Disodium L-cystinate

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:

02 July 2012 - 02 August 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity: 99.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the freezer at about -20 °C

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater
- Storage conditions / Storage length: During the holding period of three days prior to use, the sludge was aerated at room temperature
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Concentration of sludge: Prior to use, the dry weight of this diluted activated sludge was determined again and defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: The temperature during the test period was in the range 21–23 °C.
- pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum and found to be pH 7.4. The pH measured in all flasks at the end of exposure (Day 28) was 5.5 to 7.4
- pH adjusted: no
- Light conditions: The test flasks were incubated in a dark room.

TEST SYSTEM
- Culturing apparatus: 2000-mL Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The final volume was 1000 mL per flask. Each flask was loosely covered with aluminium foil to allow the exchange of air between the flask and the surrounding atmosphere. The test media were continuously stirred by magnetic stirrers.
- Identification: The test vessels were labeled with all necessary information to ensure unmistakable identification.
Test duration: 28 days

SAMPLING
- One sample of about 10 mL was taken from each test flask per sampling date. Prior to sampling, water evaporation losses were determined by weighing the flasks and were compensated by adding purified water. Deposits on the test vessels were resuspended.
- Sampling Dates (Test item and inoculum control): Exposure Day 0, 2, 5, 8, 11, 14, 21 and 28
- Sampling Dates (Procedure control): Exposure Day 0, 2, 5, 14 and 28
- Sampling Dates (Toxicity control): Exposure Day 0, 5, 14 and 28

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (DOC removal)

Value:

98

Sampling time:

28 d

Key result

Parameter:

% degradation (DOC removal)

Value:

ca. 95

Sampling time:

5 d

Details on results:

In the test flasks, containing the test item and activated sludge (inoculum), the mean concentration of DOC (dissolved organic carbon) continuously decreased from initially 29.7 mg/L on Day 0 to 0.6 mg/L on Day 28. The mean biodegradation of L-Cysteine based on DOC decrease amounted to 98%. The pass level for ready biodegradability, i.e. biodegradation of at least 70% removal of DOC in a 10-day window within the 28-day period of the test, was reached (95% removal of DOC within five days of exposure). In the procedure controls, containing the reference item sodium benzoate and activated sludge (inoculum), sodium benzoate was completely biodegraded (>90%) within five days of exposure, thus confirming suitability of the activated sludge. In the toxicity control, the test item was not inhibitory to activated sludge at the tested concentration of 100.1 mg/L because degradation was >35% within 14 days.

Dissolved Organic Carbon Concentration (DOC) in the Test Flasks
DOC (mg/L)^1
Time (days)	Test item			Procedure control			Inoculum control			Toxicity control
	Replicate No.			Replicate No.			Replicate No.			Replicate No.
	1	2	mean^2	1	2	mean^2	1	2	mean^2	1^2
0	35.6	36.2	29.7	35	35.5	29.1	5.7	6.8	6.3	58.2
2	30.2	32.4	28.4	25.1	5.1	2.2	3.3	2.6	3	n.d.
5	5.1	5.4	1.6	5.4	5.5	1.8	3.8	3.6	3.7	4.4
8	5.6	5.8	1.4	n.d.	n.d.	n.d.	4.6	4.1	4.4	n.d.
11	4.8	4.2	1.9	n.d.	n.d.	n.d.	2.2	3.1	2.7	n.d.
14	3.4	4.1	1.3	3.4	3.1	0.8	2.8	2.2	2.5	2.1
21	3.4	3.6	0.5	n.d.	n.d.	n.d.	2.8	3.2	3.0	n.d.
28	3.5	3.6	0.6	3.4	3.4	0.4	3.0	3.0	3.0	1.2
^1Mean values of at least triplicate measurements per sample
^2Values corrected for the inoculum controls
^n.d.Not determined

Percentage Biodegradation in the Test Flasks
Percentage Biodegradation^1
Time (days)	Test item			Procedure control			Toxicity control
	Replicate No.			Replicate No.			Replicate No.
	1	2	mean	1	2	mean	1
0	0	0	0	0	0	0	0
2	7	2	4	93	93	93	n.d.
5	95	94	95	94	94	94	92
8	96	95	95	n.d.	n.d.	n.d.	n.d.
11	93	95	94	n.d.	n.d.	n.d.	n.d.
14	97	95	96	97	98	97	96
21	99	98	98	n.d.	n.d.	n.d.	n.d.
28	98	98	98	99	99	99	98
^1Corrected for the inoculum controls
^n.d.Not determined

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

L-Cystine is readily biodegradable with a mean biodegradation based on DOC decrease amounted to 98%. The pass level for ready biodegradability, i.e. biodegradation of at least 70% removal of DOC in a 10-day window within the 28-day period of the test, was reached (95% removal of DOC within five days of exposure).

Executive summary:

The test item, L-Cysteine, was investigated for its ready biodegradability in a "28-Day DOC Die-Away Test" according to the OECD Guideline for Testing of Chemicals, No. 301 A (1992), the Commission Regulation (EC) No 440/2008 C.4-A and the OPPTS 835.3110 (Paragraph (l)). In the test flasks containing the test item, L-Cysteine, and activated sludge (inoculum), the mean concentrations of dissolved organic carbon (DOC) rapidly decreased by 98% of the initial values within the test period of 28 days. The pass level for ready biodegradability (70% removal of DOC in a 10-day window within a 28-day period) was reached (95% removal of DOC within five days of exposure). The test item L-Cysteine is therefore readily biodegradable under the test conditions.

The reference item sodium benzoate was readily biodegraded by an average of 94% within five days of exposure, thus confirming suitability of the activated sludge.

In the toxicity control, containing the test item, the reference item sodium benzoate and activated sludge (inoculum), the initial DOC decreased by 96% within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 100.1 mg/L because degradation was >35% within 14 days.

The test is regarded as valid since the difference of duplicate values for the degradation of the test item at the end of the 10-Day window was less than 20%, and the percentage degradation of the reference item reached the level for ready biodegradability by Day 2 (criterion: at least 70% removal of DOC in a 10-day window by Day 14).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater