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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:
14 January 2010 to 04 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Aerobic activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the sewage plant Darmstadt, Germany.
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. The sediment was re-suspended in test water and aerated for two days. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial conc.:
83 mg/L
Based on:
test mat.
Remarks:
Corresponding to an oxygen demand of about 136 mg/L (ThODNH4).
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Reconstituted Test Water: Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4x2H2O, 0.5 g NH4Cl filled up with deionised water to 1 000 mLvolume.
b) 22.5 g MgSO4x7H2O filled up with deionised water to 1 000 mL volume.
c) 36.4 g CaCI2x2H2O filled up with deionised water to 1 000 mL volume.
d) 0.25 g FeCl3x6H2O filled up with deionised water to 1 000 mL volume.
In order to avoid precipitation of iron hydroxide in the stock solution d) after storage and before use, one drop of concentrated HCI per litre was added.
10 mL of stock solution a) and 1 mL of the stock solutions b) to d) were combined and filled up to a final volume of 1 000 mL with deionised water.
- Solubilising agent: The amounts of test material and reference material were directly weighed into the test flasks. No emulsifiers or solvents were used but the solutions were dispersed by stirring to achieve a homogenous solution of the test material.
- Test temperature: 22 °C ± 1 °C. Temperature was measured each working day in the climatised room.
- pH: 7.5 - 7.6 (measured at the start of the test); 6.8 - 7.4 (measured at the end of the test). pH-values were measured in control, procedure control and a separately prepared test flask with test material at test start (to prevent loss of test material in the test flasks) and in all flasks at the end of the test using a pH-electrode WTW pH 340i.
- pH adjusted: No. The pH-value of the reconstituted test water was 7.5 and therefore no adjustment was necessary.
- Suspended solids concentration: 1.5 g/L dry mattter (final concentration 31 mg/L).
- Continuous darkness: Yes
- Other: The closed test flasks were incubated in a climatised room under continuous stirring.

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: Duplicate
- Measuring equipment: The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB Sensomat system, Aqualytic Dortmund.
- Test performed in open system: No. The flasks were closed. The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: Potassium hydroxide solution (45 %) was used for trapping the produced carbon dioxide.
- Other: The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

SAMPLING
- Sampling frequency: Daily
- Sampling method: Measurement of oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany).

CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate flasks.
- Abiotic sterile control: Single flask poisoned with HgCl2 (stock solution of 48.84 mg/mL).
- Toxicity control: Single flask.

STATISTICAL METHODS:
- Calculation of BOD: The biodegradability (% BOD = mg O2 per mg test material) exerted after each period was calculated as:

BOD = (mg O2 uptake of test material - mg O2 uptake of inoculum control) / mg test material in flask

For the toxicity control, the BOD of the treatment is the sum of test material and reference material and is calculated as the sum of test material and reference material concentration in the flask.
The percentage biodegradation of the test material and of the reference material sodium benzoate was calculated as:

% degradation = (BOD (mg O2/mg test material or reference material) / ThODNH4 (mg O2/mg test material or reference material) / 100

The ThODNH4 ofthe test material was calculated to be 1.640 mg O2/mg test material.
Reference substance:
benzoic acid, sodium salt
Remarks:
Theoretical Oxygen Demand: 1.666 mg/mg (calculated) (ThODNH4).
Parameter:
% degradation (O2 consumption)
Value:
85
Sampling time:
28 d
Details on results:
Percentage biodegradation of test material: After correction for the mean biochemical oxygen demand of the inoculum controls the mean percentage biodegradation based on ThODNH4 at the end of the 10-day window was 81 % (mean value), the 10 day window criterion was passed from day 8 to day 18.
The mean percentage biodegradation at the end of the 28-day exposure period was 85 % (ThODNH4).
The degradation rate of the test material reached 60 % within the 10-day window and after 28 days of incubation. Therefore, the test material is considered to be readily biodegradable.

Biodegradation in the toxicity control: In the toxicity control containing both the test material and reference 75 % biodegradation was noted within 14 days and 83 % biodegradation was determined after 28 days of incubation.
According to the test guidelines the test material can be assumed to be not inhibitory on the aerobic activated sludge microorganisms because degradation was > 25 % within 14 days.

Abiotic control: The oxygen demand in the abiotic control was zero. No correction of the test material degradation rates was required.
Parameter:
COD
Value:
1.64 g O2/g test mat.
Results with reference substance:
The reference compound sodium benzoate showed 76 % degradation after 7 days and 87 % degradation after 14 days.
The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

The test material contains no nitrogen, therefore the evaluation of bio degradation has to be based on THOD(NH4).

Validity Criteria of the Study

Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 35 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value: The pH-value of the test material flasks at the end of the test was within the range of pH 6.0 to 8.5 (i.e. 6.9 - 7.0) as required by the test guideline.

Reference Item: The percentage degradation of the reference material should reach the level for ready biodegradability (> 60 %) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60 % after 3 days of incubation.

Test material: The difference of duplicate values for the degradation of the test material at the plateau, at the end of the test and at the end of the 10-day window was less than 20 %

The validity criterion was fulfilled.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study the test material is considered to be readily biodegradable.
Executive summary:

The ready biodegradation of the test material was assessed according to OECD Test Guideline 301F and EC Method C.4-D in compliance with GLP using a manometric respirometry method.

The study was conducted over a period of 28 days at 22 °C in darkness. The biodegradation has been assessed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test material and functioned as a procedure control. Degradation rate of the test material was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test material loading rate was 83 mg/L corresponding to an oxygen demand of about 136 mg/L (ThODNH4)

The reference item sodium benzoate was sufficiently degraded to 87 % after 14 days and 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test material and the reference item sodium benzoate, 75 % biodegradation was noted within 14 days and 83 % biodegradation after 28 days of incubation. Thus, the test material can be assumed to be not inhibitory to the aerobic activated sludge microorganisms.

The test material contains no nitrogen therefore the evaluation of biodegradation has to be based ThODNH4· The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10 % degradation and 60 % degradation. This period should not exceed 10 days.

The mean biodegradation after 28 days of the test material was 85 % (ThODNH4); the 10-day window criterion was passed from day 8 to day 18.

Under the conditions of the study the test material is considered to be readily biodegradable.

Description of key information

Feil (2010)

Under the conditions of the study the test material is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Feil (2010)

The ready biodegradation of the test material was assessed according to OECD Test Guideline 301F and EC Method C.4-D in compliance with GLP using a manometric respirometry method. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was conducted over a period of 28 days at 22 °C in darkness. The biodegradation has been assessed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test material and functioned as a procedure control. Degradation rate of the test material was calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation. The test material loading rate was 83 mg/L corresponding to an oxygen demand of about 136 mg/L (ThODNH4)

The reference item sodium benzoate was sufficiently degraded to 87 % after 14 days and 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test material and the reference item sodium benzoate, 75 % biodegradation was noted within 14 days and 83 % biodegradation after 28 days of incubation. Thus, the test material can be assumed to be not inhibitory to the aerobic activated sludge microorganisms.

The test material contains no nitrogen therefore the evaluation of biodegradation has to be based ThODNH4· The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10 % degradation and 60 % degradation. This period should not exceed 10 days.

The mean biodegradation after 28 days of the test material was 85 % (ThODNH4); the 10-day window criterion was passed from day 8 to day 18.

Under the conditions of the study the test material is considered to be readily biodegradable.