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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-14 to 2018-03-26
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, 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 from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste. Received on 2018-01-24.
- Pretreatment: 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 test start (one day).
- Concentration of sludge: The amount of inoculum used to initiate inocluation was 4.02 mL/L (25.0 mg/L dw).
- Initial cell/biomass concentration: Colony forming units in test vessels: Approx 10^7-10^8 CFU/L
- Water filtered: yes:
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 14.7 mg/L
Based on:
test mat.
Initial conc.:
ca. 50.3 mg/L
Based on:
ThOD
Remarks:
mg O2/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral Salts Medium according to OECD 301F
- Additional substrate: Not specified
- Solubilising agent (type and concentration if used): Silicone Oil (73.5 mg test material / 10 mL silicone oil); WACKER® AR 20 SALBEN SILICONŐL (Batch no: IG1190; expiration date: 2018-11-12)
- Test temperature: Nominal: 20-24; ± 1°C; Actual: 20.9 - 21.0°C
- pH: Reults provided in Table 1.
- pH adjusted: Not specified
- CEC (meq/100 g): Not specified
- Aeration of dilution water: yes
- Suspended solids concentration: 25.0 mg/L dw
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Brown glass bottles (500 mL)
- Number of culture flasks/concentration: 2 for inoculum control (C1, C2); 2 for inoculum control with silicone oil (C1s, C2s); 1 for functional control (R1); 2 for the test material (P1, P2); 1 for the toxicity control (T1)
- Method used to create aerobic conditions: continuous stirring
- Measuring equipment: The oxygen consumption was determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 day incubation period.
- Test performed in closed vessels due to significant volatility of test substance: Yes (see below)
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with OxiTop® heads and the measuring system was activated.

SAMPLING
- Sampling frequency: Temperature in the incubator was documented continuously throughout the test. At test start, pH values of the prepared solutions in the measuring flasks (inoculum control and functional control) and the prepared solutions in the separate replicates (test material and toxicity control), were determined. At test end the pH values of the test solutions in the brown glass bottles were determined. Oxygen consumption was determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 day incubation period.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inocluated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.
- Toxicity control: For the toxicity control, 250 mL of the reference item medium was filled in the brown glass bottle of the toxicity control, using a 250 mL measuring flask. 500 µL test material - silicone oil stock solution was pipetted directly into the reference item medium
- Functional Control: The reference item was weighed out and transferred to a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and inoculum, were added. 250 mL of this solution were filled in the brown glass bottle of the functional control, using a 250 mL measuring flask.

STATISTICAL METHODS:
Theoretical oxygen demand (ThOD):

Calculation of the Theoretical oxygen demand (ThOD) of the test and reference materials was based on the formula:

ThOD = (16 x (2c + (1/2 x (h – cl – 3n) + 3s + 5/2p + 1/2na – o) mg O2/mg) / Molecular Weight (MW)

According to OECD Guideline 301, the index of the atoms of the item CcHhClclNnNanaOoPpSs were used for the calculation of the ThOD.

The oxygen depletion of the test material, functional control, and toxicity control was calculated out of the difference of the test and inoculum control replicates.

Biodegradation:
The biodegradation of the test material, functional control and toxicity control was calculated out of the BOD in relation to the ThOD according to the formula:

Biodegradation (%) = (BOD (mg O2/L) / ThOD or ThODNO3 (mg O2/L)) x 100 where:

BOD = Biological Oxygen Demand
ThOD or ThODNO3 = Theoretical Oxygen Demand as applicable
Reference substance:
benzoic acid, sodium salt
Remarks:
Purity: 99.6%
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 97
Sampling time:
28 d
Details on results:
Theoretical oxygen demand (ThOD): The biodegradation in % was calculated based on the ThOD 3.42 mg O2 / mg test material.

Colony Forming Units of the Inoculum: Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.46 x 10^9 CFU/L. The CFU concentration of the inoculum correponds to approximately 0.58 x 10^7 CFU/L in the final test solution.

Results of the Functional Control: The pass level for ready biodegradation (≥ 60% degradation) was reached within 5 days. The valifity criterion that the degradation should be ≥ 60% after 14 days was fulfilled. The adaptation phase changed to degradation phase within 2 days (degradation >10%) and the biodegradation came to a maximum of 92% on Day 26.

Results of the Toxicity Control: In the toxicity control, 75% biodegradation was achieved after 14 days, and 85% after 28 days. The validity criterion that the test material should not inhibit the biodegradation of the reference item was achieved.

Results of the Test Material: Both test material replicates reached the 10% level (beginning of biodegradation) within 3 days. The mean of replicates reached the 60% pass level on Day 8. Mean biodegradation on Day 28 was 97%.
Results with reference substance:
Biodegradation of the functional control sodium benzoate was 92% on Day 26.

Table 1. pH Values at Test Start and Test End

 

pH Value

Start

End

Inoculum Control

7.72

1) 7.57

2) 7.57

Functional Control

7.72

7.70

Inoculum Control with Silicone Oil

7.72

1) 7.44

2) 7.51

Test Material

7.67

1) 7.58

2) 7.58

Toxicity Control

7.69

7.72

1) First Replicate

2) Second Replicate

Table 2. Biodegradation (%) of the Test Material in comparison to the Functional Control and Toxicity Control

 

Replicate

Biodegradation (%)

Study Day (d)

7

14

21

28

Test Material

(14.7 mg/L Test Material)

1

59

82

92

97

2

56

79

90

97

Functional Control

(30 mg/L Reference Item)

 

75

83

88

92

Toxicity Control

(14.7 mg/L Test Material + 30 mg/L Reference Item)

 

57

75

81

85

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Both test material replicates reached the 10% level (beginning of biodegradation) within 3 days. The mean of replicates reached the 60% pass level on Day 8. Mean biodegradation on Day 28 was 97%.
 
Based on the results observed, the test material is considered to be readily biodegradable within the 10-day window within the 28-day period of the study. 
Executive summary:

In a key OECD Guideline 301F biodegradation study, the ready biodegradability of the test material (Hydrocarbons, C16-C18, Isoalkanes, <2% aromatics) was determined with non-adapted activated sludge in the Manometric Respirometry test for a period of 28 days. The test material concentration selected as appropriate was 14.7 mg/L, corresponding to a ThOD of 50.3 mg O2/L per test vessel.

 

The oxygen was depleted by the respiration of bacteria and degradation was followed by measuring the oxygen concentration. The biodegradation rate was therefore expressed as the percentage BOD and was calculated for each study day.

 

The mean oxygen depletion in the inoculum control with silicone oil was 20.4 mg O2/L and in the inoculum control 25.6 mg O2/L on Day 28.

 

Sodium benzoate was used as the functional control and the pass level of >60% was reached within 5 days. The biodegradation reached a maximum of 92% degradation on Day 26.

 

In the toxicity control containing both test and reference material, 75% degradation occurred within 14 days, and 85% after 28 days. The degradation of the reference item was not inhibited by the test material.

 

Both test material replicates reached the 10% level (beginning of biodegradation) within 3 days. The mean of replicates reached the 60% pass level on Day 8. Mean biodegradation on Day 28 was 97%.

 

Based on the results observed, the test material is considered to be readily biodegradable within the 10-day window within the 28-day period of the study. 

Description of key information

Readily biodegradable within the 10-day window and within the 28-day period of an OECD Guideline 301F study. 

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key biodegradation data is available for Hydrocarbons, C16-C18, isoalkanes, <2% aromatics (EC# 948-766-3) and is summarised below. In addition, key and supportive data is available for other members of the category C14-C20 Aliphatics [<2% Aromatics] Hydrocarbon Solvents.

 

In a key OECD Guideline 301F biodegradation study, the test substance was determined with non-adapted activated sludge, included toxicity control and performed for 28 days. Based on the strict criteria of the OECD 301F, the test achieved 97% biodegradation on Day 28, and reached the 10% level (beginning of biodegradation) within the 10 day window.

The above biodegradation data for Hydrocarbons, C16-C18, isoalkanes, <2% aromatics (EC# 948-766-3) is consistent with the biodegradation information available for other members of the category C14-C20 Aliphatics [<2% Aromatics] Hydrocarbon Solvents that have been found to be readily biodegradable. Please refer to the Category endpoint summary for further information on the biodegradation of C14-C20 Aliphatics [<2% Aromatics] Hydrocarbon Solvents.

[Type of water: freshwater]