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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-12-2009 - 30-12-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
acceptable deviations
Principles of method if other than guideline:
Minor deviations from the guidelines of the Closed Bottle test were introduced; a) ammonium chloride was omitted from the medium to prevent
oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference
compound), and b) activated sludge instead of an effluent/extract/mixture was used as inoculum.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (25-11-2009) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stock solutions
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Sodium acetate and the test substance were added to the bottles using a stock solutions and a stock suspension of 1.0 g/L. The stock suspension of N-(tallow-alkyl)-1,3-propanediamine oleates was prepared through acidification to pH 4 with 2 M HCl.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and test substance, and 6 bottles containing sodium acetate and inoculum. Theconcentrations of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was
dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were
immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark.
Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.


Calculation of the results:

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottle inoculated with river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of N-(tallow-alkyl)-1,3-propanediamine oleates, and sodium acetate were calculated from their molecular formulae and
molecular weights. The calculated theoretical oxygen demand (ThOD) of N-(tallow-alkyl)-1,3-propanediamine oleates is 2.9 mg/mg. The ThOD of
sodium acetate is 0.8 mg/mg


Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and
reference compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc is the mean oxygen level in the control bottles n days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days
after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by
the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Details on results:
Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of N-(tallow-alkyl)-1,3-propanediamine oleates to microorganisms degrading acetate is not relevant. Inhibition of the
endogenous respiration of the inoculum by the test substance was not detected. Therefore, no inhibition of the biodegradation due to the "high"
initial concentration of the test compound is expected.

Test conditions
The pH of the media was 7.0 at the start of the test. The pH of the medium at day 28 was 6.9 (control) and 7.0 (test). Temperatures were within the
prescribed temperature range of 22 to 24°C.
Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of 1.4 mg/L at day 28, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, at day 14 was 83; oxygen concentrations >0.5 mg/L in all bottles during the test
Interpretation of results:
readily biodegradable
Conclusions:
Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity criteria.
N-(tallow-alkyl)-1,3-propanediamine oleates only fulfilled one criterion for ready biodegradable compounds i.e. a biodegradation percentage in excess of 60 within 28 days. The pass level of 60% was not reached within 10 days upon achieving 10% biodegradation. The reasons for failing the 10-day window are the composition of the test substance i.e. a salt of N-(tallow-alkyl)-1,3-propanediamine and oleic acid and N-(tallow-alkyl)-1,3-propanediamine being a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of this fatty amine derivative are therefore degraded sequentially.
The time window criterion was developed on the assumption that a compound is degraded according to the “standard” growth curve in ready biodegradability tests. The degradation curve of N-(tallow-alkyl)-1,3-propanediamine oleates is the sum of growth curves of oleate and the two moieties of N-(tallow-alkyl)-1,3-propanediamine. The biodegradability of oleate and the two moieties in the Closed Bottle test may be fully in line with the time-day window criterion when judged as separate chemicals. The time-window should therefore be ignored as a pass fail criterion for N-(tallow-alkyl)-1,3-propanediamine oleates. N-(tallow-alkyl)-1,3-propanediamine oleates is therefore classified as readily biodegradable only based on the biodegradation percentage of 65% at day 28.
Executive summary:

In order to assess the biotic degradation, a ready bio­degradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

N-(tallow-alkyl)-1,3-propanediamine oleates did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. N-(tallow-alkyl)-1,3-propanediamine oleates was biodegraded 65% at day 28 in the Closed Bottle test. Hence this substance should be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 83% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 March 2003 - 21 June 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP, quality criteria are met, lack of details on test material
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
(item is adsorbed on silicagel)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge from the biological wastewater treatment plant in Abidos.
- Storage length: 1d
- Preparation of inoculum for exposure:
- Pretreatment: effluent is centifuged at approximately 20° C for 20 min. at 4000g. The base is washed in a volume of water to obtain a concentration factor of approximately 1 with respect to the sample. This inoculum is preconditioned (aeration) until flasks are seeded.
- Concentration of obtained sludge: 3.37 g/L dry weight
- Concentration of sludge in the test vessels: 30 mg dry material /l
Duration of test (contact time):
28 d
Initial conc.:
41.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The study was performed according to OECD guideline 301F .

A measured volume (250 mL) of inoculated mineral medium, containing 41.2 mg/L of INIPOL002 with a theoretical oxygen demand of 2.97 mg O2/L as the sole source of organic carbon, is stirred in a closed flask at a constant temperature of 21 ° C for up to 28 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas pressure in the respirometer flask. Degradation is followed by measuring the quantity of oxygen generated automatically by the automatic respirometer, the SAPROMAT. The CO2 generated by the ultimate biodegradation process is fixed by the soda lime in the cupel of the reactor. This results in a negative pressure which is detected by the manometer and communicated to the control unit. In return, the latter sends an electrical current into the oxygen generator which electrochemically generates a quantity of oxygen which is proportional to the strenght of the electrical current. The quantity of oxygen consumed by the test item (corrected for the quantity produced in the blank test with the inoculum) is expressed as a percentage of ThOD or COD.

The activity of the inoculum is controled by means of a reference item. Simultaneously, the posisble inhibiting effects of the test item are studied with respect to the inoculum, with a mixture of test and reference items, resulting in the following series:

Ft1, Ft2: + test item, in duplicate
Fb1, Fb2: blank, in duplicate
Fc1, Fc2: inoculum control, in duplicate
Fa1, Fa2: silica gel control, in duplicate
Fi: Inhibition
Reference substance:
benzoic acid, sodium salt
Preliminary study:
not applicable
Test performance:
- Difference of extremes of replicate values of the removal of test chemical at the plateau, at the end of the test, or at the end of the 10-d window, was less than 20%.
- Percentage biodegradation of the reference item reached a level of 83% by 14 days which is above the level of 60% for ready biodegradability.
- Oxygen uptake of the inoculum blank was about 12 mg O2/L in 28 days, which is below 60 mg O2/ L
- In the toxicity controls containing both the reference item and N-(oleyl)-1,3-propanediamine oleates, the percentage biodegradation reached 68% after 28 days. Since this value is higher than 25%, the test item is not inhibitory for inoculum.
Parameter:
% degradation (O2 consumption)
Value:
61
Sampling time:
28 d
Details on results:
The maximum degradation level of INIPOL 002 is 61% of the ThOD in 28 days. The latency phase for the degradation of the item (i.e. 10% mineralisation reached) is approximately 2 days, and the percentage of degradation after the time interval of 10 days after this latency phase is 53%.
Results with reference substance:
The percentage mineralisation of the reference item reached a level of 83% by 14 days.

Results and calculations, together with degradation curve are included in attached document: Results OECD301F 40027 -38 -1

Validity criteria fulfilled:
yes
Remarks:
study quality criteria are met
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
N-(oleyl)-1,3-propanediamine oleates is readily biodegradable (but failing 10 day window criterion) in an aerobic ready biodegradation test according to OECD TG 301F. The test item was mineralised by 61% in 28 days.
Executive summary:

The biodegradation of the substance (CAS No. 40027 -38 -1) was determined in an aerobic ready biodegradation test according to OECD TG 301F (manometric respirometry test) under GLP conditions (reliability 2). The test material (41.2 mg/L at start) was exposed to sewage sludge micro-organisms with culture medium in a closed flask at at a constant temperature of 21 ° C for 28 days.

The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas pressure in the respirometer flask by using an automatic respirometer, the SAPROMAT. The CO2 generated by the ultimate biodegradation process is fixed by the soda lime in the cupel of the reactor. The quantity of oxygen consumed by the test item (corrected for the quantity produced in the blank test with the inoculum) is expressed as a percentage of ThOD. Under the test conditions, mineralisation of the test substance reached 51% within 11 days and 61% at the end of the test (28 days). The level of mineralisation reached 53% ThOD at the end of the 10-d window (day 12). The test substance did not inhibit the micro-organisms as shown by the toxicity control flask. It can be concluded that the test substance is readily biodegradable (failing the 10 -d window).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Performed under GLP and according to OECD guideline. Guidline criteria are met. Certificate of Analysis not included.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
See principle of method if other than guideline
Principles of method if other than guideline:
Instead of an effluent / extract / mixture, activated sludge was used as an inoculm. The inoculm was taken fram an activated sludge plant.
The sludge was pre conditioned to reduce the endogenous respiration rates. To precondition the sludge (200 mg Dry weight ( DW) / l i t r e )
was aerated for a period of seven days. The sludge was diluted to a concentxrion in the B0D bottles of 2 mgram/litre. Ammonium chloride was o
ommited from test media to prevent nitrification.
GLP compliance:
yes
Oxygen conditions:
aerobic
Remarks:
Oxygen use determined electrochemically with oxygen electrode.
Inoculum or test system:
other: Secondary activated sludge.
Details on inoculum:
activated sludge was obtained from the RZWI niewegraaf in duiven. This plant treats predominantly domestic waste water.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was conducted in 280 ml BOD bottles.
Parameter:
% degradation (O2 consumption)
Value:
76
Sampling time:
28 d
Details on results:
Results equate to a readily biodegradable classification.
Results with reference substance:
See below

 Time Days  5  15  28
 Duomac T BOD/ThoD  20 50  76
 Sodium Acetate BOD/Thod  73  88  90
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Test conducted according to guidelines with silica gel to reduce toxicity to inoculum. Modifications made were for test optimization only and do not invalidate this result. This test can be considered reliable
Executive summary:

DuoMac T is biodegraded 76% in the closed bottle test OECD guidline 301D performed in conformity with the OECD principles of Good Laboratory Practices. It should therefore be classified as readily biodegradable. In this test, the presence of silica gel so the toxicity of the substance did not influence the biodegredation.

Description of key information

The substance under evaluation is an tallow alkyl-1,3 -diaminopropane salt with acetate, 2ethylhexanoate and tallate which will for a large extent be dissociated under environmental conditions.

Biodegradation screening tests are available for various structurally related alkyl-1,3-diaminopropanes. This includes oleyl- and tallow-1,3-diaminopropane acetate and oleic acid salts. For all ready biodegradability was demonstrated. The 10 day window (for OECD 301D 14 day window) was not evaluated because the test substances are UVCB’s and degrade sequentially by a consortium of microorganisms (Ginkel, 1996).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Biodegradation screening tests are available for various structurally related alkyl-1,3-diaminopropanes. This includes oleyl- and tallow-1,3-diaminopropane acetate and oleic acid salts. For all ready biodegradability was demonstrated. The 10 day window (for OECD 301D 14 day window) was not evaluated because the test substances are UVCB’s and degrade sequentially by a consortium of microorganisms (Ginkel, 1996). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The time-window should be ignored as a pass fail criterion because of these scientific reasons.

 

Tallow-1,3-propanediamine oleates (CAS no.: 1307863-78-7) did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Tallow-1,3-propanediamine oleates was biodegraded 65% at day 28 in the Closed Bottle test (Ginkel, 2010). Hence this substance is classified as readily biodegradable.

 

Oleyl-1,3-propanediamine oleates (CAS no.: 40027-38-1 or 34140-92-5) is readily biodegradable in an aerobic ready biodegradation test according to OECD TG 301F. The test item was mineralised by 61% in 28 days (Gancet, 2004). Hence this substance is classified as readily biodegradable.

 

Tallow-1,3-propanediamine acetates (CAS no.: 61791-54-6) was tested in the presence of silica gel to mitigate the toxicity to the inoculum. Tallow-1,3-propanediamine acetates was biodegraded 76% at day 28 in the Closed Bottle test (Ginkel, 1990). Hence this substance is classified as readily biodegradable.

 

Oleyl-1,3-diaminopropane (CAS no.: 7173-62-8) was biodegraded 66% at day 28 in the Closed Bottle test and should therefore be classified as readily biodegradable.

 

Based on the available studies on biodegradation, all alkyl-1,3 -diaminopropanes (C10 - C18) including their fatty acid salts, are considered to be readily biodegradable. The 10-days-window criterion does not need to be evaluated as the alkyl 1,3 -diaminopropanes are UVCB substances.