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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:
17-09-2010 - 22-10-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP, according guidelines with acceptable minor deviations, meeting quality/validity criteria.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
minor acceptable deviations
Principles of method if other than guideline:
One deviation introduced from the guidelines of the Closed Bottle test was; ammonium chloride was not added 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. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (17-09-2010). 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
(van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by
sedimentation after 1 day while aerating.


Duration of test (contact time):
28 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 0.3 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients and stock solutions
The river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2 HPO4·2H2O,
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 and the test substance were added to the bottles using aqueous stock solution of 1.0 and 0.1 g/L, respectively.

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 test substance, and 6 bottles containing 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.

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 bottle with river water.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of tert-butyl peroxy benzoate 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:
Reference compound acetic acid, sodium salt; CAS reg. No. 127-09-3; purity >99%; batch lot No. A0206783001; appearance white crystals; solubility soluble in water; storage at ambient temperature in the dark.
Test performance:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of tert-butyl peroxybenzoate is 2.14 mg/mg.
The calculated theoretical oxygen demand (ThoD) of sodium acetate is 0.78 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 tert-butyl peroxy benzoate to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the
inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test
compound is expected.

Test conditions
The pH of the river water spiked with mineral salts was 7.2 at the start of the test. The pH of the medium at day 28 was 7.3 (control and test). Temperatures
were within the prescribed temperature range of 22 to 24°C.

Validity of the test
The validity of the test is demonstrated 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%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 78. Finally, the validity of the test is
shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
28 d
Remarks on result:
other: 60% biodegradation was achieved in a period of 11 days
Details on results:
tert-Butyl peroxy benzoate is biodegraded 70% at day 28 in the Closed Bottle test . Over 60% biodegradation was achieved
in a period of 11 days immediately following the attainment of 10% biodegradation. The 10 day window can be increased to 14 days
for the Closed Bottle test and this criterion has therefore been fulfilled.

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)

Oxygen concentration (mg/L)   

 

Oc

Ot

 Oa

0

8.8

8.8

8.8 

 

8.8

8.8

8.8 

Mean (M)

8.8

8.8

 8.8

7

8.1

7.3

 4.8

 

8.2

7.3

 4.6

Mean (M)

8.2

7.3

 4.7

14

7.8

6.4

3.5 

 

7.7

6.3

3.7

Mean (M)

7.8

6.4

 3.6

21

7.4

6.0

 

 

7.5

6.0

 

Mean (M)

7.5

6.0

 

28

7.4

5.9

 

 

7.4

5.9

 

Mean (M)

7.4

5.9

 

Ot       River water with mineral nutrient solution with test material (1.0 mg/l)

Oc       River water with mineral nutrient solution   

Oa River water with mineral nutrient solution with sodium acetate (6.7 mg/l)

Table II  Oxygen consumption (mg/L) and the percentages biodegradation of tert-butyl peroxybenzoate (BOD/ThOD) and sodium acetate (BOD/ThoD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

 
 Biodegradation (%)    

Test substance

Acetate

Test substance   Acetate 

0

0.0

0.0

 0

7

0.9

3.5

42  56 

14

1.4

4.2

65  78 

21

1.5

70   
 28  1.5    70  

 

Validity criteria fulfilled:
yes
Remarks:
see test performance
Interpretation of results:
readily biodegradable
Conclusions:
Test performed under GLP, according guidelines with acceptable minor deviations, meeting quality/validity criteria.
Tert-butyl peroxybenzoate was biodegraded 70% at day 28 in the Closed Bottle test (Figure and Table II). The time window determined is 11 days (Figure). The substance should therefore be classified as readily biodegradable
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. tert-Butyl peroxy benzoate did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. tert-Butyl peroxy benzoate was biodegraded 70% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of 11 days immediately following the attainment of 10% biodegradation. Hence this substance should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 78% 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

The substance is readily biodegradable meeting the 10 day window in a closed bottle test (OECD 301D)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Four studies were available, a MITI test for which insufficient information was available on the methodology used. One valid, GLP, OECD 301D test in which a total biodegradation level of 72% within 28 days was reached. However, the 10 day window for this test was not reached. One non-GLP OECD 301D was performed in which a total biodegradation level of 70% within 28 days was reached and the criterion (for the Closed Bottle Test a 14 -day window) was fulfilled. Finally one valid, GLP, OECD 301 test was performed in which a total biodegradation level of 70% was reached. Over 60% biodegradation was achieved in a period of 11 days immediately following the attainment of 10% biodegradation. Hence this substance should be classified as readily biodegradable.