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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 16, 1992 to April 20, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
slight inoculum modification
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- CAS reg. No. 61789-71-7
- Composition:
cocobenzyldimethylammonium chloride 50.1%
amine 1.1%
water 48.8%
- Batch/lot No. 16881027
- Appearance: liquid
- Stability: stable under test conditions
- Solubility in water: soluble at test concentrations
- Storage at room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
other: Secondary activated sludge was used
Details on inoculum:
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 the sludge (200 mg Dry Weight (DW)/L) was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles. This method was described in a proposal for harmonizing ready biodegradability test protocols.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- The closed bottle test was performed according to the EEC, OECD and ISO Test Guidelines. Ten bottles containing only inoculum, 10 bottles containing test substance and inoculum, and 10 bottles containing sodium acetate and inoculum were used. The concentrations of the test compound and sodium acetate in the bottle were 4.0 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. The zero time bottles were immediately analysed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated at 21°C 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. One extension from the protocol of the closed bottle test was introduced.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
63
Sampling time:
28 d
Details on results:
The test was conducted in the presence of silica gel due to toxicity to inoculum.
The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28 . Furthermore, the differences of the replicate values at Day 28 were less than 20%.
The validity of the test was shown by oxygen concentrations being >0.5 mg/L in the bottles.
Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at Day 14 was 78.

Table 1. Biodegredation percentages (%)

   Test substance  Reference substance
 0  0  0
 7  1  69
 14  50  78
 21  66  86
 28  63  88
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study, the biodegradation of the test substance was determined to be 63% at Day 28. The test substance was considered readily biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Not GLP
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): active substance (alkyl(C12-C16)dimethylbenzylammonium chloride (ADBAC) in aqueous/ethanol solution).
- Stability under test conditions: ADBAC is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions
- Storage condition of test material: At room temperature
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
>106 CFU/mL; consisting of micro-organisms in mud collected from Station Epuration Wavre 2nd Stage, a household water treating plant
Duration of test (contact time):
28 d
Initial conc.:
5 other: mgC/L
Based on:
test mat.
Details on study design:
Test conditions:
- Test temperature: 22 ± 2°C
- pH: 7.3 at start and 7.8 at the end of study
- Aeration of dilution water: Air free from carbon dioxide was passed through the solutions using a flow of 50 to 100 mL/min
- Continuous darkness: Yes
- Other: Total organic carbon content: 62.8%
Test system:
- Culturing apparatus: 3-litre flasks mounted with an aeration tube on a magnetic stirrer
- Details of trap for CO2 and volatile organics if used: the CO2 produced in each flask was precipitated with Ba(OH2). The amount of CO2 produced was determined by titration.


Reference substance:
benzoic acid, sodium salt
Remarks:
20 mgC/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
95.5
Sampling time:
28 d
Details on results:
- The biodegradability in the test flask was determined to be 95.5% after 28 days (See Table 1 under 'Any other information on results inc. tables).
- The CO2 production in the blank (inoculum control) was 39.2 mg. The pH measured at the start and end of the test period was 7.3 and 7.8, respectively. The temperature throughout the test ranged from 20.8 to 21.4°C.
Results with reference substance:
The biodegradability in the reference flask was determined to be 88.9% after 28 days (See Table 1 under 'Any other information on results inc. tables).

Table 1. Biodegradability values:

Day

Reference substance

Test substance

% CO2Produced

% CO2 Total

% CO2Produced

% CO2 Total

0

0.00

0.00

0.0

0.0

1

14.16

14.16

1.2

1.2

3

28.54

42.69

1.0

2.2

6

21.20

63.89

9.5

11.6

8

8.77

72.67

16.0

27.7

10

3.72

76.39

20.2

47.9

13

3.26

79.65

17.4

65.3

15

2.53

82.18

7.5

72.8

17

1.47

83.65

5.1

77.9

20

2.31

85.95

3.9

81.8

22

1.15

87.11

1.6

83.5

24

0.25

87.36

4.4

87.8

28

0.16

87.52

1.8

89.6

29

1.35

88.87

5.8

95.5

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5 after 28 days (CO2 evolution). The test substance was considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5% after 28 d (CO2 evolution). The test substance was considered to be readily biodegradable (van Dievoet, 2005).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: Activated sludge domestic unadapted and adapted in SCAS test, river water, sea water, ditch water and soil (loam)
Duration of test (contact time):
3 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
3 d
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test). Half-lives were determined using inoculum from various aquatic sources. The biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, with half -lives of 0.3, 0.1 and 0.1 d, respectively. Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable (van Ginkel, 1996).

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 11, 1992 to January 11, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Principles of method if other than guideline:
- Deviations OECD 301 D: Ammonium chloride was omitted from the medium to prevent nitrification. The closed bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of Day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing the BOD bottle. The test substance was toxic to microorganisms. Therefore, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the test substance should be released slowly from the silica gel (0.5 g/bottle). Although no additional oxygen consumption was expected, controls with silica gel were carried out as well (10 bottles containing test substance, inoculum and silica gel).
- Deviations OECD 302 A: Fill and draw procedure was only six times per week instead of daily. To maintain a constant pH in the SCAS unit, phosphate buffer was added six times per week. Effluent samples were filtered using cellulose nitrate filters with pores of 8 µm to remove sludge particles.
GLP compliance:
yes
Specific details on test material used for the study:
- Chemical name: cocobenzyldimethylammonium chloride
- Physical state: liquid
- Analytical purity: 50% cocobenzyldimethylammonium chloride; free amine 1.1%; water 49%
- Lot/batch No.: 03664
- Stability under test conditions: stable under test conditions
- Water solubitlity: soluble at test concentrations
- Storage condition of test material: at room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Duration of test (contact time):
>= 28 - <= 56 d
Initial conc.:
2 mg/L
Based on:
test mat.
Key result
Parameter:
% degradation (O2 consumption)
Value:
52
Sampling time:
28 d
Remarks on result:
other: inoculated with sludge from the SCAS unit (Day 0); 62% biodegradation at Day 56
Key result
Parameter:
% degradation (O2 consumption)
Value:
77
Sampling time:
28 d
Remarks on result:
other: inoculated with activated sludge from the SCAS unit (Day 21)
Details on results:
- OECD 302A:
Before the addition of the test substance, the effluent non purgeable organic carbon (NPOC) values obtained from the test substance unit and the control unit were comparable and constant. After the first addition (Day 0), the removal of 69% was immediately accomplished due to the adsorption on the sludge, dilution and/or biodegradation. Next, the NPOC values of the units decreased and subsequently remained constant. The percentage removal after 4 weeks over 3 consecutive measurements was 99. Additional closed bottle tests were performed to draw conclusion on the removal mechanism in the SCAS test.
- OECD 301D:
In the closed bottle tests inoculated with sludge taken on Days 0, and 21 from the SCAS test unit, biodegradation of the test substance took place. The test substance was biodegraded at 52% on Day 28 in the closed bottle test inoculated with sludge from the SCAS test (Day 0). In the prolonged closed bottle test the test substance was biodegraded at 62% on Day 56. The biodegradation in the closed bottle tests did increase due to acclimatization of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 21.

The test substance was degraded in waste water treatment plant after a short acclimatization period (5 d). In an acclimatized waste water treatment plant, the test substance was probably totally mineralized. Total mineralization was demonstrated by increased biodegradation percentages in the closed bottle test inoculated with sludge taken on Day 21 compared to the closed bottle test inoculated with unacclimatized sludge. The algal growth inhibition test showed that the effluent of Day 1 was slightly toxic. This toxicity was probably caused by the test substance and/or biodegradation products present in the effluent of the test unit of the SCAS test. The results of the toxicity test of Day 23 demonstrated that acclimatized waste water treatment plants detoxified the test substance by biodegradation.

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Under the study conditions, the test substance was considered to be inherently biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guidelines 301D and 302A (closed bottle test / modified SCAS test), in compliance with GLP. The experiment was carried out using a combination of an inherent and a ready biodegradability test. In order to predict the effects of possible biodegradation products, the toxicity of effluents from a semi-continuous activated sludge (SCAS) units were assessed. The test substance caused no reduction of the biodegradation of non-purgeable organic carbon (NPOC) present in primary settled sewage. Therefore, it was considered to be non-inhibitory to activated sludge. During the test period, the substance was totally removed (99%) from the waste water by adsorption and/or biodegradation. In a second step, the distinction between biodegradation and adsorption was evaluated in closed bottle tests inoculated with approximately 2 mg/L of activated sludge collected on Days 0 and 28 from the SCAS unit fed with the test substance. With the Day 0 SCAS sample, the test substance was biodegraded by 52% within 28 d and by 62% within 56 d. The biodegradation in the closed bottle tests did increase due to acclimatization of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 28. The closed bottle test results demonstrated that the test substance was removed by biodegradation in the SCAS test. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1993).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From December 12, 1986 to February 04, 1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Some discrepancies for the identification of the test substance
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
GLP compliance:
no
Remarks:
But includes statement of inspections by GLP quality assurance officer.
Specific details on test material used for the study:
- Molecular weight 355.5
- Activity 50.15 %
- Water: 47.5%
- Ethyleneglycol 2.5 %
- COD =5.2 mg/O2/L
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
The inoculum is taken from an activated sludge plant, the municipal waste water treatment plant in Duiven (NL). The sludge is preconditioned during a week: a sludge suspension of 1 g/L is aerated in the dilution water. This modification is introduced to reduce high residual respiration rates.
Duration of test (contact time):
42 d
Initial conc.:
2.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Performance of the test:
The biodegradability test was carried out according to OECD Test Guideline 301D: Closed Bottle Test. The method was modified according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concern the inoculum, the composition of the dilution water and the analyses. The density of the inoculum in the test was 3 mg/L. On Day 0, 14, 28 and 42 the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled completely with a suspension of preconditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were carried out in a new series of three bottles.
Reference substance:
acetic acid, sodium salt
Test performance:
Adequate reference substance performance.
Adequate blank performance.
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Details on results:
- A toxicity control was carried out with 4.3 and 12.8 mg of test substance and was found not to be toxic at these concentrations although a slight inhibition was seen at 12.8 mg/L.
- Ready biodegradable
Validity criteria fulfilled:
no
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study, the biodegradation of the test substance in water was 65% at Day 28. Hence it should be classified as readily biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test).The method was adapted according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concerned the inoculum, the composition of the dilution water and the analyses. The density of the inoculum in the test was 3 mg/L. On Days 0, 14, 28 and 42, the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled completely with a suspension of pre-conditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were conducted in a new series of three bottles. Under the conditions of the study, the biodegradation of the test substance in water was 65% at Day 28. Hence, the test substance was considered readily biodegradable (Balk, 1987).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From November 06, 1991 to December 05, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): alkyl(C12-C16)dimethylbenzylammonium chloride (ADBAC))
- Physical state: Yellow liquid
- Analytical purity: 80.8% w/w a.s. in ethanol solution
- Composition of test material, percentage of components: A blend of alkyl dimethyl benzyl ammonium chlorides; active ingredient: n-alkyl (50% C14; 40% C12;10% C10)
- Lot/batch No.: C1222304
- Stability under test conditions: ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Ambient
- Other:
- TOC: 80% carbon
- TCO2: 3.13
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): acclimated activated sludge, Avondale Sewage Treatment Plant, Avondale.
- Method of cultivation and preparation of the inoculum mixture: acclimated activated sludge was obtained from the final day of a previous SCAS assay conducted with the test substance at a concentration of 10 mg active substance per litre. Approximately 150 mL of mixed liquor was collected from each duplicate units, composited and homogenized at medium speed in a blender for 2 minutes. The homogenised sample was poured into a beaker and allowed to settle for 30 mins. The supernatant was decanted and added to the flasks at a conc. of 1% (v/v). On the same day the sludge was collected a standard plate count (SPC) was performed on the inoculum. The plates were incubated at test temperature. The result was 2.3 E06 CFU/mL
- Inoculum addition: 20 mLof the above inoculum mixture was added to all test flasks.
Duration of test (contact time):
28 d
Initial conc.:
5 mg/L
Based on:
act. ingr.
Initial conc.:
10 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test conditions:
- Composition of medium: Momodified BOD water
- Test temperature: 22.1- 23.0°C
Test system:
- Culturing apparatus: four glass four-litre Erlenmeyer flasks containing two litres of modified biochemical oxygen demand (BOD) water
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: Tte test flasks were placed on a rotary shaker, connected to the scrubbing train and aerated over-night to purge the system of background CO2.
- Details of trap for CO2 and volatile organics if used: the CO2 produced in each flask reacted with 0.024 N Ba(OH)2 and precipitated as BaCO3. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 N standardized hydrochloric acid (HCl).
- Other: after 28 d, the contents of the flasks were acidified with concentrated sulfuric acid (H2SO4) and aerated overnight. One final titration was performed.
Sampling:
- Sampling frequency: on Day 2, 5, 8, 11, 14, 17, 20, 23, 28.
Control and blank system:
- Inoculum blank: one flask containing test medium and inoculum
- Reference control: one flask containing only d-glucose at a conc. of 20 mg active/L
Statistical methods:
- Gauss-newton method
Reference substance:
other: d-glucose at a concentration of 20 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
84
Sampling time:
28 d
Remarks on result:
other: at 5 mg/L concentration
Key result
Parameter:
% degradation (CO2 evolution)
Value:
82.6
Sampling time:
28 d
Remarks on result:
other: at 10 mg/L concentration
Details on results:
- Biodegradability in the test flasks was determined to be 84.0% and 82.6% within 28 d at 5 and 10 mg/L of test substance respectively.
- Biodegradation in the blank was 0%.
- Final SOC test concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose.
Results with reference substance:
Biodegradability in the reference flask was determined to be 80.6% in 28 d.

Table 1. % TCO2 over time

Day

d-glucose

Test substance

5 mg a.i./L

Test substance

10 mg a.i./L

2

20.6

9.3

1.8

5

39.2

43.8

39.1

8

50.6

58.5

54.2

11

61.0

75.4

67.9

14

64.9

85.0

76.0

17

69.2

88.5

80.2

20

73.4

87.2

81.6

23

77.0

85.6

81.9

28

80.1

85.0

82.3

28

80.6

84.0

82.6

Table 2. Final results

Test substance

Concentration

(mg a.i./L)

Final %TCO2

Final SOC (mL/L)

Control (blank)

0

0

0.4

d-glucose

20

80.6

2.2

Test substance

5

84.0

0.7

Test substance

10

82.6

0.6

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study, the test substance was considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. Flasks containing acclimated inoculum (at 10 mg/L) from a previous SCAS assay were dosed with 5 and 10 mg a.i./L of the test substance or 20 mg/L of the reference substance (d-glucose) and were maintained for 28 d. Biodegradability was calculated from the CO2 released over time in the test and reference flasks relative to that which was released in the blank control (a flask prepared without test or reference substance). The results indicated that 84.0 and 82.6% CO2 was produced in vessels dosed with 5 and 10 mg/L test substance, respectively, compared to 0% with the control and 80.6% with d-glucose. Final suspended organic carbon (SOC) concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose. Under the conditions of the study, the substance was considered to be readily biodegradable (Corby, 1992a).

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From October 30, 1991 to November 06, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): active substance (a.s.), alkyl(C12-C16)dimethylbenzylammonium chloride (ADBAC)
- Physical state: Yellow liquid
- Analytical purity: 80.8% w/w a.s. in ethanol solution
- Composition of test material, percentage of components: A blend of alkyl dimethyl benzyl ammonium chlorides; active ingredient: n-alkyl (50% C14; 40% C12;10% C10)
- Lot/batch No.: C1222304
- Expiration date of the lot/batch: August 1992
- Stability under test conditions: ADBAC, is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): acclimated activated sludge, Avondale Sewage Treatment Plant, Avondale.
- Preparation of the SCAS units: the sludge was screened through a 2 mm sieve to remove large clumps. The total suspended solids (TSS) level was determined and based on this reading the sludge was distributed among the SCAS units such that when the volume in each unit was adjusted to 1.5L with tap water the suspended solids level was approx. 2500 mg/L.
- Pre-acclimation period: the units were aerated for 23 and half hours at a rate adequate to maintain solids suspension. At the end of this period, the air was turned off and the sludge was allowed to settle for 30 mins. One liter of effluent was drawn off and replaced with 1L of influent consisting of 10 mL synthetic sewage and 990 mL tap water to bring back to 1.5L. The air was turned on. This process was repeated on a daily basis.
- Sludge acclimation period: all the units were fed 10 mL of synthetic sewage and 990 mL of tap water daily for a minimum of 4d prior to initiation of the test substance acclimation period.
- Sludge distribution: on the day where the addition of the test substance was to be started, the sludge was settled, composited, and re-distributed among the units so that each unit contained a uniform sample of the sludge.
Duration of test (contact time):
7 d
Initial conc.:
10 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
other: SOC removal
Details on study design:
Test conditions:
- Composition of medium: synthetic sewage. It was prepared using the following composition: d-glucose: 30g; nutrient broth: 20g; K2HPO4: 13g; tap water: 1L. 20 mL of synthetic sewage was added to each unit on the 1st two days of the pre-acclimation period to help maintain suspended solids at 2500 mg/L.
- Temperature: 21.9-23.2°C
- Suspended solids concentration: 2500 mg/L.
- Lighting: during daily maintenance, the SCAS units were exposed to only room lighting and not exposed to direct sunlight.
- Other:
Test substance acclimation period: the test substance was added to the test units incrementally for a 7-d acclimation period. The control units are treated in the same way except that they do not receive any of the test substance.
Testing period: the test substance was added to the test units at the specified test concentration for an additional 7d following the acclimation period. The control units were treated on the same way.

Test system:
- Culturing apparatus: SCAS aeration chambers containing 1.5L of the activated sludge
- Number of culture flasks/concentration: 2

Sampling:
- Sampling frequency: daily (i.e., on Day 1, 2, 3, 4, 5, 6 and 7)
- Effluent analysis: one liter of effluent was withdrawn daily from each unit and saved for analysis. The effluent was replaces with comparable volume of influent of synthetic sewage. An aliquot of each effluent sample was centrifuged. A subsample of centrate was acidified with conc. H2SO4, purged with N2 and submitted for soluble organin chemical analysis (SOC).

Control and blank sampling system:
- Inoculum blank: yes, two control units were maintained only on synthetic sewage

Statistical method: Analysis of variance (ANOVA)
Key result
Parameter:
other: % SOC removal
Value:
100
Sampling time:
7 d
Details on results:
- The percentage of carbon removed varied from 98.22% to 106.51% over a 7-d period between the two test vessels. The average percent SOC removal for test substance was >100%.
- The mean SOC value in mg/L was 4.1±0.3 for control 1 and 4±0.3 for control 2.

Table 1. % Carbon removed

Days

Test 1

Test 2

1

100.59

102.96

2

98.22

106.51

3

100.00

104.73

4

98.22

100.59

5

100.59

102.96

6

101.18

104.73

7

102.96

102.96

Validity criteria fulfilled:
not specified
Interpretation of results:
inherently biodegradable
Conclusions:
Under the conditions of the study, the average percent SOC removal of the test substance was >100% indicating it to be inherently biodegradable.
Executive summary:

A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 302A (Modified SCAS test), in compliance with GLP. Four chambers containing activated sludge were aerated and the suspended solid contents were adjusted to 2500 mg/L. The test substance at 1000 mg a.i./L was added to two test units for a 7 d acclimation period. This consisted of incremental additions until the final test concentration of 10 mg a.i./L was reached. Two additional units did not receive test substance and served as controls. The testing period was an additional 7 d following the acclimation period. Throughout the study, all units were fed synthetic sewage. Effluents withdrawn from each unit were analysed for SOC. Under the conditions of the study, the average percent SOC removal was >100% indicating that the test substance was inherently biodegradable (Corby, 1992b).

Description of key information

A number of reliable studies have shown that the test substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Study 1. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test), in compliance with GLP. Secondary activated sludge was used in this experiment and the percentage of degradation (O2 consumption) was measured. Since the substance was toxic to microorganisms, it was tested in the presence of silica gel to reduce the concentration in the water phase. During the test period, the substance was released slowly from the silica gel. The validity of the test was demonstrated by an endogenous respiration of 1.3 mg/L at Day 28. Furthermore, the differences between the replicate values at Day 28 were less than 20%. The biodegradation of the reference substance, sodium acetate, at Day 14 was 78%. Finally, the validity of the test was shown by oxygen concentrations being > 0.5 mg/L in the bottles. Under the conditions of the study, the biodegradation of the substance was determined to be 63% at Day 28. The substance was considered readily biodegradable (van Ginkel and Stroo, 1992).

Study 2. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301B (CO2 evolution test). Flasks containing inoculum from a household water-treating plant dosed with the equivalent of 5 mg C/L test or 20 mg C/L reference substances were maintained for 28 d. Biodegradability was calculated from the released CO2 over time in the test and reference flasks compared to the blank control (a flask prepared without test or reference substance). CO2 production in the blank (inoculum control) was 39.2 mg. Biodegradability in the reference flask was determined to be 88.9% after 28 d. Under the test conditions, the biodegradation of the test substance in water was determined to be 95.5% after 28 d (CO2 evolution). The test substance was considered to be readily biodegradable (van Dievoet, 2005).

Study 3. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test). Half-lives were determined using inoculum from various aquatic sources. The biodegradation of the test substance was 71, 69 and 60% in seawater, ditch water and river water, with half -lives of 0.3, 0.1 and 0.1 d, respectively. Under the study conditions, the biodegradation of the test substance was determined to be >60% within three days in the closed bottle test inoculated with unacclimatized sludge. The test substance was therefore considered readily biodegradable (van Ginkel, 1996).

Study 4. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guidelines 301D and 302A (closed bottle test / modified SCAS test), in compliance with GLP. The experiment was carried out using a combination of an inherent and a ready biodegradability test. In order to predict the effects of possible biodegradation products, the toxicity of effluents from a semi-continuous activated sludge (SCAS) units were assessed. The test substance caused no reduction of the biodegradation of non-purgeable organic carbon (NPOC) present in primary settled sewage. Therefore, it was considered to be non-inhibitory to activated sludge. During the test period, the substance was totally removed (99%) from the waste water by adsorption and/or biodegradation. In a second step, the distinction between biodegradation and adsorption was evaluated in closed bottle tests inoculated with approximately 2 mg/L of activated sludge collected on Days 0 and 28 from the SCAS unit fed with the test substance. With the Day 0 SCAS sample, the test substance was biodegraded by 52% within 28 d and by 62% within 56 d. The biodegradation in the closed bottle tests did increase due to acclimatization of the microorganisms in the SCAS test unit. The test substance was biodegraded at 77% on Day 28 in the closed bottle test inoculated with sludge sampled on Day 28. The closed bottle test results demonstrated that the test substance was removed by biodegradation in the SCAS test. Under the study conditions, the test substance was considered to be inherently biodegradable (van Ginkel, 1993).

Study 5. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301D (closed bottle test).The method was adapted according to the recommendations of ECETOC (1985) or Blok et al. (1985). Modifications concerned the inoculum, the composition of the dilution water and the analyses. The density of the inoculum in the test was 3 mg/L. On Days 0, 14, 28 and 42, the concentration of oxygen was measured. On Day 28, nitrite and nitrate concentrations were measured. The dilution water was the medium as prescribed by the test guideline without ammonia. This modification was introduced to minimize the consumption of oxygen for the nitrification process. Dark glass bottles of about 280 mL with glass stoppers were filled completely with a suspension of pre-conditioned activated sludge (3 mg/L) in dilution water and a concentration of the test substance equivalent to about 6 mg ThOD/L (Theoretical Oxygen Demand). The test was carried out in triplicate and at every observation time measurements of oxygen and pH were conducted in a new series of three bottles. Under the conditions of the study, the biodegradation of the test substance in water was 65% at Day 28. Hence, the test substance was considered readily biodegradable (Balk, 1987).

Study 6. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. Flasks containing acclimated inoculum (at 10 mg/L) from a previous SCAS assay were dosed with 5 and 10 mg a.i./L of the test substance or 20 mg/L of the reference substance (d-glucose) and were maintained for 28 d. Biodegradability was calculated from the CO2 released over time in the test and reference flasks relative to that which was released in the blank control (a flask prepared without test or reference substance). The results indicated that 84.0 and 82.6% CO2 was produced in vessels dosed with 5 and 10 mg/L test substance, respectively, compared to 0% with the control and 80.6% with d-glucose. Final suspended organic carbon (SOC) concentrations were 0.7 mL/L (5 mg/L) and 0.6 mL/L (10 mg/L) compared to 0.4 mL/L with the control and 2.2 mL/L with d-glucose. Under the conditions of the study, the substance was considered to be readily biodegradable (Corby, 1992a).

Study 7. A study was conducted to determine the biodegradation of the test substance in water according to OECD Guideline 302A (Modified SCAS test), in compliance with GLP. Four chambers containing activated sludge were aerated and the suspended solid contents were adjusted to 2500 mg/L. The test substance at 1000 mg a.i./L was added to two test units for a 7 d acclimation period. This consisted of incremental additions until the final test concentration of 10 mg a.i./L was reached. Two additional units did not receive test substance and served as controls. The testing period was an additional 7 d following the acclimation period. Throughout the study, all units were fed synthetic sewage. Effluents withdrawn from each unit were analysed for SOC. Under the conditions of the study, the average percent SOC removal was >100% indicating that the test substance was inherently biodegradable (Corby, 1992b).