Amides, tall-oil fatty, N-[3-(dimethylamino)propyl]

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-28 to 2010-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

It is assessed from the methods of manufacture the starting materials and the similarity of the end product compositions that EC272-047-7 is sufficiently similar in all respects to EC800-353-8 that it is a sensible approach to use a read-across approach. A comparison of the two substances and a read-across justification can be found in section 13 of this dataset.

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)

Radiolabelling:

no

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 L 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):

48 d

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 amides, rape-oil, N-[3-(dimethylamino)propyl] 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 6.7 by adding 2 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.

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 amides, rape-oil, N-[3-(dimethylamino)propyl] 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 = v¿(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 vn/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 /vn where tn is the t statistic for a two-tailed test, n-1 degrees of freedom,
P = 0.05.
Specific analyses of amides, rape-oil, N-[3-(dimethylamino)propyl] were used to determine the primary removal of the test substance.
The removal percentage of amides, rape-oil, N-[3-(dimethylamino)propyl] 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 o famides, rape-oil, N-[3-(dimethylamino)propyl] 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 amides, rape-oil, N-[3-(dimethylamino)propyl] by adsorption is
100 x Csludge/Cmax adsorption.

Reference substance:

not required

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 (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 2.4 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.

% Degr.:

105

St. dev.:

1

Parameter:

DOC removal

Remarks on result:

other: mean removal percentage from day 35 to 48 (14 measurements)

% Degr.:

> 99.997

Parameter:

test mat. analysis

Remarks:

using octadecenamide-N- [3-(dimethylamino)propyl] as representative component

Remarks on result:

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

% Degr.:

0.02

Parameter:

test mat. analysis

Remarks:

using octadecenamide-N- [3-(dimethylamino)propyl] 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:

The results demonstrate that amides, rape-oil, N-[3-(dimethylamino)propyl] biodegrade almost completely in properly operating conventional biological wastewater treatment plants.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

NPOC concentrations in the effluent of the control and test unit and the calculated removal percentages of amides, rape-oil, N-[3-(dimethylamino)propyl]. 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	11.8	105
-2	12.1	11.9	101
2	9.5	8.1	104
4	10.8	14.6	90
6	11.0	15.8	87
9	11.7	13.8	94
13	12.7	12.9	100
16	13.9	12.1	105
20	12.7	13.5	98
23	8.3	10.9	93
27	9.7	10.0	99
30	7.5	8.5	97
34	9.1	12.1	93
35	12.0	8.8	109
36	10.3	8.3	106
37	12.7	9.2	110
38	10.2	8.6	104
39	10.9	8.8	106
40	11.0	10.2	102
41	10.5	9.1	104
42	11.4	8.8	107
43	13.0	12.4	102
44	12.1	11.3	102
45	13.9	11.5	107
46	12.8	10.4	107
47	12.6	12.1	101
48	13.7	11.7	107

Concentrations of amides, rape-oil, N-[3-(dimethylamino)propyl] 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	<0.04	>99.999
45	<0.04	>99.999
46	<0.04	>99.999
47	<0.04	>99.999
48	<0.04	>99.999
Time (days)	Concentration (mg/L)	Removal (%)
	Mixed liquid suspended solids
47	0.49	0.025
48	0.03	0.0015

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 amides, rape-oil, N-[3-(dimethylamino)propyl] biodegrade almost completely in properly operating
conventional biological wastewater treatment plants. Amides, rape-oil, N-[3-(dimethylamino)propyl] are primarily removed by
biodegradation.
105±1% NPOC removal, determined in effluent samples from test unit during 14 days from day 35 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 days 44-48
0.025 % 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. Amides, rape-oil, N-[3 -(dimethylamino)propyl] were exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. Amides, rape-oil, N-[3-(dimethylamino)propyl] 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 amides, rape-oil, N-[3-(dimethylamino)propyl] calculated over 14 measurements obtained from day 35 to 48 of the test was 105 +/-1% (95% confidence interval). These high removal percentages strongly indicate that amides, rape-oil, N-[3- (dimethylamino)propyl] is biodegraded ultimately. Formation of water-soluble compounds during biological treatment of amides, rape-oil, N-[3- (dimethylamino)propyl] can be excluded.

An accurate assessment of the removal of amides, rape-oil, N-[3- (dimethylamino)propyl] was established with specific analyses. The method (LC-MS/ MS) for the determination of amides, rape-oil, N-[3- (dimethylamino)propyl] was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery and specificity. The mean removal percentage of amides, rape-oil, N-[3- (dimethylamino)propyl] in the test unit was assessed with the specific analysis from day 44 to 48 was >99.997% using octadecenamide-N- [3-(dimethylamino)propyl] as representative component. These analyses demonstrate that the removal of amides, rape-oil, N-[3-(dimethylamino)propyl] is almost complete. Amides, rape-oil, N-[3-(dimethylamino)propyl], concentrations in the sludge of the reactor sampled on days 47 and 48 were 341 and 24 µg/L. Mean removal percentages of amides, rape-oil, N-[3-(dimethylamino)propyl] from the influent through adsorption onto sludge assessed in two samples was therefore <0.02% demonstrating that amides, rape-oil, N-[3- (dimethylamino)propyl] is primarily removed by biodegradation. In conclusion, the CAS test demonstrates that amides, rape-oil, N-[3- (dimethylamino)propyl], almost completely removed from the wastewater in conventional biological wastewater treatment plants. Amides, rape-oil, N-[3- (dimethylamino)propyl], is primarily removed by biodegradation.

Additional information

The continuous activated sludge (CAS) test was performed according to ISO Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Amides, C18-unsatd., N-[3-(dimethylamine)propyl] were exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. The substance was 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 Amides, C18-unsatd., N-[3-(dimethylamine)propyl] calculated over 14 measurements obtained from day 35 to 48 of the test was 105 +/-1% (95% confidence interval). These high removal percentages strongly indicate that the substance is biodegraded ultimately. Formation of water-soluble compounds during biological treatment of Amides, C18-unsatd., N-[3-(dimethylamine)propyl] can be excluded.

An accurate assessment of the removal of Amides, C18-unsatd., N-[3-(dimethylamine)propyl] was established with specific analyses. The method (LC-MS/ MS) for the determinati on of substance was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery and specificity. The mean removal percentage of substance in the test unit was assessed with the specific analysis from day 44 to 48 was >99.997% using octadecenamide-N- [3-(dimethylamino)propyl] as representative component. These analyses demonstrate that the removal of Amides, C18-unsatd., N-[3-(dimethylamine)propyl] is almost complete. Concentrations in the sludge of the reactor sampled on days 47 and 48 were 341 and 24 µg/L. Mean removal percentages of test substance from the influent through adsorption onto sludge assessed in two samples was therefore <0.02% demonstrating that the substance is primarily removed by biodegradation. In conclusion, the CAS test demonstrates that the substance is almost completely removed from the wastewater in conventional biological wastewater treatment plants. Amides, C18-unsatd., N-[3-(dimethylamine)propyl] is primarily removed by biodegradation.