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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:
04-10-2016 till 05-12-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
slightly modified, not affecting the outcome of the study, see below
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
- Source of inoculum/activated sludge: River water was sampled from the Rhine near Heveadorp, The Netherlands (29-09-2016). The nearest plant (Arnhem-Zuid) 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 inoculum was not pre-exposed to the test substance.

Duration of test (contact time):
60 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers

- Nutrients, and stocks: 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 and the content was mixed vigorously. Subsequently 0.10 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 1.0 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 procedures: The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). 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 1.0 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. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 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 withdrawal 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 conditions: The pH of the media was 8.0 at the start of the test. The pH of the media at day 28 was 7.9 (test and controls). Temperatures were within the prescribed temperature range of 22 to 24°C.
Reference substance:
acetic acid, sodium salt
Remarks:
purity > 99%
Test performance:
- Validity of the test: The validity of the test is demonstrated by an endogenous respiration of 1.2 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 85. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Parameter:
% degradation (O2 consumption)
Value:
44
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
52
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
52
Sampling time:
21 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
59
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
59
Sampling time:
42 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
60 d
Details on results:
Biodegradability: The test substance was biodegraded by 59% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test the test substance is biodegraded by 63% at day 60. A biodegradation percentage in excess of 60 within the 60-day test period allows categorisation of the test substance as not persistent.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 85.

- Toxicity: 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:
not readily biodegradable
Conclusions:
Under the conditions of the test, the test substance was biodegraded by 59% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 63% at day 60 (enhanced biodegradability testing). A biodegradation percentage of >60 within a 60-day time period allows categorisation of the substance as not persistent.
Executive summary:

In order to assess the biodegradation of the test substance, a ready biodegradability test was performed with the Closed Bottle test according to slightly modified OECD301D guideline and under GLP conditions. In this study river water was exposed to 1 mg/L of the substance for 60 days. Ammonium chloride was not added to the river water to prevent nitrification, this modification was not considered to affect the outcome of the study. The test substance did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Furthermore, the validity criteria of the test were met. The test substance was biodegraded by 59% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 63% at day 60 (enhanced biodegradability testing). A biodegradation percentage of >60 within a 60-day time period allows categorisation of the test substance as not persistent.

Description of key information

Ready biodegradability (Closed Bottle test, OECDTG 301D): not readily biodegradable.

In order to assess the biodegradation of the test substance, a ready biodegradability test was performed with the Closed Bottle test according to slightly modified OECD301D guideline and under GLP conditions. In this study river water was exposed to 1 mg/L of the substance for 60 days. Ammonium chloride was not added to the river water to prevent nitrification, this modification was not considered to affect the outcome of the study. The test substance did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Furthermore, the validity criteria of the test were met. The test substance was biodegraded by 59% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, the test item was biodegraded by 63% at day 60 (enhanced biodegradability testing). A biodegradation percentage of >60 within a 60-day time period allows categorisation of the test substance as not persistent.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The substance just not meets the 60% criterion within 28-days. In view of the plateau reached up to 43 days and the absence of ready biodegradability in the BIOWIN models the substance will be regarded as non readily biodegradable.