Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/10/2016 - 23/11/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

minor deviation from the guideline: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by biodegradation of the reference compound)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

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).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

See annex

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

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, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L (van Ginkel and
Stroo, 1992). The inoculum was not pre-exposed to the test substance.

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, stocks and administration
The nutrient medium of the Closed Bottle test contained per liter of deionized water; 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 was added to
the bottles using a stock solution of 1.0 g/L. The test substance was administered to the bottles using a stock solution of 1.0 g/L. C12-C14 alkylmorpholine did not
dissolve at 1.0 g/L in deionized water. The test substance did dissolve in acidified deionized water with a pH of 5.5. Acidifcation was accomplished with 2 M H2SO4.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from OECD TG 301D (1992) and an ISO TG (1994). Use was made
of 10 bottles containing only inoculum, 10 bottles containing inoculum and test substance, 6 bottles containing sodium acetate and inoculum, and 6 bottles containing
test substance, sodium acetate and inoculum. The concentrations 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 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 bottles.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of N-[2-(2-hydroxyethoxy)ethyl]acetamide (containing glycerol), and sodium
acetate were calculated from their molecular formulae and molecular weights 1

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 = Mc - 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

Remarks:

Supplier: Sigma-Aldrich, St Louis, US; Purity >99% ; Batch/lot number BCBP8197V

Key result

Parameter:

% degradation (O2 consumption)

Value:

89

Sampling time:

28 d

Remarks on result:

other: readily biodegradable

Details on results:

The calculated theoretical oxygen demand (ThOD) of the substance
is 1.4 mg/mg. The ThOD of sodium acetate is 0.8 mg/mg

Toxicity
Depletion of oxygen in the bottles with the test substance and sodium acetate was slightly higher than in the bottles with only the reference substance (Table I). The additional oxygen
consumption detected in the bottles with test substance and acetate compared to the bottles with only acetate agrees with the oxygen consumption due to the degradation of the
test substance. Inhibition of microorganisms capable of degrading sodium acetate by the test item present at a concentration of 2.0
mg/L did therefore not occur. Moreover, inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7 of the test (Table I). Inhibition of the
onset of biodegradation due to the "high" initial test substance concentration of the test item can therefore be excluded.

Biodegradability
The test item was biodegraded by 89% at day 28 in the Closed Bottle test (Table II, Figure). the test item is an UVCB consisting of different constituents. Biodegradation of the constituents requires the concerted action of different microorganisms as a single organism
usually lacks the full complement of enzymatic capabilities. The biodegradation of eachconstituent may be fully in line with the time window criterion when judged as separate
chemicals. The time window should therefore not be applied to substances consisting of structurally similar constituents (OECD, 2006). Hence the test item should be classified as readily biodegradable

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80.

Oxygen consumption (mg/L) and the percentage biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test

Time (days)	Oxygen consumption		BIodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.5	4.1	18	76
14	0.9	4.3	32	80
21	1.9		68
28	2.5		89

Validity criteria fulfilled:

yes

Remarks:

endogenous respiration of 1.0 mg/L at day 28. Differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of reference compound at day 14 was 80. Oxygen concentrations >0.5 mg/L in all bottles during the test period.

Interpretation of results:

readily biodegradable

Remarks:

The test substance is an UVCB and therefore the time window should not be applied be

Conclusions:

The test item should be classified as readily biodegradable.
The test item is an UVCB consisting of different constituents. Biodegradation of the constituents requires the concerted action of different microorganisms as a single organism usually lacks the full complement of enzymatic capabilities. The biodegradation of each constituent may be fully in line with the time window criterion when judged as separate chemicals. The time window should therefore not be applied to substances consisting of structurally similar constituents (OECD, 2006).

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.

The test item at a concentration of 2.0 mg/L did not inhibit microorganisms growing on acetate. Moreover, the test substance did also not

cause a reduction in the endogenous respiration of the microorganisms at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test item biodegraded by 89% at day 28 in the OECD 301 Closed Bottle test. The test item should therefore be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 80% 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.

Description of key information

The test item biodegraded by 89% at day 28 in the OECD 301 Closed Bottle test. The test item should therefore be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater