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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:
2014-02-13 until 2014-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal wastewater treatment plant Breisgauer Bucht, Freiburg, Germany
- Sampling date: February 11th, 2014
- Storage conditions: not applicable
- Storage length: immidiately used
- Preparation of inoculum for exposure: Washed twice by settling the sludge, decanting the supernatant and re-suspending the sludge in tap water. It was aerated with CO2-free air overnight.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: organic carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
Mineral Medium
A: Potassium dihydrogenphosphate KH2PO4 8.50 g, Dipotassium hydrogenphosphate K2HPO4 21.75 g, Disodium
hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g, Ammonium chloride NH4Cl 0.50 g, are dissolved in demineralised
water and made up to 1 litre.
B: Calcium chloride dihydrate CaCl2 * 2H2O 36.4 g, is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g, is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g, is dissolved in demineralised water, stabilised with one drop of
concentrated HCl and made up to 1 litre.
For preparation of the mineral medium 10 mL of solution (A) is mixed with 800 mL demineralised water, 1 mL each of solutions
(B), (C) and (D) are added and the volume is made up to 1 litre.
CO2-absorption medium: 47.94 g NaOH was dissolved in 6 L deionised water in closed recipients (0.2 M NaOH). The inorganic carbon concentration of the
0.2 M NaOH was determined.
- Continuous darkness: no - diffuse light for 28 days

TEST SYSTEM
- Culturing apparatus: CO2-free air production system
- Number of culture flasks/concentration: In total three reactors containing the test item, three reactors containing only inoculum
(blank), three reactors containing the reference compound and two reactors containing the solvent control.
- Measuring equipment: IC measurement was performed with a total carbon analyser (TOC-5050A Shimadzu with an autosampler
ASI-5000A) by purging the inorganic carbon with H3PO4 (25%) using a non dispersive infrared (NDIR) detector.
- Other: Due to the low water solubility of the test item a stock solution was prepared with trichlormethane. One day before start of the test, a stock solution of 9.0 g/L was prepared by dissolving 179.9 mg of the test item in 20 mL trichlormethane. 4.0 mL of the stock solution were added into each test vessel, corresponding to a test item concentration of 20 mg/L organic carbon. The solvent was then evaporated to dryness under the fume hood by slewing the vessels. For complete evaporation, the test vessels were left standing under the fume hood overnight.


SAMPLING
- Sampling frequency: On the 4th, 7th, 11th, 14th, 21st and 28th day 4 mL NaOH from the first of two CO2-absorber flasks
connected in line was sampled and the IC's were determined.
- Other: The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M
hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in
both CO2-absorber flasks in line

CONTROL AND BLANK SYSTEM
- Inoculum blank: three reactors containing only inoculum
- Reference substance: A stock solution of 10 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were
added into the reference vessels corresponding to a concentration of 20 mg/L organic carbon.
- Solvent control: The solvent controls were prepared in the same way as the test vessels. Therefore 4 mL of the solvent
trichlormethane were added into the solvent control vessels. The vessels were slewed under the fume hood until the solvent had
evaporated to dryness. For complete evaporation, the test vessels were left standing under the fume hood overnight.

STATISTICAL METHODS:
- Theoretical CO2 amount of the test item: ThCO2 [mg] = weight of added test item [mg] * carbon-content [mg/mg] * 44/12
- Amount of CO2 released from the reactors: CO2 [mg/1500 mL] = IC [mg/L]* Volumeabsorber flask [L] *44/12
- Amount of CO2 removed for IC-measurement: CO2 total (i, x) = CO2 absorber flask (i, x) + S CO2 sampling (i, x-1)
- Percentage biodegradation of the reference item: BiodegradationCO2 [%]=100*(CO2 Reference reactor [mg] - CO2 Blank
reactor [mg])/ThCO2 [mg]
- Percentage biodegradation of the test item: BiodegradationCO2 [%] = 100*(CO2 Test reactor [mg] - CO2 Solvent control reactor
[mg])/ThCO2 [mg]
Reference substance:
benzoic acid, sodium salt
Test performance:
Due to a leakage in one of the test reactors, the CO2 evolution in this reactor differed significantly from the mean value of the two
other replicates (up to 21.9% on day 7). The reactor was therefore regarded as an outlier and not considered in the evaluation.
This has no effect on the validity of the study, since only two replicates are required for the test item, according to the OECD
guideline.
Parameter:
% degradation (CO2 evolution)
Value:
90
Sampling time:
28 d
Details on results:
The criterion of the 10-d window was fulfilled, since a mean degradation of ≥ 60% was already reached on day 7.
Results with reference substance:
The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item reached the pass level for ready biodegradability (60% ThCO2 and 10 day window).
Executive summary:

The biodegradability of 2 -Tetradecyl-1 -octadecanol (CAS No.:32582 -32 -4) was assessed in a CO2 -evolution test according to the OECD 301B guideline (July 1992).

A solution of the test item in mineral medium corresponding to 20 mg TOC/L was inoculated with activated sludge (30 mg d.s./L). Due to the low water solubility of the test item the stock solution was prepared with trichlormethane. The test vessels were aerated by the passage of carbon dioxide-free air and incubated under aerobic conditions in diffuse light for 28 days. Degradation was followed by determining the carbon dioxide produced and adsorbed to sodium hydroxide via IC-measurement (IC=inorganic carbon).

The temperature was 21.6 – 23.0°C throughout the whole study. The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.6 – 4.4 bubbles/second).

The highest mean CO2-evolution of the blank flasks was 31.4 mg/L within 28 days after acidification and the reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days. The highest mean CO2-evolution of the solvent control flask was 34.4 mg/L within 28 days after acidification.

All validity criteria were fulfilled:

- The IC content in the test vessel was less than 5% of the TOC introduced with the test item.

- The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.

- The difference of extremes of replicate values at the end of the 10-d window and at the end of the test was less than 20%.

- The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.

The degradation extent of the test item was 90.0% within 28 days after acidification. The 10-d window was fulfilled, since a mean degradation of ≥ 60% was already reached on day 7. Therefore the test item reached the pass level for ready biodegradability (60% ThCO2 and 10 day window).

Description of key information

2 -tetradecyloctadecan-1 -ol reached the pass level for ready biodegradability (60% ThCO2 and 10 day window) in an OECD 301 B study (Flach, 2014).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Reliable measured data show that Guerbet alcohols of chain lengths up to C32 are readily biodegradable. At carbon chain length of C16 the data show that the pass level for ready biodegradation was reached within the 10-day window with removal levels up to 96%. Shorter and longer carbon chain lengths (for example, C12 and C20) achieved ready test pass levels but not in all cases within the 10d window. The C24 Guerbet alcohol showed biodegradation rates of 84% after 28 days and reached the pass levels of the 10 day window. Guerbet alcohols with chain lengths of C32-36 showed only degradation of up to 47%. The data demonstrate a decrease in biodegradation rates with increasing chain length if the biodegradation test is conducted according to the standard procedure. It is assumed that this decrease is associated to the decrease in water solubility and the resulting decrease in bioavailability. This assumption is supported by the biodegradation test of the C24-Guerbet alcohol. In this test the substance was administered to test system by applying it on the whole surface of the test flask. With this increased surface and resulting improvement of bioavailability higher biodegradation rates were achieved than for the C20-Guerbet alcohols. Also the C32-Guerbet alcohol 2-tetradecyloctadecan-1-ol showed ready biodegradability with this method. Therefore the whole category of the Guerbet alcohols with carbon chain lengths of C12 -C32 is considered to be readily biodegradable.