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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 18, 1997 to April 29, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Lot/batch No.: 1061962182
- Physical state: Pellets
- Composition: ca. 80% alkyltrimethyl ammonium chloride
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): community wastewater treatment plant Hildesheim, Germany
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Initial conc.:
2.4 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
18
Sampling time:
28 d
Details on results:
Reference substance: 86% degradation in 28 days
Toxicity control: 67% degradation in 28 days
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of the study, the test substance was not 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. Non-adapted activated sludge was exposed to 3 mg/L test substance (corresponding to 2.4 mg a.i./L based on a purity of ca. 80%), equivalent to a theoretical oxygen demand of 6.96 mg O2/L, for 28 days. A reference substance and a toxicity control were run in parallel. The test substance degraded to 18% within 28 days. Degradation of the reference substance and the toxicity control were 86 and 67%, respectively. Under the conditions of the study, the test substance was not readily biodegradable (Noack, 1997).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From August 12, 1992 to September 15, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
acceptable deviations
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
- Lot/batch No.: 00564
- Physical state: liquid
- Composition: 49% octadecyltrimethylammoniumchloride; 0.3% free amine; 36% 2-propanol; 15% water
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Secondary activated sludge from WWTP Nieuwgraaf In Duiven, The Neatherlands. It was an activated sludge plant treating predominantly domestic wastewater.
- The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg dry wt/L) was aerated for a period of 7d. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/L.
Duration of test (contact time):
35 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Medium:
- Composition of the medium:
The nutrient medium of the closed bottle test contained per litre 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.
- Test temperature: 21+/- 1°C (not reported)
- pH: 7.1
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottles = 2 mg/L
- Continuous darkness: yes

Test system:
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
10 bottles containing only mineral nutrient solution
10 bottles containing only minteral nutrient solution and inoculum = inoculum blank
10 bottles containing mineral nutrient solution, test substance and inoculum = test
10 bottles containing mineral nutrient solution sodium acetate and inoculum = reference control
10 bottles containing only minteral nutrient solution, silica gel and inoculum = inoculum blank (silica gel)
10 bottles containing mineral nutrient solution, test substance coated on silica gel and inoculum =test
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter

Sampling:
- Sampling frequency: Day 0, 7, 14, 21 , 28 and 35

Control and blank system:
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
- Reference control: yes
- Other: inoculum blank with silica gel (to prove the non-biodegradability of silica gel).

Calculations:
THOD test substance = 2.3 mg/mg
THOD sodium acetate = 0.8 mg/mg
Oxygen consumption (mg/L) (BOD) = mean oxygen concentration (mg/L) inoculum blank - mean oxygen concentration (mg/L) test (or reference)
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
53
Sampling time:
35 d
Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of 0.95 mg/L, total mineralization of sodium acetate >0.5 mg/L O2 in bottles during the test; differences between replicates at Day 28 < 20%
Interpretation of results:
inherently biodegradable
Conclusions:
Under the study conditons and in the presence of silica gel, the test substance biodegraded to 48% at Day 28. In the prolonged closed bottle test with silica gel, the biodegradation percentage reached 53% at Day 35. Due to the large fraction of 2-propanol in the test substance (approximately 36%) it could not be concluded that the active test substance was 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. The test was performed with activated sludge, domestic and non-adapted, exposed to 4.0 mg/L test substance (49% active substance and 36% 2-propanol) for 35 days. Silica gel was added in the study setup to reduce the concentration of test substance in the water phase, thereby reducing its toxicity. Under the study conditions, the test substance in the presence of silica gel caused no reduction in the endogenous respiration and degraded up to 53% by Day 35. The test was valid as shown by an endogenous respiration of 0.95 mg/L and by the total mineralization of the reference substance. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. However, it was not possible to distinguish between the biodegradation of the test substance and 2-propanol (van Ginkel, 1993).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From April 19, 1990 to October 18, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
acceptable deviations
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (as cited in study report): Arquad 18
- Lot/batch No.: RCD GU 2892
- Physical state: solid
- Analytical purity: 92%
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): Activated sludge plant RWZI Nieuwgraaf in Duiven, treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (200 mg dry wt/L) was aerated for a period of 7d. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/L.
Duration of test (contact time):
175 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test conditions:
- Composition of medium: The nutrient medium of the closed bottle test contained per litre 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, 0.25 mg FeCl3.6H2O.
- pH: 6.9 - 7.4
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottles = 2 mg/L
- Continuous darkness: yes

Test system:
- Culturing apparatus: incubator
- Number of culture flasks/concentration:
8 bottles containing only mineral nutrient solution
8 bottles containing only minteral nutrient solution and inoculum = inoculum blank
8 bottles containing mineral nutrient solution, test substance and inoculum = test
8 bottles containing mineral nutrient solution sodium acetate and inoculum = reference control
8 bottles containing only minteral nutrient solution, silica gel and inoculum = inoculum blank (silica gel)
8 bottles containing mineral nutrient solution, test substance coated on silica gel and inoculum =test
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter

Sampling
- Sampling frequency: Day 0, 5, 15, 28 , 55, 92, 119, 175 (without silica gel); Day 0, 5, 15, 28 ,42, 56, 84 (with silica gel)
- Sampling method: the bottles from before Day 28 were discarded after the oxygen measurement and the bottles from Day 28 were used also for measuring Days > Day 28 (by using a special funel to collect the disipated medium)

Control and blank system:
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
- Reference control: yes
- Other: inoculum blank with silica gel (to prove the non-biodegradability of silica gel).

Calculations:
THOD test substance = 2.8 mg/mg
THOD sodium acetate = 0.8 mg/mg
Oxygen consumption (mg/L) (BOD) = mean oxygen concentration (mg/L) inoculum blank - mean oxygen concentration (mg/L) test (or reference)
Biodegradation (%) = BOD/THOD *100
Reference substance:
acetic acid, sodium salt
Remarks:
6.7 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
77
Sampling time:
175 d
Remarks on result:
other: without silica gel
Key result
Parameter:
% degradation (O2 consumption)
Value:
30
Sampling time:
30 d
Remarks on result:
other: with test substance coated on silica gel
Key result
Parameter:
% degradation (O2 consumption)
Value:
57
Sampling time:
84 d
Remarks on result:
other: with test substance coated on silica gel

Table 1. Percentages biodegradation of the test subsance and sodium acetate in the closed bottle test

 Time (Days)  5 15  28   55 92  119   175 
 Test substance(%BOD/ThOD) 0 28  65  77 
sodium acetate (%BOD/ThOD)   75 88  85         

Table 2. Percentages biodegradation of the test subsance on silica gel and sodium acetate in the closed bottle test

 Time (Days) 15  28  42  56 84 
 Test substance (%BOD/ThOD) 17  30  45  55  57 
 sodium acetate (%BOD/ThOD) 73  83 90       
Validity criteria fulfilled:
yes
Remarks:
endogenous respiration of 0.4-0.5 mg/L, total mineralization of sodium acetate within 14d >0.5 mg/L O2 in bottles during test, differences of replicates <20% at Day 28
Interpretation of results:
inherently biodegradable
Conclusions:
In the prolonged closed bottle test without silica gel, test substance was biodegraded 77% at Day 175. Toxicity of test substance results in a long lag phase of 55d. After 55d biodegradation starts and reaches 77% at Day 175 showing complete minteralization of test substance. In the test with silica gel, added to ensure a slow release of the test substance into the water phase, test substance was biodegraded 57% at Day 84. In this test the lag phase is reduced to 5d. The test substance has to desorp from the silica gel into the water phase to be bioavailable for the micro-organisms. Desorption and herewith bioavailability of test substance is probably the liming factor in the experiment with silica, resulting in a biodegradation percentage that levels just around the 60%. Under the conditions of the study, the test substance was considered as inherently 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. The test was performed with activated sludge, domestic and non-adapted, exposed to 2.0 mg/L test substance (92% purity) for 175 days. The test was realised with and without silica gel. In the prolonged closed bottle test without silica gel, the substance degraded up to 77% by Day 175. In the test with silica gel, (added to ensure a slow release of the test substance into the water phase), the substance was 30% biodegraded at Day 28 and reached 57% biodegradation at Day 84. Both tests were valid as shown by endogenous respirations of 0.4 and 0.5 mg/L and the total mineralization of the reference substance, sodium acetate. Under the conditions of the study, the test substance was considered to be inherently biodegradable (van Ginkel, 1990).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
inherently 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. Non-adapted activated sludge was exposed to 3 mg/L test substance (corresponding to 2.4 mg a.i./L based on a purity of ca. 80%), equivalent to a theoretical oxygen demand of 6.96 mg O2/L, for 28 days. A reference substance and a toxicity control were run in parallel. The test substance degraded to 18% within 28 days. Degradation of the reference substance and the toxicity control were 86 and 67%, respectively. Under the conditions of the study, the test substance was not readily biodegradable (Noack, 1997).

Study 2.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. The test was performed with activated sludge, domestic and non-adapted, exposed to 4.0 mg/L test substance (49% active substance and 36% 2-propanol) for 35 days. Silica gel was added in the study setup to reduce the concentration of test substance in the water phase, thereby reducing its toxicity. Under the study conditions, the test substance in the presence of silica gel caused no reduction in the endogenous respiration and degraded up to 53% by Day 35. The test was valid as shown by an endogenous respiration of 0.95 mg/L and by the total mineralization of the reference substance. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period. However, it was not possible to distinguish between the biodegradation of the test substance and 2-propanol (van Ginkel, 1993).

Study 3.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. The test was performed with activated sludge, domestic and non-adapted, exposed to 2.0 mg/L test substance (92% purity) for 175 days. The test was realised with and without silica gel. In the prolonged closed bottle test without silica gel, the substance degraded up to 77% by Day 175. In the test with silica gel (added to ensure a slow release of the test substance into the water phase), the substance was 30% biodegraded at Day 28 and reached 57% biodegradation at Day 84. Both tests were valid as shown by endogenous respirations of 0.4 and 0.5 mg/L and the total mineralization of the reference substance, sodium acetate. Under the conditions of the study, the test substance was considered to be inherently biodegradable (van Ginkel, 1990).