Technology & 

Engineering B.V 

Environmental Chemistry 

PO Box 9300, 

6800 SB 

Arnhem, 

the Netherlands

REPORT 

CONFIDENTIAL 

Title DETERMINATION OF THE PRIMARY AND ULTIMATE 

BIODEGRADATION 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. Louwerse, 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. van Ginkel, A. Louwerse, J. van Dam

Technology &PO Box 9300, 

Engi g neering B.V 6800 SB 

Environmental Chemistry Arnhem, 

the Netherlands

T09066 CAS

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T 09066 CAS 

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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 1041%

(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 as 

quantified 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 

p g N,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 

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 CAS

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STUDY ORGANISATION 

Sponsor AkzoNobel Surface Chemistry LLC 

525 West Van Buren 

Chicago, Il 60607-3823 

USA

Study monitor D Hillebold 

Study location AkzoNobel Technology & Engineering

Arnhem 

Velperweg 76

6824 BM Arnhem

The Netherlands

Study director C.G. van Ginkel

Quality Assurance Unit H. van Daalen

Management R. Borgmann-Strahsen

Experimental starting date 03-02-2010

Experimental completion 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 Surface Chemistry LLC 

525 West Van Buren

Chicago, Il 60607-3823

D Hillebold 

AkzoNobel Technology & Engineering

Arnhem

Velperweg 76

6824 BM Arnhem

The Netherlands

C.G. van Ginkel

H. van Daalen

R. Borgmann-Strahsen

AkzoNobel Technology & Engineering Arnhem 

AkzoNobel Technology & Engineering Arnhem 

T 09066 CAS

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T 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. van Ginkel

R. Borgmann-Strahsen

T 09066 CAS

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T 09066 CAS 

Page 5 of 28 

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 study Dates of 

inspection

Dates of 

reporting

Study plan 05-02-2010 05-02-2010

Test (spiking with test substance) 11-02-2010 12-02-2010

Test (extraction and sample preparation) 03-05-2010 03-05-2010

Report 09-07-2010 10-07-2010

Quality Assurance Unit

H. van Daalen 

AkzoNobel 

Technology & Engineering Arnhem.

H. van Daalen 

T 09066 CAS

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T 09066 CAS 

Page 6 of 28 

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

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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). 

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

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

propanediamine 

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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(hydrogenated tallow-alkyl)-1,3-

propanediamine 

Trade name Duomeen 2HT

CAS number Not assigned

Batch number 2268-45-1

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

propanediamine (90-95%) 

Amines (N-(hydrogenated tallow alkyl) 

trimethylenedi- (3-6%) 

Amines, tris(hydrogenated tallow alkyl) (2-

4%) 

Chemical stability not relevant in biodegradability tests

Vapor pressure low

Water solubility suspension at test concentration

Storage at room temperature in the dark 

Stability under storage 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. 

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

AkzoNobel Surface Chemistry 

(y g

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

propanediamine 

Duomeen 2HT

p p

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

propanediamine 

Amines (N-(hydrogenated tallow alkyl) ( (y

trimethylenediAmines, tris(hydrogenated tallow alkyl) 

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

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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 Cr6+ was reduced to Cr3+

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. 

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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 H2SO4 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 (CT-(Ct-Cc)) / CT. Where CT is the carbon of the test 

compound measured as NPOC added to the settled sewage, Ct is the carbon found 

as NPOC in the effluent of the CAS unit spiked with the test substance and Cc is 

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 (Xd) series were eliminated 

according to the Dixon test at a 95% probability level. From the set of 'n' paired 

observations the mean difference (Xd) and the standard deviation (Sd) 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/Sd. 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-Xd)/SL x 100 where Xd 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: tn x Sd /n where tn 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; (Is-Es)/Is x 100, where Is is 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 

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Page 11 of 28 

test substance concentration in the influent and Es is the mean of the measured 

test substance concentrations in the effluent. The concentration of the test 

substance on the activated sludge (Csludge) 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; Cmax adsorption = Is x SRT/HRT, where SRT is the sludge retention time, 

HRT is the hydraulic retention time (both expressed in days) and Is is the nominal 

test substance concentration in the influent. The % removal of N,Ndi(hydrogenated tallow-alkyl)-1,3-propanediamine by adsorption is 100 x

Csludge/Cmax adsorption.

N,N-di(hydrogenated

tallow-alkyl)-1,3-propanediamineN-[3-(dimethylamino)propyl] 

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

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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 concentrations 

measured 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 (CT) 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 removal 

percentages 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-

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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 organic 

compounds 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 in 

the 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 by 

adsorption 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. 

g

N,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-propanediamine 

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

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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). 

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Table I Oxygen concentrations, pH values in the effluent, and dry weight of the 

activated sludge in the control and test unit. 

Time

(days)

Oxygen (mg/L) Dry weight (g/L) pH

Control Test Control Test Control Test

-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

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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 (days) NPOC (mg/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.

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Table III Summary of method validation of the LC-MS analysis 

Parameter Determined Limit

Linearity (R2) 0.999 0.98 

Repeatability (CV (%)) at lowest standard 3.4  20 

Repeatability (CV (%)) at highest standard 1.5  20 

LOQ (g/L) 0.70 <500

LOD (g/L) 0.21 <500

System stability (% deviated from nominal) 3.5 10 

Recovery from sludge (%) 83 70 and 110

Recovery from influent (%) 82 70 and 110

Recovery from effluent (%) 94 70 and 110

CV = coefficient of variation 

LOQ = Limit of Quantification

LOD = Limit of Detection 

Table IV Concentrations 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 

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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. 

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-10 0 10 20 30 40 50 60 70

Time (days)

0

5

10

15

20

25

NPOC (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. 

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

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0 10 20 30 40 50 60 70

Time (days)

0

20

40

60

80

100

120

Removal (% )

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 

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Annex 1 Compliance statement 

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Annex 2 Certificate of analysis (supplied by the sponsor) 

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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 3.5μm

Column temp.: 70 °C 

Flow: 0.5 ml/min

Mobile phase: 

A= HPLC water + TFA + NH3 (25%) + acetic acid 

B= Methanol:2-propanol 50:50 (v/v) + TFA + NH3 (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. 

Injection volume: 10 μl

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

Ion source: ElectroSpray

Polarity: Positive mode

Detector mode: Multiple Reaction Mode

Delta EMV 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 

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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 MgCl26H2O in 

methanol : 2-propanol 50:50 (v/v). Dilutions of the stock solution were made in 

the 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. 

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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

Sample peak area constant

Sample concentration 
 

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 R2

 found 

was 0.99996 which is in excess of the prescribed 0.98 (n5).

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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) of

N-hexadecyl-Noctadecyl-1,3-

diaminopropane

Sample Response (counts) of

N-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 55

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

octadecyl-1,3-

diaminopropane

N-hexadecyl-Noctadecyl-1,3-

diaminopropane

N-hexadecyl-N-octadecyl-1,3-

diaminopropane 

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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 conc. (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 

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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.

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