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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:
2018-02-13 to 2018-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
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: cultured, unadapted microorganisms inoculum originated from ten different places of the country for 28 days

- Preparation of sludge: The sludge samples collected from the sampling sites were mixed by stirring in a single container, and the mixture was allowed to stand. Floating foreign matter was removed and the supernatant is filtered. The prepared mixture was transferred into a culture tank and aerated. The pH of the mixture was checked and found 6.91; therefore pH adjustment was considered as not necessary.

- Method of cultivation: The sludge sample was aerated for about 23.5 h. 30 minutes after the stopping of aeration one third of the whole volume of the filtered supernatant was discarded and an equal volume of the synthetic sewage, containing 0.1 % glucose, peptone and potassium orthophosphate (pH=7.0-7.5, set with 1N NaOH) was added to remaining portion of supernatant, and re-commence aeration. This procedure was performed once per weekday.

- Preparation of inoculum for exposure: On the day of the test (~24 h after the culturing unit has been fed) a representative volume of the activated sludge was decanted, and the dry weight content determined using an appropriate moisture content analyzer. The final concentration of the activated sludge inoculum in the test containers was 30 mg suspended solids/L.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: as given by the guideline
- Additional substrate: no
- Solubilising agent: not used
- Test temperature: 25 +/- 1 °C
- pH: 6.81 - 7.41
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Appropriate bottles with air-tight stoppers, Funnels and filter papers, Oxygen and pH meter with appropriate O2 and pH electrode, BOD (biochemical oxygen demand) measuring system (sensors, stirring units, bottles), Aeration system, Incubator with thermometer, Moisture analyzer, Balance, Centrifuge, Microscope. Temperature controlled (25 ±1 °C) environment room (and incubator) with thermometer with exclusion of light.
- Number of culture flasks/concentration:
3 bottles containing the test item (abiotic control);
9 bottles containing the test item and inoculum (test item group);
2 bottles containing the reference item and inoculum (activity control);
3 bottles containing inoculum only (inoculum control);
1 bottle containing the test item and inoculum (viability control);
3 bottles containing the test item and inoculum (nitrification control group);
2 bottles containing inoculum only (blanks for nitrification control).


SAMPLING
Please refer to analyitcal methods


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
3.85
Sampling time:
28 d
Results with reference substance:
The percentage degradation of aniline calculated from the oxygen consumption has to exceed 40 % after 7 days and 65 % after 14 days;
The percentage degradation of aniline calculated from the oxygen consumption was 74.69 % on the 7th day and 88.80 % on the 14th day.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is considered to be not readily biodegradable under the conditions of this MITI test. The percentage biodegradation of the reference item confirmed the suitability of the used method, system and inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level applied inoculum microflora.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item. The test item was exposed to cultured, unadapted microorganisms inoculum originated from ten different places of the country for 28 days. The biodegradation was followed by the oxygen uptake (biological oxygen demand, BOD) of the microorganisms during exposure. 

The test item was investigated at the concentration of 100 mg/L (proposed by the corresponding guidelines and confirmed in the preliminary experiment where the absence of toxicity of the test item on the applied inoculum culture was determined). The theoretical oxygen demand of the test item is 1.73 mg O2/ mg test item (ThODNH4-calculated according to equation given in the referred OECD 301 guideline. The test item is an UVCB substance; however based on its physico-chemical character a ThODNH4 calculation of its main constituent was considered as necessary.)

The biodegradability of the test item in the main test was calculated based on the measured biological oxygen demand (BOD) and on its ThODNH4 (main constituent) of 1.73 mg O2/ mg test item. Additionally, for possible later assessment of biodegradation, appropriate test item containing bottles, and control bottles (abiotic and inoculum control) were stored frozen (~ -20°C) in the testing laboratory with their whole content. The frozen samples are kept stored to enable additional analyses on request. Sampling was performed on day 0, on 14th day and on 28th day of the test. In parallel (under the same conditions as the test item), positive reference item, aniline at the concentration of 100 mg/L (as activity control), inoculum control (containing well-characterised cultured inoculum sample (at the concentration of 30 mg suspended solids/L), abiotic control, nitrification control (with blanks) and viability control were investigated. All validity criteria of the study were met.

 

The closed BOD system (Lovibond®, BOD-System, OxiDirect) was applied.Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of reached a mean of 3.85 % after 28 days that is expressed as the percentage oxygen uptake (measured BOD) of its ThODNH4 (calculated for main constituent).

 

Under the test conditions no abiotic degradation of this test item was noticed. 

The results of the viability determinations show that the cell numbers did not change significantly throughout the study, the obtained differences were considered as acceptable, within the biological variability of the applied test system and without any effect on the final conclusion of the study.

 

The M.I.T.I. (I) test belongs to respirometric methods with oxygen uptake; therefore the oxygen uptake of N-containing chemicals resulting from nitrification can influence the biodegradability results. In this study a closed BOD measurement system was used and nitrification inhibitor (containing N-Allylthiourea) was added to the content of each test bottle. Based on the measured nitrite and increasing nitrate concentrations in the 14-day and 28-day nitrification control (and appropriate blank) samples, presence of nitrification potential of the applied inoculum can be assumed. However, based on the appearance of higher nitrate concentrations most likely microbial life processes and technical effects (turbidity and/or discoloration) influenced the nitrate concentration determinations. Additional measurements (performed at the end of the test from each test item and/or inoculum containing bottle) confirmed the effect of nitrification inhibitor that suppressed the activity of the bacteria (by enzymatic inhibition). So that in the closed BOD system only the breakdown of organic substances in the sample was measured as the BOD value. The percentage degradation of aniline calculated from the oxygen consumption was 74.69 % on the 7th day and 88.80 % on the 14th day.

The test item is considered to be not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNH4. The percentage biodegradation of the reference item confirmed the suitability of the used method, system and inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level applied inoculum microflora.

Description of key information

The test item is considered to be not readily biodegradable under the conditions of this MITI test. The percentage biodegradation of the reference item confirmed the suitability of the used method, system and inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level applied inoculum microflora.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The purpose of this study was to determine the ready biodegradability of the test item. The test item was exposed to cultured, unadapted microorganisms inoculum originated from ten different places of the country for 28 days. The biodegradation was followed by the oxygen uptake (biological oxygen demand, BOD) of the microorganisms during exposure. 

The test item was investigated at the concentration of 100 mg/L (proposed by the corresponding guidelines and confirmed in the preliminary experiment where the absence of toxicity of the test item on the applied inoculum culture was determined). The theoretical oxygen demand of the test item is 1.73 mg O2/ mg test item (ThODNH4-calculated according to equation given in the referred OECD 301 guideline. The test item is an UVCB substance; however based on its physico-chemical character a ThODNH4 calculation of its main constituent was considered as necessary.)

The biodegradability of the test item in the main test was calculated based on the measured biological oxygen demand (BOD) and on its ThODNH4 (main constituent) of 1.73 mg O2/ mg test item. Additionally, for possible later assessment of biodegradation, appropriate test item containing bottles, and control bottles (abiotic and inoculum control) were stored frozen (~ -20°C) in the testing laboratory with their whole content. The frozen samples are kept stored to enable additional analyses on request. Sampling was performed on day 0, on 14th day and on 28th day of the test. In parallel (under the same conditions as the test item), positive reference item, aniline at the concentration of 100 mg/L (as activity control), inoculum control (containing well-characterised cultured inoculum sample (at the concentration of 30 mg suspended solids/L), abiotic control, nitrification control (with blanks) and viability control were investigated. All validity criteria of the study were met.

 

The closed BOD system (Lovibond®, BOD-System, OxiDirect) was applied. Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 3.85 % after 28 days that is expressed as the percentage oxygen uptake (measured BOD) of its ThODNH4 (calculated for main constituent).

 

Under the test conditions no abiotic degradation of this test item was noticed. 

The results of the viability determinations show that the cell numbers did not change significantly throughout the study, the obtained differences were considered as acceptable, within the biological variability of the applied test system and without any effect on the final conclusion of the study.

 

The M.I.T.I. (I) test belongs to respirometric methods with oxygen uptake; therefore the oxygen uptake of N-containing chemicals resulting from nitrification can influence the biodegradability results. In this study a closed BOD measurement system was used and nitrification inhibitor (containing N-Allylthiourea) was added to the content of each test bottle. Based on the measured nitrite and increasing nitrate concentrations in the 14-day and 28-day nitrification control (and appropriate blank) samples, presence of nitrification potential of the applied inoculum can be assumed. However, based on the appearance of higher nitrate concentrations most likely microbial life processes and technical effects (turbidity and/or discoloration) influenced the nitrate concentration determinations. Additional measurements (performed at the end of the test from each test item and/or inoculum containing bottle) confirmed the effect of nitrification inhibitor that suppressed the activity of the bacteria (by enzymatic inhibition). So that in the closed BOD system only the breakdown of organic substances in the sample was measured as the BOD value. The percentage degradation of aniline calculated from the oxygen consumption was 74.69 % on the 7th day and 88.80 % on the 14th day.

The test item is considered to be not readily biodegradable. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNH4. The percentage biodegradation of the reference item confirmed the suitability of the used method, system and inoculum. According to the test guidelines the test item can be assumed as not inhibitory at the applied concentration level applied inoculum microflora.