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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:
Experimental starting date: 31 March 2008 and Experimental completion date: 01 May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
other: Aerobic activated sludge from a freshwater treatment plant treating predominately domestic wastewater.
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
19 mg/L
Based on:
test mat.
Initial conc.:
15.2 other: mg TOC/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of Test Flasks
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask, 90 mL activated sludge inoculum was added.
The test media were aerated overnight with CO2-free air to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.
The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask.

Calculations
The absolute amount of IC produced was calculated for each sampling date from the actual IC content in the absorber flasks plus the sum of the amount of IC removed in the analytical samples up to the respective sampling date. The IC content in the absorber flasks and in the analytical samples, removed from the absorber flasks, was calculated from the product of the actual IC concentration in the absorber flasks and the actual volume of absorbent or the sample volume.
IC content in absorber flask: mg IC = ICabs × VNaOH
IC removed in analytical samples: mg ICsample = ICabs × Vsample
IC produced per test flask: mg ICprod = mg IC + Σmg ICsample

where
mg IC = IC content in absorber flask at sampling date [mg C]
ICabs = IC measured in absorber flask on sampling date [mg C/L]
VNaOH = Volume of NaOH in absorber flask on sampling date [L]
ICsample = IC removed in the analytical sample of sampling date [mg C]
Vsample = Volume of analytical sample withdrawn on sampling date [L]
mg ICprod = Absolute amount of IC produced per test flask [mg C]
Σmg ICsample = Sum of IC removed in the analytical samples up to sampling date [mg C]

The percent degradation was calculated from(2):

% degradation = (((mg IC prod in test flask) - (mg IC prod in blank )) / mg TOC) x100
where
test flask = flasks containing test item and/or reference item.
blank = flasks containing neither test item nor reference item.
TOC = mg TOC added as test and/or reference item.

The amount of IC found in the second absorber flasks on Exposure Day 14 (procedure and inoculum controls) and 28 (all test flasks) was extrapolated assuming a linear increase between each sampling. The IC formation in mg C and the percentage degradation within these time periods were calculated by the sum of the amount of IC found in the first absorber flasks and the extrapolated amount in the second absorber flasks.

(2) The calculation of the percentage biodegradation is based on the IC measurements. The conversion factor for carbon to carbon dioxide is 3.67.
Reference substance:
other: sodium benzoate
Key result
Parameter:
% degradation (CO2 evolution)
Value:
-3 - 5
Sampling time:
28 d
Remarks on result:
other: The percent biodegradation of the test item was calculated based on a total carbon content (TOC) of 0.80 mg C/mg test item.
Details on results:
The CO2 formation of the test item in the test media was in the range of the inoculum controls. Expressed as percentage biodegradation, values in the range -3% to 5% were calculated for the substance over the 28-day exposure period. Consequently, the test item was not biodegradable under the test conditions within 28 days.
Results with reference substance:
The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure control, biodegradation of the reference item was 83% by Exposure Day 14, thus confirming suitability of the activated sludge (>60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 95%.

Biodegradation in the Toxicity Control

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.

CO2 formation in the toxicity control showed a similar course over the 28-day exposure period as the procedure control, containing only the reference item. Within 14 days of exposure, biodegradation reached 33%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 19 mg/L because biodegradation in the toxicity control was >25% within 14 days of incubation.

Driving Off CO2

No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29. Consequently, no residual CO2 was present in the test solutions or suspensions at the end of the test.

pH Measurement

The pH measured in all flasks at the end of exposure (Day 28) was between 7.3 and 7.5.

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance was not biodegradable under the test conditions within 28 days.
Executive summary:

The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C (1992) and OECD Guideline for Testing of Chemicals, No. 301 B (1992).

The substance was not biodegradable under the test conditions within 28 days.

In the toxicity control, containing both test itemand the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, the substance had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 19 mg/L.

In the procedure control, biodegradation of the reference item was 83% by Exposure Day 14, thus confirming suitability of the activated sludge (>60% degradation by Exposure Day 14). By the end of the test (Exposure Day 28), biodegradation was 95%.

Description of key information

The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C (1992) and OECD Guideline for Testing of Chemicals, No. 301 B (1992).

The substance was not biodegradable under the test conditions within 28 days.

The CO2 formation of the test item in the test media was in the range of the inoculum controls. Expressed as percentage biodegradation, values in the range -3% to 5% were calculated for the test item over the 28-day exposure period. Consequently, the test item was not biodegradable under the test conditions within 28 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information