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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:
2016-05-04 to 2016-07-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Acceptable, well documented study (performed compliant with GLP), OECD guideline slightly adapted, testing period extended up to 60 days
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted 1992
Deviations:
yes
Remarks:
, slightly adapted according to Ginkel and Stroo et al. (1992); testing period extended up to 60 days; ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification;
Principles of method if other than guideline:
CG van Ginkel and CA Stroo (1992) Simple method to prolong the Closed Bottle test for the determination of the inherent biodegradability. Ecotox Environ Saf 24 319-327.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
River water was used as inoculum within the test.
River water was sampled from the Rhine near Heveadorp, The Netherlands (28-04-2016). 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 moderately aerating. The inoculum was not pre-exposed to the test substance. The river water was used undiluted.
Duration of test (contact time):
60 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Solubilising agent (type and concentration if used): Tween 80, Sigma Aldrich;
- Test temperature: 22 to 24°C;
- pH: 8.0 at the start of the test; 7.9 (sufactant control) and 8.0 (treatments and control) at day 28;

Nutrients:
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 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.

Stock solution of test item, refrence item, dispersant stock, and administration:
The test substance was administered using a stock emulsion prepared in deionized water with Tween 80. The concentration of both substances was 1.0 g/L. The emulsion was prepared by ultrasonic dispersion at 400 W of approximately 80 mL deionized water with the surfactant and test substance using a Vibra-cell (Beun de Ronde, Abcoude, The Netherlands) for 1 minute. A volume of 0.6 mL of this emulsion was added to bottles already filled with nutrient medium with inoculum and subsequently closed. Sodium acetate (reference) and Tween 80 (additional control) were added using stock solutions of 1.0 g/L.

Test procedure:
Use was made of
- 10 bottles containing only river water,
- 10 bottles containing river water and surfactant,
- 10 bottles containing river water, surfactant with test substance,
- 6 bottles containing river water and sodium acetate.
The bottles containing only river water were filled to the brim and then closed with the stoppers.
The bottles containing surfactant and river water were first filled with river water to the brim. Subsequently 0.6 mL of the stock solution with Tween 80 was dispensed into each bottle and next the bottles were immediately closed with stoppers (2 mg/L final concentration in the test).
The bottles containing the test substance and river water were also first filled with river water to the brim. Subsequently 0.6 mL of the stock suspension with Tween 80 and test substance was dispensed into each bottle and the bottles were immediately closed with stoppers (2 mg/L final concentration, each).
The bottles with reference substance were filled to the brim with river water and sodium acetate (13.4 mL of the sodium acetate stock solution in 2 L of river water; 6.7 mg/L final concentration in the test).
Next the bottles were closed with stoppers. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.
Final concentrations:
The concentrations of the test substance, Tween 80, and sodium acetate in the bottles were 2.0, 2.0 and 6.7 mg/L, respectively.

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.

Prolonged screening test (enhanced ready biodegradability test):
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).

Evaluation / calculations:
- 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): ThOD_NH3 was clalulated according to Annex IV of OECD 301.
- 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 = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or Mcs is the mean oxygen level in the control bottles with and without surfactant n-days after the start of the test.
Mt or Ma 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 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 BOD to the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Test performance:
After 28 days, the pass level criterion for ready biodegradability (60% biodegradation based on O2 consumption) was not yet reached (48%). As biodegradation proceeded steadily up to 28 days and no plateau was reached, the test was prolonged till day 60, using the 28-day bottles and the "funnel-technique" desribed in section "Details on study design".
Parameter:
% degradation (O2 consumption)
Value:
48
Sampling time:
28 d
Remarks on result:
other: river water
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
42 d
Remarks on result:
other: river water
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
60 d
Remarks on result:
other:
Remarks:
river water
Details on results:
Trinonyl benzene-1,2,4-tricarboxylate was biodegraded by 48% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test (enhanced biodegradability test) the test substance is biodegraded by 63% at day 60. The biodegradation percentage of >60% demonstrates that test substance is inherently biodegradable. Moreover, the biodegradation in excess of 60% within the 60-day test period allows classification of trinonyl benzene-1,2,4-tricarboxylate as not persistent.
Results with reference substance:
The reference substance sodium acetate was degraded to 74% within 14 days. As such, the inoculum was demonstrated to be active.

Details on results

Table 1: Dissolved oxygen concentrations (mg/L) in the closed bottles:

Time (days)

Oxygen concentration (mg/L)

 

Ocs

Ot

Oc

Oa

0

8.7

8.7

8.7

8.7

 

8.7

8.7

8.7

8.7

Mean (M)

8.7

8.7

8.7

8.7

7

7.3

7.0

8.5

4.8

 

7.3

6.8

8.5

4.9

Mean (M)

7.3

6.9

8.5

4.9

14

6.6

5.4

8.0

3.9

 

6.6

5.2

8.0

4.0

Mean (M)

6.6

5.3

8.0

4.0

21

6.3

4.2

7.6

 

 

6.3

4.4

7.6

 

Mean (M)

6.3

4.3

7.6

 

28

6.0

3.7

7.5

 

 

6.2

4.0

7.6

 

Mean (M)

6.3

3.8

7.6

 

42

6.0

2.9

 

 

 

5.8

3.1

 

 

Mean (M)

5.9

3.0

 

 

60

5.4

2.0

 

 

 

5.6

2.3

 

 

Mean (M)

5.5

2.2

 

 

Oc: Mineral nutrient solution with only inoculum.

Ocs: Mineral nutrient solution without test material but with inoculum and surfactant.

Ot: Mineral nutrient solution with test material (2.0 mg/L), surfactant, and inoculum.

Oa: Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

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

0.4

3.6

8

67

14

1.3

4.0

25

74

21

2.0

 

38

 

28

2.5

 

48

 

42

2.9

 

56

 

60

3.3

 

63

 

Toxicity:

Inhibition of the degradation of an easily-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition can be detected prior to the onset of the biodegradation through suppression of the oxygen consumption in the presence of the test substance (higher oxygen concentration in bottles with test substance compared to the concentration in the control bottles). Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected (Table 1). Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test item is expected.

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 7.9 (control with surfactant) and 8.0 (test and 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 1.1 mg/L at day 28 (Table 1). 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 76. Finally, the validity of the test is shown with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Validity criteria fulfilled:
yes
Remarks:
preliminary test results
Interpretation of results:
inherently biodegradable
Remarks:
and not persistent based on the results of the prolonged (60 days) closed bottle test
Conclusions:
The test substance failed the criteria for ready biodegradability (48% biodegradation within 28 days, i.e. less than 60%). Thus, the test was prolonged, and a degradation of 63% was achieved till day 60 based on O2 consumption. Conclusion: The test item is not persistent.
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. River water was used as the inoculum.

Trinonyl benzene-1,2,4-tricarboxylate did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The test substance was biodegraded by 48% at day 28 in the Closed Bottle test.

In the prolonged Closed Bottle test, the test item was biodegraded by 63% at day 60 (enhanced biodegradability testing). This result demonstrates that the test substance is inherently biodegradable. Moreover, a biodegradation percentage of >60 within a 60-day time period allows classification of trinonyl benzene-1,2,4-tricarboxylate as not persistent.

The test is valid as shown by an endogenous respiration of 1.1 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

Description of key information

The submission substance is not persistent: 63% biodegradation at day 60 (inoculum river water, aerobic test conditions). 

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional 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. River water was used as the inoculum.

Trinonyl benzene-1,2,4-tricarboxylate did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The test substance was biodegraded by 48% at day 28 in the Closed Bottle test.

In the prolonged Closed Bottle test, the test item was biodegraded by 63% at day 60 (enhanced biodegradability testing). This result demonstrates that the test substance is inherently biodegradable. Moreover, a biodegradation percentage of >60 within a 60-day time period allows classification of trinonyl benzene-1,2,4-tricarboxylate as not persistent.

The test is valid as shown by an endogenous respiration of 1.1 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 with oxygen concentrations remaining at >0.5 mg/L in all bottles over the test period.

According to these results on biodegradation, the submission substance does not persist in the environment.

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