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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Fresh samples of activated sludge are withdrawn on June 10th, 2014 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater. Since it was not necessary, the samples were not washed with mineral medium after the arrival at the laboratory but kept aerobic until use.The concentration used in the test was 29.6 mg dry mass/litre (7.39 mg dry mass/250 mL).
Duration of test (contact time):
28 d
Initial conc.:
2 000 mg/L
Based on:
test mat.
Initial conc.:
290 mg/L
Based on:
ThOD/L
Initial conc.:
400 mg/L
Based on:
test mat.
Initial conc.:
58 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
( a ) KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4 x 12 H2O: 67.13 g/L
NH4Cl: 0.50 g/L
adjusted to 7.4 ± 0.2 with H2SO4 (50 g/L)
( b ) CaCl2 x 2 H2O: 36.40 g/L
( c ) MgSO4 x 7 H2O: 22.50 g/L
( d ) FeCl3 x 6 H2O: 0.25 g/L.
The mineral medium applied in the test contained 10 mL/L of mineral stock solution a and 1 mL/L of the mineral stock solution b–d, respectively.
- Test temperature: 22 °C- pH: 7.4 +/- 0.2
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2 vessels containing test item (2000 mg/L) and inoculum; 2 vessels containing test item (400 mg/L) and inoculum; 2 vessels containing only inoculum; 2 vessels containing test item (2000 mg/L) and a sterilising agent
- Method used to create aerobic conditions: The suspension was aerated during the whole test.
- Measuring equipment: SAPROMAT respirometer (VOITH Inc.).

SAMPLING
- Sampling frequency: The measurement and recording of the oxygen demand was carried out continuously

CONTROL AND BLANK SYSTEM
- Inoculum blank: The blank control consists of inoculated mineral medium only
- Abiotic sterile control: An abiotic control containing test item at 2000 mg per litre sterilized mineral test medium (500 mg/250 mL) was applied. The assay was sterilized by adding HgCl2.
- Toxicity control: A toxicity control containing test item at 2000 mg per litre (500 mg/250 mL) and reference item at 100 mg per litre mineral test medium (25 mg/250 mL) was applied.

STATISTICAL METHODS:
Theoretical Oxygen Demand (ThOD):The Theoretical Oxygen Demand (ThOD) was calculated on the basis of the sum formula of the test and reference item by:
ThOD [g/g] = 16 * (2 C + 1/2 H + 5/2 N + 1/2 Na – O) / Molecular weight.
The ThOD values for Na-benzoate, the test item and the toxicity control were determined as follows:
ThODPC-2014-536: 0.145 mg O2/mg test itemThOD
Na-Benzoate: 1.665 mg O2/mg reference item
ThODtoxicity control: 0.218 mg O2/mg substance mixture
Biochemical Oxygen Demand (BOD):
The Biochemical Oxygen Demand (BOD) was calculated on the basis of the test raw data by BOD [mg/mg] = mg O2 uptake corrected by blank per mg test item.
The percent degradation was calculated according to the following formula:Dt = [(Ct –Cb) / ThOD] x 100Dt: degradation (%) at time t;
Ct: mean oxygen consumption (mg/L) in the test suspension at time t;Cb: mean oxygen consumption (mg/L) in the blanks at time t;
ThOD: Theoretical oxygen demand of the test suspension (mg/L).
Reference substance:
benzoic acid, sodium salt
Test performance:
The test item in concentrations of about 2000 and 400 mg per litre mineral medium, respectively, and sodium benzoate in concentrations of about 100 mg per litre mineral medium, were incubated with 29.6 mg dry mass inoculum per litre mineral medium in 500 mL glass vessels at a medium volume of 250 mL. The test was run for 28 days, in darkness at 22°C ± 1°C. The suspension was aerated during the whole test. Oxygen demand was carried out continuously throughout the test course.
Parameter:
% degradation (O2 consumption)
Value:
9
St. dev.:
1.5
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
53
St. dev.:
7.3
Sampling time:
28 d
Results with reference substance:
96.4 % degradation after 28 days

Oxygen consumption. Cumulated consumption (mg O2/L) after 28 days. Single and mean values of the parallel test vessels and standard deviation. Test suspension B: 32 mg/L; Test suspension A and procedural control: 100 mg/L; Toxicity control: 200 mg/L.

Vessel

Inoculum blank

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

8

31

42

7

165

214

2

8

37

36

7

172

192

Mean

8

34

39

7

169

203

SD

0

4

4

0

5

16

Percent degradation. Degradation (%) after 28 days. Single and mean values of the parallel test vessels and standard deviation.

Vessel

Test suspension A

Test suspension B

Abiotic control

Procedural control

Toxicity control

1

7.9

58.5

2.4

94.3

45.1

2

10.0

48.2

2.4

98.5

40.3

Mean

9.0

53.4

2.4

96.4

42.7

SD

1.5

7.3

0

3.0

3.4

The biodegradation of the substance after 28 days of incubation in the static test was found to be 9 % (SD = 1.5 %) and 53 % (SD = 7.3 %) in the assays with 2000 mg/L and 400 mg/L, respectively. The biodegradation within the 10-day-window was 34 % in the assays with 400 mg/L. For the 2000 mg/L assay, no 10-day-window could be calculated since the degradation maintains below 10 %. The 10-day-window in the 400 mg/L assay started at day 5 - 7. With 2 %, there was no significant abiotic degradation of the substance noticeable within the 28 days of incubation. The biodegradation of the item mixture in the toxicity control was found to be 31 % after 14 days of incubation. Thus, the demanded threshold value of 25 % is exceeded and the test item can be identified as non-toxic in a ready biodegradability test. The reference item sodium benzoate was degraded to 92 % within the first 14 days.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the test substance after 28 days of incubation in the static test was found to be 9 % (SD = 1.5 %) and 53 % (SD = 7.3 %) in the assays with 2000 mg/L and 400 mg/L, respectively
Executive summary:

The biodegradation of C16 Alkylamidopropyltrimethylammonium Chloride was investigated in a study according to OECD Guideline 301 F (1992). The biodegradation of the substance in the static test was found to be at mean 9 % with a standard deviation of 1.5 % for a concentration of 2000 mg test item per liter and at mean 53 % with a standard deviation of 7.3 % for a concentration of 400 mg test item per liter after 28 days. For a concentration of 2000 mg test item per liter no 10-day-window could be calculated since degradation rate maintained below 10 %. For a concentration of 400 mg test item per liter biodegradation within the 10-day-window was found to be 34 %. The 10-day window in the 400 mg/L assay started at day 5 - 7. The degradation of the reference substance sodium benzoate had reached 92 % within the first 14 days. The difference of extremes of replicate values of the removal of the test item at the end of the test for 2000 mg/L is less than 20%. Therefore, the test can be considered as valid. No inhibitory effects of the test item were observed (more than 25 % degradation occurred within 14 days) in the toxicity control.

According to the guideline, the substance be considered as not readily biodegradable under the chosen test conditions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-10-02 to 2018-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted July 17, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, 31137 Hildesheim, Germany (Activated sludge frorn the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.) receipt: 2018-09-28
- LPretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until three days before test start. Further treatment see section 'preparation of the test vessels'. 4.55 mL/ L of this mixture were used to initiate inoculation (25.1 mg/L dw).
Duration of test (contact time):
28 d
Initial conc.:
16 mg/L
Based on:
act. ingr.
Initial conc.:
10.8 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B I C02 Evolution Test
- Additional substrate: no
- Test temperature: Nominal 22 ± 2°C, actually measured 20.0 - 23.0 °C
- Aeration of dilution water: 30 - 100 mLlmin
- Continuous darkness: Low light conditions (brown glass bottles)
- Other: additional vessels, including controls, with 10 mg/L humic acids

TEST SYSTEM
- Culturing apparatus: Test vessels: 5000 mL, brown glass; Volume of the test medium: 3000 mL
- Number of culture flasks/concentration: 2

CONTROL AND BLANK SYSTEM
- Functional Control: 20 mg/L Sodium benzoate, 1 replicate
- Functional Control with humic acids: 20 mg/L Sodium benzoate, 10 mg/L humic acids, 1 replicate
- Toxicity control: 16 mg/L test item + 20 mg/L Sodium benzoate, 1 replicate
- Toxicity control with humic acids: 16 mg/L test item + 20 mg/L Sodium benzoate + 10 mg/L humic acid, 1 replicate
- Inoculum control: Test medium without test and/or reference item, duplicate
- Humic Acid control: Test medium without test and/or reference item + 10 mg/L humic acid, duplicate

The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via aseries of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.
The test item and reference item were weighed out. The test item were weighed out into small beakers. 10 mg/L humic acid was weighed out and was transferred into the incubation vessels for the test item, toxicity control, functional contral and humic acid control replicates. A defined amount of ultrapure water were added to the test item. The test item dispersions and the reference item were transferred to the respective incubation vessels with ultrapure water.
On day 28, 1 ml 37 % HCI was added to each of the vessels. Aeration was continued for further 24 hand the quantity of CO2 released was determined.
Determination of C02 was carried out by titration subsequent to complete adsorption of the released C02 in an alkaline solution (0.0125 mol/l Ba(OH)2). For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.
Back titration of the residual Ba(OH)2 with 0.05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly.
On day 28 the pH of all solutions was measured prior to acidification.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Remarks:
test item without humic acid
Value:
81.5
Sampling time:
16 d
Parameter:
% degradation (CO2 evolution)
Remarks:
test item with humic acid
Value:
65.5
Sampling time:
16 d
Parameter:
% degradation (CO2 evolution)
Remarks:
toxicity control without humic acid
Value:
77
Sampling time:
16 d
Parameter:
% degradation (CO2 evolution)
Remarks:
toxicity control with humic acid
Value:
63
Sampling time:
16 d
Details on results:
Both test item replicates without humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 13 days. The mean biodegradation on day 28 was 100%.
Both test item replicates with humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 16 days. The mean biodegradation on day 28 was 72%.

In the toxicity control containing both test and reference item a biodegradation of 67% was determined within 13 days, with 95% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. In the toxicity control with humic acid a biodegradation of 57% was determined within 13 days, with 70% after 28 days.
Results with reference substance:
The adaptation phase of the functional control changed within 2 days into the degradation phase (degradation >/= 10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 8 days and a maximum biodegradation of 84% on day 16.
The validity criterion degradation >/= 60% after 14 days is fulfilled.
The adaptation phase of the functional control with humic acid changed within 6 days into the degradation phase (degradation >/= 10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 13 days and a maximum biodegradation of 67% on day 28. The validity criterion deqradation >/= 60% after 14 days is fulfilled.

Table 1: Biodegradation of the Test Item without humic acid in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

Day 8

Day 16

Day 22

Day 28

Test Item, 1streplicate

38

81

92

100

Test Item, 2ndreplicate

43

82

99

100

Functional Control

71

84

84

84

Toxicity Control

test item + reference item

34

77

88

95

 

Table 2: Biodegradation of the Test Item with humic acid in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

Day 8

Day 16

Day 22

Day 28

Test Item, 1streplicate

38

63

68

69

Test Item, 2ndreplicate

40

68

74

74

Functional Control

46

65

65

67

Toxicity Control

test item + reference item

33

63

70

70

Validity criteria

The validity criteria were fulfilled according to the guideline:

• The total CO2 evolution in the inoculum control at the end of the test was 40.8 mg/L (validity criterion: < 40 mg CO2/L after 28 days) (Guideline: “…. the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/Lmedium. If values greater than 70 mg CO2/L are obtained, the data and experimental technique should be examined critically." Accordingly, the result was considered to be valid.)

• The degradation of the functional control reached the pass level of >/=60% within 8 days (degradation: 71% on day 8).

• The differences of extremes of replicate values of removal of the test item at the end of the test was less than 20% (11% difference on day 28).

• The degradation of the toxicity control reached the pass level of 25% within 8 days (degradation: 34% on day 8).

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions C16 Alkylamidopropyltrimethylammonium Chloride is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.
Executive summary:

The ready biodegradability of C16 Alkylamidopropyltrimethylammonium Chloride was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test according to OECD 301 B.

The test item was tested at a concentration of 16 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.8 mgC/Lin the test vessels with and without Humic acid. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2°C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% within 8 days and a maximum biodegradation of 84% on day 16. The functional control with Humic acid reached the pass level of 60% within 13 days and a maximum biodegradation of 67% on day 28.

In the toxicity control containing both test and reference item a biodegradation of 67% was determined within 13 days, with 95% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. In the toxicity control with humic acid a biodegradation of 57% was determined within 13 days, with 70% after 28 days.

Both test item replicates without humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 13 days. The mean biodegradation on day 28 was 100%.

Both test item replicates with humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 16 days. The mean biodegradation on day 28 was 72%.

The replicates without humic acid did show better results and are therefore used to evaluate the study. The replicates with humic acid did also fulfil the 10-day-window, but only for the mean of the replicates.

Under the test conditions the test item is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.

Description of key information

readily biodegradable (100% after 28 d; 16 mg test item/L; >/=60% degradation within 10-d-window); OECD Guideline 301 B (Modified Sturm Test); GLP; RL1

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Two tests are available which adress biodegradation of C16 Alkylamidopropyltrimethylammonium Chloride.

The biodegradation of C16 Alkylamidopropyltrimethylammonium Chloride was investigated in a study according to OECD Guideline 301 F (1992). The biodegradation of the substance in the static test was found to be at mean 9 % with a standard deviation of 1.5 % for a concentration of 2000 mg test item per liter and at mean 53 % with a standard deviation of 7.3 % for a concentration of 400 mg test item per liter after 28 days. For a concentration of 2000 mg test item per liter no 10-day-window could be calculated since degradation rate maintained below 10 %. For a concentration of 400 mg test item per liter biodegradation within the 10-day-window was found to be 34 %. The 10-day window in the 400 mg/L assay started at day 5 - 7. The degradation of the reference substance sodium benzoate had reached 92 % within the first 14 days. The difference of extremes of replicate values of the removal of the test item at the end of the test for 2000 mg/L is less than 20%. Therefore, the test can be considered as valid. No inhibitory effects of the test item were observed (more than 25 % degradation occurred within 14 days) in the toxicity control.

According to the guideline, the substance is considered as not readily biodegradable under the chosen test conditions.

As the results were slightly below the pass level of 60% biodegradation, the substance may be regarded as inherently biodegradable in accordance with the Guidance on information requirements and chemical safety assessment, Chapter R.7b, Endpoint specific guidance.

The second test was performed with a lower test material concentration.

The ready biodegradability of C16 Alkylamidopropyltrimethylammonium Chloride was determined with a non-adapted activated sludge over a test period of 28 days in the Modified Sturm Test according to OECD 301 B.

The test item was tested at a concentration of 16 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.8 mgC/Lin the test vessels with and without Humic acid. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2°C.

The biodegradation of the test item was followed by titrimetric analysis of the quantity of CO2 produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last titration was made on day 29 after residual CO2 had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2 production (ThCO2) of the test item. The biodegradation was calculated for each titration time.

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% within 8 days and a maximum biodegradation of 84% on day 16. The functional control with Humic acid reached the pass level of 60% within 13 days and a maximum biodegradation of 67% on day 28.

In the toxicity control containing both test and reference item a biodegradation of 67% was determined within 13 days, with 95% after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. In the toxicity control with humic acid a biodegradation of 57% was determined within 13 days, with 70% after 28 days.

Both test item replicates without humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 13 days. The mean biodegradation on day 28 was 100%.

Both test item replicates with humic acid reached the 10% level (beginning of biodegradation) within 6 days. Both test item replicates reach the 60% pass level within 16 days. The mean biodegradation on day 28 was 72%.

The replicates without humic acid did show better results and are therefore used to evaluate the study. The replicates with humic acid did also fulfil the 10-day-window, but only for the mean of the replicates.

Under the test conditions the test item is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.

In the Manometric respirometry Test the test item concentrations of 400 and 2000 mg/L were above the recommended concentration of 100 mg/L, although the ThOD/L was in the recommended range for the lower concentration. In contrast, the test item concentration (16 mg/L) in the Modified Sturm test was within the recommended range (10 - 20 mg/L). Thus, the latter study is given more weight in this weight of evidence. Overall, the substance can be regarded as readily biodegradable.