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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:
8th of January 2014 until 10th of March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study according to OECD guideline, without deviations that influence the results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Omission of ammonium chloride from the medium to prevent nitrification. The omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound.
GLP compliance:
yes
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (02-01-2014) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles to 2.0 mg/L (van Ginkel and Stroo, 1992).
Duration of test (contact time):
<= 60 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 nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Accurate administering of the Pinyl iso butyraldehyde 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 Pinyl iso butyraldehyde 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 inoculum, 10 bottles containing medium with inoculum and silica gel, 10 bottles containing medium with inoculum and silica gel with test substance, 6 bottles containing sodium acetate and medium with inoculum, and 6 bottles containing test substance, sodium acetate and medium with inoculum. 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. 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 7.2 at the start of the test. The pH of the medium at day 28 was 7.2 (tests and controls). Temperatures were within the prescribed temperature range of 22 to 24°C.
Reference substance:
acetic acid, sodium salt
Remarks:
purity > 99%
Preliminary study:
A study was run parallel to this study to investigate the influence of the source of inoculum and test substance dose (AkzoNobel, 2014, F14040 CG).
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 93% of its theoretical oxygen demand after 14 days. Degradation of sodium acetate was not inhibited by the test substance. 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:
3
Sampling time:
28 d
Remarks on result:
other: 2 mg/l test substance concentration
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Remarks on result:
other: 2 mg/l test substance concentration
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
14 d
Remarks on result:
other: 2 mg/l test substance concentration
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
21 d
Remarks on result:
other: 2 mg/l test substance concentration
Parameter:
% degradation (O2 consumption)
Value:
29
Sampling time:
60 d
Remarks on result:
other: 2 mg/l test substance concentration
Details on results:
The test substance is biodegraded by 3% at day 28 in the Closed Bottle test. 29% biodegradation was achieved after 60 days. Pinyl iso butyraldehyde should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that pinyl iso butyraldehyde is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 93%.

Depletion of oxygen in the bottles with the test substance and sodium acetate and the bottles with only the reference substance was comparable. Inhibition of microorganisms capable of degrading sodium acetate by pinyl iso butyraldehyde present at a concentration of 2 mg/L did therefore not occur. However, slight inhibition of the endogenous respiration of the inoculum by the test substance was detected during the first two weeks of the test. Inhibition of the onset of biodegradation due to the "high" initial test substance concentration can therefore not be excluded.

Validity criteria fulfilled:
yes
Remarks:
Validity of the test is demonstrated by: an endogenous respiration of 1.4 mg/L at day 28, oxygen concentrations >0.5 mg/L in all bottles during the test period, and the differences of the replicate values at day 28 were less than 20%.
Interpretation of results:
other: not readily biodegradable
Conclusions:
The substance is biodegraded by 3% at day 28 in the Closed Bottle test and should therefore be classified as not readily biodegradable.
Executive summary:

The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D under GLP. Activated sludge is exposed to 2 mg/l of test substance. At day 7, 14, 21 and 28 days the following degradation rates were measured 0, 0. 2 and 3%, respectively. Pinyl iso butyraldehyde should therefore be classified as not readily biodegradable. Pinyl iso butyraldehyde caused a slight inhibition of the endogenous respiration. This test in activated sludge was prolonged (non-GLP) and the test substance was biodegraded by 29% at day 60.

Description of key information

Several Ready test results are available which will be presented one by one in the additional information section.

Closed Bottle test OECD TG 301D: Not ready biodegradable

Extended Closed Bottle test (OECD TG 301D, non-GLP: Not persistent

MITI test (OECD TG 301C: Not readily biodegradable but full primary degradation into the substance respective acid and alcohol

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
other: activated sludge

Additional information

Ready biodegradability test, Closed Bottle test in activated sludge

The ready biodegradability was determined in the Closed Bottle test (OECD TG 301D under GLP. Activated sludge is exposed to 2 mg/l of test substance. At day 7, 14, 21 and 28 days the following degradation rates were measured 0, 0. 2 and 3%, respectively. The substance should therefore be classified as not readily biodegradable. The substance caused a slight inhibition of the endogenous respiration. This test in activated sludge was prolonged (non-GLP) and the test substance was biodegraded by 29% at day 60.

Ready biodegradability test, Closed Bottle test in river water and activated sludge

Introduction and method: The ready biodegradability of Pinyl isobutyraldehyde was investigated in a series of Closed Bottle tests according to OECD TG301D, not under GLP. The goal of this study was to investigate the influence of the source of inoculum and test substance concentration on the biodegradability of the substance. Tests were performed with activated sludge and river water as inocula, and with test substance concentrations of 1 and 2 mg/L.

Results: Inhibition of the endogenous respiration of the inoculum during the first 2 weeks was detected in the presence of the substance independent on the type of inoculum and concentration, except in river water and 1 mg/l concentration no inhibition was seen. The following results were seen at day 7,14, 21, 28, 42, 56 and 70.

Activated sludge 2 mg/l: -2, -2, 1, 5, 12, 32, 66%, respectively;

River water 2 mg/l: -3,-1, 6, 14, 32, 47 and 66%, respectively;

River water 1 mg/l: -1, 0, 2, 18, 54, 73 and last time point not measured, respectively.

Discussion and Conclusion: Biodegradation percentages of < 20 were found at day 28 with both river water and activated sludge as inocula. The substance is therefore not readily biodegradable. After 56 to 70 days biodegradation percentage in excess of 60 were found in a prolonged Closed Bottle test. Higher biodegradation rates were found with river water compared to the tests performed with activated sludge as inoculum. A result of >60% was obtained with river water and an initial test substance concentration of 1.0 mg/L and therefore the substance is not persistent.

Ready biodegradability in MITI test:

In order to assess the biodegradation of the substance a modified MITI test according to OECD 301C was performed and a "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances", was applied under GLP conditions. In this study a mixture of activated sludge (containing samples from surface water and surface soil of rivers, lakes and inland sea, and return sludge from sewage plants) was exposed to 100 mg/L of the substance for 28 days. The validity criteria of the test were met. Though it needs to be noted that in the concentration of the test item in the test exceeded its water solubility (100 versus 10 mg/l, respectively) and the pH was not measured to limit the volatilisation from the test vessels. The test item was measured and ca 85% could be recovered, the ca 15% loss was probably due to volatilisation. The test item was not biodegraded (-1%) during the 28 -days and is therefore not readily biodegradable. The substance slightly decreased the oxygen consumption of the bacteria, which is an indication of the toxicity of the substance or its metabolites. The test item was fully primary degraded and the metabolites were identified: the aldehyde functional group was converted to an acid and/or an alcohol. The concentration of these two could not be quantified and not be distinguished. The pH was not measured to limit the volatilisaton of the test item.