Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-06-04 to 2014-07-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study. The deviations were not considered to have an adverse effect on the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
No note of the room number where study performed
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Remarks:
The toxicity control failed the validation criteria but is not obligatory for the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Off white solid block
- Analytical purity: 100% product
- Expiration date of the lot/batch: 24 March 2016
- Storage condition of test material: room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK (aeration stage)
- Preparation of inoculum for exposure:
The activated sewage sludge sample was washed twice 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 °C 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 preweighed 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 10mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C 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 1.8 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 other: mg carbon/L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test Item
Prior to use the test was heated to 50°C in order to aid weighing. An amount of test item (762 mg) was dissolved in 10 mL of acetone with the aid of approximately 2 minutes ultrasonication to give a 762 mg/10 mL stock solution. An aliquot (500µL) of this stock solution was dispersed onto filter paper and the solvent allowed to evaporate to dryness for approximately 15 mins. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (ca. 7500 rpm, 5 mins) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 litres to give a final concentration of 12.7 mg/L, equivalent to 10 mg carbon/L The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution..
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Reference Item
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.
A filter paper was added to each vessel in order to maintain consistency between the test and procedure control vessels. The filter paper was treated in the same manner as the test material but without the test item added.
Toxicity Control
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 (48.3 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 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 16.1 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.
TEST CONDITIONS
- Composition of medium: OECD mineral medium
- Test temperature: 20 - 23°C
- pH: 7.4± 0.2
- pH adjusted: yes if necessary
- Suspended solids concentration: 30 mg ss/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 25.7 mL of inoculum and aerated overnight. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- Number of culture flasks per concentration: Inoculated control, procedure control, test item all in duplicate; toxicity control one vessel only.
- Method used to create aerobic conditions: 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.
- Test performed in open system: No, culture vessels were sealed.
- Details of trap for CO2 and volatile organics, if used: 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
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29.
- The appearence of the test preparations was recorded on Days 0, 5, 12, 19 and 26.
CONTROL AND BLANK SYSTEM
- An inoculated control consisting of inoculated mineral medium.
- The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
- 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.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 28.7 mg/L. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and the difference between values for the CO2 production at the end of the test for the replicate vessels was <20%. The test therefore satisfied the validation criterion given in the OECD Guidelines.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. 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.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
74
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
87
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
97
Sampling time:
28 d
Remarks on result:
other: 102% degradation on Day 29. Day 29 values corrected to include carry-over CO2 in second absorber vessel if detected.
Details on results:
The test item attained 102% biodegradation after 28 days and satisified the 10-day window criterion, whereby 60% biodegradation must be attained within 10 days of biodegradation exceeding 10%. The substance is therefore readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.
The toxicity control showed no degradation at all over the 28 days of the test. However, as the test item was readily biodegradable, this indicates that the test item does not show any inhibitory effects. The results for the toxicity control were therefore considered to be erroneous.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 80% biodegradation after 14 days and 87% after 28 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 102% biodegradation after 28 days and satisified the 10-day window criterion, whereby 60% biodegradation must be attained within 10 days of biodegradation exceeding 10%. The substance is therefore readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B
Executive summary:

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. 30IB, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

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 21 - 23 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and in the published literature (Handley et al 2002), the test item was dissolved in an auxiliary solvent prior to be ing absorbed onto a filter paper and subsequent dispersal in test media. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

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 used for validation purposes.

Results

The test item attained 102% biodegradation after 28 days and satisified the 10-day window criterion, whereby 60% biodegradation must be attained within 10 days of biodegradation exceeding 10%. The substance is therefore readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.