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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-12-14 - 1995-05-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

OECD Guideline for Testing of Chemicals; adopted 17 July 1992; "Ready Biodegradability, Closed Bottle Test" 301 D. Organisation for Economic Cooperation and Development, Paris

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

EC Guideline 92/69/EEC, Annex 1, Method C.4-E.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient room temperature, keep dry in closed vessels
- Stability under test conditions: stable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sewage plant effluent from municipal sewage plant Karlsruhe/Neureut

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
The test item is virtually insoluble in water. For this reason silica gel is used as a carrier (with reference to Annex III, No. 5 of OECD-Guideline 301D a permissible method):
366.95 g Silicagel 60 is heated for 1d at 250 °C. Following this pretreatment 37 ml (10%) water is added to the silica gel in a flask and the mixture is homogenized for three days by rotation. Yield: 403.95 g. 100 g is taken for the test blank. The test item is added to the remaining inactivated silica gel (303.61 g). The mixture is homogenized for 1 day by rotation. Concentration: 0.60 mg test item / g silica gel.
Two series of flasks with an overall volume of quite accurately 300 ml are prepared: one with addition of 1 g spiked silica gel per flask and one with 0.5 g spiked silica gel addition. This corresponds to test item concentrations of 2.0 mg/l and 1.0 mg/l test water. A blank with 1 g inactivated silica gel is performed, too. Subsequently, all the flasks are filled up to volume with test water.

TEST SYSTEM
- Culturing apparatus: flasks with an overall volume of quite accurately 300 ml
- Number of culture flasks/concentration: 1
- Measuring equipment: oxygen content measured electrometrically with O2 electrode compensation oxygenmeter WTW OXI 196， Wissenschaftlich-Technische Werkstatten GmbH, D-82362 Weilheim, according to DEV DIN 38 408-G22, November 1986

SAMPLING
- Sampling frequency: every 7 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 flask containing 1g inactivated silica gel
- Toxicity control: A test for inhibitory effects on the development of the bacteria is performed by cultivation of the inoculum in a culture medium (Trypton Soya Broth) in presence of the test substance with the highest concentration made use of in the closed bottle test (36 hours; 23°C).
The development of the bacteria is compared visually with a control without test material.

Reference substance:

other: sodium acetate and potassium hydrogen phthalate

Preliminary study:

There are no inhibitory effects to the development of the bacteria by the test material in its highest concentration or by test blank used in the Closed Bottle Test.

Test performance:

Within 7 days the biodegradability of the reference item sodium acetate has been reached a plateau. It clearly exceeds the required minimum degradation of 60 % within 14 days. Hence, the validity criteria are met.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other:

Remarks:

Due to the missing biological degradation of the test item the oxygen consumption shows little indifferent values close to zero. Within the measuring accuracy these values are equal to an oxygen consumption of zero. The nitrification of the degradation products was not analysed due to the missing degradation.

Details on results:

Evaluation of the Test Control
The Inhibition Test did not show any inhibitory effects on the development of the microorganisms bythe test item in the test concentration of 2.0 mg/l. In the Closed Bottle Test the test substance concentrations of 2.0 mg/l and 1.0 mg/l are analysed three times each.
The biological degradabilities of sodium acetate and potassium hydrogen phthalate as reference substances are clearly above the 60 % level after 14 days . Measured temperatures and O2 consumption of blank solutions are within the range demanded by the guideline.
The test controls document the correct run of the Closed Bottle Test.
Evaluation of the Degradation of Test Material
The chemical oxygen demand of the test item is 3005 mg O2/g substance regarding a complete degradation.
The biological degradation in the Closed Bottle Test is calculated by the BOD/COD ratio.
Test materials giving a measured BOD greater than 60 % of the TOD/COD within 28 days should be regarded as readily biodegradable. This level must be reached within 10 days of biodegradation exceeding 10 %.
There is no biological degradability of the test item during 28 days measurable by this method. Due to the missing degradation no analysis of possible nitrification was performed. Therefore, the criterion for "ready degradability" of the test item under aerobic conditions is not met according to OECD Guideline 301D.

Results with reference substance:

Within 7 days the biodegradability of sodium acetate has been reached a plateau. It clearly exceeds the required minimum degradation of 60 % within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test was performed according to OECD TG 301D with minor deficiencies, hence, the results can be considered sufficiently reliable. Positive controls gave appropriate results. The biochemical oxygen demand (BOD) of the test substance is determined to 0 mg O2 after 28 days. The biological degradation of the test substance (BOD/COD) is 0 %. Hence, the test item is not "readily biodegradable".

Executive summary:

The biological degradability of the test item was determined by the Closed Bottle Test according to OECD Guideline 301 D under GLP.

The test controls document the correct progress of the Closed Bottle Test.

The inhibition test doesn't show any inhibitory effects on the development of the microorganisms. The biological degradability of the reference material (sodium acetate and potassium hydrogen phthalate) is clearly above the 60 % level within 14 days.

The determination of the chemical oxygen demand (COD) of the test substance gives 3002 mg O₂/g.

The biochemical oxygen demand (BOD) of the test substance is determined to 0 mg O₂after 28 days. The biological degradation of the test substance (BOD/COD) is 0 % .

The result is that the test item is not "readily biodegradable" by the Closed Bottle Test, according to OECD Guideline 301D.

Description of key information

Biodegradation in water: Screening tests: No biodegradation observed, 0% (O2 consumption) in 28 days (OECD TG 301D, GLP)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed