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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:
24 January 2017 - 23 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according 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
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on inoculum:
- Source of 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.
- Preparation of inoculum for exposure: 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 conc.:
ca. 2 mg/L
Based on:
test mat.
Remarks:
coated on silica gel
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST ITEM PREPARATION: The test substance is poorly water soluble. The test substance was therefore administered with the help of silica gel. To this end, approximately 70 mg of test substance was dissolved in a few mL of dichloromethane (DCM). The DCM with test substance was dosed on an appropriate amount of silica gel in a 50-mL serum flask to give 3.0 mg test substance/g silica gel. Next DCM was evaporated for 4 hours. After evaporation the content of the serum flask was thoroughly mixed. An amount of 0.20 g of this silica gel was added to each test bottle. Although, no additional oxygen consumption was expected an additional control with bottles containing silica gel (DCM added and evaporated) was included. 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 bottles using a stock solution of 1.0 g/L.

TEST CONDITIONS
- Composition of medium: 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.
- Test temperature: 22-24°C
- pH of the media: at the start: 8.0, at day 28: 7.9 (bottles containing test item) and 8.0 (control and control with silica gel)
- pH adjusted: no
- Suspended solids concentration: not determined
- Continuous darkness: yes

TEST SYSTEM
- Test vessel: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Fill volume: ca. 300 mL
- Number of culture flasks/concentration: 10 bottles containing only river water (blank control), 10 bottles containing river water and silica gel (silica control), 10 bottles containing river water and silica gel with test substance, 6 bottles with river water and sodium acetate (reference substance).
- Method used to create aerobic conditions: inoculum was aerated for 7 days prior to the test

SAMPLING
- Sampling frequency: every 7 days
- Sampling method: 2 duplicate bottles of each series were withdrawn for analyses of the dissolved oxygen concentration (determined electrochemically using an oxygen electrode and meter).
- Sample storage before analysis: not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 bottles
- Abiotic sterile control: none
- Toxicity control: none
- Other: silica control (10 bottles)

STATISTICAL METHODS: none
Reference substance:
acetic acid, sodium salt
Remarks:
Purity >99%
Test performance:
The validity of the test is demonstrated by an endogenous respiration <1.5 mg/L (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 80%. Oxygen concentrations were >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
28 d
Details on results:
- The test substance biodegraded for 70% after 28 days, passing the 60% criterium for readily biodegradability. The 10-d window was not applied since the test substance is poorly soluble in water. Poorly soluble substances are expected to affect biodegradation kinetics because slow desorption and dissolution rates of the test substance present at high concentrations may limit the biodegradation in ready biodegradability tests.
- The ThOD of the substance was calculated to be 3.0 g oxygen/g test substance, ThOD of the reference item was calculated to be 0.8 mg/mg.
- No toxicity control was used in this test since possible toxicity of the test substance to microorganisms degrading acetate is considered to be not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table 1). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.
- Functioning of the test system was checked by testing the reference item (sodium acetate), which showed a normal biodegradation curve (see attached illustration).
Results with reference substance:
80% after 14 days.

Table 1 Biodegradation (%) of the test substance and the reference substance (sodium acetate)

Time (days)

Biodegradation (%)

 

Test substance

Reference substance

0

0

0

7

8

76

14

57

80

21

67

 

28

70

 

Validity criteria fulfilled:
yes
Remarks:
Endogenous respiration was 1.4 mg/L at day 28, the differences of the replicate values at day 28 were <20%, the reference item was degraded by 80% of its ThOD after 14 days and oxygen concentrations were >0.5 mg/L in all bottles throughout the test.
Interpretation of results:
readily biodegradable
Conclusions:
In a Closed Bottle Test for readily biodegradability, according to OECD 301D, the test substance was bioegraded for 70% after 28 days. The test substance is therefore classified as readily biodegradable under the experimental conditions of this study.
Executive summary:

The readily biodegradability of the substance is tested in a Closed Bottle Test according to OECD 301D and under GLP conditions. In this study river water was used as inocolum, which was exposed to 2.0 mg/L of the test substance for 28 days. All validity criteria were met. The test substance was biodegraded at 8, 57, 67 and 70% at day 7, 14, 21 and 28, respectively, also fulfilling the 14 -day time window and should therefore be classified as readily biodegradable.

Description of key information

The readily biodegradability of the substance is tested in a Closed Bottle Test according to OECD 301D and under GLP conditions. In this study river water was used as inocolum, which was exposed to 2.0 mg/L of the test substance for 28 days. All validity criteria were met. The test substance was biodegraded at 8, 57, 67 and 70% at day 7, 14, 21 and 28, respectively, also fulfilling the 14 -day time window and should therefore be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information