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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
EU Method C.5 (Degradation: Biochemical Oxygen Demand)
Version / remarks:
BOD test in large volume
Principles of method if other than guideline:
BOD test in large volume. Measurement of oxygen levels, TOC, bacterial growth
GLP compliance:
Study conducted before GLP development

Test material

Constituent 1
Chemical structure
Reference substance name:
Oxalic acid
EC Number:
EC Name:
Oxalic acid
Cas Number:
Molecular formula:
oxalic acid
Test material form:

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Settled raw wastewater from a St. Louis Metropolitan Sewer District manhole on the Washington University Campus
- Preparation of inoculum for exposure: the raw wastewater was settled and filtrated
- Pretreatment: none
- Concentration of sludge: very low: the raw wastewater was settled and filtrated
- Initial cell/biomass concentration: total bacteria count circa 6x10^4 organisms/ml.
Duration of test (contact time):
20 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
O2 consumption
O2 measured by azide modification of Winkler method
Parameter followed for biodegradation estimation:
TOC removal
Beckman infrared carbonaceous analyzer
Parameter followed for biodegradation estimation:
other: COD removal acc. to ASTM method
Parameter followed for biodegradation estimation:
other: bacterial growth, spot plate technique on tryptone glucose extract agar
Details on study design:
- Composition of medium: According to Standard Methods (for BOD) for the examination of Water and Wastewater, Am. Pub. Health Assn., New York 1965
- Additional substrate: none
- Test temperature: 19.5 - 20.5 degree C
- pH: no data (reference to ASTM BOD test)
- CEC (meq/100 g): no data (reference to ASTM BOD test)
- Aeration of dilution water: yes
- Suspended solids concentration: not given. Inoculum given as circa 6 x 10^4 organisms/ml

- Culturing apparatus: BOD apparatus of Orford et al. Two 9-L Pyrex bottles with a connecting siphon, one above the other. The lower one is the test bottle that is sampled. The lower one was kept full and sealed to exclude air.
- Number of culture flasks/concentration: single dilution technique (1 bottle)
- Method used to create aerobic conditions: aeration
- Method used to create anaerobic conditions:
- Measuring equipment: DO analysis according to azide modification of the Winkler method, titration with 0.0125 N sodium thiosulphate.
COD: low-level DCOD test recommended for samples having < 100 mg/l, acc. to ASTM.
TOC by Beckman IR C-analyzer. Samples were acidified with concentrated HCl and carbonates were stripped by nitrogen gas. Also performed on soluble and cellular fractions that had been separated by centrifugation.
Nitrite: to differentiate between carbonaceous and nitrogenous oxygen utilisation. Diazotation test (to detect nitrites)
Spot plate technique: Sterile petri dishes with tryptone glucose extract agar, spotted with 5 0.01 ml droplets of sample. Incubation for 24 h at 37 degree C. Counting by Quebec colony counter.

- Sampling frequency: on day 0, 1, 2, 3, 4, 5, 7, 9, 12, 16, 20
- Sampling method: Samples were taken from the lower bottle by opening the hose clamp on a siphon. The volume of the sample was replaced continuously through the siphon from the second bottle which was covered but had access to the atmosphere.
Samples for dissolved oxygen: 50 ml; for COD and TOC analysis 30 to 40 ml; centrifugation 40 ml (also for TOC);

- Inoculum blank: included (no C-source added)
- Abiotic sterile control: not included
- Toxicity control: not included
- Other: reference substance: glucose and glutamic acid
Reference substance
Reference substance:
other: glucose-glutamic acid solution, 10 mg/l

Results and discussion

Preliminary study:
no data provided
Test performance:
The test system performed well as indicated by the performance of the blank and of the reference (see below). In the blank, the oxygen utilisation proceeded at a low and relatively constant rate throughout the test period, attaining a level of 1.2 mg/l after 5 days and 3.3 mg/l after 20 days. Initial COD and TOC levels were very low and they were depleted to insignificant levels after 5 days. Bacterial growht reached a peak of 4x10^5 organism/ml after 2 days. Growth declined to a fairly constant level of about 1 to 2 x 10^4 org./ml to the end of testing.
% Degradationopen allclose all
% degradation (O2 consumption)
Sampling time:
5 d
Remarks on result:
other: BOD5 = 0.16 mg/mg
% degradation (O2 consumption)
Sampling time:
20 d
Remarks on result:
other: BOD20 = 0.16 mg/mg
Details on results:
Lag time before start of degradation was 0 days.
The peak of bacterial growth was noted at day 2.
Respiration to synthesis ratio (C released as CO2/C utilised in cel synthesis): on day 1: 2.7; on day 9: 25. A high ratio indicates most of the carbon is used in respiration.

BOD5 / COD results

BOD5 / CODopen allclose all
0.16 g O2/g test mat.
Results with reference substance:
The reference with glucose-glutamic acid solution (10 mg/l)showed immediate uptake of oxygen at the start., attaining its maximum rate within 5 dyas and thereafter the slope of the oxygen utilisation curve matched that of the seed blank. BOD5 1.49 mg/mg (compared to 1.45 +/- 0.07 mg/mg reported in the 'Standard Methods'. Bacterial growth showed a peak of about 2 x 10^6 org/ml on day 2, decreasing to apprx. 1x10^4 afterwards. COD and TOC immediately decreased to a low level at day 4.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
No specific criteria for this test. There are no replicates to compare the repeatability of the results. Yet the degradation is so fast there is no doubt. The reference substance degrades well and the blank control gives a low oxygen consumption.
Interpretation of results:
readily biodegradable
The test falls under the strict conditions defined for screening for ready biodegradability. The test substance completely mineralises within 5 days, without a lag time. This implies the test substance is readily biodegradable, meeting the 10d-window.
Executive summary:

A test method was described that can be considered as a pre-development of the various OECD screening test methods for ready biodegradation (OECD 301). The test conditions were as strict as in those screening tests: a low amount of non-adapted inoculum and a relatively low level of test substance. Oxalic acid completely mineralised in 5 days, indicating that the substance is readily biodegradable.