Amines, di-C16-18-alkyl

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From February 3, 2010 to May 12, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See section 13.2 for the read-across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.10 (Biodegradation: Activated Sludge Simulation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO 11733: Activated sludge simulation test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on source and properties of surface water:

Not applicable

Details on source and properties of sediment:

No data

Details on inoculum:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): Wastewater treatment plant (WWTP) Nieuwgraaf in Duiven, The Netherlands
- Storage conditions: Frozen
- Storage length: 1 Week
- Other:
- 0.35 L of secondary activated sludge containing approximately 3 g/L dry weight was used as an inoculum for each CAS unit

Duration of test (contact time):

ca. 42 d

Initial conc.:

5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

other: Analysing the non-purgeable organic carbon (NPOC) content

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 1 L
- Composition of stock solution: 7.3 g of test substance to 1.0 L of deionized water and mixing for a few hours on a magnetic stirrer. This stock suspension was subsequently diluted 10 times to a concentration of 0.73 g/L. 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 5.0 mg/L
- pH adjusted: yes
- Aeration of dilution water:

TEST SYSTEM
- Culturing apparatus: Hussmann-type units constructed of glass
- Method used to create aerobic conditions: Aeration was achieved through a capillary on the bottom of the aeration section at a rate of 7-8 L/h of air
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Test performed in open system: No

SAMPLING
- Sampling frequency: Day 1, 5, 8, 12, 15, 19, 20, 22, 26, 27, 29, 33, 34, 36, 40, 41, 42

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 0.8 um 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 (Shimadzus 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.

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 HCl to approximately pH 2. The samples were stored in the refrigerator until analysis.

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 quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides 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 quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides were used to determine the primary removal of the test substance. The removal percentage of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides 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 quaternary ammonium compounds, di- C12-18-alkyldimethyl, chlorides 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 quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides by adsorption is
100 x Csludge/Cmax adsorption.

STATISTICAL METHODS:
t-test for the statistical significance of the observed difference

Deviation and amendments
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 are three amendments to the study plan; 1) An additional test unit fed with 5.0 mg/L of test substance was included after three weeks because the unit fed with 50 mg/L was still turbid after this period. Turbidity of the effluent indicates that the test substance is toxic to microorganisms at the load used. 2) The test unit fed with 50 mg/L of test substance was still turbid after 6 weeks of operation. An influent concentration of 50 mg/L was therefore considered not representative although the removal of organic carbon from the waste water was still high. Operation of the unit fed with 50 mg/L was therefore stopped. 3) A method to determine the concentrations of the test substance, in aqueous and sludge samples was issued. 4) The information on the test substance has been changed.

Reference substance:

other: Ammonium chloride,

Reference substance:

other: Potassium hydrogen phthalate

Reference substance:

other: Sodium nitrite

Test performance:

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

Key result

% Degr.:

> 99.9

Parameter:

other: based on LC-MS/MS analysis

Remarks:

using C12 DAQ as representative

Sampling time:

42 d

Remarks on result:

other: (Total mean removal)

Key result

% Degr.:

4

Parameter:

other: based on LC-MS/MS analysis

Remarks:

using C12 DAQ as representative

Sampling time:

42 d

Remarks on result:

other: (via sorption)

Key result

% Degr.:

96

Parameter:

other: based on LC-MS/MS analysis

Remarks:

using C12 DAQ as representative

Sampling time:

42 d

Remarks on result:

other: (degraded)

Transformation products:

not measured

Details on transformation products:

Not applicable

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

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 2.7 to 3.0 g/L. The performance of the control unit was checked by measuring the COD removal at Day 14 and at day 42 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 516 and 35 mg/L, respectively. At day 42, the COD levels in the influent and effluent were 472 and 34 mg/L, respectively. COD removal percentages at both days were 93. The ammonium-N and nitrite-N concentrations in the effluent at Day 14 were 0.7 and <0.6 mg/L. These results demonstrate that the test is valid.

Biodegradation in the CAS test
At day 0 the control unit had been in operation for 28 days. The test unit was preconditioned for 3 days before day 0. At day 0 administration of the test substance at a concentration of 5.0 mg/L was started (first and second amendment to the study plan). The test substance was introduced with a syringe pump. The sludge wasting resulted in a sludge retention time of 10 days. The calculated carbon content of test substance in the influent of the reactor was 3.8 mg/L. The measured concentration of the non purgeable organic carbon (NPOC) in an aqueous solution with 5.0 mg/L of test substance was 3.4 mg/L. After filtration through a 0.45 µm filter the NPOC was 3.3 mg/L. 3.8 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 i.e. 74 were immediately accomplished. These high removal percentages have to be ascribed to the adsorption and/or biodegradation of test substance. The biodegradation percentages were high during the entire test period except for days 15 and 42. From Day 25 to 42, 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 25 to 42 were used in a t-statistic. The mean difference between the NPOC in the effluents of control and test unit was -1.7 ± 0.6 mg/L (95 per cent confidence interval). The mean removal percentage calculated with this mean difference was 145 ± 11 (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 test substance removes >100% of the organic carbon of the test substance. These results strongly indicate that test substance are degraded by micro-organisms. The removal percentages demonstrate that formation of recalcitrant water-soluble substances is unlikely during the biodegradation process. The biodegradation percentage in excess of 100% shows that the removal of organic compounds present in the domestic wastewater are removed better when quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides are present in the influent. Removal percentages calculated with NPOC values are not very accurate. An accurate assessment of the removal of quaternary ammonium compounds, di-C12-18- alkyldimethyl, chlorides was therefore established with specific analyses.

The concentrations of quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides in the effluent of the test unit using didodecyldimethylammonium as representative component were 3 µg/L (day 38), 4 µg/L (day 39), 3 µg/L (day 40), 4 µg/L (day 41) and 5 µg/L (day 42) µg/L. These concentrations correspond with 99.9 % removal. The mean quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides concentrations in the sludge of the reactor monitored on days 41 and 42 were 6.0 and 9.7 mg/L, respectively. This was also determined with didodecyldimethylammonium as representative component. Using 7.9 mg/L (mean of day 41 and 42), 4.0% removal through adsorption onto sludge of the quaternary ammonium compounds, di-C12-18- alkyldimethyl, chlorides present in the influent of the test unit was calculated. These results demonstrate that quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides biodegrades 96% in properly operating conventional biological wastewater treatment plants.

Results with reference substance:

- At Day 14 the COD contents (means of two measurements) in the influent and effluent were 516 and 35 mg/L, respectively. At day 42, the COD levels in the influent and effluent were 472 and 34 mg/L, respectively.
- COD removal percentages at both days were 93. The ammonium-N and nitrite-N concentrations in the effluent at Day 14 were 0.7 and <0.6 mg/L.

Results

Table 1. 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
1	3.7	3.7			7.2	7.2
5	4.1	3.8			7.3	7.2
6			2.9	3.0
8	4.2	4.1			7.2	7.0
12	3.7	3.9			7.0	7.0
13			2.8	2.9
15	4.2	4.7			7.0	7.2
19	4.2	4.9			7.1	7.1
20			2.7	3.0
22	4.5	4.5			7.4	7.1
26	5.1	4.1			7.3	7.2
27			2.8	2.9
29	4.8	4.9			7.2	7.0
33	4.3	4.1			7.1	7.1
34			2.9	2.9
36	4.8	4.8			7.2	7.3
40	3.9	5.0			7.0	7.1
41			2.8	2.9
42	4.5	5.0			7.2	7.0

Table 2. NPOC concentrations in the effluent of the control and test unit and the calculated removal percentages of test substance

Time (days)	NPOC (mg/L)		Removal (%)
	Control	Test
1	11.8	12.8	74
5	11.5	9.9	142
8	12.6	9.3	187
12	11.7	9.6	155
15	12	14.5	35
19	9.8	9.3	118
21	12.1	13	76
22	13.1	11	155
25	13.1	11.7	136
26	10.6	9.3	134
27	11.5	9.7	147
28	10.8	10.4	111
29	12.5	10.3	156
32	11.4	8.8	168
33	11.3	9.3	153
34	12.7	11.2	139
35	11.9	10.5	137
36	13.4	9.3	208
38	9.1	8.6	113
39	12.5	10.1	163
40	12.3	10.3	153
41	12.1	11.3	121
42	13.2	15	53

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the test substance was almost completely removed from wastewater in conventional biological wastewater treatment plants primarily by biodegradation.

Executive summary:

A study was conducted to determine the biodegradation of the test substance, C12-18 DAQ (99.6% active) using a continuous activated sludge (CAS) test, according to OECD guideline 303 A, EU method C10 and ISO guideline 11733, in compliance with GLP. The test substance spiked at a nominal influent concentration of 5.0 mg/L (3.8 mg/L carbon; calculated) was exposed to micro-organisms maintained by addition of domestic waste water for a period of 42 d and included a control fed with domestic wastewater only. Based on non-purgeable organic carbon (NPOC)), the mean organic carbon removal between Day 25 and 42 (14 measurements) was 145 ± 11% (95% confidence interval). The biodegradation percentage in excess of 100% shows that the removal of organic compounds present in the domestic wastewater are removed better when the test substance is present in the influent. However, as removal percentages calculated with NPOC values are not very accurate, an accurate assessment of the removal of the test substance was therefore established with specific analyses using LC-MS/MS. The LC-MS/MS method for the determination of test substance was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), precision and specificity. Based on specific analysis of the test compound, the mean removal percentage from Day 38 to 42 was 99.9%. Test substance in the mixed liquid suspended solids of the reactor sampled on Day 41 and 42 were 6.0 and 9.7 mg/L, respectively. The mean removal of test substance from the influent through adsorption onto sludge assessed with two samples was therefore 4.0% demonstrating that, it is primarily removed by biodegradation. In the study, the test substance biodegraded 96% in properly operating conventional biological wastewater treatment plants. Under the study conditions, the test substance was almost completely removed from wastewater in conventional biological wastewater treatment plants primarily by biodegradation (Van Ginkel, 2010).

Reference 2

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

28-10-2009 - 05-02-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See section 13.2 for the read-across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

minor acceptable 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 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):

48 d

Initial conc.:

50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

DOC removal

test mat. analysis

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 oleyl bis(2-hydroxyethyl)amine 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 5.5 by adding HCl. A suspension was obtained by stirring for a few hours on a magnetic stirrer. 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 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 oleyl bis(2-hydroxyethyl)amine 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 oleyl bis(2-hydroxyethyl)amine were used to determine the primary removal of the test substance. The removal percentage of
oleyl bis(2-hydroxyethyl)amine 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 in the mixed liquid suspended solids (adsorbed 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 oleyl bis(2-hydroxyethyl)amine 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 oleyl bis(2-hydroxyethyl)amine by adsorption is;
removal (%) = 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 7.4.
The oxygen concentrations measured in both units were always ≥3.9 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. 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 (Table I).
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 (mean 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.:

ca. 100

St. dev.:

0.8

Parameter:

DOC removal

Remarks on result:

other: 15 measurements from day 34 - 48

% Degr.:

> 99.99

Parameter:

test mat. analysis

Remarks on result:

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

% Degr.:

0.16

Parameter:

test mat. analysis

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 high NPOC removal percentages strongly indicate that oleyl bis(2-hydroxyethyl)amine is biodegraded completely. Formation of water soluble
compounds during biological treatment of oleyl bis(2-hydroxyethyl)amine can be excluded.

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 oleyl bis(2-hydroxyethyl)amine. 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	11.4
-2	12.1	10.9
2	9.5	11.4	95
0	10.8	18.2	80
6	11.0	11.3	99
9	11.7	10.7	103
13	12.7	10.2	107
16	13.9	16.2	94
20	12.7	12.1	102
23	8.3	8.8	99
27	9.7	11.8	94
30	7.5	9.4	95
34	9.1	8.2	102
35	12.0	9.5	107
36	10.3	8.0	106
37	12.7	9.3	109
38	10.2	8.8	104
39	10.9	8.2	107
40	11.0	11.3	99
41	10.5	10.2	101
42	11.4	10.5	102
43	13.0	14.9	95
44	12.1	12.2	100
45	13.9	13.7	101
46	12.8	12.3	101
47	12.6	12.5	100
48	13.7	13.9	100

Concentrations ofoleyl bis(2-hydroxyethyl)aminemeasured 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	<0.06	>99.999
45	<0.06	>99.999
46	<0.06	>99.999
47	<0.06	>99.999
48	<0.06	>99.999
Time (days)	Concentration (mg/L)	Removal (%)
	Mixed liquid suspended solids
47	3.2	0.16
48	3.2	0.16

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 oleyl bis(2-hydroxyethyl)amine almost completely removed from the wastewater in conventional biological
wastewater treatment plants. Oleyl bis(2-hydroxyethyl)amine is primarily removed by biodegradation.
102±0.8% NPOC removal, determined in effluent samles from test unit during 15 days from day 34 to 48
>99.999% removal of test substance calculated with specific analysis in effluent samples taken from day 44-48
0.16 % removal of test substance through 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.Oleyl bis(2-hydroxyethyl)amine was exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test.

Oleyl bis(2-hydroxyethyl)amine was spiked at a nominal influent concentration of 50 mg/L (37.0 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 oleyl bis(2-hydroxyethyl)amine calculated over 15 measurements obtained from day 34 to 48 of the test was 102±0.8% (95% confidence interval). These high removal percentages strongly indicate that oleyl bis(2-hydroxyethyl)amine is biodegraded completely. Formation of water soluble compounds during biological treatment ofoleyl bis(2-hydroxyethyl)amine can be excluded.

An accurate assessment of the removal of oleyl bis(2-hydroxyethyl)amine was established with specific analyses. The method (LC-MS/MS) for the determination of oleyl bis(2-hydroxyethyl)amine was valid with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), limit of detection (LOD), recovery and system stability. The mean removal percentage of oleyl bis(2-hydroxyethyl)amine in the test unit was quantified with the specific analysis from day 44 to 48 was >99.999% using octadecenyl bis(2-hydroxyethyl) amine as representative component as a worst-case. These analyses demonstrate that the removal of oleyl bis(2-hydroxyethyl)amine is complete. Oleyl bis(2-hydroxyethyl)amine concentrations in the mixed liquid suspended solids (activated sludge) of the reactor sampled on days 47 and 48 were 3.2 mg/L. Mean removal percentages of oleyl bis(2-hydroxyethyl)amine reaction from the influent through adsorption onto sludge assessed in two samples was therefore 0.16 % demonstrating that oleyl bis(2-hydroxyethyl)amine is primarily removed by biodegradation.

In conclusion, the CAS test demonstrates that oleyl bis(2-hydroxyethyl)amine almost completely removed from the wastewater in conventional biological wastewater treatment plants.Oleyl bis(2-hydroxyethyl)amineis primarily removed by biodegradation.

Description of key information

The removal of dialkylamine sin biological wastewater systems through read-across is assumed >99.8% removal from the wastewater for dialkylamines. The removal by biodegradation in biological treatment systems will probably range from 96% (low bioavailability) to 99.999% (high bioavailability).

Key value for chemical safety assessment

Half-life in freshwater:

15 d

at the temperature of:

12 °C

Half-life in marine water:

50 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

30 000 d

at the temperature of:

12 °C

Half-life in marine water sediment:

30 000 d

at the temperature of:

12 °C

Additional information

The Kpsed was determined as 14000 L/kg. The suggested maximum half-life for a readily degradable substance with a Kpsed in the range >1000 and < 10000 L/kg is 3000 days for (at 12 ºC). These values are considered as extremely conservative but in the absence of measured data will be used in the exposure assessment as a worst-case. The half-life of the bioavailable fraction of dialkylamines in the water phase of sediments is expected to be in the order of a few days, which is based on experiments with dialkyldimethylammonium salts (van Ginkel et al, 2003).

Biological wastewater treatment.

 

Dialkylamines have not been tested in the continuously-fed activated sludge (CAS) test. However, the fate of a number of chemically related substance i.e. oleyl bis (2-hydroxyethyl)amine (high bioavailability) and dicocodimethylammonium chloride (low bioavailability) have been assessed in CAS tests (Akzo Nobel 2010^a, Akzo Nobel 2010^b). Comparable biodegradation pathways of fatty amine derivatives i.e. oxidation of the alkyl chain (primary degradation) and the bioavailability are the keys to justification of the use of read-across of the results obtained CAS units fed with domestic wastewater spiked with other fatty amine derivatives. An overview of the removal percentages of the parent compounds from the influent and removal by biodegradation in the CAS units are given below.

Surfactant	Removal from wastewater (%)	Removal by biodegradation (%)
Dicocodimethylammonium chloride (CAS 68424085-1)	99.9	96
Oleyl bis(2-hydroxyethyl)amine (CAS25307-17-9)	99.999	99.84

The removal of dialkylamines in biological wastewater systems through read-across is assumed >99.8% removal from the wastewater for dialkylamines. The removal by biodegradation in biological treatment systems will probably range from 96% (low bioavailability) to 99,999% (high bioavailability).