the motor activity measurement showed lower activity {total movements and 

ambulations) at 450 mg/kg/day. 

• Animals administered 450 mg/kg/day showed lower body weight gains and reduced 

body weights compared to control animals. As a result of the poor health status of the 

animals at 450 mg/kg/day, changes were observed in hematology, clinical biochemistry, 

organ weights and histopathology. These findings are considered to be a secondary 

effect unrelated to any specific systemic toxicity of the test substance. 

• Atrophy of the uterus, cervix and vagina were observed at 150 mg/kg/day accompanied 

by the macroscopicaily visible reduced size of ovaries and uterus at 450 mg/kg/day; In 

males at 450 mg/kg/day, the prostate gland was considered to be small but with normal 

histology. At this level, the contents in the seminal vesicles and coagulating glands were 

considered to be reduced. A lower absolute and/or relative-prostate and seminal vesicle 

weight was noted at the lowest dose level of 50 mg/kg/day, but these observations were 

not supported by any histological findings, and are not considered to be toxicologicaily 

relevant. 

• The effects on liver and kidney were noted at 450 mg/kg/day: centrilobular hypertrophy 

in the liver, discoloration of kidney and adrenals and enlarged adrenal glands in females, 

with microscopically observed vacuolization of the zona fasciculata in the adrenal 

glands. 

• Effects were observed in the gastrointestinal tract and associated lymphatic system, but 

these effects are considered to be secondary findings related to the physical properties 

of the test material and not due to any specific systemic toxicity of the test substance. 

Study Conclusion: 

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for Wistar 

rats exposed to 2-Propanol, 1-[bis[3-(dimethyiamino)propyl]amino]- over 28 consecutive days is 

considered to be 50 mg/kg/day, based on atrophy of the female reproductive organs observed 

at 150 mg/kg/day. 

As always, if I can provide any additional information on the above study, please call me at 

(281) 719-3017, or contact me via e-mail at: Ray_Papciak@Huntsman.com. 

Regards, 

Raymond J. Papciak 

Manager, Product Safety 

mi T 
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Authorization and Restriction of Chemical Substances (REACH), without the express written permission of Huntsman 

International LLC is unauthorized and strictly prohibited. 

FINAL REPORT 

Study Title 

DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES OF 

P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILINE 

Validation of an analytical method 

Melting and boiling temperature 

Density 

Vapour pressure 

Water solubility 

Flash-point 

Auto-ignition temperature 

Hydrolysis as a function of pH 

Oxidizing properties 

Adsorption coefficient 

Dissociation constant 

Viscosity 

Author 

Dr. Ir. E. Baltussen 

Test Facility 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Netherlands 

Laboratory Project Identification 

NOTOX Project 496981 

NOTOX Substance 203277/A 

- Page 1 of 76 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

1. CONTENTS 

1. CONTENTS 2 

2. STATEMENT OF GLP COMPLIANCE 6 

3. QUALITY ASSURANCE STATEMENT 7 

4. SUMMARY 8 

5. INTRODUCTION 10 

5.1. Preface 10 

5.2. Aim of the study 10 

5.3. Storage and retention of records and materials 10 

6. MATERIALS 11 

6.1. Test substance 11 

6.2. Electronic data capture 11 

6.3. List of deviations 12 

7. VALIDATION OF AN ANALYTICAL METHOD 13 

7.1. Guideline 13 

7.2. Reagents 13 

7.3. Performance of the study 13 

7.4. Analytical method 14 

7.5. Formulas 14 

7.6. Results 15 

7.7. Conclusion 17 

7.8. Figures , 18 

8. MELTING AND BOILING TEMPERATURE 19 

8.1. Guidelines 19 

8.2. Performance of the test 19 

8.3. Analytical method 20 

8.4. Interpretation 20 

8.5. Results 20 

8.6. Conclusion 20 

8.7. Figures 21 

9. DENSITY 23 

9.1. Guidelines 23 

9.2. Reagents 23 

9.3. Performance of the study 23 

9.4. Interpretation 24 

9.5. Results 24 

9.6. Conclusion 24 

10. VAPOUR PRESSURE 25 

10.1. Guidelines 25 

10.2. Performance of the test 25 

10.3. Analytical method 25 

10.4. Reference substances 26 

10.5. Interpretation 27 

10.6. Results 28 

10.7. Conclusion 28 

10.8. Figures 29 

11. WATER SOLUBILITY 30 

11.1. Guidelines 30 

11.2. Reagents 30 

11.3. Performance of the study 30 

11.4. Analytical method 31 

11.5. Interpretation 31 

11.6. Results 32 

11.7. Conclusion 32 

Final report - Page 2 -
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p-(2,3-epoxypropoxy)-N1N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

12. FLASH-POINT 33 

12.1. Guidelines 33 

12.2. Performance of the test 33 

12.3. Interpretation 33 

12.4. Results 34 

12.5. Conclusion 34 

13. AUTO-IGNITION TEMPERATURE 35 

13.1. Guidelines 35 

13.2. Performance of the test 35 

13.3. Interpretation 36 

13.4. Results 36 

13.5. Conclusion 37 

14. OXIDIZING PROPERTIES 38 

14.1. Guideline 38 

14.2. Statement 38 

15. HYDROLYSIS AS A FUNCTION OF PH 39 

15.1. Guideline 39 

15.2. Reagents 39 

15.3. Buffer solutions 39 

15.4. Performance of the study 39 

15.5. Analytical method 41 

15.6. Interpretation 41 

15.7. Results 43 

15.8. Conclusion 53 

15.9. Figures 54 

16. ADSORPTION COEFFICIENT 57 

16.1. Guidelines '. 57 

16.2. Reagents , 57 

16.3. Performance of the study' 57 

16.4. Analytical method 58 

16.5. Formulas 59 

16.6. Results 59 

16.7. Conclusion 60 

16.8. Figures 61 

17. DISSOCIATION CONSTANT - 62 

17.1. Guidelines 62 

17.2. Reagents 62 

17.3. Buffers 62 

17.4. Performance of the study 64 

17.5. Spectrophotometry method 64 

17.6. Performance of the test 64 

17.7. Formulas 65 

17.8. Results 66 

17.9. Conclusion 68 

17.10. Figures 69 

18. VISCOSITY 74 

18.1. Guidelines 74 

18.2. Performance of the test 74 

18.3. Interpretation 74 ' 

18.4. Results 75 

18.5. Conclusion 75 

Final report - Page 3 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

TABLES 

Table 1 Statistical parameters of the calibration curve 15 

Table 2 Accuracy samples 16 

Table 3 Stability of the analytical system and end solutions 16 

Table 4 Stored accuracy samples 17 

Table 5 Density of the test substance 24 

Table 6 Results of the isothermal TGA analysis 28 

Table 7 Vapour pressure of the test substance 28 

Table 8 Main study - water solubility of the test substance 32 

Table 9 Main study - flash-point of the test substance 34 

Table 10 Main study-auto-ignition temperature of the test substance 37 

Table 11 Preliminary test - hydrolysis of the test substance at pH 4, pH 7 and pH 9 ". 43 

Table 12 Recoveries 43 

Table 13 Main test - hydrolysis of the test substance at 20°C 44 

Table 14 Main test - hydrolysis of the test substance at 40°C 45 

Table 15 Main test - hydrolysis of the test substance at 50°C 45 

Table 16 Recoveries 46 

Table 17 Statistical parameters of the regression curves 46 

Table 18 Rate constants (k0bs) and half-life time (t M ) 46 

Table 19 Main test - hydrolysis of the test substance at 20°C 47 

Table 20 Main test - hydrolysis of the test substance at 40°C 48 

Table 21 Main test - hydrolysis of the test substance at 50°C 48 

Table 22 Recoveries , 49 

Table 23 Statistical parameters of the regression curves 49 

Table 24 Rate constants (k obs ) and half-life time (t%) 49 

Table 25 Main test - hydrolysis of the test substance at 20°C 50 

Table 26 Main test - hydrolysis of the test substance at 40°C 51 

Table 27 Main test - hydrolysis of the test substance at 50°C 51 

Table 28 Recoveries , 52 

Table 29 Statistical parameters of the regression curves 52 

Table 30 Rate constants (k obs ) and half-life time (t % ) 52 

Table 31 Ko c of the test substance 60 

Table 32 Actual pH values and optical absorbances at 249 nm 67 

Table 33 Optical absorbances at 249 nm 68 

Table 34 Viscosity of the test substance 75 

Final report - Page 4 -
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FIGURES 

Figure 1 HPLC-UV chromatogram of a 2.00 mg/l test substance solution [res. id. 1509] 18 

Figure 2 HPLC-UV chromatogram of the blank accuracy sample [res. id. 1529] 18 

Figure 3 Regression line: response of the test substance as a function of concentration [cal. curve 

id. 1497] 18 

Figure 4 TGA curve of the test substance 21 

Figure 5 DSC curve of the test substance obtained with the first experiment : 21 

Figure 6 DSC curve of the test substance obtained with the second experiment 22 

Figure 7 Weight loss curve of the test substance 29 

Figure 8 Plot of log PT of the test substance versus 1 U 29 

Figure 9 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C [middle] 

and 50°C -{bottom] at pH4 54 

Figure 10 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C [middle] 

and 50°C [bottom] at pH7 55 

Figure 11 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C [middle] 

and 50°C [bottom] at pH9 56 

Figure 12 HPLC chromatograms of the 14.5 mg/l test solution [top; res. id. 1571] and corresponding 

blank [bottom; res. id. 1572] 61 

Figure 13 The regression line of the reference substances: log k' versus log Ko c 61 

Figure 14 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 0 69 

Figure 15 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 1 69 

Figure 16 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 2 69 

Figure 17 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 3 70 

Figure 18 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 4 70 

Figure 19 Absorption spectrum of a 20 mg/I solution of test substance in buffer pH 5 70 

Figure 20 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 6 71 

Figure 21 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 7 71 

Figure 22 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 8 71 

Figure 23 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 9 72 

Figure 24 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 10 72 

Figure 25 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 11 72 

Figure 26 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 12 73 

Figure 27 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 13 73 

APPENDICES 

Appendix 1 GLP certificate 76 

Final report - Page 5 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

2. STATEMENT OF GLP COMPLIANCE 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

The study described in this report has been correctly reported and was conducted in compliance with: 

The Organization for Economic Co-operation and Development (OECD), OECD Principles on Good 

Laboratory Practice (GLP) (as revised in 1997), ENV/MC/CHEM (98) 17. 

The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test substance 

information unless determined by NOTOX. 

Dr. Ir. E. Baltussen Dr. Ir. T.H.M. Noij 

Study Director Head of Chemistry 

Final report - Page 6 -
p. 10 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

3. QUALITY ASSURANCE STATEMENT 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

This report was inspected by the NOTOX Quality Assurance Unit (QAU) to confirm that the methods 

and results accurately and completely reflect the raw data. 

During the on-site process inspections, procedures applicable to this type of study were inspected. 

The dates of Quality Assurance inspections are given below. 

Type of 

Inspections 

Study 

Phase/Process 

Protocol 

Protocol Amendment 01 

Analysis 

Observations 

/measurements chemical 

properties 

Report 

Protocol Amendment 02 

Start 

Inspection 

date 

19-Aug-11 

15-Nov-11 

17~Nov-11 

17-Nov-11 

17~Dec-11 

11-Jan~12 

End 

Inspection 

date 

19-Aug-11 

15-Nov-11 

17-Nov-11 

17- Nov-11 

18- Dec-11 

11-Jan-12 

Reporting 

date 

19-Aug-11 

15-Nov-11 

17-NOV-11 

17-Nov-11 

19-Dec-11 

11-Jan-12 

Analytical and physical 

Process chemistry 

Test Substance Handling 

Obse rvations/Measu reme nts 

08-Aug-11 16-Aug-11 16-Aug-11 

Analytical and physical 

chemistry 31-Oct-11 10-Nov-11 10-Nov-11 

Test Substance Handling 

Observations/Measurements 

NOTOX B.V. 

C.J. Mitchell B.Sc. 

Head of Quality Assurance 

Final report - Page 7 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

4. SUMMARY 

The results of the physico-chemical properties of the test substance are given below. 

Parameter Guideline(s) Result Comment 

Analytical method Sanco 3029 successfully validated 

Melting temperature EC A.1 

OECD 102 

-39°C (234K) Glass transition 

Boiling temperature EC A.2 

OECD 103 

no boiling temperature reaction and/or 

decomposition of the 

test substance at 

> 250°C (> 523K) was 

observed 

Density EC A.3 

OECD 109 

D l ° :1.22 

Vapour pressure EC A.4 

OECD 104 

20°C 5.5 x 10' 7Pa = 

4.1 x 10'9 mm Hg 

25°C 1.3 x 10* Pa = 

9.9 x 10"9 mm Hg 

isothermal TGA 

effusion method 

Water solubility EC A.6 

OECD 105 

3.34 g/l at 20°C by the 

flask method 

Flash-point EC A.9 

UN p. 32.4.1 

ISO 2719 

ASTM D 93 

228°C Pensky-Martens 

closed cup method 

Auto-ignition temperature EC A.15 

DIN 51794 

I EC 79-4 

410aC at 1012.6-1013.9 

hPa 

Oxidizing properties EC A.21 

UN 0.2 

not oxidizing statement 

Hydrolysis as a function of 

PH 

EC C.7 

OECD 111 

pH 4: t« = 2.2 days 

pH 7: X% - 2.3 days 

pH 9: tj4 = 2.6 days 

at 25°C 

Final report - Page 8 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

Adsorption coefficient EC C.19 

OECD 121 

log KoC: 1.93 HPLC method based 

on soil-adsorptionreference data 

Dissociation constant OECD 112 pKg value : 3.25 

(basic) 

spectro photometric 

method 

Viscosity OECD 114 

ISO 3104 

ASTM D 445-09 

20°C: 840.6 mm2/s 

40°C: 138.9 mm2/s 

Final report - Page 9 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ariiline NOTOX Project 496981 

5. INTRODUCTION 

5.1. Preface 

Sponsor Huntsman Advanced Materials GmbH 

Klybeckstrasse 200 

4057 BASEL 

Switzerland 

Study Monitor Dr. H. Wilbers 

Test Facility NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Netherlands 

Study Director Dr. Ir. E, Baltussen 

Study Plan Start 

Completion 

24 August 2011 

01 December 2011 

5.2. Aim of the study 

The aim of the study was to determine the following physico-chemical properties for 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline: 

• Validation of an analytical method 

• Melting and boiling temperature 

• Density 

• Vapour pressure 

• Water solubility 

• Flash-point 

• Auto-ignition temperature 

• Hydrolysis as a function of pH 

• Oxidizing properties 

• Adsorption coefficient 

• Dissociation constant 

• Viscosity 

5.3. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the NOTOX archives 

for a period of at least 10 years after finalization of the report. After this period, the sponsor will be 

contacted to determine how the records and materials should be handled, NOTOX will retain 

information concerning decisions made. 

Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as the 

quality of the specimens permits evaluation but no longer than three months after finalization of the 

report. 

NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

Final report - Page 10 -

ir 
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

6. MATERIALS 

6.1. Test substance 

6.1.1. Test substance information 

NOTOX Project 496981 

Identification 

Structure 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

O. 

Molecular formula 

Molecular weight 

CAS Number 

EC Number 

Description 

Batch 

Purity 

Test substance storage 

Stability under storage conditions 

Expiry date 

C1 5Hi gN04 

277.3 

5026-74-4 

225-716-2 

Yellow liquid 

AA00373400 

94.33% 

In refrigerator (2-8°C) in the dark 

Stable 

28 February 2013 

6.1.2. Study specific test substance information 

There was no study specific test substance information necessary for this study. 

6.2. Electronic data capture 

System control, data acquisition and data processing were performed using the following programmes: 

- Cary WinUV pharma version 3.1 (Varian, Mulgrave, Australia) 

- Empower version 7.00 (Waters, Milford, MA, USA) 

- Advantage Integrity Software version 3.0 (TA Instruments, New Castle, DE, USA) 

Temperature and/or relative humidity and/or pressure during sample storage and/or performance of 

the studies were monitored continuously using the following programme: 

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA) 

Final report - Page 11 -
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6.3. List of deviations 

6.3.1. List of protocol deviations 

1. Chapter 13 "Hydrolysis as a function ofpH", paragraph 13.4 "Performance of the study": 

During the main studies - Tier 2 at all pH values at 20°C, the temperature was controlled within 

± 0.6°C (criterion was ± 0.5°C). 

Evaluation; The temperature ranges were slightly higher than the criterion. This was considered 

not to be of significantly influence on the determined half-life time of the test substance at the 

temperatures. 

The study integrity was not adversely affected by the deviations. 

6.3.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures (SOPs) were evaluated and filed in the study file. 

There were no deviations from SOPs that affected the integrity of the study. 

Final report - Page 12 -
p. 16 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

7. VALIDATION OF AN ANALYTICAL METHOD 

7.1. Guideline 

The study was based on the following guideline: 

European Commission: Guidance for Generating and Reporting Methods of Analysis in Support of 

Pre-registration Data Requirements for Annex II (Part A, section 4) and Annex III {Part A, section 5) of 

Directive 91/414, SANCO/3029/99 rev. 4 (11/07/00). 

7.2. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA) 

Acetonitrile Biosoive, Valkenswaard, The Netherlands. 

All reagents were of analytical grade, unless specified otherwise. 

7.3. Performance of the study 

A high performance liquid chromatographic method with spectrophotometric detection (HPLC-UV) for 

the quantitative analysis of the test substance in water was developed. Validation of the analytical 

method was performed for the following parameters: 

Specificity 

A test substance solution and blank accuracy sample were analysed by single injection. The analytical 

method was found to be specific if the blank chromatogram showed no response for the test 

substance or a response of < 30% of the limit of quantification. 

Linearity 

Calibration solutions were analysed in duplicate. The response of the calibration solutions was 

correlated with concentration using regression analysis with a 1/concentration2 weighting factor. A 

calibration curve with a coefficient of correlation (r) of > 0.99 and back calculated accuracies of the 

calibration solutions in the range 85-115% was accepted. 

Accuracy and repeatability 

Accuracy samples were analysed by single injection into the analytical system. The analytical method 

was considered applicable for the determination of the test substance if the mean accuracy was in the 

range 70-110% and the coefficient of variation was £ 20%. 

Limit of quantification 

The limit of quantification (LOQ) is defined as the lowest concentration level at which an accuracy in 

the range 70-110% and a repeatability of < 20% is demonstrated. The LOQ was obtained from the 

data of the accuracy- and repeatability test. 

Stability of the analytical system and end solutions 

Calibration solutions were injected throughout the validation sequence including the beginning and 

end. The analytical system and/or end solutions were found to be stable if the coefficient of variation 

on the responses of the solutions was £ 20%. 

Stability of stock solutions 

Stock solutions of the test substance were stored at room temperature, for at least 12 hours. Additional 

calibration solutions were prepared and analysed by single injection. The stock solutions were found to 

be stable if the coefficient of variation on the response factors of the calibration solutions prepared with 

fresh and stored stock solutions was £ 10%. 

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p-(2,3-e poxy pro poxy)-N,N-bis(2,3-e poxy propyl) aniline NOTOX Project 496981 

Storage stability of samples 

Additional accuracy samples were prepared and stored in the freezer for at least 16 hours. On the day 

of analysis, the samples were defrosted at room temperature and treated identically as freshly 

analysed accuracy samples. The stored samples were found to be stable if the mean accuracy was in 

the range 70-110%. 

7.4. Analytical method 

7.4.1. Analytical conditions 

Instrument 

Detector 

Column 

Column temperature 

Injection volume 

Mobile phase 

Flow 

UV detection 

7.4.2. Preparation of solutions 

Stock and spiking solutions 

Stock solutions of the test substance were prepared in acetonitrile at concentrations of 1712 -

2420 mg/l. 

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of 

the spiking solutions was acetonitrile. 

Calibration solutions 

Six calibration solutions in the concentration range of 0.04 - 2 mg/l were prepared from two stock 

solutions. The end solution of the calibration solutions was 30/70 (v/v) acetonitrile/water. 

Accuracy samples 

2 ml water was spiked with the test substance at a target concentration of 0.1 or 100 mg/l. The 

accuracy samples were diluted in a 1:1 (v;v) ratio with 60/40 (v/v) acetonitrile/water and analysed. If 

necessary, the samples were further diluted with 30/70 (v/v) acetonitrile/water to obtain concentrations 

within the calibration range. 

The blank accuracy sample was prepared and treated similar to the accuracy samples. 

7.5. Formulas 

Response (R) Peak area of the test substance [units] 

Response factor (Rf) Rf = — 

CN 

where: 

CN = nominal concentration [mg/l] 

Calibration curve R = a C N + b 

where: 

a = slope [units x l/mg] 

b = intercept [units] 

Alliance Separation Module 2695 

(Waters, Milford, MA, USA) 

Dual X Absorbance Detector 2487 (Waters) 

Symmetry Shield RP-18, 100 mm x 4.6 mm i.d., dp = 3.5 urn 

(Waters) 

40°C±1°C 

100 pi 

30/70 (v/v) acetonitrile/water 

1.0 ml/min 

253 nm 

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Analysed concentration (CA) (R-b ) 

xd [mg/I] 

a 

where: 

d = ( dilution factor 

Accuracy x100 [%] 

7.6. Results 

7.6.1. Specificity 

Chromatograms of a test substance solution and the blank accuracy sample are shown in Figure 1 

and Figure 2, respectively. 

Several small peaks and one large peak were observed in the chromatogram of the test substance 

solution. It was assumed that the large peak derives from the major component of the test substance, 

whereas the small peaks derive from impurities. The peak area of the major component was used as 

response in the calculations. 

The chromatogram of the blank sample showed no peak at the retention time of the test substance. 

Since no interferences were detected, the specificity requirements were met and the analytical method 

was found to be specific for the test substance. 

7.6.2. Linearity 

The calibration line was constructed using all data points. Figure 3 illustrates the calibration curve and 

Table 1 shows the statistical parameters. There was a linear relationship between response and test 

substance concentration in the range of 0.0401 - 2.00 mg/l (in end solution). Since the coefficient of 

correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range 85-

115% the calibration line was accepted. 

Table 1 Statistical parameters of the calibration curve 

Slope 

Intercept 

Weighting factor 

r 

2.03 x 105 

-1.75 x 103 

1/concentration2 

0.997 

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7.6.3. Accuracy and repeatability 

The results of the accuracy samples are given in Table 2. Since the mean accuracy at each 

concentration level fell in the criterion 85-115% and the coefficient of variation was £ 20% the 

analytical method was accepted for the analysis of the test substance in water in the target 

concentration range of 0.1 -100 mg/l. 

Table 2 Accuracy samples 

Target 

concentration 

[mg/ll 

Nominal 

concentration 

[mg/ll 

Analysed 

concentration 

rmg/n 

Accuracy 

r%i 

Mean 

accuracy 

[%1 

Coefficient of 

variation 

[%l 

0.1 0.0995 0.109 109 107 2.4 

0.0995 0.104 104 

0.0995 0.103 104 

0.0995 0.108 108 

0.0995 0.107 108 

100 99.5 105 106 101 3.3 

99.5 98.7 99 

99.5 97.7 98 

99.5 104 104 

99.5 99.4 100 

7.6.4. Limit of quantification 

The limit of quantification (LOQ) was assessed at 0.1 mg/l in water. 

7.6.5. Stability of the analytical system and end solutions 

The results of the stability of the analytical system and end solutions are given in Table 3. Since the 

coefficient of variation at both concentration levels was < 20% the analytical system and end solutions 

were stable over at least a 26.7 hour time interval. 

Table 3 Stability of the analytical system and end solutions 

Nominal concentration Elapsed time Coefficient of variation 

[mq/n [hoursl f%l 

0.0401 28.3 17 (n=6) 

0.0901 28.1 14 (n=6) 

2.00 26.7 6.2 (n=6) 

7.6.6. Stability of stock solutions 

The coefficient of variation on the response factors of the calibration solutions prepared with fresh and 

stored stock solutions was 2.2%. Since the value was £ 10% the stock solutions were stable when 

stored at room temperature for at least 16 days. 

Final report - Page 16 -
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7.6.7. Storage stability of samples 

The results of the storage stability of the test substance in the freezer are given in Table 4. Since the 

mean accuracy of the frozen samples fell in the criterion 70-110% the samples were stable when 

stored in the freezer. 

Table 4 Stored accuracy samples 

Target 

concentration 

rmg/ll 

Nominal 

concentration 

fmg/ll 

Analysed 

concentration 

fmg/ll 

Accuracy 

[%l 

Mean accuracy 

r%i 

0.1 0.0995 0.0766 77 77 

0.0995 0.0761 76 

100 99.5 80.8 81 80 

99.5 78.3 79 

7.7. Conclusion 

The analytical method was validated for the following parameters: 

Specificity specific 

Linearity r = 0.997 

Accuracy 101 and 107% 

Repeatability 2.4 and 3.3% 

Limit of quantification 0.1 mg/l 

Stability analytical system and end solutions stable 

Stability stock solutions stable 

Storage stability stable 

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p. 21 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

7.8. Figures 

NOTOX Project 496981 

OOKh 

o.cca 

0.038' 

0 024' 

0.0)2 

0.030 

0.033' 

0.034 

0.022' 

, 0.020' 

' 0 018 

0.018' 

0.014 

0.012 

0.010' 

0.008 

0.009 

0.004' 

0,002 

0.0W - - • 

o.oo o n 

0JM0-] 

0.035' 

0.036' 

0.034' 

0.032' 

0.030-

0.025 

OJKO 

0024 

0.022 

, 0.020' 

' 0 01S' 

0.018' 

0.014 

0.012' 

0.010' 

0.008' 

0.0O9' 

0.004 

0.002 

O.OOJ 

1.00 ' ' 'i.bo' " ' 2.00ihaih o t$o'4.00 ' ' ' '4.50' 'she ' ' s.bo 's.io' ' 'ego ' iha ' '?»' BOO ' ' MO' ' ' V60' ' ' b.io' ' ' iico 

Mnu(4f 

Figure 1 HPLC-UV chromatogram of a 2.00 mg/l test substance solution [res. id. 1509]. 

i.oo' ' ' 'tha ' ' '2.00 :.feo' MO' 1S0 4,00' Vto '5.00' 's.w' ' eh 

Mnutos 

0.00 oso 7.00 7J50 (.00 5.50 8.60 »Jo ' ' io!o0 

Figure 2 HPLC-UV chromatogram of the blank accuracy sample [res. id. 1529]. 

3C0C001 

4SOCC0 

400000-

3W0OO30CO0O-

• 2OXO0-

150KO1CO0OO90000 

IOO ' b.io 0.20 ' oho o.W ' ' b.io' ' ' o.w ' ' bjo' ' ' oho ' ' bio 1^0 no ' 1.20' ' ' "1 io i.« ' i.bo 'ieo ' 1.70 ' 1.80' 'i.oo ' 2.00 

Aitaurt 

Figure 3 Regression line: response of the test substance as a function of concentration [cal. 

curve id. 1497]. 

Final report - Page 18 -
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8. MELTING AND BOILING TEMPERATURE 

8.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no. 102: "Melting Point / Melting Range", July 27, 1995. 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.1: "Melting/Freezing Temperature", Official Journal of the European 

Union no. L142, May 31, 2008. 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no. 103: "Boiling Point", July 27, 1995. 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.2: "Boiling Temperature", Official Journal of the European Union no. 

L142, May 31, 2008. 

8.2. Performance of the test 

The melting and boiling temperature of the test substance were determined using differential scanning 

calorimetry (DSC). This technique records the difference in heat flow to two crucibles, one filled with 

test substance and the other filled with an inert reference material, while these crucibles are subjected 

to a controlled temperature programme. When the test substance undergoes a transition, such as 

melting or evaporation, it is indicated by a deviation from the base line of the heat-flow record. 

Because melting and evaporation are processes in which heat is consumed, for these processes the 

deviation from the base line is in the endothermic direction. The melting temperature or boiling 

temperature (if any) of the test substance is evaluated from the DSC curve as the extrapolated onset 

temperature of the endothermic melting or evaporation peak. 

As a safety precaution for the DSC cell a preliminary test was performed using a thermogravimetric 

analyzer (TGA). 

Preliminary test 

6.69 mg test substance was heated with a rate of 20°C/minute from 25°C to 432°C. At this point 70% 

weight loss of the test substance was observed. After the experiment, the sample was cooled to 50°C 

and the consistency of the test substance was determined. 

Main study 

An overview with the experimental conditions of the main study is given beiow. 

Experiment First Second 

Sample amount (mg) 6.97 5.97 

Initial temperature (°C) 25 25 

Rate (°C/minute) 5 5 

Temperature 2 (*C) -90 -90 

Rate (°C/minute) 20 20 

End temperature (°C) 345 50 

The experiments were performed under a flow of nitrogen and at atmospheric pressure. 

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8.3. Analytical method 

Instrument Q100 differential scanning calorimeter (TA Instruments, New 

Castle, DE, USA) 

aluminum; closed with a lid in which a small hole was drilled 

empty sample container 

Sample container 

Reference 

Preliminary test 

Instrument 

Sample container 

Q50 thermogravimetric analyzer (TGA) (TA Instruments) 

aluminum; closed with a lid in which a small hole was drilled 

8.4. Interpretation 

The melting temperature is defined as the temperature at which the phase transitioh from solid to 

liquid state takes place at normal atmospheric pressure. This temperature ideally corresponds to the 

solidification or freezing temperature. 

The standard boiling temperature is defined as the temperature at which the vapour pressure of a 

liquid is the same as the standard pressure (1013.25 hPa). 

8.5. Results 

8.5.1. Preliminary test 

The TGA-curve of the test substance is shown in Figure 4. From 325°C upwards the weight of the 

sample decreased significantly. At 345°C the sample weight had decreased by 25%. 

After the experiment, black residue was observed on the lid of the sample container and a black 

molten residue remained in the sample container (original colour: yellow). The change of the colour 

indicated reaction and/or decomposition of the test substance. 

8.5.2. Main study 

The DSC-curve obtained with the first experiment is shown in Figure 5. 

During cooling, a glass transition was observed between -40°C and -60°C (results are archived in the 

raw data). 

During heating, a glass transition between -50°C and -25°C was observed (see Figure 5). The 

inflection point of the glass transition (melting point) of the test substance was -39.42°C. An 

exothermic peak was observed from 250'C. The effect was due to reaction and/or decomposition of 

the test substance. After the experiment, dark brown residue was observed on the lid of the sample 

container and a dark brown molten residue remained in the sample container. The change of the 

colour indicated reaction and/or decomposition of the test substance. 

The second experiment was performed to determine the duplicate glass transition temperature of the 

test substance. Figure 6 shows the DSC-curve obtained. Similar results to the first experiment were 

obtained. The inflection point of the glass transition was -39.53°C. After the experiment, the sample 

appeared to have been unchanged, I.e. no decomposition and/or evaporation were observed. 

8.6. Conclusion 

The melting and boiling temperature of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline were 

determined using DSC. 

The melting temperature (glass transition) of the test substance was -39°C (234K). 

Reaction and/or decomposition of the test substance was observed at temperatures of > 250°C (> 

523K). Boiling of the test substance was not observed below the temperature at which reaction and/or 

decomposition started. Based on this, the test substance has no boiling temperature. 

Final report - Page 20 -

Tin T i 1 
p. 24 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

8.7. Figures 

NOTOX Project 496981 

File. NOTOX2010 13201 S4713 

. 34 5.4 3" C 25.00% Loss 

\ 

\ 

432.23" C 70.00% Los» 

200 300 

Temperature (°C) 

40Q 500 

Univsrwt V*.7A TA Inalnimflnto 

Figure 4 TGA curve of the test substance. 

Fila NOTOX2010 13202 £6673 

100 200 

Temperature (°C) 

400 

Unlvtml V4.7A TA Imtnimwiti 

Figure 5 DSC curve of the test substance obtained with the first experiment. 

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Fife. NOTOX2010 13202 26622 

1 

0-

- 4 H , . . , , , , ; , , , , , . . , , . 1 1 . . 1 , , , 1 , , , 1 

-100 -30 -60 -40 -20 0 2D 40 60 

E>oup Temperature ('C) univarwi W A TA iratrumonts 

Figure 6 DSC curve of the test substance obtained with the second experiment. 

Final report - Page 22 -
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9. DENSITY 

9.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guideline for the Testing 

of Chemicals no. 109: "Density of Liquids and Solids", July 27, 1995. 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.3: "Relative Density", Official Journal of the European Union no. 

L142, May 31,2008. 

9.2. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA). 

9.3. Performance of the study 

The density (p) and relative density (DTM) of the test substance were determined using a pycnometer 

with a volume of 10 ml. With this method the density of the test substance is calculated from the 

difference in weight between the full and empty pycnometer and its volume. 

The volume of the pycnometer was determined first. The pycnometer was weighed empty. Then it was 

filled with water, dried on the outside and weighed again. The temperature of the water was 

measured. The procedure was performed in duplicate. The mean volume of the pycnometer was 

calculated. 

The pycnometer was filled with test substance, dried on the outside and weighed. The temperature of 

the test substance was measured. The test was performed in duplicate. The mean density and relative 

density of the test substance were determined. 

Handlings were performed at 20.0 ± 0.5°C. Weighings were performed on an analytical balance with 

an accuracy of 0.1 mg. 

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9.4. Interpretation 

The density (p) of a substance is the quotient of its mass and its volume and is expressed in SI units of 

kg/m3. 

The relative density (DTM) is the ratio between the mass of a volume of a substance to be examined, 

determined at 20°C, and the mass of the same volume of water, determined at 4°C (pjj a 0 = 

1000 kg/m3). The relative density has no dimension. 

Volume of the pycnometer (V) V = ——— [cm3] 

P

2 0 

"H2O 

where: 

A = weight of the empty pycnometer [g] 

B = weight of the pycnometer with water [g] 

p ^ 0 = density of water at 20°C i.e. 0.9982 g/cm3 (CRC 

Handbook of Chemistry and Physics, 1981) 

Density (p) p = ° " Amaa n [g/cm3] 

^meai i 

where: 

C = weight of the pycnometer with test substance [g] 

9.5. Results 

The results of the tests on the determination of the density of the test substance are given in Table 5. 

The individual measurements were obtained with an accuracy of < 0.01 g/cm3. According to the 

guideline, the density of the test substance is given as the mean value. 

Table 5 Density of the test substance 

Testl Test 2 Mean 

Mass empty pycnometer (A) 

Mass pycnometer with water (B) 

30.9325 g 

40.6660 g 

30.9310 g 

40.6585 g 

30.93175 g 

Volume of the pycnometer 9.7511 cm3 9.7450 cm3 9.7481 cm3 

Mass pycnometer with test substance (C) 42.7754 g 42.7787 g 

Density 1.215 g/cm3 1.215 g/cm3 1.22 g/cm3 

9.6. Conclusion 

A pycnometer was used for the determination of the density and relative density of 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

The density of the test substance at 20°C was 1.22 g/cm3 (1.22 x 103 kg/m3). 

The relative density was 1.22. 

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10. VAPOUR PRESSURE 

10.1. Guidelines 

European Community (EC), EC no. 761/2009, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.4: "Vapour Pressure", Official Journal of the European Union no. 

L220, August 24, 2009. 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no.104: "Vapour Pressure", March 23, 2006. 

10.2. Performance of the test 

The vapour pressure of the test substance (PT) was determined by the isothermal thermogravimetric 

effusion method, The method is validated in the range 10"8 -103 Pa using a set of five reference 

substances with known vapour pressures (see paragraph 10.4). The validity of the method was 

verified maximum one month before this study using hexachlorobenzene (>99%, Fluka Chemie, 

Buchs, Switzerland) as reference control substance. The logarithm of the evaporation rate at 20°C (log 

vT, 20) deviated < 10% from the average value obtained during the validation test. From this, it was 

possible to apply the constants obtained with the validation test for the determination of the vapour 

pressure of the test substance. 

Approximately 8.24 - 13.3 mg of the test substance was applied to the surface of a roughened glass 

plate as a homogeneous layer. The weight loss of the test substance was measured continuously as a 

function of time. The following temperature program was used. 

Start temperature 100°C 

Isothermal intervals 10 minutes 

Increment steps 10°C 

End temperature 240°C 

The weight loss of the test substance was measured continuously as a function of time at defined 

isothermal temperatures. The evaporation rate of the test substance (vT) was calculated from the 

weight loss of the compound. The PT values at the defined isothermal temperatures were determined 

using the vapour pressure equation with the constants c and d specific for the experimental 

arrangement. Linear regression analysis of the log PT values and 1/T was performed using a least 

squares method. The PT values of the test substance at 20°C and 25°C were determined using the 

vapour pressure regression curve. 

10.3. Analytical method 

Instrument Q50 thermogravimetric analyzer (TGA) (TA Instruments, New 

Castle, DE, USA) 

Surface of the glass plate 0.50 cm2 

The experiments were performed under a flow of nitrogen and at atmospheric pressure. 

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10.4. Reference substances 

Validation of the method is performed at least once every year (NOTOX project 495910). 

Each references substance was measured in duplicate by TGA using a temperature program that was 

specific for the substance. Plots of log v T obtained at elevated temperatures and 1/T were inter- or 

extrapolated to determine the log v T values at 20°C (log vTl2o)- The log v T 2 0 values were plotted against 

the logarithm of the vapour pressure at 20°C in Pascal units (log PT.M)- Linear regression analysis 

using the least squares method yielded an equation of log PT,2o = 1.16 x log vT,2o

 + 4.34. The 

coefficient of correlation (r) was > 0.99, The constants c and d specific for the experimental 

arrangement were 1.16 and 4.34, respectively. 

Reference' PT values Corresponding PT values Literature 

20°C 25°C 

Reference substance [Pal [mm Hgl fPa] rmm Hgl 

Benzo(ghi)perylene 1.33 x itr 8 1.00 x 10' 1 0 6.46 x 10" 8 # 4.85 x10- 1 0 * 1 

Chrysene 8.40 x 10"7 6.30 x 10"9 5.76 x 10' 7 # 4.32 x 10" 9 # 2 

Hexachlorobenzene 1.47 x 10"3 1.10 x 10"5 4.21 x 10' 3 # 3.16 x 10- 5" 1 

Naphthalene 7.56 x 10° 5.67 x 10"a 1.15 x 101 8.61 x 10"2 3 

Water 2.34 x 103 1.75 x 101 3.17 x 103 2.38 x 101 3 

Determined by the vapour pressure regression curve of the validation study, No literature value was available. 

1 Verschueren, K., Handbook of environmental data on organic chemicals, 4l h edition, Wiley Chemistry 

2. Borges, H T , Formal toxicity summary for Chrysene, The Risk Assessment Information System (RAIS), 1994 

3. Lide, D R , CRC Handbook of Chemistry and Physics, 79"' edition, CRC Press LLC, Boca Raton, FL, USA, 1998 

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10.5. Interpretation 

10.5.1. Specifications 

The vapour pressure is a function of the temperature and is specified in Pascal (Pa) or in mm Hg, 

10.5.2. Formulas 

Evaporation rate (vT) v T = [g/cm2/h] 

where: 

Am = weight loss of the test substance [g] 

F ~ surface of the sample plate [cm2] 

t = elapsed time for the weight loss [h] 

Vapour pressure equation logPj = c iogv T + d 

where: 

c = constant specific for the experimental arrangement 

d = constant specific for the experimental arrangement 

Vapour pressure regression curve log PT = a 1/T + b 

where: 

T = temperature [K] 

a = slope [Kj 

b - intercept 

Final report - Page 27 -

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10.6. Results 

A representative weight loss curve of the test substance is shown in Figure 7. The results of the 

isothermal TGA analysis for the test substance and the vapour pressure at 20°C and 25°C are given in 

Table 6 and Table 7. 

The plot of the log PT of the test substance as function of the reciprocal temperatures is shown in 

Figure 8. The equation of the curve was: log PT,2o = -6573 x 1/T + 16.16 (r = 0.997, n = 8). 

Table 6 Results of the isothermal TGA analysis 

Temperature 

PCI 

Weight loss 

fpq/minl 

v T 

[q/cm2/hl 

log v T log PT PT 

[Pal 

150 5.003 5.97 x 10"4 -3.22 0.61 4.1 

5.111 6.10 x 10- 4 -3.21 0.62 4.2 

160 10.42 1.24 x 10'3 -2.91 0.98 9.6 

11.37 1.36 x 10* -2.87 1.03 11 

170 20.46 2.44 x 10'3 -2.61 1.32 21 

22.58 2.70 x 10"3 -2.57 1.37 23 

180 36.74 4.39 x 10"3 -2.36 1.61 41 

41.75 4.80 x 10"3 -2.30 1.68 48 

Table 7 Vapour pressure of the test substance 

Temperature log PT PT PT 

rci [Pa] [mm Hgl 

20 -6.26 5.5 x 10- 7 4.1 x 10'9 

25 -5.88 1.3 x 10* 9.9 x 10"9 

10.7. Conclusion 

The isothermal TGA effusion method was applied for the determination of the vapour pressure of 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

The vapour pressure of the test substance at 20°C (293K) and 25°C (298K) was: 

20 

[Pa] 

°C 

[mm Hq] 

25 

[Pal 

°C 

[mm Hg] 

Test substance 5.5 x 10"7 4.1 x 109 1.3 x 10* 9.9 x 10"9 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

10.8. Figures 

File; NOTOX201013202 Z47Z2 

6400 

-10.«|ifl/mln 

160,00*C 

1SO.0CC -20.46 tig/m In 

-5.003|ifj/mln 17Q.O0'C 

-36.74(jg/m/n 

lao.oo-c' 

Tim e (min ) Universal V4.7A TA Ina&urnflnti 

Figure 7 Weight loss curve of the test substance. 

0.4 -

0.2 -

0 -I 1 1 1 1 1 1 1 1 1 1 

0.00218 0.0022 0.00222 0.00224 0.00226 0.00228 0.0023 0.00232 0.00234 0.00236 0.00238 

1/T [K-1] 

Figure 8 Plot of log PT of the test substance versus 1/T. 

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11. WATER SOLUBILITY 

11.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no. 105: "Water Solubility", July 27, 1995. 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.6: "Water Solubility", Official Journal of the European Union no. 

L142, May 31, 2008. 

11.2. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA) 

Double distilled water Waldeck, Munster-Roxel, Germany 

Acetonitrile VWR International, Leuven, Belgium 

All reagents were of analytical grade, unless specified otherwise. 

11.3. Performance of the study 

Two methods are available for the determination of the water solubility of a test substance: 

1. Column elution method 

The method is based on the elution of the test substance with water from a column which is charged 

with an inert support material, such as bare silica particles, coated with an excess of the test 

substance. It can be applied to test substances with a water solubility of < 10"2 g/l. 

2. Flask method 

The mass concentration of the test substance in aqueous solutions saturated with the test substance 

will be determined after stirring or shaking at a specific temperature for a specific period of time. The 

method can be used for test substances with a water solubility of £ 10"2 g/l. 

A preliminary test was carried out as a range-finding test prior to performance of the column elution 

method or flask method. 

Preliminary test 

Test substance was stirred with double distilled water at a nominal concentration of 2.02 g/l at 

20.2 ± 0.6°C for 23 hours (1 day). After stirring, the water phase was centrifuged, treated and 

analysed. 

The centrifugation method was checked and was found to be sufficient to remove all undissolved test 

substance. 

In the preliminary test the water solubility of the test substance was £ 10"2 g/l. Therefore, the flask 

method was chosen for the main study. 

Main study 

Three aliquots between 1.44 and 1.48 mg of the test substance were weighed into glass vessels. To 

each vessel, 100 ml double distilled water was added. The vessels were placed on a magnetic stirring 

device in a climate room at 19.9 ± 0.7°C where the content of each vessel was stirred for 24, 48 or 

72 hours. As the blank, 100 ml double distilled water was stirred for 72 hours. 

After the stirring period, the water phases-were two-times centrifuged at 16421 g and 20°C for 

5 minutes. The aqueous phases were diluted in a 7:3 (v:v) ratio with acetonitrile and analysed. If 

necessary, the samples were further diluted with 30/70 (v/v) acetonitrile/water to obtain concentrations 

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within the calibration range. The pH of each of the water mixtures (except for the blank) was 

determined after removal of undissolved test substance. 

11.4. Analytical method 

11.4.1. Analytical conditions 

Quantitative analysis was performed according to the analytical method as validated in chapter 7. 

11.4.2. Preparation of the calibration solutions 

Stock solutions 

Stock solutions of the test substance were prepared in acetonitrile at concentrations of 1712 -

2236 mg/l. 

Calibration solutions 

Calibration solutions in the concentration range of 0.0401 - 2.00 mg/l (preliminary study) or 0.0401 -

59.9 mg/l (main study) were prepared from two stock solutions. The end solution of the calibration 

solutions was 30/70 (v/v) acetonitrile/water. 

11.4.3. Sample injections 

Calibration solutions were injected in duplicate. Test samples were analysed by single injection. 

11.4.4. Calibration curves 

Calibration curves were constructed using five concentrations. For each concentration, two responses 

were used. Linear regression analysis was performed using the least squares method with a 

1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve. 

11.5. Interpretation 

11.5.1. Specifications 

The water solubility of a test substance is specified by the saturation mass concentration of the test 

substance in water at a given temperature and is expressed in g/l. 

11.5.2. Formulas 

Response (R) Peak area of the test substance [units] 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

Maximum difference 

highest value-lowest value x1Q0% 

meanvalue 

where: 

'mean value' is the mean of the highest and lowest value. 

Calibration curve R = a C N + b 

where: 

CN = nominal concentration [mg/l] 

a = slope [units x l/mg] 

b = intercept [units] 

Analysed concentration (CA) (R-b ) 

x d x 10 - 3 [g/i] 

a 

where: 

d = ( dilution factor 

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11.6. Results 

11.6.1. Preliminary test 

The content of test substance dissolved in the water sample was 2.93 g/l. 

11.6.2. Main study 

The results for the samples taken at 24, 48 and 72 hours are given in Table 8. 

The maximum difference (MD) of the concentrations determined in the 24, 48 and 72 hour test 

samples was £ 15%. Based on this, the water solubility of the test substance is given as the mean 

value of the analysed concentrations. 

No test substance was detected in the pretreated sample from the blank water mixture. 

Table 8 Main study - water solubility of the test substance 

Stirring time 

[hours] 

Analysed 

concentration 

[g/i] 

Mean 

[g/i] 

MD 

[%] 

pH 

24 3.27 3.34 3.1 9.5 

48 3.36 9.1 

72 3.38 8.3 

11.7. Conclusion 

The flask method was applied for the determination of the water solubility of p-(2,3-epoxypropoxy)-

N,N-bis(2,3-epoxypropyl)aniline. 

The water solubility of the test substance at 20°C was 3.34 g/l. 

The pH of the aqueous samples was 8.3 - 9.5. 

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12. FLASH-POINT 

12.1. Guidelines 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.9: "Flash-point", Official Journal of the European Union no. L142, 

May 31, 2008. 

United Nations (UN), UN no. ST/SG/AC.10/11/Rev.5: Recommendations on the Transport of 

Dangerous Goods, Part III: Classification Procedures, Test Methods and Criteria Relating to Class 2, 

Class 3, Class 4, Division 5.1, Class 8 and Class 9, Paragraph 32.4.1: "Non-Viscous Flammable 

Liquids", 2009. 

The International Organization for Standardization (ISO), ISO Guide 2719: "Determination of Flash 

Point - Pensky Martens Closed Cup method", 20.02. 

ASTM International, ASTM D 93: "Standard Test Methods for Flash Point by Pensky-Martens Closed 

Cup Tester", December 10, 2002. 

12.2. Performance of the test 

The flash-point of the test substance was determined using a model APM-7 Pensky-Martens closed 

cup automatic flash-point tester (Tanaka Scientific Limited, Tokyo, Japan). 

12.2.1. Preliminary test 

The test cup was filled with approximately 70 ml test substance and placed in the heating block of the 

flash-point tester. The lid with a thermocouple for the determination of the flash-point and a resistance 

thermometer for measuring the temperature was placed on the cup. The test cup was heated with a 

rate of approximately 5°C/minute. The sample was stirred during heating. The flame igniter was 

applied so that it was lowered into the vapour space of the cup in 0.5 seconds, left in its lowered 

position for 1 second, and quickly raised to its high position again. The sample was not stirred when 

the igniter was applied. 

The temperature of the test substance at the start of the test was 16.2°C. Starting at 20°C, an attempt 

was made to ignite the vapour of the test substance for every 2°C temperature rise. 

12.2.2. Main study 

Two tests were performed. The temperature of the test substance at the start of the test was 19.3°C 

and 22.rc , respectively. Starting at 206°C, the flame igniter was applied for every 2°C temperature 

rise. 

The atmospheric pressure was 1014 hPa. 

12.3. Interpretation 

The flash-point of the test substance is the lowest temperature, corrected to a barometric pressure of 

1013 hPa, at which the test substance evolves vapours in such amount that a flammable vapour/air 

mixture is produced in the test vessel. 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

Flash point (Tc) Tc =T 0 +0.025(1013-p) 

where: 

To = 

P 

flash point of the test substance at ambient atmospheric 

pressure [°C] 

ambient atmospheric pressure [hPa] 

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p. 37 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

12.4. Results 

12.4.1. Preliminary test 

The estimated flash-point of the test substance was 228°C. 

12.4.2. Main study 

The results of the main study on the determination of the flash-point of the test substance are given in 

Table 9. 

The difference between the results obtained with the two tests was £ 6°C. 

Table 9 Main study - flash-point of the test substance 

Test Flash-point Lowest flash-point 

[°C1 rci 

1 228 228 

2 228 

12.5. Conclusion 

The Pensky-Martens closed cup method was applied for the determination of the flash-point of 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

The flash-point of the test substance was 228°C. 

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13. AUTO-IGNITION TEMPERATURE 

13.1. Guidelines 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.15: "Auto-Ignition Temperature (Liquids and Gases)", Official Journal 

of the European Union no. L142, May 31, 2008. 

Deutsches Institut fur Normung (DIN), DIN Guide 51794: "Determining the Ignition Temperature of 

Petroleum Products", May 2003. 

International Electrotechnical Commission (IEC), IEC standard 79-4: "Electrical Apparatus for 

Explosive Gas Atmospheres, Part 4, Method ofTestfor Ignition Temperature", 1975. 

13.2. Performance of the test 

The auto-ignition temperature of the test substance was determined using a commercially available 

auto-ignition temperature apparatus (Chilworth Technology, Southampton, UK). The method is 

applicable to test substances with an auto-ignition temperature in the range 75°C -650°C. 

In every ignition test, liquefied test substance was introduced into a preheated test vessel using a 

volumetric pipette. It was observed visually whether a reaction (clearly perceptible flame and/or 

explosion) of the sample within five minutes after introduction took place at the prevailing temperature. 

After each ignition test, any vaparizable components left in the flask were blown out with air (Air 

Products, Amsterdam, The Netherlands). 

13.2.1. Preliminary test 

Starting at 200°C, for every 20°C temperature rise, 70 pi test substance was introduced into the test 

vessel until ignition of the test substance was first observed (i.e. at 440°C). 

Starting at 455°C, for every 5°C decrease, 70 pi test substance was tested until no ignition of the test 

substance had been observed. The temperature at which ignition was last observed is the estimated 

auto-ignition temperature. 

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13.2.2. Main study 

Three tests were performed. The sample volumes applied and temperature range of each test are 

given below. Starting at the "high temperature", for every 2°C decrease, the prevailing volume of test 

substance was tested until no ignition of the test substance had been observed. The temperature at 

which ignition was last observed is the auto-ignition temperature at the sample volume applied. This 

procedure was repeated with the other volumes until a minimum auto-ignition temperature for each 

test was obtained. With these results the auto-ignition temperature of the test substance was 

determined. 

The atmospheric pressure during the performance of the main study was between 1012.6 and 1013.9 

hPa. 

Test Volume Temperature (°C) 

[ull start end 

Testl 70 432 420 

250 410 404 

650 425 417 

Test 2 85 428 410 

175 427 405 

350 423 415 

Test 3 60 431 419 

200 420 408 

450 430 416 

13.3. Interpretation 

The degree of auto-ignitability is expressed in terms of the auto-ignition temperature. The auto-ignition 

temperature is the lowest temperature at which the test substance ignites when mixed with air under 

the conditions defined in the test method. 

13.4. Results 

13.4.1. Preliminary test 

The estimated auto-ignition temperature was 425°C. 

13.4.2. Main study 

The results of the main study on the determination of the auto-ignition temperature of the test 

substance are given in Table 10. 

Ignition of the test substance within 5 minutes after introduction was observed with each sample 

volume and with each test a minimum auto-ignition temperature was obtained. The maximum 

deviation between the three values was < 20°C. According to the guidelines, no further testing was 

required. 

The lowest temperature at which ignition of the test substance occurred was 410°C, This temperature 

was a multiple of 5°C and therefore given as the auto-ignition temperature of the test substance. 

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Table 10 Main study - auto-ignition temperature of the test substance 

Test Volume 

run ' 

Auto-ignition 

temperature 

m 

Ignition delay 

Tsl 

Colour of the 

flame 

Minimum 

auto-ignition 

temperature 

PCI 

1 70 426 11 Orange 410 

250 410 18 Orange 

650 423 13 Orange 

2 85 416 19 Orange 411 

175 411 21 Orange 

350 421 27 Orange 

3 60 425 25 Orange 414 

200 414 16 Orange 

450 422 21 Orange 

s

 13.5. Conclusion 

Commercially available auto-ignition temperature apparatus was used for the determination of the 

degree of auto-ignStability of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

The test substance is auto-ignitable with an auto-ignition temperature of 410°C at 1012.6 -

1013.9 hPa. 

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14. OXIDIZING PROPERTIES 

14.1. Guideline 

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.21: "Oxidizing Properties (Liquids)", Official Journal of the European 

Union no. L142, May 31, 2008. 

United Nations (UN), UN no. ST/SG/AC.10/11/Rev.5: Recommendations on the Transport of 

Dangerous Goods, Part III: Classification Procedures, Test Methods and Criteria Relating to Class 2, 

Class 3, Class 4, Division 5.1, Class 8 and Class 9, Test 0.2: "Test for Oxidizing Liquids", 2009. 

United Nations (UN), UN no. ST/SG/AC.10/11/Rev.5: Recommendations on the Transport of 

Dangerous Goods, Appendix 6: "Screening Procedures", 2009. 

14.2. Statement 

A test substance is considered an oxidizing substance when a mixture of the substance and cellulose 

in a 1:1 ratio (by mass) spontaneously ignites or the mean pressure rise time of the mixture is less 

than or equal to mean pressure rise time of a 1:1 reference mixture (by mass) of 65% aqueous nitric 

acid and cellulose. 

The molecular structure of the test substance is shown in paragraph 6.1. The test substance does not 

contain groups that act as an oxidizing agent. The oxygen atoms that are present in the molecular 

structure of the test substance are chemically bonded to carbon or hydrogen. The molecular structures 

of impurities at <, 6% were not taken into account but these are not expected to be of influence on the 

oxidizing properties of the test substance. 

In conclusion, p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline has no oxidizing properties. 

Final report 

Tin 

- Page 38 -

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15. HYDROLYSIS AS A FUNCTION OF PH 

15.1. Guideline 

European Community (EC), EC no. 440/2008, Part C: Methods for the Determination of Ecotoxicity, 

Guideline C.7: "Degradation - Abiotic Degradation: Hydrolysis as a Function of pH", Official Journal of 

the European Union no. L142, May 31, 2008. 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no. 111: "Hydrolysis as a Function of pH", April 13, 2004. 

15.2. Reagents 

Water 

Acetonitrile 

Sodium acetate 

Acetic acid, 100% 

Potassium d/-hydrog en phosphate 

Boric acid 

Potassium chloride 

Sodium azide 

Sodium hydroxide 

All reagents were of analytical grade, 

15.3. Buffer solutions 

Acetate buffer pH 4, 0.1 M 

Phosphate buffer pH 7, 0.1 M 

Borate buffer pH 9, 0.1 M 

15.4. Performance of the study 

The rate of hydrolysis of the test substance as a function of pH was determined at pH values normally 

found in the environment (pH 4-9). 

Preliminary test - Tier 1 

Test substance solutions were prepared in the buffer solutions at a target concentration of 1000 mg/l, 

after stirring for at least 60 minutes the test substance was not completely dissolved. Each solution 

was filter-sterilised through a 0.2 pm FP 30/0.2 CA-S filter (Whatman, Dassel, Germany) and 

transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 

5 minutes. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 ml test 

solution and placed in the dark in a temperature controlled environment at 49.9°C ± 0.1 °C. 

Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA) 

Biosolve, Valkenswaard, The Netherlands 

Merck, Darmstadt, Germany 

Merck 

Merck 

Merck 

Merck 

Merck 

Boom, Meppel, The Netherlands 

unless specified otherwise. 

solution of 16.6% 0.1 M sodium acetate and 83.4% 0.1 M acetic 

acid. The buffer contains 0.0009% (w/v) sodium azide. 

solution of 0.1 M potassium dihydrogenphosphate adjusted to 

pH 7 using 10 N sodium hydroxide. The buffer contains 

0.0009% (w/v) sodium azide. 

solution of 0.1 M boric acid and 0.1 M potassium chloride 

adjusted to pH 9 using 10 N sodium hydroxide. The buffer 

contains 0.0009% (w/v) sodium azide. 

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The concentration of the test substance in the test samples was determined immediately after 

preparation (t=0) and after 5 days. The samples taken at t=5 days were cooled to room temperature 

using running tap water. The samples were diluted in a 1:1 (v:v) ratio with 60/40 (v/v) acetonitrile/buffer 

solution (the same buffer as where test substance solution in was prepared) and analysed. If 

necessary, the samples were further diluted with 30/70 (v/v) acetonitrile/buffer solution to obtain 

concentrations within the calibration range. 

Blank buffer solutions were treated similarly as the test samples and analysed at t-0. 

The pH of each of the test solutions (except for the blanks) was determined at each sampling time. 

Main study-Tier 2 

The buffer solutions were filter-sterilised through a 0.2 urn FP 30/0.2 CA-S filter (Whatman, Dassel, 

Germany) and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through 

the solution for 5 minutes. The test substance was spiked to the solutions at a target concentration of 

500 mg/l using a spiking solution in acetonitrile. The obtained solution was filtered again through a 0.2 

urn FP 30/0.2 CA-S filter to remove possibly present undissolved test substance. For each sampling 

time, duplicate sterile vessels under vacuum were filled with 6 ml test solution and placed in the dark 

in a temperature controlled environment. 

Note: the spiking volume was < 1% of the sample volume. Nominal concentrations were not corrected 

for the spiking volume. 

The concentrations of the test substance were determined immediately after preparation (t=0) and at 

several sampling points after t=0. The concentration of the bulk solution was also determined. 

The samples taken after t=0 were cooled to room temperature using running tap water (except the 

samples stored at 20°C). The samples were diluted to 30/70 (v/v) acetonitrile/borate buffer pH 9 as 

test substance stability was found to be optimal at pH 9. 

Blank buffer solutions containing a similar content of blank spiking solution were treated similarly as 

the test samples and analysed att=0. 

The pH of each of the test solutions (except for the blanks) was determined at least at the beginning 

and at the end of the test. 

The study was performed at the following temperatures: 

pH code • Temperature I Temperature II Temperature III 

pH4 20.2°C ± 0.6°C 40.0°C±0.1°C 49.9°C±0.1°C 

pH 7 20.2°C±0.6°C 40.0 Q C±0.1°C 49.9°C±0.1°C 

pH9 20.2°C ± 0.6°C 40.o°c±o.rc 49.8°C ± 0.2°C 

Identification of hydrolysis products - Tier 3 

Research to investigate the identity or nature and rates of formation and decline of hydrolysis products 

was not required since the studies were conducted according to the testing guidelines provided in 

Annex VIII (volume 10-100 tons/year). 

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15.5. Analytical method 

15.5.1. Analytical conditions 

Quantitative analysis was performed according to the analytical method as validated in chapter 7. 

15.5.2. Preparation of the calibration solutions 

Stock solutions 

Stock solutions of the test substance were prepared in acetontrile at concentrations of 2000 -

4385 mg/l. 

Calibration solutions 

Calibration solutions in the concentration range 0.04 - 2 mg/l were prepared from two stock solutions. 

The end solution of the calibration solution was: 

pH code Preliminary test Main test 

pH4 30/70 (v/v) acetonitrile/acetate buffer pH4 30/70 (v/v) acetonitrile/borate buffer pH9 

pH 7 30/70 (v/v) acetonitrile/phosphate buffer pH7 30/70 (v/v) acetonitrile/borate buffer pH9 

pH 9 30/70 (v/v) acetonitrile/borate buffer pH9 30/70 (v/v) acetonitrile/borate buffer pH9 

15.5.3. Sample injections 

Calibration solutions were injected in duplicate. Test samples were analysed by single injection. 

15.5.4. Calibration curves 

Calibration curves were constructed using at least four concentrations. For each concentration, two 

responses were used. Linear regression analysis was performed using the least squares method with 

a 1/concentration2 weighting factor. If necessary, the calibration line was excluding one data point 

since the back calculated accuracy was > 15% from the nominal concentration. The coefficient of 

correlation (r) was > 0.99 for each curve. 

15.6. Interpretation 

Response (R) Peak area of the test substance [units] 

Calibration curve R = a C N + b 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

where: 

CN = r nominal concentration [mg/l] 

slope [units x l/mg] 

intercept [units] 

a = 

b = 

Analysed concentration (CA) (R-b ) 

xd [mg/l] 

a 

where: 

d = dilution factor 

Recovery x 100% 

where: 

CN = nominal concentration [mg/l] 

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p. 45 

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Degree of hydrolysis 

meanCo - C( 

meanC0 

x100% 

where: 

C0 = concentration at t=0 

Ct = concentration at t=5 days 

Relative concentration (Cr) Cr = 

meanC0 

•x100% 

where: 

C0 ~ concentration at t=0 

Ct = concentration at t^x hours 

Pseudo-first order curve 10 logCr = -ax t + b 

where: 

t = time [hours] 

a = slope [hours'1] 

b = intercept 

Rate constant (k obs ) =-ax2.30 3 [hours' 1] 

where: 

2.303 is the conversion factor between natural and base 

10 logarithms 

Arrhenius equation Inkobs =^= p + !nA R x T 

where: 

A plot of In k o b s versus 1/T gives a linear relationship with a 

slope of - E/R and intercept In A 

Half-life time (ty a ) tya = [hours] 

kobs 

Final report - Page 42 -

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15.7. Results 

15.7.1. Preliminary test - Tier 1 

The analytical results of the preliminary test are given in Table 11 and Table 12. 

At pH 4, pH 7 and pH 9 a degree of hydrolysis of £ 10% after 5 days was observed. According to the 

guideline, the higher Tier test was required to determine the half-life time of the test substance. 

No test substance was detected in the blank buffer solutions pH 4 and pH 7. 

In the blank buffer solutions of pH9 a small response was observed at the test substance retention 

time, the contribution base on area to the samples at t=0 was 0.09%, this was negligible. 

The mean recoveries of the buffer solutions fell within the criterion range of 70-110%. It demonstrated 

that the analytical method was adequate to support the hydrolysis study on the test substance. 

Table 11 Preliminary test - hydrolysis of the test substance at pH 4, pH 7 and pH 9 

pH code Sampling time Analysed 

concentration 

[mg/l] 

Degree of hydrolysis 

[%] 

pH code Sampling time Analysed Actual pH 

concentration 

[mg/l] Individual Mean 

Actual pH 

pH4 0 hours 786 4.0 

782 4.0 

5 days 0.0776

 1 100 100 4.0 

0.0616

 1 100 4.0 

pH 7 0 hours 876 7.1 

891 7.1 

5 days 0.0412

 1 100 100 7.1 

0.115 100 7.1 

pH9 0 hours 978 9.0 

956 9.0 

5 days 0.248 100 100 9.0 

n.d. 100 9.0 

n.d. not detected 

Extrapolated value 

Table 12 Recoveries 

pH code Nominal 

concentration 

[mg/ll 

Analysed 

concentration 

[mg/ll 

Recovery 

[%l 

Mean 

recovery 

[%l 

pH4 1122 786 70 70 

1122 782 70 

pH7 994 876 88 89 

994 891 90 

pH9 1055 978 93 92 

1055 956 91 

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15.7.2. Main study - Tier 2 

PH4 

The analytical results of the main study are given in Table 13 - Table 16. 

No test substance was detected in the blank buffer solutions. 

The mean recoveries of the buffer solutions relative to the stock solution fell within the criterion range 

of 90-110%. It demonstrated that the analytical method was adequate to support the hydrolysis study 

on the test substance. 

Table 13 Main test - hydrolysis of the test substance at 20°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/]" - [%] 

0 462 100 2.00 4.0 

0 461 100 2.00 4.0 

47 318 69 1.84 4.0 

47 323 70 1.85 4.0 

71 275 60 1.78 4.0 

71 276 60 1.78 4.0 

120.5 186 40 1.61 4.0 

120.5 189 41 1.61 4.0 

144 135 29 1.47 4.0 

144 136 29 1.47 4.0 

167.5 117 25 1.40 4.0 

167.5 117 25 1.40 4.0 

191 98.0 21 1.33 4.0 

191 97.7 21 1.33 4.0 

215 85.7- 19 1.27 4.0 

215 87.1 19 1.28 4.0 

294.5 56.2 12 1.09 3.9 

294.5 56.2 12 1.09 3.9 

359 32.3 7.0 0.85 4.0 

359 31.4 6.8 0.83 4.0 

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Table 14 Main test - hydrolysis of the test substance at 40°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] [%] 

0 126 100 2.00 4.0 

0 126 100 2.00 4.0 

2 109 86 1.93 4.0 

2 109 86 1.94 4.0 

4.5 91.7 73 1.86 4.0 

4.5 90.7 72 1.86 4.0 

7 73.0 58 1.76 4.0 

7 75.5 60 1.78 4.0 

7.6 73.1 58 1.76 4.0 

7.6 73.3 58 1.76 4.0 

22.75 25.5 20 1.31 4.0 

22.75 25.2 20 1.30 4.0 

25.5 20.0 16 1.20 4.0 

25.5 20.4 16 1.21 4.0 

28.25 17.0 13 1.13 4.0 

28.25 17.3 14 1.14 4.0 

31 14.9 12 1.07 4.0 

31 14.3 11 1.05 4.0 

47.5 4.82 3.8 0.58 4.0 

47.5 4.84 3.8 0.58 4.0 

Table 15 Main test - hydrolysis of the test substance at 50°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] [%] 

0 474 102 2.01 4.0 

0 459 98 1.99 4.0 

1 385 83 1.92 4.0 

1 396 85 1.93 4.0 

2.25 310 66 1.82 4.1 

2.25 305 65 1.82 4.0 

3 269 58 1.76 4.0 

3 274 59 1.77 4.1 

4 228 49 1.69 4.1 

4 228 49 1.69 4.1 

5 197 42 1.63 4.1 

5 192 41 1.61 4.1 

6.25 151 32 1.51 4.1 

6.25 152 33 1.51 4.1 

7 133 28 1.45 4.1 

7 132 28 1.45 4.1 

23.5 7.56 1.6 0.21 4.1 

23.5 7.26 1.6 0.19 4.1 

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Table 16 Recoveries 

Temperature 

(°C) 

Bulk solution 

concentration 

frng/ll 

Analysed 

concentration 

rmg/ll 

Recovery

 1 

[%1 

Mean 

recovery 

[%1 

20 453 462 102 102 

461 102 

40 122 126 103 103 

126 104 

50 455 474 104 102 

459 101 

Recovery was determined relative to the concentration of the prepared bulk solution. A relatively high spiking 

concentration had to be used to allow dilution of the samples to the end solution {30/70 (v/v) acetonitrile/borate buffer 

pH 9) to ensure sample stability during chromatographic analysis and sufficient analytical sensitivity. 

For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 

10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The 

half-life times of the test substance were determined according to the model for pseudo-first order 

reactions. 

All logarithms of the relative concentrations were correlated with time using linear regression analysis. 

Figure 9 illustrates the regression curves and Table 17 shows the statistical parameters. 

Table 17 Statistical parameters of the regression curves 

Temperature 

(°C) 

Slope 

fhours"1l 

Intercept Coefficient of 

correlation 

20 -3.21 x 10'3 1.98 0.996 

40 -3.00 x 10'2 1.99 0.9997 

50 -7.65 x 10'2 2.00 0.99991 

The rate constant (k ot)S) and half-life time of the test substance at each temperature was obtained and 

the Arrhenius equation was used to determine the rate constant and half-life time at 25°C {see Table 

18). 

Table 18 Rate constants (k obs ) and half-life time (tV]) 

Temperature 

PCI 

kobs 

[hours"11 

20 7.39 x 10'3 3.9 days 

25 1.33 x 10"2 2.2 days 

40 6.90 x 10"2 10 hours 

50 1.76 x 10"1 3.9 hours 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

oH7 

The analytical results of the main study are given in Table 19 - Table 22. 

No test substance was detected in the blank buffer solutions. 

The mean recoveries of the buffer solutions relative to the stock solution fell within the criterion range 

of 90-110%, It demonstrated that the analytical method was adequate to support the hydrolysis study 

on the test substance. 

Table 19 Main test - hydrolysis of the test substance at 20°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] [%] 

0 253 100 2.00 7.1 

0 252 100 2.00 7.1 

7 249 99 1.99 7.1 

7 256 101 2.01 7.1 

71 156 62 1.79 7.1 

71 155 61 1.79 7.1 

95 133 53 1.72 7.1 

95 134 53 1.73 7.1 

144.5 94.5 37 1.57 7.1 

144.5 94.2 37 1.57 7.1 

168 68.5 27 1.43 7.1 

168 69.5 28 1.44 7.1 

191.5 59.8 24 1.37 7.1 

191.5 59.9 24 1.38 7.1 

215 50.5 20 1.30 7.1 

215 50.5 20 1.30 7.1 

239 44.3 18 1.24 7.1 

239 44.9 18 1.25 7.1 

318.5 29.6 12 1.07 7.1 

318.5 30.1 12 1.08 7.1 

383 17.5 6.9 0.84 7.1 

383 17.5 6.9 0.84 7.1 

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Table 20 Main test - hydrolysis of the test substance at 40°C 

Sampling time 

[hours] 

Analysed 

concentration 

[mg/] 

Relative 

concentration 

[%] 

Logarithm relative 

concentration 

Actual pH 

0 328 100 2.00 7.1 

0 326 100 2.00 7.1 

2.75 301 92 1,96 7.1 

2.75 306 93 1.97 7.1 

5 257 79 1.90 7.1 

5 261 80 1.90 7.1 

6.5 250 76 1.88 7.1 

6.5 245 75 1.87 7.1 

23.25 69.2 21 1.33 7.1 

23.25 r 68.9 21 1.32 7.1 

25.75 57.3 18 1.24 7.1 

25.75 58.3 18 1.25 7.1 

28.25 49.4 15 1.18 7.1 

28.25 50.6 15 1.19 7.1 

30.75 41.9 13 1.11 7.1 

30.75 41.3 13 1.10 7.1 

47.25 15.4 4.7 0.67 7.1 

47.25 15.4 4.7 0.67 7.1 

Table 21 Main test - hydrolysis of the test substance at 50°C 

Sampling time 

[hours] 

Analysed 

concentration 

[mg/] 

Relative 

concentration 

[%1 

Logarithm relative 

concentration 

Actual pH 

0 433 100 2.00 7.1 

0 433 100 2.00 7,1 

1 373 86 1.93 7.1 

1 386 89 1.95 7.1 

2.25 328 76 1.88 7.1 

2.25 323 75 1.87 7.1 

3 280 65 1.81 7.1 

3 287 66 1.82 7.1 

4.25 233 54 1.73 7.1 

4.25 236 54 1.74 7.1 

5.25 195 45 1.65 7.1 

5.25 196 45 1.66 7.1 

6.25 162 37 1.57 7.1 

6.25 165 38 1.58 7.1 

7.25 138 32 1.50 7.1 

7.25 139 32 1.51 7.1 

24.75 8.08 1.9 0.27 7.1 

24.75 8.05 1.9 0.27 7.1 

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Table 22 Recoveries 

Temperature 

(°C) 

Nominal 

concentration 

Fmg/ll 

Analysed 

concentration 

rmg/ll 

Recovery1 

r%i 

Mean 

recovery 

f%l 

20 263 253 96 96 

252 96 

40 342 328 96 95 

326 95 

50 442 433 98 98 

433 98 

Recovery was determined relative to the concentration of the prepared bulk solution. A relatively high spiking 

concentration had to be used to allow dilution of the samples to the end solution (30/70 (v/v) acetonitrile/borate buffer 

pH 9) to ensure sample stability during chromatographic analysis and sufficient analytical sensitivity. 

For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 

10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The 

half-life times of the test substance were determined according to the model for pseudo-first order 

reactions. 

All logarithms of the relative concentrations were correlated with time using linear regression analysis. 

Figure 10 illustrates the regression curves and Table 23 shows the statistical parameters. 

Table 23 Statistical parameters of the regression curves 

Temperature 

(°C) 

Slope 

[hours"1l 

Intercept Coefficient of 

correlation 

20 -3.06 x 10"3 2.00 0.996 

40 -2.95 x 10*2 2.03 0.998 

50 -7.06 x 10"2 2.02 0.9997 

The rate constant (k obs ) and half-life time of the test substance at each temperature was obtained and 

the Arrhenius equation was used to determine the rate constant and half-life time at 25°C (see Table 

24). 

Table 24 Rate constants (k obs ) and half-life time (tVj) 

Temperature 

PCI 

kobs 

[hours"1l 

t« 

20 7.05 x 10'3 4.1 days 

25 1.27 x 10'2 2.3 days 

40 6.79 x 10- 2 10 hours 

50 1.63 x 10"1 4.3 hours 

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pH 9 

The analytical results of the main study are given in Table 25 - Table 28. 

No test substance was detected in the blank buffer solutions. 

The mean recoveries of the buffer solutions relative to the stock solution fell within the criterion range 

of 90-110%. It demonstrated that the analytical method was adequate to support the hydrolysis study 

on the test substance. 

Table 25 Main test - hydrolysis of the test substance at 20°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] E%] 

0 403 100 2.00 9.1 

0 403 100 2.00 9.1 

7 415 103 2.01 9.0 

7 411 102 2.01 9.0 

24 394 98 1.99 9.0 

24 397 99 1.99 9.0 

95 236 59 1.77 9.0 

95 231 57 1.76 9.0 

168.5 144 36 1.55 9.0 

168.5 145 36 1.56 9.1 

192 108 27 1.43 9.1 

192 109 27 1.43 9.1 

215 95.2 24 1.37 9.0 

215 95.0 24 1.37 9.0 

239 79.0 20 1.29 9.0 

239 79.8 20 1.30 9.0 

263 74.8 19 1.27 9.0 

263 76.6 19 1.28 9.0 

288 58.2 14 1.16 9.1 

288 59.0 15 1.17 9.1 

342 54.2 13 1.13 9.0 

342 54.7 14 1.13 9.0 

407 36.6 9.1 0.96 9.0 

407 36.7 9.1 0.96 9.0 

Final report - Page 50 -
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Table 26 Main test - hydrolysis of the test substance at 40°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] [%] 

0 462 99 2.00 9.0 

0 471 101 2.00 9.0 

3 435 93 1.97 9.0 

3 425 91 1.96 9,0 

5.25 377 81 1.91 9,0 

5.25 373 80 1.90 9.0 

7 338 73 1.86 9.0 

7 344 74 1.87 9.0 

23.5 121 26 1.41 9.0 

23.5 123 26 1.42 9.0 

26 105 23 1.35 9.0 

26 106 23 1.36 9.0 

28.5 94.1 20 1.31 9.0 

28.5 94.9 20 1.31 9.0 

31 82.1 18 1.25 9.0 

31 80.6 17 1.24 9.0 

47.5 37.9 8.1 0.91 9.0 

47.5 37.9 8.1 0.91 9.0 

Table 27 Main test - hydrolysis of the test substance at 50°C 

Sampling time Analysed Relative Logarithm relative Actual pH 

concentration concentration concentration 

[hours] [mg/] [%] 

0 377 100 2.00 9.1 

0 381 100 2.00 9.1 

1 341 90 1.95 9.1 

1 338 89 1.95 9.1 

2 300 79 1.90 9.1 

2 303 80 1.90 9.1 

3 271 72 1.85 9.1 

3 267 71 1.85 9.1 

4 240 63 1.80 9.1 

4 244 65 1.81 9.1 

5 214 56 1.75 9.1 

5 218 58 1.76 9.1 

6 187 49 1.69 9.1 

6 190 50 1.70 9.1 

7 168 44 1.65 9.1 

7 167 44 1.65 9.1 

24.5 19.1 5.0 0.70 9.1 

24.5 19.4 5.1 0.71 9.1 

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Table 28 Recoveries 

Temperature 

(°C) 

Nominal 

concentration 

[mg/ll 

Analysed 

concentration 

[mg/l] 

Recovery1 

[%l 

Mean 

recovery 

[%l 

20 410 403 98 98 

403 98 

40 514 462 90 91 

471 92 

50 387 377 97 98 

381 98 

Recovery was determined relative to the concentration of the prepared bulk solution. A relatively high spiking 

concentration had to be used to allow dilution of the samples to the end solution (30/70 (v/v) acetonitrile/borate buffer 

pH 9) to ensure sample stability during chromatographic analysis and sufficient analytical sensitivity. 

For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 

10% and 90% were plotted against time. At all temperatures linear relationships were obtained. The 

half-life times of the test substance were determined according to the model for pseudo-first order 

reactions. 

All logarithms of the relative concentrations were correlated with time using linear regression analysis. 

Figure 11 illustrates the regression curves and Table 29 shows the statistical parameters. 

Table 29 Statistical parameters of the regression curves 

Temperature 

rc) 

Slope 

[hours"1l 

Intercept Coefficient of 

correlation 

20 -2.74 x 10"3 2.01 0.992 

40 -2.42 x 10"2 2.01 0.997 

50 -5.30 x 10"2 2.01 0.9998 

The rate constant (k obs ) and half-life time of the test substance at each temperature was obtained and 

the Arrhenius equation was used to determine the rate constant and half-life time at 25°C (see Table 

30). 

Table 30 Rate constants (k obs ) and half-life time (t%) 

Temperature 

rci 

kobs 

[hours'1] 

tw 

20 6.30 x 10'3 4.6 days 

25 1.11 x 10"2 2.6 days 

40 5.58 x 10'2 12 hours 

50 1.22 x 10"1 5.7 hours 

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15.8. Conclusion 

The preliminary test (Tier 1) and main study (Tier 2) were performed for the determination of the rate 

of hydrolysis of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline at pH values normally found in 

the environment (pH 4-9). 

The half-life times of the test substance were: 

pH4 pH7 pH9 

Temperature Temperature Temperature 

[°C] [°ci r°ci 

20 3.9 days 20 4.1 days 20 4.6 days 

25 2.2 days 25 2.3 days 25 2.6 days 

40 10 hours 40 10 hours 40 12 hours 

50 3.9 hours 50 4.3 hours 50 5.7 hours 

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15.9. Figures 

Time [hours] 

Figure 9 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C 

[middle] and 50°C [bottom] at pH4. 

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Time [hours] 

Figure 10 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C 

[middle] and 50°C [bottom] at pH7. 

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\ 0.60 -

0.40 -I , 1 1 . • , , 

0 100 200 300 400 500 600 700 800 

Time [hours] 

0.60 -I . , , , , , 

0 5 10 15 20 25 30 

Time [hours] 

Figure 11 Plot of the logarithms of relative concentration against time at 20°C [top], 40°C 

[middle] and 50°C [bottom] at pH9. 

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16. ADSORPTION COEFFICIENT 

16.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guideline for the Testing 

of Chemicals no. 121: "Estimation of the Adsorption Coefficient (Ko c ) on Soil and on Sewage Sludge 

using High Performance Liquid Chromatography (HPLC)", January 22, 2001. 

European Community (EC), EC no. 440/2008, Part C: Methods for the Determination of Ecotoxicity, 

Guideline C.19: "Estimation of the Adsorption Coefficient (Ko c ) on Soil and on Sewage Sludge using 

High Performance Liquid Chromatography (HPLC)", Official Journal of the European Union no. L142, 

May 31, 2008. 

16.2. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Miilipore, Bedford, MA, USA) 

Methanol VWR International, Leuven, Belgium 

All reagents were of analytical grade, unless specified otherwise. 

16.3. Performance of the study 

The principle of the test method is similar to that of the OECD guideline no. 117; "Partition coefficient 

(n-octanol/water), high performance liquid chromatography (HPLC) method". While passing through 

the column along with the mobile phase the test substance interacts with the stationary phase. As a 

result of partitioning between mobile and stationary phases, the test substance is retarded. The dual 

composition of a cyanopropyl stationary phase, having polar and non-polar sites allows for interaction 

of polar and non-polar groups of a molecule in a similar way as is the case for organic matter in soil or 

sewage sludge matrices. This enables the relationship between the retention time on the column and 

the Koc on organic matter to be established. 

According to the guidelines, the determination of the Ko c for test substances that are ionized for at 

least 10% within pH 5.5 to 7.5 should be performed with both the ionized and non-ionized form. 

Therefore, the pKa values of the test substance were calculated using the Perrin calculation method 

(pKalc 5.0, module in Pallas 3.0, CompuDrug International San Francisco, CA, USA). Based on the 

calculations, the HPLC analysis was performed at neutral pH. 

Solutions of reference substances with known log KQC values based on soil adsorption data and the 

test substance were analysed. The capacity factor (k1) of each compound was calculated from its 

retention time. The log k' values of the references substances were plotted against the known log Koc 

values. A linear regression program was used to calculate the calibration curve. Linear regression 

analysis was performed using the least squares method. The coefficient of correlation (r) was 

calculated. The log Ko c value for the test substance was calculated by substituting its mean log k' in 

the calibration curve. The value of log Ko c obtained from duplicate measurements was within ± 0.25 

log units. 

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16.4. Analytical method 

16.4.1. Analytical conditions 

Column temperature 

Mobile phase 

Flow 

Injection volume 

UV detection 

Instrument 

Detector 

Column 

Alliance Separation Module 2695 (Waters, Milford, MA, USA) 

Dual A Absorbance Detector 2487 (Waters) 

Hypersil BDS-CN, 150 mm x 4.6 mm i.d., dp = 5 pm (Thermo 

Fisher Scientific, Waltham, MA, USA) 

35°C±1°C 

30/70 (v/v) methanol/water 

1 ml/mih 

10 pi 

210 nm 

16.4.2. Preparation of the solutions 

Solution of the unretained compound 

A 4.81 g/l stock solution of formamide (99.8%, [75-12-7], Acros Organics, Geel, Belgium) in methanol 

was used. 500pl of the stock solution + 667 pi water was diluted in a 5 ml volumetric flask. The 

volumetric flask was filled up to the mark with 30/70 (v/v) methanol/ water. 

The formamide blank solution was 30/70 (v/v) methanol/water. 

Reference substance solutions 

Stock solutions of the reference substances at concentrations of approximately 1 g/l in methanol were 

used. The stock solutions were diluted. Two reference substance solutions were made. Reference 

solution containing Acetanilide, Atrazine, Benzoic acid phenylester and Phenanthrene was made as 

followed: 50 pi of each reference substance + 267 pi water were diluted in a 5 ml volumetric flask. The 

volumetric flask was filled up to the mark with 30/70 (v/v) methanol/ water. The second reference 

substance solution containing Monuron, 2,5-Dichloroaniline and Fenthion was made as followed: 50 pi 

of each reference substance + 200 pi water were diluted in a 5 ml volumetric flask. The volumetric 

flask was filled up to the mark with 30/70 (v/v) methanol/ water. 

The blank solution for the mixture of reference substances was 70/30 (v/v) methanol/water. 

Reference substance Purity CAS number Supplier log Koc 

Acetanilide > 99.9% 103-84-4 Sigma-Aldrich 1.26 

Atrazine 97.5% 1912-24-9 Sigma-Aldrich 1.81 

Monuron 99.9% 150-68-5 Sigma-Aldrich 1.99 

2,5-Dichloroaniline 99.8% 95-82-9 Sigma-Aldrich 2.55 

Benzoic acid phenylester 99.9% 93-99-2 Sigma-Aldrich 2.87 

Fenthion 98.3% 55-38-9 Sigma-Aldrich 3.31 

Phenanthrene 98.1% 85-01-8 Acros Organics 4.09 

"values according to the OECD 121 guideline based on soil adsorption data 

Acros Organics, Geel, Belgium 

Sigma-Aldrich, Stein heim, Germany 

Test solution 

A 1445 mg/l stock solution of the test substance was prepared in methanol. The stock solution was 

diluted to obtain an end solution of 30/70 (v/v) methanol/water. The final concentration of the test 

substance solution was 14.5 mg/l. 

The test substance blank solution was 30/70 (v/v) methanol/water. 

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16.4.3. Injections 

The reference substance and test substance solutions were injected in duplicate. Blank solutions were 

analysed by single injection. 

16.5. Formulas 

(t - t ) 

Capacity factor (k') k = —^ _ 

to 

where: 

tr = retention time 

t 0 = mean column dead time 

Calibration curve logk* = alogKo c +b 

where: 

a = slope 

b = intercept 

16.6. Results 

16.6.1. Calculation of pKa values 

The following pKa values in the logarithm range of 1 -14 for acidic and basic groups in the molecular 

structure of the test substance were calculated using the Perrin calculation method: 

Acidic Basic 

none RARN(R1R2) pKa4.21 

ROR pKa -3.28 

ROR pKa -3.88 

ROR pKa -4.37 

Based on the calculated pKa values the study was performed at neutral pH. 

16.6.2. Determination of the Ko c 

HPLC chromatograms of the test substance solution and corresponding blank are shown in Figure 12. 

In the chromatogram of the test solution, one major peak and several small test substance peaks were 

observed. 

The results of the HPLC method are given in the Table 31. Figure 13 shows the calibration curve of 

the log k1 of the reference substances as function of log K^. The equation of the regression line was: 

log k' = 0.502 x log K<,c - 0.930 (r = 0.96, n = 14). 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy.)aniline NOTOX Project 496981 

Table 31 Ko c of the test substance 

Substance tr,1 tp.2 mean tr log Ko c Koc Area 

[mini [mini (n=2) % 

Formamide (to) 1.997 1.995 1.996 

Acetanilide 2.853 2.853 1.26 

Atrazine 4.262 4.257 1.81 

Monuron 4.148 4.147 1.99 

2,5-Dichloroaniline 5.380 5.382 2.55 

Benzoic acid phenylester 9.522 9.529 2.87 

Fenthion 21.314 21.347 3.31 

Phenanthrene 19.634 19.691 4.09 

Impurity I 1.969 1.968 1.969 * * 0.28 

Impurity II 2.946 2.939 2.943 1.21 1.6x 101 0.67 

Impurity III 3.672 3.648 3.660 1.70 5.0 x 101 1.1 

Test substance 4.169 4.163 4.166 1.93 8.4 x 101 96 

Impurity IV 4.766 4.760 4.763 2.14 1.4 x 102 0.91 

Impurity V 5.005 5.004 5.005 2.21 1.6 x 102 0.77 

Impurity VI 5.173 5.169 5.171 2.26 1.8 x 102 0.52 

' Since the retention time of Impurity I was less than that of formamide, a Koc and log K0!; value could not be calculated. 

16.7. Conclusion 

The HPLC method using soil-adsorption-reference data was applied for the determination of the 

adsorption coefficient (Ko c ) of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

The Koc and log KoC value of the test substance and impurities with peak areas > 1% at neutral pH 

was: 

log Koc Area 

% 

Test substance 8.4 x 101 1.93 96 

Impurity III 5.0 x 101 1.70 1.1 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

16.8. Figures 

NOTOX Project 496981 

0.03S' 

0.0M' 

! 

• 0.015 

0.010-

0.0W 

0.000 

0.030-

0.OW-

} 

:

 O.OItV 

0.010-

o.oosOO00-

Figure 12 HPLC chromatograms of the 14.5 mg/l test solution [top; res. id. 1571] and 

corresponding blank [bottom; res. id. 1572]. 

1.5 

1 -

o 

0.5 -

0 -

-0.5 -

-1 

1.5 2.5 3 3.5 4 4.5 

log Ko c 

Figure 13 The regression line of the reference substances: log k' versus log Ko c . 

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p. 65 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

17. DISSOCIATION CONSTANT 

NOTOX Project 496981 

17.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of 

Chemicals, Guideline no. 112: "Dissociation Constants in Water" Adopted May, 12 1981. 

17.2. Reagents 

Citric acid monohydrate 

Sodium chloride (NaCI) 

Triethylamine 

Acetic acid 

Disodium tetraborate decahydrate 

(Na2B4O7*10H2O) 

Hydrochloric acid (HCI) 

Hydrochloric acid (HCI) 

Sodium hydroxide (NaOH) 

Sodium hydroxide 

Potassium dihydrogen phosphate 

(KH2P04 ) 

Disodium hydrogenphosphate dodehydrate 

(Na2HP04 *12H2 0) 

Potassium chloride 

Acetonitrile 

Water 

Merck, Darmstadt, Germany 

Merck 

Merck 

Merck 

Merck 

37% Merck 

1M, Merck 

pellets, Merck 

1M, Merck 

Merck 

Merck 

Merck 

Biosolve, Valkenswaard, The Netherlands 

Tap water purified by a Milli-Q water purification 

system (Millipore, Bedford, MA, USA). 

All reagents were of analytical grade, unless specified otherwise. 

17.3. Buffers 

All buffers were prepared in water and adjusted to the required pH with the chemical written after the 

sign"/". 

Buffer pH 0: 1M HCI 

Buffer pH 1: 0.488 g HCI + 0.372 g KCI in 1000 ml water/adjusted to pH 1 with 1 M HCI 

Buffer pH 2: 3.17 g Citric acid monohydrate + 0.158 g HCI + 1.80 g NaCI in 1000 ml water/ 

adjusted to pH 2 with 1 M HCI 

Buffer pH 3: 4.21 g Citric acid monohydrate + 0.445 g NaOH + 1,81 g NaCI in 1000 ml water 

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Buffer pH 4: 5.86 g Citric acid monohydrate + 1.35 g NaOH + 1.30 g NaCI in 1000 mi water/ 

adjusted to pH 4 with 1 M NaOH 

Buffer pH 5: 0.6 g Acetic acid in 1000 ml water / adjusted to pH 5 with 1 M NaOH 

Buffer pH 6: 4.21 g Citric acid monohydrate + 2.02 g NaOH in 1000 ml water / adjusted to pH 6 

with 1 M NaOH 

Buffer pH 7: 3.56 g KH2P04 + 14.68 g Na2HP04 *12H2 0 in 1000 ml water / adjusted to pH 7 with 

1 M HCI 

Buffer pH 8: 4.96 g Na2B4O7*10H2O + 0.78 g HCI in 1000 m! water / adjusted to pH 8 with 

1 M HCI 

Buffer pH 9: 4.95 g Na2B4O7*10H2O + 0.16 g HCI in 1000 ml water / adjusted to pH 9 with 

1 M HCI 

Buffer pH 10: 4.96 g Na2B4 07 *10H2O + 0.76 g NaOH in 1000 ml water / adjusted to pH 10 with 

1 M HCI 

Buffer pH 11: 1.02 g Triethylamine in 1000 ml water / adjusted to pH 11 with 1 M HCI 

Buffer pH 12: 0.39 g NaOH in 1000 ml water / adjusted to pH 12 with 1 M NaOH 

Buffer pH 13: 4.05 g NaOH in 1000 ml water / adjusted to pH 13 with 1 M NaOH 

Buffer pH 2.6: 100 ml buffer pH 3 adjusted to pH 2.6 with 1 M HCI 

Buffer pH 2.8: 100 ml buffer pH 3 adjusted to pH 2.8 with 1 M HCI 

Buffer pH 3.0: 100 ml buffer pH 3 adjusted to pH 3.0 with 1 M HCI 

Buffer pH 3.2: 100 ml buffer pH 3 adjusted to pH 3.2 with 1 M NaOH 

Buffer pH 3.4: 100 ml buffer pH 3 adjusted to pH 3.4 with 1 M NaOH 

Buffer pH 3.6: 100 ml buffer pH 3 adjusted to pH 3.6 with 1 M NaOH 

Buffer pH 3.8: 100 ml buffer pH 3 adjusted to pH 3.8 with 1 M NaOH 

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17.4. Performance of the study 

Three methods are available for the determination of the dissociation constant(s) (pKa) of the test 

substance. The principle of each method is described below: 

1. Titration method 

A test solution of the test substance is titrated with standard acid or base, as appropriate. The pKa 

values are determined based on the titration curve obtained. The method is not suitable for low 

solubility compounds. 

2. Spectrophotometric method 

The spectrophotometric method involves the determination of the relative concentrations of the ionised 

and unionised forms of the test substance and their pH dependence. The method is only applicable to 

compounds having appreciably different UV/Vis adsorption spectra for the ionised and unionised 

forms. This method may be suitable for low solubility compounds. 

3. Calculation method 

Calculation of the pKa values from the structural formula is performed using the Perrin calculation 

method (pKalc 5.0, module in Pallas 3.0, CompuDrug International, San Francisco, CA, USA). 

The spectrophotometric method was selected for the determination of the pKa values of the test 

substance. The calculation method was used for a preliminary estimation of the Po w of the test 

substance. 

17.5. Spectrophotometric method 

17.5.1. Test system 

Spectrophotometer Single-beam, Varian Gary 50 UVA/IS, Varian, Mulgrave, 

Victoria, Australia 

Slit 1.5 nm 

Scan speed 600 nm/min 

Probe Ultra mini immersion probe with titanium tube and stainless 

steel handle. Prism made from quartz, pathlength = 10 mm 

Type of cuvette Quartz 

Background-correction Reference solution 

17.6. Performance of the test 

17.6.1. Search for spectra of the pure molecule and the pure anion 

The -N group in the test substance can dissociate. The pKa value to be determined is thus for a basic 

group. 

A 4385 mg/l solution of the test substance was prepared in acetonitrile and further diluted in 

acetonitrile prior to use. Then, fourteen test solutions (all 20 mg/l) were prepared by diluting the stock 

solution with various buffers (pH 0, pH 1, pH 2, pH.3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 

11, pH 12and pH 13). 

Reference solutions were prepared by diluting acetonitrile the same as the test solution with each of 

the above mentioned buffers. 

From each of the test solutions, an absorption spectrum between 200 and 900 nm was recorded with 

the appropriate reference solution as reference. The pH of each solution (test and reference solution) 

was measured using a pH meter. 

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17.6.2. Choice of an analytical wavelength 

The analytical wavelength was chosen from the absorption spectra recorded from the test substance 

between pH 0 and pH 13. 

r 

17.6.3. Search for an approximate pKa value 

The optical absorbances at the analytical wavelength were obtained from the absorption spectra 

recorded from 20 mg/l solutions between pH 0 and pH 7. From these results, an approximate pKa 

value was calculated. 

17.6.4. Exact determination of pKa 

Three different stock solutions of the test substance in acetonitrile; 3770 mg/l, 4385 mg/l and 

15376 mg/l were prepared and diluted with acetonitrile prior to use. Nine test solutions (all 20 mg/l) 

were prepared by diluting each stock solution with buffers pH 0, pH 2.6, pH 2.8, pH 3, pH 3.2, pH 3.4, 

pH 3.6, pH 3.8 and pH 6. 

Reference solutions were prepared by diluting acetonitrile the same as the test solution with each of 

the above mentioned buffers. 

The optical absorbances at the analytical wavelength of all nine test solutions were measured after 

zero-adjustment with the appropriate reference solution. The pH of each solution (test and reference) 

was measured. 

17.7. Formulas 

For an basic group, the following equation is used if Ai > Am: 

A-A m 

pKa = pH + log 

Ai - A 

If Am > Ai: 

Am - A 

pKa = pH + log 

A-Ai 

Where Am is the optical absorbance of the neutral molecule. Ai is the optical absorbance of the ion 

and A is the optical absorbance of the mixture of ion and neutral molecule. pH is the actual pH. 

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17.8. Results 

17.8.1. Calculation method 

The following pKa values'in the logarithm range of 1 -14 for acidic and basic groups in the molecular 

structure of the test substance were calculated using the Perrin calculation method: 

Acidic Basic 

none RARN(R1R2) pKa4.21 

ROR pKa -3.28 

ROR pKa -3.88 

ROR pKa -4.37 

17.8.2. Search for spectra of the pure molecule and the pure anion 

The -N group in the test substance can dissociate. The pKa value to be determined is thus for an 

basic group. 

The spectra recorded at pH 0 and pH 1 were comparable and so were the spectra recorded at pH 5 till 

pH 13. The spectra recorded at pH 2, pH 3 and pH 4 had a form somewhere between those at pH 1 

and pH 5 and therefore called intermediate spectra. It was concluded that spectra recorded at pH < 1 

are of the ionized test substance, whereas spectra recorded at pH > 5 are of the non-ionized test 

substance. The intermediate spectra are of a combination of both species. Absorption spectra at pH 0 

to 13 are shown in Figure 14 to Figure 27. 

17.8.3. Choice of an analytical wavelength 

From the spectra of the ionized substance with the spectra of the non-ionized substance, it was 

concluded that the difference in optical absorbance is maximal at 249 nm. Hence 249 nm was chosen 

as the analytical wavelength. 

17.8.4. Search for an approximate pKa value 

The optical absorbances at 249 nm were determined. As well as the actual pH values of the test 

solutions and actual pH values of the reference solutions. From these absorbances and the actual pH 

value, the approximate pKa value was calculated. All results are summarized in Table 32. 

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Table 32 Actual pH values and optical absorbances at 249 nm. 

Concentration 

stock [mg/l] 

actual pH values of 

the reference 

solutions 

actual pH values of 

the test solutions 

Absorbance for the 

test solution 

[unitsl 

pKa value 

4385 0.39 0.36 0.0138 

0.97 1.01 0.0179 3.25 

1.97 1.99 0.0824 2.97 

3.02 3.04 0.3550 3.08 

4.12 4.12 0.6398 3.29 

5.08 5.08 0.7211 3.32 

6.10 6.11 0.7334 

7.12 7.11 0.7284 

7.99 8.02 

9.02 9.04 

9,97 10.01 

10.78 10.8 

11.63 11.65 

12.68 12.71 

The approximate pKa value was calculated to be 3.18. 

17.8.5. Exact determination of the pKa value 

The actual pH values of the reference solutions were 0.30, 2.62, 2.81, 3.03, 3.24, 3.45, 3.64, 3.84 and 

5.70. 

The optical absorbances at 249 nm of all nine test solutions and the seven calculated pKa values are 

summarized in Table 33. 

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Table 33 Optical absorbances at 249 nm. 

Concentration stock 

[mg/l] 

Actual pH Absorbance for 

the test solution 

funitsl 

pKg value 

4385 0.30 0.0170 

2.62 0.1662 3.23 

2.81 0.2328 3.21 

3.04 0.3137 3.23 

3.24 0.3945 3.24 

3.45 0.4783 3.26 

3.64 0.5488 3.27 

3.84 0.6134 3.27 

5.70 0.7732 

3770 0.29 0.0230 

2.62 0.1719 3,24 

2.82 0.2353 3,24 

3.04 0.3229 3.23 

3.24 0.4024 3.25 

3.45 0.4925 3.25 

3,64 0.5680 3.25 

3.84 0.6304 3.26 

5.75 0.7896 

15376 0.29 0.0230 

2.62 0.1844 3.22 

2.82 0.2465 3.23 

3.04 0.3316 3.24 

3.24 0.4144 3.26 

3.46 0.5073 3.28 

3.64 0.5806 3.28 

3.84 0.6486 3.29 

5.78 0.8240 

The twenty one pKa values were averaged, resulting in the pKa value of 3.25. The standard deviation 

is 0.02. 

The test was performed in a room where the temperature (20.4 ± 0.6°C) was measured continuously 

using a validated computerised system (Rees Scientific, NJ, USA). 

17.9. Conclusion 

The pKa value for p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was determined to be 3.25 

using the spectrophotometric method. This pKg value corresponds to dissociation of a basic group in 

the molecule. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

17.10.Figures 

NOTOX Project 496981 

< 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 14 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 0. 

1.5-

< 

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 15 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 1. 

< 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 16 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 2. 

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p. 73 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 17 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 3. 

1.5-

< 

1.0-

0.5-

0.0-

200 

— i 1 

400 600 

Wavelength (nm) 

800 

Figure 18 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 4. 

1.5-

£1 

< 

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 19 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 5. 

Final report - Page 70 -

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496981 

200 400 600 800 

Wavelength (nm) 

Figure 20 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 6. 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 21 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 7. 

< 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 22 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 8. 

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p. 75 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)aniline NOTOX Project 496981 

V) 

< 

1.5-

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 23 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 9. 

1.5-

S3 

< 

1.0-

0.5-

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 24 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 10. 

1.5-

« 1.0H 

< 

0.5H 

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 25 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 11, 

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400 600 

Wavelength (nm) 

800 

Figure 26 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 12. 

1.5-

« 1.0-1 

XI 

< 

0.5J 

0.0-

200 400 600 

Wavelength (nm) 

800 

Figure 27 Absorption spectrum of a 20 mg/l solution of test substance in buffer pH 13. 

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18. VISCOSITY 

18.1. Guidelines 

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing 

of Chemicals no. 114: "Viscosity of Liquids", May 12,1981. 

International Organization for Standardization (ISO), ISO 3104: "Petroleum Products - Transparent 

and Opaque Liquids - Determination of Kinematic Viscosity and Calculation of Dynamic Viscosity", 

1994. 

ASTM International, ASTM D 445-09: "Standard Test Method for Kinematic Viscosity of Transparent 

and Opaque Liquids (and Calculation of Dynamic Viscosity)", July 1, 2009. 

18.2. Performance of the test 

The viscosity of the test substance was determined at 20°C and 40°C using a glass capillary 

viscometer (Tamson Instruments, Bleiswijk, The Netherlands). 

Based on appearance of the test substance, the Ubbelohde viscometers with constants 0.4655 and 

0.4685 were selected. The viscometer was charged with the test'substance by tilting it about 30° from 

the vertical position. When the viscometer was returned to the vertical the meniscus of the test 

substance was between the filling marks of the reservoir bulb. 

The viscometer was placed in the thermostatic water bath and remained in the bath for at least 

30 minutes to reach the test temperature. 

The test substance was drawn by vacuum through the timing tube to about 5 mm above the upper 

timing mark. The vacuum was released. The time required for the meniscus to pass from the upper to 

the lower timing mark was measured and the kinematic viscosity of the test substance was 

determined. 

The kinematic viscosity of the test substance was determined at 20 + 0.1 °C and 40 ± 0.1 °C. Each test 

was performed in duplicate. 

18.3. Interpretation 

18.3.1. Specifications 

The kinematic viscosity is defined as the resistance to flow of a fluid under gravity. The kinematic 

viscosity is expressed in mm2/s. 

18.3.2. Formulas 

Kinematic viscosity o = C x t 

where: 

C = calibration constant of the viscometer ((mm2/s)/s| 

t = flow time [s] 

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18.4. Results 

The results for the kinematic viscosity of the test substance at 20°C and 40°C are given in Table 34. 

Table 34 Viscosity of the test substance 

Temperature 

PC] 

Test Calibration constant 

[(mm2/s)/s] 

Flow time 

[s] 

Kinematic viscosity 

[mm2/s] 

Mean 

[mm2/s] 

20 1 0.4685 1790.9 839.1 840.6 

il 0.4655 1809.1 842.2 

40 1 0.4655 294.5 137.1 138.9 

II 0.4685 300.4 140.7 

18.5. Conclusion 

The viscosity of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was determined using a glass 

capillary viscometer. 

The kinematic viscosity of the test substance was: 

Kinematic 

[mrr 

20°C 

viscosity 

i

2

/s] 

40°C 

Test substance 840.6 138.9 

Final report - Page 75 -
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Appendix 1 GLP certificate 

ENDORSEMENT OF COMPLIANCE 

WITH THE OECD PRINCIPLES OF 

GOOD LABORATORY PRACTICE 

Pursuant to the Netherlands GLP Compliance Monitoring Programme and 

according to Directive 2004/9/EC, the conformity with the OECD Principles of GLP 

was assessed on 21 - 25 March, 2011 at 

It is herewith confirmed that the afore-mentioned test facility is currently operating 

in compliance with the OECD Principles of Good Laboratory Practice in the 

following area of expertise: Physical-chemical testing, Toxicity studies; Mutagenicity 

studies; Environmental toxicity studies on aquatic and terrestrial organisms; Studies 

on behaviour in water, soil, and air, bioaccumulation; Residue studies; Analytical and 

clinical chemistry testing; Kinetic and metabolism studies, Safety Pharmacology. 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD's Hertogenbosch 

Den Haag, 19 May 2011 

Food and Consumer Product Safety Authority (VWA) 

Catharijnesmgel 59 

3511GG Utrecht. The Netherlands 

Final report - Page 76 -

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REPORT 

DETERMINATION OF 'READY* BIDDEGRADABILITY: 

CARBON DIOXIDE (C02) EVOLUTION TEST (MODIFIED STURM TEST) 

WITH 

TK 12759, ARALDITE MY 0510 

NOTOX Project 169875 

NOTOX Substance 59652 

Ciba Project 964625 

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p. 81 

TK 12759, ARALOITE MY 0510 NOTOX Project 169875 

Ciba Project 964625 

REPORT APPROVAL 

STUDY DIRECTOR: Ing. M.O.E. Desmares-Koopmans 

MANAGEMENT: Ing. E.J. van de Waart 

Section Head, Genetic & 

Eco-Toxicology 

rC. Enninga 

;al director 

Date: 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169875 

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SUMMARY 

TK 12759, ARALDITE MY 0510 was tested for its ready blodegradablllty in the 

Carbon Dioxide (C02) Evolution Test (Modified Sturm Test) at ca. 39 mg per 

2 litres, corresponding to ca. 12 mg TOC/1. 

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, 

and OECD guideline No. 301 B July 17, 1992. 

The Theoretical C02 production (ThCOo) Of TK 12759, ARALDITE MY 0510 

(MF: C15H1gN04, MW: 277 g) was calculated to be 2.383 mg C02/mg. 

The relative degradation values calculated from the measurements performed 

during the test period revealed no significant degradation of TK 12759, 

ARALOITE MY 0510 in test bottle A (significant: >10*). 

In test bottle B 30^ degradation was measured on day 29. This result was 

not comparable to the results of test bottle A and the toxicity control, 

and i t was assumed that the produced C02 in test bottle B was not test 

substance related, but the result of a technical failure. Therefore, the 

degradation was based on the data of day 27 and considered as not 

significant. 

Since all further acceptability criteria were met, this study was 

considered to be valid. 

In conclusion, TK 12759, ARALDITE. MY 0510 was not readily biodegradable 

under the conditions of the modified Sturm test presently performed. 

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TK 12759, ARALOITE MY 0510 NOTOX Project 169675 

Ciba Project 964625 

PREFACE 

Sponsor Ciba-Geigy Ltd. 

Polymers Division 

R-1006.520 

CH-4002 BASEL 

Switzerland 

Study Monitor Dr. E.G. Semadeni 

Testing Facilit Facility NOTOX B.V. 

Hambakenwetering 3 

5231 DO 's-Hertogenbosch 

The Netherlands 

Study Director Ing. M.3.E. Desmares-Koopmans 

Technical Coordinator Mrs. C.J. van Leeuwen-van Drunen 

Study Plan Start: February 14, 1996 

Completion: March 14, 1996 

TEST SUBSTANCE 

Identification TK 12759, ARALDITE MY 0510 

Description Clear yellow liquid {Determined at NOTOX) 

Batch 50008M 

Purity > 95% 

Test substance storage In freezer in the dark 

Stability under storage 

conditions Stable 

Expiry date May 01, 1998 

Stability in water At least 96 h 

The sponsor is responsible for the completeness and GLP Compliance of all 

test substance data. 

PURPOSE 

The purpose of the study is to evaluate a non-volatile test substance for 

i ts ready biodegradability in an aerobic aqueous medium with microbial 

activity introduced by inoculation of activated sludge. 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169875 

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GUIDELINES 

The study procedure described in this report was based on the following 

guidelines: 

European Economic Community (EEC), EEC directive 92/69, Part C: Methods for 

the determination of ecotoxicity, Publication No. L383, December 1992, 

C.4. "Biodegradation: determination of the 'ready* biodegradability, 

C.4-C: Carbon dioxide (CO2) evolution test (Modified Sturm Test). 

Organisation for Economic Co-operation and Development (OECD), OECD 

guidelines for Testing of Chemicals, Section 3, Degradation and 

Accumulation, guideline No. 301 B: "Ready Biodegradability: C02 Evolution 

Test" adopted July 17, 1992. 

DEFINITIONS 

Readily biodegradable: Test substances giving a result of at least 

yield of C02 (within 28 days). This pass level must 

be reached within 10 days of biodegradation 

exceeding 1096. 

ThC02 : Theoretical carbon dioxide (mg) is the quantity of 

carbon dioxide calculated to be produced from the 

known or measured carbon content of the test 

substance when fully mineralized; also expressed as 

mg carbon dioxide evolved per mg test substance. 

TOC : Total organic carbon of a sample is the sum of the 

organic carbon in solution and in suspension. 

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PREPARATION OF TEST SOLUTIONS 

After addition of milli-Q water to each weighing bottle containing TK 

12759, ARALDITE MY 05.10 (A: 38.7 mg, 6: 39.5 and toxicity control: 39.0 

mg), followed by ultra sonication (15 min.), the resulting mixture was 

quantitatively added to the test media. The test solutions were 

continuously stirred during the test. 

TEST SYSTEM 

Source 

Treatment 

Reason for selection 

The source of test organisms was activated 

sludge freshly obtained from a municipal 

sewage treatment plant: 'Waterschap de 

Maaskant', *s-Hertogenbosch, the Netherlands. 

The sludge was kept under continuous aeration 

until further treatment. The concentration of 

suspended solids was 3.2 g/l 1n the 

concentrated sludge. Before use, the sludge 

was allowed to settle for at least 30 minutes 

and the liquid decanted for use as inoculum at 

the amount of 10 ml/1 of mineral medium. 

The test has been accepted internationally 

(EEC, OECD) for determining the 'ready' 

biodegradability of test substances under 

aerobic conditions. 

TEST PROCEDURE AND CONDITIONS 

Test duration 

Test vessels 

Milli-Q water 

Stock solutions of 

mineral components 

28 days (last C02~measurement on the 29th 

day). 

2 litr e all-glass brown coloured bottles. 

Tap-water purified by reverse osmosis and 

subsequently passed over activated carbon and 

ion-exchange cartridges (Millipore Corp,, 

Bedford, Mass., USA). 

A) 8.50 g KH2P04 

21.75 g K2HP04 

67.20 g Na2HP04.12H20 

0.50 g NH4C1 

dissolved in 1 1 Milli-Q water, pH 7.4+0.2 

B) 22.50 g MgS04.7H20 dissolved in 1 1 

Milli-Q water. 

C) 36.40 g CaCl2..2H20 dissolved in 1 1 

Milli-Q water. 

D) 0.25 g FeCl3.6H20 dissolved 1n 1 1 

Milli-Q water. 

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Mineral medium 

Barium hydroxide, 0.0125 M 

C02-free air 

Test concentration 

1 1 mineral medium contains: 10 ml of 

solution (a), l ml of solutions (b) to (d) 

and Milli-Q water. 

4 g Ba(0H)2.8H20 per litr e Milli-Q water was 

filtered through filte r paper and stored in a 

sealed vessel to prevent absorption of C02 

from the air. 

A mixture of oxygen (21%) and nitrogen (79%) 

was led through a bottle, containing 

0.5 - 1 litr e 0.0125 M Ba(0H)2 solution to 

trap C02 which might be present in small 

amounts. The C02-free air was sparged through 

the scrubbing solutions at a constant rate. 

The test substance was tested in duplicate at 

ca. 39 mg per 2 litres, corresponding to ca. 

12 mg TOC/l. The organic carbon content was 

based on the molecular formula. 

Preparation of bottles: 

Pre-incubation medium 

Type and number of 

bottles 

Preparation 

Mineral components, Milli-Q water (ca. 80% 

total volume) and Inoculum (1% final volume) 

were added to each bottle. This mixture was 

aerated with C02-free air overnight to purge 

the system of C02. 

Test suspension: containing test substance 

and inoculum (2 bottles). 

Inoculum blank: containing only inoculum (2 

bottles) 

Positive control: containing reference 

substance (ca. 40 mg/l sodium acetate (Merck 

art. 6268, batch 049 TA933768), T0C= 12 mg/l) 

and inoculum (1 bottle). 

Toxicity control: containing test substance, 

reference substance and inoculum ( l bottle). 

The test substance and positive control were 

added to the bottles. 

The volumes of suspensions were made up in 

a ll bottles by the addition of Milli-Q water 

previously aerated with C02*-free air, 

resulting in the mineral medium described 

before. 

Three C02-absorbers (bottles filled with 100 

ml 0.0125 M Ba(0H)2) were connected in series 

to the exit air line of each test bottle. 

Start of the incubation The test was started by bubbling C02-free air 

through the solution at a rate of 

approximately 1-2 bubbles per second 

(ca. 30-100 ml/min). 

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p. 87 

DETERMINATION OF C02 

Experimental C02 production The C02 produced in each test bottle reacted 

with the barium hydroxide In the gas 

scrubbing bottle and precipitated out as 

barium carbonate. The amount of C02 produced 

was determined by titrating the remaining 

Ba(0H)2 with 0.05 M standardized HCI. 

Measurements Titrations were made every second or third 

day during the firs t 10 days, and thereafter 

at least every fift h day until the 28th day. 

Each time the C02-absorber nearest to the 

test bottle was removed for titration; each 

of the remaining two absorbers was moved one 

position in the direction of the test bottle. 

A new C02~absorber was placed at the far end 

of the series. Phenolphthalein was used as 

pH-indicator. 

On the 28th day, the pH of the test 

suspensions was measured and 1 ml of 

concentrated HCI was added to each bottle. 

The bottles were aerated overnight to drive 

off C02 present in the test suspension. The 

final titration was made on day 29. 

Theoretical C02 production The theoretical C02 production was calculated 

from the molecular formula. 

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DATA EVALUATION 

ThC02, expressed as mg C02/mg test substance, that can be generated by a 

test substance was calculated as follows: 

No. of carbon in test substance x MW C02 

ThC02 = MW test substance 

The first step in calculating the amount of C02 produced is to correct for 

background (endogeneous) C02 production. Thus the amount of C02 produced 

by a test material is determined by the difference (in ml of titrant) 

between the experimental and blank Ba(0H)2 traps. 

The amount of 0.05 N HCI titrated is converted into mg of C02 produced: 

(0.05 x ml HCI titrated^ 

mg C02 « 2 x 44 = l. i x ml of HCI titrated. 

Calculations were based on the actual normality without rounding off. 

Relative degradation values was calculated from the cumulative C02 

production relative to the total expected C02 production based on the 

total carbon content of the amount of test material present in the test 

bottles. They were plotted versus time together with the relative 

degradation of the positive control. 

ACCEPTABILITY OF THE TEST 

The results of the biodegradation test were considered to be valid when: 

- the total C02 evolution in the inoculum blank at the end of the test did 

not normally exceed 40 mg/l. I f values greater than 70 mg C02/1 are 

obtained, the data and experimental technique should be examined 

critically. 

- the difference of duplicate values for the %-degradation of the test 

substance at the plateau, at the end of the test or at the end of the 

10-day window, as appropriate, was less than 20. 

- the percentage degradation of the reference substance reached the level 

for ready biodegradability (60%) by 14 days. 

Because of the stringency of the method, low values do not necessarily mean 

that the test substance is not biodegradable under environmental 

conditions, but indicates more work will be necessary to establish 

biodegradability. 

Toxicity control: i f less than 35% degradation (based on DOC) or less than 

25* degradation (based on ThC02) occurred in 14 days, the test substance 

can be assumed to be inhibitory. 

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RESULTS 

Theoretical C02 production 

The Theoretical C02 production (ThC02) of TK 12759, ARALDITE MY 0510 

(MF: Ci5H19N04, MW: 277 g) was calculated to be 2.383 mg C02/mg. 

The concentration was 38.7 (A) and 39.5 mg (B) TK 12759, ARALDITE MY 0510 

in 2 litre s test medium. Hence, the theoretical C02 production following 

complete degradation was 92.2 mg for A and 94.1 mg per 2 litre s for B. 

The positive control contained 80.1 mg sodium acetate (ThC02* 1,073 mg 

C02/mg) resulting in a theoretical C02 production following complete 

degradation of 85.9 mg per 2 litres . 

The toxicity control contained 80.1 mg sodium acetate and 39.0 mg TK 12759, 

ARALOITE MY 0510 in 2 litre s of test medium. Hence, the theoretical C02 

production following complete degradation of TK 12759, ARALDITE MY 0510 

plus sodium acetate was 178.9 mg per 2 litres . 

Biodegradation 

The results are summarized 1n Tables 1-4, and Figure 1. 

The relative degradation values calculated from the measurements performed 

during the test period revealed no significant degradation of TK 12759, 

ARALOITE MY 0510 in test bottle A (significant: >10%). 

In test bottle 6 30% degradation was measured on day 29. This result was 

not comparable to the results of test bottle A and the toxicity control, 

and i t was assumed that the produced C02 in test bottle B was not test 

substance related, but the result of a technical failure. Therefore, the 

degradation was based on the data of day 27 and considered as not 

significant. 

In the toxicity control more than 25% degradation occurred in 14 days 

(based on ThC02). Therefore, the test substance was assumed to be not 

inhibitory. 

Monitoring of temperature and pH 

The temperature recorded during the study varied between 20 and 23°C. 

The pH values of the different test media were: 

TK 12759, ARALDITE MY 0510 NOTOX Project 169875 

Ciba Project 964625 

Just before the 

start of the test: 

On day 28: 

81ank control (A) 

Blank control (B) 

Positive control 

7.5 

7.5 

7.5 

7.6 

7.6 

7.6 

7.5 

7.5 

7.8 

7.7 

7.6 

7.9 

TK 12759, ARALDITE MY 0510 (A) 

TK 12759, ARALDITE MY 0510 (B) 

Toxicity control 

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Acceptability of the test 

The positive control substance was degraded 79% in 14 days (see Table 1 and 

Figure 1). 

The total CQ2 release In the blank reached a total value of 20 mg C02 per 2 

litres of medium (see Table 4). 

Except for day 29, the difference of duplicate values for ^-degradation of 

TK 12759, ARALDITE MY.0510 was always less than 20. Since the degradation 

on day 29 in test bottle B was considered to be due to a technical failure, 

and the final judgement for this bottle was based on the data of day 27, 

the difference of duplicate values on day 29 had no effect on the outcome 

of this study. 

TK 12759, ARALDITE MY 0510 was found to be not inhibitory. 

CONCLUSION 

TK 12759, ARALOITE MY 0510 was not readily biodegradable under the 

conditions of the modified Sturm test presently performed. 

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Table 1: CO, evolution and percentage of biodegradation of the positive control substance. 

Day ml titran t ml titran t Produced C02 Normality Produced Cumulative Degradation 

Blank (mean) C02 trap i n ml HCI HCI mg C02 ng C02 % 

2 49.07 40.27 6.80 0.048 9.29 9.29 10.8 

5 48.90 22.86 26.02 0.048 27.48 36.77 42.8 

7 47.94 33.57 14.37 0.048 15.17 51.94 60.5 

9 46.85 40.73. 6.15 0.048 8.61 60,55 70.5 

14 47.23 40.36 6.67 0.048 7.25 67.80 78.9 

19 47.90 41,46 6.42 0,049 6.92 74.72 87.0 

23 48.51 41.59 6.92 0.049 7.46 82.18 93.7 

27 45.60 38.15 7.45 0.049 8.03 90.21 105.0 

29 43.95 42.12 1.83 0.049 1.97 92.16 •107.3 

29 45.20 44.80 0.40 0.049 0.43 92.61 107.6 

29 48.26 46.50 0.00 0.049 0.00 92.61 107.6 

Theoretical C02 production sodium acetate; 83.9 »g C02 /21 

Table : 2a: C02 evolution and percentage of biodegradation of the tes t substance (bottl e A) 

Oay ml titran t (nl titran t Produced C02 Normality Produced Cumulative Degradation 

Blank (mean) C02 trap ml HCI N tng CO 2 mg C02 31 

2 49.07 48.32 0.55 0.O48 0.56 0.58 0.6 

5 48,90 48.70 0.20 0.048 0.21 0.79 0.9 

7 47.94 48.71 0.00 0.048 0.00 0.79 0.9 

9 48.88 48.74 0.14 0.048 0.15 0.94 i .o 

14 47.23 47.84 0.00 0.048 0.00 0-94 1.0 

)9 47.90 47.96 0.00 0.049 0.00 0.94 1-0 

23 48.51 47.89 0.62 0.049' 0.67 1.61 1.7 

27 43.60 45.26 0.34 0,049 0.37 1.98 2,1 

29 43.95 43.77 0.00 0.049 0.00 1.96 2.1 

29 45.20 44.15 1.03 0.049 1.13 3.11 3.4 

29 48.26 46.62 0.00 0.049 0.00 3.11 3.4 

Theoretical C02 production tes t substance: 92.2 ng C02 

121 

Table 2b: co2 evolution and percentage of biodegradeition of the tes t substance (bottl e B) 

Day ml titran t ml titran t Produced CO, Normality Produced Cumulative Degradation 

Blank (mean) CQ2 trap ml HCI N mg co 2 mg C02 % 

2 49.07 48.69 0.38 0.048 0.40 O.AO 0.4 

5 48.90 49.00 0.00 0.048 0.00 O.AO 0.4 

7 47.94 48.49 0.00 0.046 0.00 O.AO 0.4 

9 4S.88 47.33 1.33 0.048 1,62 2.02 2.1 

14 47 .23 48.63 0.00 0.048 0.00 2.02 2.1 

39 47.90 SB.26 0.00 0.049 0.00 2.02 2.1 

23 48.51 48.37 0.14 0.049 0.15 2.17 2.3 

27 45.60 45.28 0-32 0,049 0.34 2.31 2.? 

29 43.95 37.69 6.26 0.049 6.75 9-26 9.8 

29 45.20 29.47 15.73 0.049 16.96 26.22 27.9 

29 46.26 46.33 1.91 0.049 2.06 28.26 30.1 

Theoretical CO2 production tes t substance: 94.1 »g CO2 /21 

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Table 3: C02 evolution end percentage of biodegradation of the toxicity control. 

Day ml titran t ml titran t Produced C02 Normality Produced Cumulative Degradation 

Blank (mean) C02 trap »1 HCI N Big C02 »g co2 

X 

2 49.07 33 ,01 14.06 0.048 14.85 14. B3 6.3 

5 48.90 26.70 22.20 0.046 23.44 38.29 21.4 

7 47.94 38.77 9.17 0.048 9.68 47.97 26.8 

9 48.88 44.58 4.30 0.046 4.54 52.31 29.4 

14 47,23 42.48 4.73 0.046 5.02 37.53 32.2 

19 • 47.90 42.96 4,94 0.049 3.33 62.86 35.1 

23 . 48.31 41.76 6.75 0.049 7.26 70.14 39.2 

27 45.60 32.36 13.24 0.049 14,27 64.41 47.2 

29 43.95 41.82 2.13 0.049 2.30 86.71 48.5 

29 43.20 44.61 0.39 0.049 0.64 67.35 48.8 

29 46.26 48.42 0.00 0.049 0.00 87.33 48.6 

Theoretical co2 production test substance: 92.9 mg C02/21 

Theoretical C02 production sodium acetate: 65.9 ng C02/2l 

Theoretical C02 production test substance plus sodium acetate: 176.9 mg C02/21 

Note: Since calculations Mara performed without rounding off a deviation In the last 

figure of the theoretical C02 production per 2 litre s may be observed. 

Table 4: co2 evolution I n the blank. 

Day Stand. ml titran t Produced C02 Normality Produced cumulative 

Ba(0H)2 Blank »1 HCI tl mg C02 *g co2 

ml HCI * 5 (mean value) 

2 9.90 49.07 0.43 0.046 0.45 0-45 

5 9.81 48.90 0.13 0.048 0.16 0.61 

7 9.81 47.94 1.11 0.048 . 1.17 1.79 

9 ,9.83 48.88 D.27 0.048 0.29 2.07 

14 9.81 47.23 1.62 0.048 1.92 3.99 

19 9.84 47.90 1.30 0.049 1.40 5.39. 

23 9.84 46.31 0.69 0.049 0.74 6.13 

27 9.85 45.60 3,53 0.049 3.83 9.96 

29 9.80 43.95 3.05 0.049 5.44 15.40 

29 9.80 45.20 3.80 0.049 4.10 19.30 

29 9.80 48.26 0.74 0.049 0.60 20.30 

Figure 1; Biodegradation of TK 12759, ARALDITE HY 0510 In the modified Sturm test: 

- : positive control o : TK 12759, ARALDITE MY 0310 (A)' 

[ ] : Toxicity control x : TK 12739, ARALDITE HY 0510 (B) 

0 2 4 6 « 10 12 14 16 IS 20 22 24 2C 7.0 

C0UK5E OF OlQOtGRAQAHON IN TIME (DAYS) 

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» 

Mil I I ~~ 
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FINAL REPORT 

Study Title 

FRESH WATER ALGAL GROWTH INHIBITION TEST 

WITH 

P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILINE 

Author 

Ir. LM. Bouwman 

Test Facility 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Nethedands 

Laboratory Project Identification 

NOTOX Project 496987 

NOTOX Substance 203277/A 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

1. CONTENTS 

1 CONTENTS 2 

2. STATEMENT OF GLP COMPLIANCE 4 

3. QUALITY ASSURANCE STATEMENT 5 

4. SUMMARY 6 

5. INTRODUCTION 7 

5.1. Preface 7 

5.2. Aim of the study 7 

5.3. Guidelines 7 

5.4. Storage and retention of records and materials 7 

5.5. Definitions 8 

6. MATERIALS AND METHODS • 8 

6.1. Test Substance 8 

6.1.1. Test substance information 8 

6.1.2. Study specific test substance information 8 

6.1.3. Reference substance 9 

6.2. Test System 9 

6.3. Fresh water algae culture 9 

6.4. Preparation of test solutions 10 

6.5. Combined limit/range-finding test 10 

6.6. Final test 10 

6.6.1. Test concentrations 10 

6.6.2. Test procedures and conditions 10 

6.6.3. Sampling for analysis of test concentrations 11 

6.6.4. Measurements 11 

6.6.5. Recording of cell densities 11 

6.7. Electronic data capture 11 

6.8. Interpretation 12 

6.8.1. Data handling i 12 

6.8.2. Acceptability of the test 1 13 

6.9. List of deviations 13 

6.9.1. List of protocol deviations 13 

6.9.2. List of standard operating procedures deviations 13 

7. RESULTS 13 

7.1. Combined limit/range-finding test 13 

7.2. Final test 14 

7.2.1. Measured test substance concentrations 14 

7.2.2. Mean cell densities 14 

7.2.3. Reduction of growth rate and inhibition of yield 15 

7.2.4. Determination of effect concentrations 16 

7.2.5. Experimental conditions 16 

8. CONCLUSION 17 

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TABLES 

Table 1 Mean cell densities (x104 cells/ml) during the combined limit/range-finding test 14 

Table 2 Percentage reduction of growth rate and inhibition of yield during the combined 

limit/range-finding test 14 

Table 3 Mean cell densities (x 104 cells/ml) during the final test :..15 

Table 4 Percentage reduction of growth rate (total test period) and percentage inhibition 

of yield during the final test : 16 

Table 5 Percentage reduction of growth rate at different time intervals during the final test....16 

Table 6 Effect parameters 16 

Table 7 pH levels recorded during the final test 17 

Table 8 Individual cell densities 18 

Table 9 Calculation of growth rate and yield 19 

Table 10 Calculation of growth rate (section-by-section) 20 

Table 11 EC-values for growth rate reduction 23 

Table 12 EC-values for yield inhibition 24 

FIGURES 

Figure 1 Growth curves at different concentrations of p-(2,3-epoxypropoxy>-N,N-bis(2,3-

epoxypropyl)aniline 15 

Figure 2 Percentage reduction of growth rate as function of the log concentration (mg/l) of 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 23 

Figure 3 Percentage inhibition of yield as function of the log concentration (mg/l) of p-(2,3-

epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 24 

APPENDICES 

APPENDIX 1 WORKSHEET DATA 18 

APPENDIX 2 STATISTICS: GROWTH RATE (0-72 HOURS) 21 

APPENDIX 3 STATISTICS: YIELD (0-72 HOURS) 22 

APPENDIX 4 EC-VALUES 23 

APPENDIX 5 REFERENCE TEST 25 

APPENDIX 6 ANALYTICAL REPORT 26 

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2. STATEMENT OF GLP COMPLIANCE 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

The study described in this report has been correctly reported and was conducted in compliance with: 

Yhe Organization for Economic Co-operation and Development (OECD) Principles of Good Laboratory 

Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17. 

The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test substance 

information unless determined by NOTOX. 

NOTOX B.V. 

Final Report 

—

 i in-

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3. QUALITY ASSURANCE STATEMENT 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

This report was inspected by the NOTOX Quality Assurance Unit to confirm that the methods and. 

results accurately and completely reflect the raw data. 

During the on-site process inspections, procedures applicable to this type of study were inspected. 

The dates of Quality Assurance inspections are given below. 

Type of 

Inspections 

Phase/Process Start 

Inspection 

date 

End 

Inspection 

date 

Reporting 

date 

Study Protocol 

Protocol Amendment 01 

Report 

02-Aug-11 

09-Aug-11 

24-Nov-11 

02-Aug-11 

09-Aug-11 

24-Nov-11 

02-Aug-11 

09-Aug-11 

24-Nov-11 

Process Environmental Toxicology 

Test Substance Handling 

Exposure 

Observations/Measurements 

18-JuMI 22-JU.I-11 22-Jul-11 

Analytical and physical 

chemistry 

08-Aug-11 16-Aug-11 16-Aug-11 

Test Substance Handling 

Observations/Measurements 

NOTOX B.V. 

C.J. Mitchell B.Sc. 

Head of Quality Assurance 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

4. SUMMARY 

Pseudokirchnerieila subcapitata, Fresh Water Algal Growth Inhibition Test with p-(2,3-epoxypropoxy)-

N,N-bis(2,3-epoxypropyl)aniline. 

The study procedures described in this report were based on the OECD guideline No. 201, 2006. In 

addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) 

No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 

2004 and the OECD series on testing and assessment number 23, 2000. 

The batch of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline tested was a yellow liquid with a 

purity of 94.33% and the substance was not completely soluble in test medium at loading rates of 

1.0 mg/l and higher. 

A final test was performed based on the results of a preceding combined limit/range-finding test. All 

test solutions were prepared separately applying two days of magnetic stirring to reach maximum 

solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize 

for 2 hours where after the Water Accommodated Fractions (WAFs) were siphoned off and used for 

testing. The final test solutions were all clear and colourless. 

Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at loading 

rates of 4.6, 10, 22, 46 and 100 mg/l. Simultaneously, six replicates were exposed in a control group. 

The initial cell density was 104 cells/ml and the total test period was 72 hours. Samples for analytical 

confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and 

at the end of the test. 

At the start of the test, the actual test concentrations were 3.2, 3.3, 5.5, 33 and 34 mg/l in the WAFs 

prepared at loading rates of 4.6, 10, 22, 46 and 1Q0 mg/l, respectively. These concentrations 

remained stable during the first 24 hours of exposure (82-87% of initial) but decreased to 59-66% of 

initial at the end of the test period. Note that actual concentrations being almost similar is related to 

medium solubility and the preparation of WAFs. Based on these results, the average exposure 

concentrations were calculated to be: 2.5, 2.6,4.2, 26 and 26 mg/l. 

The study met the acceptability criteria prescribed by the protocol and was considered valid. 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline reduced growth rate of this fresh water algae 

species significantly at an average exposure concentration of 26 mg/l. 

The EC5 0 for growth rate reduction (E R C5 0 : 0-72h) was 13 mg/l with a 95% confidence interval ranging 

from 8.3 to 19 mg/l. 

The EC5 0 for yield inhibition (EYC5 0 : 0-72h) was 7.0 mg/l with a 95% confidence interval ranging from 

5.3 to 9.3 mg/l. 

The NOEC for growth rate reduction was 4.2 mg/l, while the NOEC for yield inhibition could only be 

estimated to be <2.5 mg/l. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!ine 

5. INTRODUCTION 

5.1. Preface 

NOTOX Project 496987 

Sponsor 

Study Monitor (Tox and ecotox) 

Test Facility 

Study Director 

Technical Coordinator 

Principal Scientist 

Study Plan 

5.2. Aim of the study 

Huntsman Advanced Materials GmbH 

Klybeckstrasse 200 

4057 BASEL 

Switzerland 

Dr. P. Dollenmeier 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Netherlands 

Ir. L.M. Bouwman 

R.W.A.M. Coolen 

Dr. Ir. E. Baltussen 

Start 

Completion 

: 19 September 20fl 

: 13 October 2011 

The purpose of the study was to evaluate the test substance for its ability to generate toxic effects in 

Pseudokirchneriella subcapitata during an exposure period of at least 48 and at most 96 hours and, if 

possible, to determine the EC5 0 for both reduction of growth rate and inhibition of yield. 

5.3. Guidelines 

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 201: 

"Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006. 

In addition, the procedures were designed to meet the test methods prescribed by the following 

guidelines and guidance document: 

• Commission regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination 

of ecotoxicity, Publication No. L142, C3: "Algal Inhibition Test"; Amended by EC No. 761/2009 of 

23 July 2009, Publication No. L220. 

• ISO International Standard 8692: "Water quality - Freshwater algal growth inhibition test with 

unicellular green algae", Second edition, 01 October 2004. 

• Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series 

on testing and assessment number 23, December 14, 2000. 

5.4. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the NOTOX archives 

for a period of at least 10 years after finalization of the report. After this period, the sponsor will be 

contacted to determine how the records and materials should be handled. NOTOX will retain 

information concerning decisions made. 

Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as the 

quality of the specimens permits evaluation but no longer than three months after finalization of the 

report. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Cell density is the number of cells per millilitre. 

Growth rate is the increase in cell density per unit time. It is derived from the slope of the growth 

curve in a logarithmic plot. Following from the mathematical nature of exponential growth, the measure 

of the specific growth rate is preferable over biomass or yield. The ERCSO is the concentration of test 

substance that results in a 50% reduction in growth rate relative to the control. 

Yield is defined as the biomass at the end of the exposure period minus the biomass at the start of the 

exposure period. The EyC5 0 is the concentration of test substance that results in a 50% inhibition of 

yield relative to the control. 

No Observed Effect Concentration (NOEC) is the highest concentration tested at which the 

measured parameter(s) show(s) no significant effect on algal growth relative to control values. 

6. MATERIALS AND METHODS 

6.1. Test Substance 

6.1.1. Test substance information 

Identification 

Structure 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

O. 

0 

Molecular formula 

Molecular weight 

CAS Number 

EC Number 

Description 

Batch 

Purity 

Test substance storage 

Stability under storage conditions 

Expiry date 

C1 5H1 9N04 

277.3 

5026-74-4 

225-716-2 

Yellow liquid 

AA00373400 

94.33% 

In refrigerator (2-8°C) in the dark 

Stable 

28 February 2013 

6.1.2. Study specific test substance information 

Volatile 

Stability in water 

Solubility in water 

No 

No 

No 

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6.1.3. Reference substance 

This report includes the results of the most recent reference test with potassium dichromate 

(APPENDIX 5). 

6.2. Test System 

Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1 

Source In-house laboratory culture. 

Reason for selection This system is an unicellular algal species sensitive to toxic 

substances in the aquatic ecosystem and has been selected 

as an internationally accepted species. 

6.3. Fresh water algae culture 

Stock culture Algae stock cultures were started by inoculating growth 

medium with algal cells from a pure culture on agar. The 

suspensions were continuously aerated and exposed to light 

in a climate room at a temperature of 21-24°C. 

Light intensity 60 to 120 uE/m2/s when measured in the photosynthetically 

effective wavelength range of 400 to 700 nm. 

Stock culture medium M1; according to the NPR 6505 ("Nederlandse Praktijk 

Richtlijn no. 6505") formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, 

Mass., USA) with the following composition: 

NaN03 500 mg/l 

K2HP04 .3H2 0 52 mg/l 

MgSO,.7HaO 75 mg/l 

NaaCO3 .10H2O 54 mg/l 

C9Ha 07 .H2 0 6 mg/l 

NH4NO3 330 mg/l 

CaCI 2 .2H2 0 35 mg/l 

CeH6Fe07 .xH:0 6 mg/l 

H3B03 2.9 mg/l 

MnCI2.4HzO 1.81 mg/l 

ZnCI; 0.11 mg/l 

CuS04 .5H2 0 0.08 mg/l 

(NH<)sMo7024.4H20 0.018 mg/l 

Pre-culture 3 days before the start of the test, cells from the algal stock 

culture were inoculated in culture medium at a cell density of 

1 x 104 cells/ml. The pre-culture was maintained under the 

same conditions as used in the test. The cell density was 

measured immediately before use. 

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using 

Milli-Q water (tap water purified by reverse osmosis (MilliRO) and subsequently passed over activated carbon and 

ion-exchange cartridges: Milli-Q water; Millipore Corp., 

Bedford, Mass., USA) preventing precipitation and with the 

following composition: 

NR.CI 15 mg/l 

MgCh.6HiO 12 mg/l 

CaCI22H20 18 mg/l 

MgSO,.7H20 15 mg/l 

KH2PO, 1.6 mg/l 

FeCla.6HaO 64 (jg/l 

NaaEDTA.2H20 100 pg/l 

H3B03 185 pg/l 

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MnCl2.4H20 

ZnCI2 

CoCI2.6H20 

CuCI2.2H20 

Na2Mo04.2H20 

NaHC03 

Hardness (Ca+Mg) 

pH 

415 pg/l 

3 pg/l 

1.5 pg/l 

0.01 pg/l 

7 ug/i 

50 mg/l 

0.24 mmol/l (24 mg CaCOs/l) 

8.1 ±0.2 

6.4. Preparation of test solutions 

The standard test procedures required generation of test solutions, which contained completely 

dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The 

testing of concentrations that would disturb the test system was prevented as much as possible (e.g. 

film of the test substance on the water surface). 

The batch of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline tested was a yellow liquid with a 

purity of 94.33% and the substance was not completely soluble in test medium at loading rates of 

1.0 mg/l and higher. 

All test solutions with loading rates of 1.0 mg/l and higher were prepared separately applying two days 

of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting 

aqueous mixtures were left to stabilize for 1-2 hours where after the Water Accommodated Fractions 

(WAFs) were siphoned off and used for testing. One lower test concentration for the combined 

limit/range-finding test was prepared by subsequent dilution of the 1.0 mg/l WAF in test medium. The 

final test solutions were all clear and colourless. 

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. 

Subsequently, 1 ml of an alga! suspension was added to each replicate providing a cell density of 

104 cells/ml. | 

6.5. Combined limit/range-finding test 

The study started with a combined limit/range-finding test. Six replicates of exponentially growing 

algae were exposed to a control and a WAF prepared at a loading rate of 100 mg/l. Test procedure 

and conditions were similar to those applied in the final test with the following exceptions: 

• Three replicates per concentration were exposed to WAFs prepared at 1.0 and 10 mg/l and to 

a 10% dilution of the WAF prepared at 1.0 mg/l; 

• One extra test vessel per concentration without algae was used as background for the 

determination of the algal cell density at each time interval; 

• pH was only measured in the control and the highest test concentration. 

6.6. Final test 

6.6.1. Test concentrations 

p-(2,3-epoxypropoxy)-

N,N-bis(2,3-epoxypropyl)aniline 

Controls 

Replicates 

WAFs prepared at loading rates of 4.6, 10, 22, 46 and 

100 mg/l 

Test medium without test substance or other additives 

3 replicates of each test concentration; 

6 replicates of the control; 

1 extra replicate of the control and each test concentration 

for sampling purposes; 

2 replicates of the highest concentration without algae. 

6.6.2. Test procedures and conditions 

Test duration 72 hours 

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Test type Static 

Test vessels 100 ml, all-glass, containing 50 ml of test solution 

Medium M2 

Cell density An initial cell density of 1 x 104 cells/ml. 

Illumination Continuously using TLD-lamps of the type 'Cool-white' of 

30 Watt, with a light intensity within the range of 77 to 

90 uE.rTf2.s'1. 

Incubation Capped vessels were distributed at random in the incubator 

and as such were daily repositioned. During incubation the 

algal cells were kept in suspension by continuous shaking. 

6.6.3. Sampling for analysis of test concentrations 

Samples for possible analysis were taken from all test concentrations and the control according to the 

schedule below. The method of analysis is described in the appended Analytical Report 

(APPENDIX 6). 

Frequency at t=0 h, t=24 h and t=72 h 

Volume 2 ml 

Storage Samples were stored in a freezer until analysis. 

At the end of the exposure period, the replicates with algae were pooled at each concentration before 

sampling. 

Compliance with the Quality criteria regarding maintenance of actual concentrations was 

demonstrated by running a test vessel at the highest substance concentration but without algae and 

samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test 

period. 

Additionally, reserve samples of 2 ml were taken from all test solutions for possible analysis. If not 

already used, these samples were stored in a freezer for a maximum of three months after delivery of 

the draft report, pending on the decision of the sponsor for additional analysis. 

6.6.4. Measurements 

pH At the beginning and at the end of the test. 

The pH of the solutions should preferably not deviate by 

more than 1.5 units during the test'. 

Temperature of medium Continuously in a temperature control vessel. 

6.6.5. Recording of cell densities 

At the beginning of the test, cells were counted using a microscope and a counting chamber. 

Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm 

using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as 

blank. 

6.7. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following 

programme(s): 

- Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software (Shimadzu, Kyoto, 

Japan): Algal cell density. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA): Temperature. 

6.8. Interpretation 

6.8.1. Data handling 

Calibration curve 

Quantification of cell densities was based on a calibration curve. Cell density was plotted versus 

extinction using spectrophotometric measurements of a minimum of six dilutions of an algal 

suspension with different cell densities. The calibration curve was composed using linear regression. 

The software automatically calculates the cell densities based on this curve for the spectrophotometric 

measurements at the various points in time during the test period. 

Comparison of average growth rates 

The average specific growth rate for a specific period is calculated as the logarithmic increase in the 

biomass from the equation for each single vessel of controls and treatments: 

InX, -lnX f , 

M H = _ . '(day 1) 

Where: /J 4 = the average specific growth rate from time i to j 

Xj = the biomass at time i 

Xj = the biomass at time j 

The average growth rate at each test substance concentration is then compared with the control value 

and the percentage reduction in growth rate is calculated: 

% l r = VC-V T X1Q O 

Where: %l r = percent inhibition in average specific growth rate 

f j c = mean value for average specific growth rate in the control group 

u r = average specific growth rate for thetreatment replicate 

Yield 

The percent inhibition in yield is calculated for each treatment replicate as follows: 

%/ y =

 Yc

~

Y r x1Q 0 

Where: %l y = percent inhibition of yield 

Yc = mean value for yield in the control group 

YT = value for yield for the treatment replicate 

Determination of the average exposure concentrations 

24 x JC t = 0 x C 1=2 4 + 48 x JC,_24 x C t = „ 

The average exposure concentrations were calculated as: - — -—— —, 

being the Time Weight Average (TWA) of the concentrations of p-(2,3-epoxypropoxy)-N, N-bis(2,3-

epoxypropyl)aniline measured in the samples taken at the start (Cl=0 ), after 24 hours (Ct=24) and the 

end of the test (Ct =72 ). 

Determination of the NOEC and calculation of the1 ECTM 

For determination of the NOEC and the EC5 0 the approaches recommended in the OECD guideline 

201 were used. An effect was considered to be significant if statistical analysis of the data obtained for 

the test concentrations compared with those obtained in the negative control revealed significant 

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reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, 

D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC1 0 was determined to meet the 

recommendations as'put down in "A Review of Statistical Data Analysis and Experimental Design in 

OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC5 0 and 

EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction 

and the percentages of yield inhibition versus the logarithms of the corresponding average exposure 

concentrations of the test substance. 

6.8.2. Acceptability of the test 

1. In the control, cell density increased by an average factor of >16 within two days. 

2. The mean coefficient of variation for section-by-section specific growth rates in the control 

cultures did not exceed 35%. 

3. The coefficient of variation of average specific growth rates during the whole test period in 

replicate control cultures did not exceed 7%. 

6.9. List of deviations 

6.9.1. List of protocol deviations 

1. Combined limit range/finding test; After 48 hours of exposure one out of the six replicate vessels 

for the highest test concentration broke and could therefore no longer be used for measurement 

of algal cell density. 

Evaluation: The combined limit/range-finding test could still be used as a range-finding finding 

test to determine the range of concentrations used in the final test. Results were based on five 

instead of six replicates. 

2. Final test: After 72 hours of exposure one out of the six replicate vessels for the control group 

broke and could therefore not be used for measurement of algal cell density at the end of the test. 

Evaluation: Results were based on five instead of six replicates. According to the OECD guideline 

the number of replicate vessels for the control group should be at least three. Therefore, the test 

can still be used to determine the effect parameters (EC5 0 and NOEC). 

3. Final test: No microscopic observations of the algal cells were made at the end of the test. 

Evaluation: The study could still be used to determine EC5o values, which was the main goal. 

The study integrity was not adversely affected by the deviations. 

6.9.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures were evaluated and filed in the study file. There 

were no deviations from standard operating procedures that affected the integrity of the study. 

7. RESULTS 

7.1. Combined limit/range-finding test 

The mean cell densities measured during the combined limit/range-finding test are presented in 

Table 1. Table 2 presents the percentages growth rate reduction and yield inhibition per 

concentration. Based on these results samples taken from the WAFs prepared at 1.0 and 100 mg/l 

were analysed. The initial concentrations were 0.72 and 60 mg/l. These concentrations remained 

stable during the first 24 hours of exposure (80-81 % of initial) but decreased to 45-62% of initial at the 

end of the test period (see also Table 2 of the appended Analytical Report). The expected EC5 0 for 

both growth rate reduction and yield inhibition was between concentrations obtained in WAFs 

prepared at 10 and 100 mg/l. 

All test conditions were maintained within the limits prescribed by the protocol. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

Table 1 Mean cell densities (x104 cells/ml) during the combined limit/range-finding test 

. Loading rate1 

Test substance2 

(ma/I) 

. Loading rate Exposure time (hours) 1 

Test substance2 

(ma/I) 0 24 48 72 

control 1.0 11.0 54.0 227,2 

10% Of 1.0 1.0 11.2 59.2 239.5 

1.0 1.0 10.6 53.3 219.2 

10 1.0 10,9 48.0 197.7 

100 1.0 5.7 8.5 6.4 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

Table 2 Percentage reduction of growth rate and inhibition of yield during the combined 

limit/range-finding test 

Loading rate1 

Test substance2 

(mg/l) 

Loading rate Mean growth rate Yield (0-72 h) 1 

Test substance2 

(mg/l) u (0-72 h) Reduction (%) x104 cells/ml Inhibition (%) 

control 0.07532 226.19 

10% of 1.0 0.07608 -1.0 238.50 -5.4 

1.0 0.07482 0.7 218.24 3.5 

10 0.07342 2.5 196.70 13.0 

100 0.02576 65.8 5.41 97.6 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

7.2. Final test 

7.2.1. Measured test substance concentrations 

The results of analysis of the samples taken during the final test are described in Table 3 of the 

appended Analytical Report. 

At the start of the test, the actual test concentrations were 3.2, 3.3, 5.5, 33 and 34 mg/l in the WAFs 

prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/l, respectively. These concentrations 

remained stable during the first 24 hours of exposure (82-87% of initial) but decreased to 59-66% of 

initial at the end of the test period. Note that actual concentrations being almost similar is related to 

medium solubility and the preparation of WAFs. Based on these results, the average exposure 

concentrations were calculated to be: 2.5, 2.6, 4.2, 26 and 26 mg/l. 

7.2.2. Mean cell densities 

Table 3 shows mean cell densities measured at 24-hour intervals at the different concentrations of p-

(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. The respective growth curves are shown in 

Figure 1 (see APPENDIX 1 for the cell densities per replicate). Note that the mean cell density of the 

control was based on 5 replicates at the end of the test due to the fact that one vessel fall and broke. 

- Page 14 -

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Table 3 Mean cell densities (x 104 cells/ml) during the final test 

Loading rate1 

Test substance2 

(mg/l) 

Loading rate Exposure time (hours) 1 

Test substance2 

(mg/l) 0 24 48 72 

control 1.0 8.7 38.4 158.5 

4.6(2.5) 1.0 8.2 34.2 131.7 

10(2.6) ' 1.0 9.0 36.6 145.6 

22 (4.2) 1.0 8.7 31.5 108.7 

46 (26) 1.0 6.2 7.1 4.2 

100 (26) 1.0 5.5 7.6 3.5 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

() Average exposure concentration 

180 

Exposure time (hours) 

Figure 1 Growth curves at different concentrations of p-(2,3-epoxypropoxy)-N,N-bis(2,3-

epoxypropyl)aniline 

7.2.3. Reduction of growth rate and inhibition of yield 

Table 4 shows the calculation of the percentages of growth rate reduction (total test period) and the 

percentages of yield inhibition. Table 5 shows the calculation of the percentages of growth rate 

reduction at different time intervals (see APPENDIX 1 for the values of growth rate and yield per 

replicate). Statistical analysis of the data is shown in APPENDIX 2 and APPENDIX 3. 

Growth rates were in the range of the control at 2.5/2.6 mg/l during the 72-hour test period, whereas 

the growth rate of algae exposed to 4.2 and 26 mg/l were reduced. Statistically significant reduction of 

growth rate was found at a test concentration of 26 mg/l (Bonferroni t-Test, a = 0.05), that was 

measured in WAFs prepared at both 46 and 100 mg/l. 

Inhibition of yield increased with increasing concentration of p-(2,3-epoxypropoxy)-N,N-bis(2,3-

epoxypropyljaniline from 2.5/2.6 mg/l upwards resulting in almost complete inhibition at 26 mg/l. 

Statistically significant inhibition of yield was found at all test concentrations, except for 2.6 mg/l 

measured in the WAF prepared at 10 mg/l (Bonferroni t-Test, a = 0.05). No explanation can be given 

for the relatively high inhibition at the lowest loading rate. Statistical analysis does not give a clear 

output on the NOEC for yield inhibition. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani ine 

Table 4 Percentage reduction of growth 

of yield during the final test 

Loading rate1 

Test substance2 

inm 

control 

_4.6 (2.5) 

10(2.6) 

_22 (4.2) 

_46 (26) 

100 (26) 

Mean growth rate 

u (0-72 h) Reduction (%) 

0.07030 

0.06774 

0.06917 

0.06512 

0.01977 

0.01683 

3.6 

1.6 

7.4 

71.9 

76.1 

x10* cells/ml 

157 

130 

144 

107 

2.£0 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl Janiline 

() Average exposure concentration 

Table 5 Percentage reduction of growth 

test 

Loading rate1 

Test substance2 

fmg/l) 

control 

4.6 (2.5) 

_10 (2.6) 

_22 (4 2) 

46 (26) 

_100 (26) 

M(0-24h) Reduction (%> u (24-48 h) Reduction (%) p (48-72 h) Reduction {%) 

0.09011 

0.08753 

0.09126 

0.09029 

0.07573 

0.07138 

2.9 

-1.3 

-0.2 

16.0 

20.8 

0.06135 

0.05931 

0.05875 

0.05339 

0.00535 

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NOTOX Project 496987 

rate (total test period) and percentage inhibition 

Yield (0-72 h) 

50 

66 

61 

69 

Inhibition (%) 

17.0 

8.2 

31.6 

97.9 

98.4 

rate at different time intervals during the final 

Mean growth rate 

0.01260 

' WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

() Average exposure concentration 

7.2.4. Determination of effect concentrations 

Table 6 shows the effect parameters based on average exposure concentrations, see also 

APPENDIX 4. 

Table 6 Effect parameters 

Parameter Concentration 

Test substance1 

<mfl/l) 

95%-confidence 

interval 

NOERC 4.2 

72h>ERCio 3.6 2.3 - 5.4 

72h^ERC5o 13 8.3-19 

NOEyC <2.5 

72tvEYC1 0 2.4 1.8-3.2 

72h-EYC5o 7.0 5.3-9.3 

7.2.S. Experimental conditions 

Table 7 shows the pH recorded at the beginning s 

prescribed by the protocol (6.0-9.0, preferably not 

the test medium was 22.1°C at the start of the test 

measured in the incubator was maintained between 

the limits prescribed by the protocol (21-24°C, constant 

- P£ 

3.3 

4.7 

13.4 

91.3 

79.2 

0.05898 

0.05609 

0.05751 

0,05167 

0.00000 

0.00000 

4.9 

2.5 

12.4 

100.0 

100.0 

i-epo>ypropyl)aniline 

nd the end of the test. The pH was within the limits 

varying by more than 1.5 unit). The temperature of 

During the exposure period the temperature 

22.4 and 23.5°C. Temperature remained within 

within 2°C). 

ge 16-

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Table 7 pH levels recorded during the final test 

Loading rate1 

Test substance2 

(mg/l) 

Loading rate Exposure time (hours) 1 

Test substance2 

(mg/l) 0 72 

control 8.1 7.9 

4.6 (2.5) 8.2 7.9 

10 (2.6) 8.1 7.9 

22 (4.2) 8.2 7.8 

46(26} 8.1 7.9 

100 (26) 8.2 7.9 

' WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

() Average exposure concentration 

8. CONCLUSION 

Under the conditions of the present study with Pseudokirchneriella subcapitata, p-(2,3-epoxy pro poxy)-

N,N-bis(2,3-epoxypropyl)aniline reduced growth rate of this fresh water algae species significantly at 

an average exposure concentration of 26 mg/l. 

The EC5 0 for growth rate reduction (E R C5 0 : 0-72h) was 13 mg/l with a 95% confidence interval ranging 

from 8.3 to 19 mg/l. 

The EC5 0 for yield inhibition (EYC5 0 : 0-72h) was 7.0 mg/f with a 95% confidence interval ranging from 

5.3 to 9.3 mg/l. 

The NOEC for growth rate reduction was 4.2 mg/l, while the NOEC for yield inhibition could only be 

estimated to be <2.5 mg/l. 

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ine NOTOX Project 496987 

APPENDIX 1 WORKSHEET DATA 

Table 8 Individual cell densities 

Number of inoculated cells att=0: 1x10" cells/ml 

Loading rate1 Vessel Exposure time (hours) 

Test substance2 number 

(mg/l) 0 24 48 72 

control 1 1.00 8.95 39.79 165.54 

2 1.00 8.00 37.48 3 

3 1.00 8.74 41.73 169.66 

4 1.00 8.18 30.46 135.09 

5 1.00 9.80 4313 173.60 

6 1.00 8.61 37.88 148.60 

4.6 (2.5) 1 1.00 7.83 31.65 122.04 

2 1.00 8.40 34.50 144.65 

3 1.00 8.29 36.54 128.28 

10 (2.6) 1 1.00 8 66 35.69 145 46 

2 1.00 9.63 38.97 150.05 

3 1.00 8.56 35.27 141.31 

22 (4.2) 1 1.00 8.85 31.60 109.20 

2 1.00 8.68 31.40 107.03 

3 100 8.67 31.35 109.86 

46 (26) 1 1.00 5.66 5.68 3.86 

2 1.00 6.36 7.47 5.44 

3 1.00 6.48 8.09 3.42 

100 (26) 1 1 00 5.43 6.62 2.20 

2 1.00 5.72 7.69 4.17 

3 1.00 5.49 8 42 4.13 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

3

 No measurement of algal cell density at the end of the test; v issel was broken 

{) Average exposure concentration 

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APPENDIX 1 WORKSHEET DATA - continued -

Table 9 Calculation of growth rate and yield 

Growth rate 

Loading rate1 Vessel Growth rate Yield red. Yield inhib. 

Test substance2 number (M) <x104

 cells/ml) (%) (%) 

(mg/l) 0-72 h 0-72 h 0-72 h 0-72 h 

control 1 0.07096 

3 

164.54 

3 

/L 

3 0.07130 168 66 

4 0.06814 134.09 ' 

5 0.07162 172.60 

6 0.06946 147.60 

mean 0.07030 157 50 

CV 2% 

4 6 (2.5) 1 0.06673 121.04 5 23 

2 0.06909 143.65 2 9 

3 0.06742 127.28 4 19 

10(2.6) 1 0.06916 144 46 2 8 

2 0,06960 149.05 1 5 

3 0.06876 140.31 2 11 

22 (4 2) 1 0.06518 108.20 7 31 

2 0.06490 106.03 8 33 

3 0.06527 108.86 7 31 

46 (26) 1 0.01874 2.86 73 98 

2 0.02351 4.44 67 97 

3 0.01706 2.42 76 98 

100 (26) 1 0.01098 1.20 84 99 

2 0.01983 3.17 72 98 

3 0.01968 3.13 72 98 

WAF prepared at the given loading rate 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

3

 No measurement of algal cell density at the end of the test; vessel was broken 

() Average exposure concentration 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani ine 

APPENDIX 1 WORKSHEET DATA - continued -

Table 10 Calculation of growth rate (section-by-section) 

NOTOX Project 496987 

Loading rate1 Vessel Growth rate IV) Growth rate reduction {%) 

Test substance* number 

(mq/l) 0-24 h 24-48 h 48-72 h 0-24 h 24-48 h 48-72 h 

control 1 0.09131 0 06217 0.05940 

2 0.08666 0 06433 3 

. 3 0.09032 0.06515 0.05844 

4 0.08758 0.05478 0.06206 

5 0.09511 0.06173 0.05803 

6 0.08969 0.06174 0.05696 

mean 0.09011 0.06165 0.05898 

CV 3% 6% 3% 

The mean CV for section-bv-section specil cc rowth rate was: 2 1 % 

4.6 (2.5) 1 0.08576 0 05819 0.05623 5 6 5 

2 0.08869 0.05884 0.05973 2 5 -1 

3 0 08814 0.06179 0.05232 2 0 11 

10 (2.6) 1 0.08994 . 0.05901 0.05854 0 4 1 

2 0.09439 0.05823 0.05617 -5 6 5 

3 0.08946 0.05900 0.05783 1 4 2 

22 (4.2) 1 0.09083 0 05305 0.05166 -1 14 12 

2 0.09004 0.05357 0.05109 0 13 13 

3 0.09001 0.05354 0.05225 0 13 11 

46(26) 1 0.07220 0.00016 0.00000 20 100 100 

2 0 0771O 0.00666 0.00000 14 89 100 

3 0.07789 0.00923 0.00000 14 85 100 

100 (26) 1 0 07052 0.00823 0.00000 22 87 100 

2 0.07266 0.01232 0.00000 19 80 100 

3 0.07095 0.01784 0.00000 21 71 100 

2

 The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2,3-' 

3

 No measurement of algal cell density at the end of the test; 

() Average exposure concentration 

epoxyprcpyl)ani!ine 

vessel was broken 

rr 

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APPENDIX 2 STATISTICS: GROWTH RATE (0-72 HOURS) 

Chi-Square Test for Normality 

Actual and Expected Frequencies 

INTERVAL <-1.5 -1.5 to <-0.5 0.5 to 0.5 >0.5 to 1.5 >1.5 

EXPECTED 

OBSERVED 

1.3400 

0 

4.8400 

7 

7.6400 

5 

4 .8400 

8 

1.3400 

0 

Chi- Square ~ 6.6194 (P- value = 0.1574) 

Critica l Chi-Square = 13.277 (alpha = 0.01 , df = 4) 

= 9.488 (alpha = 0.05 , df = 4} 

Data PASS normality tes t (alpha = 0.01). Continue analysis. 

Levane's Test for Homogeneity of Variance 

ANOVA Table 

SOURCE DF SS MS F 

Between 5 0.0000 0.0000 0.7627 

Within (Error) 14 0.0001 0.0000 

Total 19 0.0001 

(p-value = 0.5914) 

Critica l F = 4.6950 (aloha = 0.01, df = 5,14) 

= 2.9582 (alpha = 0.05, df = 5,14) 

Since F < Critica l F FAIL TO REJECT Ho: Al l equal (alpha = 0.01) 

ANOVA Table ' 

SOURCE DF SS MS F 

Between 5 

Within (Error) 14 

0.0106 

0.0001 

0.0021 

0.0000 

346.7543 

Total 19 0.0107 

Critica l F = 4.6950 

= 2.9582 

Since F > Critica l F 

(alpha = 0.01, df = 5,14) 

(alpha = 0.05, df = 5,14) 

REJECT Ho: Al l equal (alpha 

(p-value 

= 0.05) 

= 0.0000) 

Bonferroni. t-Test TABLE 1 OF 2 Ho: Control=Treatment 

TRANSFORMED MEAN CALCULATED IN S1G 

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05 

1 contro l 0.0703 0.0703 

2 4.6 0.0677 0.0677 1 4113 

3 10 0.0692 0.0692 0 6215 

4 22 0.0651 0.0651 2 8672 

5 46 0.0198 0.0198 27 9700 * 

6 100 0.0168 0.0168 29 5975 * 

Bonferroni t critica l value = 2.9768 (2 Tailed, alpha = 0.05, df = 5,14 ) 

Bonferroni t-Test TABLE 2 OF 2 Ho: Control =Treatraent 

HUM OF MIN SIG DIFF % OF DIFFERENCE 

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL 

1 contro l 5 

2 4.6 3 0.0054 7.6 0.0025 

3 10 3 0.0054 7.6 0.0011 

4 22 3 0.0054 7.6 0.0052 

5 46 3 0.0054 7.6 0.0505 

6 100 3 0.0054 7.6 0.0535 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)an me i 

APPENDIX 3 STATISTICS: YIELD (0-72 HOURS) 

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NOTOX Project 496987 

Chi-Square Test for Normality 

INTERVAL 

EXPECTED 

OBSERVED 

<-1.5 

1.3400 

0 

Actual and Expected Frequencies 

-1.5 to <-0.5 -0. to 0.5 

4.8400 

7 

.6400 

5 

>0.5 to 1.5 

4.8400 

>1.S 

1.3400 

0 

Chi-Square = 6.6194 (p-va:.ue = 0.1574) 

Critica l Chi-Square = 13.277 (alpha == 0.01 , df = 4) 

= 9.488 (alpha f 0.05 , df = 4) 

Data PASS normality tes t (alpha = 0.01). Con :inue analysis. 

Levene's Test for Homogeneity of Variance 

ANOVA Table 

SOURCE DF SS MS 

Between 

Within (Error) 

5 

14 

433.2516 

741.9633 

86.6503 

52.9974 

F 

1.6350 

Total 19 1175.2148 

Critica l F = 4.6950 (alpha = 0.01, df = 

= 2.9582 (alpha =0.05, df = 

Since F < Critica l F FAIL TO REJECT Ho 

(p-value = 0.2150) 

5,14) 

5,14) 

A l l equal (alpha = 0.01) 

SOURCE DF 

ANOVA Table 

SS MS 

Between 

Within (Error) 

5 

14 

81995.1222 

1368.2673 

16399.0244 

97.7334 

167.7935 

Total 19 83363.3895 

Critica l F = 4.6950 (alpha = 0.01, df = 

= 2.9582 (alpha = 0.05, df = 

Since F > Critica l F REJECT Ho: Al l eqja l 

(p-value = 0.0000) 

5,14) 

5,14) 

(alpha = 0.05) 

Bonferroni t-Test TABLE 1 OF 2 Ho: Control=Treatment 

GROUP IDENTIFICATION 

contro l 

4.6 

10 

22 

46 

100 

TRANSFORMED 

MEAN 

157.4980 

130.6567 

144.6067 

107.6967 

3.2400 

2.5000 

MEAN CALCULATED IN SIG 

ORIGINAL UNITS t STAT 0.05 

157.4980 

130.6567 

144.6067 

107.6967 

3.2400 

2.5000 

3.7178 

1.7856 

6.8980 

21.3662 

21.4687 

Sonferroni t critica l value = 2.97f (2 Tailed, alpha = 0.05, df = 5,14) 

Bonferroni t-Test TABLE 2 OF 2 Ho: Control=Treatment 

GROUP IDENTIFICATION 

NUM OF 

REPS 

MIN S 

(IN 

IG DIFF % OF DIFFERENCE 

ORiTjG. UNITS) CONTROL FROM CONTROL 

contro l 

4.6 

10 

22 

46 

100 

1.4920 

1.4920 

1.4920 

1.4920 

1.4920 

13.6 

13.6 

13.6 

13.6 

13.6 

26.8413 

12.8913 

49.8013 

154.2580 

154.9980 

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APPENDIX 4 EC-VALUES 

Table 11 EC-values for growth rate reduction 

Concentration X Y 

(mg/l) Log cone, (mq/f) Reduction (%) 

2.5 0.398 5.1 

2.5 0.398 1.7 

2.5 0.398 4.1 

2.6 0 415 1.6 

2.6 0.415 1.0 

2.6 0.415 2.2 

4.2 0.623 7.3 

4.2 0.623 7.7 

4.2 0.623 7.2 

26 1.415 73.3 

26 1.415 66.6 

26 1.415 75.7 

26 1.415 84.4 

26 1.415 71.8 

26 1.415 72.0 

Slope: 72.9723 

Intercept: -30.1554 

Multiple R: 0.9872 

n = number of observations: 15 

Regression line' Y= 72.97X - 30.16 

Prediction of X values based on known Y values 

Known Y 

Reduction (%) 

1 0 Xre g 

(mg/l) 

^QX95%-

(mq/l) 

1 0X9E% + 

(mg/l) 

10 3.55 2.33 5.40 

20 4.87 3.21 7.38 

50 12.54 8.26 19 06 

Figure 2 Percentage reduction of growth rate as function of the log concentration (mg/l) 

of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

Dashed curves represent the 95% confidence limits. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

APPENDIX 4 EC-VALUES - continued -

Table 12 EC-values for yield inhibition 

Concentration X Y 

(rnq/l) Loq cone, (mq/l) Inhibition (%) 

2.5 0.398 23 2 

2.5 0.398 8.8 

2.5 0.398 19.2 

2 6 0.415 8.3 

2.6 0.415 5.4 

2.6 0.415 10.9 

4 2 0.623 31.3 

4.2 0.623 32.7 

4.2 0.623 30 9 

26 1.415 98.2 

26 1 415 97.2 

26 1.415 98,5 

26 1.415 99.2 

26 1.415 98.0 

26 1.415 98.0 

Slope: 84.6297 

Intercept. -21.5651 

Multiple R: 0.9938 

n = number of observations: 15 

Regression line: Y= 84.63X - 21.57 

Prediction of X values based on known Y values 

Known Y 

Inhibition (%) 

io Xre g 

(mg/l) 

,JQX95%-

(mq/l) (mq/l) 

10 2.36 1.76 3 17 

20 3.10 2.31 4.15 

50 7.01 5.26 9.33 

1 10 100 

Concentration (mg/l) 

Figure 3 Percentage inhibition of yield as function of the log concentration (mg/l) of p-

(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. 

Dashed curves represent the 95% confidence limits. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

APPENDIX 5 REFERENCE TEST 

NOTOX Project 496987 

Pseudokirchneriella subcapitata, strain: NIVA CHL-1. Fresh water algal growth inhibition test with 

potassium dichromate (NOTOX Project 498136). 

Start of first exposure: 10 October 2011 

Completion last exposure: 13 October 2011 

The study procedures described in this report were based on the OECD guideline No. 201, Adopted 

March 23, 2006 and ISO Standard 8692, Second edition, 01 October 2004. 

This reference test was carried out to check the sensitivity of the test system used by NOTOX to 

Potassium dichromate (Merck, Art. 1.04864, Batch K34869764 607). 

Algae were exposed for a period of 72 hours to K2Cr 2 07 (Potassium dichromate) concentrations of 

0.18, 0.32, 0.56,1.0, 1.8 and 3.2 mg/l and to a control. The initial cell density was 1.0 x 104 cells/ml. 

Results: 

Overview of % reduction of growth rate and % inhibition of yield in the reference test: 

Nominal cone. 

K2Cr 2 07 

(mq/l) 

Nominal cone. Mean growth rate Yield (0-72 h) 

K2Cr 2 07 

(mq/l) M (0-72 h) Reduction (%) x104

 cells/ml Inhibition (%) 

Control 0.06997 153.26 

0.18 0.06933 0.9 146.59 4.4 

0.32 0.06681 4.5 122.63 20.0 

0.56 0.06004 14.2 74.56 51.4 

1.0 0.04340 38.0 21.89 85.7 

1.8 0.02860 59.1 6.86 95.5 

3.2 0.02109 69.9 3.57 97.7 

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations 

of 0.56 mg/l and higher. 

The EC5 0 for growth rate reduction (ERCso: 0-72h) was 1.5 mg/I with a 95% confidence interval ranging 

from 1.1 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. 

Hence, the E R C5 0 : 0-72h for the algafculture tested corresponds with this range. 

The ECso for yield inhibition (EyC^: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 

0.34 to 0.78 mg/l. The historical ranges of the 72h-EC5o for yield inhibition lie between 0.43 and 1.1 

mg/l. Hence, the EYC5 0 : 0-72h for the algal culture tested corresponds with this range. 

The protocol, raw data and report of this study are kept in the NOTOX archives. The test described 

above was performed under GLP conditions with a QA-check. 

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p. 119 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

APPENDIX 6 ANALYTICAL REPORT 

DETERMINATION OF THE 

CONCENTRATIONS 

Author 

Dr. Ir. E. Baltussen 

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p. 120 

1. CONTENTS 

1. CONTENTS 27 

2. REPORT APPROVAL 28 

3. INTRODUCTION 29 

3.1. Preface 29 

3.2. Aim of the study 29 

4. MATERIALS AND METHODS 29 

4.1. Reagents 29 

4.2. Samples 29 

4.3. Analytical method 29 

4.3.1. Analytical conditions 29 

4.3.2. Preparation of the calibration solutions 29 

4.3.3. Sample injections 30 

4.4. Electronic data capture 30 

4.5. Formulas 30 

5. RESULTS 31 

5.1. Calibration curves 31 

5.2. Samples 31 

5.2.1. Procedural recovery samples 31 

5.2.2. Test samples 31 

6. TABLES 32 

TABLES 

Table 1 Procedural recovery samples 32 

Table 2 Concentrations of the test substance in test medium - combined limit/range-finding 

test 33 

Table 3 Concentrations of the test substance in test medium - final test 33 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

2. REPORT APPROVAL 

NOTOX B.V. 

NOTOX Project 496987 

Principal Scientist 

Analytical Chemistry 

Dr. Ir. E. Baltussen 

Date: 

Final Report - Page 28 -
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3. INTRODUCTION 

3.1. Preface 

Study plan analytical phase Start 30 September 2011 

Completion : 22 October 2011 

3.2. Aim of the study 

The purpose of the analytical phase was to determine the actual concentrations in samples taken from 

the test solutions used during the ecotoxicity test. 

4. MATERIALS AND METHODS. 

4.1. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA) 

Acetonitrile Biosolve, Valkenswaard, The Netherlands 

M2-medium see main report 

All reagents were of analytical grade, unless specified otherwise. 

4.2. Samples 

The samples were stored in the freezer (£ -15°C). Storage stability of samples under these conditions 

was demonstrated in NOTOX project 496981. 

On the day of analysis, the samples were defrosted at room temperature. The test samples were 

diluted in a 1:1 (v:v) ratio with 60/40 (v/v) acetonitrile/M2-medium and analysed. If necessary, the 

samples were further diluted with 30/70 (v/v) acetonitrile/M2-medium to obtain concentrations within 

the calibration range. 

4.3. Analytical method 

4.3.1. Analytical conditions 

Quantitative analysis was based on the analytical method validated for the test substance in NOTOX 

project 496981. 

Instrument Alliance Separation Module 2695 

(Waters, Milford, MA, USA) 

Detector Dual A Absorbance Detector 2487 (Waters) 

Column Symmetry Shield RP-18, 100 mm x 4.6 mm i.d., dp = 3.5 urn 

(Waters) 

Column temperature 40°C ± 1°C 

Injection volume 100 pi 

Mobile phase 30/70 (v/v) acetonitrile/water 

Flow 1.0 ml/min 

UV detection 253 nm 

4.3.2. Preparation of the calibration solutions 

Stock and spiking solutions 

Stock solutions of the test substance were prepared in acetonitrile at concentrations of 2415 - 3820 

mg/l. 

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of 

the spiking solutions was acetonitrile. 

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p. 123 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

Calibration solutions 

Calibration solutions in the concentration range of 0.04 - 2 mg/l were prepared from two stock 

solutions. The end solution of the calibration solutions was 30/70 (v/v) acetonitrile/M2-medium. 

Procedural recovery samples 

2 ml blank medium was spiked with the test substance at a target concentration of 0.1 or 100 mg/l. 

The accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples'). 

4.3.3. Sample injections 

Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were 

analysed by single injection. 

4.4. Electronic data capture 

System control, data acquisition and data processing were performed using the following programme: 

- Empower version 7.00 (Waters, Milford, MA, USA). 

Temperature and/or relative humidity during sample storage and/or performance of the studies were 

monitored continuously using the following programme: 

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA). 

4.5. Formulas 

Response (R) Peak area test substance [units] 

Calibration curve R = a C N + b 

where: 

CM = nominal concentration [mg/lj 

slope [units x l/mg] 

intercept [units] 

a -

b = 

Analysed concentration (CA) (R-b) 

xd [mg/l] 

a 

where: 

d = dilution factor 

Recovery 100 [%] 

Relative to initial concentration 

C A (t = x hours) 

C A (t = 0 hours) 

x100 [%] 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

5. RESULTS 

5.1. Calibration curves 

Calibration curves were constructed using five concentrations. For each concentration, two responses 

were used. Linear regression analysis was performed using the least squares method with a 

1 /concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve. 

5.2. Samples 

5.2.1. Procedural recovery samples 

The results for the procedural recovery samples are given in Table 1. 

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110%. It 

demonstrated that the analytical method was adequate for the determination of the test substance in 

the test samples. 

5.2.2. Test samples 

The results for the test samples are given in Table 2 and Table 3 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyr)aniline NOTOX Project 496987 

6. TABLES 

Table 1 Procedural recovery samples 

Date of 

preparation 

jdd-mm-yyj 

Date of 

analysis 

[dcj-mm-yy] 

Target 

concentration 

Jmg/rj 

Nominal 

concentration 

rmavTj 

Analysed 

concentration 

Jm.g/IJ 

Recovery 

f%) 

Mean 

recovery 

I%] 

30-09-11 30-09-11 0.1 0.100 0.0924 92 98 

0.100 0.103 103 

30-09-11 30-09-11 100 100 101 101 101 

100 101 101 

22-10-11 22-10-11 0.1 0.0993 0.0867 87 84 

0.0993 0.0808 81 

22-10-11 22-10-11 100 99.3 95.9 97 98 

99.3 99.6 100 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496987 

Table 2 Concentrations of the test substance in test medium - combined limit/rangefinding test 

Time of 

sampling 

[hours! 

Date of 

sampling 

[dd-mm-yyl 

Date of 

analysis

 1 

[dd-mm-yyl 

Loading rate

 2 

rmg/ll 

Concentration 

analysed 

[mq/ll 

Relative to 

initial 

r%i 

0 19-09-11 30-09-11 1 0.728 

100 60.0 

1003 56.4 

24 20-09-11 30-09-11 1 0.580 80 

100 48.8 81 

1003 51.4 91 

72 22-09-11 30-09-11 1 0.325 45 

100 37.0 62 

1003 33.7 60 

Samples were stored in the freezer (s -15°C) until the day of analysis. 

A water accommodated fraction (WAF) prepared at the loading rate. 

Without algae. 

Table 3 Concentrations of the test substance in test medium - final test 

Time of Date of Date of Loading rate1 Concentration Relative to 

sampling sampling analysis1 analysed initial 

fhoursl rdd-mm-yyl [dd-mm-yyl [mg/ll [mg/ll [%1 

0 10-10-11 22-10-11 0 n.d. 

4.6 3.23 

10 3.30 

22 5.48 

46 32.6 

100 33.8 

1003 36.4 

24 11-10-11 22-10-11 0 n.d. n.a. 

4.6 2.82 87 

10 2.74 83 

22 4.59 84 

46 27.1 83 

100 27.6 82 

1003 28.5 78 

72 13-10-11 22-10-11 0 n.d. n.a. 

4.6 1.89 59 

10 2.03 61 

22 3.22 59 

46 21.4 66 

100 21.3 63 

1003 22.8 62 

' Samples were stored in the freezer (s -15°C) until the day of analysis 

2

 A water accommodated fraction (WAF) prepared at the loading rate. 

3

 Without algae. 

n.d. Not detected, 

n.a. Not applicable. 

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FINAL REPORT 

Study Title 

DAPHNIA MAGNA, REPRODUCTION TEST WITH 

P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILINE 

(SEMI-STATIC) 

Author 

Ir. L.M. Bouwinan 

Test Facility 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Netherlands 

Laboratory Project Identification 

NOTOX Project 496989 

NOTOX Substance 203277/A 

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p. 128 

p-(2,3-epoxypropoxy)~N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496983 

1. CONTENTS 

1. CONTENTS 2 

2. STATEMENT OF GLP COMPLIANCE 4 

3. QUALITY ASSURANCE STATEMENT , 5 

4. SUMMARY , , , 6 

5. INTRODUCTION 7 

5.1. Preface 7 

5.2. Aim of the study 7 

5.3. Guidelines 7 

5.4. Storage and retention of records and materials 7 

5.5. Definitions 8 

6. MATERIALS AND METHODS 3 

6.1. Test Substance 8 

6.1.1. Test substance information 8 

6.1.2. Study specific test substance information 9 

6.2. Test system 9 

6.3. Breeding 9 

6.4. Preparation of test solutions 10 

6.5. Preliminary test .10 

6.6. Reproduction test .........11 

6.6.1. Test concentrations.... ....11 

6.6.2. Test procedure and conditions 11 

6.6.3. Sampling for analysis of test concentrations 12 

6.6.4. Measurements and recordings 12 

6.7. Interpretation 12 

6.7.1. Data handling , 12 

6.7.2. Electronic data capture 13 

6.7.3. Acceptability of the test 13 

6.8. List of deviations 13 

6.8.1. List of protocol deviations 13 

6.8.2. List of standard operating procedures deviations 14 

7. RESULTS 14 

7.1. Preliminary test 14 

7.2, Reproduction test.. 14 

7.2.1. Measured concentrations 14 

7.2.2. Condition of parental daphnids 15 

7.2.3. Reproduction 15 

7.2.4. Body length 16 

7.2.5. Determination of effect concentrations 17 

7.2.6. Experimental conditions 17 

8. CONCLUSION 17 

- Page 2 -

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p"(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)aniline NOTOX Project 498989 

TABLES 

Table 1 Mortality and reproductive potency during the preliminary test 14 

Table 2 Cumulative mortality of parental daphnids during the 21 -day exposure period 15 

Table 3 Average body lengths (mm) of parental daphnids at the end of the test 16 

Table 4 Effect parameters 17 

Table 5 Cumulative scores of living newborn per parent in the control 18 

Table 6 Cumulative scores of living newborn per parent at 0.040 mg/l , 18 

Table 7 Cumulative scores of living newborn per parent at 0.096 mg/l 19 

Table 8 Cumulative scores of living newborn per parent at 0.42 mg/l 19 

Table 9 Cumulative scores of living newborn per parent at 1.7 mg/l 20 

Table 10 Cumulative scores of living newborn per parent at 4.8 mg/l 20 

Table 11 Individual values for body length of the surviving parental daphnids ,.21 

Table 12 pH values in freshly prepared media (F) and old media (O) 26 

Table 13 Dissolved oxygen concentrations (mg/l) in freshly prepared media (F) and old 

media (O) , 26 

Table 14 Temperature (nC) in freshly prepared media (F) and old media (O) 27 

Table 15 Hardness (mg/l CaC03 ) in freshly prepared media (F) and old media (O) 27 

FIGURE 

Figure 1 Cumulative mean number of living young per parent at the various average 

concentrations of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)aniline during 

the 21-day test period 16 

APPENDICES 

APPENDIX 1 CUMULATIVE SCORES OF LIVING NEWBORN DAPHNIDS PER PARENT 18 

APPENDIX 2 PARENTAL BODY LENGTH 21 

APPENDIX 3 STATISTICS; PARENTAL MORTALITY 22 

APPENDIX 4 STATISTICS; REPRODUCTION 23 

APPENDIX 5 EC-VALUES 24 

APPENDIX 6 EXPERIMENTAL CONDITIONS 26 

APPENDIX 7 ANALYTICAL REPORT 28 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

2. STATEMENT OF GLP COMPLIANCE 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

The study described in this report has been correctly reported and was conducted in compliance with: 

The Organization for Economic Co-operation and Development (OECD) Principles of Good Laboratory 

Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17. 

The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test substance 

information unless determined by NOTOX. 

NOTOX B.V. 

Final Report - Page 4 -
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3. QUALITY ASSURANCE STATEMENT 

NOTOX B.V., 's-Hertogenbosch, The Netherlands 

This report was inspected by the NOTOX Quality Assurance Unit to confirm that the methods and 

results accurately and completely reflect the raw data. 

During the'on-site process inspections, procedures applicable to this type of study were inspected. 

The dates of Quality Assurance inspections are given below. 

p-{2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

Type of 

Inspections Phase/Process 

Start End 

Inspection Inspection Reporting 

date date date 

Study Protocol 

Protocol Amendment 01 

Protocol Amendment 02 

Report 

02-Aug-11 02-Aug-11 

09-Aug-11 09-Aug~11 

23-Dec-11 23~Dec-11 

28-Feb-12 29-Feb-12 

02-Aug-11 

09-Aug-11 

23-Dec~11 

29-Feb-12 

Process Environmental Toxicology 17-OcM1 21-Oct~11 

Test Substance Handling 

Exposure 

O bservation s/ Measu re me nts 

21-Oct-11 

Analytical and physical 

chemistry 

Test Substance Handling 

Observations/Measurements 

31-oct-n IO-Nov-11 10-Nov-11 

NOTOX B.V. 

C.J. Mitchell B.Sc. 

Head of Quality Assurance 

Date:J3.Ti^rrr:&>J.Z 

Final Report - Page 5 -
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p. 132 

p-(213-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)aniline NOTOX Project 496989 

4. SUMMARY 

Daphnia magna, 21-day reproduction study with 

p~(2,3-epoxypropoxy)-N,N-bis{2,3~epQxypropyl)aniline. 

The study procedures described in this report were based on the OECD guidelines for Testing of 

Chemicals: Guideline No, 211, 2008. In addition, the procedures were designed to meet the test 

methods and validity criteria of the ISO International Standard 10706, 2000, the Commission 

Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000. 

The batch of test substance tested was a yellow liquid with a purity of 94.33%. 

A reproduction test was performed based on the result of a preceding 7-day preliminary test. 

Preparation of test solutions started with a loading rate of 10 mg/l applying two days of magnetic 

stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous 

mixture was left to stabilize for 1-1.5 hours where after the Water Accommodated Fraction (WAF) was 

siphoned off and used as the highest test concentration, Ail lower test concentrations were prepared 

by subsequent dilutions of the WAF in test medium. The final test concentrations contained 1.0, 3.2, 

10, 32 and 100% of the WAF prepared at a loading rate of 10 mg/l. 

The reproduction test was performed in a semi-static system, included 10 vessels per test 

concentration and 20 vessels for an untreated control group. Each of the vessels contained one 

neonate (<24h old) Daphnia magna in 50 ml test medium. The study duration was 21 days and the 

test solutions were renewed every 48 hours. The daphnids were fed on a daily basis with a Chlorelta 

pyrenoidosa suspension. During the test samples for analytical confirmation of actual exposure 

concentrations were taken at the start and the end of four intervals of 48 hours. 

Analysis of the sample taken from the WAF prepared at 10 mg/l at the start of four renewal periods 

showed that the actual test concentration ranged between 4.2 and 6.6 mg/l. Test concentrations 

slightly decreased during the 48 hour renewal periods (to 70-82% of initial). Test concentrations 

containing 10 and 32% of the WAF proved to have been prepared properly and also slightly decreased 

during the 48 hour renewal periods (to 63-79% of initial). The test concentration containing 1.0% of the 

WAF was below the lowest calibration standard at all times, while the test concentration containing 

3.2% of the WAF varied between being below or just above the lowest calibration standard. Given 

these results effect parameters were based on the average exposure concentrations: 0.040, 0.096, 

0.42, 1.7 and 4.8 mg/l. 

Three out of the twenty parental daphnids in the control group died during the test period. Mortality in 

the test concentrations was not statistically significant when compared to the control. 

The average cumulative number of young per female in the control group after 21 days was 136 ± 16. 

The reproduction curves recorded at 0.040, 0.096 and 0.42 mg/l closely followed the curve of the 

control. The curve recorded at 1.7 mg/l stayed behind from the start and dapnids exposed to 4.8 mg/l 

did not reproduce at all. There were some recordings of immobile young and/or aborted eggs in the 

control and the three lowest test concentrations, while significant amounts of aborted eggs were 

recorded at the two highest test concentrations throughout the 21 -day test period. Statistical analysis 

showed that the reproduction of the daphnids was significantly reduced at 1.7 mg/| and higher. 

Mean parental body length was not significantly reduced at any of the test concentrations. 

The study met the acceptability criteria prescribed by the protocol and was considered valid. 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not affect reproduction of Daphnia magna at 

an average concentration of 0.42 mg/l after 21 days of exposure (NOEC). 

Exposure to average concentrations of 1.7 mg/l and higher induced significant inhibition of the 

reproductive capacity of the parental daphnids (LOEC). The 21d-EC5 0 for reproduction was calculated 

to be 1.4 mg/l with a 95% confidence interval ranging from 0.91 to 2.2 mg/l. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)aniline NOTOX Project 496989 

5. INTRODUCTION 

5.1, Preface 

Sponsor Huntsman Advanced Materials GmbH 

Klybeckstrasse 200 

4057 BASEL 

Switzerland 

Study Monitor 

Test Facility 

Dr. P. Dollenmeier 

NOTOX B.V. 

Hambakenwetering 7 

5231 DD 's-Hertogenbosch 

The Netherlands 

Study Director 

Technical Coordinator 

Principal Scientist 

Ir. L.M. Bouwman 

ing. V.E. Carolus 

Dr. Ir. E. Baltussen 

Study Plan Start : 20 September 2011 

Completion : 29 December 2011 

5.2. Aim of the study 

The purpose of the test was to evaluate the effects of the test substance on the mobility, the growth 

and the reproductive capacity of Daphnia magna. For this purpose, test organisms were exposed to 

aqueous solutions containing the test substance at various concentrations. The time of the first 

production of young, the number of young born, immobility and other signs of intoxication observed 

were compared with corresponding parameters in the controls. 

5.3. Guidelines 

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 211: 

"Daphnia magna, Reproduction Test", Adopted: October 2008. 

In addition, the procedures were designed to meet the test methods prescribed by the following 

guidelines and guidance document: 

• Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination 

of ecotoxicity, Publication No. L142, C.20. "Daphnia magna Reproduction Test". 

« ISO International Standard 10706: "Determination of long term toxicity of substances to Daphnia 

magna Straus (Cladocera, Crustacea)", 2000-03-30. 

• Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series 

on testing and assessment number 23, December 14, 2000. 

5.4. Storage and retention of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the NOTOX archives 

for a period of at least 10 years after finalization of the report. After this period, the sponsor will be 

contacted to determine how the records and materials should be handled. NOTOX will retain 

information concerning decisions made. 

Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as the 

quality of the specimens permits evaluation but no longer than three months after finalization of the 

report. 

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NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Parent animals are those female daphnids present at the start of the test and of which the 

reproductive output is the object of study. 

Offspring are the young daphnids produced by the parent animals in the course of the test. 

The Lowest Observed Effect Concentration (LOEC) is the lowest tested concentration at which the 

substance is observed to have a statistically significant effect on reproduction and parent mortality (at 

p < 0.05) when compared with the control, within a stated exposure period. However, all test 

concentrations above the LOEC must have a harmful effect equal to or greater than those observed at 

the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the 

LOEC (and hence the NOEC) has been selected. 

The No Observed Effect Concentration (NOEC) is the test concentration immediately below the 

LOEC, which when compared with the control, has no statistically significant effect (p < 0.05), within a 

stated exposure period, 

ECX is the concentration of the test substance dissolved in water that results in a x per cent reduction 

in reproduction of Daphnia magna within a stated exposure period. 

Mortality. An animal is recorded as dead when it is immobile, i.e. when it is not able to swim, or if 

there is no observed movement of appendages or postabdomen, within 15 seconds after gentle 

agitation of the test vessel. 

6. MATERIALS AND METHODS 

6.1. Test Substance 

6.1.1. Test substance information 

Identification p-(2,3~epoxypropoxy)-N,N-bis(2,3~epoxypropyl)aniline 

Structure O. 

p~(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

O 

Molecular formula 

Molecular weight 

CAS Number 

EC Number 

Description 

Batch 

Purity 

Test substance storage 

Stability under storage conditions 

Expiry date 

C1 6H,gN04 

277.3 

5026-74-4 

225-716-2 

Yellow liquid 

AA00373400 

94.33% 

In refrigerator (2-8°C) in the dark 

Stable 

28 February 2013 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

6.1.2. Study specific test substance information 

NOTOX Project 496989 

Volatile 

Stability in water 

Solubility in water 

6.2. Test system 

Species 

Source 

Reason for selection 

Validity of batch 

Characteristics 

6.3. Breeding 

Start of each batch 

Maximum age of the cultures 

Monitoring of the individual cultures 

Temperature of medium 

Feeding 

Validity of the cultures 

Medium 

No 

No 

No 

Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at 

least third generation, obtained by acyclical parthenogenesis 

under specified breeding conditions. 

In-house laboratory culture with a known history. 

This system has been selected as an internationally 

accepted invertebrate species. 

Daphnids originated from a healthy stock, 2n d to 5,M brood, 

showing no signs of stress such as mortality >20%, 

presence of males, ephippia or discoloured animals and 

there was no delay in the production of the first brood. 

To initiate the test, young daphnids < 24 hours old were 

selected, from parental daphnids greater than two weeks 

old. 

With newborn daphnids, i.e. less than 3 days old, by placing 

them individually in 50 ml M7-medium. 

4 weeks 

Three times a week the young are counted and the parental 

daphnids are transferred to new media. 

18-22X 

Daily, a suspension of fresh water algae 

Historical data on the reproductive capacity are based on the 

numbers of living young counted three times a week in the 

individual cultures and tested to meet the validity criteria for 

survival and reproduction. 

M7, as prescribed by Dr. Elendt-Schneider 

(Elendt, B,-P., 1990: Selenium deficiency in Crustacea. An 

ultrastructural approach to antennal damage in Daphnia 

magna Straus. Protoplasma 154, 25-33). 

Composition of medium M7 

Adjusted ISO medium1 the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (ROwater, GEON Waterbehandeling, Berkel-Enschot, The Netherlands) 

Macro salts. CaCI;,2H;0 2115 mg/l 

MgSOiJHiO 88.8 mg/l 

NaHCO; 46.7 mg/l 

KC1 4.2 mg/l 

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p. 136 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following 

concentrations. 

Trace elements. B 0.125 rng/l 

Fe 0 05 mg/l 

Mn 0.025 mg/l 

U, Rb and Sr 0.0125 mg/l 

Mo 0.0063 mg/l 

Br 0.0025 mg/l 

Cu 0.0016 mg/l 

Zn 0.0063 mg/l 

Co and I 0.0025 mg/l 

Se 0,0010 mg/! 

V 0.0003 mg/l 

Na.;EDTA,2H:0 2.5 mg/l 

Macro nutrients Na;,Si0.j. 9H;.0 10.0 mg/l 

NaN03 0.27 mg/l 

KH^PO-i 0,14 mg/l 

K 2 HPO J 0.13 mg/l 

Vitamins" Thiamine 75.0 pg/l 

Bt i 1.0 (jg/l 

Biotin 0.75 ug/l 

The hardness' 180 mg/l expressed as CaCO^ and the pH: 7,7 t 0.3. 

6.4. Preparation of test solutions 

The standard test procedures required generation of test solutions, which should contain completely 

dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The 

testing of concentrations that disturb the test system should be prevented (e.g. precipitate or a film of 

the test substance on the water surface). 

The batch of p~(2,3~epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline tested was a yellow liquid with a 

purity of 94.33% and the substance was not completely soluble in test medium at loading rates of 

1.0 mg/l and higher. 

Preliminary test 

All test solutions with loading rates of 1.0 mg/l and higher were prepared separately applying two days 

of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting 

aqueous mixtures were left to stabilize for 1 hour where after the Water Accommodated Fractions 

(WAFs) were siphoned off and used for testing. One lower test concentration was prepared by 

subsequent dilution of the 1.0 mg/l WAF in test medium. The final test solutions were all clear and 

colourless. 

Reproduction test 

Preparation of test solutions started with a loading rate'of 10 mg/l applying two days of magnetic 

stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous 

mixture was left to stabilize for 1-1.5 hours where after the Water Accommodated Fraction (WAF) was 

siphoned off and used as the highest test concentration. All lower test concentrations were prepared 

by subsequent dilutions of the WAF in test medium. 

6.5. Preliminary test 

A preliminary test was performed to define the effects of p-(2,3-epoxypropoxy)~N,N-bis(2,3-

epoxypropyl)aniiine on mobility and mortality of Daphnia magna to be tested in the reproduction test. 

Test procedure and conditions were similar to those applied in the reproduction test with the fallowing 

exceptions: 

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p-(2,3-epoxypropoxy)~N,N~bis{2,3-epoxypropyl)ani!ine NOTOX Project 496989 

• Ten daphnids per concentration (two replicates, five daphnids per replicate) were exposed to 

a control, WAFs prepared at loading rates of 1.0, 10 and 100 mg/l and to a dilution containing 

10% of the WAF prepared at a loading rate of 1.0 mg/l; 

• Test duration was 7 days; 

• Test solutions were renewed on days 1, 3 and 6; 

• No body length was recorded at the end of the test; 

• Samples for analysis were taken at days 0, 1 and 3 from the freshly prepared solutions and at 

days 1, 3 and 6 from the old solutions; 

• Samples were taken from solutions containing the daphnia and food (algae), but also from an 

extra vessel incubated under the same conditions, but without daphnia and algae. 

6.6. Reproduction test 

6.6.1. Test concentrations 

p-(2,3-epoxy pro poxy) -

N,N-bis(2,3-epoxypropyl)aniline 

Controls 

6.6.2. Test procedure and conditions 

Test duration 21 days 

Test type Semi-static 

Frequency of renewal Every 48 hours 

1,0, 3.2, 10, 32 and 100% of a WAF prepared at a loading 

rate of 10 mg/I 

Test medium without test substance or other additives 

Test vessels 

Medium 

Volume: 60 mi (6x 0 3.5 cm), all-glass covered with a 

Perspex plate. 

M71 

Experimental design 

Light 

Feeding 

At the start of the experiment (nominal day 0) 10 neonate 

daphnids, less than one day old, per group were divided 

over ten vessels each containing a minimum of 50 ml test 

medium. The control group consisted of 20 daphnids. 

16 h photoperiod daily; 

intensity at the start: 624-708 lux (8.8-10 uE.m

 2 .s' 1

) 

intensity at the end: 612-710 lux (8.8-9.9 pE.rrfV1) 

Daily, 0.5 ml of Chlorella pyrenoidosa suspension2 

containing 0.40-0.54 x103 cells/ml was added as feed for 

the daphnids. This daily ration corresponded to 0.2 mg 

C/Daphnia/day, which is the recommended value for daily 

feeding per daphnid in the reproduction test according to the 

OECD Guideline 211. 

Total (dissolved) organic carbon of the M7 medium used in the reproduction study was measured at the start of the test. 

Measurements were performed with a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V auto 

sampler (Shimadzu Kyoto, Japan). The sample volume was 40 ml and the number of repeats was at least 3. 

2 

Algae suspensions were made by inoculating growth medium with Chloreila pyrenoidosa from a pure culture. The suspensions 

were continuously aerated and exposed to light (6000-10000 lux; 60-120 uE,m .s':) in a climate room at a temperature of 23 ± 2"C 

for 2-3 weeks. After this period, the TOC concentration of the batch algal suspension was measured before use as feed tn the 

reproduction study. The exact volume to be added per test vessel was then calculated. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)aniiine 

6.6.3. Sampling for analysis of test concentrations 

NOTOX Project 496989 

Samples for analysis were taken from all test concentrations and the control. The method of analysis 

is described in the appended Analytical Report (APPENDIX 7). 

Sampling: Frequency 

Volume 

Storage 

At the beginning and at the end of four intervals of 48 hours 

(nominal days 0 and 2, 6 and 8, 12 and 14, 18 and 20) 

2 ml 

Samples were stored in a freezer until analysis. 

At the end of the refreshment period, the replicates were pooled at each concentration before 

sampling. 

Additionally, reserve samples of 2 ml were taken from all test solutions for possible analysis. If not 

already used, these samples were stored in a freezer for a maximum of three months after delivery of 

the draft report, pending on the decision of the sponsor for additional analysis. 

6.6.4. Measurements and recordings 

Parental daphnids 

Condition Every workday and upon renewal on non-workdays, the 

number of living, immobile and dead parental daphnids was 

recorded. Dead daphnids were removed when observed. 

Every workday and upon renewal on non-workdays. 

At the end of the test. 

When observed. 

Every workday and upon renewal on non-workdays, the 

number of newborn young was counted and the condition of 

the young recorded. Thereafter the young were removed. 

When observed. 

When observed. 

Presence of eggs in the brood pouch 

Body length 

Offspring 

Appearance first brood 

Newborn daphnids 

Presence of unhatched eggs 

Incidence of immobility 

Test, medium 

Temperature, oxygen and pH 

Hardness 

Light 

6.7. Interpretation 

6.7.1. Data handling 

The values for reproduction observed at various concentrations of the test substance were expressed 

as mean number of living young per parent. The mean values for reproduction at each concentration 

were compared to those recorded in the control on the various days of recording. Further, the length of 

the parental daphnids (day 21) exposed to the test substance were compared to the control. 

At the start of the test and just before and after each renewal 

in one of the vessels of each test group with surviving 

daphnids. 

Once a week in fresh and old media from the control and the 

highest test concentration 

At the start and the end of the test 

- Page 12 -
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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 

Exposure concentrations 

The exposure concentrations were calculated as 

NOTOX Project 496989 

4 

being the mathematical means of the concentrations of p-(2,3-epoxypropoxy)-N,N~bis(2,3-

epoxypropyl)aniline measured in the samples taken from the freshly prepared solutions 

(Ct=o, C,=s, and C^ s ) and from the 48-hour old solutions (Ct=2, C(=a, Ct=i4 and Ct=2n). 

In case concentrations measured were below the lowest calibration solution, the final exposure 

concentration(s) were taken as a factor of 2 below the lowest calibration solution. This procedure is 

based on the OECD "Guidance document on the use of the harmonised system for the classification of 

chemicals which are hazardous for the aquatic environment". 

Statistical analysis 

Parental survival on day 21 was statistically tested using Fisher's exact test (TOXSTAT Release 3.5, 

1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Total reproduction on day 21 was tested for normality 

and for homogeneity of variance and statistically tested using an ANOVA test followed by a mean 

comparison test (Bonferroni t-Test, TOXSTAT Release 3.5, 1996). The respective threshold levels of 

effect (LOEC) and the respective NOECs were determined on basis of these statistics. 

EC-values 

The 21d-EC5Q for reproduction was based on log-linear regression analysis of the percentages of 

reproductive reduction versus the logarithms of the corresponding average exposure concentrations of 

the test substance. 

6.7.2. Electronic data capture 

Observations/measurements in the study were recorded electronically using the following 

programme(s): 

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA) 

- Shimadzu TOC-Control V version 2.10 (Shimadzu, Kyoto, Japan): carbon analysis. 

6.7.3. Acceptability of the test 

1. The mortality of the parent animals (female Daphnia) did not exceed 20% at the end of the test 

(15%). 

2. The average cumulative number of young per female in the controls after 21 days was >60 

(136 ± 16). 

6.8. List of deviations 

6,8.1. List of protocol deviations 

1. The condition of parental daphnids was not recorded on the first day of exposure. 

Evaluation: No parental daphnids died during the first refreshment period. In addition no adverse 

effect on mobility of the daphnids were observed. 

2. Inadvertently, oxygen concentrations and pH were not measured in the old solutions on the 

second day of the reproduction test. 

Evaluation: No parental daphnids died during the first refreshment period. In addition no adverse 

effect on mobility of the daphnids were observed. 

The study integrity was not adversely affected by these deviations. 

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p. 140 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

6.8.2. List of standard operating procedures deviations 

Any deviations from standard operating procedures were evaluated and filed in the study file. There 

were no deviations from standard operating procedures that affected the integrity of the study. 

7. RESULTS 

7.1. Preliminary test 

Table 1 contains the daily observations on mortality and reproductive potency of surviving parental 

daphnids during the 7-day preliminary test. All daphnids exposed to the highest test concentration died 

within 24 hours of exposure. No (significant) mortality was observed at the three lowest test 

concentrations and the control. Note that a maximum of 20% mortality is acceptable for the control and 

therefore not considered significant. Almost all surviving daphnids in the control and the three lowest 

test concentrations carried eggs in their brood pouch on day 7 of exposure. Overall, no effects on 

survival or time of egg development were observed at the three lowest test concentrations when 

compared to the control. 

Analysis of the samples taken from the WAFs prepared at 1.0 and 10 mg/l at the start of the three 

renewal periods showed that the actual test concentrations ranged between 0.66 and 0.97 mg/l for the 

1.0 mg/l WAF and between 6.6 and 7.3 mg/l for the 10 mg/l WAF. These test concentrations remained 

(relatively) stable during the 24 and 48 hour renewal periods (79-90% of initial), but decreased to 61-

68% of initial during the 72 hour renewal period. See also Table 2 of the appended Analytical Report, 

Test conditions were maintained within the limits prescribed by the protocol. 

Based on the results of this preliminary test, it was decided to perform a reproduction test with a WAF 

prepared at a loading rate of 10 mg/l as the highest test concentration. 

Table 1 Mortality and reproductive potency during the preliminary test 

Loading rate1 

Test substance2 

(mg/l) 

Number 

Daphnia 

exposed 

Cumulative number of dead parental daphnids and parents 

with eggs in the brood pouch between parentheses 

Loading rate1 

Test substance2 

(mg/l) 

Number 

Daphnia 

exposed Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 

control 10 0(0) 0(0) 0(0) 0(0) 0(0) 0(1) 0(10) 

10% of 1.0 10 0(0) 0(0) 0(0) 0(0) 0{0) 0(1) 0(9) 

1.0 10 0(0) 

0(0) 

0(0) 

0 (0) 

0(0) 

o (b) 

10 

0(0) 0(0) 1 (5) 1 (9) 

10 10 0 (10) 

0(0) 

0(0) 

0(0) 

0 (0) 

0(0) 

o (b) 

10 

0(0) 0(0) 0(2) 

1 (9) 

0 (10) 

100 10 10(0) 10 

0(0) 

o (b) 

10 10 10 10 10 

I WAF prepared at the given loading rate 

" The test substance is p-(2,3-epoxypropoxy)-N,N-bis(2l3-epoxypropyl)aniline 

7.2. Reproduction test 

7.2.1. Measured concentrations 

The results of analysis of the samples taken during the reproduction test are described in Table 3 of 

the appended Analytical Report. 

Analysis of the sample taken from the WAF prepared at 10 mg/l at the start of four renewal periods 

showed that the actual test concentration ranged between 4.2 and 6,6 mg/l. Test concentrations 

slightly decreased during the 48 hour renewal periods (to 70-82% of initial). Test concentrations 

containing 10 and 32% of the WAF proved to have been prepared properly and also slightly decreased 

during the 48 hour renewal periods (to 63-79% of initial. The test concentration containing 1.0% of the 

WAF was below the lowest calibration standard at all times, while the test concentration containing 

3.2% of the WAF varied between being below or just above the lowest calibration solution. Given 

these results effect parameters were based on the average exposure concentrations: 0.040, 0,096, 

0.42, 1.7 and 4,8 mg/l. 

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p-(2,3-epoxypropoxy}~N,N-bis(2,3-epoxypropyl)8niline NOTOX Project 496989 

7.2.2. Condition of parental daphnids 

Three out of the twenty parental daphnids in the control died during the test period (see Table 2). 

Hence, parental mortality did not exceed 20% in the control. Mortality in all p~(2,3-epoxypropoxy)-N,Nbis(2,3-epoxypropyl)aniline concentrations tested was lower or slightly higher than in the control but 

not statistically significant (Fisher's exact test, a=0.05, see APPENDIX 3). 

In the control group, the presence of eggs in the brood pouch was recorded for the first time on day 4-

6 and the first brood appeared between day 7 and day 9. The first recording of the presence of eggs in 

the brood pouch was similar to the control in treated solutions up to and including 4.8 mg/l. Hence, no 

delay in appearance of the first brood was observed in the treatments when compared to the control. 

Table 2 Cumulative mortality of parental daphnids during the 21-day exposure period. 

Average cone. 

Test substance1 

(mg/l) 

Number 

exposed 

Cumulative number of dead parental daphnids on day: Mortality 

% 

Average cone. 

Test substance1 

(mg/l) 

Number 

exposed 2 4 7 10 11 12 14 16 18 19 20 21 

Mortality 

% 

control 20 0 0 0 0 0 0 1 1 2 2 3 3 15 

0,040 10 0 0 0 0 0 0 0 0 0 ' 1 1 1 10 

0.096 10 0 0 0 0 0 0 0 0 0 0 0 0 0 

0.42 10 0 0 0 0 1 1 1 1 1 1 1 1 10 

1.7 10 0 0 0 2 2 2 2 2 2 3 3 3 30 

4.8 10 0 0 0 1 1 1 1 1 1 1 1 1 10 

1

 The test substance is p-(2,3-epoxypropDxy)-N.N-bis(2,3-epoxypropyl)aniline 

7.2.3. Reproduction 

Records of cumulative mean number of living offspring produced per parent during the test period are 

summarised in Figure 1 with a data table. The data recorded per parent are presented in 

APPENDIX 1. 

The average cumulative number of young per female in the control group after 21 days was 136 with a 

coefficient of variation of 11 %. The reproduction curves recorded at 0.040, 0.096 and 0.42 mg/l closely 

followed the curve of the control. The curve recorded at 1.7 mg/l stayed behind from the start and 

daphnids exposed to 4.8 mg/l did not reproduce at all. There were some recordings of immobile young 

and/or aborted eggs in the control and the three lowest test concentrations, while significant amounts 

of aborted eggs were recorded at the two highest test concentrations throughout the 21-day test 

period. 

Statistical analysis showed that the reproduction of the daphnids was significantly reduced at 1.7 mg/l 

and higher (Bonferroni t-Test, a=Q.05, see APPENDIX 4). 

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p. 142 

p-(2,3-epoxypropoxy)-N,N~bis(2,3-epoxypropyt)anilme NOTOX Project 496989 

160 

c 

o 

£ 

33 

C 

C 

ro 

Day7 Day8,Dav1C Day 11 'Day 12 Dayl3 Day 14 Day15 Day16jDay!8 Day 13 | Day 20 Day21 

-•-Control 1.3 4.1 25.5 29.2 49,1 51 3 Sfe.3 32 5 84~2 ' 109 6 118.8 126,3 136 2 

—13—0.0-10 mg/l 3.3 12,0 2S.9 33.2 43.3 S3.8 S2.1 76.1 82.1 10Q.4 110.3 125,0 131.6 

- H - 0.036 mg/l 4,4 3.1 23.9 28.3 44.4 56.7 60.4 81 3 93.0 103.1 117.1 124.6 137,9 

-v- 0 42 mg/l 8.1 9,5 31,1 34.2 47.1 61 A 54,9 35 1 ' 109.1 124.9 123,3 135 2 

--•-1.7 mg/l 1.3 1 3 "ia,a 20.4 32.4 " 33 4 " 35.4 46, S 48.3 " 62.0 " " S2A SA6 56,6" 

-»!• -4.9 rmj/l 0,0 0.0 0,0 0,0 0.0 00 0.0 0.0 00 0.0 0.0 00 o'.o 

Days of exposure 

Figure 1 Cumulative mean number of living young per parent at the various average 

concentrations of p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline during 

the 21 -day test period 

7.2.4. Body length 

The average body lengths of the surviving daphnids per concentration measured at the end of the test 

and the relative reduction of body lengths compared to the control are summarised in Table 3. See 

APPENDIX 2 for the individual body lengths of the surviving parental daphnids. Statistical analysis of 

the data was not needed as the % reduction recorded was not biologically significant (<10%). 

Table 3 Average body lengths (mm) of parental daphnids at the end of the test 

Concentration 

4,4'-DDS 

(mg/l) 

Average 

length (mm) 

SD % 

Reduction 

control 4.32 (n=17) 0.12 

0.040 4.29 (n=9) 0,11 1 

0.096 4.40 (n-10) 0,10 -2 

0.42 4.39 (n=9) 0.10 -2 

1.7 4.31 (n=7) 0.12 0 

4.8 4.18 (n=9) 0.14 4 

The test substance is p-(2,.3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilme 

n= number of daphnids surviving the test period 

SD= standard deviation 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 498989 

7.2.5. Determination of effect concentrations 

Table 4 shows the effect parameters based on mean measured concentrations, see also 

APPENDIX 5. 

Table 4 Effect parameters 

Parameter Concentration 95%-

Test substance1 confidence 

(mg/l) interval 

21-day LCso (parental mortality) >4.8 

21-day NOEC for parental mortality3 4.3 

21-day EC50 for reproduction2 1.4 0.91 -2.2 

21-day LOEC for reproduction4 1.7 

21-day NOEC for reproduction4 0.42 

21-day LOEC for parental body length >4.8 

21-day NOEC for parental body length 4.8 

the test substance is p-(2,3~epoxypropaxy]-N.N-bi5(2,^ 

2

 Log-linear regression analysis 

3

 Fisher's exact test (Toxstat) 

4

 Bonferroni t -Test (Toxstat) 

7.2.6. Experimental conditions 

The pH values recorded during the test are presented in APPENDIX 6, Table 12. The pH remained 

within the range of 7.5 to 8.4 throughout the test and thus was maintained within the limits prescribed 

by the protocol (6.0-9.0, constant within 1.5 units). 

The dissolved oxygen concentrations measured during the study are presented in APPENDIX 6, 

Table 13. The oxygen concentration in all test solutions remained within the range of 8.2 to 9.7 mg/l 

during the exposure period and thus complied to the requirements as laid down in the protocol 

(>3 mg/l). 

Temperatures recorded in the test media are shown in APPENDIX 6t Table 14. The temperatures in 

the test media varied between 19.4 and 20.5°C. The temperature continuously measured in a 

temperature control vessel varied between 19.3 and 20.3°C during the test, and complied with the 

requirements as laid down in the protocol (18-22°C, constant within 2°C), 

The results of the measurements of total hardness are presented in APPENDIX 6, Table 15. Total 

hardness varied between 179 and 214 mg calcium carbonate per litre, and thus complied to the 

requirements as laid down in the protocol (>140 mg CaG03 per liter). 

The total dissolved organic carbon content of the M7 medium was 0.42 mg/l. This complied with the 

requirements as laid down in the guidelines (TOC <2 mg/l). 

8. CONCLUSION 

Under the conditions of the present study p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did 

not affect reproduction of Daphnia magna at an average concentration of 0.42 mg/l after 21 days of 

exposure (NOEC). 

Exposure to average concentrations of 1,7 mg/l and higher induced significant inhibition of the 

reproductive capacity of the parental daphnids (LOEC). The 21 d-ECSQ for reproduction was calculated 

to be 1,4 mg/l with a 95% confidence interval ranging from 0.91 to 2.2 mg/l. 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

APPENDIX 1 CUMULATIVE SCORES OF LIVING NEWBORN DAPHNIDS PER PARENT 

Table 5 Cumulative scores of living newborn per parent in the control 

Parent Day 7 Day 8 _Day_10_ Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1 0 2 7 31 31 34 (2i) + 

P2 0 0 31 31 56 56 56 90 90 90 126 126 126 

P3 25 29 ~JzL 63 63 87 87 87 119 119 161 161 161 

P4 0 0 27 27 48 48 48 84 84 109 122 122 122 

P5- 0 0 20 20 45 45 45 31 31 117 117 117 140 

P6 0 0 29 29 56 56 56 89 89 129 129 129 129 

P7 0 0 33 33 52 52 52 84 84 + 

P8 0 0 26 26 66 66 66 101 101 141 141 141 168 

P9 0 13 13 39 39 39 72 72 72 100 100 135 135 

P10 0 0 19 19 41 41 41 63 68 89 89 89 105 

P11 0 18 18 41 41 41 70 70 70 99 99 137 137 

P12 0 0 22 22 48 48 48 82 82 82 117 117 117 

P13 0 0 27 27 52 52 52 88 88 126 126 126 152 

P14 0 0 25 25 55 55 55 91 (1i) 91 129 129 129 129 

P15 0 0 29 29 62 62 62 93 93 93 133 133 133 

P16 0 0 20 20 50 50 __

 5? 85J1'L 85 121 121 121 147 (1i) 

P17 0 0 24 24 53 53 53 35 (1i) 35 125 125 125 144 

P18 0 19 30 30 30 58 63 63 63 92 92 136 136 

P19 0 0 22 22 45 45 45 77 (1i) 77 109 109 + 

P20 0 0 25 25 48 48 48 77 77 103 103 103 134 

Mean 1.3 4.1 25.5 29.2 49,1 51.8 56.3 82.5 84,2 109.6 118.8 126,3 136 2 

SD 5.6 8.5 10,3 9.9 9.7 11,4 11,4 9.5 12.6 17.0 18.1 15,7 15,5 

n 20 20 20 20 20 20 19 19 19 18 18 17 17 

+ Parental daphnid was found dead 

() Number of immobilised young (i) on the respective day (not cumulative) 

Table 6 Cumulative scores of living newborn per parent at 0.040 mg/l 

Parent Day 7 Day8 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1_ 18 18 47 47 47 78 73 78 106 106 106 138 138 

P2 0 0 12 12 39 39 39 78 78 106 106 107 107 

P3 0 23 23 50 50 50 78 78 78 97 97 130 130 

P4 15 15 23 23 23 63 63 63 95 95 124 124 124 

P5 0 0 25 25 50 50 50 85 85 118 118 118 118 

P6 0 24 24 46 46 46 68 68 68 100 

P7 0 16 39 39 3S 68 72 72 72 102 102 139 139 

P8 0 0 28 28 53 53 53 85 (1il 85 124 124 124 154 

P9 0 24 24 48 48 48 77 77 77 108 108 137 137 

P10 0 0 14 14 43 43 43 77 77 103 108 108 137 

Mean 3.3 12,0 25.9 33.2 43.8 53,8 62,1 76.1 82.1 106.4 110,3 125.0 131.6 

SD 7.0 10,8 10.4 14.6 8.7 12,2 14.9 6.9 11.3 .3-0 9.5 12.2 13.7 

n 10 10 10 10 10 10 10 10 10 10 9 9 9 

+ Parental daphnid was found dead 

() Number of immobilised young (i) on the respective day (not cumulative) 

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APPENDIX 1 CUMULATIVE SCORES OF LIVING NEWBORN DAPHNIDS PER PARENT -

continued -

Table 7 Cumulative scores of living newborn per parent at 0.098 mg/l 

Parent Day 7 DayS Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1 0 0 28 28 65 65 65 103 103 137 137 137 154 

P2 0 0 25 25 49 49 50 •85 85 85 113 113 113 

P3 0 11 36 36 36 73 73 73 101 101 101 143 143 

P4 16 16 37 37 37 62 62 62 83 88 88 121 121 

P5 0 0 19 19 45 45 45 93 93 119 119 119 147 

P6 _ _?o 20 49 49 49 82 82 32 114 115 149 (1e) 149 149 

P7 8 8 • • 26 26 26 54 54 54 84 84 112 112 112 

P8 0 26 26 56 43 43 79 79 79 116 116 116 148 

P9 0 0 20 20 45 45 45 90 91 124 124 124 152 

PtO 0 0 23_ 23 49 49 49 92 92 112 112 112 140 

Mean 4.4 8.1 28.9 28.9 44.4 56.7 60.4 81.3 93.0 108.1 117.1 124 6 137 9 

SD 7.6 9.8 9.2 9.2 10.3 13.3 13.9 14.9 10.4 18,0 17.1 13.6 16,2 

n 10 10 10 10 10 10 10 10 10 10 10 10 10 

() Number of unhatched eggs (e) on the respective day (not cumulative) 

Table 8 Cumulative scores of living newborn per parent at 0.42 mg/l 

Parent Day 7 Day 8 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1 0 0 29 + 

P2 0 0 24 24 56 56 56 91 91 129 129 129 159 

P3 0 0 23 23 49 49 49 88 88 83 111 111 111 

P4 0 0 26 26 57 57 57 99 99 135 135 135 135 

P5 17 17 40 40 40 67 67 67 95 95 120 124 124 

P6 18 18 39 39 39 72(11) 72 72 100 100 132 (2e) 132 132 

PT 0 0 23 23 50 50 50 82 82 105 105 105 137 

P8 21 21 47 47 47 30 ao 80 120 120 149 149 149 

P9 0 14 14 40 40 40 71 71 71 100 100 127 127 

P10 25 25 46 46 46 82 82 (1ej 110 110 143 

Mean 8.1 9.5 31 1 34.2 47.1 61.4 64.9 81.3 95.1 109.1 124.9 128.3 135.2 

SD 10.7 10.4 11.2 10.1 6.7 14.6 12.4 10.3 14.6 15.9 17,0 14.0 14.2 

n 10 10 10 9 9 9 9 9 9 9 9 9 9 

+ Parental daphnid was found dead 

() Number of immobilised young (i) or unhatched eggs (e) on the respective day (not cumulative) 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyt)aniline NOTOX Project 496989 

APPENDIX 1 CUMULATIVE SCORE S OF LIVING NEWBORN DAPHNIDS PE R PARENT -

continued ~ 

Table 9 Cumulative scores of living newborn per parent at 1.7 mg/l 

Parent Day 7 Day 8 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1 0 0 + 

P2 0 0 

P3 0 0 0 13 13 13 29 29 29 36 36 62(1 e) 62 

P4 13 13 15 15 15 22 (20 22 22 34 (4e) 34 34 

37 

(10i, 9e) 37 

P5 0 0 26 26 48 49 49 68 68 63 68 (10e) 68 (1i) 68 

P6 0 0 27 27 43 43 48 70 70 78 (7e) 78 78 

85 

(2i, 8e) 

P7 0 0 23 23 27 27 27 49 49 51 (11e) 51 51 51 (1e) 

PS 0 0 23 28 40 40 40 48 (9i) 48 58 

58 

(7i, 3e) 53 58 

P9 0 0 14 14 31 31 31 

39 

(6i, 4e1 39 42 (13e) 42 42 

49 

(3t, 7e) 

P10 0 0 17 17 37 37 37 49 49 49 (10e) + 

Mean 1.3 1.3 186 20.4 32.4 33.4 35.4 46.8 48.3 52.0 52.4 56.6 53.6 

SD 4.1 4,1 9.4 6.3 13,5 12.6 9.8 16.9 14.3 15.4 16.6 14.4 15.3 

n 10 10 8 a 8 8 8 8 8 7 7 7 

+ Parental daphnid was found dead 

() Number of immobilised young (i) or unhatched eggs (e) on the respective day (not cumulative) 

Table 10 Cumulative scores of living newborn per parent at 4.8 mg/l 

Parent Day 7 Day 3 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 18 Day 19 Day 20 Day 21 

P1 0 0 0 0(1 Se) 0(3e) 0(3e) 

0 

(11, 21 e) 0 0 0 (23e) 0 

0 

(11, 27*) 0 

P2 0 0 0 0 0 (9e) 0 (18e) 0 0 0 (34e) 0 0 

0 

(111, 14e) 0 

P3 0 0 0 0 (17e) 0 0 0 (I9e) 0 0 0 (24e) 0{2e) 

0 

(2i, 13e) 0 

P4 0 0 0 0 0(16e) 0 0 (4e) 0 (45e) 0 0 (31e) 0(7e) 0(1i) 0 

P5 0 0 0 0 0 (22e) 0 0 0 (24e) 0 0 (34e) 0 0|1e) 0 (11e) 

P6 0 (17e) 0 0 0 0(4e) 0 (23e) 0 0 0 (43e) 0 0(8e) 

0 

(5i, 16e) 0 

P7 0 0 0 0 0 

0 

(2i, 25e) 0 0 0 (23e) 0(1 Oe) 0 (32e) 0 0 

P8 0 0 0 0 (22e) 0(2e) 0(2e) 0(17e) 0 0 0 (33e) 0(16) 

0 

(2i, 23e) 0 

P9 0 0 0 0 (12e) 0 0(1e) 

0 

(1i, I6e) 0 0 0 (28e) 0 0 (28e) 0(1e) 

P10 0 (10e) 0 + 

Mean 0.0 0.0 0.0 0.0 0,0 0.0 0.0 0.0 0.0 0.0 0.0 0,0 0,0 

SD 0.0 0.0 0.0 0.0 0,0 0,0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 

n 10 10 9 9 9 9 9 9 9 9 9 9 9 

+ Parental daphnid was found dead 

() Number of imniobilised young (0 or unhatched eggs (e) on the respective day (not cumulative) 

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APPENDIX 2 PARENTAL BODY LENGTH 

Table 11 Individual values for body length of the surviving parental daphnids 

Parent 

Control 

(mm] 

0.040 mg/l 

(mm) 

0.096 mg/l 

(mm) 

0,42 mg/l 

(mm) 

1.7 mg/l 

(mm) 

+ 

4.8 mg/l 

(mm) 

P1 + 4.45 4.30 + 

1.7 mg/l 

(mm) 

+ 4.38 

92 4.45 4,30 4.22 4.38 + 4.14 

P3 4.38 4.38 4.45 4,22 4.06 4.30 

P4 4.14 4.22 4,45 4.45 4 38 4.14 

P5 4.22 4.14 4.45 4.45 4.45 3.91 

P6 4,45 + 4.53 4.45 4.38 4.22 

P7 + 4.14 4.38 4.30 4.30 4.06 

P8 4 30 4,30 4.45 4.30 4.30 4.22 

P9 4, OS 4.30 4.30 4.45 4.30 4.06 

P10 4.45 4.38 4.45 4.53 + + 

P11 4.45 

P12 4.22 

P13 4.22 

P14 4,30 

P15 4.38 

P16 4.30 

P17 4.45 

P18 4.30 

P19 + 

P20 4,38 

Mean 4.32 4.29 4,40 4.39 4.31 4.16 

SD 0.12 0.11* 0.10 0.10 0.12 0.14 

n 17 9 10 9 7 9 

+ Parental daphnid was found dead 

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p-{2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!ine NOTOX Project 496989 

APPENDIX 3 STATISTICS: PARENTAL MORTALITY 

Summary of Fisher' 3 Exact 

NUMBER NUMBER 3IG 

GROUP I DENT ] F.E CATION EXPOSED DEAD 0.05 

CONTROL 20 ;? 

1 0.040 1 0 1 

2 0.09 b 10 o 

0.42 10 1 

4 i . 7 3 0 3 

S 4.8 i ' l 1 

I'll 

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APPENDIX 4 STATISTICS: REPRODUCTION 

Chi-Square Test for Normality 

Actua l and Expected FirequencJ es 

INTERVAL < - i . 5 -1.5 t o -0.5 0.5 t o 0.5 >0.5 t o 1.5 >1.5 

EXPECTED 4.0*370 1-5.7 620 2 3. 3020 1-1.7620 4.0370 

OBSERVED 5 10 29 12 5 

Chi-Square =- 3.3542 {p-value - 0.4261) 

Critica l Chi-Square - 13.277 £alpha - 0.01 , df - 4) 

- 9.43d (alpha -- 0.05 , df - 4) 

Data PASS normality tes t (alpha - 0.01). Continue- analysis. 

Levene s Teat fa r Homogeneity of Variance 

AN'OVA Table 

SOURCE DF MS F 

954.9390 190.9978 2.0135 

W H" hi. n [Error) '35 521.7. 33 8 S 9-1.8 607 

Tota l 60 6172 . 3279 

(p-valu e - 0.0910) 

C r i r.3 ca l F --- i . 3700 (alpha 0.01, d f =-5,55) 

= 2.3&28 (alpha - 0.05, d f -5,55) 

S.i nue F <- Critica l F FAIL TO PEJECT Ho A l l equal (alpha -• 0 01.) 

ANOVA Tabl e 

SOURCE: DF S3 MS ir 

Between 

WJ t h Ln (Srsoi ) 

5 

55 

l

I:.60o5.2029 

10739.8627 

,U21 3 

195 

04 Oo 

2520 

159.8602 

Tota l SO 166804.0656 

(p-value - 0.0000) 

Critica l F ~ 3. 3 700 (alpha = 0.01 , d f - 5,55) 

- 2.3S29 (alpha = 0.05, d£ ~ 5,55) 

Since F > Critica l F REJECT Ho: Al l equal (.alpha - 0.05) 

Botr erron i L-Test TABLE i OF 2 Ho: Control - Trea inti'-int 

TRANSFORMED MEAN CALCULATED IN 

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0,05 

1 contco l .1.36.1765 13c.17 65 

2 0.040 131.iy.56 U l . 5556 0 8022 

3 0.096 1.37.T-000 137.5000 0 3095 

4 0. 42 135.2222 135.2222 0 1657 

1 .7 S&.5714 bS.b H4 12 3669 * 

S 4.8 0.0000 0.0000 23 *4Q9 * 

Boi-iCetri.on i t cri t ica l va l ue =• 2 , 6682 [2 Tailed , alpha ~ 0.05, d f * 5,55) 

Bonferroni t-Tes t TABLE 2 OF 2 Ho: Contro l -Trea tment 

NUN OF 3 DIFF ?, OF IHFFKRENCH: 

GROUP IDENT 1FICATIOH REPS [ IN o: . I'G •NITS CONTROL FROM CONTROL 

1 contro l 17 

2 0.O40 9 15 11.3 4.6209 

3 0.0 96 .10 14 8585 10. 9 1.7235 

4 0,42 9 15 3 6P5 11.3 0.954 2 

5 I . 7 7 1 " 7 4 37 12.3 7"'. SO50 

6 4.8 Q 15 .36SS 11.3 L36.1765 

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APPENDIX 5 EC-VALUES 

Concentration X Y 

(mg/l) Log cone, (mg/l) Reduction (%) 

0.040 -1.3 

0.040 21.4 

0.040 * 4.5 

0.040 8.9 

0.040 * 13.3 

0.040 - -2.1 

0.040 -13.1 

0.040 - -0.6 

D.040 * -0.6 

0.09S * -13.1 

0.096 - 17.0 

0,096 

• 

-5.0 

0.096 

• 

11.1 

0.096 • -7.9 

0.096 * -9.4 

0.096 * 17.8 

0.096 • -8.7 

0.096 -11.6 

0.096 • -2.8 

0.42 -0.377 -16.8 

0.42 -0.377 18.5 

0.42 •0.377 0.9 

0.42 -0 377 8.9 

0.42 -0.377 3.1 

0.42 -0.377 -0.6 

0.42 -0.377 -9,4 

0.42 -0.377 6.7 

0 42 -0.377 -5.0 

1.7 0.230 54.5 

1.7 0,230 72.8 

1.7 0.230 50.1 

1.7 0.230 37.6 

1,7 0.230 62.5 

1.7 0.230 57.4 

1.7 0.230 64.0 

4.8 0.681 100.0 

4.8 0.631 100.0 

4.8 0.6S1 100.0 

4.8 0.681 100,0 

4.8 0,681 100,0 

4.8 0.681 100,0 

4.8 0.631 100.0 

4.8 0.631 100.0 

4.8 0.681 100.0 

Slope- 93.8018 

Intercept: 35.3742 

Multiple R: 0.9822 

n = number of observations 25 

Regression line. 

93.80 X 35.8? 

Prediction of X values based on known Y values 

Known Y 

Reduction (%} 

1 0 X r O g 

(mg/l) 

1 0 X3S% -

(mg/l) (mg/l) 

10 0.53 0.34 0.83 

20 0.68 0.44 1.05 

50 1.41 0.91 2.19 

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APPENDIX 5 EC-VALUES - continued -

Concentration (mg/l) 

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APPENDIX 6 EXPERIMENTAL CONDITIONS 

Table 12 pH values in freshly prepared media (F) and old media (O) 

Nominal day 0 2 2 4 4 6 6 8 a 10 10 12 12 

Group (mg/l) F O F O F O F O F 0 F O F 

control 8.0 7.9 3.1 7.9 8,0 7,9 7.9 3,0 7.8 8.0 7.9 7.9 

0.040 8,0 7.9 8.1 7.9 8.1 7.9 7.9 8.0 7.9 8.0 7.9 7.9 

0.096 8.0 7.9 8.2 7.9 8.1 7.9 7.9 a.o 7.8 8.0 7.9 7.9 

0,42 8.1 7.9 8.1 7.9 8.0 7.9 7,9 8.0 7.8 7.9 7.9 7.9 

1.7 _Jil_ 

7.9 JA 8,0 7.9 7.9 8.0 7.8 7.9 7,9 7,9 _ 

4.8 8.1 7.9 8.1 7.9 8.1 7.9 7.9 3.0 8.0 7.9 8.0 8.0 

Nomina! day 14 14 16 16 18 18 20 20 

Group (mq/l) O F O F O F 0 . p 

control 8.0 7.9 7.9 8,0 7.9 8.0 7.5 8.4 

0.040 8.0 8.0 7.8 7.9 7.8 8.0 7.6 8.4 

0.096 8.0 8,0 7.8 7,9 7.8 8.0 7.6 8.3 

0.42 8.0 3.0 7.7 8.0 7.8 8.0 7.6 8.3 

1.7 8.0 8.0 7.3 3.0 7.8 8.0 7.7 8.2 

4 8 8.1 8.0 7.8 8,0 7.8 80 7 8 8.2 

Table 13 Dissolved oxygen concentrations (mg/l) in freshly prepared media (F) and old 

media (0} 

Nominal day 0 2 2 4 4 6 6 8 8 10 10 12 12 

Group (mg/l) F 

__° . JL_ 

O F O F O F 0 F O F 

control 9.1 3.3 9.2 9.0 9.2 9.4 8.7 9.0 8.4 9.1 8.6 9.2 

0.040 9.0 8.9 9.2 9.0 9.4 9 3 8.6 9.0 8.6 9.1 8.8 9.1 

0.096 9.0 8.8 9.4 9,0 9.5 9.3 8.6 9.0 8.7 9.1 8.8 9,1 

0.42 8 9 8,9 9.1 8.9 9,4 9.3 8.6 9 0 8.5 9.1 8.8 9.0 

1.7 8.8 8.9 9.0 9.0 

. 9-1 9,3_ 8,7 9.0 S.5 9.0 8.9 9.1 

4,8 8.7 9.0 9.0 9.0 9.7 9.3 8.7 9.0 9.0 9.0 9.1 9.0 

Nominal day 14 14 16 16 18 18 20 20 

Group (mg/l) O F O F 0 F O F 

control 9.3 9.2 8.9 9,4 8.4 9,2 8,7 9.3 

0,040 

I H .9,2 _ _a3_ ... *L4 _

 9 :1 8 3 9.2 8.5 9.3 

0.096 9.3 9.3 8.3 9.4 8.5 9.2 3.6 9.3 

0.42 9.3 9.2 8.5 9.4 3.3 9.2 8.7 9,2 

1,7 9.6 9 2 3.2 9.3 8.7 9.2 8.9 9.2 

4.8 9.7 9.1 8.5 9.2 8.6 9,1 9,0 9,1 

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APPENDIX 6 EXPERIMENTAL CONDITIONS - continued -

Table 14 Temperature (DC) in freshly prepared media (F) and old media (O) 

Nominal day 0 2 2 4 4 6 6 8 8 10 10 12 12 

Group (mg/l) F O F O F O F O F O F O F 

control 19,8 19.6 19.7 19.8 19.6 20.0 19.9 19.9 19.9 19.9 19.7 19.5 19,8 

0,040 19.9 19.8 19.8 19.6 19,5 20.0 19.9 19.9 19,9 19,9 19.8 19.8 19.9 

0.096 19.9 19.8 19.8 19.5 19.4 20.0 19.9 19.9 19.9 19.9 19.8 19.8 19.9 

0.42 20.0 19.8 19.8 19.5 19.4 20.0 19.9 20.0 19.9 19.9 19.9 19.9 19.3 

1.7 20.0 19.9 20 0 19.5 19,5 20.0 20.0 

20.1 

20.0 19.9 19.9 20.0 19.8 20.0 

4.8 20.2 20.0 20 5 19.5 19.6 20.0 

20.0 

20.1 20.0 20.0 19,9 20.1 19,9 20.2 

Nominal day 14 14 16 16 18 18 20 20 

Group (mg/t) O F 0 F 0 F O F 

control 20.0 19.9 20.2 20.2 20.0 19.8 19.5 19.4 

0,040 20.2 19.9 20 2 20.3 20,1 19.7 19.7 19,4 

,0,096 _ 

0,42 

20.2 

20.2 

20.0 

20.1 

,0,096 _ 20.2 20.2 20.1 19.8 19,8 19.5 

0,42 

20.2 

20.2 

20.0 

20.1 20.3 20.2 20,0 19.8 19.8 19.6 

1.7 20.2 20.3 20.3 20.2 20.0 19.9 19.3 19.7 

4.8 20 1 20 5 20.3 20.1 20.1 20.1 19.7 198 

Table 15 Hardness (mg/l CaC03 ) in freshly prepared media (F) and old media (O) 

Nominal day 4 4 12 12 20 20 

Group (mg/l) O F O F 0 F 

control 179 179 196 196 214 214 

4.8 179 179 196 196 214 214 

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APPENDIX 7 ANALYTICAL REPORT 

DETERMINATION OF THE 

CONCENTRATIONS 

Author 

Dr. ir. E. Baltussen 

IV 

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1. CONTENTS 

1. CONTENTS 29 

2. REPORT APPROVAL 30 

3. INTRODUCTION , 31 

3.1. Preface 31 

3.2. Aim of the study 31 

4. MATERIALS AND METHODS 31 

4.1. Reagents 31 

4.2. Samples 31 

4.3. Analytical method 31 

4.3.1. Analytical conditions 31 

4.3.2. Preparation of the calibration solutions 31 

4.3.3. Sample injections 32 

4.4. Electronic data capture 32 

4.5. Formulas 32 

5. RESULTS 33 

5.1. Calibration curves 33 

5.2. Samples 33 

5.2.1. Procedural recovery samples 33 

5.2.2. Test samples , 33 

6. TABLES....... 34 

TABLES 

Table 1 Procedural recovery samples 34 

Table 2 Concentrations of the test substance in test medium - preliminary test 35 

Table 3 Concentrations of the test substance in test medium - reproduction test 36 

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2. REPORT APPROVAL 

NOTOX B.V, 

Principal Scientist E. Baltussen, PhD 

Analytical Chemistry 

Date: .,}2~....th*rcK .%£>j2r. 

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3. INTRODUCTION 

3.1. Preface 

Study plan analytical phase Start : 22 August 2011 

Completion : 07 January 2012 

3.2. Aim of the study 

The purpose of the analytical phase was to determine the actual concentrations in samples taken from 

the test solutions used during the ecotoxicity test. 

4. MATERIALS AND METHODS 

4.1. Reagents 

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA) 

Acetonitrile Biosolve, Valkenswaard, The Netherlands 

M7-medium see main report 

All reagents were of analytical grade, unless specified otherwise. 

4.2. Samples 

The samples were stored in the freezer (< -15°C). Storage stability of samples under these conditions 

was demonstrated in NOTOX project 496981. 

On the day of analysis, the samples were defrosted at room temperature. The test samples were 

diluted in a 1:1 (v:v) ratio with 60/40 (v/v) acetonitrile/M7-medium and analysed. If necessary, the 

samples were further diluted with 30/70 (v/v) acetonitrile/M7-medium to obtain concentrations within 

the calibration range. 

4.3. Analytical method 

4.3.1. Analytical conditions 

Quantitative analysis was based on the analytical method validated for the test substance in NOTOX 

project 496981. 

Instrument Alliance Separation Module 2695 

(Waters, Milford, MA, USA) 

Detector Dual A Absorbance Detector 2487 (Waters) 

Column Symmetry Shield RP-18, 100 mm x 4.6 mm i.d., dp = 3.5 pm 

(Waters) 

Column temperature 40°C ± 1 °C 

Injection volume 100 pi 

Mobile phase 30/70 (v/v) acetonitrile/water 

Flow 1.0ml/min 

UV detection 253 nm 

4,3.2. Preparation of the calibration solutions 

Stock and spiking solutions 

Stock solutions of the test substance were prepared in acetonitrile at concentrations of 2064 -

2572 mg/l. 

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p. 158 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)aniline NOTOX Project 496989 

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of 

the spiking solutions was acetonitrile. 

Calibration solutions 

Calibration solutions in the concentration range of 0.0400 - 2.00 mg/l were prepared from two stock 

solutions. The end solution of the calibration solutions was 30/70 (v/v) acetonitnle/M7-medium. 

Proceduraj.recovery samples 

2 ml blank medium was spiked with the test substance at a target concentration of 0,1 or 10 mg/l. The 

accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples'). 

4.3,3. Sample injections 

Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were 

analysed by single injection. 

4.4. Electronic data capture 

System control, data acquisition and data processing were performed using the following programme: 

- Empower version 7.00 (Waters, Milford, MA, USA). 

Temperature and/or relative humidity during sample storage and/or performance of the studies were 

monitored continuously using the following programme: 

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, 

USA). 

4.5. Formulas 

Response (R) 

Calibration curve 

Peak area test substance [units] 

R = aC N + b 

where: 

C N = nominal concentration [mg/l] 

a = slope [units x l/mg] 

b = intercept [units] 

Analysed concentration (CA) C A _ -

(

- _

b

> x d [mg/l] 

a 

where: 

d ~ dilution factor 

Recovery ^x10 0 [%] 

Relative to nominal concentration • x 1 oo [%] 

Relative to initial concentration Cill^lx^ O [%] 

CA (t--=0days) 

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p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

5. RESULTS 

5.1. Calibration curves 

Calibration curves were constructed using five concentrations. For each concentration, two responses 

were used. Linear regression analysis was performed using the least squares method with a 

1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve. 

5.2. Samples 

5.2.1. Procedural recovery samples 

The results for the procedural recovery samples are given in Table 1. 

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110% except for 

the high concentrations level on 29-09-11 where one of the duplicate results deviated above 110%. It 

was assumed that this was probably an outlier and since it occurred during the range-finding test it has 

no influence on the study. The procedural recovery samples demonstrated that the analytical method 

was adequate for the determination of the test substance in the test samples. 

5.2.2. Test samples 

The results for the test samples are given in Table 2 and Table 3. 

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p. 160 

p-(2,3-epoxypropoxy)-N,N-bis(213-epoxypropyl)aniline NOTOX Project 496989 

6. TABLES 

Table 1 Procedural recovery samples 

Date of 

preparation 

[dd-mm-yyj 

Date of 

analysis 

[dd-mm-yyl 

Target 

concentration 

[rng/l] 

Nominal 

concentration 

, [mg./!l 

Analysed 

concentration 

[mg/ll 

Recovery 

[%] 

Mean 

recovery 

[%] 

29-09-11 29-09-11 0.1 0.100 0.102 102 104 

0.100 0.105 105 

29-09-11 29-09-11 10 10.0 12.1 121 115 

10.0 10.9 109 

17-12-11 17-12-11 0.1 0.100 0.0788 79 80 

0.100 0.0820 82 

17-12-11 17-12-11 10 10.0 8.44 84 82 

10.0 7.98 80 

06-01-12 06-01-12 0.1 0.101 0.0875 86 n.a. 

0.101 0.0480 47

 1 

06-01-12 06-01-12 10 10.1 8.73 86 85 

10.1 3.54 84 

This result was identified as an outlier based on a Dixon's Q-tesL 

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p-(2,3-epoxypropoxy)-N,N~bis(2,3-epoxypropy!)aniiine NOTOX Project 496989 

Table 2 Concentrations of the test substance in test medium ~ preliminary test 

Time of 

sampling 

TMJ£ayjl__ _ 

Date of 

sampling 

[dd-mm-yy] 

Date of 

analysis

 1 

[dd-mm-yy] 

Percentage of 

WAF filtrate2 

E%] 

Analysed 

concentration 

fm.g/ll 

Relative to 

initial 

0 20-09-11 29-09-11 1 0.811 

(fresh) 10 7.34 

1 21-09-11 29-09-11 1 0.732 - 90 

(old) 10 6.33 86 

1

3 0.746 92 

1 0 3 6.55 89 

1 21-09-11 29-09-11 1 0.646 

(fresh) 10 6.60 

3 23-09-11 29-09-11 1 0.509 79 

(old) 10 5.21 79 

1

a 0.513 79 

10

 3 5.28 80 

3 23-09-11 29-09-11 1 0.966 

(fresh) 10 7.16 

6 26-09-11 29-09-11 1 0.593 61 

(old) 10 4.85 68 

1

: i 0.656 68 

1Q3 5.14 72 

Samples were stored in the free2er (<. -1 S"C) until the day of analysis. 

Percentage of a water accommodated fraction (WAF) prepared at a loading rate of 10 mg/l. 

Without algae. 

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p. 162 

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniIine NOTOX Project 496989 

Table 3 Concentrations of the test substance in test medium - reproduction test 

Time of Date of Date of Percentage of Analysed Relative to 

sampling sampling analysis

 1 WAF filtrate 2 concentration 3 initial 

fdaysl [dd-mm-yy] [dd-mm-yy] [%l [mg/l] [%] 

0 08-12-11 17-12-11 0 n.d. 

(fresh) 1 < 0.0800 

3.2 0.145 

10 0.566 

32 2.42 

100 6.55 

2 10-12-11 17-12-11 0 n.d. n.a. 

(old) 1 < 0.0800 n.a. 

3.2 0.122 84 

10 0.443 78 

32 1.92 79 

100 5.35 82 

6 14-12-11 17-12-11 0 n.d. 

(fresh) 1 < 0.0800 

3.2 0.152 

10 0.553 

32 2.23 

100 6.12 

8 16-12-11 17-12-11 0 n.d. n.a. 

(old) 1 < 0.0800 n.a. 

3.2 0.110 72 

10 0.418 76 

32 1.74 78 

100 4.75 78 

12 20-12-11 06-01-12 0 n.d. 

(fresh) 1 < 0.0800 

3.2 < 0.0800 

10 0.390 

32 1.45 

100 4.16 

14 22-12-11 06-01-12 0 n.d. n.a. 

(old) 1 < 0.0800 n.a. 

3.2 < 0.0800 n.a. 

10 0.244 63 

32 1.05 72 

100 2.89 70 

18 26-12-11 . 06-01-12 0 n.d. 

(fresh) 1 < 0.0800 

3.2 0.172 

10 0.502 

32 1.91 

100 5.23 

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p-(2,3~epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTOX Project 496989 

Table 3 Concentrations of the test substance in test medium - reproduction test 

(continued) 

Time of Date of Date of Percentage of Analysed Relative to 

sampling sampling analysis

 1 WAF filtrate 2 concentration 3 initial 

|days] [dd-mm-yy] [dd-mm-yy] {%] [mg/l] [%] 

20 28-12-11 06-01-12 0 n.d. n.a. 

(old) 1 < 0.0800 n.a. 

3.2 < 0.0800 n.a. 

10 0.330 66 

32 1.27 67 

100 3.87 74 

Samples were stored in the freezer (s -15"C) until the day of analysis, 

Percentage of a water soluble fraction (WSF) prepared at a loading rate of 10 mg/l. 

In several samples test substance was detected at a concentration beiow the lowest calibration solution. These sampfes 

were reported as < 0.0800 mg/l (i.e. a concentration analysed lower than the lowest calibration standard taken the 

dilution factor of the samples (2) into account). 

n.d. Not detected, 

n.a. Not applicable. 

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REPORT 

96-HOUR ACUTE TOXICITY STUDY IN CARP 

WITH 

TK 12759, Araldite MY 0510 

NOTOX Project 169548 

CIBA test number 964623 

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TK 12759, AraTdite MY 0510 NOTOX Project 169548 • 

REPORT APPROVAL 

STUOV DIRECTOR: 

MANAGEMENT: 

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TK 12759, AraldUe MY 0510 NOTOX Project 169548 

SUMMARY 

96-Hour Acute Toxicity Study in Carp with TK 12759, AraldUe MY 0510. 

The study procedures described in this report were based on the EEC 

directive 92/69, Part C.l. "Acute toxicity for fish"; and the OCCO 

guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992. 

The definitive test was performed with carp exposed to concentrations 

ranging from 1.0 to 10 mg/l in a static system. Acetone was used as a 

solvent in the stock solutions. 

The 96h-LC50 for carp is 4.2 mg/l with 0 % mortality at 3.2 ngn and 100 % 

mortality at 5.6 mg/l. 

The BWZ of TK 12759, AraldUe MY 0510 for acute toxicity in fish is 5.4. 

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p. 167 

TK 12759, Araldite MY 0510 NOTOX Project 169548 

PREFACE 

Sponsor 

Study Monitor 

Testing Facility 

Study Director 

Study Plan 

Ciba-Geigy Ltd. 

Polymers Division 

R-1006.520 

P.O. Box 

CH-4002 BASEL 

Switzerland 

Dr. E.G. Semadeni 

Additives Division 

R-1002.2.62 

NOTOX 8.V. 

Hambakenwetering 3 

5231 DO 's-Hertogenbosch 

The Netherlands 

Ors. M. Sogers 

Start : 25 March, 1996' 

Completion ; 19 April, 1996 

TEST SUBSTANCE (NOTOX Substance Code: 59652) 

Identification 

Description 

Batch 

Purity 

Test substance storage 

Stability under storage 

conditions 

Expiry date 

Stability in vehicle 

TK 12759, Araldite MY 0510 

Clear yellow liquid (Determined at NOTOX) 

50008M 

> 95* 

In freezer in the dark 

Stable 

May 01, 1998 

Water: at least 96 h 

Acetone: at least 96 h 

PURPOSE 

The purpose of the study was. to evaluate the test substance for it s ability 

to generate acute toxic effects in Cyprinus carpio during an exposure 

period of 96 hours. 

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TK 12759, Araldite MY 0510 NOTOX Project 169548 

GUIDELINES 

The study procedures described in this report were based on the following 

guidelines: 

European'Economic Community (EEC), EEC directive 92/69, Part C: Methods for 

the determination of ecotoxicity, Publication No. L383, December 1992, C.l. 

"Acute toxicity for fish". 

The OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish 

Acute Toxicity Test", Adopted 17 July, 1992. 

ARCHIVING 

NOTOX 8.V. will archive the following data for at least 10 years: report, 

and raw data. 

DEFINITIONS 

Fish were considered to be dead when no reaction was observed after 

touching the caudal peduncle and visible breathing movements were absent. 

The LC50 is the concentration killing 5096 of the fish after a defined 

period of exposure. 

TEST SPECIES 

Species 

Source 

Mean length 

Mean weight 

Characteristics 

Reason for selection 

Total fish used 

Carp (Cyprlnus caroio. Teleostei, cyprinidae) 

(Linnaeus, 1758) 

Zodiac, proefacc, "De Haar Vissen", L.U. 

Wageningen, the Netherlands. 

Range-finding test: 1.90 + 0.27 cm 

Final test: 1.96 + 0.10 ciii 

Range-finding test: 0.30 + 0.07 g 

Final test: 0.21 ± 0.03 cm 

Fl from a single parent-pair bred in untreated water. 

This system has been selected as an 

internationally accepted species. 

79 

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p. 169 

TK 12759, Araldite MY 0510 NOTOX Project 169548 

HOLDING 

Quarantine/Acclimation 

Medium' 

Measurements 

Feeding 

Control of sensitivity 

'i 

Validity of batch 

At least 12 days after delivery. 

ISO-medium, formulated using Milli-Ro water 

(tap-water purified by reverse osmosis; 

Millipore Corp., Bedford, Mass., USA) with the 

following composition: 

Ca2+ 80 mg/l 

12 mg/l 

15 mg/l 

3 mg/l 

145 mg/l 

49 mg/l 

47 mg/l 

Mg 24 

Na+ 

K

+ 

cr 

S0„2 

HC0 Hardnes 3 s 1s 250 mg CaC03/l 

Oxygen concentration, pH, nitrate and nitrite 

concentration and ammonia concentration: once 

a week. Temperature; every day. 

Daily with Trouvit. 

A reference test with pentachlorophenol (PCP, 

SIGMA) is carried out within a period of 3 

months. The results of this test are appended 

to the report. 

In the batch of fish used for the test, 

mortality during the seven days prior to the 

start of the test was less than 5%. 

RANGE-FINDING TEST 

A range-finding tjest was performed to provide information about the range 

of concentrations! to be used Iri the final test: 

three fish per concentration were exposed to a concentration range of 0.1 

to 10 mg/l with an increasing factor of 10, while seven fish were exposed 

to 100 mg/l and the blank and solvent control. 

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TK 12759, Araldite MY 0510 NOTOX Project 169548 

FINAL STUDY: TEST PROCEOURE AND C0N0ITI0NS 

Test duration 

Test type 

Test vessels 

Test medium 

Number of fish 

Loading 

lllumination 

Aeration 

Feeding 

Introduction of fish 

Euthanasia 

96 hours 

Static 

5 litres, all-glass. 

ISO-medium, aerated until the dissolved oxygen 

concentration had reached saturation and the 

pH had stabilized.. After aeration the hardness 

was 250 mg CaCOs per litr e and the pH was 8.0 

+ 0.2. 

7 fish per concentration and controls. 

0.74 g fish/litre, i.e. 7 fish per 2 litres of 

test medium. 

16 hours photoperiod daily 

The test media were aerated continuously. 

No feeding from 24 hours prior to the test and 

during the total test period. 

Directly after preparation of the test media. 

At the end of the test the surviving fish were 

rapidly killed by exposing them to ca. 1.2% 

ethylene glycol monophenylether in water. 

PREPARATION OF TEST SOLUTIONS 

Stock solutions were prepared in acetone at 100, 56, 32, 18 and 10 mg/ml. A 

volume of 0.2 ml was taken from each stock and mixed with 2 litres of test 

medium providing the test concentrations of 10, 5.6, 3.2, 1.8 and 1.0 mg/l. 

The final test solutions were all clear without precipitation. 

MEASUREMENTS AND RECORDINGS 

Mortality and other effects At (2), 24, 48, 72 and 96 hours following the 

start of exposure. Dead fish were removed when 

observed. 

Dissolved oxygen content, 

pH and Temperature Daily, beginning at the start of the test. 

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p. 171 

TK 12759, Araldite MY 0510 NOTOX Project 169548 

RESULTS 

Range-finding test: 

Incidence and total mortality: 

Concentration 

(mg/l) 

initia l 

number 

of fis h 

Cumulative mortality Total 

Mortality 

(%) 

Concentration 

(mg/l) 

initia l 

number 

of fis h 24 h 48 h 72 h 96 h 

Total 

Mortality 

(%) 

Blank-control 7 0 0 0 0 0 

Solvent-control 7 0 0 0 0 0 

0.1 3 0 0 0 0 0 

1 3 0 0 0 0 0 

10 3 0 3 3 3 100 

100 7 7 7 7 7 100 

Definitive study: 

i 

Incidence and total mortality; 

Concentration 

(mg/l) 

Initia l 

number 

of fis h 

Cumulative mortality Total 

Mortality 

(%) 

Concentration 

(mg/l) 

Initia l 

number 

of fis h 24 h 48 h 72 h 96 h 

Total 

Mortality 

(%) 

Blank-control 7 0 0 0 0 0 

Solvent-control 7 0 0 0 0 0 

1.0 7 0 0 0 0 0 

1.8 7 0 0 0 0 0 

3.2 • 7 0 0 0 0 0 

5.6 7 0 3 * 7 7 100 

10 7 0 7 7 7 100 

* Surviving fish showed severe disturbance of swimming behaviour. 

pH, dissolved oxygen and temperature: 

Concentration Day 0 Day 1 Day 2 Day 3 Day 4 

(mg/l) pH DO pH DO pH DO pH DO pH DO 

Blank-control 8 1 9. 1 8.0 9.0 7.8 8 8 7 9 8,9 7 9 8 9 

Solvent-control 8 1 9. 0 8.0 8,8 7.8 8 7 7 9 8.8 7 8 3 8 

1.0 8 1 9. 0 8.0 8.8 7.8 8 8 7 8 8.9 7 8 8 6 

1.8 8 0 9. 2 8.0 8.8 7.8 9 0 7 8 8.9 7 7 8 8 

3.2 8 0 9 1 7.9 8.7 7.8 8 8 7 9 8.9 7 6 8 7 

5.6 , 8 0 9 1 8.0 8.9 7.8 8 9 8 0 8.9 

10 8 0 9 2 8.0 8.8 

Temperature 20 .5 20 .4 20 5 20. 5 20 4 

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TK 12759, Araldite MY 0510 NOTOX Project 169548 

CONCLUSION 

TK 12759, Araldite MY 0510: 

The 96h-LC50 for carp Is 4.2 mg/l with 0 % mortality at 3.2 mg/l and 100 % 

mortality at 5.6 mg/l. 

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p. 173 

[PS + R, PO 2.1 

(Kopie ; 

I Ablags 'j 

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REPORT 

PSEUDOMOHAS CELL MULTIPLICATION INHIBITION TEST 

WITH 

TK 12759, ARALDITE MY 0510 

NOTOX Project 169706 

Ciba Project 964624 

NOTOX Substance 59652 

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p. 175 

TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

REPORT APPROVAL 

STUDY DIRECTOR: I r , Y.H.M. van Erp 

Date: WVv^ ^ x 

MANAGEMENT: Ing, E.J. van de tfaart 

Section Head, Genetic & 

Eco-Toxicology 

Date: ^a, 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

SUMMARY 

TK 12759, ARALDITE MY 0510 was investigated for its ability to inhibit the 

cell multiplication of the bacteria species Pseudomonas putida according to 

DIN 38412 Part L 8 (March 1991) and NEN-EN-ISO 10712 (1996), 

Suspensions of Pseudomonas putida were exposed to concentrations ranging 

from 312.5 to 10,000 yg TK 12759, ARALOITE MY 0510 per litre . Acetone was 

used as vehicle. 

After incubation the extinction values of the bacterial cell suspensions 

were measured. No toxicity was observed. 

Based on the results of the test presently performed, the toxicity 

threshold value (EC10) of TK 12759, ARALDITE MY 0510 was over 10 mg/l. 

The "Bewertungszahl" (Assessment figure or value for bacterial toxicity) is 

therefore < 4.9 (Bewertung wassergefahrdender Stoffe, 1989). 

The EC50 value for the reference substance, 3,5-dichlorophenol, tested 

during the same experiment was 20 rag/1. Since this was within the accepted 

range of 10-30 mg/l for the EC50 and the multiplication factor of the 

bacteria within the test was over 60, i t was concluded that the test 

conditions were optimal and the results obtained were valid. 

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p. 177 

TK 12759, ARALOITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

PREFACE 

Sponsor Ciba-Geigy Ltd. 

Polymers Division • 

Bau R-1002.2.60 

CH-4002 BASEL 

Switzerland, 

Study Monitor Dr. E.G. Semadeni 

Testing Facility NOTOX B.V. 

Hambakenwetering 3 

5231 DD 's-Hertogenbosch 

The Netherlands 

Study Director I r . Y.H.M.-van Erp 

Technical Coordinator A.M.C.. Bertens 

Study Plan Start : March 28, 1996 

Completed : March 29, 1996 

TEST SUBSTANCE, 

Identi fication 

Description 

Batch 

Purity 

Test substance storage 

Stability under storage 

conditions 

Expiry date 

Stability in vehicle 

TK 12759, Araldite MY 0510 

Clear yellow liquid (Determined at NOTOX) 

50008M 

> 95% 

In freezer in the dark 

Stable 

May 01, 1998 

Water ; at least 96 h 

Acetone: at least 96 h 

GUIDELINES • ! 

| 

The study procedure .described in this report was based on the following 

guidelines: 

DIN 38412 Part L 8, March 1991: Determination of the Inhibition Effect of 

Water Constituents on Bacteria (Pseudomonas Cell Multiplication Inhibition 

Test) with minor; modifications. 

NEN-EN-ISO 10712, 1996: Water quality - Pseudomonas putida growth 

inhibition test (pseudomonas cell multiplication inhibition test). 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

C1ba Project 964624 

OBJECTIVE 

Purpose of the study 

The objective of this study is to evaluate the" test substance for its 

ability to inhibit the cell multiplication of the bacterial species 

Pseudomonas putida. Furthermore, the toxicity threshold value (EC10) and 

EC50 value (i f possible) of the test substance for Pseudomonas putida is 

determined. 

Pseudomonas putida. an aerobic gram-negative bacterial species from the 

genus Pseudomonas, is a natural inhabitant of surface waters. This 

bacterium is a model water organism involved in biological selfpurification. The inhibition of multiplication of bacterial cells of 

Pseudomonas putida 1s the selected parameter for dissolved toxic water 

ingredients. 

DEFINITIONS 

Effective concentration fEClQ. EC501: the concentration of test substance 

determined after a test period of 16 + 1 hour from the concentration-effect 

relationship at which cell multiplication is inhibited by 10 % or 50 % 

compared to that of the control. EC is expressed in mg test substance per 

litre . 

Formazine nephelometric unit (FNU1: Formazine turbidity units. The optical 

density of a bacterial suspension at X=436 nm, measured as FNU. 

Biomass: culture of bacterial cells after a certain time, expressed as FNU. 

"Bewertunqszahl": Assessment figure or value for bacterial toxicity 

expressed as the negative logarithms of the EC10. 

Example: EC10: 150 mg/l « 150 ppm » 1.5 . 10"4 = Bewertungszahl 3.8. 

TEST SYSTEM 

Test system Pseudomonas putida. 

Strain MIGULA, Berlin 33/2 (DSM 50026), 

Source BGA, Berlin, Germany. 

Rationale Recognized by international guidelines. 

Stock cultures of the bacteria are stored in liquid nitrogen (~I96°C). 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

TEST SUBSTANCE PREPARATION 

Since the test substance was poorly soluble in water and no homogeneous 

suspension 1n water could be obtained, a stock solution (A) of 100 g per 

litr e in acetonewas prepared. Thorough mixing was performed to enhance the 

dissolving of the test substance. Solution A was diluted 1:1 with acetone 

(stock B, 50 g/l). In the same way stock C (25 g/l) to stock F (3.1 mg/l) 

were prepared. 

REFERENCE SUBSTANCE 

3,5 ~ Dichlorophenol (97 %, Aldrich). 

A stock solutionlof 100 mg 3,5-dichlorophenol per litr e in Milli-Q water 

was prepared. 

EXPERIMENTAL CONDITIONS AND SOLUTIONS 

Vessels i 

Incubation temperature 

Incubation time

 ; 

- preliminary suspension 

cultures i 

- exposure i 

Stock solution I 

Stock solution I I 

Stock solution II I 

Stock solution IV 

Culture medium (stock) 

100 ml Erlenmeyer flasks 

23 + 1°C 

6 + 1 hours 

16+2 hours 

10.0 g NaN03 

2.4 g K2HPO4 

1.2 g KH2P04 

1.0 g yeast extract 

Dissolved in 500 ml Milli-Q water. 

10.0 g NaN03 

2.4 g K2HP04 -

1.2 g KH2P04 

Dissolved in 500 ml Milli-Q water. 

40,0 g D(+")- glucose 

Dissolved in 500 ml Milli-Q water. 

0.01 g Fe (III ) citrate 

4.0 g MgS04 . 7H20 

Oissolved in 1 1 M1111-Q water. 

Nutrient medium: 18 g of agar dissolved in 

water, plus 50 ml of solution I , 125 ml of 

solution II I and 100 ml of solution IV, made 

up to one litr e with water. 

- Page 6 -
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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

PERFORMANCE OF THE TEST 

Preliminary suspension cultures 

A small amount of bacteria from a maximally 7-day old stock culture of 

Pseudomonas putida was Inoculated in pre-culture medium (25 ml of stock 

solution I and II I plus 50 ml of solution IV, made up to one litr e with 

water) in Erlenmeyer flasks. After incubation the final turbidity value of 

the bacterial suspension was adjusted up to FNU/436 nm = 50. 

Exposure 

Three parallel dilution series in 100 ml Erlenmeyer flasks covered with 

aluminium caps were prepared from the formulated test substance stock 

solution as described below. 

Cone. Mi 111- Test Stock solution Bacterial total 

test water solution1 I I I I I IV suspension volume 

subst. (ml) (ul of stock) (ml) (m!) (ml) FN07436.50 

(pg'i) (ml) (•1) 

10,000 32 4 A 1 1 2 4 402 

5,000 32 4 B 1 1 2 4 40 

2,500 32 4 C 1 1 2 4 40 

1,250 32 4 D 1 1 2 4 40 

625 32 4 E 1 1 2 4 40 

312 32 4 F l 1 2 4 40 

0

3 32 4 acetone 1 1 2 4 40 

° — Tap water purified by reverse osmosis and subsequently passed over 

activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, 

Mass., USA). 

1 = Final volume of acetone was 0.0195. 2

 - pH of one of the test vessels was 8.1. 3

 m 10 control flasks were prepared. 

Six flasks were made up identically as above, only 4 ml of pre-culture medium 

was added instead of the bacterial suspension (turbidity control). 

The series of the reference substance were prepared by direct dilutions from 

the stock solution as follows: 

Cone. Milli-Q Reference Stock solution Bacterial total 

reference water stock sol I I I I I IV suspension volume 

subst. (ml) (ml) (ml) (ml) (ml) FNU/436=50 (ml) 

(mg/l) (ml) 

40 16.0 16.0 1 1 2 4 40 

28 20.8 11.2 1 1 2 4 40 

20 24.0 8.0 1 1 2 4 40 

14 26.4 5.6 1 1 2 4 40 

10 28.0 4.0 1 1 2 4 40 

7 29.2 2.8 1 1 2 4 40 

- Page 7 -
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Authorization and Restriction; of Chemical Substances (REACH), without the express written permission of Huntsman 

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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

After an appropriate time in an incubator shaker the turbidity values of the 

cell suspensions (biomasses) were measured. 

MEASUREMENTS 

Equipment 

Calibration solution 

Turbidity value ; 

bacterial suspension 

UV/VIS Spectrophotometer.Lambda 5 (Perkin Elmer, 

the Netherlands), X » 436 nm; 10 mm layer 

Formazin standard suspension 

(in Formazine Turbidity Units) : 

FNU/436 nm = 100 

FNU/436 nm « 75 

FNU/436 nm * 50 

FNU/436 nm » 25 

FNU/436 nm . 12 

FNU/436 nm . 50 

CALCULATIONS 

The percentage of cell multiplication Inhibition for each tested 

concentration was calculated as follows: 

I * Be - an * 100 

Be - 8o 

I Cell multiplication inhibition in % 

8n Measured biomass (extinction - extinction of turbidity control) at the 

end of the test period for the nt h concentration of the test material 

Be Measured biomass (extinction) at the end of the test period in the 

control batch. 

Bo Measured biomass (extinction) at the start of the test (t=0) in the 

control batch. 

The inhibition values calculated for each concentration were plotted 

against the corresponding concentration of the test substance. 

ACCEPTABILITY OFj THE TEST 

The EC50 value of the reference substance 3,5-dlchlorophenol should be in 

the accepted range of 10-30 mg/l. 

The inoculum used in the control batch should be multiplied by a factor of 

at least 60 within the test period. 

I 

The preceding criteria are not absolute and other modifying factors might 

enter into the final evaluation decision. 

- Page 8 -
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Authorization and Restriction of Chemical Substances (REACH), without the express written permission of Huntsman 

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TK 12759, ARALOITE MY 0510 ' NOTOX Project 169706 

Ciba Project 964624 

RESULTS 

Bacterial toxicity 

After incubation extinction values were measured and the cell 

multiplication inhibition for each test concentration was determined (see 

Table 1). 

Since no significant (significant: >109>) inhibition of cell multiplication, 

was observed the toxicity threshold value of TK 12759, ARALOITE MY 0510 for 

Pseudomonas putida was over 10 mg/l. 

Acceptability of the test 

The EC50 value of 3,5-dichlorophenol determined for Pseudomonas putida as 

calculated by linear regression (Y *• 6.8169 X -83.9824) was 20 mg/l (see 

Table 2). 

The inoculum used in the control batch was multiplied by a factor 77 within 

the test period. 

With respect to the accepted range of 10-30 mg/l for the EC50 of the 

reference substance and the multiplication factor of the bacteria within 

the test being over 60, 1t was concluded that the test .conditlonswere 

optimal and the results obtained were valid. 

CONCLUSION 

Based on the results of the .test presently performed, the toxicity 

threshold value (EC10) of TK 12759, ARALOITE MY 0510 was over 10,000 mg/l. 

The "BewertungszahlVis therefore < 4,9 (Bewertung wassergefahrdender 

Stoffe, 1989). 

- Page 9 -
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TK 12759, ARALDITE MY 0510 NOTOX Project 169706 

Ciba Project 964624 

TABLE 1 

i 

Extinction values of the inoculated dilution series I , II , 111, the noninoculated dilution series t (turbidity control) and the control flasks 8. 

CONCENTRATION 

of TK 12759, EXTINCTION IN FNU 436 nb CORRECTED INHIBITION 

ARALDITE MY 0510 series MEAN MEAN % 

(ug'l) I I I i n I,II,I I t 

312.5 a 291 1.219 1.292 1.267 0.004 1.263 2.8 

625 i 259 1.356 1.248 1.268 0.001 1.287 1.0 

1250 l .231 1.201 1.173 1.202 0.001 1.201 7.7 

2500 1.231 1.191 1.234 1.222 0.001 1.221 6.1 

5000 1.291 1.394 1.294 1.326 0.001 1.325 -2,0 

10000 1 .172 1.293 1.222 1.226 0.002 1.224 5.9 

CONTROL 1.520 

MEAN 1.299 

60 0.0168 

1,175 1.34*5 1,201 1.349 1.251 1.410 1.376 1.297 1.265 

TABLE 2 

Extinction values of the reference substance dilution series: 

3,5-dichlorophenol. 

CONCENTRATION EXTINCTION IN FNU 436 nm INHIBITION 

3,5-dichlorophenol sarles r X 

7 1.324 -1.9 

10 1.264 2.7 

Id 1.269 2.4 

20 0.424 68.2 

26 0.013 100.1 

40 0.016 100.0 

I 

- Page 10 -
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ed and strictly prohibited. 

PS + Ft, POZ1 

Eing. 3 >.^f c UJ 

. * i 

5. !