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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:
From 2019-01-15 to 2019-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge freshly obtained from a municipal sewage treatment plant receiving predominantly domestic sewage.
- Storage conditions: kept under continuous aeration until further treatment
- Pretreatment: Before use, sludge was coarsely sieved (1 mm).
- Concentration of sludge: After treatment concentration of suspended solids (SS) was determined to be 3.6 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 11 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
20.5 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solutions of mineral components: A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl, dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2; B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre. C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre. D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.
- Test temperature: 22 and 23 °C
- pH: 7.6-7.8
- Continuous darkness: Test media were excluded from light.
- Test duration: 28 days for inoculum blank and test item (last CO2 measurement on day 29). 14 days for procedure and toxicity control (last CO2 measurement on day 15). During the test period, test media were aerated and stirred continuously.
- Test vessels: 2 litre brown coloured glass bottles.
- Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

TEST SYSTEM
- Reason for selection: The test has been accepted internationally for determining 'ready' biodegradability of test items under aerobic conditions.
- Type and number of bottles
Test suspension: containing test item and inoculum (2 bottles).
- Details of trap for CO2 and volatile organics if used: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate.
- Determination of CO2
Experimental CO2 production: CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl)
Theoretical CO2 production: Theoretical CO2 production was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum (2 bottles)
- Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Procedure control: containing reference item and inoculum (1 bottle).
Reference substance:
other: sodium acetate
Key result
Parameter:
% degradation (CO2 evolution)
Value:
>= 0 - <= 1
Sampling time:
28 d
Details on results:
Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (0% and 1%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (38%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.
Results with reference substance:
Reference item was biodegraded by 84% within 14 days.

ThCO2 of the test item was calculated to be 2.20 mg CO2/mg.

ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In conclusion, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the study was to evaluate test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test).

The test item was tested in duplicate at a concentration of 20.5 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO2 production (ThCO2) of the test item was calculated to be 2.20 mg CO2/mg.

Since the test item was easily soluble in water test media were prepared using a stock solution of 1 g/L in Milli- RO water. Aliquots of 40 mL of the clear and colourless stock solution were added to test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms. Volumes of suspensions were made up to 2 litres with Milli- RO water. Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).

 

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (0% and 1%, based on ThCO2).

In the toxicity control, the test item was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, the test item was designated as not readily biodegradable.

Description of key information

In a study to OECD Guideline 301 B, relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (0% and 1%, based on ThCO2). The test substance is therefore not considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The objective of the study was to evaluate test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge according to OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test).

The test item was tested in duplicate at a concentration of 20.5 mg/L, corresponding to 12 mg TOC/L. Organic carbon content was based on the molecular formula. Theoretical CO2 production (ThCO2) of the test item was calculated to be 2.20 mg CO2/mg.

Since the test item was easily soluble in water test media were prepared using a stock solution of 1 g/L in Milli- RO water. Aliquots of 40 mL of the clear and colourless stock solution were added to test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms. Volumes of suspensions were made up to 2 litres with Milli- RO water. Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of the test item (0% and 1%, based on ThCO2).

In the toxicity control, the test item was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, the test item was designated as not readily biodegradable.