NITTA

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:

30 March 1999 to 28 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation is to adsorb the test material onto an inert support prior to dispersion in the test vessels. Using this method the test material is evenly distributed throughout the test medium and the surface area of test material exposed to the test organism is increased thereby increasing the potential for biodegradation.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Appearance: brown viscous liquid
- Storage conditions: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge: a mixed population of activated sewage sludge microorganisms was obtained from the aeration stage of the Severn Trent Water plc sewage treatment plant (Belper, Derbyshire, UK)
Preparation of inoculum:
The sample of activated sewage sludge was maintained on continuous aeration. A sample was taken, washed three times by settlement and re-suspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

12.7 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Medium was preprared as follows:
To 1 litre (final volume) of purified water was added to the following volumes of solution a-d.
10 mL of Solution a and 1 mL of each of Solutions b, c and d.
Solution a (KH2PO4, 8.50 g/L; K2HPO4, 21.75 g/L; Na2HPO4.2H2O, 33.40 g/L, NH4Cl, 0.50 g/L), pH 7.4
Solution b (CaCl2, 27.50 g/L)
Solution c (MgSO4.7H2O, 22.50 g/L)
Solutiond (FeCl3.6H2O, 0.25 g/L)

- Test temperature: 21 °C
- Suspended solids concentration: 30 mg suspended solids/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels each containing 3 L of solution (approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of culture medium and 37.5 mL of inoculum and aerated overnight. On day 0 the test and reference materials were added and the volume in all the vessels adjusted to 3 L by the addition of culture medium)
- Number of culture flasks/concentration:
a) a control, in duplicate, consisting of inoculated culture medium plus 100 mg silica gel
b) the reference substance (sodium benzoate), in duplicate, in inoculated culture medium plus 100 mg silica gel to give a final test concentration of 10 mg carbon/L
c) the test material plus 100 mg silica gel, in duplicate, in inoculated culture medium t give a final test concentration of 10 mg carbon/L
d) the test material and the standard material plus 100 mg silica gel in inoculated culture medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one essel only)
- Method used to create aerobic conditions: culture vessles were sealed and CO2-free air bubbled through the solution at a rate of ca. 40 mL/minute and stirred continuously by a magnetic stirrer
- Measuring equipment: Samples were analysed for CO2 using an Ionics 1555B TOC analyser and a Dohrmann DC-190 TOC analyser. Calibration was by a standard soution of sodium carbonate.
- Details of trap for CO2 and volatile organics if used: the CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorbed vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.
- Sample storage before analysis: The samples taken on days 0, 1, 2, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on days 3 and 6 were stored deep frozen at -20 °C prior to analysis. The samples taken on days 12 and 18 were also stored deep frozen at -20 °C. However, these samples were not analysed for CO2 as the results from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.
- Other: on day 28 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on day 29.

Reference substance

Reference substance:

other: Sodium benzoate

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

39

Sampling time:

28 d

Details on results:

Points of degradation plot (test substance):
0 % degradation after 3 d
5 % degradation after 6 d
9 % degradation after 10 d
18 % degradation after 20 d
39 % degradation after 28 d

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 90% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Points of degradation plot (reference substance):
48 % degradation after 3 d
63 % degradation after 6 d
67 % degradation after 10 d
79 % degradation after 20 d
90 % degradation after 28 d

Any other information on results incl. tables

The total CO₂ evolution in the control vessels on day 28 was 34.53 mg/L and therefore satisfied the validation criteria in the guidelines.

The test material attained 39 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the conditions of the study.

The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO₂ present in solution being driven off by the addition of hydrochloric acid on day 28 and resulted in an increase in the percentage degradation value for the test material from 39% on day 28 to 43% on day 29. Inorganic carbon analysis of the samples from the second absorber vessels on day 29 confirmed that no significant carry-over of CO₂ into the second absorber vessels occurred.

The TC/IC ratio of the test material dispersion was in excess of the recommended level of 5% given in the test guidelines. This is considered to be due to the low TC concentration in the test medium and hence the IC contribution is relatively large. This is not considered to affect the integrity of the study or the results obtained given that the CO₂ evolution in the control vessels satisfied the validation criteria.

The toxicity control attained 55% degradation after 28 days, which suggests that the test material was not toxic to the sewage treatment micro-organisms used in the study. The icnrease in inorganic carbon in the first absorber vessels on day 29 resulted in an increase in the percentage degradation value for the toxicity control from 55% on day 28 to 59% on day 29.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test material attained 39% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the conditions of the study.

Executive summary:

The ready biodegradability of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and EPA OPPTS 835.3110.

During the study the test material was exposed to activated sewage sludge microorganisms at a concentration of 10 mg C/L with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

In order to aid dispersion of the test material in the test system and to increase the surface area of the test maerial available to the activated sewage sludge microorganisms, the test material was adsorbed onto silica gel prior to addition to the test vessels.

The test material attained 39% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the conditions of the study.