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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 17 July, 2009 to 21 August, 2009
Reliability:
2 (reliable with restrictions)
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)
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.

As per the guidelines, activated sludge, effluent from biological wastewater treatment plant, river water, soil etc. are suggested as inocula. Possible characterization of the inoculum for instance by determining the most probable number (MPN) is not obligatory. This is because the ecosystems from which the inoculum are derived change continuously over time; hence not making it possible to have identical or constant amounts of inoculum at every sampling.
However, the use of the small inoculum can be justified based on the fact that the test substance fulfills the validity criteria ≤ 1.5 mg/L oxygen consumption in the control group at Day 28 in the Closed Bottle test. This criteria fulfilment indicates low endogenous respiration, which is due to presence of small amount of inoculum. Further, the river water was used undiluted and was not pre-exposed to the test substance after sampling, which is another critical pre-requisite for ready biodegradation tests with regard to the inoculum.
Therefore, based on the above arguments, the absence of specification of inoculum concentration will not pose as a hindrance in evaluating the ready biodegradation results of the registered substance.
Duration of test (contact time):
ca. 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.
Please refer to the tables for details under 'attached background materials'

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.

For result tables and figure, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study 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 (49% active in isopropanol and water), in water according to OECD Guideline 301D (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. The test was performed using 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, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. 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. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability of the test substance was determined to be 71% by the end of 28 days. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the test substance is considered to be readily biodegradable (van Ginkel CG, 2010).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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.
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):
ca. 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.
Key result
Parameter:
ThOD
Value:
ca. 2.9 other: mg O2/mg
Remarks on result:
other: (NH3)
Key result
Parameter:
ThOD
Value:
ca. 3.06 mg O2/g test mat.
Remarks on result:
other: (NO3)
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. Please refer to the tables appended under 'attached background materials'.

Lower test concentrations than the guideline:

The test substance was tested at 1 mg/L, due to toxicity of the substance on the inoculum which was demonstrated in the other biodegradation studies conducted with the test substance in the concentration range of 2-4 mg/L

Omission of ammonium from the test medium:

Ammonium chloride is omitted from the test medium to prevent oxygen consumption by nitrifying bacteria. The reason for this omission is to lower the endogenous oxygen consumption in the BOD bottles, thereby increasing the accuracy of the biodegradation assessment. This is reflected in the validity criterion of less than 1.5 mg/L of oxygen consumption in the control bottles at Day 28. Omission of ammonium is not considered to hamper the biodegradation of organic compounds in the Closed Bottle Test. The biodegradation of the reference substance (sodium acetate) demonstrates that nitrogen is not limiting growth and that the nitrogen introduced with the inoculum is sufficient to fulfill the nitrogen requirement of the microorganisms.

Further, due to the presence of nitrogen in the test substance, there is small likelihood of occurence of nitrification, although its probability in case of quaternary ammonium substances was found to be low (see further explanation below).

Nitrification corrections:

Therefore, the biodegradation assessment based on theroretical oxygen demand (ThODNO3) with nitrification has been additionally evaluated and found to be 72.8%, allowing classification of the substance as readily biodegradable. See below for calculation details:

 

Molecular formula

MW

ThODNH3 (g/g)

ThODNO3(g/g)

Weight (%)

C18 TMAC

C21H44NCl

348.06

2.90

3.08

0.995

The ThODNH3of the test substance is =

2.88

The ThODNO3of the test substance is =

3.06

 

 

Day

O2 consumption

BOD

ThODNH3

% biodegradation

ThODNO2

% biodegradation

7

0

0

2.88

0.0

3.06

0.0

14

0.5

0.5

17.4

16.3

21

1.5

1.5

52.1

48.9

28.00

2.23

2.23

77.4

72.8

Test conc:

1

mg/L

 

 

 

 

However, in general the use of ThODNO3is not obligatory for all nitrogen-containing test substances. The choice of the ThOD used to estimate biodegradation should not be based on possible formation of nitrite or nitrate. Tests of the OECD 301 series were developed to assess the biodegradability and mineralization of organic substances. Nitrogen-containing substances are biodegraded in ready biodegradability tests by heterotrophic micro-organisms capable of utilizing these substances as carbon and energy source. This usually results in the formation of biomass (growth), water, carbon dioxide and ammonium (mineralization). The ammonium formed may subsequently be oxidized by nitrifying bacteria. These nitrifying bacteria utilizing ammonium as energy source and carbon dioxide as carbon source (autotrophic growth) are not involved in the biodegradation of nitrogen-containing substances. Biodegradation percentages calculated with the ThODNH3therefore do represent the biodegradability and mineralization of most nitrogen-containing substances. The formation of nitrite and nitrate during the degradation of organic substances is rare and only occurs when organic nitrogen is for example present in the form of a nitro group. Organic nitrogen is always liberated by microorganisms as ammonium when nitrogen is present as primary amine (amino group), secondary amine group, tertiary amine or quaternary ammonium group.

C16-18 and C18-unsatd. TMAC has a quaternary ammonium group. To understand the metabolic basis of degradation by microorganisms, the pathway of alkyltrimethylammonium salts has been studied with a pure culture. Bacteria identified asPseudomonas spcapable of degrading alkyltrimethylammonium salts were isolated from activated sludge (van Ginkelet al.,1992; Takenakaet al.,2007). Alkyltrimethylammonium salts with octadecyl, hexadecyl, tetradecyl, dodecyl, decyl, octyl, hexyl and coco alkyl chains supported growth of the isolates, showing the broad substrate specificity with respect to the alkyl chain length. Alkanals, and fatty acids can also serve as a carbon and energy source (van Ginkelet al.,1992; Takenakaet al.,2007). In simultaneous adaptation studies,1H nuclear magnetic resonance spectrometry (1H-NMR) and GC-MS showed that acetate, alkanals and alkanoates are the main intermediates of alkyltrimethylammmonium salt degradation, indicating that the long alkyl chain is utilized for microbial growth (van Ginkelet al.,1992; Nishiyama and Nishihara, 2002; Takenakaet al.,2007). Trimethylamine is stoichiometrically produced by pure cultures of microorganisms growing with the alkyl chain of alkyltrimethylammonium chloride as the sole source of carbon. The cleavage of the C-alkyl-N bond of alkyltrimethylammonium salts resulting in the formation of trimethylamine is initiated by a mono-oxygenase (van Ginkelet al.,1992). Additional evidence of the cleavage of the C-alkyl-N bond as the initial degradation step of alkyltrimethylammonium salts was presented by Nishiyamaet al.(1995) and Takenakaet al.(2007).

Dehydrogenase activity present in cell-free extract of hexadecyltrimethylammonium chloride-grown cells catalysed the oxidation of alkanal to fatty acids. The route of the fatty acid degradation is by β-oxidation. Trimethylamine, a naturally occurring compound is readily biodegradable (Pitter and Chudoba 1990). Complete degradation of trimethylamine is demonstrated through the assessment of the biodegradation pathway. Trimethylamine is degraded by methylotrophic bacteria through successive cleavage of the methyl groups (Large, 1971; Meiberg and Harder, 1978). Consortia of microorganisms degrading the alkyl chain of alkyltrimethylammonium salts and trimethylamine are therefore capable of complete (ultimate) degradation of alkyltrimethylammonium salts. Complete degradation of alkyltrimethylammonium salts using a mixed culture has been demonstrated by Nishiyamaet al.(1995). More recently, Nishiyama and Nishihara (2002) have isolated aPseudomonas spcapable of degrading both the alkyl chain and trimethylamine.  Both the pure and mixed culture studies showed that the degradation of the alkyl chain of alkyltrimethylammonium salts results in the formation of water, carbon dioxide and ammonium (see Figure 1).

For figure 1:Biodegradation pathway of alkyltrimethylammonium salts- please refer to the attachment under 'attached background material'

In conclusion, estimation of biodegradation based on the ThODNH3 is therefore considered to be a more appropriate choice for assessment for biodegradation of C18 TMAC.

References:

  • Ginkel CG van, Dijk JB van, and Kroon AGM (1992). Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1. Appl. Env. Microbiol. 58:3083-3087.L
  • arge PJ (1971). The oxidative cleavage of alkyl-nitrogen bonds in micro-organisms. Xenobiotica, 1:457-467.
  • Meiberg JBM, and Harder W (1978). Aerobic and anaerobic metabolism of trimethyl¬amine, dimethylamine and methylamine in Hyphomicrobium X. J. Gen. Microbiol. 106:265-276.Nishiyama N, Toshima Y and Ikeda Y (1995). Biodegradation of alkyltrimethylammonium salts in activated sludge. Chemosphere 30:593-603.
  • Meiberg JBM, and Harder W (1978). Aerobic and anaerobic metabolism of trimethyl¬amine, dimethylamine and methylamine in Hyphomicrobium X. J. Gen. Microbiol. 106:265-276.
  • Nishiyama N, Toshima Y and Ikeda Y (1995). Biodegradation of alkyltrimethylammonium salts in activated sludge. Chemosphere 30:593-603.
  • Nishiyama N and Nishihara T (2002). Biodegradation of dodecyltrimethylammonium bromide byPseudomonas fluorescensF7 and F2 isolated from activated sludge. Microbes Environments 17:164-169.
  • Pitter P and Chudoba J (1990). Biodegradability of organic substances in the aquatic environment. CRC Press, Boca Raton, USA p 191.
  • Takenaka S, Tonoki T, Taira K, Murakami S and Aoiki K (2007). Adaptation ofPseudomonas spstrain 7-6 to quaternary ammonium compounds and their degradation via dual pathways. Appl. Environ. Microbiol. 173:1797-1802.

.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the biodegradation of the test substance was determined to be 77% and the test substance was therefore considered readily biodegradable (activated sludge, domestic).
Executive summary:

A study was conducted to determine the biodegradation in water of the read across substance, C18 TMAC (99.5% active) 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 read across substance. The concentrations of the read across 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 read across 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 read across 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 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results of the read across study, which is a worst case read across, the test substance can also be considered to be readily biodegradable.

Description of key information

Based on the available weight of evidence, the test substance can be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Study 1: A study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (49% active in isopropanol and water), in water according to OECD Guideline 301D (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. The test was performed using 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, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6. 7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of 0.3 L BOD bottles so that all bottles were completely filled without air bubbles. 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. The test was found to be valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 76% of its theoretical oxygen demand after 14 day. Finally, the most important criterion was met with the oxygen concentrations being > 0.5 mg/L in all bottles during the test period. Biodegradability of the test substance was determined to be 71% by the end of 28 days. Therefore, the substance can be considered readily biodegradable in water. Furthermore, the test substance did not cause a reduction in the endogenous respiration in presence of silica gel, hence was considered to be non-inhibitory to the inoculum. Under the conditions of the study, the test substance is 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 (99.5% active) 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 read across substance. The concentrations of the read across 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 read across 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 read across 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 day. Oxygen concentrations remained >0.5 mg/ L in all bottles during the test period. Under the study conditions, the read across substance can be considered readily biodegradable (van Ginkel, 2005). Based on the results from this longer chain alcohol free quaternary ammonium substance, which would represent a worst case – as longer chains tend to biodegrade more slowly than shorter chains, the test substance, which is mix of C16 and C18 alkyl chains, can be expected to be degrading faster than the read across chemical

Study 3: A preliminary study was conducted to determine the ready biodegradability of the test substance, C16-18 and C18-unsatd. TMAC (96% active), using Closed bottle test, according to the OECD Guideline 301D. The inoculum used were: (a) activated sludge obtained from the local wastewater treatment plant and diluted to 2 mg Dry Weight (DW)/in the biological oxygen demand (BOD) bottles (b) river water without particles and spiked with mineral salts of the nutrient medium was used undiluted. Ammonium chloride was omitted from the medium to prevent nitrification. The test substance (solvent free) and humic acid were dosed using an aqueous stock solution of 1 g/L in water. Isopropanol was dosed from a 0.1 g/L stock solution in demiwater. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 control bottles containing only respective inoculum, 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance), and silica gel or humic acid. For the test substance (at 2 mg/L) 3 bottles were used containing the respective inoculum and silica gel or humic acid. Silicagel and humic acid concentrations in the bottles (test and control) were 1 and 2 g /bottle and 1 and 2 mg acid/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The BOD (mg/mg) of the test substance was calculated by dividing the oxygen consumption by the concentration of the test substance in the closed bottle. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.4 and 7.2±0.1 (activated sludge) and 8.2 and 8.0±0.1 (river water) at the start and end of Day 42 of the test respectively. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. The inhibition of the endogenous respiration of the inoculum was detected only at day 7 of the test for the bottles supplemented with humic acid at a concentration of 1 mg/L. The ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.05 g/g, respectively. The biodegradation percentages at Day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. Using the conservative ThODNO3 to calculate the biodegradation of test substance still >60% biodegradation was achieved within 28 days using activated sludge as inoculum and 1 g silica gel / bottle for detoxification. Under the study conditions, the test substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020).

Study 4: Main study ongoing. The test results will be available by end of March 2020.

Overall, based on the available weight of evidence, the test substance can be considered to be readily biodegradable.