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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

Vegeflux soy can be considered as rapidly biodegradable.

Key value for chemical safety assessment

Additional information

The substance has been tested in a first experiment according to the OECD guideline 301F (biodegradation measurement by manometric respirometry, OXITOP device). The substance is multi-constituent, so ThOD calculation was not appropriate. Therefore COD was measured by a COD analyser (Metrohm) and was found to be 2.44 mg O2/mg substance. Around 30 mg DOC (12.5 mg substance) were weighed on polycarbonate strips and introduced as such in 150 ml test medium. The test vessels were inoculated with 5 ml suspension prepared from capsules of BOD seed inoculum, Polyseed, from Interlab (one capsule per 150 ml phosphate buffer). This lead to around 80 mg/l substance loading rate. The microorganisms used for Polyseed formulation areBacillusspecies endospores. The endospores were grown 4 hours in growth medium before use. No information was given about inoculum concentration obtained. However it achieved 60 % reference substance (sodium benzoate) biodegradation within 3 days, which shows it was valid for the biodegradation test. No toxicity appeared to occur from the substance, as the toxicity control vessels (20 mg/l sodium benzoate added) showed 60 % biodegradation within 14 days. The duplicate test vessels showed biodegradation curves with a relatively low slope, as currently observed for multi-constituent substances. The substance biodegradation achieved within 28 days was 46 % (mean from duplicate vessels), which lead to the conclusion that the substance is not readily biodegradable according to the specific EC rules.

However, both biodegradation curves did not reach a plateau within 28 days, suggesting a higher biodegradation could be achieved beyond 28 days. Also, these results were obtained with an inoculum obtained from a single species (Bacillus) endospore (short) growth. So failure to meet the ready biodegradation criteria may result from lack of proper microorganisms.

Therefore a new ready biodegradation experiment was launched, using a multi-species inoculum, and designed to measure biodegradation beyond 28 days. The test was performed according to OECD 301B guideline (biodegradation measurement by carbon dioxide evolution). The substance is poorly soluble in water, so DOC measurement was not appropriate. The substance carbon content was measured by elemental analysis (Carlo Erba) as 0.733 mg C/mg substance. Around 61.6 mg substance were weighed and introduced as such in 3 l test medium. This lead to around 20 mg/l substance loading rate (and around 15 mg/l C loading rate). The inoculum was obtained from activated sludge from a predominantly domestic wastewater treatment plant and added to the test vessels at 15 mg/l suspended solids. Use of larger test vessel volumes (3 l instead of 150 ml) was in favor of a better microorganism recruitment. The substance biodegradation achieved within 27 days was 57 % (mean from duplicate vessels), and 58 % within 31 days. The biodegradation curves decreased beyond 31 days, showing a decrease of carbon dioxide evolution, so a microorganisms starvation.


Weight of evidence

In a strictly regulatory approach, the substance cannot be classified as readily biodegradable, as the maximal biodegradation percentage was obtained beyond 28 days, and was slightly below 60 %.

However, in a scientific approach, given the uncertainty in biodegradation measurements, particularly when the substance tested is poorly water soluble, and multi-constituent, it is obvious that biodegradation percentages cannot be determined with two significant figures. The second figure is generally kept because it gives more precision to the first one (and so, a third figure is really irrelevant, even if reported, it is always possible to calculate with several figures, they are not necessarily relevant). So, realistically, the 57 or 58 % biodegradation obtained have to be rounded to a 60 % biodegradation level. This does not allow classifying the substance as readily biodegradable as the 10-day window was missed (low slope biodegradation curves). However, this results meets the criteria for rapid biodegradation according to the new classification rules in the new EC “CLP” regulation. The substance can be considered as rapidly biodegradable.

This conclusion is supported by results obtained on individual substance constituents or similar substances for which results can be read across :

-      Soyate methyl ester was tested according to EPA 560/6-82-003 (CO2 evolution method) (Zhang et al., 1998) in a study quoted as reliability 2 according to Klimisch criteria. In this test, soyate methyl ester was considered as readily biodegradable, with 85.54% degradation within 28 days.

-      QSAR results on the different constituents of Vegeflux soy are available and conclude that all of them are readily biodegradable. These results, generated with BIOWIN v4.10, are considered as reliable and quoted as reliability 2 according to Klimisch: BioWin is QSAR applicable to organic substance, the fragment used in this evaluation are well represented in the database and the molecular weight of the compound is in the range of the training dataset.

-      Vegeflux colza (Rapeseed oil, reaction product with methanol, oxidised), an analogue to Vegeflux Soy, was tested for biodegradability according to 'CO2-Evolution-Test' (OECD Guideline 301B) and GLP. The calculated mean degradation value of the test item was 84%. The threshold of ready biodegradability of 60% ThCO2 was achieved in both test solutions, the “10-days-window being met, too. Thus, the test item Vegeflux Colza should be regarded “readily biodegradable”.

In conclusion, based on the borderline data with Vegeflux Soy analysed with a scientific approach, and using the reliable data obtained on the individual constituents and on analogue substances, Vegeflux soy is considered as rapidly biodegradable according to the CLP criteria.