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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:
Study has been conducted between 13 April 2016 and 12 May 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands. The nearest plant treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water was spiked with mineral salts. Ammonium chloride was not added to the river water to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
as a percentage of ThOD
Details on study design:
Test bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients, stocks and administration:
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was not added to the river water to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of the test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with aluminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

Test procedure:
Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. 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. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Test conditions:
The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 7.9 (controls) and 7.8 (test). Temperatures were within the prescribed temperature range of 22 to 24°C.
Reference substance:
acetic acid, sodium salt
Remarks:
purity > 99%
Preliminary study:
Biodegradation of ≥60% was found within one week with both activated sludge and river water. For the final GLP test it is recommended to use river water as inoculum.
Test performance:
The test is valid as shown by an endogenous respiration of 1.4 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. Sodium acetate was degraded by 78% of its theoretical oxygen demand after 14 days. Inhibition of endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
72
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
82
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
86
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
88
Sampling time:
28 d
Details on results:
The substance is biodegraded by 88% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation at day 1. The test item therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds and hence the substance should be classified as readily biodegradable.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 78.

Toxicity to inoculum:

Inhibition of the degradation of a well degradable compound e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the high initial test substance concentration is expected.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The substance was biodegraded by 88% at day 28 in the Closed Bottle test, fulfilling the 14 day window criterion, and should therefore be classified as readily biodegradable.
Executive summary:

In order to assess the biodegradation of the test substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. In this study river water was exposed to 2 mg/L of the substance for 28 days. The substance did not cause a reduction in the endogenous respiration. Furthermore, the validity criteria of the test were met. The substance was biodegraded by 88% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation at day 1. Thus the substance fulfilled the 14-day time window criterion for ready biodegradable substances. The substance should therefore be classified as readily biodegradable.

Description of key information

In order to assess the biodegradation of the test substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. In this study river water was exposed to 2 mg/L of the substance for 28 days. The substance did not cause a reduction in the endogenous respiration. Furthermore, the validity criteria of the test were met.

The substance was biodegraded by 88% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 5 days immediately following the attainment of 10% biodegradation at day 1. Thus the substance fulfilled the 14-day time window criterion for ready biodegradable substances. The substance should therefore be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information