Registration Dossier

Environmental fate & pathways

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:
2009-02-12 - 2009-08-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP according guidelines with acceptable deviations. Meeting validity and quality criteria.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Deviations:
yes
Remarks:
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
A sample of Oleyl tripropylene tetramine (C18 tripropylene tetramine) was received from AkzoNobel Surface Chemistry on 2009-01-30. The following test substance data were submitted by the sponsor, who accepted full responsibility for the validity hereof.
Chemical name: Oleyl tripropylene tetramine (C18 tripropylene tetramine)
CAS number: 67228-83-5
Batch number: S001029
Composition certificate of analysis (see attached report)
11% diamine
35.6% triamine
44.6% tetraamine
5.8% pentaamine
from which 91% contains a C18 alkyl chain
and 35% of the alkyl chains is unsaturated.
Chemical stability: Stable but not relevant for the test
Vapor pressure: compounds are unlikely to evaporate
Water solubility: Not soluble at test concentration
Storage: At room temperature in the dark under nitrogen
Stability under storage conditions: stable
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 12-02-2009 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):
91 d
Initial test substance concentration
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The CAS test was performed in Husmann-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.
A suspension of Oleyl tripropylene tetramine (C18 tripropylene tetramine) 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 first acidifying the stock to a pH of 4 by adding 2 M 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 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. The CAS test was performed according to the study plan. The study plan was developed from 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 tripropylene tetramine (C18 tripropylene tetramine) ontaining wastewater and to preliminary follow the removal of the test substance during the test period. Specific analyses of Oleyl tripropylene tetramine (C18 tripropylene tetramine) were used to determine the primary removal of the test substance.

Results and discussion

Test performance:
The incubation temperature of both CAS units ranged from 19 to 21°C. The pH of the effluent of the CAS units varied from 6.0 to 7.4. The oxygen concentrations measured in both units were ≥ 3.5 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. 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 3.5 g/L . The performance of the control unit was checked by measuring the COD removal at Day 14
and at day 63 (day before prolongation 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 532 and 70 mg/L, respectively. At day 63 the COD levels in the influent and effluent were 582 and
75 mg/L, respectively. COD removal percentages at both days were 87. 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.
% Degradationopen allclose all
% Degr.:
88
St. dev.:
5
Parameter:
DOC removal
Sampling time:
44 d
Remarks on result:
other: 15 measurments obtained fromd day 44 to 60
% Degr.:
99.9
Parameter:
test mat. analysis
Sampling time:
56 d
Remarks on result:
other: using octadecyl dipropylenetriamine
% Degr.:
99.8
Parameter:
test mat. analysis
Sampling time:
56 d
Remarks on result:
other: using octadecyl tripropylenetetramine
Transformation products:
no
Details on transformation products:
Complete mineralisation based on the organic carbon removal
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
no
Details on results:
The continuous activated sludge system treating domestic wastewater spiked with Oleyl tripropylene tetramine (C18 tripropylene tetramine) removes the organic carbon of the test substance for more than 80% from wastewater. These results indicate Oleyl tripropylene tetramine (C18 tripropylene tetramine) is degraded by micro-organisms. Finally, the high removal percentages show that formation of recalcitrant water-soluble substances is unlikely during the biodegradation process.
The concentrations of the test substance was measured using LC/MS/MS. The analysis was focussed on the C18 alkyl of the tri and tetra amine because these are the main constituents of the test substance. The mean concentration of Oleyl tripropylene tetramine (C18 tripropylene tetramine) in the effluent of the test unit at day 56, 57, 58, 59 and 60 using the Octadecyl dipropylene triamine as representative component was 42 μg/L. The mean concentration of Oleyl tripropylene tetramine (C18 tripropylene tetramine) in the effluent of the test unit at day 56, 57, 58, 59 and 60 using Octadecyl tripropyletetramine as representative component was 103 μg/L.
These concentrations correspond with 99.9% and 99.8% removal . The mean Oleyl dipropylene triamine (C18 dipropylene triamine) concentrations in the sludge of the reactor monitored on days 59 and 60 were 115 mg/L determined with Octadecyl dipropylene triamine and 286 mg/L determined with Octadecyl tripropylene tetramine. Using Octadecyl dipropylene triamine 5.8% of the Oleyl tripropylene tetramine (C18 tripropylene tetramine) present in the influent of the test unit was removed by adsorption onto the sludge whereas 14.3% of the Oleyl tripropylene tetramine (C18 tripropylene tetramine) was adsorbed when Octadecyl tripropylene tetramine was used to measure the concentration. These results demonstrate that Oleyl tripropylene tetramine biodegrades almost completely in properly operating conventional biological wastewater treatment plants. The extent of adsorption of chemicals with dipropylene triamine as hydrophilic moiety is small compared to the compounds with four nitrogens.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see test performance
Conclusions:
Study performed under GLP according guidelines with acceptable deviations. Meeting validity and quality criteria.
In conclusion, the CAS test demonstrates that Oleyl tripropylene tetramine (C18 tripropylene tetramine), almost completely removed from the wastewater in conventional biological wastewater treatment plants. The substance is primarily removed by biodegradation.
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 tripropylene tetramine (C18tripropylene tetramine) was exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. Oleyl tripropylene tetramine (C18tripropylene tetramine) was spiked at a nominal influent concentration of 50 mg/L (38.4 mg/L carbon; measured) for a period of approximately 63 days and included a control fed with domestic wastewater only. The immediate high removal percentages can be attributed to adsorption and maybe some biodegradation. The mean removal percentage Oleyl tripropylene tetramine (C18tripropylene tetramine)-carbon calculated over 15 measurements obtained from day 44 to 60 of the test was 88 ± 5% (95% confidence interval). The high removal percentage over a period of more than 8 weeks indicates that Oleyl tripropylene tetramine (C18tripropylene tetramine) is biodegraded ultimately. Formation of water soluble compounds during biological treatment of Oleyl tripropylene tetramine (C18tripropylene tetramine) can be excluded. The effluent of the test unit was turbid during the 63 day test period. Turbidity of effluent points to toxicity of Oleyl tripropylene tetramine (C18tripropylene tetramine) to activated sludge. Turbidity is caused by lysis products of microorganisms. Inhibitory effects were not noted on the basis of the removal of organic carbon present in the domestic wastewater. To investigate the possible toxicity of Oleyl tripropylene tetramine (C18tripropylene tetramine), the test was prolonged for another 4 weeks. During these weeks Oleyl tripropylene tetramine (C18tripropylene tetramine) was spiked at a concentration of 12.5 mg/L. The control unit was unfortunately not operating properly during these 4 weeks. The results obtained of this period could therefore not be used. An accurate assessment of the removal of Oleyl tripropylene tetramine (C18tripropylene tetramine) was established with specific analyses. The method (HPLC-MS) for the determination of Oleyl tripropylene tetramine (C18tripropylene tetramine) was satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), precision and specificity. The mean removal percentage of Oleyl tripropylene tetramine (C18tripropylene tetramine) in the test unit was assessed with the HPLC-MS method from day 56 to day 63. The mean removal was 99.9% and 99.8% using Octadecyl dipropylene triamine and Otadecyl tripropylene tetramine as representative components, respectively. These analyses demonstrate that the removal of Oleyl tripropylene tetramine (C18tripropylene tetramine) is almost complete. Mean Oleyl tripropylene tetramine (C18tripropylene tetramine) concentrations in the sludge of the reactor sampled on days 59 and 60 were 115 mg/L (Octadecyl dipropylene triamine) and 286 mg/L (Octadecyl tripropylene tetramine). Mean removal percentages of Oleyl tripropylene tetramine (C18tripropylene tetramine) from the influent through adsorption onto sludge assessed in two samples was therefore 5.8 (Octadecyl dipropylene triamine) and 14.3 (Octadecyl tripropylene tetramine) demonstrating that Oleyl tripropylene tetramine (C18tripropylene tetramine) is primarily removed by biodegradation. In conclusion, the CAS test demonstrates that Oleyl tripropylene tetramine (C18tripropylene tetramine) almost completely removed from the wastewater in conventional biological wastewater treatment plants. The substance is primarily removed by biodegradation.