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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:
weight of evidence
Study period:
From 26 November, 1988 to 29 December, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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
Remarks:
Deviations: -Instead of an effluent/extract/mixture, activated sludge was used as an inoculums -ammonium chloride was omitted from the medium to prevent nitrification -the course of the oxygen decrease was measured in one bottle using a special funnel
Qualifier:
according to
Guideline:
other: EEC, 1984: “Official Journal of the European communities” L251, 1984.09.19. Part C. Methods for the determination of ecotoxicity, C Degradation biotic degradation: Closed bottle test.
GLP compliance:
no
Remarks:
No, although the study was not performed under GLP it was performed in a GLP lab by the same people with the GLP experience and equipment under the same circumstances as the GLP studies
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge was obtained from the RZWI Nieuwgraaf at duiven (1988.11.26). The RZWI Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water.
The inoculum taken from a preconditioned and activated sludge plant was diluted to a sludge concentration in the BOD bottle of 2 mg DW/litre.
Duration of test (contact time):
ca. 28 d
Initial conc.:
3 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test was performed in 250 to 300 mL BOD bottles. The test substance was added to an aqueous solution of mineral salts and exposed to relatively low number of microorganisms under aerobic conditions for a period of 28 days.The dissolved oxygen concentration were determined electrochemically using an oxygen electrode and meter.
Reference substance:
acetic acid, sodium salt
Remarks:
at 5 mg/L
Parameter:
% degradation (O2 consumption)
Value:
ca. 59
Sampling time:
5 d
Parameter:
% degradation (O2 consumption)
Value:
ca. 74
Sampling time:
15 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 75
Sampling time:
28 d
Details on results:
As evident from the biodegradation of 59% at Day 5 and 74% at Day 15, the plateau for ready biodegradability of the test substance was reached within 14 d of time point when 10 % degradation occurred. The 14 d window was therefore achieved.
Results with reference substance:
For the reference substance, the biodegradation was 82% after 28 d.

Oxygen consumption in the presence of the test substance and sodium acetate:

Time (days)

5

15

28

Test substance (mg O2/L)

4.9

6.1

6.2

Sodium acetate (mg O2/L)

3

3.2

3.2

Comments on removal of ammonium chloride from the test medium as well as nitrification corrections for biodegradation percentage determinations:

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.

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).Therefore, the biodegradation assessment based on theroretical oxygen demand (ThODNO3) with nitrification has been additionally evaluated and found to be 69.6%, allowing classification of the substance as readily biodegradable. See below for calculation details:

 

Molecular formula

MW

ThODNH3 (g/g)

ThODNO3(g/g)

Weight (%)

Coco TMAC ( n= C12)

C15H34NCl

263.9

2.73

2.97

0.33

 

Day

O2 consumption

BOD

ThODNH3(mg a.i./L)

% biodegradation

ThODNO2(mg a.i./L)

% biodegradation

5

4.9

1.63

2.73*(3*0.33) = 2.70

60.5

2.97*(3*0.33) = 2.94

55.5

15

6.1

2.03

75.3

69.1

28

6.2

2.07

76.5

70.3

Test conc: 3mg/L

Using C12 as the representative carbon chain, the ThODNH3 and ThODNO3 for the pure test substance was determined to be 2.73 and 2.97 g/g respectively. Considering that the test sample has only 33% active test substance, the active concentration corresponds to 0.99 mg a.i./L. ThODNH3 and ThODNO3 in the bottles are therefore determined to be 2.70 and 2.94 mg a.i./L (i.e., 0.99 mg a.i./L x 2.73 g/g or 2.97 g/g) respectively. The measured oxygen consumption (BOD) in the bottles at Day 28 was 2.07 mg/L. Therefore, the corresponding biodegradation percentage was calculated to be 77 and 70% respectively (i.e., 2.07x 2.07 = 77% and 2.94 x 2.07 = 70%) 

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.

Coco 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 ThODNH3is considered to be a more appropriate choice for quaternary ammonium substances including Coco 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 crieria:

The test does not fulfill two validity criteria i.e. not exceeding 1.5 mg/L consumption after 28 days in the inoculum blank and >0.5 mg/L residual concentration of oxygen in the test bottles at the end of the study.

- Oxygen concentration of ≥1.5 mg/L indicates presence of high number of micro-organisms in the inoculum leading to high endogenous respiration.

- An oxygen concentration of 0.1 mg/L at Day 28 in the bottles with the test substance means that the percentage biodegradation is ≥76 (ThODNH3) or ≥70 (ThODNO3) at Day 28.

Overall, despite these validity criteria deviations and given the high percentage of biodegradation obtained under the stringent test conditions, the author beleives the test substance can still be considered to meet the >60% criteria for ready biodegradation.

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

Validity criteria fulfilled:
no
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results of the read across study, the test substance is overall considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance, Coco TMAC (33% active in water), according to OECD 301D and EEC Guidelines using a closed bottle test. The read across substance at 3 mg/L was incubated with sludge from activated sludge plant treating predominantly domestic waste and O2 consumption was determined over a period of 28 d. The biodegradation was calculated as the ratio of the biochemical oxygen demand to the theoretical oxygen demand. The read across substance reached a biodegradation of 75% at Day 28. As evident from the biodegradation of 59% at Day 5 and 74% at Day 15, the plateau for ready biodegradability of the read across substance was reached within 14 d of time point when 10% degradation occurred. Further, using C12 as the representative structure for the read across substance and using ThODNO3 and ThODNH3 equations, the biodegradability of the read across substance following nitrification corrections was determined to be 77% and 70% within 28 days respectively. Under the study conditions, the read across substance is readily biodegradable (van Ginkel, 1989).  Based on the results of the read across study, the test substance is overall considered to be readily biodegradable.  

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:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Preliminary test
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)
Version / remarks:
(preliminary test)
Deviations:
not specified
GLP compliance:
no
Remarks:
Preliminary study
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, domestic, non-adapted and river water
Details on inoculum:
(a) Activated sludge wasobtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
(b) River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.
Duration of test (contact time):
ca. 42 d
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test procedures of Closed Bottle test
The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (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 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. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using a EUTECH instruments pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 um cellulose nitrate filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.
Key result
Parameter:
% degradation (O2 consumption)
Value:
> 60
Sampling time:
28 d
Remarks on result:
other: activated sludge as inoculum and 1 g silica gel / bottle for detoxification

Results

Test conditions

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 (activated sludge) and 8.2 (river water) at the start of the test. The pH was 7.2±0.1 (activated sludge) and 8.0±0.1 (river water) at day 42. Temperatures ranged from22 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. 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 Closed Bottle test results

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. From the four different detoxification methods used humic acid at a concentration of 1 and 2 mg/L were less effective compared to the other methods as demonstrated by negative biodegradation percentages at day 7.

 

In OECD 301 tests growth-linked biodegradation takes place. This means that carbon and nitrogen is built into microorganisms (new biomass). Calculating the biodegradation by using the ThODNO3 assumes that all the organic nitrogen present in a test substance is transiently degraded to ammonium nitrogen and subsequently oxidized to nitrate. The C:N ratio of quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (solvent free) is ~20:1. Due to the omission of ammonium nitrogen from the nutrient medium this organic nitrogen is the only nitrogen present for growth. With a C:N ratio of about 7:1 (not exact) for biomass (new cell formed) this means that most likely all the organic nitrogen present in the test substance will be built into new biomass. No oxidation of test substance nitrogen to nitrate is expected in the CBT. The use of ThODNH3 can be justified if the low nitrate/nitrite concentrations in the CBT can be analyzed accurately enough in order to demonstrate that no additional nitrate/nitrite is formed as a result of the biodegradation of the test substance (NO3 concentration in CBT blank ~0.7 mg/L and maximum amount of NO3 formed from organic nitrogen present in the test substance is 0.4 mg/L).

 

Using the conservative ThODNO3 to calculate the biodegradation of quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (solvent free) still >60% biodegradation was achieved within 28 days. quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (solvent free) should therefore be classified as readily biodegradable. For the final GLP test it is recommended to use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the test substance.

Table I Percentages biodegradation of quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (solvent free) in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThODNH3.

Inoculum

Sorbent

% Biodegradation percentage at day

7

14

21

28

42

Activated sludge

2 g silica gel /bottle

10

42

52

65

69

1 g silica gel / bottle

13

49

58

69

71

2 mg/L humic acid

-2

35

52

64

65

1 mg/L humic acid

-3

34

52

60

64

River water

2 g silica gel /bottle

2

37

47

50

54

1 g silica gel / bottle

4

41

48

53

60

2 mg/L humic acid

-6

36

53

57

72

1 mg/L humic acid

-4

35

53

56

71

 

Table II Percentages biodegradation of quaternary ammonium compounds, C16-C18 (even numbered) alkyl trimethyl chlorides (solvent free) in Closed Bottle tests inoculated with activated sludge and river water. Biodegradation percentages were calculated using the ThODNO3.

Inoculum

Sorbent

% Biodegradation percentage at day

7

14

21

28

42

Activated sludge

2 g silica gel /bottle

9

40

49

61

65

1 g silica gel / bottle

13

46

54

65

67

2 mg/L humic acid

-2

33

48

60

61

1 mg/L humic acid

-3

32

49

56

60

River water

2 g silica gel /bottle

2

35

44

47

51

1 g silica gel / bottle

3

39

45

50

56

2 mg/L humic acid

-5

34

50

54

67

1 mg/L humic acid

-3

33

50

53

67

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the preliminary results of the read across study, which is a worst case read across, the test substance can also be considered to be readily biodegradable.

Executive summary:

A preliminary study was conducted to determine the ready biodegradability of the read across 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 read across 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 read across substance), and silica gel or humic acid. For the read across 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 read across substance was calculated by dividing the oxygen consumption by the concentration of the read across 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 read across 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 read across 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 read across substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020). Based on the results from the preliminary study with the read across substance, the test substance is overall considered to be readily biodegradable.

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
Remarks:
See below section for details
Qualifier:
according to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Version / remarks:
Degradation-biotic degradation: Closed Bottle test
Deviations:
yes
Remarks:
See below section for details
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
Remarks:
See below section for details
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:
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.
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.

Endpoint:
biodegradation in water: ready biodegradability
Data waiving:
other justification
Justification for data waiving:
other:

Description of key information

Overall, based on the available weight of evidence, the test substane 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 read across substance, Coco TMAC (33% active in water), according to OECD 301D and EEC Guidelines using a closed bottle test. The test substance at 3 mg/L was incubated with sludge from activated sludge plant treating predominantly domestic waste and O2 consumption was determined over a period of 28 d. The biodegradation was calculated as the ratio of the biochemical oxygen demand to the theoretical oxygen demand. The test substance reached a biodegradation of 75% at Day 28. As evident from the biodegradation of 59% at Day 5 and 74% at Day 15, the plateau for ready biodegradability of the test substance was reached within 14 d of time point when 10% degradation occurred. Further, using C12 as the representative structure for the test substance and using ThODNO3 and ThODNH3 equations, the biodegradability of the test substance following nitrification corrections was determined to be 77% and 70% within 28 days respectively. Under the study conditions, the test substance is readily biodegradable (van Ginkel, 1989). Based on the results of the read across study, the test substance is overall considered to be readily biodegradable.  

Study 2: A preliminary study was conducted to determine the ready biodegradability of the read across 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 read across 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 read across substance), and silica gel or humic acid. For the read across 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 read across substance was calculated by dividing the oxygen consumption by the concentration of the read across 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 read across 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 read across 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 read across substance was determined to be readily biodegradable with >60% biodegradation after 28 days (Geerts, 2020). Based on the results from the preliminary study with the read across substance, the test substance is overall considered to be readily biodegradable.

Study 3: OECD 301D study ongoing on another read across substance, C16 -18 TMAC. The draft study report of this study will be available by 31 March 2020.

Study 4: 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.

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