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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 17 July, 2009 to 21 August, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
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)
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:
Based on the results of the read across study, the test substance, Oleyl TMAC, is considered to be readily biodegradable, with about 71% degradation in 28 days.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across 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 d. 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-d 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-d 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 d) 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). Based on the results of the read across study, the test substance, Oleyl TMAC, is considered to be readily biodegradable, with about 71% degradation in 28 d.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From October 4, 2005 to November 02, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Qualifier:
according to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Version / remarks:
Degradation-biotic degradation: Closed Bottle test
Deviations:
yes
Qualifier:
according to
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Deviations:
yes
Principles of method if other than guideline:
The test was modified to permit prolonged measurements (van Ginkel and Stroo 1992).
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract/mixture, activated sludge was used as an inoculum. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles (van Ginkel and Stroo 1992).
Duration of test (contact time):
28 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The oxygen concentration was measured with a special funnel which enabled testing without sacrificing bottles. This funnel exactly fitted into the BOD bottle. Subsequently, the oxygen electrode was inserted into the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flew back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo, 1992). The oxygen concentrations were measured in quadruplicate bottles instead of the prescribed duplicate bottle to improve accuracy. Use was therefore made of 4 bottles containing only inoculum, 4 bottles containing test substance and inoculum, and 4 bottles containing sodium acetate and inoculum. The concentrations of the test substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. The inoculum was diluted to 2 mg DW/L in the closed bottles. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles.
Reference substance:
acetic acid, sodium salt
Remarks:
concentration in the bottles: 6.7 mg/L
Preliminary study:
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. A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test substance is expected. This toxicity was the reason for testing at an initial test substance concentration of 1.0 mg/L.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
ratio BOD/ThoD
Value:
77
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Details on results:
The calculated theoretical oxygen demand of the test substance was 2.9 mg/mg. This theoretical oxygen demand is calculated by assuming formation of ammonium chloride.
The pH of the media was 7.0 at the start of the test. The pH of the medium at Day 28 was 6.8. Temperatures ranged from 19 to 21°C.
Results with reference substance:
The ThOD of sodium acetate was 0.8 mg/mg.
The biodegradation percentage at Day 14 was 66%.

Validity of the test:

The validity of the test is demonstrated by an endogenous respiration of 1.1 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 substance, sodium acetate, at day 14 was 66. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results of the read across study, the test substance, Oleyl TMAC, is considered to be readily biodegradable with 77% degradation in 28 d.
Executive summary:

A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC, according to OECD guideline 301D, EU Method C.6 and ISO 10707 (Closed Bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the test substance. The concentrations of the test substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at Day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The test substance was biodegraded by 77% at Day 28 in the Closed Bottle test. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 d. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the test substance was considered to be readily biodegradable (van Ginkel, 2016). Based on the results of the read across study, the test substance, Oleyl TMAC, is considered to be readily biodegradable.

Description of key information

Based on the results of the read across studies, the test substance, Oleyl TMAC, is considered to be readily biodegradable, with about 71-77% degradation in 28 d.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Study 1: A study was conducted to determine the ready biodegradability of the read across substance, C16-18 and C18-unsatd. TMAC, according to 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 d. Biodegradation was 71% by the end of the 28 d. 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-d 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 d) 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).

Study 2: A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC, according to OECD Guideline 301D, EU Method C.6 and ISO 10707 (closed bottle test), in compliance with GLP. The test was performed with activated sludge, domestic in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers. There were 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with the test substance. The concentrations of the test substance, and sodium acetate in the bottles were 1.0, and 6.7 mg/L, respectively. (A slight inhibition of the endogenous respiration of the inoculum by the test substance was detected at Day 7. Therefore, limited inhibition of the biodegradation due to the "high" initial concentration of the test compound is expected. This toxicity was the reason for testing at an initial test compound concentration of 1.0 mg/L). The test substance was biodegraded by 77% at Day 28 in the Closed Bottle test. The test was valid, as shown by an endogenous respiration of 1.1 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 66% of its theoretical oxygen demand after 14 d. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the test substance was considered to be readily biodegradable (van Ginkel, 2016).

Based on the results of the read across studies, the test substance, Oleyl TMAC, is considered to be readily biodegradable, with about 71-77% degradation in 28 d.