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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:
15.Nov.2007-19.Nov.2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
On demand of the sponsor, evaluation of the biodegradation behaviour of the test item was made without consideration of the 10-day-window.
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item
Name Palmitoyl proline
Batch no. 1184 JGO
Appearance Solid, white
Composition Palmitoyl proline, palmitic acid (8.6%), salts
CAS NO. 5944 1-32-6
ENECS-NO. 2617-63-5
Molecular formula unknown
Molecular weight unknown
Purity approx. 90%
Homogeneity homogeneous
Vapour pressure unkown
Stability H2O, EtOH, acetone, acetonitrile, DMSO unknown
Solubility H2O, EtOH, acetone, acetonitrile, DMSO unknown
Production date 12/2006
Expiry date 12/2007
Storage room temperature 20 +/- 5 C
Hazard information none stated
R-phrases none stated
S-phrases none stated

Storage
The test item was stored in a closed vessel at room temperature

Preparation
The organic carbon content of 71.4% was determined by elemental analysis. The test item was used directly.

Reference item
Aniline (Phenylamine, C6H5NH2, CAS-Nr. 62-53-3) was used as reference item. A stock solution containing 2.1 g/L (nominal) in deionised water was prepared and its organic carbon content was measured with 1638 ppm, resulting in an organic carbon content of the reference item of 77.9%.

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Test System
Specification Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage.
Source and Pre-Treatment
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf. Date of Collection: 09. Nov. 2007, batch no: O911 2007.
The sludge was filtrated, washed with tap water twice, then washed with and re-suspended in test medium. It was then aerated for > 12 hours. The dry matter was determined with 4180 mg suspended solids/litre.
Duration of test (contact time):
ca. 29 d
Initial conc.:
ca. 20 mg/L
Based on:
other: organic carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparations
The medium was prepared from the stock solutions. The inoculum was taken from its Source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then all flasks were aerated for more than 24 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental parameters
Flask Volume 1 500 mL
Controls 2, containing mineral medium and inoculum
Reference flaskS 2, containing reference item, mineral medium and inoculum
Test flaskS 2, containing test item, mineral medium and inoculum
Abiotic Control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, reference item, mineral medium and inoculum
Inoculum concentration: 25.1 mg/L
Temperature 22 +/- 2 °C
Duration 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L.

Apparatus
The test vessels were aerated with CO2-scrubbed air. Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25-m-NaOH. Two scrubbers containing 100 mL each Were Connected in Series to the test vessels. The initial C value of the 0.25 m-NaOH was determined as 6.85 ppm.

Sampling
From each front scrubber flask, nine samples were taken in order to determine the emitted CO2. (on days 2, 4, 6, 8, 11, 14, 18, 22 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
On day 28, 5 mL HCI 2-m. were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

CO2 determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC 5050A (Shimadzu). Each sample was diluted as necessary and then measured at least three times. The carbon analyser was calibrated with freshly prepared reference solutions (containing a mixture of Na2CO3 and NaHCO3) after every start.
Reference substance:
aniline
Test performance:
The IC content of the test item solution in medium was higher than demanded in the guideline. This is due to the fact that the test item is very poorly soluble in water: The concentration of the solution was 1.041 g/L, corresponding to an organic carbon content of 743 ppm. The carbon content was measured with 21.02 ppm, with IC (inorganic carbon) content being 3.75 ppm. The measured IC corresponds to 0.5% of nominal TC. Therefore, the result is considered valid.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 99
Sampling time:
28 d
Details on results:
The test item palmitoyl proline is considered as biodegradable.
The degree of biodegradation reached 99% after 28 days.
The abiotic degradation reached 16%.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Remarks:
without consideration of the 10 day-window
Conclusions:
All validity criteria were met.
Degradation values of more than 100% were calculated. This is due to the fact that the measurement errors of test flasks and control flasks add up when using the equation proposed in the guideline.
lf degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 86% after 14 days, the test item can be stated as "not toxic towards the inoculum in a concentration of 28 mg/L".
The test item showed biodegradation, but not the typical behaviour of a readily biodegradable Substance with exponential phase and plateau, but rather a continuous degradation within 28 days. Biodegradability is given, as the test item showed complete biodegradation within 28 days.
Both replicates of the test item showed good correspondence at the end of the test, whereas the difference between replicates was high from day 4 to day 22. The measured values in the back scrubber flasks showed significant variation, leading to the good Correspondence on day 29. Unfortunately, the IC in the back scrubber flasks is measured on day 29 only, so that it can not be stated when the CO2 was absorbed in the back scrubber. Normally, CO2 doesn't pass the front scrubber flask; only if emission is very high, part of the CO2 reaches the back scrubber. It may be that fast emission took place at the begin of the test, but in Test 1, all CO2 could be absorbed in the front scrubber flask, whereas in Test 2, part of the CO2 was caught in the back scrubber only. This could be an explanation for the difference within replicates.
Executive summary:

Study: Determination of the aerobic ready biodegradability of Palmitoyl proline in the CO2 Evolution Test following OECD 301 B resp. EU C.4.C

Sponsor: SEPPIC

Jérôme GUILBOT, Pole Synthèse 127, Chemin de la Poudrerie F-81 105 CASTRES

Monitor: Phycher Bio Développement

Dr. Richeux 18, Chemin de Lou Tribail Z.l. de Toctoucau F-33610 CESTAS

Test Facility: LAUS GmbH

Auf der Schafweide 20 D-67489 KirrWeiller

Project No.: O7091101 G605

Study Plan No.: PPO7091101 G605

Findings:

The test item Palmitoyl proline was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 28 mg Palmitoyl proline/L) in test medium following OECD 301 B and EU-Method C4-C. Aniline was chosen as reference item. Activated sludge was used as inoculum (concentration in the test 25.1 mg dry matter/L). The test was left running for 28 days. All validity criteria were met. Degradation of the reference item was 75% after six days.

The following data were determined for the test item Palmitoyl proline: degradation at the end of the test 99% pass level: 60%

Therefore Palmitoyl proline is biodegradable following OECD 301 B/EU C.4-C.


Description of key information

All validity criteria were met.

Degradation values of more than 100% were calculated. This is due to the fact that the measurement errors of test flasks and control flasks add up when using the equation proposed in the guideline.

lf degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 86% after 14 days, the test item can be stated as "not toxic towards the inoculum in a concentration of 28 mg/L".

The test item showed biodegradation, but not the typical behaviour of a readily biodegradable Substance with exponential phase and plateau, but rather a continuous degradation within 28 days. Biodegradability is given, as the test item showed complete biodegradation within 28 days.

Both replicates of the test item showed good correspondence at the end of the test, whereas the difference between replicates was high from day 4 to day 22. The measured values in the back scrubber flasks showed significant variation, leading to the good Correspondence on day 29. Unfortunately, the IC in the back scrubber flasks is measured on day 29 only, so that it can not be stated when the CO2 was absorbed in the back scrubber. Normally, CO2 doesn't pass the front scrubber flask; only if emission is very high, part of the CO2 reaches the back scrubber. It may be that fast emission took place at the begin of the test, but in Test 1, all CO2 could be absorbed in the front scrubber flask, whereas in Test 2, part of the CO2 was caught in the back scrubber only. This could be an explanation for the difference within replicates.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

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