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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:
Initiation date: 1989.09.01 Completion date: 1989.10.05
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP test with all appropriate validity criteria but the temperature is not reported. There are some minor deviations to the guideline and some informations are scarce.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Minor adaptions to protocol
Principles of method if other than guideline:
Activated Sludge was used as inoculum.The sludge was preconditioned to reduce endogenous respiration rates. Ammonium chloride was ommitted
from the medium to prevent nitrification. The test was also prolonged. The final pH is a bit low (6.7 instead of 7.4+/-0.2 recommended).
GLP compliance:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge was obtained from the RZWI Nieuwgraaf in Duiven (1989.09.01). The RZWI Nieuwgraaf is an activated sludge plant treating predominantly domestic wastewater.
- Laboratory culture: Not specified
- Method of cultivation: Not specified
- Storage conditions:
- Storage length:
- Preparation of inoculum for exposure: according to the method described in a proposal for harmonizing ready biodegradability test protocols (Blok et al, 1985) and a draft of an EEC update (Test method C9 to C10 of Annex V Doc XI/501/89, 1989)
- Pretreatment: The sludge (200 mg DW/l) was aerated for a period of six days
- Concentration of sludge: The sludge was diluted to a concentration in the BOD bottles of 2 mg DW/l.
- Initial cell/biomass concentration: Not specified
- Water filtered: Not specified
- Type and size of filter used, if any: Not specified
Duration of test (contact time):
98 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The closed bottle test was performed according to the EEC (1984) and OECD 301D (1982) test guidelines with slight modifications ( listed above ).

- Composition of medium: Not specified
- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: Not specified
- pH: 6.7 at the end of the test period
- pH adjusted: Not specified
- CEC (meq/100 g):Not specified
- Aeration of dilution water: Not specified
- Suspended solids concentration: Not specified
- Continuous darkness: Not specified
- Other: None

- Culturing apparatus: The test was performed in 280 ml BOD (Biological Oxygen Demand) bottles
- Number of culture flasks/concentration: 2 replicates for Blank control (without test material and inoculum), Blank inoculum (without test material but with inoculum), Test material (2 mg/l) with inoculum and Reference substance (Sodium acetate, 6.7 mg/l) with inoculum.
- Method used to create aerobic conditions: Not specified
- Method used to create anaerobic conditions: Not applicable
- Test performed in closed vessels due to significant volatility of test substance: Yes

Reference substance:
acetic acid, sodium salt
Sodium acetate
Preliminary study:
No information regarding preliminary studies.
Test performance:
The validity of the test is shown by the oxygen consumption in the control bottle with sodium acetate and an endogenous respiration of 0.2 mg/litre.
% degradation (O2 consumption)
Sampling time:
28 d
% degradation (O2 consumption)
Sampling time:
98 d
Details on results:
Test chemical is not biodegraded in the closed bottle test and therefore should not be classified as readily biodegradable. This lack of biodegradation is not due to the toxicity of the test compound because the endogenous respiration is not inibited by di-tert-butyl peroxide.
Results with reference substance:
87% degredation after 28 days.

Guideline suitably followed and relevant quality criteria reported except temperature. Study was conducted under GLP. Although no certificate of analysis was included purity and batch number was reported. Information over temperature and calculation of theoretical oxygen demand is lacking. Test was conducted only in duplicate and a higher degradability might be expected with more replicate.

Validity criteria fulfilled:
Oxygen depletion in the inoculum blank didn't exceed 1.5 mg dissolved oxygen/l after 28 days. The residual concentration of oxygen in the test bottles didn't failed below 0.5 mg/l at any time.
Interpretation of results:
other: Not readily Biodegradable.
The test was carried out reliably without major restrictions. The substance was not readily biodegradable in this test. Optimization of the test method or alternative may yield a higher degredation result. It can be concluded that this substance is not readily biodegradable in the closed bottle test.
This lack of biodegradation is not due to the toxicity of the test compound because the endogenous repiration is not inhibited by di-tert-butyl peroxide. The lack of biodegradation in the closed bottle test does not mean that di-tert-butyl hydroperoxide is recalcitrant in nature. The stringency of the
procedures could account for the recalcitrance of di-tert-butyl peroxide in the closed bottle test.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the closed bottle test performed according to slightly modified OECD 301D, EEC and ISO Test Guidelines. Microorganisms (Secondary activated sludge at concentration of 2 mg DW/L, obtained from the RZWI Nieuwgraaf in Duiven (1989.09.01): plant treating predominantly domestic wastewater) are inoculated into a chemically defined liquid medium containing the test substance (Di-tert-butyl at 2 mg/L) or the reference substance (Acetic acid, sodium salt or Sodium acetate at 6.7 mg/L) under aerobic conditions for a period of 98 days; the test was prolonged because the pass level was not reached at day 28. Use was made of 2 replicates for blank control (without test material and inoculum), for blank inoculum (without test material but with inoculum), for test material with inoculum, and for reference substance with inoculum. The pH was 6.7 at the end of the test period. Parameter followed for degradation estimation was O2 consumption. The dissolved oxygen concentrations were determined electrochemically using a oxygen electrode and meter at days 0, 5, 15, 28, 42, 56, 71 and 98.

The test substance caused no reduction in the endogenous respiration. Therefore, Di-tert-butyl is considered to be non-inhibitory to the inoculum. Di-tert-butyl was biodegraded 6% at day 28 under the stringent conditions in the close bottle test and, therefore should not be classified as ready biodegradable. This study was carried out under GLP and fulfilled validity criteria. Some minor deviations to the guidelines were justified but the temperature was not reported and some informations are scarce.

Description of key information

The substance is not readily biodegradable in a ready biodegradation study.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The biotic degradability of the substance was assessed in a GLP study according to OECD 301D, close bottle test (Ginkel, 1990). The study is reliable and found no significant biodegradation (6%) after 28 days while no inhibition was observed and the reference substance was successfully degraded. The study was then prolonged to 98 days without further biodegradation. It is finally concluded the substance is not readily biodegradable.