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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:
24-05-2006 to 30-05-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study to ISO Guideline or which is equivalent to recognised Guidelines performed under GLP. All relevant validity criteria were met with acceptable deviations.
Qualifier:
according to guideline
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Deviations:
yes
Remarks:
Test terminated on day 27 not day 28. This was not considered a significant deviation given the study conclusion.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
See above
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: September 2005 ; signature: February 2006
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms (Test System) was obtained from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. De Theonex 105, 1226 THONEX Geneva, Switzerland)), which treats predominantly domestic sewage.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week
- Preparation of inoculum for exposure:
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge) and suspension in culture medium. To remove any excessive amounts of dissolved organic carbon (DOC) the solution was stirred and maintained on pure oxygen at room temperature. The final inoculum concentration in test bottles was 4 mg/L dry weight activated sludge solids.
- Concentration of sludge: The sludge was diluted in the test bottles to 4 mg DW/L.
Duration of test (contact time):
28 d
Initial conc.:
23.76 mg/L
Based on:
test mat.
Remarks:
20.00 mg C/L
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 10 mL of Solution A [KH2PO4: 8.50 g/L; K2HPO4: 21.75 g/L; Na2HPO4,2H2O: 33.40 g/L; NH4Cl: 0.50 g/L]; 1 mL of Solution B [CaCl2,2H2O: 36.40 g/L]; 1 mL of Solution C [MgSO4,7H2O: 22.50 g/L]; 1 mL of Solution D [FeCl3,6H2O: 0.25 g/L] and purified water diluted to 1 L.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 23 ±2 °C
- pH: Not reported ; typically pH 7.2 – 7.4
- pH adjusted: No
- Aeration of dilution water: Not reported
- Suspended solids concentration: 4 mg/L dry weight
- Continuous darkness: No. The test was conducted in diffuse light.

TEST SYSTEM
- Culturing apparatus: 250 mL glass flasks with continuous stirring (fill volume ca. 75 mL)
- Number of culture flasks/concentration: In duplicate (test item); In duplicate (Inoculum blank); duplicate flasks (reference item) ; duplicate (Toxicity Control) ; N/A (Abiotic Sterile Control)
- Method used to create aerobic conditions: Gas tight syringe/teflon septum.
- Measuring equipment: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered prior to analysis. Analysis conducted in duplicate.

SAMPLING
- Sampling frequency: Every three or four days to day 27.
- Sampling method: Measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the sample was filtered prior to analysis. Analysis conducted in duplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate).
Reference substance:
benzoic acid, sodium salt
Remarks:
34.27 mg/L or 39.99 mg C/L
Parameter:
% degradation (inorg. C analysis)
Value:
0
Sampling time:
27 d
Remarks on result:
other: Day 27 can be considered equal to day 28 measurement.
Details on results:
Applicant assessment indicates: The test item was biodegraded by 0 % at day 28 in the CO2 evolution test. The multi-constituent test substance is therefore classified as not readily biodegradable.

References:
1. OECD (2006), Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation
2. GHS revision 4 (2011), Globally harmonized system of classification and labelling of chemicals (GHS). Chapter 4.1. Hazardous to the Aquatic Environment. United Nations, 2011
Results with reference substance:
The reference item attained 90.26% degradation at 4 days and 96.23% at day 13 thereby confirming the suitability of the inoculum and test conditions.

All validity criteria were met.

1. The blank control TIC at day 27 was 1.18 mg C/L (< 3.0 mg C/L)

2. The Reference Item attained 96.23% degradation at day 13 (> 60% degradation at day 14)

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item mean biodegradation in duplicate was 0 % at day 28.
Executive summary:

The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline under GLP. The test item, at a concentration of 23.76 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 23°C ± 2°C for 27 days. The sludge was diluted in the test bottles to 4 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 to day 27. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. Analysis conducted in duplicate. The reference item attained 96.23% degradation after 13 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 0%. Under the conditions of the study, test item is considered as not readily biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 0 % (28-days) ; eq. or similar to OECD TG 310, 2006

 

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Key Study : eq. or similar to OECD TG 310, 2006 : The ready biodegradability test was carried out according to ISO 14593 equivalent or similar to OECD TG 310 guideline under GLP. The test item, at a concentration of 23.76 mg/L was exposed in duplicate to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment which treats predominantly domestic sewage (Villette: Geneva, Switzerland) with culture medium in sealed culture vessels in diffused light at 23°C ± 2°C for 27 days. The sludge was diluted in the test bottles to 4 mg DW/L. The degradation of the test item was assessed by the regular measurement of inorganic carbon (IC) production on days 0 to day 27. This was achieved by measurement of the IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel by gas-tight syringe and needle through the teflon septum (0.5 ml) and direct injection on the TOC (Total Organic Carbon) analyser. Analysis conducted in duplicate. The reference item attained 96.23% degradation after 13 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for duplicate test flasks at 28 days for the test item was 0%. Under the conditions of the study, test item is considered as not readily biodegradable.