calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

Biodegradation in soil

Administrative data

Endpoint:

biodegradation in soil: simulation testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is readily biodegradable

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February from 04th to 18th, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

temporary certificate

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of activated sludge: ALSI (Alto Lambro Servizi Idrici) wastewater treatment plant.
- Laboratory culture: the sludge was aerated and continuously mixed by the means of a magnetic stirrer for 7 days before the start o f the test. The inoculum was held in dark conditions under room temperature until use.
- Preparation of inoculum for exposure: activated sludge inoculum was filtered before the start of the test.
- Initial cell/biomass concentration: the activated sludge inoculum was diluted in culture medium to get a final concentration of 2 ml/l.

Duration of test (contact time):

ca. 14 d

Initial conc.:

ca. 3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 ml of stock solution a) and 1 ml of stock solution b), c) and d) will be combined and made up to 1000 ml with mineral water. The pH of test water was 7.76, so adjustment was not needed.
a) KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4 x 2H2O3 3.40 g/l
NH4Cl 0.50 g/l
b) MgSO4 x 7H2O2 2.50 g/l
c) CaCl2 x 2H2O3 6.40 g/l
d) FeCl3 x 6 H2O 0.25 g/l
- Test temperature: range 20.1 - 21.0°C
- Continuous darkness: yes

TEST SYSTEM
- Inoculation: immediately before the test start of the test a proper aliquot of activated sludge inoculum was added to test medium solution in order to obtain a concentration of 2 ml/l.

TEST SOLUTIONS
- Test item: one day before the start of the test 3.5 litres of test item solution at a concentration of 3.0 mg/l was prepared by direct weighting into aqueous test medium. the solution obtained was mixed by the means of magnetic stirrer to obtain a total solubilisation until the start of the test.
- Blank control: only the inoculum was added to 3.5 litre of aqueous test medium.
- Reference item: 2.0 litres of reference item solution at 3.0 mg/l was prepared in aqueous test medium by adding a proper aliquot of a stock solution of 10 g/l.
- Toxicity control: one day before the beginning of the test 2.0 litres of test item solution of a concentration of 3.0 mg/l was prepared by direct weighing into test medium as described for test item preparation, immediately before the start of the test a proper aliquot of a stock solution of reference substance (10 g/l) was diluted into test solution and manually shaken.

EVALUATIONS
- ThOD determination: calculated on the basis of the test item molecular weight and formula.
- Determination of Nitrite and Nitrate: at the beginning, after 7 days and at the end of the test, nitrite and nitrate concentration were photometrically determined after the sample preparation by the means of specific kits.
- Temperature: temperature was registered daily by the means of a digital themqometer.
- Oxygen measurement: the oxygen concentration was done by an electrochemical method in an airtight system with and O2 electrode under constant stirring.
- BOD: it is calculated on the basis of the mg O2/l consumed ratio mg test item/l.

Reference substance:

benzoic acid, sodium salt

Test performance:

The test item has shown to be ready biodegradable under test conditions applied in a closed bottle test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 96.9

Sampling time:

14 d

Details on results:

The test item showed a very rapid biodegradation, reaching the pass level of 60 % at 7 day from start of the test period and being stable during the following 7 days.
The study was stopped on the 14th day of incubation, since both the test item and the reference item passed the biodegradation trigger percentage of 60 % after 7 days.

The oxygen consumption due to nitrification was measured and the absence of nitrification was assessed. The ThODNH3 values for test item was calculated to be equal to 1.29 mg O2/mg.

The test item did not show any inhibitory effect on the microorganism activity at the tested concentration as demonstrated by exceeding the 25 % of biodegradation in the toxicity control already at the 7th day of test period.

Results with reference substance:

The reference compound reached the biodegradation pass level (60 %) already after 7 days showing that the test conditions and the inoculum were appropriate for the use in the study.
The ThODNH3 values for reference was calculated to be equal to 1.66 1.29 mg O2/mg.

Oxygen consumption (mg O₂/l) by activated sludge in the bottles

Day of measurement	Inoculum control		Test item		Reference substance		Toxicity control
	A	B	A	B	A	B	A	B
0	7.14	6.99	7.20	6.98	6.83	6.91	6.74	6.77
7	6.83	6.80	1.33	1.89	3.57	3.61	0.75	1.41
14	5.64	5.68	2.08	1.79	0.67	1.33	0.65	0.66

BOD values and percentage biodegradation

Day of measurement	BOD	% biodegradation
Test item
7	1.74	134.8
14	1.25	96.9
Sodium benzoate
7	1.01	6.8
14	1.32	79.5
Test item plus sodium benzoate, toxicity control
7	1.81	6.2
14	1.57	53

VALIDITY OF THE TEST

The test complies with the validity criteria, since the oxygen depletion in the inoculum control after 14 days was 1.40 mg O₂/l, the residual oxygen concentration in the test flasks never drop below 0.5 mg O₂/l and the maximum difference of duplicate values for the degradation of the test item was 13.9 %. Moreover the reference compound reached the biodegradation pass level (60 %) already after 7 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Biodegradation (14 days) = 96.9 %

Executive summary:

Method

The ready biodegradability of the test item was assessed in a 28-day biodegradation test following the “Closed Bottle” method according to the EU Commission Directive 92/69/EEC, Part C.4 E (1992), and the OECD Guideline for Testing of Chemicals, No. 301 D, 1992.

For this purpose, the test item was added to activated sludge from the recirculation tank of an civil wastewater treatment plant and let to stay in contact with the sludge for 14 days (the test was stopped before the standard period of 28 days being the biodegradation plateau reached already after 7 days.

The concentration of test item was of 3 mg/L. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure, which was measured at the start of the test and after 7 and 14 days..

Observations

The test item is considered to be stable at the test conditions for a time corresponding to the exposure time. Therefore, all the reported results are related to the nominal concentrations of the test item.

The oxygen consumption due to nitrification was measured and the absence of nitrification was assessed.

The ThOD_NH3 values for test item and test reference was calculated to be equal to 1.29 mg O₂/mg and to 1.66 mg O₂/mg respectively.

The test complies with the validity criteria because the average oxygen depletion in the inoculum control (blank) at the end of the test was 1.4 mg O₂/L (trigger value = 1.5 mg O₂/L), the residual oxygen concentration in the test flasks never drop below 0.5 mg O₂/L and the maximum difference of duplicate values for the degradation of the test item was 13.9 % (maximum value = 20 %).

Results

The reference compound reached the biodegradation pass level (60.8%) already after 7 days of incubation, showing that the test conditions were appropriate. Under these conditions, the test item showed to be ready biodegradable, having reached the complete degradation after 7 days of incubation. Moreover, the test item did not show any inhibitory effect on the microorganism activity at the tested concentration as demonstrated by the 61.2 % of biodegradation reached in the toxicity control already at the 7^th day of test period.