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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-01-18 to 2018-03-21
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Analytical purity: 99.35% w/w
- Source and lot/batch No.of test material: 20141015
- Expiration date of the lot/batch: 2019-01-31
- Purity test date: 2017-01-31 (certificate of analysis release date)

- Storage condition of test material: At room temperature . Keep container tighly closed in a dry and well-ventilated place.
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source: aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage.
- Storage conditions: During the holding period of two days prior to use, the sludge was aerated at room temperature.
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant (liquid phase) and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenised final sludge suspension were weighed and dried (using a halogen moisture analyzer HS153 from Mettler Toledo) and the dry weight of the suspended solids was determined.
- Pretreatment: No pre-adaptation of the inoculum to the test item was done.
- Concentration of sludge: Prior to use, the dry weight of the sludge was again determined and the sludge was diluted with mineral medium. Defined volumes of this diluted activated sludge were added to the mineral medium in the test vessels to obtain a final concentration of 30 mg dry material per liter.
- Water filtered: The purified water used in the test was ultrapure water obtained from an ELGA PURELAB Option-Q 15 water purification system.
Duration of test (contact time):
18 d
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
ca. 243 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: test water prepared according to test guidelines, analytical grade salts dissolved in tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
* mineral stock solution 1: 8.5 g KH2PO4, 21.75 g K2HPO4, 33.40 g Na2HPO4.2H2O, 0.5 gNH4Cl pH 7.4
* mineral stock solution 2: 36.4 g CaCl2.2H2O
* mineral stock solution 3: 22.50 g MgSO4.7H2O
* mineral stock solution 4: 0.25 g FeCl3.6H2O stabilised with one drop of concentrated HCl per liter
* Final test medium: To obtain the final mineral medium, 10 mL of stock solution No. 1 and 1 mL each of stock solutions No. 2, 3 and 4 were added to approximately 800 mL ultrapure water, mixed and then made up to 1000 mL with ultrapure water. The pH was adjusted from 7.8 to exactly 7.4 with a
diluted hydrochloric acid solution.
- Additional substrate: no
- Test temperature: 21°C i a thermostatic cabinet
- pH: Prior to the start of the test (Day 0), the pH was measured in each test vessel before the addition of the inoculum, and was found to be in the
range of 7.4 to 7.5 for all vessels. At the end of incubation (Day 18), the pH was measured again in each test vessel and was found to be in the range of 7.3 to 7.7.
- pH adjusted: yes, the pH of th e final mineral medium was adjusted from 7.8 to exactly 7.4 with a diluted hydrochloric acid solution.
- Aeration of dilution water: The test solutions were continuously stirred (using an inductive stirring system OxiTop® IS 6-Var from WTW GmbH).
- Continuous darkness: The test vessels were incubated under diffused lighting.

- Culturing apparatus: Laboratory glass bottles GL45 with a nominal volume of 500 mL were used. The test vessels contained 164 mL of test suspension, a suitable volume to assure sufficient oxygen in the headspace.
- Number of culture flasks/concentration: 2
* test substance and inoculum: 2 replicates
* inoculum blank: 2 replicates
* positive control: 1 replicate
* toxicity control: 2 replicate
- Method used to create aerobic conditions: The prepared test vessels were air-tight closed with the OxiTop measuring heads
- Measuring equipment: For the measurement of the BOD, the OxiTop® Control system from WTW GmbH, Weilheim, Germany consisting of pressure measuring heads, test bottles, inductive stirring system and the OxiTop® OC 110 Controller was used.

- Sampling frequency: every three hours
- Sampling method: The biodegradation process consumed dissolved oxygen in the test vessel and generated CO2. The CO2 was absorbed by an alkali trap which caused a net reduction in gas pressure within the test vessel. The pressure reduction was continuously detected by the OxiTop pressure
measuring heads and recorded every three hours. The OxiTop OC110 controller collected the pressure values from the measuring heads and processed them to BOD values.

- Inoculum blank: yes, two replicates with only inoculum
- Toxicity control: yes, one replicate with test item, reference substance, and inoculum
- Procedure control: yes, 2 replicates with reference item and inoculum

Reference substance:
benzoic acid, sodium salt
Test performance:
In the toxicity control, the run of the curve of the oxygen consumption over the 18-day exposure period correlated very well with the oxygen demand of the two added substances, i.e. test item and reference item. Within 14 days of incubation the biodegradation reached a value of 69 % and at the end of the test period the value was 71 %.
Therefore, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 100 mg/L because biodegradation in the toxicity control was > 25 % within 14 days of incubation.
Key result
% degradation (CO2 evolution)
ca. 74
Sampling time:
18 d
Remarks on result:
other: mean of test bottle 1 and 2
Details on results:
The pass level for ready biodegradability, i.e. biodegradation of at least 60 % of the ThCO2 in a 10-day window within the 28-day period of the test, was reached.
Results with reference substance:
The percent of biodegradation of sodium benzoate was calculated based on the ThOD of 1.67 mg O2/mg.
In the procedure controls, the biodegradation of the reference item reached a value of 67 % and 88 % by Day 3 and Day 14, respectively. At the end of the test period (Day 18), the average biodegradation reached a value of 90 %.
Therefore, the suitability of the activated sludge (≥ 60 % degradation by Day 14) was confirmed.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
A 28-d ready biodegradability test (OECD 301F) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that butyl benzoate was readily biodegradable under the conditions of the test (initial concentration 100 mg/L). The test substance showed 74% biodegradation (test bottle 1 and 2, respectively, based on % ThCO2). The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.

Description of key information

One study (Eisner, 2018) is included in this dossier and regarded as a key study (Klimisch score of 1).  The biodegradability of the test substance was determined according to OECD Guideline 301F and EU Method C.4-D. Under the conditions of the test, the test substance was determined to be readily biodegradable within 18 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information