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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2018 - May 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction
Non-volatile extract of Ginger, obtained from the roots of Zingiber officinalis (Zingiberaceae) by supercritical CO2 extraction
Test material form:
Details on test material:
Batch number: 861213
Specific details on test material used for the study:
Ginger Hot Flavour CO2-to extract
Batch no. 861213

Study design

Oxygen conditions:
Inoculum or test system:
natural water: freshwater
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands. 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. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
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.

Deionized water:
Deionized water containing no more than 1.0 mg/L of organic carbon was prepared in a water purification system.

Nutrients, and stocks:
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. Silica gel (0.20 g) was added to the control bottles. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using a stock solution of
1.0 g/L.

Test procedure:
Use was made of 10 bottles containing only river water with nutrients, 10 bottles containing river water with nutrients and silica gel, 10 bottles containing river water with nutrients and silica gel with test substance, 6 bottles with river water with nutrients 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.

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 (controls and test). The temperature ranged from 22.6 to 23.0°C which is within the prescribed temperature range of 22 to 24°C.
Reference substance
Reference substance:
acetic acid, sodium salt
Purity: >99%

Results and discussion

Test performance:
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 82. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
28 d
% degradation (O2 consumption)
Sampling time:
21 d
% degradation (O2 consumption)
Sampling time:
14 d
% degradation (O2 consumption)
Sampling time:
7 d
% degradation (O2 consumption)
Sampling time:
0 d
Details on results:
Ginger hot flavor CO2 extract (UVCB) was biodegraded by 58% at day 28 in the Closed Bottle test.

BOD5 / COD results

Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 82 %

Any other information on results incl. tables


Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of Ginger hot flavor extract (UVCB) 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 test substance concentration is expected.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The substance was biodegraded by 58% at day 28 in the Closed Bottle test and should therefore not be classified as readily biodegradable.
Executive summary:

In order to assess the biodegradation of Ginger hot flavor extract (UVCB), a screening test was performed according to OECD TG 301D (Closed Bottle test) and under GLP conditions. In this study, river water was exposed to 2 mg/L of the substance for 28 days. Ginger hot flavor extract did not cause a reduction in the endogenous respiration.The test substance is therefore considered to be non-inhibitory to the inoculum. Furthermore, the validity criteria of the test were met.

Ginger hot flavor extract was biodegraded by 58% at day 28 in the standard Closed Bottle screening test and should therefore be classified as not readily biodegradable.

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