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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:
key study
Study period:
26 February - 14 April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):

Activated sludge was obtained from three locations on 26 February 2009; Totnes Sewage
Treatment Works (STW), Devon, UK, Buckland STW, Newton Abbot, Devon UK, and
Countess Weir STW, Exeter, Devon, UK. These STWs all treat sewage of predominantly
domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature.

- Preparation of inoculum for exposure:

The solids concentrations of the three sludges was determined on the day of collection.
Appropriate volumes of each sludge were mixed in order to give similar concentrations of
suspended solids from each sewage treatment works in the final inoculum. The sludge was
then diluted in medium (Section 4.2) to give a total sludge solids concentration in the test of
30 mg/l. This solution was added to test vessels and aerated at test temperature for five days
until required for use.
Reference substance:
benzoic acid, sodium salt
Remarks:
radiolabelled
Parameter:
% degradation (CO2 evolution)
Value:
80
Sampling time:
28 d
Remarks on result:
other: maximum degradation
Results with reference substance:
In the benzoic acid vessel more than 60% degradation was achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. By day 35 approximately 83% of the applied radioactivity was measured as [14C]carbon dioxide.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
[14C]C22-ATQ was shown to be readily and ultimately biodegradable, based on carbon dioxide evolution, and based on the criteria for Regulation (EC) 648/2004 (Ref 3)
Executive summary:

[14C]C22-ATQ [14C]C22-ATQ
+ LAS
Ref. substance,
[14C]benzoic
acid
Lag time Days 0 to 3 Days 0 to 3 Days 0 to 1
Degradation time Days 3 to 28 Days 3 to 28 Days 1 to 25
Maximum level of degradation 80% 75% 83%
Readily biodegradable, in compliance with criteria of Regulation (EC) 648/2004 yes yes yes

Substantial mineralisation of [14C]C22-ATQ was measured in all the test vessels. The addition of LAS made no discernable difference to the observed degradation. The concentration of [14C]C22-ATQ was below that which would cause inhibition to the activated sludge bacteria, so there was no amelioration of inhibition by the LAS. Benzoic acid was shown to be readily biodegradable, with at least 60% of the applied radioactivity being converted to carbon dioxide by day 7.

Mass Balance:

The mass balance was calculated using the [14C]carbon dioxide evolution values measured during the study and the additional sampling on Day 35 detailed in Section 4.6, and is shown in Table 2. The total radioactivity recovered from each vessel was between 89 and 102% of the applied radioactivity, with the exception of one of the benzoic acid vessels, where a mass balance of 136% was measured. The sodium hydroxide from this vessel’s carbon dioxide trap had been accidentally added to the sodium hydroxide from one of the test vessels on day 21. The amount of [14C]carbon dioxide evolved from this vessel between days 14 and 21 was calculated from the difference between the measurements done on days 14 and 21, but because much more radioactivity was applied to the[14C]C22-ATQ vessels than to the benzoic acid (approximately 100 times more), this calculated value was subject to large errors. Therefore, the results from only one of the benzoic acid vessels was used for results calculations.

1.1.1                 Radioactivity measured in sludge solids

At the end of the study approximately 18% of the applied radioactivity was measured in the sludge solids from the[14C]C22-ATQ vessels, and approximately 11% in the sludge solids from the[14C]C22-ATQ plus LAS vessels.

 

In the benzoic acid reference substance vessels approximately 15% of the applied radioactivity was measured in the sludge solids.

1.1.2                 Radioactivity measured in the filtrate

At the end of the study less than 5% of the applied radioactivity was measured in the filtrate from any of the test or reference substance vessels.

 

1.1.3                 Radioactivity evolved as [14C]carbon dioxide

A significant amount of mineralisation was measured in all the vessels, as shown in Tables 2 and 3, and Figures 2 to 4.

In the benzoic acid vessel more than 60% degradationwas achieved within the 10 day window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms (Figure 3). By day 35 approximately 83% of the applied radioactivity was measured as[14C]carbon dioxide.

 

The following phases (approximate) were observed for benzoic acid:

 

Phase

Time period

Lag phase

days 0 to 1

Degradation phase

days 1 to 25

10-day window

days 1 to 11

 

where;

 

Lag phase

Is the period from inoculation until the degradation has increased to 10%

Degradation phase

Is the time from the end of the lag phase to the time when 90% of the maximum level of degradation has been reached

10-day window

The 10 days immediately following the attainment of 10% degradation

 

In the[14C]C22-ATQ vessels a significant amount of the applied radioactivity was evolved as[14C]carbon dioxide during the study, reaching between 75 and 80% in the vessels with and without LAS, with no discernable difference between the two treatments. Over 60% biodegradation was observed within 28 days, as shown in Figure 4, so[14C]C22-ATQ can be classified as “readily biodegradable”, based on the criteria for Regulation (EC) 648/2004 (Ref 3).

 

The following phases (approximate) were observed, for[14C]C22-ATQ, with and without LAS:

 

Phase

Time period

Lag phase

days 0 to 3

Degradation phase

days 3 to 28

10-day window

days 3 to 13

Mass balance (recovered radioactivity shown as measured value (Bq) and percentage of applied)

Vessel contents

Applied radio-activity (Bq)

Recovered radioactivity

Carbon dioxide traps

Filtrate

Sludge solids

Total

Bq

%

Bq

%

Bq

%

Bq

%

[14C]C22‑ATQ

3140462

2502356

79.7

124376

3.96

593141

18.9

3219873

103

3140462

2342399

74.6

129043

4.11

565757

18.0

3037199

96.7

[14C]C22‑ATQ plus LAS

3191703

2376303

74.5

135814

4.26

369177

11.6

2881294

90.3

3191703

2381277

74.6

159218

4.99

301235

9.4

2841730

89.0

[14C]Benzoic acid

40370

-a

-a

1652

4.09

7929

19.6

-a

-a

40370

33610

83.3

1752

4.34

4553

11.3

39915

98.9

 

a      No result, due to carbon dioxide trap sampling error

Carbon dioxide evolution (% of applied radioactivity)

Vessel contents

Day 3

Day 7

Day 14

Day 21

Day 28

Day 35

[14C]C22‑ATQ

7.1

26

48

61

72

80

11

26

48

52

67

75

[14C]C22‑ATQ plus LAS

8.4

27

51

54

68

74

8.9

28

49

54

68

75

[14C]Benzoic acid

52

65

75

107

110

112

47

60

70

69

80

83

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26.03.1992 - 23.04.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Remarks:
GLP stamp of German authorities
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
5 mg/L
Based on:
test mat.
Initial conc.:
10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
7
Sampling time:
14 d
Remarks on result:
other: 10 mg/L test conc.
Parameter:
% degradation (CO2 evolution)
Value:
21
Sampling time:
28 d
Remarks on result:
other: 10 mg/L test conc.
Parameter:
% degradation (CO2 evolution)
Value:
14
Sampling time:
14 d
Remarks on result:
other: 5 mg/L test conc.
Parameter:
% degradation (CO2 evolution)
Value:
40
Sampling time:
28 d
Remarks on result:
other: 5 mg/L
Validity criteria fulfilled:
yes
Interpretation of results:
other: at these substance concentration not readily biodegradable but biodegradable. Conditions were ready biodegradation is achieved see KEY study with [14C]-C22 ATQ.
Conclusions:
The test substance is after 28d not readily biodegradable under the test conditions. It is important to note that the degradation rate is higher at lower test concentration.
Executive summary:

The test substance was tested for ready biodegradability in an OECD 301B CO2 Evolution test over 28d and with non-adapted inoculum. Test concentration were 10 mg/L and 5 mg/L. Both concentrations showed no inhibition of the sludge respiration. After 28d the degradation rate was 21% for the test concentration of 10 mg/L and 40% for 5 mg/L test concentration. It is important to note that lowering the test concentration enhances the ready and ultimate biodegradation of the test substance. The viability of the inoculum was tested with the reference substance sodium acetate which was biodegraded after 28d to 100%.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Nov 2007 - March 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Concentration reduced and time extended
Qualifier:
equivalent or similar to guideline
Guideline:
other: ISO 10634 - testing poorly soluble compounds
Deviations:
yes
Remarks:
Use of silicone oil
Principles of method if other than guideline:
The test method is based on the OECD 301 manometric test, modified as follows;
1. The test concentration was reduced to 0.5 to 2 mg/l
2. The time of assessment was extended to 60 days
3. Solubilisers and de-toxifiers were also added - e.g. silicone oil, silica gel, humic acids and lignosulphonic acid.
GLP compliance:
no
Remarks:
Test was conducted to international guidelines but with out GLP - the tests conducted were research in character.
Oxygen conditions:
aerobic
Inoculum or test system:
other: see details on inoculum
Details on inoculum:
Two sources of inncoulum were used:
1. activated sludge from a WWTP of treating predominantly domestic wastewater and
2. natural water from the river Rhine.
In neither case was an acclimation stage introduced.
Duration of test (contact time):
ca. 28 - <= 60 d
Initial conc.:
0.5 - <= 2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter:
% degradation (O2 consumption)
Value:
15
Sampling time:
28 d
Remarks on result:
other: Tested at 2 mg/l with Humic acid present
Parameter:
% degradation (O2 consumption)
Value:
64
Sampling time:
60 d
Remarks on result:
other: Tested at 2 mg/l with Humic acid present
Parameter:
% degradation (O2 consumption)
Value:
50
Sampling time:
28 d
Remarks on result:
other: Tested at 0.5 mg/l in Rhine water with Silica Gel present
Parameter:
% degradation (O2 consumption)
Value:
64
Sampling time:
60 d
Remarks on result:
other: Tested at 0.5 mg/l in Rhine water with Silica Gel present

The substance tested, C20- and C22-alkyltrimethylammonium chlorides, causes reduction in the endogenous respiration of the inocula down to concentrations as low as 0.5 - 1 mg/l. The test substance is therefore inhibitory to the innocula under the conditions assessed in the Closed Bottle, OECD 301D test. Nevertheless, by utilising solubilising agents, e.g. silicone oil, and other agents that allow for the susbtance to be available but with reducedtoxicity, e.g. silica gel, humic acids and lignosulphonic acid, C20- and C22-alkyltrimethylammonium chlorides, degraded to greater than 60% within 60 days (but not 28d). Consequently while C20- and C22-alkyltrimethylammonium chlorides are not readily biodegradable under the given test conditions, they cannot be be considered to be Persistent (as defined by the REACH criteria).

Validity criteria fulfilled:
yes
Interpretation of results:
other: Under the test conditions not readily biodegradable but biodegradable. Conditions for ready biodegradability are described in the OECD 301B test with [14C]-C22ATQ.
Conclusions:
The substance biodegrades depending upoon its bioavailability, which also impacts its toxicity to microorganisms. At 0.5 mg/l the substance was seen to biodegrade in the range 7 - 50% at day 28 and 50 - 65% at day 60.
Executive summary:

The test substance is not readily biodegradable under the given test conditions, primarily due to limited bioavailability and toxicity. However, in the extended ready biodegradation test at a test concentration of 0.5 mg/l, with sludge as the source of the innoculum the test substance biodegradation at day 28 was 7- 20% and by day 60 51 - 59%. With river water as the source of the innoculum these values were 7 - 50% at day 28 (depending on how the substance was suspended in the test system) and 50 - 65% at day 60.

These values are for ultimate biodegradation, thus the substance is not persistent, having a (primary) half life < 15 d in freshwater.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 4, 2005 to November 02, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Version / remarks:
Degradation-biotic degradation: Closed Bottle test
Deviations:
yes
Qualifier:
according to guideline
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
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:
(based on ThoDNH3)
Value:
77
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
73
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 test 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 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% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. 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 test substance can be considered readily biodegradable (van Ginkel, 2005).

Description of key information

[14C]C22-ATQ was shown to be readily and ultimately biodegradable, based on carbon dioxide evolution, and based on the criteria for Regulation (EC) 648/2004 (Ref 3)

A second study was conducted to determine the biodegradation in water of the test 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 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% and 73% by the end of 28 days using and ThODNH3 and ThODNO3 equations respectively. 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 test substance can be considered readily biodegradable (van Ginkel, 2005).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:
freshwater

Additional information