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Environmental fate & pathways

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:
19-10-2011 to 29-01-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: February 2010 ; signature: March 2010
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Rhine near Heavedrop, The Netherlands (14-10-2011). The nearest sewage treatment plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Not reported, assumed ambient
- Storage length: 8 days
- Preparation of inoculum for exposure: The river water was aerated for 7 days before use to reduce the endogenous respiration. Particles were removed by sedimentation after 1 day while moderately aerating. river water used in the Closed Bottle test was spiked per liter of water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O.
- Pretreatment: N/A
- Concentration of sludge: N/A
- Initial cell/biomass concentration: N/A
- Water filtered: no
- Type and size of filter used, if any: N/A
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: River water spiked with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O per litre.
- Additional substrate: N/A
- Solubilising agent (type and concentration if used):N/A
- Test temperature: 22 - 24 °C
- pH: 8.2 (starting pH) and 8.2 on day 28 in controls
- pH adjusted: no
- CEC (meq/100 g): not measured
- Aeration of dilution water: 7 days before use
- Suspended solids concentration: N/A
- Continuous darkness: yes
- Other: Ammonium chloride was not added to the river water to prevent nitrification.

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: Use was made of 10 bottles containing only river water, 10 bottles containing river water and silica gel, 10 bottles containing river water and test substance (2.0 mg/L), and 6 bottles containing river water and sodium acetate (6.7 mg/L).
- Method used to create aerobic conditions: 7 days of aeration before use
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: 0, 7, 14, 21, and 28 plus day 42, 60 and 100 days.
- Sampling method: Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28 plus day 42, 60 and 100.
- Sterility check if applicable: N/A
- Sample storage before analysis: ambient temperature, in the dark
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: river water only and river water and silica gel
- Abiotic sterile control: N/A
- Toxicity control: Sodium acetate
- Other: N/A

STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD)
The ThODs of the test substance, and sodium acetate were calculated from their molecular formulae and molecular weights as follows.

ThODNH3(mgO2 /mg)= 16(2C+12 (H-Cl-3N)+3S+2(0.5*P)+(0.5*Na-O))/ MW

Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc or cs is the mean oxygen level in the control bottles with and without silica gel n-days after the start of the test.

Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.
The BOD mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages:
The biodegradation was calculated as the ratio of the BOD to the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Remarks:
6.7 mg/L
Test performance:
1. The endogenous respiration was 1.5 mg/L at day 28 (i.e. < 30 mgO2/L after 28 days)
2. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled.
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels)
4. Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.
5. Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test item in the Closed Bottle test was not determined because possible toxicity of the test item to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test item could not be excluded.
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
24
Sampling time:
60 d
Parameter:
% degradation (O2 consumption)
Value:
64
Sampling time:
100 d
Results with reference substance:
Sodium Acetate attained 83% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The mean biodegradation was 3% at day 28, 24% at day 60 and 64 % at day 100 (within the enhanced biodegradation screening test).
Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The test item at a concentration of 2 mg/L was exposed to river water which was spiked with nutrients, in sealed BOD vessels and in the dark at 23°C ± 1°C for 28 days and then extension of the exposure for up to 100 days in an enhancement to the test method. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28, plus 42, 60 and 100. Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 1.5 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels). Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 3%. Under the conditions of the study, test item is not considered to be readily biodegradable.In the prolonged phase of the Closed Bottle test, the test item was biodegraded by 24% at day 60 and 64% at day 100. The biodegradation found demonstrates that this test item should be classified as inherently biodegradable. The biodegradation percentage of 64% reached at day 100 offers evidence that the test item is ultimately (completely) biodegradable.

Description of key information

Biodegradation: not readily biodegradable, mean biodegradation 3% (28 -days), 24% (60 -days) and 64% (100 -days), OECD TG 301D, enhanced closed bottle test, 2012

Key value for chemical safety assessment

Type of water:
freshwater

Additional information

Key study: OECD TG 301D, 2012 : The ready biodegradability test was carried out according to OECD TG 301D guideline under GLP. The test item at a concentration of 2 mg/L was exposed to river water which was spiked with nutrients, in sealed BOD vessels and in the dark at 23°C ± 1°C for 28 days and then extension of the exposure for up to 100 days in an enhancement to the test method. Degradation was assessed by the measurement of oxygen consumption via dissolved oxygen concentration measurement. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28, plus 42, 60 and 100. Control solutions with inoculum and the reference substance: sodium acetate, together with a toxicity control (where applicable) were used for validation purposes. The endogenous respiration was 1.5 mg/L at day 28. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. The pH at day 28 was in the range of 6.0 to 8.5 (actual 8.2 for controls and test item vessels). Sodium Acetate attained 83% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions. The mean biodegradation for the test item at 28 days was 3%. Under the conditions of the study, test item is not considered to be readily biodegradable.In the prolonged phase of the Closed Bottle test, the test item was biodegraded by 24% at day 60 and 64% at day 100. The biodegradation found demonstrates that this test item should be classified as inherently biodegradable. The biodegradation percentage of 64% reached at day 100 offers evidence that the test item is ultimately (completely) biodegradable.

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