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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July to August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EPA, etc)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Principles of method if other than guideline:
The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301 B "Ready Biodegradability; CO2 Evolution Test" referenced as Method CA-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).
Note: In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO 1995) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. High shear mixing was also applied to break up the filter paper containing the test item. 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.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Substance type: White powder
- Physical state: Solid
- Analytical purity: 99.5 wt% obtained by proton NMR
- Purity test date: 99.5 wt%
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: 28 February 2015
- Storage condition of test material: room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Details on properties of the test item are available in chapter 4 : physical and chemical properties.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Mixed population of activated sewage sludge micro-organisms was obtained on 25 July 2011 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
- Method of cultivation: 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: 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 deionised 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.
- Concentration of suspended solids: 2.8 g/l prior to use
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
5 mg/L
Based on:
other: carbon
Initial conc.:
7.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: as recommended in the OECD guideline.
- Additional substrate: no
- Solubilising agent (type and concentration if used): the test item has been dissolved in chloroform.
- Test temperature: 21°C
- pH: 7.65
- pH adjusted: not mentioned in the report
- CEC (meq/100 g): not mentioned in the report
- Aeration of dilution water: culture medium aerated onvernight 24 hours prior to add the test item and the reference items to tthe test vessels.
- Suspended solids concentration: Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter glass culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration:
a) A control, in duplicate, consisting of inoculated culture medium plus a filter paper (Whatman GF/A (70 mm diameter) with chloroform evaporated off)
b) The reference item (sodium benzoate), in duplicate, in inoculated culture medium plus a filter paper (Whatman GF/A (70 mm diameter) with chloroform evaporated off) to give a final concentration of 10 mg carbon/L.
c) The test item on a filter paper (Whatman GF/A (70 mm diameter) with chloroform evaporated off), in duplicate, in inoculated culture medium to give a final concentration of 5 mg carbon/L.
d) The test item on a filter paper ((Whatman GF/A (70 mm diameter) with chloroform evaporated off) plus the reference item in inoculated culture medium to give a final concentration of 15 mg carbon/L to act as a toxicity control (one vessel only).
- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 ml of culture medium and 32.1 ml of inoculum and aerated overnight. On Day 0 the test and reference items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
- Test performed in closed vessels: CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute 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.


SAMPLING
1) CO2 analysis:
- Sampling frequency:
* For the control, reference and test item: days 0, 2, 6, 8, 10, 14, 21, 28 and 29
* For the toxicity control: days 0, 2, 6, 8, 10 and 14
Note: The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.
- Sampling method: 2 mL - 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.
- Measuring equipment: Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VcsH TOC analyser. Samples (300 or 50 µI) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2C03). Each analysis was carried out in triplicate.
- Sample storage before analysis: Analyzed immediately

2) Dissolved organic carbon (DOC) analysis:
30 ml were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media.
- Measuring equipment: The samples were analysed for DOC using a Shimadzu TOC-VcPH TOC Analyser. Samples (50 µI) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. 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 (CaH5K04) and sodium carbonate (Na2C03) in deionised water. Each analysis was carried out in triplicate.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Prepared with a filter paper with chloroform evaporated to dryness added to maintain consistency with the test item vessel (as dissolved into chloroform).
- Toxicity control: 7.3 mg test item/L + 17.1 mg sodium benzoate/L, equivalent to a total of 15 mg carbon/L.


% DEGRADATION
* The percentage of Theoretical Amount of Carbon Dioxide (ThC02) produced is calculated by substituting the inorganic carbon values in the following equation:
% ThCO2( = % degradation) = ((mg IC in test flask - mg IC in control)/mg TOC as test chemical) x 100

* The percentage degradation from the results of the DOC analysis is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.
% degradation = [1 - (mg DOC in test flask on Day 28/mg DOC in test flask on Day 0)] x 100%

* The total C02 evolution in the control vessels at the end of the test is calculated from the equation below.
Total CO2 evolution = mg IC in control x 100/%C of CO2 x 1/test volume

STATISTICAL METHODS
Stduent's t-test - SAS computer software package (SAS 1999-2001).
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
C6H5COONa

Results and discussion

Preliminary study:
An initial experiment was conducted at a concentration of 10 mg Carbon/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.
Test performance:
* The total CO2 evolution in the control vessels on Day 28 was 24.15 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
* 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 hence satisfied the validation
criterion given in the OECD Test Guidelines.
* The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
* no toxic effect of the test item on the sewage sludge micro-organisms.
- The toxicity control attained 39% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
- Sodium benzoate attained 72% degradation after 14 days and 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
14 d
Remarks on result:
other: % Degradation Sodium benzoate
Parameter:
% degradation (CO2 evolution)
Value:
39
Sampling time:
14 d
Remarks on result:
other: % Degradation Test item plus Sodium benzoate Toxicity Control
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
14 d
Remarks on result:
other: % Degradation Test item
Parameter:
% degradation (CO2 evolution)
Value:
87
Sampling time:
28 d
Remarks on result:
other: % degradation Sodium benzoate
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Remarks on result:
other: % degradation test item
Details on results:
- Major results on CO2 evolution:
* The toxicity control attained 39% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
* Sodium benzoate attained 72% degradation after 14 days and 87% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

- Major results on dissolved organic carbon:
Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P~ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
Note: Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification 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 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

Any other information on results incl. tables

Note: It was not possible to test at concentrations below 5 mg C/L as below this concentration it was not possible to distinguish between background CO2 evolution from the inoculum and C02 evolution due to biodegradation using inorganic carbon analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item, 6H-Dibenz [c,e] [1,2] oxaphosphorin, 6,6'( 1 ,2-ethanediyl) bis-6,6'-dioxide, attained 0% degradation after 28 days when looking at the CO2 evolution and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.