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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement. This study was reliable with restrictions because all validity criteria could not be verified. No information on oxygen depletion in the inoculum blank is available.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Tests were modified to include studies on the effect of reducing agents and to allow measurements after 28 days.
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
Tests were performed under internal quality systems.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data
Oxygen conditions:
aerobic
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 and primary settled sewage were collected from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic sewage.
- Laboratory culture: no data
- Method of cultivation: no data
- Storage conditions: The primary settled sewage was collected weekly and stored at -20°C until required.
- Storage length: no data
- Preparation of inoculum for exposure: 150 mL of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit.
- Pretreatment: none
- Concentration of sludge: approximately 2 g DW/L of suspended solids
- Initial cell/biomass concentration: no data
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS

The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4•2H2O, 22.5 mg MgSO4•7H2O, 27.5 mg Ca¬Cl2, 0.25 mg FeCl3•6H2O. Ammonium chloride was omitted from the medium to prevent nitrifi-cation.

- Composition of medium: the deionized water used contained no more than 0.01 mg Cu/L. This water was prepared in a water purification system.
- Additional substrate: no
- Solubilising agent (type and concentration if used): yes (see other information on materials and methods, below)
- Test temperature: Temperature ranged from 19 to 21°C
- pH: The pH of the media was 7.0 at the start of the test. The pH of the medium at day 28 was 6.8 to 7.0.
- pH adjusted: no
- CEC (meq/100 g): no data
- Aeration of dilution water: yes
- Suspended solids concentration: 2 g DW/L of suspended solids
- Continuous darkness: no data
- Other: none

TEST SYSTEM

- Closed Bottle test:
Use was made of bottles containing only inoculum, and bottles containing the test substance. The concentration of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, tert butanol and 2,5-dimethyl-2,5-hexanediol (the potential decomposition products) in the bottles was 2 mg/L. The inoculum was diluted to 2 mg DW/L in the closed bottles. The inocula were derived from the communal wastewater treatment plant (unacclimatized) and the SCAS unit (acclimatized). Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. Subsequently the bottles were closed and incubated in the dark. Bottles of all series were withdrawn for analyses of the dissolved oxygen concentration. The course of the oxygen decrease in the bottles was measured using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After with-drawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).
Test performance:
Inhibition of the endogenous respiration of the inoculum by the test substance was not detected (data not shown). Inhibition of the biodegradation due to the "high" initial concentration of the test substance is therefore not expected. The pH of the media with sludge was 7.0±0.2. The pH of the river water at day 28 was 8.0 ±0.2. Temperatures ranged from 22 to 24°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Parameter:
% degradation (DOC removal)
Value:
ca. 0
Remarks on result:
other: after average 60 days
Details on results:
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

See Figure in the "Attached background material".
2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane is not biodegraded in the prolonged Closed Bottle test inoculated with unacclimated sludge.
Biodegradation of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was also not noted in the prolonged Closed Bottle test inoculated with sludge from the SCAS test. The sludge from the SCAS test was exposed to the test substance for 8 weeks allowing acclimatization. Based on these results it is concluded that it is very unlikely that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is biodegraded by microorganisms capable of growing on this test substance under completely aerobic conditions.
Results with reference substance:
Not applicable

2,5-Dimethyl-2,5-hexanediol and tert butanol are potential intermediates in the biodegradation process of the organic peroxide. 2,5-Dimethyl-2,5-hexanediol and tert butanol are degraded >80% within 56 days in the Closed Bottle test (See Figure in the "Attached background material"). Both potential intermediates were also as expected degraded in Closed Bottle tests inoculated with sludge exposed to 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. The degradation of 2,5-dimethyl-2,5-hexanediol with sludge exposed to the peroxide started almost immediately whereas with unacclimated sludge, lag periods of 2 to 4 weeks were noted. The absence of a lag period indicates that 2,5-dimethyl-2,5-hexanediol is formed in the SCAS unit fed with 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. This strongly indicates that 2,5-dimethyl-2,5-hexanediol was produced from 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the SCAS unit. It is hypothized that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is converted chemically in the SCAS unit during the settle period or with the help of chemicals excreted by the activated sludge present in the unit. During the settle period anaerobic conditions (reducing conditions) are created because aeration is stopped. Conversion of highly oxidized peroxide bonds is more likely under reducing anaerobic conditions. It is also possible that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane-2,5-di(tert-butylperoxy)hexane is reduced by microorganisms possessing reductases (enzymes) capable of inactivating toxic lipid peroxides. Microorganisms are present at very low concentrations in ready biodegradability tests. At these low concentrations, activity of these reductases may be insufficient to reduce the organic peroxides administered in the Closed Bottle test.

Validity criteria fulfilled:
no
Remarks:
all validity criteria could not be verified.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. However, potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge.
Executive summary:

This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement.

2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. This result demonstrates that microorganisms do not have the ability to grow on this organic peroxide under aerobic conditions. Potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge. The length of the lag period found with 2,5-dimethyl-2,5-hexanediol with acclimated sludge was short compared to the period obtained with unacclimated sludge. This result suggests that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is cleaved in the SCAS unit.

Description of key information

One key study was available on the biodegradation of Trigonox 101. Trigonox 101 was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. However, potential decomposition products of Trigonox 101 i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge (>80% within 56 days).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

One key study was available on the biodegradation of Trigonox 101. This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement. In the SCAS test, Trigonox 101 was exposed to activated sludge maintained by daily addition of primary settled sewage for a period of 8 weeks. The test substance caused no reduction of the biodegradation of the organic compounds present in primary settled sewage. Therefore the test substance is considered to be non-inhibitory to the activated sludge.

Trigonox 101 was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. This result demonstrates that microorganisms do not have the ability to grow on this organic peroxide under aerobic conditions. Potential decomposition products of the test substance i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge (> 80% within 56 days).The length of the lag period found with 2,5-dimethyl-2,5-hexanediol with acclimated sludge was short compared to the period obtained with unacclimated sludge. This result suggests that the peroxide bond of Trigonox 101 is cleaved in the SCAS unit.