5-amino-2-nitrobenzoic 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:

21 August - 18 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-2-nitrobenzoic acid

EC Number:

236-283-4

EC Name:

5-amino-2-nitrobenzoic acid

Cas Number:

13280-60-9

Molecular formula:

C7H6N2O4

IUPAC Name:

5-amino-2-nitrobenzoic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid

Details on test material:

Batch / Lot No. 10044431

Specific details on test material used for the study:

Batch no.: 10044431
Storage: 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 waste water 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 waste water 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. On the day of the test start, this sludge suspension was diluted down to 60 mg/l dry matter, i.e. twice as high as the final test concentration.

- 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.:

63.4 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 cabinet in the dark

- pH: 7.4±0.2 (measured prior to testing and if necessary adjusted with NaOH or HCl (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. The bottles are equipped with stirring rods and butyl rubber quivers which contain two 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 59.9 mg/L reference substance (99.7 mg ThOD/L)
X: Toxicity control (one replicate): containing inoculum, test medium, 64.0 mg/L test item and 59.9 mg/L reference substance (total 201 mg THOD/L)
A: Abiotic sterile control (one replicate): containing inoculum, ultra-pure water, 0.04 mM HgCl2 (sterilizing agent to prevent microbial degradation) and 64.0 mg/L test item (101 mg ThOD/L)


SAMPLING
The test vessels were stirred by an inductive stirring system for a period of 28 days. During the test the O2 uptake was continuously measured with a manometric BOD measuring device. Temperature was recorded with a data logger.
At the end of the test, the pH was measured in all flasks except the abiotic sterile control (A).
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:

-2

Sampling time:

28 d

Remarks on result:

other: test flask 1 after 28 d: -2% test flask 2 after 28 d: -2%

Details on results:

Based on O2 consumption, no significant biodegradability of 5-Amino-2-Nitrobenzoic Acid was observed after 28 days, compared to the theoretical oxygen demand (ThOD).

Based on the determination of the DOC at the end of the test, the total elimination was calculated to be 9% for 5-Amino-2-Nitrobenzoic Acid and 99% for sodium benzoate. For sodium benzoate, this data is in line with the biodegradation based on O2 consumption. For 5-Amino-2-Nitrobenzoic Acid, however, no significant biodegration was found based on O2 consumption. The discrepancy in this case is most likely due to the analytical uncertainty of the DOC measurements.

Any other information on results incl. tables

Procedure control:

The procedure control with sodium benzoate reached a biodegradation of 79% 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 64.0 mg/L.

Abiotic steril control:

No significant degradation (0-2%) was observed in the abiotic sterile control. Therefore, it is 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:

not readily biodegradable

Conclusions:

5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) is not readily biodegradable in the Manometric Respirometry Test 301 F within 28 days, as it did not reach the pass level of 60% biodegradation within the 10-day window.

Executive summary:

The biodegradability of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) 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. Both the biodegradation over the course of the test (based on O2 consumption compared to the theoretical oxygen demand ThOD) and the total elimination at the end of the test (based on the determination of the dissolved organic carbon, DOC) were assessed.

Based on O2 consumption, no significant biodegradation of 5-Amino-2-Nitrobenzoic Acid was observed after 28 days.

The procedure control with sodium benzoate reached a biodegradation of 79% after 14 days, thus confirming suitability of inoculum and test conditions.

Based on the determination of the DOC at the end of the test, the total elimination was calculated to be 9% for 5-Amino-2-Nitrobenzoic Acid and 99% for sodium benzoate (see Table 5). For sodium benzoate, this data is in line with the biodegradation based on O2 consumption. For 5-Amino-2-Nitrobenzoic Acid, however, no significant biodegration was found based on O2 consumption. The discrepancy in this case is most likely due to the analytical uncertainty of the DOC measurements.

5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) is not readily biodegradable in the Manometric Respirometry Test 301 F within 28 days, as it did not reach the pass level of 60% biodegradation within the 10-day window.

All validity criteria were fulfilled.