ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

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:

key study

Study period:

From February 14, 2003 to May 8, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

L.A.E.
Appearance: White solid
Batch: 10234
Expiry date: 19 March 2004

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from Eye sewage treatment works, which treats predominantly domestic waste. Aliquots (25 mL) of a homogenised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GF/C filter papers. The filters were dried for
one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solid concentration of 30 mg/L.

Duration of test (contact time):

29 d

Initial conc.:

10 other: mgC/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salt medium (MSM).
- Inoculum: Active Sludge collected from Eye sewage treatment works.
- Suspended solids concentration: 30 mg solids/L
- Continuous darkness: Yes, clear glass culture bottles were wrapped in clack plastic bags in order to protect their contents from light for the duration of the test.
- Temperature: 20-24ºC.

TEST SYSTEM
- Culturing apparatus: 5 L clear glass culture bottles.
- Number of culture flasks/concentration: 6 ( 1, 2: Control plus inoculated mineral salts medium (MSM); ; 3: Reference-MSM; 4, 5: Test substance-MSM; 6: Sodium benzoate-MSM-Test substance).
- Method used to create aerobic conditions: The mixtures were aerated overnight with a supply of air that had been treated to remove carbon dioxide by passing it through cylinders containing fused calcium chloride, Carbosorb AS and a trap containig 1M sodium hydroxide solution.
- Details of trap for CO2 and volatile organics if used: Air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025 N nominal barium hydroxide (100 ml).
- Measuring equipment: The residual concentrations of Barium hydroxide were determined at intervals by duplicate titration of 20 ml samples with hydrochloric acid (0.05N), using phenolphthalein indicator.

SAMPLING
- Sampling frequency: The CO2 evolution was determined on Days 1, 2, 4, 5, 7, 8, 11, 14, 20, 28, 29.
- Sampling method: Air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025 N nominal barium hydroxide (100 ml). The residual concentrations of Barium hydroxide were determined at intervals by duplicate titration of 20 ml samples with hydrochloric acid (0.05N), nominal barium hydroxide (100 ml).
- Sample storage before analysis: The samples were analysed on the same day.

CONTROL AND BLANK SYSTEM
- Number of culture flasks/concentration: 6 ( 1, 2: Control plus inoculated mineral salts medium (MSM); ; 3: Reference-MSM; 4, 5: Test substance-MSM; 6: Sodium benzoate-MSM-Test substance).
- Inoculum blank: Flask 1, 2
- Control Reference substance: Flask 3
- Toxicity control: Flask 6

Reference substance:

benzoic acid, sodium salt

Test performance:

The air flow rate on day 4 through one culture containing the test substance was 25 ml/minute and, therefore, slightly below the recommended minimum for this type of test (30 ml/min).

This was not considered to be a significant nor to have affected the integrity of the test.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

88

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
3 % degradation after 1 d
13 % degradation after 2 d
37 % degradation after 4 d
45 % degradation after 5 d
56 % degradation after 7 d
64 % degradation after 8 d
71 % degradation after 11 d 7
77 % degradation after 14 d
82 % degradation after 20 d
88 % degradation after 28 d
89 % degradation after 29 d.

Results with reference substance:

Points of degradation plot (reference substance):
11 % degradation after 1 d
28 % degradation after 2 d
60 % degradation after 4 d
66 % degradation after 5 d
72 % degradation after 7 d
77 % degradation after 8 d
81 % degradation after 11 d
85 % degradation after 14 d
88 % degradation after 20 d
90 % degradation after 28 d
92% degradation after 29 d

Table 1: Biodegradation as percentage of the theoretical CO2 yield.

Day	LAE
	Mean % TCO2
1	3
2	13
4	37
5	45
7	56
8	64
11	71
14	77
20	82
28	88
29	89

Validity criteria fulfilled:

yes

Remarks:

(the difference of extremes of replicate values of the removal of the test chemical are less than 20%, the percentage degradation of the reference compound reached the pass level by day 14)

Interpretation of results:

readily biodegradable

Conclusions:

The biodegradation of the substance was 88% in 28 days.

Executive summary:

The ready biodegrability test of the N^α-Lauroyl-L-argine,-ethyl ester monohydrochloride (L.A.E) was performed in the CO2 Evolution test according to OECD 301B guideline and EU method C.4 -C.

The parameter determined in this test is the cumulative amount of carbon dioxide produced by mixtures containing LAE, mineral salts medium and activated sludge inoculum after 29 days of incubation at temperatures in the range 20 to 24ºC.

Test, control and references mixtures were aereted for 29 days with air that had been treated to remove carbon dioxide (CO2). The CO2 produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration.

By the end of the test study on day 29, biodegradation of LAE had achieved 89%. LAE can, therefore, be considered to be readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From February 4, 2000 to August 7, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

L.A.E.
Appearance: White solid
Batch: 2625
Expiry date: April 2001
Purity: 92-94%

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A sample of secondary effluent was collected on the day of the test from Thorndon Sewage Treatment Works, a trickling-filter plant which treats predominantly domestic waste. It was maintained under aerobic conditions in the laboratory, then, immediately before use, filtered through glass wool and the filtrate used as the inoculum (1 ml filtrate/litre test medium)

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salt medium (MSM).
- Temperature: BOD bottles (20.3-23.2ºC); incubator (21.3-22.8ºC)
- pH: 6.9-7.9
- Inoculum: Sample of secondary effluent was collected on the day of the test from Thorndon Sewage Treatment Works..
- Continuous darkness: Yes, clear glass culture bottles were wrapped in clack plastic bags in order to protect their contents from light for the duration of the test.
- Temperature: 18-24ºC.

TEST SYSTEM
- Culturing apparatus: BOD bottles of 277 ml each.
- Number of culture flasks/concentration: Three groups of eighteen bottles. A fourth group of four bottles for the inhibitory assay.

SAMPLING
- Sampling frequency: The Dissolved Oxygen (DO) evolution was determined on Days 0, 5, 7, 11, 14, 18, 21, 25 and 28.
- Sampling method: YSI dissolved oxygen meter fitted with a self-stirring DO/temperature bottle probe.
- Sample storage before analysis: The samples were analysed on the same day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: MSM+inoculum
- Toxicity control: MSM+inoculum+L.A.E (3 mg/L)+sodium benzoate (5 mg/L)
- Control reference substance: MSM+inoculum+sodium benzoate (5 mg/L)

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (TOC removal)

Remarks:

Theoretical Oxygen Demand

Value:

ca. 45

Sampling time:

28 d

Details on results:

A biodegradation plateau at approximately 45% was thought to have been achieved after 5 days of incubation.

The five-day microbial inhibition assay was conducted to determine whether L.A.E., at a concentration of 3 mg/l, inhibited the normal degradative activity of the microbial inoculum on the reference substance, sodium benzoate. The presence of the test substance at this concentration was not inhibitory.

Results with reference substance:

Sodium benzoate had been degraded to 55% of its TOD after 5 days of incubation and 68% by Day 11.

Interpretation of results:

other: The substance cannot be considered to be readily biodegradable.

Conclusions:

Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) biodegradation plateau at approximately 45% was thought to have been achieved after 5 days of incubation and maintained under the range above mentioned until the end of the test on day 28. L.A.E. cannot, therefore, be considered to be readily biodegradable.

Executive summary:

The ready biodegradability of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was assessed in the Closed Bottle Test; OECD Procedure 301D and EU method C.4 -E.

Mean oxygen consumption in bottles containing L.A.E alone ranged between 0.83 and 0.93 MgO2/mg (44 -49% of its ThOD) during the test. A biodegradation plateau at approximately 45% was thought to have been achieved after 5 days of incubation and maintained under the range above mentioned until the end of the test on day 28. L.A.E. cannot, therefore, be considered to be readily biodegradable.

Description of key information

Key study: Test method according to OECD 301B and EU method C.4 -C. GLP study. The biodegradation of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was 89% in 29 days (and 88% in 28 days).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Supporting study:

The ready biodegradability of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was assessed in the Closed Bottle Test; OECD Procedure 301D and EU method C.4 -E.

Mean oxygen consumption in bottles containing L.A.E alone ranged between 0.83 and 0.93 MgO2/mg (44 -49% of its ThOD) during the test. A biodegradation plateau at approximately 45% was thought to have been achieved after 5 days of incubation and maintained under the range above mentioned until the end of the test on day 28. L.A.E. cannot, therefore, be considered to be readily biodegradable.