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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October - 09 November 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
according to guideline
EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report) : 2-(2-Hydroxyethyl)-1-methylpiperidin
- Physical state : clear, colourless liquid
- Analytical purity : 99.4% (GC)
- Lot/batch No. : 06040032/001
- Expiration date of the lot/batch : 2005-12-31
- Storage condition of test material : room temperature, protected from light

Study design

Oxygen conditions:
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 test substance concentration
Initial conc.:
40 other: mg C/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Pre-Treatment :
A stock solution containing 3034 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 1937 mg/L, corresponding to an organic carbon content of 63.8%.

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.

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.
-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% (HgC12, 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 T005050A
♦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
Reference substance:
benzoic acid, sodium salt
3,63 mg/l

Results and discussion

Test performance:
Two measured DOC values ( 1 from reference and 1 from test item) 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 .
% Degradation
Key result
% degradation (DOC removal)
< 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.

BOD5 / COD results

Results with reference substance:
Degradation of the reference item Aniline was 99% after 2 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
The difference within replicates was 7.3 % (criterion: <=20) ; The reference item reached the pass level on day 2. (criterion: <=14)
Interpretation of results:
other: not readily biodegradable
♦No biodegradation above 10% was detected after 28 days.
♦A 10d-window could not be detected.
Therefore 2-(2-Hydroxyethyl)-1-methylpiperidin 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 test item was tested using a concentration of 40 mg organic carbon/L (nominal) in test medium following OECD 301 E/EU-Guideline C.4-B. Aniline was chosen as reference item. Effluent from a sewage treating plant was used as inoculum (concentration: 0.5 ml/L). The test was left running for 28 days. To measure the degradation of test and reference item, the content of dissolved organic carbon (DOC) in the test vessels was measured nine times during the test. All validity criteria were met. Degradation of the reference item was 99% after 2 days.

The following data could be determined for the test item 2-(2 -Hydroxyethyl)-1-methylpiperidin:

-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 2-(2-Hydroxyethyl)-1-methylpiperidin must be classified as not readily biodegradable following OECD 301 E/EU C.4-B.