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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:
From 17 July, 2009 to 21 August, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
minor deviations: (a) ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and b) river water was used as inoculum.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Physical state: Yellowish liquid
- Solubility in water: Soluble at test concentrations
- Analytical purity: 49%
- Composition of test material, percentage of components: Tallowtrimethylammonium chloride (TMAC) (49%); 2-propanol (33%); Water (15%)
- Purity test date: 21 January 2010
- Lot/batch No.: S001510
- Storage condition of test material: At ambient temperature in the dark under nitrogen gas
Oxygen conditions:
aerobic
Inoculum or test system:
other: River water without particles
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands 1707-2009). The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation.
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
- Nutrients and stock solutions: The river water used in the closed bottle test was spiked per liter of water with 8.5 mg KH2P04, 21.75 mg K2HP04, 33.3 mg Na2HP04·2H20, 22.5 mg MgS04·7H20, 27.5 mg CaCI2, 0.25 mg FeCl3·6H20. Ammonium chloride was not added to prevent nitrification. Sodium acetate and the test substance were added to the bottles using stock solutions of 1.0 g/L.
- Deionized water: Deionized water containing no more than 0.01 mg/L Cu (Sterlab certified; nonGLPanalysis) was prepared in a water purification system.
- Test temperature: 22(21.8) to 24°C.
- pH: 7.9


TEST SYSTEM
- Test bottles: The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
- Number of culture flasks/concentration: 10 bottles containing only river water (inoculum and medium), 10 bottles containing river water and silica gel (2 g/bottle), 10 bottles containing river water, silica gel and test substance (2 g/L), and 6 bottles containing sodium acetate (6.7 mg/L) and river water. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.

SAMPLING
- Sampling frequency: The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at Day 7, 14, 21, and 28.

ANALYSES
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode (WTW Trioxmatic EO 200) and meter (WTW OXI 530) (Retsch,
Ochten, The Netherlands). The pH was measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Berlin, Germany). The temperature was
measured and recorded with a sensor connected to a data logger.
Reference substance:
acetic acid, sodium salt
Remarks:
at 6.7 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
71
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Details on results:
THEORETICAL OXYGEN DEMAND (ThOD):
The calculated ThOD of the test substance was 2.9 mg/mg. The ThOD of the test substance composed of the active substance, 2-propanol and water was 2.3 mg/mg. The ThOD of sodium acetate was 0.8 mg/mg.

TOXICITY:
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because
possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance tested in the presence of silica gel was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected.

VALIDITY OF THE TEST:
The validity of the test was demonstrated by an endogenous respiration of 1.0 mg/L at Day 28. Furthermore, the differences of the replicate values at Day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 76. Finally, the validity of the test was shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

BIODEGRADABILITY:
The test substance only fulfilled one criterion for ready biodegradable compounds i.e. a biodegradation percentage in excess of 60 within 28 d. The pass level of 60% was not reached within 10 d upon achieving 10% biodegradation. The reasons for failing the 10-d window are the composition of the test substance (TMAC and 2-propanol) and TMAC being a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms, as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of this fatty amine derivative are therefore degraded sequentially. The time window criterion was developed on the assumption that a compound is degraded according to the "standard" growth curve in ready biodegradability tests.

The degradation curve of the test substance is the sum of growth curves of 2-propanol and the two moieties. The biodegradability of 2-propanol and the two moieties in the closed bottle test may be fully in line with the timeday window criterion when judged as separate chemicals. The time-window should therefore be ignored as a pass fail criterion for TMAC contained in the test substance. The test substance was therefore classified as readilybiodegradable only based on the biodegradation percentage of 71% at Day 28.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 76.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the test substance is considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C16 -18 and C18 -unsatd. TMAC, according to the OECD Guideline 301 D (closed bottle test), in compliance with GLP. The test substance at 2 mg/L was incubated with inoculums from river water and O2 consumption was followed over a period of 28 days. Biodegradation was 71% by the end of the 28 days. The authors noted that the presence of a hydrophobic and hydrophilic moiety (surfactant) in the substance structure, requires sequential, and thus a slower rate of, biotic degradation, since the complete biodegradation of surfactants requires many bacteria due to the limited metabolic capacities of individual microorganisms.The authors concluded that the 10-day time-window criterion was not relevant to this UVCB surfactant substance and concluded that the substance was readily biodegradable. In support, the ECHA technical guidance document Chapter R.7b Endpoint specific guidance, states that the 10-day window does not apply to surfactants. The study was considered to be valid as shown by an endogenous respiration of 1.0 mg/L, total mineralization of the reference compound, sodium acetate (i.e., 76% degradation after 14 days) and oxygen concentrations >0.5 mg/L in all bottles during the study. Under the test conditions, the test substance was considered to be readily biodegradable (van Ginkel CG, 2010).

Description of key information

C16-18 and C18-unsatd. TMAC is considered to be readily biodegradable after 28 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A study was conducted to determine the ready biodegradability of the test substance, C16 -18 and C18 -unsatd. TMAC, according to the OECD Guideline 301 D (closed bottle test), in compliance with GLP. The test substance at 2 mg/L was incubated with inoculums from river water and O2consumption was followed over a period of 28 days. Biodegradation was 71% by the end of the 28 days. The authors noted that the presence of a hydrophobic and hydrophilic moiety (surfactant) in the substance structure, requires sequential, and thus a slower rate of, biotic degradation, since the complete biodegradation of surfactants requires many bacteria due to the limited metabolic capacities of individual microorganisms.The authors concluded that the 10-day time-window criterion was not relevant to this UVCB surfactant substance and concluded that the substance was readily biodegradable. In support, the ECHA technical guidance document Chapter R.7b Endpoint specific guidance, states that the 10-day window does not apply to surfactants. The study was considered to be valid as shown by an endogenous respiration of 1.0 mg/L, total mineralization of the reference compound, sodium acetate (i.e., 76% degradation after 14 days) and oxygen concentrations >0.5 mg/L in all bottles during the study. Under the test conditions, the test substance was considered to be readily biodegradable (van Ginkel CG, 2010).