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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:
Experimental Starting Date: 26 June 2012. Experimental Completion Date: 27 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 August 2011
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
Soil/Sediment Filtrate:
A mixed population of soil micro-organisms was obtained on 20 June 2012 from a wooded area in Allestree Park, Derby, Derbyshire, UK. The soil surface was cleared of leaf litter and a sample of soil (approximately 150 g) collected from a depth of up to 20 cm below the soil surface.

A further mixed population of river sediment micro-organisms was obtained from the River Derwent, Belper, Derbyshire. The river bank was cleared of leaf litter and a sample of soil sediment (approximately 150 g) collected from the side of the river bank. A sample of river water (1 liter) from the River Derwent was also collected.

Upon arrival in the laboratory a 50 g aliquot of soil was mixed with a 50 g aliquot of river sediment in 1 liter of river water. This suspension was filtered through glass wool (pre-conditioned in mineral medium) and refrigerated at approximately 4 C for 5 days


Activated Sewage Sludge Micro-organisms:
A mixed population of activated sewage sludge micro-organisms was obtained on 25 June 2012 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

Preparation of Activated Sewage Sludge:
The activated sewage sludge sample was washed two times by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.9 g/L prior to use.

* Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven
Duration of test (contact time):
28 d
Details on study design:
TEST CONDITIONS
For the purpose of the test, the test item was dispersed directly in mineral medium.

An amount of test item (42.0 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 14.0 mg/L, equivalent to 10 mg carbon/L.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution

Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An amount of test item (42.0 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 14.0 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.



TEST SYSTEM
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).


Each test 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 at approximately 21 °C, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 31.0 mL of inoculum 3.0 mL of soil/sediment filtrate and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. If necessary the pH was adjusted to pH 7.4 ± 0.2 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.

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 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.


SAMPLING
CO2 Analysis:
Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 11, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29. The samples taken on Days 0, 2, 6, 8, 10, 11, 14, 21, 28 and 29 were analyzed for CO2 immediately. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

The samples were analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyzer and a Shimadzu TOC-VCSH TOC analyzer and a Shimadzu TOC-LCSH TOC analyzer. Each analysis was carried out in triplicate.


Inorganic Carbon/Total Carbon Analysis:
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis (see
Table 3).

IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.

The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. Total carbon analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.



CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
85
Sampling time:
28 d
Remarks on result:
other: The test was carried out in a temperature controlled room at approximately 21 °C, in darkness.
Details on results:
The test item attained 85% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Results with reference substance:
The toxicity control attained 94% degradation after 14 days and 96% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 81% degradation after 14 days and 83% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Remarks:
The total CO2 evolution in the inoculum control vessels on Day 28 was 22.99 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 85 % degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % degradation must be attained within 10 days of the degradation exceeding 1 0%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B

Description of key information

The test item attained 85% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%.  The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Introduction

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 OECD Guidelines for Testing of Chemicals (1992) No. 301 B, “ “Ready Biodegradability; CO2Evolution Test” referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

Methods

The test item, 9 -decenoic acid, methyl ester, at a concentration of 10 mg Carbon/L, was exposed to inoculum consisting of sewage treatment micro-organisms and soil/ sediment filtrate with mineral medium in sealed culture vessels in the dark at approximately 21 °C for 28 d.

The degradation 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 used for validation purposes.

Results

The test item attained 85 % degradation after 28 d and satisfied the 10-Day window validation criterion, whereby 60 % degradation must be attained within 10 d of the degradation exceeding 10 %. The test can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.