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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:
2009-09-21 to 2009-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
minor deviation from the guideline: ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by biodegradation of the reference compound)
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge (16-09-2009) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Ne¬therlands. 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 (van Ginkel and Stroo, 1992).

. The inoculum was not pre-exposed to the test substance.
Duration of test (contact time):
28 d
Initial conc.:
2 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, 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. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L. The test substance was administered 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 OECD TG 301D (1992) and an ISO TG (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and test substance, 6 bottles containing sodium acetate and inoculum, and 6 bottles containing test substance, sodium acetate and 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.

Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottles.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test item and sodium acetate were calculated from their molecular formulae and molecular weights

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mc - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma

Mc is the mean oxygen level in the control bottles, n-days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.

The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Remarks:
Supplier: Acros organics, Geel, Belgium; Purity >99% ; Batch/lot number A0206783001
Key result
Parameter:
% degradation (O2 consumption)
Value:
84
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Details on results:
The calculated theoretical oxygen demand (ThOD) of the substance are 2.5 (NH3) and 3.1 (NO3) mg/mg. The ThOD of sodium acetate is 0.8 mg/mg

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test item to micro¬organisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the ino¬culum by the test substance was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected.

Biodegradability
The test item is biodegraded 84% at day 28 in the Closed Bottle test (Figure and Tables I-II), and should therefore be classified as readily biodegradable.
The test item] is a surfactant. Surfactants are chemicals in which a hydrophilic group is linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of fatty amine derivatives are degraded sequentially. Ammonium formed through degradation of the substance is oxidized by nitrifying bacteria. Nitrifying bacteria are not involved in the biodegradation of the substance]. The biodegradation of the two moieties of the substance and the oxidation of ammonium may be fully in line with the 10-day window criterion when judged as separate chemicals whereas the sum of the biodegradation curves is not. The biodegradation curves ofthe test item should therefore not be used to assess a 10-day effect.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 76.

Oxygen consumption (mg/L) and the percentage biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test

 Time (days) Oxygen consumption     Biodegradation (%)    
   Test substance Acetate  Test substance ThODNO3 Acetate 
 0.0  0.0  0  0
 7  2.6  3.7  42  69
 14  5.0  4.1  81  76
 21  5.1    82  
 28

 5.2

 

 84

 

Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of 1.3 mg/L at day 28. Differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of reference compound at day 14 was 76. Oxygen concentrations >0.5 mg/L in all bottles during the test period.
Interpretation of results:
readily biodegradable
Remarks:
The test substance is an UVCB and therefore the time window should not be applied be
Conclusions:
The test item should be classified as readily biodegradable.

The test item is an UVCB consisting of different constituents. Biodegradation of the constituents requires the concerted action of different microorganisms as a single organism usually lacks the full complement of enzymatic capabilities. The biodegradation of each constituent may be fully in line with the time window criterion when judged as separate chemicals. The time window should therefore not be applied to substances consisting of structurally similar constituents (OECD, 2006).
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, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item at a concentration of 2.0 mg/L did not inhibit microorganisms growing on acetate. Moreover, the test substance did also not cause a reduction in the endogenous respiration of the microorganisms at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test item biodegraded by 84% at day 28 in the OECD 301 Closed Bottle test. The test item should therefore be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

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, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item at a concentration of 2.0 mg/L did not inhibit microorganisms growing on acetate. Moreover, the test substance did also not cause a reduction in the endogenous respiration of the microorganisms at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test item biodegraded by 84% at day 28 in the OECD 301 Closed Bottle test. The test item should therefore be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information