N-glycylglycine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June - 15 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

Regulation (EC) No 440/2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals, Revised Introduction to the OECD Guidelines for Testing of Chemicals

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-glycylglycine

EC Number:

209-127-8

EC Name:

N-glycylglycine

Cas Number:

556-50-3

Molecular formula:

C4H8N2O3

IUPAC Name:

N-glycylglycine

Test material form:

solid: crystalline

Details on test material:

Batch Number: S119 0711
Storage conditions: room temperature

Specific details on test material used for the study:

Batch no.: S119 0711
Storage conditions: At room temperature, protected from light

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological wastewater treatment plant.

- Preparation of inoculum for exposure: The sludge was pre-conditioned to reduce the amount of O2 consumed in the blank controls. In the pre-conditioning procedure, the sludge was washed twice with tap water and once with test medium right after sampling from the wastewater treatment plant. After centrifugation, the sludge was suspended in test medium at about 3-5 g/l dry matter and kept under constant aeration and stirring without feeding for 6 days, until test start. Before the start of the test the actual dry matter content of this suspension was determined.

- Pretreatment: The activated sludge was used after sampling from the treatment plant without adaptation.

- Concentration of sludge: 30 mg/L dry matter in the final mixture.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

59.1 mg/L

Based on:

other: nominal test item concentration based on 100 mg ThOD/L

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of test medium: Analytical grade salts were dissolved in ultra-pure water:
* mineral stock solution A: 8.5 g/L KH2PO4, 28.49 g/L K2HPO4.3H2O, 33.4 g/L Na2HPO4.2H2O, 0.50 g/L NH4Cl
* mineral stock solution B: 36.4 g/L CaCl2.2H2O
* mineral stock solution C: 22.5 g/L MgSO4.7H2O
* mineral stock solution D: 0.25 g/L FeCl3.6H2O
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium
The pH value of the test medium was adjusted to 7.4±0.2.

- Test temperature: 22±2 °C, controlled at ±1 °C, in a thermostat chamber in the dark.

- pH: 7.4±0.2, measured prior to testing and if necessary adjusted, except in the abiotic sterile control.

- Continuous darkness: yes, test bottles were in a thermostat cabinet.

TEST SYSTEM
- Test units: 510 ml glass bottles (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 ml. These flasks are equipped with stirring rods and butyl rubber quivers which contain 2 pellets of sodium hydroxide each to absorb the produced CO2 from the head space.

- Test performed in duplicate (two test flasks): The test item was applied by stock solution (test item in test medium; double concentrated to account for the addition of the sludge suspension).

CONTROL AND BLANK SYSTEM
B: Blank control (two replicates): containing incolum and test medium
P: Procedure control (two replicates): containing inoculum, test medium and 60.1 mg/L reference substance (100 mg ThOD/L)
X: Toxicity control (one replicate): containing inoculum, test medium, 59.5 mg/L test item and 60.1 mg/L reference substance (total 201 mg ThOD/L)
A: Abiotic sterile control (one replicate): containing ultra-pure water, test item, 0.04 mM HgCl2 (sterilising agent to prevent microbial degradation) and 59.0 mg/L test item (100 mg ThOD/L)


SAMPLING
The test vessels were stirred by an inductive stirring system for the whole test period. During the test the O2 uptake (and by extension the biodegradation) was continuously measured with a manometric BOD measuring device. The temperature was recorded with a data logger.
At the end of the test, the pH was measured, and samples for DOC analysis were taken, if applicable.
Total elimination was determined in each test vessel (test suspension, blank and procedure control) based on the theoretical carbon content and the analysis of the dissolved organic carbon (DOC) concentrations at the start and at the end of the test.

STATISTICAL METHODS:
No statistical analysis was performed.

Reference substance

Reference substance:

other: Sodium benzoate

Remarks:

CAS 532-32-1

Results and discussion

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

87

Sampling time:

28 d

Remarks on result:

other: test flask 1 after 28 d: 85% test flask 2 after 28 d: 89%

Details on results:

Based on O2 consumption the biodegradability of Glycyl-glycine was calculated to be 87% after 28 days of the ThOD and 80% at the end of the 10-day window (i.e. within 10 days after attainment of 10% degradation).

Biodegradation of the test item was observed after a lag-phase of about two days, which corresponds to the time span until 10% biodegradation was reached.

Based on the determination of the DOC at the end of the test, the total elimination was calculated to be 99% for Glycyl-glycine and 99% for sodium benzoate. This data is in line with the degradation calculated based on O2 consumption.

Any other information on results incl. tables

Procedure control:

The procedure control with sodium benzoate reached a biodegradation of 80% after 14 days, thus confirming suitability of inoculum and test conditions.

Toxicity control:

According to the OECD guideline 301 a substance is considered having inhibitory (i.e. toxic) effects on the inoculum if less than 25% degradation after 14 days are observed in the toxicity control. Since the biodegradation exceeded this pass level, it can be concluded that the test item does not have any significant toxic effects on the microbial population at the applied initial test concentration of 59.5 mg/l.

Abiotic steril control:

No significant degradation (0%) was observed in the abiotic sterile control. Therefore, it can be concluded that the test item is not degraded by oxygen consuming processes in the absence of microorganisms.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Glycyl-glycine (CAS no. 556-50-3) reached the pass level of 60% biodegradation in the manometric respirometry test within the 10-day window and, therefore, can be termed as readily biodegradable.

Executive summary:

The biodegradability of Glycyl-glycine (CAS no. 556-50-3) exposed to microorganisms derived from activated sludge of a municipal sewage treatment plant was investigated under aerobic static exposure conditions following the OECD guideline 301 F. The biodegradation was assessed based on O2 consumption compared to the theoretical oxygen demand ThOD.

Based on O2 consumption the biodegradation of Glycyl-glycine was calculated to be 87% of ThOD after 28 days and reached 80% at the end of the 10-day window (i.e. within 10 days after attainment of 10% degradation).

Biodegradation of the test item was observed after a lag-phase of about two days.

The procedure control with sodium benzoate reached a biodegradation of 80% after 14 days, thus confirming suitability of inoculum and test conditions.

Based on the determination of the dissolved organic carbon (DOC) at the end of the test, the total elimination was calculated to be 99% for Glycyl-glycine and 99% for sodium benzoate. This data is in line with the degradation calculated based on O2 consumption.

Glycyl-glycine reached the pass level of 60% biodegradation in the Manometric Respirometry Test within the 10-day window and therefore can be termed as readily biodegradable.

All validity criteria were fulfilled.