Amines, di-C16-18-alkyl

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Principles of method if other than guideline:

The sludge (200 mg Dry Weight (DW)/l) was aerated for one week. The siudge was diluted to a concentration of 2 mg DW/I in the BOD bottles. This method was described in a proposal for harmonizing ready biodegradability test protocols. Ammonium chloride was omitted from medium to prevent nitrification.

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

3.2 mg/L

Based on:

test mat.

Parameter:

% degradation (O2 consumption)

Value:

17

Sampling time:

140 d

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

28 d

Details on results:

Armeen 2HT was biodegraded 4% at day 28 in the closed bottle test. In the prolonged closed bottle test this compound was biodegraded 17% at day 140 which demonstrates that this test compound should not be classified as readily biodegradable.

The biological oxygen demand (BOD) mg/mg of the test compound 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. The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD]. The calculated theoretical oxygen demand of Armeen 2HT is 3.3 mg/mg. The theoreticaloxygen demand of sodium acetate is 0.8 mg/mg.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

Although test results seem indicate that this product is not readily biodegradable. The limitations of bioavailability due to extremely poor
solubility must be considered here as a possible explination for poor degradation result.. No evidence of a toxic effect of test substance was
observed. The test met all important quality criteria .

Executive summary:

The test substance caused no reduction in the endogenous respiration. Therefore, Armeen 2HT is considered to be non-inhibitory to the inoculum. Armeen 2HT was biodegraded 4% at day 28 in the closed bottle test. In the prolonged closed bottle test this compound was biodegraded 17% at day 140 which demonstrates that this test compound should not be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 1.05 mg/l and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 77% 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.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

prelonged colsed bottle condiucted

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter:

% degradation (O2 consumption)

Value:

20

Sampling time:

28 d

Details on results:

Classified as not readily biodegradable.

Stock solutions the sodium acetate was added to the bottles using a stock solution of 1.0 gllitre. For the application of the test substance on silica gel, a stock solution of 1.0 gllitre in dichloromethane was used. Oxygen, pH measurement.

The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The ThOD of the test compound is 3.3 g 02/g test substance. The BOD of Armeen 2C and sodium acetate were calculated from the oxygen concentrations in the bottles without and with test substance but with silica gel and the bottles without and with sodium acetate but without silica.

Interpretation of results:

inherently biodegradable

Conclusions:

Although results point towards this substance not being readily biodegradable. There is a high possibility of poor bio-availability due to the very low solubility of this compound preventing further degredation at the tested concentration this is important to consider here as an explination of this result.

Executive summary:

Armeen 2C is biodegraded 20% at day 28 in the closed bottle test (Figure and Tables I - 111). Further, in the prolonged closed bottle test the test compound is biodegraded 18% at day 42. The validity of the test is shown by the oxygen consumption in the control bottle with sodium acetate (Figure, Tables I - 111) and an endogenous respiration of 0.8 mgllitre at day 28. The pH of the medium at day 28 was 7.7.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

key study

Study period:

19-07-2005 - 23-08-2005

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 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

minor

Principles of method if other than guideline:

Ammonium chloride was omitted from the medium to prevent nitrification
Dioctadecylamine was dissolved in dichloromethane (1.0 g/L). The test substance in dichloromethane (0.15 mL) was directly added to 4 bottles.
Approximately 5 mL of dichloromethane was added to both control and test bottles. The solvent was allowed to evaporate by placing the bottles on a roller
bank in a ventilated hood for 24 hours. The roller bank was also used to achieve an even distribution of the test substance on the wall
of the bottles. The tests were performed in 300-ml BOD (biological oxygen demand) bottles with glass stoppers. Use was made of 4 bottles containing only
dichloromethane and 4 bottles containing test substance in dichloromethane.

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Sodium acetate anhydrous, Lot 1\10. V 19146, was used as a reference substance in the Closed Bottle test.
This compound was purchased from J.T. Baker BV, Deventer, The Netherlands.

All other chemicals used were of reagent grade quality.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands (13-07-2005). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced:
instead of an effluent/extract/mixture, activated sludge was used as an inoculum. 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 to a
concentration of 2 mg DW/L in the BOD bottles (van Ginkel and Stroo 1992).

Duration of test (contact time):

28 d

Initial conc.:

0.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to the study plan and an amendment. The study plan was developed from ISO Test Guidelines (1994). Use was made of 4 bottles containing inoculum and a stirrer bar, 4 bottles containing test substance, inoculum and a stirrer bar, 6 bottles containing inoculum and 6 bottles containing sodium acetate and inoculum. The concentrations of the test compound and sodium acetate in the bottles were
0.5 and 6.7 mg/L, respectively. The inoculum was diluted to 2 mg DW/L in the closed bottles. 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 without stirrer bars were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles without stirrer bars were closed and incubated. Two
duplicate bottles of the series without stirrer bars were withdrawn for analyses of the dissolved oxygen concentration at day 7, and 14. The bottles
with stirrer bars were incubated on magnetic stirrer plates. The consumption of oxygen in the bottles with stirrer bar was measured using a special
funnel (study plan amendment). This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD
bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen
electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The bottles were incubated at 20±1°C

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of dioctadecylamine and of the sodium acetate were calculated from their molecular formulae and molecular weights.

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) = Mc - Mt or a
Mc = the mean oxygen level in the control bottles inoculated with activated sludge n days after the start of the test.
Mt or a = the mean oxygen concentration in the bottles containing the test compound (t) or the reference compound, sodium acetate (a), and inoculated with
activated sludge n-days after the start of the test.

The biological oxygen demand (BOD) mg/mg of the test compound 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

Parameter:

% degradation (O2 consumption)

Value:

68

Sampling time:

28 d

Details on results:

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined
because possible toxicity of dioctadecylamine 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 7.0. Temperatures ranged from 19 to 21°C.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 0.9 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at
day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 73. Finally, the validity of the test is
shown by oxygen concentrations >0.5 mg/L in all bottles during the test period

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Test performed under GLP according guidelines, with minor acceptable deviation, meeting quility and validity criteria.
Dioctadecyfamine is biodegraded 68% at day 28 in the Closed Bottle test and should therefore be classified as readily biodegradable.

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. Dioctadecylamine did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The validity of the test is demonstrated by an endogenous respiration of 0.9 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 73. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Dioctadecylamine was biodegraded 68% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

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

Adequacy of study:

supporting study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

minor

Principles of method if other than guideline:

The Closed Bottle test was performed according to ISO Test Guidelines (1994). Four deviations from the Guidelines were introduced;
1) an initial test substance concentration 0.5 mg/L instead of the usual 2 mg/L,
2) oxygen consumption at various time intervals was measured in the same bottles (bottles were not sacrificed),
3) the oxygen concentration was measured in triplicate (not in duplicate) and
4) the content of the bottles was mixed continuously.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Ne¬therlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum. 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 to a concentration of 2.0 mg DW/L in the BOD bottles (van Ginkel and Stroo 1992).

Duration of test (contact time):

28 d

Initial conc.:

0.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test was performed according to ISO Test Guidelines (1994). Four deviations from the Guidelines were introduced; 1) an initial test substance concentration 0.5 mg/L instead of the usual 2 mg/L, 2) oxygen consumption at various time intervals was measured in the same bottles (bottles were not sacrificed), 3) the oxygen concentration was measured in triplicate (not in duplicate) and 4) the content of the bottles was mixed continuously. The test substance in dichloromethane (approximately 5 ml) was directly added to bottles. The solvent was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 24 hours. The roller bank was also used to achieve an even distribution of the test substance on the walls of the bottles. The tests were performed in 300-ml BOD (biological oxygen demand) bottles with glass stoppers. Use was made of 3 bottles con¬taining only inoculum, and 3 bottles containing test substance and inoculum. This was performed in duplicate with inocula collected at different dates from the activated sludge plant. The inocula were diluted to 2.0 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were com¬pletely filled without air bubbles. The bottles were incubated at 20±1°C on magnetic stirrer plates. The consumption of oxygen in the bottles was measured using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The test was carried out in duplicate (on 07-07-2004 and 27-10-2004) to assess the
reproducibility.

Reference substance:

other: Sodium Acetate

Test performance:

The validity of the tests is demonstrated by an endogenous respiration of 0.93 and 1.40 mg/L at day 28 . Furthermore, the differences of the replicate values at day 28 were less than 20%. Finallly, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Parameter:

% degradation (O2 consumption)

Value:

79

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

73

Sampling time:

28 d

Details on results:

Dioctadecylamine is biodegraded 73 and 79% at day 28 in duplicate Closed Bottle tests (Figure and Tables I-II), and should therefore be classified as
readily biodegradable. The test was repeated to assess the reproducibility.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Reliable with restrictions mentioned. No GLP No Analysis certificate , No official report, No quality control Report in word Format.
Dioctadecylamine was biodegraded 73 and 79% at day 28 in duplicate Closed Bottle tests. Hence this compound should be classified as readily
biodegradable

Executive summary:

Dioctadecylamine was biodegraded 73 and 79% at day 28 in duplicate Closed Bottle tests. Hence this compound should be classified as readily bio­degradable.

Description of key information

Based on the broad substrate specificity of micro-organisms degrading fatty acids with respect to the alkyl chain length it is unlikely that the

biodegradability of dialkylamines differs significantly with varying alkyl chain lengths.

The valid ready biodegradability test results obtained with didodecylamine and dioctadecylamine, and the scientific evidence that fatty amine

derivative degrading bacteria degrade these substances though b-oxidation lead to the conclusion that all dialkylamines are readily biodegradable.

Biodegradation in excess of 60% was not achieved in a few tests. The low biodegradability test results in these tests should be attributed to the limited bioavailability under the stringent test conditions and should consequently be ignored.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Biodegradation in water; screening tests

 

Significant degradation of didodecylamine as measured by oxygen consumption in the Closed Bottle test did occur within 28 days (van Ginkel et al 1995). Didodecylamine was also found to be readily biodegradable in the MITI test (Yoshimuraet al,1980).Biodegradation in excess of 60% is more difficult to achieve with longer chain dialkylamines. Nevertheless, dioctadecylamine was also found to be readily biodegradable (Akzo Nobel, 2005). Long-chain dialkylamines like dioctadecylamine are not soluble in water and as consequence especially not bioavailable in ready biodegradability tests. Adsorption of long-chain dialkylamines onto solids probably further decreases their bioavailability. Decreased bioavailability through adsorption was demonstrated with Closed Bottle tests inoculated with soil (contains solids). The rate of degradation in the test with soil was low compared to the test with river water probably containing little particles (van Ginkel et al, 1995). Adequate availability of the test substance to microorganisms is key for demonstrating the true biodegradability of dialkylamines. A ready biodegradability test result could be achieved using a very low test substance concentration and agitation of the content of the closed bottles (Akzo Nobel 1995; van Ginkel et al, 2008) and the use of silicone oil van Ginkel et al (2008). Limited bioavailability usually results in linear instead of the well-known logarithmic biodegradation curves (van Ginkel et al 1995, 2003, 2008). Inadequate bioavailability is responsible for false-negative results obtained in ready biodegradability tests with dialkylamines (Akzo, 1992, 1993).

Chemically dialkylamines have two alkyl chains linked directly to a nitrogen atom. The alkyl chains may be derived from different sources like dodecyl, coco, or tallow. Biodegradation of surfactants refers to the reduction in complexity of the chemical through metabolic activity of micro-organisms utilizing the substance as carbon and energy source.If a surfactant is to serve as a carbon and energy source for aerobic micro-organisms then it has to be converted into a form that can enter the central metabolism of micro-organisms. Normally this involves converting the surfactant into one, or more, low molecular weight intermediates of the tricarboxylic acid (TCA) cycle or compounds that feed into it. These conversions are described in biodegradation pathways for cationic surfactants(van Ginkel, 2004). Although micro-organisms capable of degrading surfactants are immensely diverse, the central metabolism (b-oxidation and TCA cycle) is remarkably similar. Kluyver and Donker (1926) first described this similarity known as the unity of biochemistry. This unity is the key to justification of the use of read-across of biodegradability test results.

Dialkylamines have not been studied in detail. Most fatty amine derivative-degrading microorganisms initiated biodegradation through cleavage of the alkyl-N bond (van Ginkel, 2004). The alkyl chains are liberated enzymatically as alkanals. The oxidation of the alkanals to fatty acids is catalysed by another enzyme. The fatty acids are degraded through the b-oxidation cycle. In each cycle, the alkyl chain is progressively shortened by two carbons yielding one molecule of acetyl-CoA. The acetyl-CoA generated through b-oxidation enters the TriCarboxylic Acid cycle, where it is further oxidised to carbon dioxide and water. A single micro-organism can degrade both saturated and unsaturated chains with varying chain lengths. The alkyl chains are therefore completely degraded by micro-organisms with comparable potential (biodegradability).

Based on the broad substrate specificity of micro-organisms degrading fatty acids with respect to the alkyl chain length it is unlikely that the biodegradability of dialkylamines differs significantly with varying alkyl chain lengths.The valid ready biodegradability test results obtained with didodecylamine and dioctadecylamine, and the scientific evidence that fatty amine derivative degrading bacteria degrade these substances though b-oxidationlead to the conclusion that all dialkylamines are readily biodegradable.