Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

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:

09-11-2007 till 25-01-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP, Performed according to OECD 303 guideline, NPOC (non purgeable organic carbon) and specific chemical analyses. The degree of COD elimination in the control unit is >80% after two weeks and there were no unusual observations. The ammonium and nitrite concentrations in the effluent at Day 14 were <2.5 and <2.0 mg/L, respectively. The low ammonium and nitrite concentrations demonstrate almost complete nitrification. These results demonstrate that the test is valid.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

In addition the fraction sorbed is measured.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum and settled sewage
Secondary activated sludge was collected on 09-11-2007 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 (minor deviation from the guidelines).

Duration of test (contact time):

60 d

Initial conc.:

50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

The CAS test was performed according to ISO (2004), EC (1988) and OECD (2001) 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. The domestic sewage supply was pumped into the units were kept at a temperature of approximately 4°C for one day at most. The CAS units were always operated with dry weight concentration between 1 and 3 g/L as recommended in the ISO Guidelines (2004). The effluent samples (50 mL) were taken from the settler. After one week of operation the pair of units were sufficiently steady (stabilized) and subsequently the test substance was introduced to one of the units with a syringe pump (day 0). The test substance was added by spiking the influent with the test substance at a concentration of approximately 50 mg l-1 NPOC. At this time, sludge wastage was also started daily discarding approximately 35 ml of mixed liquor to give a sludge retention time of 10 days. Prior to sludge wastage, the units were brushed daily to prevent microbial growth on the walls of the CAS units.
CAS unit
The CAS test was performed in Husmann-type units constructed of glass. The units consisted of an aeration vessel capable of holding 0.35 litre from which the liquor was passed continuously to a settler of 0.30 litre capacities (minor deviation from the guideline). 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.

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 the CAS units varied from 6.2 to 7.5. The oxygen concentrations measured in both units were > 2 mg/L (Table I). These test conditions are believed to allow biodegradation of hydrogenated tallow-1,3-diaminopropane by micro-organisms present in activated sludge. The CAS test was started with a high concentration of aerobic micro-organisms (2.9 g/L dry weight) maintained by the daily addition of primary settled sewage. 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 3.1 g/L (Table I). The performance of the control unit was checked by measuring the COD removal at Day 14 and at Day 60 (the last day of the test) and the concentrations of ammonium and nitrite in the effluent (Day 14). At Day 14 the COD contents in the influent and effluent were 580 and 53 mg/L, respectively. At Day 60, the COD levels in the influent and effluent were 583 and 47 mg/L, respectively. The COD removal percentages were 91 and 92. The ammonium and nitrite concentrations in the effluent at Day 14 were <2.5 and <2.0 mg/L, respectively.
The low ammonium and nitrite concentrations demonstrate almost complete nitrification.
These results demonstrate that the test is valid.

Compartment:

other: water, material (mass) balance

% Recovery:

0.6

St. dev.:

0

Compartment:

other: sediment, material (mass) balance

% Recovery:

4.1

% Degr.:

99.4

Parameter:

test mat. analysis

Sampling time:

56 d

Remarks on result:

other: Total Parent removal

% Degr.:

95.3

St. dev.:

0.06

Parameter:

test mat. analysis

Sampling time:

56 d

Remarks on result:

other: Removal via biodegradation

% Degr.:

4.1

Parameter:

test mat. analysis

Sampling time:

56 d

Remarks on result:

other: Removal via sorption

Transformation products:

no

Details on transformation products:

Based on the NPOC removal, the specific analyses of the fraction sorbed to sludge and concentration in effluent, 95.3% is degraded to CO2, H2O and NO3.

Evaporation of parent compound:

no

Volatile metabolites:

not measured

Residues:

yes

The method (HPLC-MS/MS, external standards) for the determination of hydrogenated tallow-1,3-diaminopropane was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), precision and specificity. The content and recovery for the samples spiked with hydrogenated tallow-1,3-diaminopropane are given in the table below.

Sample	Measured concentration (mg/L)	Recovery (%)
Effluent of the control of the CAS test of Day 60 spiked with 50 mg/L	52.8	106
Influent of the CAS test of day 59 (domestic wastewater spiked with 50 mg/L)	47.3	94.6
Influent of the CAS test of day 60 (domestic wastewater spiked with 50 mg/L)	46.4	92.7
MLSS of the control of the CAS test of day 60 spiked with 500 mg/L	550	110

The removal percentage of hydrogenated tallow-1,3-diaminopropane was determined with the following equation:

% removal = (Is - Es) / Is × 100

Where:

Is : is the nominal test substance concentration in the influent

Es : is the mean of the measured test substance concentrations in the effluent

The concentration of the test substance in the activated sludge (Csludge) was used to assess the removal of the test substance by adsorption. Provided that the test substance is not biodegraded nor evaporated, the test substance concentration in the activated sludge (Cmax adsorption) would have been:

Cmax adsorption = Is * SRT/HRT

Where:

SRT : is the sludge retention time,

HRT : is the hydraulic retention time (both expressed in days)

Is : is the nominal test substance concentration in the influent.

The % removal of the parent compound by adsorption is:

% removal = Csludge/Cmax adsorption x 100

During the last week of the test, the mean removal percentage in the test unit assessed with the HPLC-MS/MS method was 99.4%. These analyses demonstrate that the primary biodegradation of hydrogenated tallow-1,3-diaminopropane is almost complete. Hydrogenated tallow-1,3-diaminopropane concentrations in the sludge of the reactor sampled on Days 59 and 60 were 93.2 and 67.9 mg/L, respectively. Removal of hydrogenated tallow-1,3-diaminopropane from the influent through adsorption onto sludge as assessed in two samples was therefore only 4.7 and 3.4% demonstrating that hydrogenated tallow-1,3-diaminopropane is primarily removed by biodegradation.

Concentrations of Effluent and Activated Sludge

Effluent taken from test reactor and activated sludge from aeration vessel

	Sample No.	Sample Name	Meas. conc. [mg/L]
Effluent Control	1	T07034 CAS Effluent Control 11/01 (Day 56)	LOQ
	2	T07034 CAS Effluent Control 12/01 (Day 57)	LOQ
	3	T07034 CAS Effluent Control 13/01 (Day 58)	LOQ
	4	T07034 CAS Effluent Control 14/01 (Day 59)	3.97 x 10^-3 1)
	5	T07034 CAS Effluent Control 15/01 (Day 60)	4.89 x 10^-3
Effluent	6	T07034 CAS Effluent Test 11/01 (Day 56)	0.272
	7	T07034 CAS Effluent Test 12/01 (Day 57)	0.312
	8	T07034 CAS Effluent Test 13/01 (Day 58)	0.314
	9	T07034 CAS Effluent Test 14/01 (Day 59)	0.263
	10	T07034 CAS Effluent Test 15/01 (Day 60)	0.317
Sludge	15	T07034 CAS MLSS Test 14/01 (Day 59)	93.2
	16	T07034 CAS MLSS Test 15/01 (Day 60)	67.9

Meas. conc. = Measured concentration, mean value of 2 injections

LOQ = Limit of quantification (0.6 µg/L effluent; 100 µg/L sludge)

1) Original (glass) container was broken. Sample was recollected in a plastic container and transferred to new

glass bottle.

Validity criteria fulfilled:

yes

Conclusions:

The CAS test demonstrates that hydrogenated tallow-1,3-diaminopropane is biodegraded for about 95.3 % and sorbed for about 4.1 % in conventional biological wastewater treatment plant. The total removal will be 99.4%.

Executive summary:

The continuous activated sludge (CAS) simulating biological plants treating domestic wastewater was performed according to ISO Guidelines 11733 (1995) and in compliance with the OECD principles of Good Laboratory Practice. In the continuous activated sludge (CAS) unit fed with domestic wastewater spiked with 50 mg/L hydrogenated tallow-1,3-diaminopropane (CAS No. 68603-64-5) removal percentages of approximately 100% were immediately achieved. These removal percentages were calculated using non purgeable organic carbon (NPOC) analysis. At the end of the test, 99.4% removal was determined using specific chemical analysis (LC-MS/MS). Only 4.1% (3.4 – 4.7) of hydrogenated tallow-1,3-diaminopropane is removed by adsorption onto the sludge. These results demonstrate that hydrogenated tallow-1,3-diaminopropane is removed primarily by biodegradation (mineralization).

Description of key information

A waste water treatment simulation test was performed with Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine (133779-11-0). 99.4% removal was observed using specific chemical analyses (LCMS/MS) of the effluent during day 56 to 60 (n=5). Besides to that the sorption to sludge was measured and this accounted for 4.1% of the total removal which means that 95.3% is removed by biodegradation. Based on the readily biodegradability of all diamines (C10-18) and the fact that the simulation test has been performed with the diamine with the lowest bioavailability, it is considered justified to use the same results for the other more bioavailable diamines as for these a higher total removal and lower removal via sorption are anticipated.

Additional information

The removal of Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine was simulated in a continuous activated sludge (CAS) unit (Akzo Nobel, 2008b + 2008c; Dr U. Noack Laboratoriën, 2008). One unit was fed with domestic wastewater spiked with 50 mg/L of Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine, the control unit was fed with domestic wastewater only. The aeration vessel had a capacity of 0.36 L, the flow rate was 1.1 L/day (i.e. the hydrolic retention time was 6 hours). The sludge concentration ranged from 2.4 to 3.1 g dw/L, The sludge retention time was 10 days. The units were maintained at 20 to 22°C. The test units were preconditioned for one week after which sludge wasting was started. After introduction of the test substance at day 0, removal of Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine was monitored by non purgeable organic carbon (NPOC) analyses. Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine was readily adsorbed by the sludge particles which lead to an immediate removal of approximately 100%.

During the last week of the test, 5 LC/MS measurements of the test substance were conducted and 99.4% removal of Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine was observed. The removal determined in CAS tests is equal to the sum of degradation and adsorption onto sludge.

To assess the contribution of each removal process the removal due to sorption was quantified by taking test samples from the sludge on two days during the last week and subjecting them to chemical analysis. Al specific chemical analyses were performed using methods developed and validated according to SANCO/3029/99 rev.4 (11/07/00). Based on these measurements it is concluded than only 4.1% (3.4 – 4.7) of Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine is removed by adsorption onto the sludge. The results demonstrate that Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine is primarily removed by biodegradation (mineralization). In conclusion, the CAS test demonstrates that Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine will be biodegraded almost completely in a conventional biological wastewater treatment plant.

These results as observed for Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine

can in general be considered to represent the worst-case removal for diamines (C10 -18) in an STP. Shorter alkyl chain diamines will have a similar or higher removal than compared to the C16 -18 -alkyl (evennumbered) because of the higher bioavailability for biodegradation. Shorter alkyl chain diamines will be removed to a lower extend via sorption. It is therefore considered justified to use the results as observed for a

mines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine also for other members of the diamines category.