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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Remarks:
Preliminary screening
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
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
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
(preliminary test)
Deviations:
not specified
GLP compliance:
no
Remarks:
Preliminary non-GLP study; main study has been planned to be conducted under GLP conditions.
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.
The Colony forming units (CFU) of the preconditioned river water inoculum and diluted preconditioned activated sludge inoculum was determined by a colony count method based on the ISO 6222 (1999) guideline. The preconditioned and diluted inoculum as used in the closed bottles was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.9 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively.
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. To perform these “negative control” tests without deviations from the guideline 0.5 mg/L ammonium chloride was included in the sorbent free tests. Test substance 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. In the tests without sorbent use was made of 3 bottles with the test substance and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 µg/L isopropanol (to correct for the small amount of isopropanol still present in the test substance). In the sorbent modified tests use was made of 3 bottles containing the test substance, the respective inoculum and silica gel or humic acid, and 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. The tests without sorbent were used to demonstrate the toxic effects of the test substance to the inoculum in the Closed Bottle test and to illustrate the positive detoxifying effects of the sorbents. 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)
Remarks:
(based on ThODNO3)
Value:
44
Sampling time:
28 d
Remarks on result:
other: activated sludge as inoculum and without sorbent
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
61
Sampling time:
28 d
Remarks on result:
other: 65% after 42 d; (using activated sludge as inoculum and 2 g silica gel / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
65
Sampling time:
28 d
Remarks on result:
other: 67% after 42 d; (using activated sludge as inoculum and 1 g silica gel / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
60
Sampling time:
28 d
Remarks on result:
other: 61% after 42 d; (using activated sludge as inoculum and 2 mg/L humic acid / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
56
Sampling time:
28 d
Remarks on result:
other: 60% after 42 d; (using activated sludge as inoculum and 1 mg/L humic acid/ bottle for detoxification)
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
35
Sampling time:
28 d
Remarks on result:
other: river water as inoculum and without sorbent
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
47
Sampling time:
28 d
Remarks on result:
other: 51% after 42 d; (using river water as inoculum and 2 g silica gel / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
50
Sampling time:
28 d
Remarks on result:
other: 56% after 42 d; (using river water as inoculum and 1 g silica gel / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
54
Sampling time:
28 d
Remarks on result:
other: 67% after 42 d; (using river water as inoculum and 2 mg/L humic acid / bottle for detoxification)
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(based on ThODNO3)
Value:
53
Sampling time:
28 d
Remarks on result:
other: 67% after 42 d; (using river water as inoculum and 1 mg/L humic acid / 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 and by differences of less than 20% for the triplicate values at day 28. Furthermore, the endogenous respiration at day 28 was <1.5 mg/L in the tests were ammonium chloride was omitted from the medium. In the tests with ammonium chloride in the medium (the “negative control” test) the endogenous respiration was 1.5 and 1.6 mg/L for the activated sludge and river water inoculum, respectively. 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. Hampering of the biodegradation by inhibition of the endogenous respiration of the inoculum was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. Silica gel and humic acid were added as sorbent for detoxification of the test substance.The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial test substance concentration is expected in the presence of silica gel (1 and 2 g/bottle).

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 3.06 g/g, respectively. 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 test substance (solvent free) is ~20:1. In the sorbent modified tests the organic nitrogen of the test substance is the only nitrogen present for growth due to the omission of ammonium nitrogen from the nutrient medium. With a C:N ratio of about 7:1 for growth of biomass (not exact) this would mean 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 therefore expected in the CBT. The use of ThODNH3 can however only be justified if the low nitrate/nitrite concentrations in the CBT can be demonstrated by analysis. However, analysis is probably not accurate enough to demonstrate this small possible increase in nitrate/nitrite concentrations (maximum theoretical amount of NO3formed from organic nitrogen present in the test substance is 0.4 mg/L and the “background” NO3 concentration in CBT blank with activated sludge and river water is ~0.7 mg/L and ~10 mg/L, respectively). The biodegradation of test substance (solvent free) is for this reason calculated with the “worst-case approach” assuming a complete oxidation of the organic nitrogen to nitrate (using the ThODNO3). The biodegradation percentages at day 28 using activated sludge as inoculum were slightly higher compared to results achieved with river water. More than 60% biodegradation was still achieved within 28 days using activated sludge as inoculum. Test substance (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 test substance (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

No sorbent*

-6

17

42

47

-

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

No sorbent*

-10

-4

21

37

-

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

* NH4Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

 

Table II Percentages biodegradation of test substance (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

No sorbent*

-5

16

39

44

-

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

No sorbent*

-9

-4

20

35

-

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

* NH4Cl (0.5 mg/L) was included in the nutrient medium as prescribed in the OECD 301D guideline.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Based on the results of the read across study, a similar biodegradation profile can be expected for the test substance.

Executive summary:

A preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the read across substance, C16-18 and C18-unsatd. TMAC (96% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the read across substance was evaluated using detoxification methods through the addition of the sorbents silica gel and humic acid at two different concentrations and two different inocula (activated sludge, river water).In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’ and to demonstrate the toxicity of the read across substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively.

The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the read across substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the read across substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the read across substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, 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 theoretical oxygen demand (ThOD) of read across substance was calculated from its molecular formula and molecular weight. 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 ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 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.

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 and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial read across substance concentration is expected in the presence of silica gel (1 and 2 g/bottle).

Under the study conditions, the read across substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the read across substance was considered further for the main study (Geerts, 2020). Based on the results of the read across study, a similar biodegradation profile can be expected for the test substance.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 08, 2020 to June 10, 2020
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
Remarks:
Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hour at 103.7 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.1 g/L.

The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in aerated Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

The preconditioned and diluted inoculum as used in the closed bottles (2 mg/L dry weight) was diluted 10x and 100x in a sterile peptone solution (1 g/L). Subsequently 1 ml of the peptone dilutions was transferred on a sterile petri dish and yeast extract agar was added. The yeast extract agar contained per liter of water 6 g tryptone, 3 g yeast extract and 15 g agar. Yeast extract agar plates were incubated for 68 hours at a temperature ranging from 22.7 – 22.8 °C. Only CFU counts between 30 and 300 were regarded as accurate and accepted for calculation of the CFU content. The inoculum concentration in the BOD bottles determined by colony count was 1.106 CFU/L.
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 2 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Reference substance and chemical
Sodium acetate anhydrous was used as a reference substance in the Closed Bottle test. This compound was purchased from Sigma-Aldrich, St Louis, US.

Reference compound - acetic acid, sodium salt
CAS reg. No. - 127-09-3
Purity - 99.9%
Batch/lot number - BCBP8197V
Appearance - white powder
The silica gel Davisil grade 636, pore size 60A, 35-60 mesh particle size (MKCH4201) was purchased from Sigma-Aldrich. All other chemicals used were of reagent grade quality.

Deionized water
Deionized water containing <1.0 mg/L of organic carbon was prepared in a water purification system.

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
Deionized water used in the Closed Bottle test contained per liter of water 8.51 mg KH2PO4, 21.75 mg K2HPO4, 33.42 mg Na2HPO4·2H2O, 22.50 mg MgSO4·7H2O, 27.51 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. The test substance and sodium acetate were added to the bottles using aqueous stock solution of 1 g/L. Silica gel was added as sorbent in the test bottles for detoxification of the test substance at a concentration of 1 g silica gel / bottle. Next the bottles were filled with nutrient medium with inoculum and closed.

Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentration of the test substance and sodium acetate in the bottles was 2.0 mg/L and 6.7 mg/L, respectively.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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.

Analyses
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(Based on ThODNO3)
Value:
ca. 65
Sampling time:
28 d
Remarks on result:
other: readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
(Based on ThODNH3)
Value:
ca. 70
Sampling time:
28 d
Remarks on result:
other: readily biodegradable

Results

Theoretical oxygen demand (ThOD) 

The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.77 and 3.00 g oxygen/g active ingredient, respectively. These ThODs were calculated with an average molecular formula of the active ingredient (of the solvent free quaternary ammonium compounds, C12-C18 (even numbered) alkyltrimethyl chloride)) using an average alkyl chain length. The average alkyl chain length is calculated from the alkyl chain distribution. The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

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 substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected. 

Test conditions 

The pH of the media at the start of the test was 7.0 for the control and reference substance and was 6.9 for the test substance and control with silica gel. The pH of the medium in the reference bottles measured at day 14 was 7.1. The pH of the medium at day 28 was 6.8 for the control and 6.7 for the test substance and control with silica gel. The temperature ranged from 22.7 to 22.8 °C which is within the prescribed temperature range of 22 to 24 °C. 

Validity of the test 

The validity of the test is demonstrated by an endogenous respiration of 1.15 mg/L at day 28. Furthermore, the differences in the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. 

Biodegradability 

Solvent free quaternary ammonium compounds, C12-18 (even numbered) alkyltrimethyl chloride was biodegraded by 70% (based on ThODNH3) at day 28 in the Closed Bottle test. Assuming complete nitrification, and calculating the biodegradation based on the ThODNO3 the test substance was biodegraded by 65% in the Closed Bottle test at day 28. Solvent free quaternary ammonium compounds, C12-18 (even numbered) alkyltrimethyl chloride is classified as readily biodegradable based on the >60% biodegradation reached at day 28.  

Table I Dissolved oxygen concentrations (mg/L) in the closed bottles. 

Time (days) 

Oxygen concentration (mg/L) 

 

Oc 

Oa 

Ocs 

Ot 

0 

8.9 

8.9 

8.9 

8.9 

 

8.9 

8.9 

8.9 

8.9 

Mean (M) 

8.90 

8.90 

8.90 

8.90 

7 

8.4 

4.6 

8.5 

7.6 

 

8.4 

4.6 

8.5 

7.2 

Mean (M) 

8.40 

4.60 

8.50 

7.40 

14 

8.2 

4.0 

8.2 

5.4 

 

8.2 

4.0 

8.2 

6.0 

Mean (M) 

8.20 

4.00 

8.20 

5.70 

21 

8.0 

 

8.2 

5.4 

 

7.9 

 

8.2 

4.6 

Mean (M) 

7.95 

 

8.20 

5.00 

28 

7.7 

 

8.0 

4.1 

 

7.8 

 

7.9 

4.2 

Mean (M) 

7.75 

 

7.95 

4.15 

Oc Mineral nutrient solution with only inoculum. 

Ocs Mineral nutrient solution with inoculum and silica gel 

Ot Mineral nutrient solution with inoculum, test substance (the dry content is used as the maximum amount of active ingredient in the test substance. The test substance concentration of 2.0 mg/L = 1.95 mg/L active ingredient) and silica gel. 

Oa Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L). 

Table II Oxygen consumption (mg/L) and the calculated percentages biodegradation (BOD/ThOD) of sodium acetate and the test substance in the Closed Bottle test. Biodegradation of the test substance is calculated both without nitrification (BOD/ThODNH3) and with nitrification (BOD/ThODNO3). 

Time (days) 

Oxygen consumption (mg/L) 

Biodegradation (%) 

Test substance  

Acetate 

Test substance 

Acetate 

ThODNH3 

ThODNO3 

0 

0.00 

0.00 

0 

0 

0 

7 

1.10 

3.80 

20 

19 

73 

14 

2.50 

4.20 

46 

43 

80 

21 

3.20 

 

59 

55 

 

28 

3.80 

 

70 

65 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was determined to be readily biodegradable with 65% biodegradation after 28 days.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C12-18 TMAC (96.8 % active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, Netherlands. The measured dry weight of the inoculum was 3.1 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.8°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.77 and 3.00 g oxygen/g active ingredient, respectively. According to the results of this study, the endogenous respiration of the inoculum by the test substance at day 7 was not detected. The test substance was biodegraded by 70% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 65% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.15 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 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the study conditions, the test substance was determined to be readily biodegradable with 65% biodegradation after 28 days (Geerts, 2020).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 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
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
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.  

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 preliminary non-GLP study was conducted to determine the best test conditions for conducting the closed bottle ready biodegradation study with the read across substance, C16-18 and C18-unsatd. TMAC (96% active), according to the OECD Guideline 301D. Due to the well-known toxicity of the quaternary substances, the read across substance was evaluated using detoxification methods through the addition of the sorbents silica gel and humic acid at two different concentrations and two different inocula (activated sludge, river water).In addition, a sorbent free test group without any deviations from the guideline was included as a ‘negative control’ and to demonstrate the toxicity of the read across substance and to demonstrate the positive detoxifying effects of the sorbents. Ammonium chloride was omitted from the medium to prevent nitrification for all groups except the sorbent free group. The inoculum concentration in the bottles determined by colony count was 7E+5 CFU/L and 6E+5 CFU/L for the river water and activated sludge inoculum, respectively. The tests were performed in triplicates using 0.3 L BOD bottles with glass stoppers. In the tests ‘without sorbent’ use was made of 3 bottles with the read across substance (at 2 mg/L) and the respective inoculum and 3 control bottles only containing the respective inoculum and 36 μg/L isopropanol (to correct for the small amount of isopropanol still present in the read across substance). In the ‘sorbent modified’ tests use was made of 3 bottles containing the read across substance (at 2 mg/L), the respective inoculum and silica gel or humic acid, and 3 control bottles containing only respective inoculum, 36 μg/L isopropanol, 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 theoretical oxygen demand (ThOD) of read across substance was calculated from its molecular formula and molecular weight. 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 ThODNH3 and ThODNO3 of the active ingredient (active with average chain length) used to calculate the biodegradation percentages was 2.86 g/g and 3.06 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. 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 and this was clearly detected until day 7-14 in the sorbent free ready biodegradation tests. The humic acid sorbent still showed an inhibition of the endogenous respiration (negative biodegradation percentages) at Day 7. Detoxification was most successful by the silica gel sorbents and no inhibition of the biodegradation due to the “high” initial read across substance concentration is expected in the presence of silica gel (1 and 2 g/bottle). Under the study conditions, the read across substance was determined to be readily biodegradable and the use activated sludge as inoculum and 1 g silica gel /bottle for detoxification of the read across substance was considered further for the main study (Geerts, 2020).

Study 2: A main study was conducted to determine the ready biodegradability of the test substance, C12-18 TMAC (96.8 % active), using Closed bottle test, according to the OECD Guideline 301D, in compliance with GLP. Secondary activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, Netherlands. The measured dry weight of the inoculum was 3.1 g/L. The activated sludge was preconditioned to reduce the endogenous respiration rates. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles. The inoculum concentration in the BOD bottles determined by colony count was 1E+6 CFU/L. The test substance (2 mg/L) was exposed to activated sludge, which was spiked to a mineral nutrient solution, dosed in closed bottles supplemented with 1 g silica gel/bottle as sorbent for detoxification of the test substance, and incubated in the dark at 22.7 to 22.8°C for 28 days. Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum, silica gel and isopropanol, 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 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. Endogenous respiration, theoretical oxygen demand (ThOD), biochemical oxygen demand (BOD) and biodegradation were calculated. The degradation of the test substance was assessed by the measurement of oxygen consumption. The ThODNH3 and ThODNO3 of the test substance used to calculate the biodegradation percentages is 2.77 and 3.00 g oxygen/g active ingredient, respectively. According to the results of this study, the endogenous respiration of the inoculum by the test substance at day 7 was not detected. The test substance was biodegraded by 70% (based on ThODNH3), at Day 28. Assuming a complete nitrification of the organic nitrogen present in the test substance and using a correction for the oxygen consumption by the nitrification, the test substance was biodegraded by 65% at day 28 (based on ThODNO3). The validity of the test is demonstrated by an endogenous respiration of 1.15 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 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period. Under the study conditions, the test substance was determined to be readily biodegradable with 65% biodegradation after 28 days (Geerts, 2020).

Study 3: 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.  

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