Amides, from C8-10-fatty acids and tetraethylenepentamine

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-10-2009 - 03-02-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test performed under GLP according the guidelines with some minor acceptable deviations. Test meets all validity criteria.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

Deviations:

yes

Remarks:

acceptable minor deviations

Principles of method if other than guideline:

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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, reaction products with N-(2-aminoethyl)piperazine

EC Number:

295-532-5

EC Name:

Tall oil, reaction products with N-(2-aminoethyl)piperazine

Cas Number:

92062-17-4

Molecular formula:

not available

IUPAC Name:

not available

Details on test material:

Test material
A sample of tall oil, reaction products with N-(2-aminoethyl)piperazine was received from AkzoNobel Surface Chemistry on 29-09-2009.
The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity thereof.
Chemical name tall oil, reaction products with N-(2-aminoethyl)piperazine
Trade name Wetfix N422
CAS number 92062-17-4
Batch number S000923
Composition (annex 2) 94%
Chemical stability not relevant in test
Vapor pressure compounds are unlikely to evaporate
Water solubility soluble 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 tall oil, reaction products with N-(2-aminoethyl)piperazine.

Radiolabelling:

no

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge to inoculate the test at the start was collected on 28-10-2009 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.

Duration of test (contact time):

49 d

Initial test substance concentration

Initial conc.:

50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

TOC removal

Details on study design:

CAS unit
The CAS test was performed in Hussmann-type units constructed of glass . 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 approximately 8 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 suspension of tall oil, reaction products with N-(2-aminoethyl)piperazine 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 approximately 0.7 L of deionized water. A homogenous suspension was obtained by acidifying the stock to a pH of 7 by adding HCl. The final stock suspension was made up to 1.0 L with deionized water giving a
concentration of 7.3 g/L. Part of this suspension did float. Floating of test material required continuous agitation of the stock in the
syringe. Agitation in the syringe was realised with a stirrer bar in the syringe and a magnetic stirrer under the syringe. 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.

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 tall oil, reaction products with
N-(2-aminoethyl)piperazine 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 (see attached report). The statistical significance of the observed
difference was then assessed from the t-statistics given by the following equation: (see attached report). 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 /SQRT(n) where tn is the t statistic for a two-tailed test, n-1 degrees of freedom,
P = 0.05.
Specific analyses of tall oil, reaction products with N-(2-aminoethyl)piperazine were used to determine the primary removal of the test substance.
The removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine was determined with the following equation; (Is-Es)/Is x 100, where Is is the nominal 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 tall oil, reaction products with N-(2-aminoethyl)piperazine adsorbed onto the sludge is;
Cmax adsorption = Is xSRT/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 tall oil, reaction products with N-(2-aminoethyl)piperazine by adsorption is
100 x Csludge/Cmax adsorption.

Reference substance

Reference substance:

not required

Results and discussion

Test performance:

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 7.3.
The oxygen concentrations measured in both units were always >=3.7 mg/L . 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. 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 2.4 to 3.0 g/L. The performance of the control unit
was checked by measuring the COD removal at Day 14 and at day 48 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 416 and 43 mg/L, respectively.
At day 48, the COD levels in the influent and effluent were 461 and 38 mg/L, respectively. COD removal percentages at both days were 90 and 92.
The ammonium and nitrite concentrations in the effluent at Day 14 were <2.5 and <2.0 mg/L. These results demonstrate that the test is valid.

% Degradation

% Degradation 1

% Degr.:

89

St. dev.:

1

Parameter:

DOC removal

Remarks on result:

other: mean removal percentage from day 34 to 48 (15 measurements)

% Degradation 2

% Degr.:

> 99.999

Parameter:

test mat. analysis

Remarks:

using octadecenyl amide of N-(2-aminoethyl)piperazine as representative component

Remarks on result:

other: analysed in effluent of test unit from day 44 to 48

% Degradation 3

% Degr.:

0.04 - 0.044

Parameter:

test mat. analysis

Remarks:

using octadecenyl amide of N-(2-aminoethyl)piperazine as representative component

Remarks on result:

other: removal from influent throug adsorption onto sludge assessed in two samples day 47 and 48

Transformation products:

no

Details on transformation products:

These organic carbon removal percentages in excess of the pass level of 80% indicate that the test substance is completely removed.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

Any other information on results incl. tables

NPOC concentrations in the effluent of the control and test unit and the calculated removal percentages of tall oil, reaction products with N-(2 -aminoethyl)piperazine. The data in grey part of the table are used to calculate the biodegradation percentage.

Time (days)	NPOC (mg/L)		Removal (%)
	Control	Test
-4	13.7	11.3
-2	12.1	11.6
2	9.5	9.0	101
0	10.8	17.9	81
6	11.0	17.2	83
9	11.7	19.5	79
13	12.7	17.6	87
16	13.9	18.5	87
20	12.7	17.4	87
23	8.3	15.4	81
27	9.7	17.2	80
30	7.5	14.9	80
34	9.1	14.6	85
35	12.0	15.3	91
36	10.3	16.0	84
37	12.7	14.9	94
38	10.2	16.4	83
39	10.9	14.4	90
40	11.0	15.1	89
41	10.5	14.9	88
42	11.4	15.0	90
43	13.0	17.1	89
44	12.1	17.6	85
45	13.9	17.1	91
46	12.8	17.2	88
47	12.6	16.7	89
48	13.7	18.4	87

Concentrations of tall oil, reaction products with N-(2 -aminoethyl)piperazine

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 (μg/L)	Removal (%)
	Effluent
44	<0.014	>99.999
45	<0.014	>99.999
46	<0.014	>99.999
47	<0.014	>99.999
48	<0.014	>99.999
Time (days)	Concentration (mg/L)	Removal (%)
	Mixed liquid suspended solids
47	0.88	0.044
48	0.80	0.040

Applicant's summary and conclusion

Validity criteria

Validity criteria fulfilled:

yes

Remarks:

COD removal in the control unit at Day 14 and day 48 of 90 and 92% resp. The ammonium and nitrite concentrations in the control effluent at Day 14 were <2.5 and <2.0 mg/L. These results demonstrate that the test is valid

Conclusions:

The CAS test demonstrates that tall oil, reaction products with N-(2-aminoethyl)piperazine biodegrade almost completely in properly operating
conventional biological wastewater treatment plants. Tall oil, reaction products with N-(2-aminoethyl)piperazine is primarily removed by
biodegradation
89±1% NPOC removal, determined in effluent samles from test unit during 15 days from day 34 to 48 (These organic carbon removal percentages in excess of the pass level of 80% indicate that the test substance is completely removed)
>99.999% removal of test substance calculated with specific analysis in effluent samples taken from day 44-48
0.04-0.044 % removal of test substance by adsorption onto sludge, calculated with specific analysis in sludge samples taken on day 47 and 48.

Test performed under GLP according guidelines with a few accpetable (minor) deviations, meeting all validity criteria

Executive summary:

The continuous activated sludge (CAS) test was performed according to ISO Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Tall oil, reaction products with N-(2-aminoethyl)piperazine were exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. Tall oil, reaction products with N-(2-aminoethyl)piperazine were spiked at a nominal influent concentration of 50 mg/L (36.5 mg/L carbon; calculated) for a period of 48 days and included a control fed with domestic wastewater only. The immediate high removal percentages can be attributed to adsorption and probably biodegradation. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine calculated over 15 measurements obtained from day 34 to 48 of the test was 89 +/-1% (95% confidence interval). These organic carbon removal percentages in excess of the pass level of 80% indicate that the test substance is completely removed. An accurate assessment of the removal of tall oil, reaction products with N-(2- aminoethyl)piperazine was established with specific analyses. The method (LCMS/ MS) for the determination of tall oil, reaction products with N-(2- aminoethyl)piperazine was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery and specificity. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine in the test unit was assessed with the specific analysis from day 44 to 48 was >99.999% using octadecenyl amide of N-(2-aminoethyl)piperazine as representative component. These analyses demonstrate that the removal of tall oil, reaction products with N-(2-aminoethyl)piperazine is complete. Tall oil, reaction products with N-(2-aminoethyl)piperazine concentrations in the sludge of the reactor sampled on days 47 and 48 were 0.88 and 0.80 mg/L. The mean removal percentage of tall oil, reaction products with N-(2-aminoethyl)piperazine from the influent through adsorption onto sludge assessed in two samples was therefore 0.04 -0.044%. This percentage demonstrates that tall oil, reaction products with N-(2-aminoethyl)piperazine are almost completely removed by biodegradation. The CAS test demonstrates that tall oil, reaction products with N- (2-aminoethyl)piperazine almost completely removed from the wastewater by biodegradation in conventional biological wastewater treatment plants.