Substituted amino acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

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:

July 2018 - September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Ammonium chloride was omitted from the medium to prevent nitrification.

Principles of method if other than guideline:

Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301 D) 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) river water instead of an effluent/extract/mixture was used as inoculum.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Remarks:

river water instead of an effluent/extract/mixture was used as inoculum.

Details on inoculum:

River water was sampled from the Rhine near Heveadorp, The Netherlands (13-07-2018). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.

Duration of test (contact time):

60 d

Initial conc.:

4 mg/L

Based on:

test mat.

Remarks:

27.8% purity

Initial conc.:

1.112 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The Closed Bottle test (OECD TG 301 D) 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 river water with nutrient, 6 bottles containing river water with nutrients and sodium acetate, 10 bottles containing river water with nutrients and test substance. The concentrations of the test substance (active), and sodium acetate in the bottles were 4.0 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. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 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).

Reference substance:

other: Not included in the screening test

Test performance:

Test conditions
The pH of the media was 7.6 (test) and 7.8 (control and reference) at the start of the test.
The pH of the medium at day 28 was 7.8 (test and control). The temperature ranged from 22.5 to 22.9°C which is within the prescribed temperature range of 22 to 24°C.

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

33

Sampling time:

60 d

Details on results:

Theoretical oxygen demand (ThOD)
The theoretical oxygen demand (ThOD) of the active ingredient of the Substitued amino acid (2) solution is 1.15 g oxygen /g test substance. The ThOD of sodium acetate is 0.78 g/g

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 the test
substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table I).
Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 1.2 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 82 (Table II and Figure). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability
Substituted amino acid (2) solution was not biodegraded (2%) after 28 days) in the Closed Bottle test (Figure and Table I) and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test this test substance was biodegraded by 33% at day 60. The test item should therefore not be classified as readily biodegradable. The biodegradation percentage achieved at day 60 demonstrates that the test item is inherently biodegradable. The test substance is degraded partially at the end of the test and could be degraded further in time because a plateau has not been reached. The lack of complete biodegradation in the Closed Bottle test does not mean that part of the test item is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)	Oxygen concentration (mg/L)
	Oc	Ot	Oa
0	8.7	8.7	8.7
	8.7	8.7	8.7
Mean (M)	8.7	8.7	8.7
7	8.0	8.1	4.1
	8.1	8.1	4.2
Mean (M)	8.1	8.1	4.2
14	7.6	7.5	3.1
	7.6	7.4	3.4
Mean (M)	7.6	7.5	3.3
21	7.5	7.5
	7.6	7.4
Mean (M)	7.6	7.5
28	7.5	6.9
	7.4	7.1
Mean (M)	7.5	7.0
42	7.1	6.0
	7.2	6.1
Mean (M)	7.2	6.1
60	6.8	5.4
	7.0	5.3
Mean (M)	6.9	5.4

O_c         River water with nutrients.

O_t         River water with nutrients, and test substance (active) (4.0 mg/L). 

O_a         River water with nutrients and sodium acetate (6.7 mg/L).

 

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

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.0	0.0	0	0
7	0.0	3.9	0	75
14	0.1	4.4	2	85
21	0.1		2
28	0.5		11
42	1.1		24
60	1.5		33

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

Valid test performed according to guideline OECD 301D with minor acceptable deviations applying GLP conditions.

Executive summary:

In order to assess the biotic degradation of Substituted amino acid (2) solution, 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 did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Sodium hydrogen substituted amino acid (2) solution was not biodegraded (2% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test this test substance was biodegraded by 33% at day 60. The biodegradation reached at day 60 demonstrates that this substance is inherently biodegradable. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is

recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 82% 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 ecotoxicity data in this dossier from Substituted amino acid are read across from Substituted amino acid (2) solution. This read across is considered to be justified because Substituted amino acid (2) solution contains about 50% Substituted amino acid and no ecotoxicity (EC50>100 mg/L) was observed for Substituted amino acid (2) solution in the acute daphnia and algae test. It is therefore considered extremely unlikely that any significant aquatic ecotoxicity will be observed for substituted amino acid.

Both stereo isomers of Substituted amino acid only the order in which the different groups are placed around the chiral carbon is different. A ready biodegradability test with Substituted amino acid is currently running to evaluate if the 33% biodegradation observed at day 60 in the test with Substituted amino acid (2) solution is caused by persistency one of both enantiomers  

The biotic degradation of Substituted amino acid (2) solution was evaluated in a ready biodegradability test 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 substance did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Substituted amino acid (2) solution was not biodegraded (2% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test this test substance was biodegraded by 33% at day 60. The biodegradation reached at day 60 demonstrates that this substance is inherently biodegradable. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is

recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

In order to assess the biotic degradation of Substituted amino acid (2) solution, 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 did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Substituted amino acid (2) solution was not biodegraded (2% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. In the prolonged Closed Bottle test this test substance was biodegraded by 33% at day 60. The biodegradation reached at day 60 demonstrates that this substance is inherently biodegradable. The lack of complete biodegradation in the Closed Bottle test does not mean that the test item is

recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 82% 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.