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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:
12 October - 09 November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
Version / remarks:
1997-11-26
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)
Version / remarks:
Directive 2004/10/EC, 2004-02-11
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
Source :
The effluent was taken from the effluent channel of the ESN (Stadtentsorgung Neustadt) sewage treatment plant (Im Altenschemel, NW-Lachen-Speyerdorf,Germany) . Date of taking : 12.10.2005, batch no: 12102005 .
Duration of test (contact time):
28 d
Initial conc.:
40 other: mg C/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Pre-Treatment :
A stock solution containing 3059 mg/L was prepared. Its TOC (total organic carbon) was determined in order to estimate the amount to be added to the test flasks. The TOC was 1037 mg/L, corresponding to an organic carbon content of 33.9%.

Reference Item :
Aniline (Phenylamine, C6H5NH2, CAS-Nr. 62-53-3) was used as reference item. A stock solution containing 2.1 g/L was prepared and its DOC measured.

Medium
Stock solutions
Solution a
-potassium dihydrogenephosphate (KH2PO4) : 8.5 g
-di-potassium hydrogenephosphate (K2HPO4) : 21.75 g
-di-sodium hydrogenephosphate hydrate (Na2HPO4*2H20) : 33.4 g
-ammonia chloride (NH4CI) : 0.5 g
-aqua demin. ad : 1000 ml
The pH was adjusted to 7.4 ± 0.1.
Solution b
-calcium chloride dihydrate (CaCl2*2H2O) : 36.4 g
-aqua demin. ad : 1000 ml
Solution c
-magnesium sulfate heptahydrate (MgSO4*7H2O) : 22.5 g
-aqua demin. ad : 1000 ml
Solution d
-iron(III) chloride hexahydrate (FeCI3*6H2O) : 0.25 g
-di-sodium-ethylen-diamin-tetraacetate dihydrate (Na2EDTA*2H2O) : 0.4 g
-aqua demin. ad : 1000 ml
Trace Element Solution
-Mangane sulfate tetrahydrate (MnSO4*4H2O) : 39.9 mg
-Boric acid (H3BO3) : 57.2 mg
-Zinc sulfate heptahydrate (ZnSO4*7H2O) : 42.8 mg
-Ammonium heptamolybdate ((NH4)6(Mo7O24)) : 34.7 mg
-Iron-chelate (FeCI3-EDTA) : 100.0 mg
-H2O demin. ad : 1000.0 ml
Vitamine Solution
-Yeast extract : 15 mg
-H2O demin. : ad 100 ml
The solution was freshly prepared.

Test medium
The medium was freshly prepared.
Composition:
-Solution a : 10 mL
-Solution b : 1 mL
-Solution c : 1 mL
-Solution d : 1 mL
-Trace Element Solution : 1 mL
-Vitamine Solution : 1 mL
-aqua demin. ad : 1000 mL

Chemicals und Reagents :
-Mercury(I1)chloride, 1% (HgCl2, 10 g/L, used to sterilize the abiotic test flask)
-Sodium hydroxide, 1-molar (NaOH, 40 g/L, for pH-adjustment)
-Hydrochloric acid, 1-molar (HCI, 36.5 g/L, for pH-adjustment)

Vessels :
-Narrow-neck glass flasks (volume 250 ml) were used as test vessels, closed with perforated aluminium foil.

Instruments :
The following instruments and devices were used in the performance of the study:
♦Glass thermometer LAUS No.: 20020912-15
♦Scales Mettler Toledo AB 184 SA and Sartorius 1403
♦Adjustable pipettes with one-way tips LAUS No. 24, 29
♦Carbon analyzer T0C5050A
♦Orbital shaker GFL 3017
♦Glass measuring flasks
♦pH-meter 340i wtw

Preparations :
The medium was prepared from the stock solutions. The inoculum was taken from its source and aerated.

Experimental Parameters :
The test was performed with a start concentration of 40 mg carbon/L (nominal concentration).
Two controls, two flasks containing aniline as reference item, two flasks containing test item, one flask containing test item and 10 ml of a solution with 1% HgCl2 (without inoculum, testing for abiotic degradation), one flask containing test item and 10 ml of a solution with 1% HgCl2 (with inoculum, testing for adsorption on flasks or bacteria) and one flask containing test item and aniline (testing for toxic effects) were tested. Each flask except for the abiotic flask was inoculated with 0.5 ml effluent/L.
Each flask contained 200 ml.
The test was performed at room temperature (21 ± 1°C) without direct lighting. Start of the test was Okt. 12th 2005
Duration of the test was 28 days. The test vessels were closed with perforated aluminium foil to enable aeration. They were put on an orbital shaker.

Sampling and Measurement
From each flask, samples of 5 ml were taken on days 0.125; 2; 5; 7; 9; 12; 14; 20 and 28. The samples were membrane filtrated and the DOC was measured. Measurement of DOC was performed with a TOC5050A (SHIMADZU).
Reference substance:
aniline
Remarks:
2.1 g/l
Test performance:
DOC-values printed in italics ( table 1 at "overall remarks, attachments") were considered as outliers and were not used for the calculation of degradation. The deviating values might be due to improperly cleaned vessels or inefficient homogenisation. The outliers did not have any effect on the assessment of biodegradation of the test item, because it can be doubtlessly stated that no biodegradation took place.
The measured values show an increase in DOC in all flasks containing test item between Day 0 and day 4. This observation led to the assumption that the test item shows reversible adsorption on the test flasks. Therefore, the theoretical values for d = 0 were used for the calculation of the degradation .
Key result
Parameter:
% degradation (DOC removal)
Value:
< 10
Sampling time:
28 d
Remarks on result:
other: The test item showed negative degradation results. This is probably due to evaporation effects as well as to the variation of DOC in the blanks and measurement uncertainties.
Details on results:
♦The test item must be considered as " not readily biodegradable".
♦No biodegradation above 10% was detected after 28 days.
♦A 10d-window could not be detected.
Results with reference substance:
Degradation of the reference item Aniline was 99% after 2 days.
Validity criteria fulfilled:
yes
Remarks:
The difference within replicates was 10.5 % (criterion: ≤20) ; The reference item reached the pass level on day 2. (criterion: ≤14)
Interpretation of results:
not readily biodegradable
Conclusions:
The following data could be determined for the test item 1-(2-Hydroxy-3-sulfopropyI)- pyridinium-betain:
-10-day-window : not detectable
-Degradation at the end of 10-day-window : not detectable
-Degradation at the end of the test : no degradation
-Pass level following guideline : 70% at the end of 10-d-window
Therefore 1-(2-Hydroxy-3-sulfopropyI)- pyridinium-betain must be classified as not readily biodegradable following OECD 301 E/EU C.4-B.
The study was conducted under GLP compliance and a well documented study report is available.
Executive summary:

The biodegradability of the substance 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain was investigated according to OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test), EU method C.4 -B. The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). Effluent from a sewage treatment plant which treats predominantly domestic sewage was used as inoculum. The test material was used in a concentration of 40 mg organic carbon/L (nominal) for the study. Two controls, two flasks containing aniline as reference item, two flasks containing test item, one flask containing test item and 10 ml of a solution with 1% HgCl2 (without inoculum, testing for abiotic degradation), one flask containing test item and 10 ml of a solution with 1% HgCl2 (with inoculum, testing for adsorption on flasks or bacteria) and one flask containing test item and aniline (testing for toxic effects) were tested. Each flask except for the abiotic flask was inoculated with 0.5 ml effluent/L. The test was performed at room temperature (21 ± 1°C) without direct lighting. Duration of the test was 28 days. From each flask, samples of 5 ml were taken on days 0.125; 2; 5; 7; 9; 12; 14; 20 and 28. The samples were membrane filtrated and the DOC was measured. Measurement of DOC was performed with a TOC5050A (SHIMADZU). All validity criteria were met. Degradation of the reference item was 99% after 2 days thereby confirming the suitability of the inoculum and test conditions. The toxicity control attained 44 % degradation at the end of the test which confirms that the test material was not toxic to the sewage treatment microorganisms used in this experiment.

The following data could be determined for the test item 1-(2-Hydroxy-3-sulfopropyI)- pyridinium-betain :

-10-day-window : not detectable

-Degradation at the end of 10-day-window : not detectable

-Degradation at the end of the test : no degradation

-Pass level following guideline : 70% at the end of 10-d-window

Therefore 1-(2-Hydroxy-3-sulfopropyI)- pyridinium-betain must be classified as not readily biodegradable following OECD 301 E/EU C.4-B.

Description of key information

Biodegradation water: screening tests: Not readily biodegradable: no degradation observed (DOC) (OECD 301E, EU method C.4-B, GLP)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

[Type of water: freshwater]