Technology & Engineering B.V Environmental Chemistry PO Box 9300, 6800 SB Arnhem, the Netherlands



## REPORT

CONFIDENTIAL

Title 

### DETERMINATION OF THE PRIMARY AND ULTIMATEBIODEGRADATION OF N,N-DI(HYDROGENATED TALLOWALKYL)-1,3-PROPANEDIAMINE IN A CONTINUOUSLY-FED ACTIVATED SLUDGE UNIT TREATING DOMESTIC WASTEWATER

Authors C.G. van Ginkel, A. L ouwerse, J. van Dam
Date 04-08-2010 Document No. 2.421.206 Study code T09066 CAS 

### N,N-DI(HYDROGENATED TALLOWALKYL)-1,3-PROPANEDIAMINE

C

. G . v a n G inkel , A

. L ou werse , J . van D am Technology & PO Box 9300, E n gi neer i n g B

. V 6800 SB

g Environmental C hemistr y Arnhem, th e N et h er l an ds

T 09 0 66 C A S 

# SANITIZEDT 09066 CAS

Page 2 of 28 

### ABSTRACT

The continuous activated sludge (CAS) test was performed according to ISO Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was spiked at a nominal influent concentration of 50 mg/L (40.5 mg/L carbon; calculated) for a period of 63 days and included a control fed with domestic wastewater only. The immediate high organic carbon removal percentages can be attributed to adsorption and probably biodegradation. The mean organic carbon removal percentage of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine calculated over 15 measurements obtained from day 46 to 63 of the test was 104 1% (95% confidence interval). This high removal percentage strongly indicates that N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine is biodegraded ultimately.Formation of water-soluble compounds during biological treatment of N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine can be excluded. An accurate assessment of the removal of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine was established with specific analyses. The method (LC-MS/MS) for the determination of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery, and specificity. The mean removal percentage of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine in the test unit asquantified with the specific analysis from day 59 to 63 was >99.999.% using Nhexadecyl-N-octadecyl-1,3-diaminopropane as representative component. These analyses demonstrate that the removal of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine from the wastewater is almost complete. N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine concentrations in the sludge of the reactor sampled on days 62 and 63 were 257 and 247 mg/L. The mean removal percentage of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine from the influent through adsorption onto sludge assessed is therefore 1.3%. The low removal percentage by adsorption demonstrates that N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine is primarily removed by biodegradation.In conclusion, the CAS test demonstrates that N,N-di(hydrogenated tallowalkyl)-1,3-propanediamine, almost completely removed from the wastewater in conventional biological wastewater treatment plants. N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine is primarily removed by biodegradation. p pp N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine
N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine N,Np g di(hydrogenated tallow-alkyl)-1,3-propanediamine N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine Nhexadecyl-N-octadecyl-1,3-diaminopropane N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine N,N-di(hydrogenated tallowalkyl)-1,3-propanediamine, N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine T 09066 C A S



# SANITIZEDT 09066 CAS

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### STUDY ORGANISATION

S p onsor AkzoNobel Surface Chemistry LLC 525 West Van Buren Chicago, Il 60607-3823 USA

Stud y monitor D Hillebold

Stud y location AkzoNobel Technolo gy& En g ineerin g Arnhem Vel p erwe g 76 6824 BM Arnhem The Netherlands

Stud ydirecto r C.G. van Ginkel

Q ualit yAssurance Uni t H. van Daalen

Mana g emen t R. Bor g mann-Strahsen

Ex p erimental startin g date 03-02-2010 Ex p erimental com p letion date 11-05-2010



### GLP COMPLIANCE STATEMENT

The project file including the final report, amendments to the final report, the study plan, amendments to the study plan, records of quality assurance inspections, all letters, memos and notes and raw data pertaining to the study will be retained in the archives of AkzoNobel Technology & Engineering Arnhem for a period of ten years. Other records including master schedule sheet, laboratory notebooks, logbooks, records of the maintenance and calibration of equipment, summary of training, curricula vitae and job descriptions of the personnel involved in the study, records related to location and storage of the test substance will also be kept in the AkzoNobel Technology & Engineering Arnhem archives for a period of ten years. Test material will be stored deep-frozen under the sample code T09066 for ten years or only as long as the quality of the test substance permits evaluation. AkzoNobel Surf ace ChemistryLLC 525 West V a n B u ren Chica g o, Il 60607-3823

D Hillebold

AkzoNobel Technology & Engineering Arnhem Vel p erwe g 7 6 6 8 24 BM Arnhe m The Netherl a n ds

C

. G . v a n G in ke l

H . v a n D aa le n

R

. B or g man n
- S tr a h se n

AkzoNobel Technology & Engineering Arnhem

AkzoNobel Technology & Engineering Arnhem T 09066 C A S



# SANITIZEDT 09066 CAS

Page 4 of 28 

### GLP COMPLIANCE STATEMENT

The study reported here was carried out according to the study plan in compliance with the OECD Principles of Good Laboratory Practice excluding test substance data submitted by the sponsor. The report contains an accurate description of the results.

Study director C.G. van Ginkel

Management R. Borgmann-Strahsen y C

. G . v a n G inkel

R . Bor g mann-Strahsen T 09066 C A S



# SANITIZEDT 09066 CAS

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### QUALITY ASSURANCE STATEMENT

This report was audited by the Quality Assurance Unit contracted by AkzoNobel Technology & Engineering Arnhem. It is considered to be an accurate presentation of the methods and procedures applied in the course of the study and an accurate reproduction of the data recorded. Listed below are the phases of the study, the dates of inspection of this study by the Quality Assurance Unit and the dates on which its findings were reported to Study Director and Management.

Phase of the stud y Dates of inspection Dates of reporting Stud y p lan 05-02-2010 05-02-2010 Test ( s p ikin g with test substance ) 11-02-2010 12-02-2010 Test ( extraction and sam p le p re p aration ) 03-05-2010 03-05-2010 Re p ort 09-07-2010 10-07-2010

Quality Assurance Unit H. van Daalen AkzoNobel Technology & Engineering Arnhem.

H . v a n D aa len T 09066 C A S


# SANITIZEDT 09066 CAS

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### CONTENTS

ABSTRACT 2 STUDY ORGANIZATION 3 ARCHIVING AND STORAGE 3 GLP COMPLIANCE STATEMENT 4 QUALITY ASSURANCE STATEMENT 5 INTRODUCTION 7 MATERIALS AND METHODS 8 RESULTS AND DISCUSSION 12 AMENDMENTS AND DEVIATIONS 14 REFERENCES 14 TABLES 15 FIGURES 18 ANNEXES 21 T 09066 C A S


# SANITIZEDT 09066 CAS

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### INTRODUCTION

For the assessment of hazards associated with the manufacture, use and disposal of a chemical substance, information on the biodegradability of a substance is required. The biodegradation of a chemical refers to the reduction in complexity of the chemical through metabolic activity of microorganisms. Under aerobic conditions microorganisms oxidise organic substances to carbon dioxide, water and biomass. Biodegradation tests are performed to assess the biodegradability of organic compounds. The Continuous Activated Sludge (CAS) test is an official method simulating the behaviour of organic substances in conventional activated sludge plants.In this CAS test, activated sludge is exposed to a specified concentration of N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine. Analysing the non-purgeable organic carbon (NPOC) content and N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine with LC-MS/MS allows assessment of the removal of the test substance. This test was performed according to ISO (1995), EC (1988) and OECD (1981) Guidelines, and in compliance with the OECD principles of Good Laboratory Practice (Annex 1). N,Ng p di(hydrogenated tallow-alkyl)-1,3-propanediamine. N,N-di(hydrogenated tallow-alkyl)-1,3- g propanediamine T 09066 C A S



# SANITIZEDT 09066 CAS

Page 8 of 28 

### MATERIALS AND METHODS

Test material A sample of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was received from AkzoNobel Surface Chemistry on 22-12-2009. The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof. Chemical name N,N-di ( h y dro g enated tallow-alky l ) -1,3- propanediamine Trade name Duomeen 2HT CAS numbe r Not assi g ned Batch number 2268-45-1 Com p osition N,N-di ( h y dro g enated tallow-alky l ) -1,3- propanediamine (90-95%) Amines (N-(hydrogenated tallow alkyl) trimethylenedi- (3-6%) Amines, tris(hydrogenated tallow alkyl) (2- 4%) Chemical stabilit y not relevant in biode g radabilit ytests Va p or p ressure low Water solubilit y sus p ension at test concentration Stora g e at room tem p erature in the dark Stabilit yunder stora g e conditions stable The concentrations cited in this report are expressed in terms of the as received N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine.

Reference substance and chemicals Ammonium chloride Lot no 04610BC-415 and potassium hydrogen phthalate, Lot no. 027K3733 used as the standard in the non purgeable organic carbon (NPOC) determination were obtained from Sigma Aldrich. Sodium nitrite Lot no 1296174 was purchased from Fluka. All other chemicals used were of reagent grade quality.

Deionized water The deionized water used contained no more than 0.01 mg/L Cu. This water was prepared in a water purification system.
Inoculum and settled sewage Secondary activated sludge to inoculate the test at the start was collected on 03- 02-2010 from the wastewater treatment plant (WWTP) Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. 0.35 liter of secondary activated sludge containing approximately 3 g/L dry weight was used as an inoculum for each CAS unit. This dry weight was obtained by diluting the sludge obtained from the treatment plant. The primary settled sewage was collected from the same plant weekly and stored frozen until required. N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine p AkzoNobel Surface Chemistry (y g

N , N

- di ( h y dro g enated tallow

- alky l )

- 1 , 3

- propanediamine Duo meen 2 H T p p

N , N

- di ( h y dro g enated tallow

- alky l )

- 1 , 3

- propanediamine Amines (N-(hydrogenated tallow alkyl) ( (ytrimethylenediAmines, tris(hydrogenated tallow alkyl)

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine. T 09066 C A S



# SANITIZEDT 09066 CAS

Page 9 of 28 CAS unit The CAS test was performed in Hussmann-type units constructed of glass (Figure 1). The units consisted of an aeration vessel capable of holding 0.35 liter from which the liquor was passed continuously to a settler of 0.3 liter. The domestic waste water liquor in a cooled vessel was supplied with a pump. The liquor passed through the aeration vessel and settler and treated effluent left the apparatus to be collected in a vessel. Aeration was achieved through a capillary on the bottom of the aeration section at a rate of 8-9 L/h of air. Sludge accumulating around the top of the aeration vessel was returned in the system once a day by brushing.
Stock suspension A solution of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine of 7.3 g/L in deionized water was directly added to the test unit using a syringe pump. The stock was prepared by adding 7.3 g of test substance to 0.7 L of deionized water. A homogenous suspension was obtained by acidifying the stock to a pH of approximately 5 by adding 3 M HCl. The final stock suspension was made up to 1.0 L with deionized water giving a concentration of 7.3 g/L. The particles in this suspension did not precipitate. The stock suspension was administered to the test unit with a syringe pump. The flow rate of the syringe pump was 9.6 mL/day giving a nominal concentration of the test substance in the influent of the unit of 50 mg/L at a sewage supply rate of 1.4 L/day.

Analyses Before the determination of the non-purgeable organic carbon (NPOC) content in the effluents of the CAS units, the effluents were filtered using Schleicher and Schüll (cellulose nitrate) filters with pores of 8.0  m to remove sludge particles. To ensure that the membrane filters used did not release carbon nor adsorb the test substance, the NPOC of a diluted stock solution was determined before and after filtration. Filtered samples were acidified prior to injection in a TOC apparatus (Shimadzu, s’Hertogenbosch, The Netherlands). The chemical oxygen demand (COD) of the influent and effluent was determined by oxidation with an acid-dichromate mixture in which Cr 6+ was reduced to Cr 3+ using Hach Lange test kits (LCK 114 and 314). The reaction vials were sealed and placed in a heating block and the contents heated at a temperature of 148°C for two hours. Ammonium and nitrite were measured with LCK 303 and LCK 342 Dr Lange test kits, respectively. The spectrophotometer (Xion 500) and heating block used were obtained from Hach Lange, Duesseldorf, Germany. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Berlin, Germany). The temperature was measured with a Tegam thermometer Model 820 (Applikon, Schiedam, The Netherlands). The dry weight (DW) of the inoculum was determined by filtering the activated sludge over a pre weighed 12 μm Schleicher and Schüll filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. DW was calculated by subtracting the weight of the filters and by dividing this difference by the filtered volume. T 09066 C A S



# SANITIZEDT 09066 CAS

Page 10 of 28 Collected influent and effluent samples were centrifuged at 8.000 g for 15 minutes. Effluent and sludge samples from the control unit collected in the same period were spiked with the test substance. These samples were used to assess the recovery and storage stability of the test substance. Subsequently the samples were acidified with H 2 SO 4 to approximately pH 2. The samples were stored in the refrigerator until analysis. The method of the specific analysis of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine in sludge and water are given in annex 3.

Procedures of the CAS test The CAS test was performed according to ISO (1995), EC (1988) and OECD (1981) test guidelines. The test and control unit were not coupled. The units were started with activated sludge. The aeration was achieved by operating an air-lift. The aeration rate was regulated so that the activated sludge was kept in suspension and the dissolved oxygen concentration was at least 2 mg/L. This oxygen concentration in the aeration vessel was measured at least two times a week. The domestic sewage supply was supplied at a rate of approximately 1.4 L/day to give a hydraulic retention time of 6 hours. The flow was checked by measuring the total volume of effluent over a 24-hour period. After brushing, 35 mL of sludge was daily removed from the aeration tank to maintain a sludge retention time of 10 days. The effluent samples (50 mL) were taken from the settler. The NPOC values were primarily used to assess the performance of biological treatment system fed with N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine containing wastewater and to preliminary follow the removal of the test substance during the test period. NPOC values of the last period of the test were used to calculate the mean removal percentage. The daily removal percentages were calculated by the following equation: 100 x (C T -(C t -C c )) / C T . Where C T is the carbon of the test compound measured as NPOC added to the settled sewage, C tis the carbon found as NPOC in the effluent of the CAS unit spiked with the test substance and C cis the carbon found as NPOC in the effluent of the control CAS unit. The analysis values in the test and control unit were treated as paired observations. Outliers of the mean difference (X d ) series were eliminated according to the Dixon test at a 95% probability level. From the set of ' n ' paired observations the mean difference (X d ) and the standard deviation (S d ) were calculated. The Sd is calculated with the following formula Sd =   (  x -X)/n-1. The statistical significance of the observed difference was then assessed from the t-statistics given by the following equation: Xd x  n/S d . The critical value of t at the required confidence level was obtained from statistical tables for a one tailed test with n-1 degrees of freedom. The percentage biodegradation/removal was given by; (SL-X d )/SL x 100 where X d the mean difference and SL is the spiking level, both values being expressed in mg/L carbon. The 95% confidence interval was calculated as follows: t n x S d /  n where t n is the t statistic for a two-tailed test, n-1 degrees of freedom, P = 0.05. Specific analyses of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine were used to determine the primary removal of the test substance. The removal percentage of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was determined with the following equation; (I s -E s )/I s x 100 , where I sis the nominal N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine p y N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine T 09066 C A S 

# SANITIZEDT 09066 CAS

Page 11 of 28 test substance concentration in the influent and E sis the mean of the measured test substance concentrations in the effluent. The concentration of the test substance on the activated sludge (C sludge ) and the theoretical maximum concentration on sludge are used to assess the removal of the test substance by adsorption. Provided biodegradation nor evaporation of the test substance occurs in the system, the theoretical maximum concentration of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamineN-[3-(dimethylamino)propyl] adsorbed onto the sludge is; C max adsorption= I s x SRT/HRT, where SRT is the sludge retention time, HRT is the hydraulic retention time (both expressed in days) and I sis the nominaltest substance concentration in the influent. The % removal of N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine by adsorption is 100 x C sludge /C max adsorption . N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamineN-[3-(dimethylamino)propyl]

N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine T 09066 C A S


# SANITIZEDT 09066 CAS

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### RESULTS AND DISCUSSION

Test conditions and validity of the test The incubation temperature of both CAS units ranged from 19 to 21°C. The pH of the effluent of both CAS units varied from 7.0 to 7.4. The oxygen concentrationsmeasured in both units were always 3.7 mg/L (Table I). These test conditions are believed to allow biodegradation by micro-organisms present in activated sludge. The CAS test was started with a high concentration of aerobic micro-organisms (3.0 g/L dry weight) maintained by the daily addition of primary settled sewage and sludge from a full-scale treatment plant (Duiven). The daily removal of 35 mL of activated sludge from the aeration vessel resulted in a sludge retention time of 10 days. The dry weight in the CAS units ranged from 2.5 to 3.0 g/L (Table I). The performance of the control unit was checked by measuring the COD removal at Day 14 and at day 63 and the concentrations of ammonium and nitrite in the effluent (Day 14). At Day 14 the COD contents (means of two measurements) in the influent and effluent were 521 and 48 mg/L, respectively. At day 63, the COD levels in the influent and effluent were 473 and 34 mg/L, respectively. COD removal percentages at both days were 91. The ammonium and nitrite concentrations in the effluent at Day 14 were 0.9 and <0.6 mg/L. These results demonstrate that the test is valid.
Biodegradation in the CAS test Both units were preconditioned for one week (Table II, Figure 2). After this week sludge wastage was started and the test substance was introduced to one of the units with a syringe pump. The sludge wasting resulted in a sludge retention time of 10 days. The test substance was added by spiking the influent with the test substance to a predetermined concentration (50 mg/L). The calculated carbon content of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine in the influent of the reactor was 40.5 mg/L. The measured concentration of the non purgeable organic carbon (NPOC) in an aqueous solution with 50 mg/L of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine was 36.5 mg/L. After filtration through a 8.0 m filter the NPOC was 37.5 mg/L. 40.5 mg/L (C T ) was used to calculate the carbon removal percentages. After the introduction of the test substance at day 0, high removal percentages of NPOC were immediately accomplished (Table II; Figure 3). These high removalpercentages have to be ascribed to the adsorption and/or biodegradation of N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine. The biodegradation percentages were >100 during the entire test period. From Day 46 to 63 (end of test), samples were taken to assess a mean of the removal percentage with NPOC contents. No outliers were identified using the Dixon test. Subsequently, the data obtained from Day 46 to 63 were used in a t-statistic. The mean difference between the NPOC in the effluents of control and test unit was 1.5 ± 1.2 mg/L (95 per cent confidence interval). The mean removal percentage calculated with this mean difference was 104 ± 1 (95% confidence). The t-statistic (n = 15) did exceed the critical value and the mean difference is therefore statistically significant. The results demonstrate that the continuous activated sludge system treating domestic wastewater spiked with N,N-di(hydrogenated tallow-alkyl)-1,3- N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine
N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine.

g y N,N-di(hydrogenated tallow-alkyl)-1,3- T 09066 C A S



# SANITIZEDT 09066 CAS

Page 13 of 28 propanediamine removes >100% of the organic carbon of the test substance. Biodegradation percentages in excess of 100% shows that the removal of organiccompounds present in the domestic wastewater are removed better when N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine is present in the influent. The results indicate that N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine is degraded by micro-organisms. The removal percentages demonstrate that formation of recalcitrant water-soluble substances is unlikely during the biodegradation process. Removal percentages calculated with NPOC values are not very accurate. An accurate assessment of the removal of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine was therefore established with specific analyses. The linearity, repeatability, LOD, LOQ, and recovery of the LC-MS analysis are given in Table III and in detail in Annex 3. The concentration of N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine inthe effluent of the test unit from day 59 to 63 using N-hexadecyl-N-octadecyl1,3-diaminopropane as representative component was  0.21 g/L (Table IV). This concentration corresponds with  99.999% removal of the test substance from the influent. The concentrations of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine in the mixed liquid suspended solids (activated sludge) of the reactor monitored on days 62 and 63 were 257 and 247 mg/L, respectively. Based on the observed in the mixed liquid suspended solids of 253 mg/L (mean), 1.3% removal of the N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine byadsorption is calculated (Table IV). The results demonstrate that N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine biodegrade almost completely in properly operating conventional biological wastewater treatment plants. gN,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamine

N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine
q p g( N,N-di(hydrogenated tallow-alkyl)-1,3-propanediamineN,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine T 09066 C A S


# SANITIZEDT 09066 CAS

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### AMENDMENTS AND DEVIATIONS

Guidelines A few minor deviations to the guidelines were introduced. The primary settled sewage was collected weekly and stored in the refrigerator until required instead of a daily collection of wastewater. The units consisted of aeration vessels capable of holding only 0.35 L from which the liquor was then passed continuously to settler of 0.30 liter capacities.
Study plan There is one deviation to the study plan; issue of method to determine the concentrations of the test substance, in aqueous and sludge samples. There are three deviations to the study plan; 1) the effluent samples were filtrated through 8.0 m instead of 0.8 m filters because the suspension of the test substance did not pass the 0.8 m filter completely, 2) the batch number in the study plan originally provided did not match with the number on the container and certificate of analysis and 3) method to analyse the test substance with the LC-MS/MS was improved by preventing entering of high salt concentrations into the MS .



### REFERENCES

ISO Guideline Water quality - Evaluation of the elimination and biodegradability of organic compounds in an aqueous medium - Activated sludge simulation test ISO 11733 (1995). EC Official Journal of the European Communities, Part C: Methods for the determination of ecotoxicity. Biodegradation Activated sludge simulation tests L133, (1988). OECD Guidelines for testing chemicals, section 3: Degradation and accumulation No 303 A Paris Cedex France (1981). T 09066 C A S



# SANITIZEDT 09066 CAS

Page 15 of 28 Table I Oxygen concentrations, pH values in the effluent, and dry weight of the activated sludge in the control and test unit. T ime ( da y s ) Ox yg en ( m g /L ) Drywei g ht (g /L ) p H

Control T est Control T est Control T est -6 2.3 4.0 7.1 7.1 -2 3.5 3.7 7.2 7.0 -1 2.7 2.9 1 4.1 4.7 7.3 7.0 5 3.9 3.9 7.1 7.2 6 2.6 2.7 8 2.5 4.3 7.3 7.3 12 3.0 4.7 7.2 7.3 13 2.8 2.7 15 3.6 4.1 7.3 7.4 19 4.1 5.1 7.4 7.1 20 3.0 2.7 22 3.7 3.9 7.2 7.3 26 4.1 4.5 7.3 7.0 27 2.9 2.6 29 4.2 4.7 7.2 7.1 33 3.7 4.8 7.0 7.3 34 2.8 2.7 36 4.2 3.9 7.0 7.1 40 4.2 4.7 7.1 7.0 41 2.7 2.5 43 4.5 4.1 7.4 7.0 47 5.1 4.7 7.3 7.0 48 2.8 2.7 50 4.8 3.9 7.2 7.1 54 4.3 4.0 7.1 7.1 55 2.9 2.6 57 4.8 4.5 7.2 7.2 61 3.9 4.1 7.0 7.0 62 2.8 2.6 63 4.5 4.6 7.2 7.2 T 09066 C A S


# SANITIZEDT 09066 CAS

Page 16 of 28 Table II NPOC concentrations in the effluent of the control and test unit and the calculated removal percentages of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine. The data in grey part of the table are used to calculate the biodegradation percentage. Time ( da y s ) NPOC ( m g /L ) Removal ( % ) Control Test -6 17.1 15.0 -2 16.5 16.6 1 19.9 19.3 101 5 12.5 8.2 111 8 15.1 9.4 114 12 12.0 8.7 108 15 15.3 9.3 115 19 16.4 6.8 124 22 11.8 9.8 105 26 11.5 9.9 104 29 12.6 9.3 108 33 11.7 9.5 105 36 12.0 8.9 108 40 9.8 8.0 104 42 12.1 11.2 102 43 13.1 10.9 105 46 13.1 9.3 109 47 10.6 9.4 103 48 11.5 10.4 103 49 10.8 9.5 103 50 12.5 11.6 102 53 11.4 10.7 102 54 11.3 9.6 104 55 12.7 10.7 105 56 11.9 9.1 107 57 13.4 9.8 109 59 9.1 10.0 102 60 12.5 12.2 101 61 12.3 11.6 102 62 11.1 10.9 100 63 13.2 12.5 102 N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine. T 09066 C A S


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Page 17 of 28 Table III Summary of method validation of the LC-MS analysis Paramete r Determined Limit Linearit y (R 2 ) 0.999 0.98 Re p eatabilit y ( CV ( % )) at lowest standard 3.4  20 Re p eatabilit y ( CV ( % )) at hi g hest standard 1.5  20 LO Q (g /L ) 0.70 <500 LOD (g /L ) 0.21 <500 S y stem stabilit y ( % deviated from nominal ) 3.5 10 Recovery from slud g e ( % ) 83 70 and 110 Recoveryfrom influent ( % ) 82 70 and 110 Recoveryfrom effluent ( % ) 94 70 and 110 CV = coefficient of variation LOQ = Limit of Quantification LOD = Limit of Detection

Table IVConcentrations of N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine measured in the effluent and mixed liquid suspended solids (adsorption onto sludge) and removal percentages from the influent and by adsorption onto sludge, respectively. Time (days) Concentration (g/L) Removal (%) Effluent 59 <0.2 g/L >99.999 60 <0.2 g/L >99.999 61 <0.2 g/L >99.999 62 <0.2 g/L >99.999 63 <0.2 g/L >99.999 Time (days) Concentration (mg/L) Removal (%) Mixed Liquid Suspended Solids 62 257 mg/L 1.3 63 247 mg/L 1.2 N,N-di(hydrogenated tallow-alkyl)-1,3- propanediamine T 09066 C A S


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Page 18 of 28 Figure 1 Husmann-type continuous flow unit in operation. (A) storage vessel, (B) dosing pump (C) aeration section (D) settling section (E) collecting vessel, (F) air supply. T 09066 C A S



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## -10 0 10 20 30 40 50 60 70
Time (days) 0 5 10 15 20 25NPOC (mg/L)

Figure 2 NPOC values of the effluent from the test unit fed with N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine ( ) and of the effluent from the control unit (  ) plotted against time. First addition was on Day 0. N,Ng di(hydrogenated tallow-alkyl)-1,3-propanediamine T 09066 C A S



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## 0 10 20 30 40 50 60 70 Time (days) 0 20 40 60 80 100 120Removal (% )

Figure 3 Biodegradability/removal curve of N,N-di(hydrogenated tallow-alkyl)- 1,3-propanediamine in the CAS test determined with NPOC. The line represents the mean removal percentage of 104% from day 46 to 63. N,N-di(hydrogenated tallow-alkyl)- 1,3-propanediamine T 09066 C A S



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Page 21 of 28 Annex 1 Compliance statement T 09066 C A S 

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Page 22 of 28 Annex 2 Certificate of analysis (supplied by the sponsor) T 09066 C A S



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Page 23 of 28 Annex 3 Description of the analytical procedure for Duomeen 2 HT the quantification in aqueous and sludge samples using a LC-MS/MS system

Introduction
A method to determine the concentration of Duomeen 2 HT in aqueous and sludge samples is described. Procedures and instrumentation were based on High Performance Liquid Chromatography combined with mass spectrometry (LC-MS). The main component i.e. N-hexadecyl-N-octadecyl-1,3-diaminopropane was treated as representative for the test substance. Samples were quantified using a calibration curve.
Analytical procedure
The following conditions were found to be suitable for the determination of the test substance, given in the Tables 3.I to 3.III.
Table 3.I Parameters for HPLC (Agilent Technologies 1200 series)

Column: Zorbax SB OCTADECYL 2.1x30 mm 3.5 μ m Column tem p .: 70 °C Flow: 0.5 ml/min

Mobile phase: A= HPLC water + TFA + NH 3 ( 25% ) + acetic acid B= Methanol:2-propanol 50:50 (v/v) + TFA + NH 3 (25%) + acetic acid

Gradient mobile phase: 0 min. 50 % A 50 % B 3 min. 0 % A 100 % B 6 min. 0 % A 100 % B post time 3 min.

In j ection volume: 10 μ l

Table: 3.II Parameters for MS (Agilent Technologies G 6410B, triple quad)

Ion source: ElectroS p ra y Polarit y : Positive mode Detector mode: Multi p le Reaction Mode Delta EM V 300 V

Source parameters: Gas temperature: 350 °C Gas flow: 12 l/min Nebulizer: 60 psi Capilary voltage: 2500 V Duomeen 2 HT

Duomeen 2 HT

N-hexadecyl-N-octadecyl-1,3-diaminopropane T 09066 C A S 

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Page 24 of 28 Table 3.III MS acquisition parameters

Compound Precursor Ion MS 1 RES Product Ion MS 2 RES Dwell time (ms) Fragm. (V) Collision Energy (V) C18, C16 551.3 Unit 534.3 Unit 250 180 35

High salt concentrations in the samples necessitated to waste the flow from the LC for the first 4 minutes. The flow was from LC was directed to the MS from 4 min. to 5.6 min. where the test substance elutes after 5.1 min. In this configuration most of the salt did not enter the MS, where the salt may precipitate. This may affect the stability of the system. Furthermore between each injection the MS was rinsed for 1.5 minutes with 2-propanol: HPLC water 30:70 (v/v) at a flow of 2 ml/min. to remove the salts that did precipitate in the MS (deviation of the study plan).
Preparation Mobile phase Mobile phase A 1 liter of HPLC water was added to a 1000 ml measuring cylinder, approximately 3⁄4 of this volume was added to a glass 1 liter bottle. Subsequently 0.65 ml of trifluoroacetic acid, 0.75 ml ammonium hydroxide solution (25% v/v), 1.15 ml acetic acid and the remaining HPLC water in the measuring cylinder was added. Content of the bottle was mixed thoroughly.

Mobile phase B 500 ml Methanol was added to a 1 liter glass bottle, subsequently 0.65 ml of trifluoroacetic acid, 0.75 ml ammonium hydroxide solution (25% v/v), 1.15 ml acetic acid and 500 ml 2-propanol was added. Content of the bottle was mixed thoroughly.

Preparation of standards and samples

Standards ; a stock solution of the test substance (364 mg/L) was prepared in leaching solution. This leaching solution contains 100 g/L MgCl 2 6H 2 O in methanol : 2-propanol 50:50 (v/v). Dilutions of the stock solution were made inthe range of 0 – 500 μg/l for the calibration standards (n 5). Control standards from the middle range of the calibration series were analyzed at a minimum rate of one per ten samples or at least after each test series.
Aqueous samples: Aqueous samples were diluted 50:50 (v/v) with leaching solution in HPLC-vials before analysis. Further dilutions were made with leaching solution when the concentration was outside the concentration range of the calibration curve. The vials/volumetric flasks were mixed thoroughly and analyzed using the LC-MS/MS settings described above.
Activated sludge samples; An amount of 10 mL of activated sludge was transferred into a centrifuge tube and centrifuged for 10 minutes at 3000 rpm. T 09066 C A S



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Page 25 of 28 The supernatant of the sludge sample was diluted with a solution of 2-propanol : HPLC water : formic acid 50 : 48 : 2 (v/v/v) and analyzed using LC/MS-MS. The sludge residue in the centrifuge tube was extracted with 2-propanol : saturated ammonium chloride solution in HPLC water : HPLC water at a ratio of 50 : 50 : 11 (v/v/v) (extraction solution). Extraction was carried out by adding 10 mL of extraction solution in the centrifuge tube. The tubes were subsequently thoroughly mixed with a mechanical shaker (Vortex) to suspend the residue. Next, the tubes with suspensions were ultrasonified for 15 minutes in a bath. After ultrasonication, the centrifuge tubes were placed on a shaker for 15 minutes at 100 rev/min. Finally, the tubes were centrifuged, for 10 minutes at 3000 rpm. After this final centrifugation step the supernatant of a tube was collected in a 25-mL volumetric flask. The second extraction was carried out by adding another 10 ml of extraction solution to the centrifuge tube and the sludge and repeating the procedure described above. The supernatant obtained after centrifugation was transferred to the same volumetric flask. The volumetric flask was brought up to volume with the extraction solution. Further dilutions of the content of the 25 ml volumetric flask were done with leaching solution and analyzed using LC/MS-MS.

Calculation of concentrations
Quantification was carried out by measuring peak areas. The concentrations of the test substance in the samples were calculated from the relation between concentration of standards (Cs) and peak area (PAs) obtained with linear regression analysis:
PAs = Cs * Slope + constant

slope constant area peak Sample ion concentrat Sample

Method validation

Linearity; The analytical method was found to be linear over the range of 0 – 500 μg/L with the system setting described above (Figure 3.1). The R 2 foundwas 0.99996 which is in excess of the prescribed 0.98 (n5). T 09066 C A S 

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Page 26 of 28 Figure 3.1 Calibration curve of N-hexadecyl-N-octadecyl-1,3-diaminopropane (06-05-2010). The concentrations given on the X-axis are in μg/L of Duomeen 2HT.
Repeatability; Six aliquots from vials with the lowest and highest standard concentration prepared from a single homogenous sample were analyzed. Mean values, standard deviations and coefficients of variation are given in Table 3.IV.
Table 3.IV repeatability lowest and highest standard concentration

Sample Response (counts) ofN-hexadecyl-Noctadecyl-1,3- diaminopropane Sample Response (counts) ofN-hexadecyl-Noctadecyl-1,3- diaminopropane 5 μg/L 1 835.0 500 μg/L 1 71374.9 5 μg/L 2 797.9 500 μg/L 2 73076.0 5 μg/L 3 775.4 500 μg/L 3 71737.6 5 μg/L 4 759.2 500 μg/L 4 70698.3 5 μg/L 5 771.8 500 μg/L 5 70268.2 5 μg/L 6 801.7 500 μg/L 6 70534.0 Mean 790.2 Mean 71281.5 SD 27.2 SD 1035.6 CV (%) 3.4 CV (%) 1.5 SD = standard deviation CV = coefficient of variation

LOD/LOQ; The Limit of Detection (LOD) was calculated using the signal to noise ratio (S/N). The LOD was calculated as 3 * S/N and the Limit of Quantification (LOQ) was calculated as 10 * S/N of the lowest calibration standard. (signal definition; peak height, noise definition; peak to peak, noise region; retention time 4.5-5.0 min.) The lowest calibration standard i.e. 5 μg/L from the calibration range analyzed had a S/N ratio of 71.9. The LOD and LOQ of N-hexadecyl-N-octadecyl-1,3- diaminopropane were 0.21 and 0.70 μg/L, respectively. C18,C16 - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs

Concentration (g/L) -50 -25 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 5 5 4 x10

0 1 2 3 4 5 6 7 y = 140.1606 * x + 715.0000 R^2 = 0.99996204

N-hexadecyl-N-octadecyl-1,3-diaminopropane Duomeen

p p

2HT.

N-hexadecyl-N- hex octadec y l-1,3

- diamino p ro p ane N -hexadec y l-Noctadec y l-1,3

- diamino p ro p ane

N-hexadecyl-N-octadecyl-1,3- diaminopropane T 09066 C A S


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Page 27 of 28 Recovery and stability of test substance ; Spiked aqueous and sludge samples were analyzed to determine the recovery and stability of the substance during storage. Fortified aqueous samples were prepared at a concentration of 50 mg/L Duomeen 2 HT and fortified sludge samples were prepared at a concentration of 500 mg/L Duomeen 2HT (Table 3.V). The concentration of the test substance adsorbed on the activated sludge was calculated by adding up the concentrations found in the supernatant and extraction liquid. Recoveries were within the prescribed 70-110% of the nominal concentrations, validating the extraction procedure and proving that the test substance was stable during storage.
Table 3.V Recovery results sludge and water samples Sample Measured con c. (mg/L ) Recovery (%) Influent spiked with 50 mg/L 41.1* 82.3 Effluent spiked with 50 mg/L 48.0* 95.9 Sludge spiked with 500 mg/L 418.4* 83.7 * Mean of two samples

System stability ; To determine the systems stability during the analyses run, control standards were analyzed at a minimum rate of every ten samples (Table 3.VI). The analyzed control standards (150 μg/L) were compared with the standard of the calibration curve which had a response of 21641. The control standards deviated  3.5% compared to this calibration standard. This is within the prescribed 10%.
Table 3.VI control standards during analysis run (06-05-2010). Control Response (counts) % of calibration standards Control standard I 20888 96.5 Control standard II 21027 97.2 Duomeen 2 HT Duomeen 2HT T 09066 C A S


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Page 28 of 28 Samples
Table 3.VII analyses of the samples Sample Concentration (mg/L) Effuent 11-04-2010 (day 59) < LOD Effuent 12-04-2010 (day 60) < LOD Effuent 13-04-2010 (day 61) < LOD Effuent 14-04-2010 (day 62) < LOD Effuent 15-04-2010 (day 63) < LOD Influent test 14-04-2010 (day 62 spiked with 50 mg/L) 40.7 Influent test 15-04-2010 (day 63 spiked with 50 mg/L) 41.6 MLSS test supernatant 14-04-2010 (day 62) 3.7 MLSS test extractie 14-04-2010 (day 62) 253.0 MLSS test supernatant 15-04-2010 (day 63) 1.7 MLSS test extractie 15-04-2010 (day 63) 245.0 Effluent control 11-04-2010 (day 59 spiked with 50 mg/L) 51.2 Effluent control 15-04-2010 (day 63 spiked with 50 mg/L) 44.7 MLSS control supernatant 14-04-2010 (day 62 spiked with 500 mg/L) 25.9

MLSS control extractie 14-04-2010 (day 62 spiked with 500 mg/L) 386.8

MLSS control supernatant 15-04-2010 (day 63 spiked with 500 mg/L) 23.4

MLSS control extractie 15-04-2010 (day 63 spiked with 500 mg/L) 400.8

MLSS Mixed liquid Suspended Solids
Example chromatogram; Figure 3.2 is an example of the peak generated in MRM mode when analyzing the main component N-hexadecyl-N-octadecyl-1,3- diaminopropane.

Figure 3.2 Calibration standard 50 μg/L N-hexadecyl-N-octadecyl-1,3- diaminopropane. T 09066 C A S


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