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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:
07 February 2017 - 10 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Qualifier:
equivalent or similar to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
Qualifier:
equivalent or similar to
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Version / remarks:
1994
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (on 02-02-2017). 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):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Remarks:
(on silica gel carrier)
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 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 0.20 g of silica gel with the test substance 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 positive control bottles using a stock solution of 1.0 g/L.

Test procedure:
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 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.
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.

Test conditions:
The pH of the media was 8.0 at the start of the test. The pH of the medium at day 28 was 8.0 (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:
The test is considered 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 85% of its theoretical oxygen demand after 14 days. Finally, oxygen concentrations were >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
10
Sampling time:
28 d
Details on results:
The substance is biodegraded for 10% at day 28 in the Closed Bottle test and for 17% at day 60 in the prolonged Closed Bottle test. The substance did not fulfill the 60% pass level criterion for ready biodegradable compounds and hence should be classified as not readily biodegradable.
Results with reference substance:
The biodegradation percentage of the reference substance, sodium acetate, at day 14 was 85%.

Toxicity to inoculum:

Inhibition of the degradation of a well degradable substance 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 concentration of the test substance is expected.

Table 1       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

7.9

7.6

7.9

3.8

 

7.9

7.5

7.8

3.5

Mean (M)

7.9

7.6

7.9

3.7

14

7.6

7.1

7.6

2.9

 

7.5

7.0

7.6

3.0

Mean (M)

7.6

7.1

7.6

3.0

21

7.5

7.1

7.5

 

 

7.5

7.0

7.4

 

Mean (M)

7.5

7.1

7.5

 

28

7.4

6.9

7.4

 

 

7.3

6.8

7.4

 

Mean (M)

7.4

6.9

7.4

 

 42  6.8 6.2    
   6.8 6.3    
 Mean (M)  6.8 6.3    
 60  6.6 5.6    
   6.4 5.8    
 Mean (M)  6.5 5.7    

Ocs        River water with nutrients and silica gel.
Ot         River water with nutrients, test material (2.0 mg/L) and silica gel.

Oc         River water with nutrients.

Oa          River water with nutrients and sodium acetate (6.7 mg/L).

Table 2       Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (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

0

7

0.3

4.2

6

78

14

0.5

4.6

10

85

21

0.4

 

8

 

28

0.5

 

10

 

 42 0.5   10  
 60 0.8   17  
Validity criteria fulfilled:
yes
Remarks:
Oxygen concentrations were >0.5 mg/L in all bottles during the test period, oxygen depletion in the inoculum blank was 1.4 mg dissolved oxygen/L, differences between replicates at day 28 were <20%, biodegradation of reference compound was 85% at day 14.
Interpretation of results:
not readily biodegradable
Conclusions:
The substance is biodegraded by 10% at day 28 in the Closed Bottle test and should therefore be considered as not readily biodegradable.
Executive summary:

In order to assess the biodegradation of the substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and GLP. River water was exposed to 2 mg/L of the substance on silica gel for 28 days and the study was prolonged to 60 days. The substance did not cause a reduction in the endogenous respiration at day 7. The substance is therefore considered to be non-inhibitory to the inoculum. The validity criteria of the test were met. The test substance was biodegraded for 10% at day 28 and should therefore be considered not readily biodegradable under the conditions of this test.

Description of key information

In order to assess the biodegradation of the substance, a screening test was performed according to OECD TG 301D (Closed Bottle test) and GLP. River water was exposed to 2 mg/L of the substance on silica gel for 28 days and the study was prolonged to 60 days. The substance did not cause a reduction in the endogenous respiration at day 7. The substance is therefore considered to be non-inhibitory to the inoculum. The validity criteria of the test were met. The test substance was biodegraded for 10% at day 28 and should therefore be considered not readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information