3-aminopyrazine-2-carboxylic acid

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:

23 January 2013 - 14 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

Cas Number:

1623405-26-4

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Identification: Amiloride Compound 5
- Molecular formula: C5H5N3O2
- Molecular weight: 139.11
- CAS Number: 5424-01-1
- Description: Tan powder
- Batch: 203-2
- Purity/Composition: 100 area%
- Test substance storage: In refrigerator (2-8°C) protected from light, desiccated
- Stability under storage conditions: Stable
- Expiry date: 13 March 2014 (Retest date)
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store desiccated
- Volatile: No
- Reactivity: Reactive to light, moisture and oxygen
- Test substance handling: No specific handling required for moisture and oxygen
- Use amber-coloured glassware or wrap container in aluminium-foil
- Stability in water: Unknown
- Solubility in water: Insoluble

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.

- Treatment: The freshly obtained sludge was used immediately. The concentration of suspended solids was 5.0 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (53 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

28 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

other: Total Organic Carbon (TOC)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water
Stock solutions of mineral components
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl; dissolved in Milli-Q water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-Q water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-Q water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-Q water and made up to 1 litre.

- Test temperature: between between 22 and 23°C.
- pH:
At t=0 d: 7.5
At t=28 d: 7.5 – 8.0
- pH adjusted:no
- Aeration of dilution water: Not before the test, the test is aerated continously
- Suspended solids concentration: The freshly obtained sludge was used immediately. The concentration of suspended solids was 5.0 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (53 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
- Method used to create aerobic conditions:
Synthetic air (a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%)) was sparged through the solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:
CO2 was trapped in barium hydroxide solution. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampul). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made at least 14 days.


SAMPLING
- Sampling frequency: Titration were made on day: 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: Titration of the whole volume of CO2-absorber

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

16

Sampling time:

29 d

Remarks on result:

other: HCl added on the 28th day (last CO2-measurement on the 29th day)

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

20

Sampling time:

29 d

Remarks on result:

other: HCl added on the 28th day (last CO2-measurement on the 29th day)

Details on results:

The relative biodegradation values calculated from the measurements performed during the test period revealed 16% and 20% biodegradation of Amiloride Compound 5, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control more than 25% biodegradation occurred within 14 days (41%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

BOD5 / COD results

Results with reference substance:

Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.
The positive control substance was biodegraded by at least 60% (69%) within 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable (16-20%)

Conclusions:

Amiloride Compound 5 was not readily biodegradable under the conditions of the modified Sturm test presently performed.