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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989.09.01 - 1989.10.05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test performed under GLP, according to guidelines with acceptable deviations but no information reported on test medium and temperature. Due to the poor solubility of the substance in water, it was dissolved in a solvent which was allowed to evaporate in a ventilated hood for 3 hours. An hypothetical loose of test substance by volatility was considered but it would resulted anyway in an under estimation of the biodegradability.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Deviations:
not specified
Principles of method if other than guideline:
- Instead of an effluent/extract mixture, activated sludge was used as an inoculum.
- Ammonium chloride was omitted from the medium to prevent nitrification
- The test compound is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane. The test substance in dichloromethane was added to silica gel weighed in a glass petri dish. The solvent was allowed to evaporate by placing the petri dish in a ventilated hood for 3 hours, and the entire contents were then transferred to the BOD bottles.
GLP compliance:
yes
Remarks:
certificate 1990-05-17
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge was obtained from the plant RZWI Nieuwgraaf in Duiven treating predominantly domestic wasteater.
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg DW/liter) was aerated for a period of 6 days. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg DW/liter.
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 CONDITIONS
- Composition of medium: not specififed. Deionnised water containing no more than 0.01 mg Cu/l was prepared in an Elgastat water purification system (Elga, Breukelen, The Netherlands).
- Test temperature: not specified
- pH: 7.3 in the medium at the end of the test period
- pH adjusted: No
- Aeration of mineral medium: Yes
- Suspended solids concentration: 2 mg DW/l of sludge
- Continuous darkness: Yes

PREPARATION OF TEST SUBSTANCES
- The test substance is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane (stock solution of 1000mg/l) . The test substance in dichloromethane (0.56 ml) of was added to 2g silica gel (100-200 mesh) weighed in a glass petri dish. The solvent was allowed to evaporate by placing the petri dish in a ventilated hood for 3 hours, and the entire contents were then transferred to the BOD bottles. Although no additional oxygen consumption was expected, controls with silica gel were carried out as well. This technique was described in "N. Nyholm, C. Seiero: Biodegradability testing of poorly soluble compounds by means of manometric respirometry, ISO/TC 147SC5/WG4, 1989".
- The sodium acetate was added to the bottles using a stock solution of 1000 mg/l.

TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
2 replicates containing only inoculum = inoculum blank
2 replicates containing no inoculum and no test substance = abiotic control
2 replicates containing test substance on silica gel and inoculum =test
2 replicates containing silica gel and inoculum = inoculum and silica gel blank
2 replicates containing evaporated silica gel and inoculum = inoculum and evaporated silica gel blank
2 replicates containing sodium acetate and inoculum = reference control
- Method used to create aerobic conditions: aeration with pressured air


SAMPLING
- Sampling frequency: day 0, 5, 15,28

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
- Reference control: yes



CALCULATIONS:
THOD test substance (pure active ingredient) = 2.69 mg O2/mg test substance
THOD sodium acetate = 0.8 mg O2/mg sodium acetate
Oxygen consumption (mg/l) (BOD) = mean oxygen concentration (mg/l) inoculum blank - mean oxygen concentration (mg/l) test (or reference)
Biodegradation (%) = BOD/THOD *100
The oxygen consumption for the test substance is calculated with the inoculum blank with silica gel.
The oxygen consumption for the reference is calculated with the inoculum blank without silica gel.


Reference substance:
acetic acid, sodium salt
Remarks:
sodium acetate at 6.7 mg/l
Preliminary study:
None
Test performance:
mg:l- Difference of extremes of replicate values of the removal of the test chemical at the end of the test is 4% (<20%).
- Percentage degradation of the reference substance has reached 90% (>60%) by day 14.
- Oxygen depletion in the inoculum blank is 0.9 mg O2/l after 28 days (<1.5 mg O2/l).
- The residual concentrations of oxygen in the test bottles don't fall below 0.5 mg/l at any time.
Parameter:
% degradation (O2 consumption)
Value:
39
Sampling time:
5 d
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
15 d
Parameter:
% degradation (O2 consumption)
Value:
61
Sampling time:
28 d
Details on results:
Falling 10 days window .
Results with reference substance:
Sodium acetate was biodegraded 94% in the closed bottle test at day 28
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Dilauroyl peroxide was biodegraded 61% in the closed bottle test at day 28 and therefore should be classified as readily biodegradable but falling the 10-days window criteria. Because the biodegradation curve is only made of 3 measuring points it is difficult to determine the 10 day window and therefore it is not possible to say if this criteria is met.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allowed 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 301D, EU Method C.6 Guideline. Microorganisms (Secondary activated sludge at concentration of 2 mg DW/L, obtained from the RZWI Nieuwgraaf in Duiven: plant treating predominantly domestic wastewater) are inoculated into a chemically defined liquid medium containing the test substance (Dilauroyl peroxide at 2 mg/L) or the reference substance (Sodium acetate at 6.7 mg/L) under aerobic conditions for a period of 28 days. The test substance is a poorly soluble substance in water and therefore the test substance was first dissolved in dichloromethane, added to silica gel weighed in a glass petri dish. Use was made of 2 replicates containing only inoculum (inoculum bank), 2 replicates containing no inoculum and no test substance (abiotic control), 2 replicates containing test substance on silica gel and inoculum (test), 2 replicates containing silica gel and inoculum (inoculum and silica gel blank), 2 replicates containing evaporated silica gel and inoculum (inoculum and evaporated silica blank), and 2 replicates containing sodium acetate and inoculum (reference control). The pH was measured, it was 7.3 in the medium at the end of the test period. Parameter followed for degradation estimation was O2 consumption. The dissolved oxygen concentrations were determined electrochemically using a oxygen electrode and meter. The percentages biodegradation of Dilauroyl peroxide in the closed bottle test were 61% for 28 days. The percentages biodegradation of sodium acetate in the closed bottle test were 94% for 28 days. Dilauroyl peroxide is biodegraded in the closed bottle test and therefore should be classified as readily biodegradable but failing the 10 day window.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-12-2010 - 21-01-2011
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
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. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (17-12-2010). 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.:
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 0.3 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 was added to the bottles using aqueous stock solution of 1.0 g/L. Accurate administration of
water-insoluble dilauroyl peroxide was accomplished by dissolving the test substance in dichloromethane (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). The test substance in dichloromethane (0.6 ml) was directly added to bottles. Dichloromethane (0.6 ml) was added to a series of control bottles. The solvent was allowed
to evaporate by placing the bottles on a roller bank in a ventilated hood for 24 hours to obtain an even distribution of the test substance on the walls of the bottles. Use was made of 10 bottles containing only river water (not agitated), 6 bottles containing river water and sodium acetate (not agitated), 10 bottles containing river water and test substance (agitated), and 10 bottles treated with dichloromethane (added and evaporated) containing river water. 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. The contents of bottles with silicone oil were agitated with magnetic stirrers at 600 rpm. 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 2.7 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 dilauroyl peroxide 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 8.0 at the start of the test. The pH of the medium at day 28 was 7.9 (agitated control and test) and
8.0 (control). 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 1.3 mg/L at day 28 (Table I). 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 74. 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:
80
Sampling time:
28 d
Remarks on result:
other: 60% biodegradation was achieved in a period of less than 8 days
Details on results:
Dilauroyl peroxide is biodegraded 80% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of
less than 8 days immediately following the attainment of 10% biodegradation. The 10 day window was therefore met.

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

Time (days)

Oxygen concentration (mg/L)      

 

Ocs

Ot

 Oc

 Oa

0

8.8

8.8

8.8   8.8

 

8.8

8.8

8.8   8.8

Mean (M)

8.8

8.8

 8.8 8.8 

7

8.1

5.1

 8.0  4.1

 

8.0

4.9

8.0  4.4

Mean (M)

8.1

5.0

 8.0  4.3

14

7.7

3.7

7.7  3.7

 

7.6

3.5

7.7  3.6

Mean (M)

7.7

3.6

7.7  3.7

21

7.5

3.4

7.6   

 

7.5

3.2

 7.6  

Mean (M)

7.5

3.3

7.6   

28

7.5

3.1

 7.6  

 

7.4

3.3

 7.5  

Mean (M)

7.5

3.2

 7.5  

Ot       River water with mineral nutrient solution with test material (2.0 mg/l). content was agitated 

Oc       River water with mineral nutrient solution   

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

Ocs River water without test material but with inoculum in bottles treated with dichloromethane. content was agitated

Table II  Oxygen consumption (mg/L) and the percentages biodegradation of dilauroyl peroxide (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

3.1

3.7

57 69 

14

4.1

4.0

76 74

21

4.2

78  
 28  4.3    80  

 

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.
Dilauroyl peroxide is biodegraded 80% at day 28 in the Closed Bottle test . Over 60% biodegradation was achieved in a period of
less than 8 days immediately following the attainment of 10% biodegradation. The 10 day window was therefore met. Dilauroyl peroxide 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. Dilauroyl peroxide did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Dilauroyl peroxide was biodegraded 80% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of less than 8 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 1.3 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 74 % 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

Dilauroyl peroxide was biodegraded 80% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of less than 8 days immediately following the attainment of 10% biodegradation. Hence this substance should be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information