2,2'-(octadec-9-enylimino)bisethanol

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:

28-10-2009 - 05-02-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH
Category document is included in Chapter 13

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
[Describe why the read-across can be performed]

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
[Summarise here based on available experimental data how these results verify that the read-across is justified]

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

Based on the available studies on biodegradation, the substances are classified as readily biodegradable. The 10-days-window criterion should not be evaluated for a UVCB thus there is no requirement for performing a surface water or sediment biodegradation simulation test (Annex IX. 9.2.1.2 and 4).

Therefore no simulation tests for degradation rates under environmental conditions are available for primary fatty amine ethoxylates. For a very similar substance (hexadecyl amine) however the half-life in soil has been quantified and these results will be used for read-across to primary fatty amine ethoxylates (2EO). For soil and aerobic sediment this is a half-life value of 17 days at 12 °C.

In addition a waste water treatment simulation test was performed with 2,2’(octadec-9-enylimino)-bisethanol (25307-17-9). More than 99.999% removal was observed using specific chemical analyses (LCMS/MS) of the effluent during day 44 to 48 (n=5). Also  the sorption to sludge was measured and this accounted for 0.16 % of the total removal which means that 99.83% is removed by biodegradation.

Key value for chemical safety assessment

Half-life in freshwater:

15 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

17 d

at the temperature of:

12 °C

Additional information

Biodegradation in a sewage treatment simulation test:

More than 99.999% removal is observed for 2,2’(octadec-9-enylimino)-bisethanol (25307-17-9) in waste water treatment simulation test. From this removal 99.83% is removed via biodegradation and 0.16 % via sorption. The test result can be used for the other primary fatty amine ethoxylates as a worst-case because all primary fatty amine ethoxylates

The removal ofoctadecenyl bis(2-hydroxyethyl)amine in biological treatment plants was simulated in a continuously-fed activated sludge (CAS) unit fed with domestic wastewater spiked with octadecenyl bis(2-hydroxyethyl)amine (AkzoNobel, 2010). Octadecenyl bis(2-hydroxyethyl)amine was exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. Octadecenyl 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 octadecenyl 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 octadecenyl bis(2-hydroxyethyl)amine is biodegraded completely. Formation of water soluble compounds during biological treatment of octadecenyl bis(2-hydroxyethyl)amine can be excluded. An accurate assessment of the removal of octadecenyl bis(2-hydroxyethyl)amine was established with specific analyses. The mean removal percentage of octadecenyl bis(2-hydroxyethyl)amine in the test unit was quantified from day 44 to 48 and was more than 99.999% usingoctadecenyl bis(2-hydroxyethyl) amineas representative component as a worst-case. These analyses demonstrate that the removal of octadecenyl bis(2-hydroxyethyl)amine is complete. Octadecenyl 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 octadecenyl bis(2-hydroxyethyl)amine from the influent through adsorption onto sludge assessed in two samples was therefore 0.16% demonstrating that octadecenyl bis(2-hydroxyethyl)amine is primarily removed by biodegradation.

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

 

Biodegradation in sediment:

Default half-life’s

For the derivation of the default half-life’s the bioavailability is taken into account via the sorption properties of the substance. This is realistic for soluble non-toxic substances. For poorly soluble/strongly sorbing substances however, the degradation rate in a standard ready test is limited by the dissolution rate and in many cases not 60% degradation is achieved within 28 days but in a slightly longer time frame. Such a substance is in fact completely degraded to CO₂and H₂O and thus completely biodegradable. The bioavailable fraction is readily biodegradable but the due to the stringency of the test setup the substance this cannot be observed.

The Kpsoil was determined as 4526 L/kg. The suggested maximum half-life for a readily degradable substance with a Kpsoil in the range >1000 and < 10000 L/kg is 3000 days for soil (at 12 ºC). These values are considered as extremely conservative but in the absence of measured data can be used in the exposure assessment as a worst-case. The half-life of the bioavailable fraction of primary fatty amine ethoxylates in the water phase of soils is expected to be in the order of a few days, which is based on experiments with dialkyldimethylammonium salts (van Ginkel et al, 2003).

Read across from primary alkyl amines

For hexadecylamine[1-14C] a substance which isvery similar to 2,2'-(C12-18 evennumbered alkyl imino) diethanol (CAS no 71786-60-2) there is an OECD 307 aerobic soil transformation study available and the results of this study will be used for read across to the primary alkyl amine ethoxylates for both the soil and sediment compartment. Although this C16 amine is strongly sorbing to soil (median Kp soil of 3875 L/kg at lowest measured concentration) the following half-life’s at 20 °C were determined for three soils: Soil 1 t1/2 = 9.0 d; Soil 2 t1/2 = 8.1 d; Soil 3 t1/2 = 8.9 d.

The median Half-life of 8.9 d at 20 °C corresponds to a median Half-life of 16.9 d at an environmental temperature of 12 °C (see REACH Guidance). This study demonstrates that 1-Hexadecanamine (C16 amine) is rapidly degraded in various soils and the results indicate that the assumption of low degradation rates for strongly sorbing substances could be unjustified.