Environmentally friendly properties of vegetable oil methyl esters

1 Loire 2i.S, 21, rue de la Poterie, 44640 Saint-Jean de Boiseau, France  2 BFB Oil Research, Parc Scientifique Crealys, rue Phocas Lejeune 10, B-5032 Gembloux, Belgique  3 Onidol, 12, avenue George V, 75008 Paris, France  4 Onidol, 12, avenue George V, 75008 Paris, France  5 Iterg, Rue Monge, Parc Industriel, 33600 Pessac, France  Abstract: Measurements were carried out on Vegetable Oil Methyl Esters (VOME or FAME) answering the most recent specifications. The products tested are RME (Rapeseed oil Methyl Ester), ERME (Erucic Rapeseed oil Methyl Esters), SME (Sunflower oil Methyl Esters), and HOSME (High Oleic Sunflower oil Methyl Esters). They contain more than 99.5% of fatty acid mono esters. The compositions are given. VOME are not volatile and they are not easily flammable. They are not soluble in water and they are biodegradable. According to the methods implemented for the determination of the German classification of substances hazardous to waters WGK, they are not toxic on mammals and unlike diesel fuel they are not toxic on fish, daphnia, algae and bacteria. The RME is not either toxic for shrimps. According to tests on rabbits, RME and SME are not irritating for the skin and the eyes. VOME display particularly attractive environmental properties.

In 2005, France will produce approximately 450,000 T of vegetable oil methyl esters (VOME/FAME) for diesel fuel use.The data published on the biodegradability and ecotoxicity of VOMEs are incomplete and often obsolete.Measurements were sometimes carried out on products whose composition is far from the specifications required.Taking into account new emerging markets for VOMEs (lubricants and solvents) besides that of diesel fuels, it was necessary to bring these data up to date.That is why Onidol (National Inter Professional Oilseed Organisation) asked an independent laboratory (BFB Oil Research) to analyse four VOMEs and compare them to a reference diesel fuel CEC RF-73-A-93: -Rapeseed oil Methyl Esters (RME), -Erucic Rapeseed oil Methyl Esters (ERME), -Sunflower oil Methyl Esters (SME), -High Oleic Sunflower oil Methyl Esters (HOSME).Some other test results on RME coming from the Marine Biology Laboratory of Concarneau are also provided here.

Composition of VOMEs
VOMEs are manufactured by oil transesterification with methanol, so the fatty acid distribution is the same in esters and oils (tables 1 and 2).Samples of RME, SME and HOSME result from industrial productions with distillation and fulfil the standards imposed on the European biofuels market.ERMEs were manufactured by ITERG (French Institute of Fatty Substances) in a small pilot unit.This explains its water content (300 ppm).

Physical-chemical characteristics of the reference fuel CEC RF-73-A-93 (tables 3 and 4)
The reference fuel is used to test the future diesel engines.Its characteristics comply with the specifications of table 3. The aromatics content of the fuel was measured by HPLC according to the IP391method.

Vapour emissions of VOMEs
VOMEs are non volatile products: the initial distillation point at atmospheric pressure is above 200 °C.During the manufacturing process, methanol is eliminated by flash evaporation or distillation, avoiding alcoholic vapours formation during use.This study was an opportunity to revisit some data.At ambient temperature, the saturated vapour pressure of fatty acid methyl esters is very weak and difficult to measure directly.The measurement of the vapour pressure of RME is published here for the first time.It was obtained with an original gas saturation method with GC-FID detection [1]: P RME (20 ± 0.1 °C) = (1.09± 0.11) 10 -3 Pa Therefore, measured volatility of RME is 10,000 times weaker than the VOC directive threshold (P ≥ 10 -2 kPa at 20 °C).That value confirms the very low volatility of VOME and is in accordance with literature.At 25 °C, the reference [2] indicates 0.005 Pa for methyl palmitate (C16:0) and 0.001 Pa for all the C18 (stearate, oleate, linoleate and linolenate) methyl esters.The corresponding molar fractions of gaseous esters (0.01 to 0.05 ppm) in free atmosphere are 1,000 to 10,000 times weaker than mean limit values specified for the majority of industrial solvents.It can be concluded that the handling of VOME at moderate temperatures does not give rise to VOC (Volatile Organic Compounds) emissions.

Flammable atmospheres
The flash point is the temperature at which a liquid should be heated so that it can ignite under the passage of a pilot flame.The flame dies out when the pilot flame is withdrawn, except if the temperature has reached the fire point (table 5).VOMEs are combustible but, by their low volatility, it is necessary to heat them strongly to make them flammable.So they show better safety characteristics than the majority of industrial solvents.

Solubility in water
The C18 hydrocarbons solubility is lower than one ppm [3], but as the tested VOMEs are industrial products it was necessary to carry out a measurement.Two independent laboratories, BFB Oil Research and IFP (French Institute of Petroleum) were asked by Onidol to implement that delicate measurement with RME.

Result from BFB Oil Research [4]
Determination of RME solubility in water is carried out using guidelines from OECD 105, ASTM D 6081 and ISO 9377.A bidistilled rapeseed oil methyl ester is put down in excess on the surface of neutral pure water and mixed with a magnetic stirrer at a speed so that the resulting vortex is between 10-15% of total liquid height.After 30 minutes equilibrium time, water dissolved components are extracted and concentrated.The quantification is performed using an FID gas chromatograph under a calibration curve.Precautions have been taken to avoid biodegradation and hydrolysis.The average of several determinations, with and without internal standard, have led to a hydrosolubility of 124 lg/l at 20 °C.

Result from IFP [5]
The aqueous solubility of a rape oil methyl ester solution has been assessed both using OECD method 105 and biphasic contact.The aqueous concentration of the different fatty acid methyl ester composing a bidistilled rape oil methyl ester has been analysed by solid phase micro extraction using a 100 lm PDMS (polydimethylsiloxane) fibre from Supelco.The methyl esters of the following compounds (C16:0, C18:1, C18:2 and C18:3) were individually quantified after gas chromatography separation and flame ionisation detection.Calibration curves were obtained on standard solutions and have been established using the optimised analytical protocol.For the sum of the methyl esters quantified, the results obtained at 20 °C with the OECD test were 121 lg/l (± 17%) using a percolation water flow That value is far from the 1 ppm (e.g. 1 mg/l) considered as threshold (low solubility) for the ecological impact assessment on water resources.

Selected toxicity tests
The tests of the German standard "Blue Angel" were retained for the 4 VOMEs (table 6) and a toxicity test on shrimps was also applied to RME.

Oral toxicity on mammals according to the OECD 401 method
This analysis was carried out on rats for BFB by "Hygiene Institute of Gelsenkirchen".Rats are fed with calculated amounts of the substance tested.The LD 50 is the concentration which causes the death of 50% of the animals.It is expressed by the ratio between the weight of substance and the weight of animal (mg/kg).

Sample preparation for aquatic toxicity testing and methodology WAF (Water Accommodated Fraction)
A certain quantity of VOME is added to a nutritional solution.The amount is determined by the desired nominal exposure load [6].For all the substances, loads of 10, 1, 0.1, 0.01 and 0.001% were tested.Each mixture is stirred for 24 hrs and allowed to settle for 4 hrs.The solution collected after decantation is noted WAF and is used for fish and daphnia toxicity tests.
Components dissolved in the water phase can be stable droplets or emulsion whose presence causes problems for optical density reading.This is the case for toxicity tests on algae and bacteria.For these tests, WAF is filtered through 2.3 lm then 0.45 lm filters to give a solution called WSF (Water Soluble Fraction).
For each method, the concentration resulting in a toxic effect is obtained by interpolation of the results obtained with the loads mentioned above.A methodology is specified for that, with reference to a pilot test.

Fish toxicity tests according to the OECD 203 method (ISO 9439)
The method consists in determining the concentration (WAF-Water Accommodated Fraction) which kills 50% of Brachydanio rerio (Teleostei, Cyprinidae) (Hamilton-Buchanan) also known as Zebrafish in 48 hrs.This lethal concentration is indicated by LC 50-48h (WAF mg/ml).
10 fish were used for each concentration.

Daphnia toxicity test according to the OECD 202 method (ISO 6341)
The principle consists in determining the concentration present at the beginning of the test which, in 48 hrs, immobilizes 50 % of Daphnia Magna put in experimentation.This effective concentration is indicated by EC 50-48h (WAF mg/ml).20 daphnia were used for each concentration, divided into 4 batches of 5.

Algae toxicity test according to the OECD 201 method (ISO 8692)
The method consists in determining inhibition of the growth of Selenastrum capricornutum.72 hours incubation is carried out for solutions of different concentrations and an identical culture without toxic substance.
The inhibition of growth is followed by optical density.The EC 50-72h (WAF mg/ml) result is the extrapolated concentration giving 50% of growth inhibition.

Bacteria toxicity test according to ISO 10712 method
The method consists in determining inhibition of growth of Pseudomonas putida.16 hrs incubation is carried out with solutions of different concentrations and an identical culture without toxic substance.The inhibition of growth is followed by optical density.The EC 0-16h (WAF mg/ml) result is the extrapolated concentration giving 0% of inhibition.

Ultimate biodegradability test according to OECD 301 B method (ISO 9439)
The modified Sturm test was applied.In a chemical solution without organic carbon, the substance to be tested is mixed with inoculums coming from activated sludge sampled from a domestic sewage treatment plant.Wavre city water treatment plant was selected because it is free from industrial effluents.CO 2 released during 28 days is captured by a Ba(OH) 2 solution in excess and CO 2 amount is determined by back titration After comparison with a reference, the quantity of produced CO 2 is translated into the percentage of total organic carbon of the substance.
The given results are the average of 2 tests at 2 different concentrations of about 20 mg of carbon per litre.

Results and comments
The results of the six tests referred to above are reported in table 7.
The 4 VOMEs, like the diesel fuel, are not orally toxic on mammals.
For the other toxicity tests (bacteria, fish, daphnia, algae), it should be noted that diesel fuel always gives very bad results.Concerning toxicity on daphnia, the significant differences between VOMEs and diesel fuel are confirmed in reference [7].This study also reports rainbow trout behaviour.Those die after 72 hrs with 1,200 ppm diesel fuel, while after 96 hours with 7,500 ppm of RMEs, all trouts are still alive.
Concerning biodegradability, the results obtained confirm the results indicated in the literature, namely a very good biodegradability of VOMEs (> 87 %) whereas diesel fuel must be considered according to OECD 301 guidelines as not biodegradable (38.7 %).
In reference [8] according to the same method, rape and soy methyl esters lead to 88% biodegradation whereas a 2D diesel fuel (US fuel) leads to a value of 18%.
Determination of the WGK according to the method in effect before 2000 BZ (beziehungsweise in German) means literally "respectively".BZ S , BZ B and BZ F, , are calculated based on toxicity tests on mammals, bacteria and fish according to the formula: BZ = 6 -log 10 (concentration in ppm).
From their average value (BZ S + BZ B + BZ F )/3, one point is withdrawn if the product is more than 80% biodegradable and one point is added if there is bio-accumulation.The total gives WGZ.According to the value obtained, a hazardous to waters classification WGK (WGK = Wasser Gefährdungs-Klassen) is determined.Before 2000, the classification was the following: WGZ WGK 0-1.9 0 non-hazardous to waters 2-3.9 1 slight hazard to waters 4-5.9 2 hazard to waters ≥ 6.0 3 severe hazard to waters Diesel fuels and are bio-accumulable.Table 8 gives the WGZ and WGK values calculated based on the preceding results.In this classification, the four esters obtain a zero value for WGK: they are in the class of "non-hazardous to waters" product class.The reference diesel fuel with a WGK of 2 is in the "hazard to waters" product class.

Determination of the WGK according to the new method since 2000
Since 2000, WGK = 0 does not exist any more and has been replaced by meet "substances non-hazardous to waters".In order to qualify for it, the substance must fulfil all the following criteria: -Total points must be 0.
-No toxicity at saturation level (tested with at least two organismsfishes, daphnia or algae).
-Easily biodegradable.The new classification is as follows: Total number of points WGK 0 (and all criteria above) substance non-hazardous to waters 0-4 1 low hazard to waters 5-8 2 hazard to waters ≥ 9 3 severe hazard to waters The attribution of the points is carried out on the basis of following tests (tables 9 and 10): -Toxicity on mammals, -The most significant data of toxicity on daphnia, fish or algae, -Biodegradability and -Potential for bioaccumulation.
According to test results in table 7 and taken into account the potential for bioaccumulation, the number of points attributed to RME is 0 which is the first criteria to fulfil a 'non-hazardous to waters' substance.The complementary analyses have been performed on bidistilled RME showing a hydrosolubility at 20 °C of 120 lg/kg (lower than 10 mg/l).
Acute toxicity tests conducted using WAF or Soja lecithin to be sure to reach the saturation level concentration showed no toxicity at saturation level on at least two organisms (fishes and daphnia).RME is readily biodegradable.
In conclusion, taken into account the data above, the RME must be considered as non-hazardous to waters.Diesel fuel stays in a WGK equal to 2.

Tests on batches of shrimps
To be approved for the cleaning of coastlines spoiled by hydrocarbon spills, the wash products must comply with a procedure recommended by the CEDRE (French Research and Experimentation Centre on accidental pollution of water).This procedure was applied to RME.Analyses were carried out by the Marine Biology Laboratory of Concarneau [9].The method consists in leaving shrimps for 6 hours in tanks containing the products being tested at various concentrations.Afterwards, the shrimps are put in sea water and mortality is examined at the end of 24 hours.
To avoid the influence of the physiological state of the animals, a preliminary test of sensitivity is carried out with lauryl-dimethyl-benzylammonium chloride marketed under the name of Noramium DA50 [supplier CECA (ATO FINA)] with concentrations ranging between 20 and 120 mg/l.For all the tests, the shrimps come from the same batch.
The results are given in table 11.
The lethal amount of RME is 1,000 times higher than that of the reference product.It can be underlined that to be approved by the CEDRE, a ratio higher than 10 is enough.

Contact with skin and eyes
The tests were carried out according to the OECD 404 and 405 methods on male New Zealand Albino Rabbits.

Results of the tests
Each test was carried out on 3 rabbits (table 14).The following tables present the various notes obtained for RME and SME.By forming only one hardly perceptible erythema which disappears quickly, RME is not irritating for the skin (table 15).RME injection causes redness at once but they disappea: SME is neither irritating nor corrosive for the eyes.Within the limits of the assay, SME is not irritating for the skin (table 16).SME injection causes redness and tumefaction at once but they disappear: SME is neither irritating nor corrosive for the eyes (table 17).

Conclusion
The whole of the results obtained shows that VOME resulting from various oleaginous seeds have a better biodegradability and a lesser ecotoxicity than a Diesel reference fuel.These results are very interesting for the biofuel market.They can also be emphasized for applications in other sectors (solvents and lubricants) where the environmental advantages of VOME will bring an undeniable advantage.
Another significant environmental advantage of these esters was not mentioned in this text.It is their favourable impact on greenhouse effect.Many publications deal with this point.We will underline only that of Etienne Poitrat [10] where the use of one tonne of EMC as diesel fuel is estimated to save 2.1 T of CO 2 .These environmental benefits combined with their functionality mean that the derivatives of vegetable oils will play a significant role in the chemistry of the 21st century.

Table 4 .
Aromatic content of reference fuel.

Table 1 .
Composition in fatty acids (%) of the 4 VOMEs./h and 115 lg/l (± 4%) with a water flow 9,4 ml/h.These values are consistent with those obtained on biphasic contacts.Both studies point out that the solubility of RME in water is approximately: 120 lg/l (20 °C).

Table 6 .
Tests of the standard "Blue Angel".

Table 7 .
Results of the tests.