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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:
2010-03-15 - 2010-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Water solubility: 1.4 g/L at 40°C
Purity: 99.5%
State of aggregation: liquid
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Type: mixed population of aquatic microorganisms
- Origin: Secondary effluent of a domestic sewage treatment plant (Wupper area water authority, STP Odenthal)
- Preconditioning: separation of coarse particles by filtration, aeration of mineral medium plus inoculum for 1 day- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
- Concentration of sludge: 5 mL/L (5.8 mL/1.16 L)
- Date of collection: 2010-03-17
Duration of test (contact time):
28 d
Initial conc.:
0.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium
- Additional substrate: no
- Solubilising agent (type and concentration if used): none
- Test temperature: 22 ± 2 °C
- pH: 6.7 - 7
- pH adjusted: not specified
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus:Flask of 1.16 L
- Number of culture flasks/test concentration: 18
- Number of culture flasks/procedure control:10
- Number of culture flasks/Inoculum blank:18
- Number of culture flasks/Toxicity control:10
- Method used to create aerobic conditions: not specified
- Measuring equipment:Infrared spectrometry
- Test performed in closed vessels: yes


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes


CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
Reference substance:
benzoic acid, sodium salt
Remarks:
100.1%, Batch No. 1373379, 2.9 mg/L
Parameter:
% degradation (O2 consumption)
Value:
83
Sampling time:
28 d
Details on results:
Thymol rn. showed:
4 % degradation after 4 days
25 % degradation after 7 days
81 % degradation after 11 days
89 % degradation after 14 days
87 % degradation after 19 days
87 % degradation after 21 days
88 % degradation after 25 days
83 % degradation after 28 days
Results with reference substance:
The reference compound sodium benzoate showed 78 % degradation after 14 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Remarks:
according to EU GHS
Conclusions:
The study was conducted under GLP according to EU Method C.4-E on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Hence, the results can be considered as reliable to assess the ready biodegradability of the test substance.
A degradation of 83 % within 28 days was observed for the test item thymol; and hence thymol is considered readily biodegradable.
Executive summary:

The objective of this study was to assess the ready biodegradability of the test material thymol. The study was conducted according to EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test) under GLP requirements.

The test item thymol in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions at 22°C for 28 days in the dark. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of theoretical oxygen demand (ThOD).

A degradation of 83 % within 28 days was observed for thymol; and hence thymol is considered readily biodegradable (Currenta 2010). The reference compound sodium benzoate showed 78 % degradation after 14 days.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Principles of method if other than guideline:
batch system (comparable to Zahn-Wellens Test OECD TG 302B)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
- Source of inoculum/activated sludge: from a sewage plant, activated sludge,
- Pretreatment: Adapted for 20 days, fed by glucose and peptone
- Laboratory culture: cultivated in a 1000 ml cylinder
- Method of cultivation: The mixture aerated with pressure air. Every day 200 ml of the mixture driven off so that sludge age is 5 days.
- Preparation of inoculum for exposure: After sedimentation ca. 600 ml of the liquid phase driven off. The residue (200 ml of the thickened activated sludge) diluted with tap water to the volume of ca. 800 ml and 600 mg/L of starch or glucose, 600 mg/L of pepetone, 25 ml of a phosphate buffer and the solution of the tested compund were added. Then the mmixtire in the cylindder is made up to 1000 mL with tap water and aerated for 23 h. After this period the procedure is repeated.
- Concentration of sludge: 100 mg/L (dry matter)

Duration of test (contact time):
5 d
Initial conc.:
200 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
MsSO4Pretreatment: The mixture in the cilinder made up to 1000 ml with tap water and aerated for 23 h. After this period the procedure repeated, and the concentration of the test substance gradually increased so that after 20 days of adaptation it reaches the value of 200 mg/L COD.

TEST CONDITIONS
- Composition of medium: In ca. 800 mL of distilled water solutions of calcium chloride (27.5 g CaCl2 in 1 L distilled water), magnesium sulphate (22.5 g MgSO4: 7 H2O in 1 L distilled water), and ferric chloride (0.25 g FeCl3: 6H2O in 1 L distilled water) are added in 1 mL portions each. Then 5 mL ammonium sulphate solution [10g (NH4)2SO4 in 1 L distilled water], 20 mL of a phophate buffer of pH 7.2 (8.5 g KH2PO2, 21.8 g K2HPO4 and 44.7 g Na2HPO4: 12 H2O in 1 L distilled water), and 100 mL of tap water for securing the content of trace elements are added. The solution thus prepared is made up to 1000 mL with distilled water.
- Additional substrate: no
- Solubilising agent (type and concentration if used): none
- Aeration: Magnetic stirring
- Test temperature: 20 +/- 3°C
- pH: 7.2
- pH adjusted :not reported
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: beaker
- Number of culture flasks/concentration: not reported
- Test performed in open system: yes, oxygeneration capacity is 11 mg oxygen h/L at 800 rev/min

SAMPLING
- Sampling frequency: dependenent on biodegradability rate, but normally once or twice a day
- Sampling method: 50-80 mL are taken, filtration through a filter paper of medium porosity
- Sample storage before analysis: direct measurement


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: Occasional biological analysis is made and the change of biocenosis is evaluated to decrease the initial concentration in case of toxic substances

Reference substance:
aniline
Remarks:
ethylene glycol, diethylene glycol
Parameter:
% degradation (DOC removal)
Value:
94.6
Sampling time:
5 d
Details on results:
The measured COD in samples was compared with those of the blank test and standard compound decomposition.
The substance tested can be considered to be biologically decomposable, since 90 % of the initial COD of the substance is removed in 120 h of incubation under the experimental conditions (the degradation rate being over 15 mg of COD g/h).
Inition degradation rate: 15.6 mg COD g/h
Results with reference substance:
Aniline: 94.5 % removed (based upon COD), biodegradation rate: 19 mg COD g/h
Ethylene glycol: 96.8 % removed (based upon COD), biodegradation rate: 41.7 mg COD g/h
Diethylene glycol: 95 % removed (based upon COD), biodegradation rate: 13.7 mg COD g/h
Validity criteria fulfilled:
yes
Remarks:
results of the reference substances > 70% degradation
Interpretation of results:
inherently biodegradable
Remarks:
according to EU GHS
Conclusions:
The test item thymol showed a degradation of 94.6% DOC in 5 days and is thus considered inherently biodegradable.
Executive summary:

A test on inherent biodegradation comparable to OECD Guideline 302 B was performed with the registered substance itself. The method is to be considered scientifically reasonable with only minor deficiencies in documentation. Hence, the results can be considered as reliable to assess the inherent biodegradability of the test substance.

The test was performed in an open batch system under earobic condition. Adapted, activated sludge (100 mg/L dry matter) from a sewage plant was incubated at 20+/-3°C in the dark under continuous stirring with mineral medium and the test substance solution (initial concentration 200 mg/l COD) for a duration of 5 days (until no further decrease in COD). The test compound was the sole source of carbon. Samples were filtered or centrifuged before analysis at suitable intervals (once or twice a day). An inocumulm blend and a standard substance (reference substance) test were carried out in parallel.

94.6 % DOC removal was measured for thymol during the test period. Therefore, thymol is considered being inherently biodegradable or even ready biodegradable based on the evaluation criteria cited in the report for adapted microorganisms (Pitter, 1976).

Description of key information

Ready biodegradation screening test:

EU Method C.4 -E (equivalent to OECD 301 D), test substance concentration 0.8 mg/L, ThOD 2.77 mg/mg TS/L, 60 % threshold passed: 83% biodegradation in 28 days; readily biodegradable (Currenta, 2010)

Inherent biodegradation screening test:

equivalent to OECD Guideline 302B, initial concentration 200 mg/L COD: 94.6% DOC removal in 5 days; inherently biodegradable (or even redy biodegradable based on the evaluation criteria cited in the report) (Pitter, 1976)

No further simulation tests are considered necessary, since thymol is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ready biodegradation screening test:

In the key study on ready biodegradation according to EU Method C.4 -E (Currenta, 2010) it was concluded that the test item thymol was ready biodegradable with meeting the 10-day window.

The initial test substance concentration in this study was 0.8 mg/L, and therefore lower than the recommended concentration outlined in EU Method C.4 -E of 2 -10 mg/L. Also the test substance concentration based on the theoretical oxygen demand (ThOD) of 2.770 mg per mg DM per liter was outside the range recommended in the guideline (5-10 mg/mg/L). As not explicitly stated in the report (Currenta, 2010) it can be assumed that this lower test substance concentration was chosen based on an initial experiment (Bayer, 1986, re-written Currenta, 2009) on ready biodegradation according to OECD Guideline 301 D with concentrations of 0.8 mg/L and 2.4 mg/L showing only 57% biodegradation of thymol at 2.4 mg/L. Although at 0.8 mg/L about 61% biodegradation was observed, it is not possible to conclude whether or not 10-day window criterion was fulfilled based on the available data. Toxicity to microorganisms is a valid explanation for the inconclusive results of the Bayer study, 1986 and the observed non-biodegradation in a MITI study from 1996. In this study the test concentration (100 mg/L) used was ca. 3 times higher than the 3h-EC50 of thymol (39.6 mg/L) observed on activated sludge (Currenta, 1986) and a respiration inhibition on activated sludge is suspected. Thus it is concluded that the lower test concentration in the key study for ready biodegradation (Currenta, 2010) is appropriate to meet the requirements outlined in EU Method C.4-E taking into account an inhibitory toxic effect on effluent microorganisms and is therefore suitable as key study.