Influence of fertilisation on furanocoumarins content in two celeriac varieties
V. Schulzová, J. Hajšlová , L. Babička, L. Kouřimská
Key words: celeriac, fertilisation system, furanocoumarins, food safety
Abstract
The aim of this study was to investigate the effect of way of celeriac cultivation on the content of naturally occurring toxicants - furanocoumarins. Their levels have been shown to be strongly affected by individual variety and also fertilization strategy. Climatic conditions in particular crop years play also an important role in the furanocoumarins occurrence.
Introduction 
Besides of various biologically active constituents such as vitamins and phenolic antioxidants, many cultivated plants contain specific secondary metabolites which are classified as toxicants or antinutrients in humans and/or farm animals. Risk associated with an occurrence of natural toxicant in human diet is of growing concern both of scientists and regulators (Hajslova, 2004). 
Furanocoumarins are natural toxic chemicals occurring in edible food plants such as celery, parsnip, parsley, carrot etc. belonging to the Apiaceae family, lower levels of these phytochemicals are also contained in citrus fruits and other crops representing the Rutaceae family (Søborg, 1996). Since their presence in human diet represents food safety issue of concern, more knowledge is needed to reduce consumers´ exposure. There are various factors influencing furanocoumarin levels in fresh and processed vegetables. Growing attention is paid to the quality of crops from organic farming and its comparison with conventionally grown products. 
More than 50 plant furanocoumarins are known at present (Søborg, 1996), considering their chemical structure, two subgroups can be recognised. The first one involves linear furanocoumarins - psoralen, bergapten, xanthotoxin, trioxsalen, isopimpinellin and bergamottin etc.; the second one is represented by angular furanocoumarins such as angelicin, pimpinellin, sphondin, isobergapten etc. Typical levels of furanocoumarins in root vegetable are in celeriac 4 - 40 mg/kg, in parsnip 3 - 60 mg/kg and in carrot <0.004 - 2 mg/kg. Under stress conditions such as attack of insects or fungi, mechanical damage, unfavourable climatic/storage factors etc furanocoumarins levels may increase significantly. Concentrations as high as 45 mg/kg, 145 mg/kg and 112 mg/kg were found in celery, parsnip and parsley, respectively (Schulzova, 2007). 
Furanocoumarins are phototoxic compounds, yielding reactive intermediates under UV light irradiation, formation of adducts with DNA can take place (Llano, 2003; Søborg, 1996). In addition, mutagenic and carcinogenic effects have been demonstrated in experimental animals when exposed to high doses of furanocoumarins. The lowest observed adverse effect level (LOAEL) was estimated in the range 0.14 – 0.38 mg/kg body weight. Although the data on the consumption of root vegetable of Apiaceae family are available the estimation of dietary exposure is rather difficult due to large variability of furanocoumarins levels. The only information on average daily dietary intake of phototoxic furanocoumarins from vegetables was provided by Nordic group who estimated it 0.5 mg/kg body weight (Søborg, 1996). In any case, dietary furanocoumarins should be considered as potentially health risk for consumers´, therefore their intake should be minimised.  
Materials and methods
Celeriac samples (Apium graveolens rapaceum) were obtained from field trials managed by Czech University of Agriculture Prague in years 2005 and 2006. Two varieties of celeriac Albin (A) and Kompakt (K) were examined. Plants were grown from pre-plant seedlings, either of variety was grown in each variants of farming in four repetitions (farming sites 0.5 x 0.4 m). Representative samples consisting of 8 bulbs were taken for analysis.
The fertilization regimes employed in this experiment were as follows: 
N - control farming (not fertilized with mineral and/or organic fertilizers)
O - organic farming (9.1 L m-2 of slurry before planting and  9.1 L m-2 43 days after  planting)
M - mineral farming (22.7 g m-2 of ammonium sulphate, 52 g m-2  of superphosphate and 4.7 g m-2  of potassium chloride before panting and 9.1 L m-2 of slurry 43 after planting)
C - combined farming (4.5 L m-2 of slurry, 11.4 g m-2  of ammonium sulphate, 26 g m-2   of superphosphate and 2.3 g m-2  of potassium chloride before panting and 4.5 L.m-2 of slurry and 11.4 g m-2  of superphosphate 43 days after planting)
Gas chromatography coupled with mass spectrometry (GC/MS) operated in an electron impact ionisation mode was employed for determination of furanocoumarins in ethyl acetate extract. Furanocoumarins were separated on capillary column DB-5MS (60m x 0.25mm x 0.25µm). Quantification (selected ion monitoring) was achieved by comparing the peak areas of the target analytes with the corresponding standard calibration plot. Detection limits (LODs) of angelicin, psoralen, bergapten, xanthotoxin, trioxsalen, isopimpinellin, sphondin, pimpinellin and isobergapten obtained by this analytical method were in the range 0.01-0.08 μg g-1, recovery 89-97% (spiking level 10 mg/kg), the repeatability of measurements expressed as relative standard deviation was in the range 3.9 - 4.7% (for detail information see Peroutka, 2007).
Results and discussion
While in all analysed celeriac samples the presence of linear furanocoumarins psoralen, bergapten, xanthotoxin and isopimpinellin was documented, none of angular furanocoumarins (angelicin, sphondin, isobergapten) and linear trioxalen was detected. The total furanocoumarins content was relatively low, corresponding with literature data.  Higher levels (approximately 2 times) were found in celeriac harvested in 2006 (2.4 – 23.5 mg/kg) compared to 2005 (1.7-13.0 mg/kg).  Maximum furanocoumarins content was found in variety Kompakt grown in combined farming system (C). In 2005 mean furanocoumarin levels were 3.3 mg/kg for variety Albin and 9.3 for variety Kompakt, next year (2006) 3.5 mg/kg for Albin and 19.1 mg/kg for Kompakt. Total furanocoumarins content was significantly lower (t-test, α=0.05) in variety Albin (in crop year 2005 3 times and in crop year 2006 5 times. Variety Albin was more resistant for climatic changes between individual crop years (Figure 1). 

Figure1: Content of furanocoumarins in two celeriac varieties (Albin - A, Kompakt - K) grown in four farming systems in crop years 2005 and 2006 (mg/kg fresh weight)
It should be noted, that mechanical injury or any other stress factors cause large increase of furanocoumarin levels. Interestingly, this effect is more pronounced in crops from conventional farming (Schulzova, 2002).
Conclusions 
Differences between celeriac varieties in inherent furanocoumarin levels were shown in our study. Inter annual variation is mainly due to climatic conditions in particular crop year. The average levels of furanocoumarins determined in hardy fresh celeriac roots were 8.8 mg/kg, ranging from 1.7 to 23.5 mg/kg. Content of targeted toxicants was relatively low and obviously do not present health risk for consumers. 
Slurry, organic fertilizer with high fertilization efficiency, is a good alternative to mineral fertilization in terms of certain agricultural parameters such as crop yield. However, the distinct impact on furanocoumarin levels was not found i.e. no significant relationships between the levels of monitored toxic compounds and the way of fertilization were observed.
Follow up experiments have been initiated since only long term studies may provide information needed for unambiguous assessment of the influence of farming practices on furanocoumarin levels in particular celeriac variety and other similar vegetable. Field experiments have been running also in 2007.  
Acknowledgments
This study was carried out with support from the Ministry of Education, Youth and Sports, Czech Republic - partly from the project MSM 6046137305, partly within the project COST OC 924.
References 
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