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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:
03 August 2017 to 15 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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)
Specific details on test material used for the study:
- Purity: > 90%
- Description: Brown viscous liquid
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 20 November 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Sewage sludge/treatment micro-organisms were selected following the recommendations of guidelines. Pre-adaption of the inoculum was not performed.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
other: suspended solids
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter glass test culture vessels each containing 3 liters of solution:
a) Duplicate inoculated control vessels consisting of inoculated mineral medium, plus a filter paper (Whatman GF/A; 70 mm diameter)
b) Duplicate procedure control vessels containing the reference item (sodium benzoate) in inoculated mineral medium plus a filter paper to give a final concentration of 10 mg carbon/L.
c) Duplicate test item vessels containing test item adsorbed onto a filter paper in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) A single toxicity control consisting of the test item adsorbed onto a filter paper plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L.

One additional vessel containing water only was incubated under the same conditions as the test vessels to allow the temperature to be checked on each sampling occasion. A filter paper was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels. Each vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room, in darkness, and the temperature of the additional vessel containing water which was incubated under the same conditions as the test vessels ranged from 23 to 24 °C.

Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 21.4 mL of inoculum and aerated overnight.
On Day 0, the test and reference items were added and the pH of all vessels measured using a handheld meter. The pH was adjusted using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.
For the duration of the study, the test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The CO2 produced by degradation was collected in two Dreschel bottles containing 350 mL of 0.05 M NaOH.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
8
Sampling time:
28 d
Results with reference substance:
The reference item, sodium benzoate, attained 86% biodegradation after 14 days and a maximum biodegradation value of 113% over the study period, thereby satisfying the validation criterion given in the OECD Test Guideline 301B and confirming the suitability of the inoculum and test conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation relative to the inoculum control. The toxicity control attained 51% biodegradation after 14 days and a maximum biodegradation value of 54% over the study period, thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Acidification of the test vessels on Day 28 followed by the analysis on Day 29 was conducted according to the methods specified in the test guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28.

 

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in inorganic carbon in all replicate vessels with the exception of inoculum control Replicate 1 and procedure control Replicate 1.

 

Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

 

The test item attained a maximum biodegradation value of 8% biodegradation over the 28-day study period and therefore cannot be considered to be readily biodegradable according to the criteria set forth in OECD Guideline No. 301B.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained a maximum biodegradation value of 8% biodegradation over the study period and therefore cannot be considered to be readily biodegradable according to the criteria set forth in OECD Guideline No. 301B.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the standardized guidelines OECD 301B and EU Test Method C.4-C, under GLP conditions.

 

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 23 and 24 °C for 28 days.

 

Based on the results of a preliminary solubility/dispersibility trial and following the recommendations of the International Standards Organisation (ISO 10634, 1995) and the published literature (Handley et al, 2002), the test item was adsorbed onto filter paper prior to the addition of the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

 

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were included for validation purposes.

 

All validation criteria were met in this study. 

 

The test item attained a maximum biodegradation value of 8% biodegradation over the 28-day study period and therefore cannot be considered to be readily biodegradableaccording to the criteria set forth in OECD Guideline No. 301B.

Description of key information

Biodegradation in water: Screening

Key Study:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the standardized guidelines OECD 301B and EU Test Method C.4-C, under GLP conditions. The test item attained a maximum biodegradation value of 8% biodegradation over the study period and therefore cannot be considered to be readily biodegradable according to the criteria set forth in OECD Guideline No. 301B (Envigo, 2018).

Key value for chemical safety assessment

Additional information