Marie Janska

Monika Tomaniova

Jana Hajslova

Vladimir Kocourek

come

Optimisation of procedure for determination of polycyclic aromatic hydrocarbons and their derivatives in fish tissue; estimation of measurements uncertainty

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Calibration of syringe 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 Transferring into 0.5 ml acetonitrile u 7 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 of PAHs inputs (these may result from industry incineration, transport, uncontrolled spills, 33 surface runoff and/or atmospheric deposition), their fluxes and fate, particularly in aquatic 34 environment, have been initiated [START_REF] Hellou | Partitioning of polycyclic aromatic hydrocarbons between water and particles compared to bioaccumulation in mussels: a harbour case[END_REF], Varanasi 1989[START_REF] Koh | Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea[END_REF], Ross et 35 al 2004, Shi et al. 2004[START_REF] Reeves | Partitioning and desorption behavior of polycyclic aromatic hydrocarbons from disparate sources[END_REF], Chen et al. 2004, Grimalt et al. 2004, Oost et (Jaouen-Madoulet et al. 2000[START_REF] Frankenius | Extraction of PAHs in Soil[END_REF], batch extraction 1 enhanced by sonication [START_REF] Wenzel | Accelerated Solvent Extraction of Semivolatile Organic Compounds from Biomonitoring Samples of Pine Needles and Mosses[END_REF][START_REF] Wenzel | Immission patterns of airborne pollutants in Argentina and Germany II. Biomonitoring of organochlorine compounds and polycyclic aromatics[END_REF]) and alkaline saponification 2 followed by re-extraction [START_REF] Chen | Formation of Polycyclic Aromatic Hydrocarbons during Processing of Duck Meat[END_REF] represent common methods in routine 3 laboratories. The use of supercritical fluid extraction (SFE) has also been described in some 4 studies (Taylor 1996, Luque de [START_REF] Castro | Analytical Supercritical Fluid Extraction[END_REF][START_REF] Librando | Supercritical fluid extraction of polycyclic aromatic hydrocarbons from marine sediments and soil samples[END_REF]), however, its routine 5 use for processing of biotic samples is limited, probably due to strong dependence of optimal 6 parameters setting on sample composition (fat, moisture) what is the cause of a rather low 7 robustness of SFE-based procedures. At present, a growing interest in alternative extraction 8 techniques such as pressurised liquid extraction (PLE) [START_REF] Wenzel | Accelerated Solvent Extraction of Semivolatile Organic Compounds from Biomonitoring Samples of Pine Needles and Mosses[END_REF], Dean 1996, Saim 9 Nor 'ashikin et al. 1997, Schantz et al. 1997, Lopez-Avila et al. 1997) and microwave assisted 10 extraction (MAE) (Saim Nor'ashikin et al. 1997, Lopez-Avila et al.1997[START_REF] Leray | Microwave-oven extraction procedure for lipid analysis in biological samples[END_REF], 11 Saim et al. 1997, Budzinski et al. 1996) can be noticed. Organic solvents such as methanol, 12 acetone [START_REF] Knulst | Airborne organic micropollutant concentrations in mosses and humus as indicators for local versus long-range sources[END_REF][START_REF] Franzaring | Active biological monitoring of polycyclic aromatic hydro-carbons using kale (Brassica oleracea) as a monitor-species[END_REF], chloroform [START_REF] Cejpek | Simplified extraction and cleanup procedure for the determination of PAHs in fatty and protein-rich matrices[END_REF], 13 Tomaniová et al. 2000[START_REF] Tomaniová | Microwave-assisted solvent extraction -a new method for isolation of polynuclear aromatic hydrocarbons from plants[END_REF], dichloromethane [START_REF] Howsam | PAHs associated with the leaves of three deciduous tree species. I -Concentrations and profiles[END_REF], 14 Marvin et al. 1999[START_REF] Gratz | Interlaboratory comparison of HPLC-fluorescence detection and GC/MS: analysis of PAH compounds present in diesel exhaust[END_REF], hexane [START_REF] Weiss | Regional aspects and statistical characterisation of the load with semivolatile organic compounds at remote Austrian forest sites[END_REF] and cyclohexane (Brouwer 15 et al. 1994) or mixtures of them, e.g. hexane-acetone [START_REF] Shu | Analysis of polycyclic aromatic hydrocarbons in sediment reference materials by microwave-assisted extraction[END_REF][START_REF] Budzinski | Optimisation of the microwave-assisted extraction in open cell of polycyclic aromatic hydrocarbons from soils and sediments: Study of moisture effect[END_REF], 16 Chee et al. 1996[START_REF] Lopez-Avila | Microwave-Assisted Extraction as an Alternative to Soxhlet, Sonication, and Supercritical Fluid Extraction[END_REF], dichloromethane-acetone (Popp et al. 1997, 17 Fischer et al. 1997[START_REF] Tao | Determination of PAHs in wastewater irrigated agricultural soil using accelerated solvent extraction[END_REF], hexane-acetone-toluene [START_REF] Berset | Comparison of different drying, extraction and detection techniques for the determination of priority polycyclic aromatic hydrocarbons in background contaminated soil samples[END_REF], hexane-18 dichloromethane [START_REF] Hubert | Accelerated Solvent Extraction -More Efficient Extraction of POPs and PAHs from Real Contaminated Plant and Soil Samples[END_REF]) are typically used for performance of extraction step. 19

Extraction enhanced by sonication

hexane-acetone (1:1, v/v) chloroform hexane-dichloromethane (1:1, v/v)
u7 2 = σ7 2 σ σ σ σ 6 2 σ σ σ σ 5 2 u6 2 = σ6 2 -σ7 2 -σ8 2 σ σ σ σ 4 2 u5 2 = σ5 2 -σ6 2 σ σ σ σ 3 2 σ σ σ σ 2 2 σ σ σ σ 1 2 u4 2 = σ4 2 u3 2 = σ3 2 -σ4 2 -σ5 2 u 2 2 = σ2 2 -σ3 2 u1 2 =
When analysing complex samples such as plants, biota or foodstuffs, many other matrix 20 components are unavoidably co-isolated together with target analytes. Oils, waxes, essential 21 oils and natural pigments like carotenoids and chlorophylls are the most typical matrix 22 components occurring in extracts obtained from plant samples. In animal tissues, lipids 23 represent the major class of co-extracts. Effective removing of these substances, which can 24 adversely affect identification and/or quantification of PAHs, is the crucial prerequisite for 25 obtaining credible results. However, not only good separation of target analytes from co-26 extracts but also the overall throughput of clean-up step (which is largely dependent on the 27 possibility of its automatisation) is important criterion considered when choosing an optimal 28 procedure. At present, gel permeation chromatography (GPC) employing fractionation 29 according to differences in the molecular size (or more exactly effective molecular volume) of 30 impurities and target contaminants together with adsorption chromatography based on 31 differences in polarity/chemical structure of sample components, represent nowadays 32 dominating purification techniques applied in processing of primary extracts in PAHs 33 analysis. GPC procedure may employ several types of gels for purification of primary 34 extracts, e.g. styrene divinylbenzene copolymer gels Bio-Beads S-X3 [START_REF] Kaupp | A method for analysing polycyclic aromatic hydrocarbons (PAHs) in plant samples[END_REF], 35 Czuczwa et al. 1989), S-X12 [START_REF] Fernández | Selective enrichment procedures for the determination of polychlorinated biphenyls and polycyclic aromatic hydrocarbons in environmental samples by gel permeation chromatography[END_REF]), XAD-2 [START_REF] Codina | Comparison of various extraction and clean-up methods for the determination of polycyclic aromatic hydrocarbons in sewage sludge-amended soils[END_REF], PL gel 36 (Schantz et al. 1997) and Phenogel [START_REF] Nyman | Comparison of two clean-up methodologies for the gas chromatographic/mass spectrometric determination of low nanogram/gram levels of polynuclear aromatic hydrocarbons in seafood[END_REF]). Elution of sample components is 37 usually carried out by different elution solvents, e.g. toluene (Kaupp et al. 1996), 38 dichloromethane (Schantz et al. 1997), mixture of dichloromethane-cyclohexane (Kaupp et al. 39 1996) and ethyl acetate-cyclohexane [START_REF] Czuczwa | Optimized gel permeation chromatographic cleanup for soil, sediment, wastes, and oily waste extracts for determination of semivolatile organic pollutants and PCBs[END_REF]). As regards the adsorption 40 chromatography, the use of following sorbent/eluent combinations was reported in literature: 41 (i) silica with cyclohexane [START_REF] Tao | Determination of PAHs in wastewater irrigated agricultural soil using accelerated solvent extraction[END_REF][START_REF] Karl | Determination of polycyclic aromatic hydrocarbons in smoked fishery products from different smoking kilns[END_REF], Poppet al. 1997), hexane (Chee 42 et al. 1996[START_REF] Berset | Comparison of different drying, extraction and detection techniques for the determination of priority polycyclic aromatic hydrocarbons in background contaminated soil samples[END_REF][START_REF] Falcón | Enrichment of benzo[a]pyrene in smoked food products and determination by high-performance liquid chromatography-fluorescence detection[END_REF], pentane [START_REF] Budzinski | Optimisation of the microwave-assisted extraction in open cell of polycyclic aromatic hydrocarbons from soils and sediments: Study of moisture effect[END_REF]), (ii) Florisil 43 with hexane [START_REF] Wenzel | Immission patterns of airborne pollutants in Argentina and Germany II. Biomonitoring of organochlorine compounds and polycyclic aromatics[END_REF], dichloromethane [START_REF] Wang | Accelerated Solvent Extraction and Gas Chromatography / Mass Spectrometry for Determination of Polycyclic Aromatic Hydrocarbons in Smoked Food Samples[END_REF] and their mixtures 44 [START_REF] Chen | Formation of Polycyclic Aromatic Hydrocarbons during Processing of Duck Meat[END_REF] In the first part of our study the attention was paid to the optimisation of crude extracts clean-3 up. Based on our previous experience with purification of lipid-rich extracts obtained from 4 animal tissues when analysing liphophilic pollutants in biota, GPC was identified as the 5 method of choice. 6

u c (y(x 1 ,x 2 ,...)) = ∑ ∑ = = = ∂ ∂ n i i n i i i x y u x u x y , 1 2 , 1 2 2 ) , ( ) ( ) / ( (1
Three different elution systems employing two types of polystyrene divinylbenzene 7 copolymer gels (PSDVB), one "soft" gel -Bio-Beads S-X3 and two "rigid" gels -PL gel and 8

Envirogel were tested (experimental configurations and procedure codes are listed in Table 9 III). The latter type of gel (routinely used in our laboratory for clean-up of pesticide samples) 10 offers some practical advantages since it is resistant to higher operating pressures and it's 11 fluctuation, hence the column lifetime is higher. Moreover, the overall ruggedness of clean-up 12 step when employing rigid gel columns is improved. Another advantage of using rigid gels 13 for GPC fractionation is a possibility to operate a column at different elution solvents without 14 a risk of gel volume changes (swelling after switching between two miscible eluents). 15

Attention was also paid to the selection of optimal GPC mobile phase. In our earlier 16 studies chloroform was used as an elution solvent in clean-up step employing BioBeads SX-3 17 column [START_REF] Cejpek | Simplified extraction and cleanup procedure for the determination of PAHs in fatty and protein-rich matrices[END_REF][START_REF] Tomaniová | Focused Microwave (FMW) Extraction of polycyclic aromatic hydrocarbons (PAHs) from plant bioindicators, comparison with classical techniques[END_REF][START_REF] Tomaniová | Microwave-assisted solvent extraction -a new method for isolation of polynuclear aromatic hydrocarbons from plants[END_REF] Considering the 18 work place hazard as well as ecological aspects posed by this solvent ethyl acetate-19 cyclohexane (1:1, v/v) solvent mixture, which is commonly used for purification of extracts in 20 pesticide residue analysis was tested as an alternative for its replacement. For evaluation of 21 performance of clean-up procedure, several parameters were considered: (i) efficiency of 22PAHs separation from co-extracts (purity of PAHs fraction), (ii) column capacity for co-23 extracts (sample equivalent that can be safely loaded into GPC system), (iii) the total solvent 24 volume needed to accomplish GPC clean-up and (iv) time needed for complete elution of 25 PAHs fraction. 26

Besides fish oil also rape seeds oil representing plant lipids was used to characterize the 27 elution curves of these matrix constituents. The maximum load of lipids used for comparison 28 of tested systems i.e. amount injected onto GPC column was 300 mg. The first set of 29 experiments employing GPC-BB-C system revealed rape seed oil as less resolved from PAHs 30 elution band (compared to fish oil and/or triolein). Representing the candidate for the worst 31 separation scenario, rape seed oil was employed as model lipid for construction of elution 32 profiles in the other GPC systems. Differences in elution profiles of rape seed oil and fish oil 33 might be attributed to different types of triacylglycerols (contrary to plant matrices high 34 amounts of long chain polyene fatty acids with 20 -24 carbon atoms are present in fish oil) 35 and other natural liphophilic compounds such as waxes, vitamins, sterols…) and/or 36 triacylglycerols breakdown products possibly occurring in these materials. In GPC-BB-C 37 system elution of pure triolein was also measured. Elution curve of this pure chemical was 38 fairly narrower as compared to fish oil, the elution band of which was rather tailing, probably 39 due to the presence of some lower molecular weight components (see above). 40

Based on results summarised in Table III, the columns packed with gel Bio-Beads S-X3 41 and/or PL gel operated with ethyl acetate-cyclohexane (1:1, v/v) as a mobile phase were 42 identified as the most promising. 43

Similar trends in separation efficiencies of PAHs and co-extracts were recognised in the 44 both soft gel systems. In GPC-BB-EC 8% of lipids weight loaded onto column occurred in 45

PAHs fraction and in the system GPC-BB-C 6% of lipids weight loaded onto column 46 occurred in PAHs fraction. However, it should be noted that the elution volume of PAHs 47 fraction in ethyl acetate-cyclohexane (1:1, v/v) was higher -about by 30% than in chloroform. 48

No differences in elution profiles between fish oil and rape seeds oil were observed anymore 49 The column packed with rigid PL gel operated in chloroform was found fairly unsuitable 3 for purification purpose due to the severe overlap of PAHs and lipids fraction (about 30% of 4 injected lipids co-eluted with PAHs). On the other hand a complete separation of PAHs and 5 oil was achieved on PL gel column when using ethyl acetate-cyclohexane (1:1, v/v) as a 6 mobile phase. Unfortunately elution volume of PAHs fraction was more than twice higher as 7 compared to GPC-P-C. It should be also noted that in both GPC systems employing ethyl 8 acetate-cyclohexane (1:1, v/v) as a mobile phase (GPC-B-EC and GPC-P-EC), even 9 separation of PAH groups occurred. Complete separation of 3-and 5-ring, 3-and 6-ring and 10 also 4-and 6-ring PAHs on the PL gel column was obtained. Contrary to size exclusion 11 theory, the elution volume of PAHs increased with increasing amount of aromatic rings. The 12 reason of this reversed elution order ("small" PAHs eluted before those with higher molecular 13 weight) might be caused by strong unspecific interactions of analytes with gel structure, e.g. π 14 -π interactions. No adsorption phenomena occurred for lipids; their elution order follows the 15 principles of size exclusion chromatography only. 16

Regarding other tested systems consisting from column packed with rigid gel Envirogel 17 and mobile phase chloroform, under conditions listed in Table I complete separation from co-18 extracts was obtained, however, PAHs were eluted at volume as large as 75 -115 ml (i.e. 40 19 ml), which is three times higher compared to GPC-BB-C. On this account (good separation in 20 small solvent volume i.e. in a shorter time) the latter system was used throughout all 21 following experiments. On the other hand, it should be also noted, that higher amount of 22 sample is possible to load on this clean-up column and thus limits of detection/quantitation of 23 procedure could be set lower. 24

Considering the throughput of sample and purification efficiency, currently used 25 automated system GPC-BB-C (gel Bio-Beads S-X3 and mobile phase chloroform), was found 26 as the most effective for routine processing of crude PAHs extracts. In this system, PAHs 27 derivatives and PAHs sulphur analogues are eluted in the same fraction as the un-substituted 28

PAHs. Although the volume of chloroform used for clean-up step is very low compared to for 29 example extraction step, the current trend is to replace chlorinated solvents completely due to 30 their toxicity and ecological reasons. Therefore the system GPC-BB-EC (gel Bio-Beads S-X3 31 and mobile phase ethyl acetate-cyclohexane, 1:1, v/v) could be recommended as an 32 alternative. It should be noted that the total time needed for a sample is about 50% longer than In general, the use of certified reference material (CRM) provides the best way to assess the 41 trueness of respective procedure. Unfortunately, at present no certified reference material 42 relevant to matrices involved in our study is commercially available. For this reason the 43 optimisation of extraction step was performed with fish tissue containing naturally incurred 44

PAHs. The evaluation of individual experiments efficiency was carried out on the relative 45 basis taking the highest result as the reference concentration (100%). However, it should be 46 noted that the evaluation of all experiments was somehow difficult because of low levels of 47 target analytes naturally present in the testing fish sample. The levels of some analytes were 48 close to the limits of quantification, which ranged, depending on the individual PAH, from 49 0.03 to 0.12 µg kg -1 . 50 For majority of all target analytes an extraction mixture hexane-acetone (1:1, v/v) was found 9 as the most efficient isolation solvent, which is in the agreement with studies already 10 mentioned [START_REF] Shu | Analysis of polycyclic aromatic hydrocarbons in sediment reference materials by microwave-assisted extraction[END_REF][START_REF] Budzinski | Optimisation of the microwave-assisted extraction in open cell of polycyclic aromatic hydrocarbons from soils and sediments: Study of moisture effect[END_REF][START_REF] Chee | Microwave-assisted solvent elution technique for the extraction of organic pollutants in water[END_REF], Lopez-Avila et al. 1995). 11 For all other validation experiments only this solvent mixture was used. 12 13

F
[Insert Table IV The "Bottom-up" approach. The comprehensive "bottom-up" approach (Ellison et al. 2000) 20 in the first phase requires identification and quantification of the individual relevant sources 21 of uncertainty and in the next phase combination of the individual uncertainty estimates 22 follows (see Equation ( 1)). Although this approach is rather complicated and laborious, it 23 enables to distinguish, which sources of uncertainty are the most important and which are 24 insignificant and can be neglected. 25

The process of the total uncertainty calculation is shown in Figure 3, where the analytical 26 procedure is divided into specific blocks representing individual steps. 27

Generally, uncertainty contributions can be classified into two groups. The first one 28 includes random and the second one systematic components of uncertainty. In the later case, 29 the uncertainty contribution is assessed by considering a recovery and also information from 30 other sources, including manufacture's specifications, calibration certificates or general 31 knowledge about the behaviour and properties of relevant materials or instruments, etc. 32

Both random and systematic uncertainty components of most individual steps cannot be 33 determined directly as it is not possible to measure them in a separate way without 34 considering contributions of the subsequent sample handling steps and the determinative 35 HPLC/FLD procedure (see Figure 3). To determine the uncertainty (u), which can be assigned 36 to each individual step, subtraction of variances (σ 2 ) of the following steps from the variance 37 calculated for this particular step must be performed. In this text the symbol u 1-8 is used for 38 uncertainty associated with only one individual procedure step and symbol σ 1-8 is applied for 39 variance involving also contributions of the subsequent steps. For the calculation of the uncertainty of the "make-up to 10 ml" procedure, the 17 repeatability (u RSD4 ), uncertainty of volumetric glass calibration (u VOL ) and the influence of 18 temperature-related solvent volume expansion (u ex ) were taken into account. Repeatability 19 was estimated as a relative standard deviation of 10 results obtained by making-up of 10 ml 20 volumetric flask to the mark with chloroform and subsequent weighing (Equation ( 4)). 21

Uncertainty of volumetric flask volume was calculated as 22 3 a u VOL = (9) 23 where a is a tolerance provided by manufacturer (±a), i.e. 0.25 % for 10 ml flask. 24

The uncertainty of the solvent volume expansion was calculated as 25

100 × ∆ = V V u ex (10) 26
where ∆V (ml) is a volume expansion of 10 ml of solvent calculated for temperature range 27 from 20 to 24 °C and V is volume of the solvent (10 ml). 28

The variance ( (iii) making-up of this solution (see (ii)) to 10 and 25 ml (calculation of uncertainty 1 comprises of repeatability, calibration of volumetric flask and influence of 2 temperature on acetonitrile volume expansion; data for selected standard solution 3 at level 3-83 ng ml -1 were used) 4

(iv) repeatability of 20 µl injection which comprises also fluctuation of detector 5 response 6

For the calculation of uncertainty of these components the same approach as was shown in the 7 previous text was applied, i.e.: 8 ad (i): for the calculation the Equation ( 5) was used 9 ad (ii) and (iii): for the calculation the Equations (4, 9, 10, 11) were used 10 ad (iv): for the calculation the Equation ( 4) was used. 11

The combined uncertainty σ 8 was calculated using Equation (1).

12 13

[Insert Figure 3 about here] 14 15

Based on a detailed analysis of uncertainty budget, extraction step and both evaporation 16 steps (RVO 1 and RVO 2) followed by GPC clean-up were identified as the most critical 17 phases of the analytical procedure (see Figure 4; Table 8) More detailed analysis of individual contributions to total uncertainty was carried out for 28 HPLC/FLD determinative step (see Figure 5). The repeatability of injection together with 29 uncertainty of standard dilution was identified as the most significant contributions to total 30 uncertainty of this step. 31 32

[Insert Figure 5 about here] 33 34

Comparing the total uncertainties obtained by both approaches (see Tables VII andVIII) 35 it is evident that there is no significant difference between the values of combined 36 uncertainties calculated by either the "bottom-up" or "top-down" strategy. The former 37 approach provided expanded uncertainty for individual PAHs measurements within the range 38 of 12 -43%, the latter one from 20 to 42%. Based on these results it can be concluded that in 39 practice for the assessment of analytical result uncertainty simpler "top-down" approach is 40 possible to apply. 41

Reduction of uncertainties of isolation and, possibly, purification steps, could be 42 accomplished e.g. by utilization of automated extraction device (e.g. pressurised liquid 43 extraction, PLE). Relative standard deviations and also recoveries thus could be improved. To 44 minimize losses of more volatile analytes during vacuum evaporation and to improve 45 repeatability of this step, a keeper, e.g. 1-hexanol and 1-octanol [START_REF] Matthiessen | Use of a keeper to enhance the recovery of volatile polycyclic aromatic hydrocarbons (PAH) in HPLC analysis[END_REF]) might be 46 used. Based on our experience, addition of 20% mixture of 1,2-propandiol in 2-propanol to 47 the sample solution prior to its evaporation is also a feasible option. Since uncertainties of the 48 most critical procedure steps are usually given by used instrumentation and by operator 49 (human factor), further improvement is difficult to control. 50 analogues from fish samples as compared to sonication and saponification. To demonstrate 9 purification efficiency of several conceivable GPC systems, the elution profiles of the main 10 co-extracts and PAHs were constructed. As the most convenient, clean-up procedure 11 employing column filled by gel BioBeads S-X3 and mobile phase chloroform was chosen. 12 For most of target PAHs comparable results are obtained by optimised analytical procedure 13 regardless the estimation of uncertainty is carried out using either "bottom-up" or "top-down" 14 approaches. While the "bottom-up" approach is based on the constructon of an "uncertainty 15 budged" with estimates of each source of error, a simplified "top-down" approach uses 16 experimental determination of the accuracy of a measurement method by calculating 17 measures of repeatability, reproducibility and intermediate precision. In or study the 18 application of "top-down" approach was shown to be more convenient option, which can be 19 used in the case that data from method validation studies or proficiency testing are available.

  was calculated on fresh weight (moisture of sample was 62.5 %).

FigureFigure 2 .

 2 Figure 1. Procedure flow chart

  cyclohexane (1:1, v/v) mobile phase. Diverse separation capabilities of tested GPC 1 systems are documented. 2

  . The examples of recognized 18 contributions of individual procedure steps to the uncertainty of various PAHs classes 19 measurement are shown in Figure 4 (representatives of 3-ring -Phe; 4-ring -Chr; 5-ring -20 B[b]F and 6-ring -B[ghi]P PAHs were selected for comparison). It can be seen that with the 21 increasing molecular weight of analytes (what is accompanied by volatility decrease) the 22 contribution of the evaporation step to the total uncertainty declines, while the contribution of 23 uncertainty associated with the extraction step raises.

  an extraction efficiency of three routinely used extraction techniques were 6 critically assessed. The Soxhlet extraction with hexane-acetone (1:1, v/v) was identified as the 7 most efficient procedure for isolation of PAHs (including their methyl derivatives and sulphur 8

Table I .

 I Characterisation of GPC systems used PAHs -co-extract separation

	GEL	column dimensions (mm)	particle size/ pore size	mobile phase	flow rate of mobile phase (ml min -1 ) #	Code of procedure
	Bio-Beads S-X3	500 x 8	200-400 mesh ∼ (37 -75 µm)	chloroform EtAc-CyH (1:1, v/v) *	0.6 0.6	GPC-BB-C GPC-BB-EC
	PL gel	500 x 7.5	10 µm/50 Å	chloroform EtAc-CyH (1:1, v/v) *	0.6 0.6	GPC-P-C GPC-P-EC
		450 x 19
	Envirogel	(150 x 19 &	15 µm/100 Å	chloroform	3	GPC-E-C
		300 x 19)
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Table II .

 II FLD settings for PAHs determination

	PAHs	Time window (min)	λ λ λ λ excitation (nm)	λ λ λ λ emission (nm)
	Naph, 1-MeNaph	0-1.0	217	338
	Ace, Fln	1-10.6	250	341
	DBT, Phe	10.6-12.1	240	360
	Ant	12.1-14.0	248	405
	Flt	14.0-16.0	232	445
	Pyr	16.0-17.4	236	389
	2-MeAnt	17.4-19.3	250	397
	1-MePyr, B[ [ [ [a] ] ] ]A, Chr	19.3-21.9	270	401
	5-MeChr, B[ [ [ [b] ] ] ]N[ [ [ [d] ] ] ]T	21.9-25.5	270	369
	B[ [ [ [e] ] ] ]P, B[ [ [ [b] ] ] ]F, B[ [ [ [k] ] ] ]F, B[ [ [ [a] ] ] ]P DB[ [ [ [ah] ] ] ]A, B[ [ [ [ghi] ] ] ]P I[ [ [ [1,2,3-cd] ] ] ]P F o r	25.5-27.5 27.5-35.0 35.0-38.7	266 295 248	425 405 484
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Table III .

 III Summary of results obtained by different GPC systems employed for separation efficiency of PAHs and co-extracts testing purity of PAHs fraction is expressed as % of lipid fraction from total lipids loaded on column which is coeluted with PAHs fraction

	Code of		volume of PAHs	volume of co-extracts	Total time needed for	"purity" of PAHs
	procedure		fraction (ml)	fraction (ml)	fractionation (min)	fraction *
	GPC-BB-C		15.5 -30 (14.5)	6 -16 (10)	50	6 % co-elution of PAHs and lipids
	GPC-BB-EC	15 -45 (30)	6 -18 (12)	75	8 % co-elution of PAHs and lipids
	GPC-P-C		15 -36 (21)	9 -21 (12)	60	30 % co-elution of PAHs and lipids
	GPC-P-EC		21 -57 (36)	3 -15 (12)	57	complete separation of PAHs and lipids
	GPC-E-C	75 -115 (40) F o r	50 -75 (25)	40	complete separation of PAHs and lipids
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*

Table IV .

 IV Efficiency of different extraction solvents for isolation of PAHs from fish (n = 6; sonication was used as a testing extraction technique)

	Analytes	Chloroform Relative efficiency (%) * RSD (%)	Hexane -dichloromethane (1:1, v/v) Relative efficiency (%) * RSD (%) Analyte content (µ µ µ Hexane -acetone (1:1, v/v) µg/kg) # RSD (%)
	Naph	86		25		87	20	4.30	28
	1-MeNaph	78		13		56	18	1.89	12
	Ace	87		8		81	16	1.25	7
	Fln	119		12		93	13	11.92	8
	DBT	56		14		55	27	21.32	7
	Phe Ant Flt Pyr 2-MeAnt	62 80 70 61 80 F o r	19 22 20 24 19		62 97 62 48 45	27 21 16 20 21	11.61 2.82 1.52 1.83 1.12	6 7 6 12 10
	1-MePyr B[a]A Chr 5-MeChr	57 98 74 57	15 23 19 P 8 e		48 71 74 44	18 20 20 7	0.38 0.21 0.21 0.04	12 11 7 11
	B[b]N[d]T B[e]P B]b]F	47 64 73		14 15 11 e r	40 39 46	18 20 25	0.42 0.85 0.12	14 12 7
	B[k]F B[a]P DB[ah]A B[ghi]P I[cd]P	77 60 65 62 60		9 28 39 28 10	44 41 41 R 43 44 e v i 23 17 20 21 12 e	0.06 0.20 0.04 0.19 0.15	4 8 9 6 8
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Table V .

 V Efficiency of different extraction techniques for isolation of PAHs from fish (n = 6)

		Sonication		Saponification		Soxhlet extraction
	Analytes	Relative efficiency (%) * RSD (%)	Relative efficiency (%) * RSD (%)	Analyte content (µ µ µ µg/kg) #	RSD (%)	Recovery (%)
	Naph	106	24	101	16	4.05	54	70
	1-MeNaph	99

Table VI .

 VI Uncertainties of optimised analytical procedure obtained by "top-down" approach (%)

			Repeatability (n=10 )		0.05		0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
	Weighting of sample	Accuracy of balance		0.00005 0.00005 0.00005 0.00005 0.00005 0.00005 0.00005 0.00005 0.00005 0.00005	0.00005 0.00005
	Analytes	u 1	u REC	u RSD	0.05	u std	0.05	u 0.05	U 0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
	Phe Ant Extraction Flt	2.0 Repeatability (n=6) 3.8 Recovery (n=6) 4.3 u 2 Repeatability (n=5)	14.1 9.2 17.1	14.1 2.0 14.2 13	0.5 0.7 0.4	9.2 3.8 9.8 8	14.2 17.1 9.8 4.3 17.5 17.5 17	28.3 9.0 4.1 19.6 9.9 35.1 9	11.5 1.6 11.6 7	9.8 6.3 11.6 7	17.3 4.0 17.8 7	21.9 4.2 22.3 8	12.1 4.6 12.8 10	17.1 5.0 17.7 7	13.7 5.8 14.9 7	14.2 4.8 15.0 9
	Pyr RVO 1		4.1 Recovery (n=5)	9.0	2.0	0.4	4.0	9.9 2.9	29.8 1.4	1.4	0.6	2.0	1.4	2.3	0.6	1.2	0.6
	B[a]A		u 3	1.6	11.5	13.2	0.3	9.0	11.6 17.2	23.2 8.6	6.7	6.5	7.3	8.1	10.3	7.0	7.1	9.0
	Chr B[b]F B[k]F Make-up to 10 ml (chloroform)	6.3 Repeatability (n=10) 4.0 4.2 Calibration of volumetric flask 9.8 17.3 21.9 Temperature volume expansion of solvent (for 22±2°C)	1.1 0.14 0.3	0.3 0.3 0.4	1.1 0.14 0.3	11.7 1.1 17.8 22.3 0.14 0.3	23.3 1.1 35.5 44.6 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3	1.1 0.14 0.3
	B[a]P		u 4	4.6	12.1	1.13	0.5	1.13	12.9 1.13	25.8 1.13	1.13	1.13	1.13	1.13	1.13	1.13	1.13	1.13
	DB[ah]A B[ghi]P GPC I[cd]P	5.0 Repeatability (n=5) 5.8 Recovery (n=5) 4.8 u 5 Repeatability (n=5)	17.1 13.7 14.2	8.1 4.9 9.5 8.3	1.8 1.0 0.6	7.6 4.0 8.6 7.1	17.8 7.7 14.9 0.1 15.0 7.7 7.3	35.6 8.3 0.2 29.8 8.3 30.0 6.9	6.2 1.4 6.4 6.2	5.8 1.4 6.0 5.8	6 1.4 6.2 5.4	7.1 1.5 7.9 7.2	9.2 1.6 9.3 9.1	5.8 1.6 6.5 5.9	5.9 1.3 6.0 4.8	8 1.6 8.5 8.0
	Captions: RVO 2	Recovery (n=5)		1.4		4.0	1.2	0.9	0.6	0.5	0.1	1.1	0.5	0.9	0.2	0.3
	u REC uncertainty of recovery of analytical procedure, Eq. (3) u 6 8.4 8.2	7.4	7.0	6.2	5.8	5.4	7.3	9.1	6.0	4.8	8.0
	u RSD repeatability of the procedure (n=6), Eq. (4) Repeatability (n=10)
	u std uncertainty of purity of the standard solution, Eq. (5)
	u	total uncertainty -for calculation see Eq. (1)
	U	expanded uncertainty -for calculation see Eq. (2); coverage factor k = 2

Total uncertainty u 2 = u 1 2 + u 2 2 + u 3 2 + u 4 2 + u 5 2 + u 6 2 + u 7 2 + u 8 2

			σ1 2
	F o r
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	Concentration of standards / purity Preparation of working standard solution Repeatability (injection volume, detector response)	e v i e
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	Figure 3. Flow-chart of the total uncertainty calculation	O n l y

Phenanthrene (3 ring PAH) Chrysene (4 ring PAH) C 28% F 25% G 2% H 10% A 0% B 17% E 14% D 4% B 43% D 5% E 6% F 25% H 8% G 2% C 11% A 0% Benzo[b]fluoranthene (5 ring PAH) Benzo[ghi]perylene (6 ring PAH)

	F 16% D 4% E 9%	C 12% G 2% F H 5% o A 0% r P e	B 52%	F 16% E 13%	D 4%	G 2%	H 6% C 13%	A 0%	B 46%
	Figure 4. Contributions of partial uncertainties of the individual steps of procedure to the total uncertainty (A -weighting of sample, B -extraction, C -RVO 1, D -make-up to 10 ml, E -e r
	GPC, F -RVO 2, G -transferring into acetonitrile, H -HPLC) R
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Phenanthrene (3 ring PAH) Chrysene (4 ring PAH) A 11% B 33% C 56% C 48% B 43% A 9% Benzo[b]fluoranthene (5 ring PAH) Benzo[ghi]perylene (6 ring PAH)

		A			A
	C 45% F o r	9%	B 46%	C 37%	25% B
	38% Figure 5. Contributions of partial uncertainties to the total uncertainty of HPLC/FLD step (A P
	-concentration of standard solution, B -diluting of working standard solution, C -e repeatability) e r
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1 Optimisation of procedure for determination of polycyclic aromatic hydrocarbons and 1 their derivatives in fish tissue; estimation of measurements uncertainty

	2
	3
	4
	5	Abstract
	6	Three alternative procedures were employed for isolation of polycyclic aromatic
	7	hydrocarbons (PAHs; 15 of 16 US EPA priority pollutants and benzo[e]pyrene), their methyl-
	8	derivatives and sulphur analogues from fish tissue: (i) Soxhlet extraction, (ii) batch extraction
	9	enhanced by sonication and (iii) saponification of sample followed by reextraction of analytes
	10	into hexane. Soxhlet extraction using hexane-acetone (1:1, v/v) was found as the most
	11	efficient extraction technique, with analyte recoveries in the range 70-108%. Within
	12	optimisation of clean-up step, several types of gel permeation chromatography (GPC) systems
	13	were tested: two types of polystyrene divinylbenzene copolymer gels (PSDVB), both "soft"
	14	gel type (Bio-Beads S-X3) and "rigid" gels type (PL gel and Envirogel) in combination with
	15	various mobile phases were compared. Bio-Beads S-X3 and mobile phase chloroform were
	16	found as the most appropriate for purifying of crude extracts prior to the final determinative
	17	step. High performance liquid chromatography with fluorimetric detection (HPLC/FLD) was
	18	used for identification and quantification of PAHs in purified fish extracts. The uncertainties
	19	of PAHs measurements were estimated employing two alternative approaches. Both provided
	20	similar results: the expanded uncertainties obtained for individual PAHs by "top-down"
	21	approach were in the range 9-53 %, their values resulting from application of "bottom-up"
	22	were in the range 16-52 %.
	23
	24	Keywords: polycyclic aromatic hydrocarbons (PAHs), uncertainty, clean-up, gel permeation
	25	chromatography (GPC), extraction, fish tissue
	26
	27	Introduction
	28
	29	Polycyclic aromatic hydrocarbons (PAHs) represent a group of ubiquitous environmental
	30	contaminants originating from various emission sources. Their control in the environment and
	31	in the food chain is required due to the mutagenic and carcinogenic potential of some PAHs,
	32	exhibited in vertebrates. During the recent decade, many studies concerning with monitoring

  al. 36 2003). It should be noted that the list of 16 US EPA PAHs is different from that established 37 recently by European Union (EU, 2005). 38 Exposure of habitant biota to PAHs is often assessed through measurement of these 39 hazardous chemicals and/or PAHs metabolites in their tissues. Since the concentrations of 40 target analytes are typically very low, fine tuning of analytical procedure is needed to generate 41 reliable data. Within the optimisation of analytical process, the availability of reference

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	42
	43	materials with certified concentrations of target analytes incurred in relevant matrix is of a
	44	great importance for accuracy control. Unfortunately, with very few exceptions, appropriate
	45	certified reference materials (CRMs) enabling analytical quality control (AQC) in monitoring
	46	studies concerned with occurrence of PAHs in various biotic matrices, are not commercially
		available.

47 Similarly to analysis of other organic pollutants, extraction of PAHs from respective 48 matrix represents a critical step in their determination scheme. Various techniques have been 49 applied for isolation of these substances from biotic and/or abiotic samples. Particularly 50 2 Soxhlet extraction

  The presented study concerning PAHs analysis was focused on following tasks(i) critical 1 assessment of extraction efficiencies of most conceivable isolation techniques represented by 2 Soxhlet extraction, batch extraction enhanced by sonication and saponification of sample 3 followed by re-extraction; (ii) to find a suitable experimental configuration of GPC for 4 purifying of crude extracts within determination of PAHs and some of their derivatives in fish An automated GPC (gel permeation chromatography) system consisting of 305 MASTER 1 pump, fraction collector, automatic regulator of loop 231 XLI, microcomputer (software 731 2 PC via RS232C), dilutor 401C (GILSON, France) and stainless steel columns (i) 500 x 8 mm 3 i.d. packed with gel Bio-Beads S-X3, 200 -400 mesh ∼ 37-75 µm (Bio-Rad Laboratories, 4 USA) and (ii) 500 x 7.5 mm i.d. packed with PL gel, particles size 10 µm, pores size 50 Å

	1 1	evaporator at 40 °C just to dryness and quantitatively transferred into a 5-ml volumetric flask Optimisation of clean-up step
	2 2	by chloroform.
	3
	4	Clean-up. Experimental set-ups of all gel / mobile phase combinations used for optimisation
	5 5 5	of crude extract purification are summarised in Table I.
	6 6 6	samples; (iii) estimation of uncertainties of optimised methods frequently employed in food (Polymer Laboratories, U.K.) and (iii) two columns connected in series packed with
	7 7 7	analysis (using "bottom-up" and "top-down" approaches) with respect to their practical use. Envirogel, with dimensions 150 x 19 mm and 300 x 19 mm i.d., particles size 15 µm, pores [Insert Table I about here]
	8 8 8	size 100 Å (Waters, USA) were used for clean-up of extracts.
	9 9 9	Materials and methods A Vacuum evaporator (Büchi Rotavapor R-114 a Waterbath B-480, Switzerland) was Within optimisation of isolation step, the clean-up procedure carried out by gel
	10 10 10	used for concentration of extracts. permeation chromatography (GPC) employing gel Bio-Beads S-X3 and mobile phase
	11 11 11	Experimental materials A high performance liquid chromatographic system (HPLC) Hewlett-Packard 1100 Series chloroform was used. Flow rate of the mobile phase was 0.6 ml/min and injection volume 1
	23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 12 13 14 15 12 13 14 15 16 16 17 18 19 20 21 22 17 18 19 20 21 22 23 23 24 25 26 27 28 29 30 31 32 24 25 26 27 28 29 30 31 32 33 33 34 35 36 37 38 39 50 50 49 49 48 48 47 47 46 46 45 45 44 44 43 43 42 42 41 41 40 34 35 36 37 38 39 40	2000). In analytical chemistry an expanded uncertainty (U) is commonly used rather than standard uncertainty. Its value defines an interval within which the value of the measurand lies with a known level of confidence. U is obtained by multiplying the combined standard uncertainty (u) by a coverage factor (k): U = k.u (2) A coverage factor, k, is typically in the range from 2 to 3. The choice of this factor is based on a level of confidence required for particular purpose. For example, for an approximate level of confidence of 95 %, k = 2. Several concepts were developed for the estimation of uncertainties related to results. Nevertheless, in practice two main different approaches to uncertainty estimation are used -"bottom-up" described in EURACHEM/CITAC document (Ellison et al. 2000) and "top-down" recommended in the recently published ISO 21748:2004 (ISO/TS 21748 2004) which gives a guidance for the estimation of measurement uncertainty using the data obtained from interlaboratory studies performed in an accordance with ISO 5725-2 (determination of repeatability and reproducibility of a standard measurement method) (ISO 5725-2 1994). Currently, under discussion there is a draft ISO standard (ISO/CD 15753, 2004), which describes the interlaboratory validated method for determination of 15 US EPA PAHs in animal and vegetable oils from where data on reproducibility could be taken (provided that this particular standardised procedure is used for sample analysis) Alternative concepts aimed at practical and understandable way of measurement uncertainty calculations, based mainly on already existing quality control and validation data, namely the use of certified reference materials (CRM), participation in interlaboratory comparisons (proficiency testing) and recovery tests are described in the Handbook for calculation of measurement uncertainty in environmental laboratories (Magnusson et al. 2003). F o r P e e r R e v i e w O n l y The sample of smoked fish (mackerel) used for optimisation of analytical procedure was obtained from the common market of the Czech Republic. Before homogenization in a blender, the skin and bones were removed. Homogenised sample (3 kg of fish fillets) was stored at -20 °C. Chemicals and materials Chloroform and acetone (analytical reagent grade, Lachema Brno, Czech Republic) were redistilled in glass before use. Acetonitrile (gradient grade, for chromatography, Merck, Germany), hexane (for organic trace analysis, Merck, Germany), dichloromethane (for gas chromatography, Merck, Germany), methanol (gradient grade, for chromatography, Merck Germany) cyclohexane (for gas chromatography, Merck, Germany) and ethyl acetate (for pesticide residue analysis, Scharlau, Spain) were used as supplied. Deionised water was obtained from Milli-Q water purification system (MILLIPORE, USA). Anhydrous sodium sulphate (Penta Praha, Czech Republic) was dried at 500 °C for 5 hours and then stored in a tightly capped glass bottle. Potassium hydroxide (analytical reagent grade, Penta Praha, Czech Republic) was used for saponification. The standard mixture 1647d of 16 priority PAHs -naphthalene (Naph), acenaphthene (Ace), fluorene (Fln), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flt), pyrene (Pyr), benz[a]anthracene (B[a]A), chrysene (Chr), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), dibenz[a,h]anthracene (DB[ah]A), benzo[g,h,i]perylene (B[ghi]P) and indeno[1,2,3-cd]pyrene (I[cd]P) dissolved in acetonitrile was supplied by National Institute of Standards and Technology (NIST, USA). Standards of individual PAHs derivatives -1-methylnaphthalene (1-MeNaph), dibenzothiophene (DBT), 2-methylanthracene (2-MeAnt), 1-methylpyrene (1-MePyr), 5-methylchrysene (5-MeChr), benzo[b]naphtho[2,1-d]thiophene (B[b]N[d]T), benzo[e]pyrene and (B[e]P) dissolved in acetonitrile were supplied by Dr. Ehrenstorfer (Germany). Purity of individual standards was not less than 95 %. Working standard solutions were prepared in acetonitrile and stored at 4 °C. Before the use, all glassware was washed with detergent, rinsed with distilled water and acetone and then heated. Equipment A laboratory blender (WARING blender, 38BL-40, Waring Commercial, USA) was used for homogenization of samples, an ultrasonic bath Sonorex RK 510 (Bandeline, Germany) and Soxhlet extractor with cellulose extraction thimbles (Whatman, U.K) were used for sample extraction. F o r P e e r R e v i e w O n l y composed of quarternary pump system with degasser, autosampler, column thermostat, fluorescence detector (FLD) (Hewlett Packard, USA) and a LiChroCART 250-4 (250 × 4 mm i.d.) column and guard column 4-4 (4 × 4 mm i.d.) both with the sorbent LiChrospher PAHs  (Merck, Germany), was used for PAH analysis. F o r ml. After discarding the first 15.5 ml of eluate, the next 14.5 ml were collected. The purified extracts were subsequently subjected to concentration by rotatory vacuum evaporator at 40°C just to dryness. The residue after evaporation was immediately dissolved in 0.5 ml of acetonitrile and the solution was transferred into a 2-ml amber vial. F o r Analytical procedures Procedure flow chart is shown in Figure 1. [Insert Figure 1 about here] Soxhlet extraction. Twenty grams of homogenised sample mixed with 60 g of anhydrous P e e r Identification and quantification. The HPLC/FLD determination was carried out under the following conditions: gradient elution (A -acetonitrile, B -water; 0 min -55 % A, 40 min -100 % A, 42 min -100 % A), mobile phase flow rate 1 ml min -1 , injection volume 20 µl, P column temperature 35°C, FLD settings are shown in Table II. e An example of PAHs determination in standard solution and fish sample by HPLC/FLD is demonstrated in Figure 2. e r sodium sulphate in a grinding mortar were placed into the extraction cellulose thimble, covered with glass wool and inserted into the Soxhlet extractor. Thimbles were pre-extracted for 2 hours with an extraction solvent to obtain lower PAHs procedure blank. Extraction was carried out with 170 ml of extraction solvent (hexane-acetone, 1:1, v/v) for 6 hours (10 cycles/hour). The Soxhlet apparatus was covered with an aluminium foil to avoid access of daylight (to prevent the risk of photodegradation). The extraction solvent was then carefully evaporated by rotatory vacuum evaporator at 40°C just to dryness and quantitatively transferred into a 10-ml volumetric flask by chloroform. Batch extraction enhanced by sonication ("Sonication"). Twenty grams of homogenised R e v i e w [Insert Table II about here] [Insert Figure 2 about here] R e Recovery experiments. Because of unavailability of reference fish tissue with certified content of PAHs, alternative strategy represented by spiking the matrix by target analytes was used for trueness check. It should be emphasised that spiking may affect the recovery information generated this way due to the potential difference between extractability of naturally incurred v i e and in solvent spiked target analytes (in the latter case extraction of analytes is more straightforward). In our experiments samples spiked at four different levels (50, 100, 150 and w sample dried with 60 g of anhydrous sodium sulphate in a grinding mortar were transferred into an Erlenmeyer flask with 100 ml of extraction solvent (chloroform; hexane-acetone, 1:1, v/v and hexane-dichloromethane, 1:1, v/v were tested). The flask was covered with an aluminium foil to avoid access of daylight (to prevent the risk of photodegradation) and placed for 20 min into an ultrasonic bath. The extract was then filtered through a layer of anhydrous sodium sulphate. Extraction was repeated twice with 50 and 30 ml of extraction solvent, in either case for 20 min. Combined filtrates were evaporated by rotatory vacuum evaporator at 40 °C just to dryness and quantitatively transferred into a 10-ml volumetric flask by chloroform. Saponification. Ten grams of homogenised sample were transferred into a flask with 50 ml of 2 M methanolic solution of potassium hydroxide and boiled under reflux for 2 hours. Apparatus was covered with an aluminium foil to avoid access of daylight (to prevent the risk of photo degradation). The content of flask was filtered to a separatory funnel and then extracted by intensive shaking for 5 min with 50 ml of hexane. The extraction was repeated with separated water layer under the same conditions. Combined hexane layers were filtered through a layer of anhydrous sodium sulphate, carefully evaporated by rotatory vacuum measurement is made are described in detail. addition, all relevant considerations on the basis of which the estimation of uncertainties of identification/quantification are documented; the choice of optimal strategy is discussed. In approaches to conduct isolation and purification procedures preceding HPLC/FLD out prior to the final determinative step. In following paragraphs the most common alternative compare and critically assess conceivable strategies of sample handling that is to be carried derivatives in various matrices (see Introduction), only few of published studies attempted to Regardless of a large number of existing methods dedicated for analysis of PAHs and their O n l y 200 % of 0-spike level i.e. natural PAHs content) were analysed; the measured and theoretical values were compared by linear regression. Recovery was obtained as a slope of this curve multiplied by 100. The incubation time of the spike (in acetone solution) in a sample was 16 hours. For the purpose of uncertainty calculation, four repeated measurements of level spiked at 150% of natural PAHs content (0-spike level) were done. O n l Results and discussion y

) 21

where y(x 1 ,…x n ) is a function of parameters x 1 ,…x n , δy/δx i is the partial differential of y with 22 respect to x i and u(y,x i ) is the uncertainty y derived from the uncertainty x i (Ellison et al.

  Procedure blank samples were handled together with extracts of tested material in the same 1 way as real sample. The values of PAHs determined in blanks were subtracted from obtained 2 results to compensate contamination from the external environment.

	3
	4
	5	Choice of a suitable extraction solvent. To identify the most efficient extraction solvent,
	6	experiments employing sonication for PAHs isolation were carried out. Chloroform, hexane-
	7	acetone (1:1, v/v) and hexane-dichloromethane (1:1, v/v) were used for this purpose. Relative
	8	efficiencies obtained within individual extraction experiments are summarised in Table IV.
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Béatrice ; Sangare-Tigori

Béatrice Cote D'ivoire Sangare-Tigori

H Kouadio

James

Betbeder

Anne-Marie Dano

Djédjé Sébastien

E Creppy

Edmond

Prof E E Creppy

Food Additives and Contaminants 1 CO-OCCURRENCE OF AFLATOXIN B 1 , FUMONISIN B 1 , OCHRATOXIN A AND ZEARALENONE IN CEREALS AND PEANUTS IN

Keywords: Survey, Chromatography -HPLC, Clean-up -affinity columns, Immunoassays Mycotoxins, Aflatoxins, Mycotoxins -ochratoxin A, Mycotoxinsfusarium Food Types: Peanuts, Rice, Cereals and grain aflatoxin B 1, fumonisin B1, zearalenone, ochratoxin A, co-occurrence, Côte

The present survey examined 30 samples of rice (n=10), maize (n=10) and peanuts (n=10) from Côte d'Ivoire for aflatoxin B 1 , fumonisin B 1 and zearalenone using immunoassays, and ochratoxin A using a validated HPLC method with fluorescence detection. Similarly to some other countries it appeared that in Côte d'Ivoire several mycotoxins are present in the same commodities. These mycotoxins are from different structural families: aflatoxin B 1 , fumonisin B 1 , zearalenone, and ochratoxin A. There are normally produced by fungal species from Aspergillus, Penicilium and Fusarium genera. Some samples contained four mycotoxins (86%). Four peanuts samples did not show ochratoxin A (14%) whereas they contained aflatoxin B 1 concentrations, above the EU regulatory limits. The concentrations of ochratoxin A, zearalenone and fumonisin B 1 were low and may not cause problem per se however fears remain that the tolerable daily intake might be exceeded due to eating habits and that synergistic effects might be important with the combination of several mycotoxins.

Investigations in this direction are underway together with isolation and characterization of fungal species involved.

Introduction

The European Union (EU) has established regulatory limits for mycotoxins in crops and foodstuffs, (EU Commission Directive, 2005) this implies that crops exported from any country to Europe have to be analysed for mycotoxins. In Côte d'Ivoire, a preliminary survey conducted by us showed the presence of ochratoxin A in maize, millet rice and peanuts [START_REF] Sangare-Tigori | Preliminary survey of ochratoxin A in millet, maize, rice and peanuts in Côte d'Ivoire from 1998 to 2002[END_REF]. Other authors have found aflatoxins in several foods samples, [START_REF] Iarc | IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins[END_REF]Creppy, 2002a). The question was then; do we have multi-mycotoxin contamination in Côte d'Ivoire? The present study aimed to determine the presence and levels of ochratoxin A, aflatoxins, fumonisins and zearalenone in cereals (maize and rice) and peanuts in order to give an answer to the above question.

Materials and methods

Samples

Every time the samples were taken according to the state of the art, with four identical sampling sizes at four different levels of each bulk (top to bottom). Five kg samples were

Chemicals

Toluene (Prolabo), HPLC-grade methanol (Scharlau), HPLC-grade acetonitrile (Scharlau), glacial acetic acid (Chromanorm-Prolabo), anhydrous magnesium chloride approx.98%

(Sigma), sodium acetate (Merck) and hydrochloric acid( Prolabo) were all provided by Merck Eurolab France. Ultrapure water, MilliQ R Plus was obtained using Millipore (France) system in tandem with USF ELGA Prima (France) system. Sep-Pak Plus Silica Cartridges were from Waters (France) (3 cm long, 1 cm diameter). Ochratoxin A was purchased from Sigma-Aldrich (USA). The standard solutions of ochratoxin A were made in methanol according to the concentration established using a UV spectrophotometer (€ = 5500 mol-1 cm-1). The required quantity was evaporated to dryness and dissolved in the mobile phase indicated under chromatographic conditions.

OCHRATOXIN A was assayed by an HPLC method with fluorescence detection and the others mycotoxins by immunoassays (Rhône Diagnostic Technologies, France).

Each sample of rice, maize and peanut was analysed separately for each family of mycotoxin.

Four different extracts were obtained for ochratoxin A, aflatoxin B 1 , fumonisin B 1 and zearalenone, [START_REF] Abdelhamid | Occurrence of some mycotoxins (aflatoxin, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds[END_REF][START_REF] Wood | Mycotoxins in foods and feeds in the United States[END_REF], Shephard et al. 1996[START_REF] Scott | Methods of analysis for ochratoxin[END_REF], Creppy 2002a). The rotatory evaporator, Heidolf WB 2000, was from Prolabo (France). The chromatographic system consisted of a Bischoff pump Model A 2200 and an Alcott Autosampler Model RF-551 Fluorescence Monitor. To measure the peak area, a PIC3 ® software from ICS was used.

Apparatus

A pre-packed stainless steel column( 250 x 4.6 mm) with 10 µm Spherisorb ODS1 and a guard column ( 30 x 4.6 mm) packed with 10 µm Lichrosorb C-18 were used.

Extraction and purification of OCHRATOXIN A:

A solution containing 10 g of mixed ground sample, 50 ml of 0.4 M MgCl 2 and 30ml of 2N hydrochloric acid were extracted twice with 50 ml of toluene. The two phases were separated by centrifugation (5 min at 3000 g). The organic phase was evaporated to dryness under vacuum and evaporated extract was dissolved in 1 ml of methanol and loaded onto the cartridge. The cartridge was then washed with 10 ml toluene-acetic acid (90:10; v/v). The flow rate during washing and elution of the Sep Pak cartridge was maintained at 1 ml/min.

The elute was evaporated to dryness and dissolved in 1ml of the mobile phase [START_REF] Ospital | Ochratoxine A dans les vins[END_REF].

Chromatographic conditions for ochratoxin A:

The mobile phase (0.45 µm filtered and degassed) consisted of 48% acetonitrile and 52% 4mM sodium acetate acetic acid (19:1 v/v). The flow rate was 1 ml/min, the column was at room temperature and the injection volume was 50 µl. The run time for one cycle was 12 min, fluorescence detection wavelengths were excitation 330 nm and emission 470 nm. The retention time of OCHRATOXIN A under the conditions described was approximately 7 min.

Analytical data were collected stored and processed using the PIC 3 software developed by ICS. The quantification of OCHRATOXIN A was achieved automatically by the computer according to the areas under the curve of four standard solutions in the range of 0.6 ng/ml to

OCHRATOXIN A confirmation

Out of the 1 ml final extract, 0.5ml was evaporated to dryness and 0.4 ml of the buffer 0.04M

Tris in 1 M NaCl adjusted to pH 7.5 with HCl was added to the evaporated extract. To this solution 100 µl of carboxypeptidase (Sigma, Ref C-0261, 100 U/ml) was added and the mixture incubated for 3h at 37°C. Fifty µl of this solution was injected into the chromatographic system for separation and quantification of OT alpha, under the same conditions as described for OCHRATOXIN A. The quantification of OT alpha was achieved automatically by the computer according to the areas under the curve of five standards of OT alpha (elution time 5.1min). All the positive samples were confirmed using this procedure that shows moreover disappearance of the peak of OCHRATOXIN A..

Other toxins:

Aflatoxins, fumonisin and zearalenone were purchased from Sigma-Aldrich (USA). They were dissolved in methanol and utilized similarly to the standards solutions provided with the kits. Zearalenone ZEARAPLATE ® (Rhône Diagnostics, France) was used to assayed zearalenone. It is similar to AFLAPLATE ® or FUMONIPLATE ®, but highly specific to zearalenone.

Aflatoxin

Quality control

Each matrix analysed in this survey was used for validation, i.e. ochratoxin A and aflatoxin B 1 free maize, rice, peanuts were spiked in the ranges of regulatory limits up to three times these limits and extracted afterward to evaluate the recovery rates. This was in addition performed three times for each matrix using uniformly tritiated toxins (isotopic mixture 1/1000 with a minimum of 1µCi/ assay). However, higher concentration of ZEA and fumonisin B 1 , 10, 40 80 and 100 µg/kg, were used to spiked toxins free matrixes that were extracted afterwards.

For every five samples of cereals and peanuts, OCHRATOXIN A-free foods provided in France and previously analysed for the mycotoxins concerned were spiked with OCHRATOXIN A at levels of 0.2; 0.6; 2; 6; 20; 40 and 60 ng/g (µg/kg) were extracted and analysed sequentially. The recovery was in the range of 85 to 95% for peanuts and 90 to 95% Similar quality control was performed for the other toxins without radioactivity however. The recoveries were in the range of 89 to 99% for aflatoxin B 1 (spiked at levels of 1.5, 5, 10 and 20 µg/kg) and zearalenone (spiked at levels of 20, 50, 200, and 500 µg/kg) and 85 to 95% for fumonisin B 1 (spiked at levels of 0.3, 0.5, 1.5 and 3 mg/kg).

Negative control samples from toxin-free crops samples were routinely analysed along with spiked samples

Statistical analysis of data

Results are presented as individual values in µg/kg (ppb) for each type of matrix. For the same mycotoxin, concentrations are compared to those found in different matrix. In order to evaluate the contribution of each matrix in human contamination, all individuals values were compared from one matrix to the other using the non-parametric Wilcoxon Rank Sum Test 21 (WRST) with p fixed at p=0.01.

Results and Discussion

Concerning matrix effects, the lowest recovery was always obtained for peanut samples (n=10), whereas the highest was obtained for rice and maize, 80±5% in peanuts and 98±3%

Maize contamination by ochratoxin A:

In a previous survey ochratoxin A was detected in cereals, millet, maize and rice and in peanuts from Côte d'Ivoire [START_REF] Sangare-Tigori | Preliminary survey of ochratoxin A in millet, maize, rice and peanuts in Côte d'Ivoire from 1998 to 2002[END_REF]. The present data thus confirm that ochratoxin A is a frequent contaminant of foodstuffs in this country. All maize samples were ochratoxin A-contaminated and the concentrations ranged from 0.09 to 0.86 µg/kg, Table 2.

These concentrations are far less than those found formerly [START_REF] Dem | Contribution à l'étude des mycotoxines en Côte d'Ivoire : recherche et dosage de l'ochratoxine A dans les céréales[END_REF]Creppy, 2002b of toxinogenic species, interactions with micro organisms and insects as suggested [START_REF] Beti | Effects of maize weevils (Coleoptera: Curculionidae) on production of aflatoxin B1 by Aspergillus flavus in stored corn[END_REF]. We just do not know the most relevant reason for this low OCHRATOXIN A contamination in the year 2002. But, we suspect that in 2002 the cereal traders imported from neighbour countries maize usually found and eaten locally. This is actually in line with information given by the ministry of agriculture and the ministry of trade in 2003, which also indicated that freshly imported yellow corn was available for human consumption. This

Rice contamination by ochratoxin A:

Rice samples of the year 2002 (November) were analysed for ochratoxin A and the concentration ranged from 0.16 to 0.912 µg/kg with an overall mean value of 0.44 ± 0.032.

These values are lower than those reported [START_REF] Abdelhamid | Occurrence of some mycotoxins (aflatoxin, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds[END_REF]) from Egypt where several samples of foods, including rice, had been assayed and ochratoxin A was found at concentrations ranging from 4 to 577 µg/kg with an average of 58.2 ± 22.9 µg/kg.

Peanuts contamination by ochratoxin A:

In four peanuts samples, ochratoxin A was not detected at the limit of detection of the method. Ochratoxin A concentrations ranged from 0.203 to 0.642 µg/kg in the other samples (Table 4). In Tunisia for example, ochratoxin A contaminations ranged from 5 to 46000µg/kg [START_REF] Bacha | Mycotoxins and mycotoxicosis in Tunisia: what do we know and what do we need to know?[END_REF]. Tunisia has a warm and humid climate that favours fungal development especially because peanuts are stored for long periods since Tunisia does not produce peanuts.

This could explain extremely high values such as 46000 µg/kg in this country.

In fact ochratoxin A production depends on conditions of humidity and temperature [START_REF] Pitt | Penicillium viridicatum, Penicillium verrucosum and production of ochratoxin A[END_REF]. The optimal temperature of ochratoxin A production by Aspergillus ochraceus is 28°.

This production decreases at 15° or 37° [START_REF] Trenk | Production of ochratoxin in different cereal products by Aspergillus ochraceus[END_REF]. Besides, Penicillium verrucosum formerly viridicatum grows at temperatures ranging from 4 to 30° with water activity of about 22% [START_REF] Mislivec | Temperature and relative humidity requirements of species of Penicillium isolated from yellow dentcorn kernels[END_REF][START_REF] Pitt | Penicillium viridicatum, Penicillium verrucosum and production of ochratoxin A[END_REF]. In winter times this large variation of temperature may be encountered in Tunisia rendering the situation more favourable to fungi adapted to these conditions. It is then assumed that ochratoxin A is more produced in winter times by [START_REF] Pohland | Ochratoxin A, a review[END_REF][START_REF] Varga | Ochratoxin production by Aspergillus species[END_REF]. Thus, combination of all these factors may explain the high concentrations exceptionally found in Tunisia.

After confirmation of the presence of ochratoxin A in most of the samples let us examine the case of other mycotoxins, successively aflatoxin B 1 , fumonisin B 1 and zearalenone.

Aflatoxins

Aflatoxins in maize:

Maize samples were contaminated by aflatoxin B 1 and the levels ranged from <1.5 µg/kg to 20 µg/kg, Table 2. These values are similar to those reported in foodstuffs from Egypt (3 to 12 µg/kg), from Europe and Tunisia (11.5-180 µg/kg) in corn, wheat, oats, and, corn and barley from Korea [START_REF] Abdelhamid | Occurrence of some mycotoxins (aflatoxin, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds[END_REF][START_REF] Bacha | Mycotoxins and mycotoxicosis in Tunisia: what do we know and what do we need to know?[END_REF][START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom[END_REF]Creppy, 2002a;[START_REF] Park | US perspective on mycotoxin regulatory issues[END_REF]. These values are interestingly in the range of those reported from a very similar west-African country from the viewpoint of the climate, Bénin, [START_REF] Bouraïma | Mise en evidence de la contamination des cereals par les aflatoxines et l'ochratoxine A au Bénin[END_REF].

Rice contamination by aflatoxins:

Aflatoxin B 1 concentrations ranged from <1.5 µg/kg to 10 µg/kg in rice samples, Table 3.

According to the EU regulatory limits for aflatoxin B 1 four of these samples (40%) would not be accepted for human consumption. Aflatoxin B 1 belongs to the mycotoxins family the most studied in the world. In an eastern African country like Kenya, rice may be contaminated at levels ranging from 294 to 1050 µg/kg [START_REF] Jelinek | Review of mycotoxin contamination. Worldwide occurrence of mycotoxins in foods and feeds an update[END_REF][START_REF] Iarc | IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins[END_REF], Creppy et al., 2002a). In USA, aflatoxin B1

Peanuts contamination by aflatoxin B 1 :

Ten samples of peanuts from Côte d'Ivoire were assayed for aflatoxins and results showed that aflatoxin B 1 concentrations ranged from 1.5 to 10 µg/kg, Table 4. All the peanuts samples were contaminated with an average of 4.8 µg/kg. These concentrations are similar to those found in Egypt: 3 to 12 µg/kg, [START_REF] Abdelhamid | Occurrence of some mycotoxins (aflatoxin, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds[END_REF] and were far less than those encountered in Sudan, 250 µg/kg, [START_REF] Jelinek | Review of mycotoxin contamination. Worldwide occurrence of mycotoxins in foods and feeds an update[END_REF][START_REF] Bacha | Mycotoxins and mycotoxicosis in Tunisia: what do we know and what do we need to know?[END_REF][START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom[END_REF]Creppy, 2002a;[START_REF] Park | US perspective on mycotoxin regulatory issues[END_REF].

Aflatoxins are produced by at least three fungal species including, Aspergillus flavus which produces aflatoxins B 1 and B 2 and Aspergillus parasiticus which produces all four alfatoxins B 1 , B 2 , G 1 and G 2 and Aspergillus niger. The dryness, temperature, type of grain and their contaminants may influence the growth of the mould. After the harvest, the conditions of storage and transportation of grains or foods may enhance aflatoxin B 1 concentrations suddenly. The most favourable conditions for growth and production aflatoxins by A. flavus are quite similar to that leading to production of ochratoxins by A ochraceus, aliaceus or other Aspergilli [START_REF] Christensen | Results of feeding tests with rations containing grain invaded by a mixture of naturally present fungus plus Aspergillus flavus[END_REF]. It is thus not surprising that most of samples collected in the present survey are contaminated by aflatoxin B 1 and ochratoxin A.

Fumonisins

All maize, rice and peanuts samples were analysed for fumonisin B 1 .

Maize contamination by fumonisin B 1 :

All the samples were contaminated and the concentrations ranged from 0.3 to 1.5 µg/kg with a mean value of 0.9 µg/kg. Fumonisins are the most prevalent mycotoxins in the world [START_REF] Shepard | Worldwide survey of fumonisin contamination of corn and corned-based products[END_REF] and these mycotoxins contaminate essentially maize. The real problem is the fact that maize cobs, although very contaminated, have the appearance of noncontaminated cobs. And some people for example from Transkei in South Africa prefer mouldy maize. Some other people do prefer traditional beers made at home with mouldy maize, because of its particular taste [START_REF] Norred | Fumonisins-Mycotoxins produced by Fusarium moliniforme[END_REF]. Fumonisins are essentially produced by fungi called Fusarium moniliforme and Fusarium proliferatum and Fusarium culmorum which contaminate the cobs without any physical sign. In South Africa sometimes 100% of maize samples are found contaminated with maximal concentrations reaching 1890 µg/kg [START_REF] Sydenham | Fumonisin contamination of commercial corn-based human foodstuffs[END_REF]. In Benin, a western African country, 9 of 11 samples of maize were fumonisins-contaminated and the maximal concentrations were 2.3 µg/kg [START_REF] Doko | Incidence of levels of fumonisins in contamination in maize genotypes grown in Europe and Africa[END_REF].

In France 23 of 25 samples were contaminated with a maximal concentration of 3.35 µg/kg (De Nijs et al., 1999b). The concentrations found in maize in Côte d'Ivoire are not apparently different from those found in Bénin or in France.

Rice contamination by Fumonisin B 1

Unexpectedly rice samples collected in November 2002 were not contaminated by fumonisin B 1 . No explanation could be given for that at present except that this rice could be a freshly imported one, however some other mycotoxins such as aflatoxin B 1 and ochratoxin A were found. Only seven samples analysed were found with appreciable concentrations of fumonisin B 1 , Table 4. The concentrations ranged from <0.3 to 6 µg/kg.

Zearalenone

Zearalenone, (ZEA) also called F2-toxin, is a non-steroïdal estrogenic mycotoxin produced as a secondary metabolite by several species of Fusarium, particularly F.graminearum or Gibberella zea (Stob et al.,1962), F. semitectum, F.equiseti, F.crookwellense andF.culmorum (IARC, 1993). This mycotoxin may be also produced by F.tricinctum, F.moniliforme, F.oxysporum, F.sporotrichoïdes and F.laterium [START_REF] Mirocha | F-2 (zearalenone) estrogenic mycotoxin from Fusarium[END_REF].

The production of zearalenone by these Fusarium species is favoured by low temperatures ranging from 10 to 15° [START_REF] Caldwell | Zearalenone production by Fusarium species[END_REF] at certain period of fungal infestation of crops.

In fact, the maximum of mycotoxin is produced at such low temperatures that are considered stressful to fungi (Sherwood andPeberdy, 1972, 1974). The water activity in grains is also a crucial factor in zearalenone production by Fusarium species. The presence of oxygen is important too, for example F.graminearum cannot grow neither produce zearalenone in anaerobia conditions of storage [START_REF] Escoula | Fusarium graminearum dans les ensilages. Production de zéaralenone[END_REF]. For all these reasons and especially because of low temperature required during fungal development on crops the presence of ZEA was questionable in foodstuffs collected in Côte d'Ivoire.

Maize contamination by zearalenone:

All the maize samples were contaminated by zearalenone at levels up to 50 µg/kg, Table 2.

These zearalenone concentrations in maize could not endanger people's health considering zearalenone alone [START_REF] Castegnaro | Les mycotoxines: des contaminants omniprésents dans l'alimentation humaine et animale, risqué et prevention[END_REF]. Indeed these concentrations are under the EU

Rice contamination by zearalenone:

All rice samples were zearalenone-contaminated and the concentrations ranged from 50 ppm to 200 µg/kg, Table 3. In France and in Europe, the maximal concentration admitted for human foods and animals feed are 200 µg/kg, [START_REF] Castegnaro | Les mycotoxines: des contaminants omniprésents dans l'alimentation humaine et animale, risqué et prevention[END_REF], EU Commission Directive, 2005).

Peanuts contamination by zearalenone:

All peanuts samples were contaminated by zearalenone and the concentrations ranged from 50 to 200 µg/kg. Although zearalenone was detected in samples collected in Côte d'Ivoire, concentrations do not exceed EU regulatory limits and were rather low in cereals produced and commercialized in Côte d'Ivoire. This country is located in western Africa where temperatures being always between 24 to 34 ° do not permit Fusarium species to grow and produce zearalenone in large amounts. However it is present and could worsen the toxic effects of other mycotoxins such as aflatoxin B 1 , ochratoxin A and fumonisin B 1 .

The literature already reported cases of combination of ochratoxin and aflatoxin or ochratoxin and fumonisin. Cases of combination of four to five mycotoxins in the same commodities have been reported with combined effects in vivo and/or synergistic effects [START_REF] Dawlatana | The occurrence of mycotoxins in key commodities in Bengladesh: surveillance results from 1993 to 1995[END_REF][START_REF] Diaz | Ochratoxin A and fumonisin B1 natural interaction in pigs. Clinical and pathological studies[END_REF][START_REF] Gobel | Simultaneous determination of aflatoxins, ochratoxin A and zearalenone in grains by new immunoaffinity column/liquid chromatography[END_REF][START_REF] Park | US perspective on mycotoxin regulatory issues[END_REF] ochratoxin A increasingly involved in hormonal disorders, one may fear that whenever zearalenone is combined with ochratoxin A and/or fumonisin B 1 this could enhance hormonal disorders in relation with zearalenone and nephrotoxic effect of ochratoxin A, [START_REF] Benesic | Nephritogenic ochratoxin A interferes with hormonal signalling in immortalized human kidney epithelial cells[END_REF].

Concerning the possibility that this mixture of mycotoxins, some of them being genotoxic such as aflatoxins, ochratoxins and zearalenone, could affect immune response in human, the situation should be carefully evaluated through epidemiological studies. One possibility is the morbidity linked to infectious diseases such as bacterial or viral ones. It is believed that mycotoxins may participate in premature death of children in case of bacterial-induced diarrhoea. In this purpose the childhood seems to be a crucial period when the food available should be preserved from high contamination since this may worsen malnutrition, kwachiorkor and possible Reye's syndrome. It seems to be a valuable duty to survey for these diseases and to determine the severity of malnutrition and human exposure to mycotoxins mainly for pregnant women and children during the period of lactation since several toxins including those detected in the present study are known to contaminate breast milk of exposed people. The fungi that produce the mycotoxins detected should be further isolated cultured and characterized by mycologists.

The weekly diet of inhabitants of Cote d'Ivoire contains approximately 500g of maize, 3kg of rice and 150g of peanuts. Using data in table1 the weekly intake can be calculated for aflatoxin B 1 , ochratoxin A, fumonisin B 1 and zeralenone which are 41.5µg (Daily Intake, DI= 99 ng/kg bw/day), 3.29 µg (DI= 7.8 ng/kg bw/day), 1.65 mg (DI= 3.9 µg/kg bw/ day) and 655 µg (DI= 1.56 µg/kg bw/day) respectively. These features appear to be largely above the tolerable daily intake set by WHO for respectively aflatoxins (< 1ng/kg bw/day), fumonisin

  taken each time and mixed to form the sample for analysis. 5 kg from the mixture of 20 kg were ground and 200 g of powder were taken after cautious homogenization of the samples, which were then stored at -20°C until subsequent extraction and analysis. Maize, rice and peanuts were obtained from markets in Abidjan, Côte d'Ivoire, especially in Adjamé, Abobo and Treichville. These markets represent the significant areas which provide feeds to other markets in Côte d'Ivoire.Samples for analysis were assigned a number from 1 to 10 for each type of foodstuffs. Each sample was composed of five sub-samples of equal size of 5 kg mixed carefully prior to a tochratoxin Al of 50 samples were analysed simultaneously for each type of foodstuffs. All the samples were taken from locally produced crops by the end of November 2002, they were stored at -20°C and analysed from January 2003.

  ml of OCHRATOXIN A, run sequentially, during the course of analysis. They were linearly related to the amount of OCHRATOXIN A injected. The correlation coefficient was always between 0.997 and 0.998.

  B 1 AFLAPLATE ® (Rhône Diagnostics, France) was used. It is an immunoassay based on a direct competition ELISA provided with multi-wells plates. It is a very specific, sensitive and The reaction with antibody develops a colour inversely proportional to the concentration of aflatoxin B 1 . The development of the colour is stopped by 1 N sulfuric acid solution and the absorbance of each sample measured at 450 nm in a plate reader. Fumonisin B 1 FUMONIPLATE ® (Rhône Diagnostics, France) was used to assayed fumonisin B 1 .

  rice samples. This was confirmed by using [ 3 H]-OCHRA TOXIN A randomly during the course of extractions, at least one time for each matrix (n=5)

Table 1 :

 1 Different mycotoxins assayed and detected in the same commodities in Côte d'Ivoire

	Mycotoxins		Food samples
		Maize	Rice	Peanuts
	Aflatoxin B1	n=10	n=10	n=10
	( g/kg)	<1.5-20	<1.5-10	1.5-10
	Range (positive %)	(100%)	(100%)	(100%)
	Fumonisin B1	n=10	Not detected	n=10
	(mg/kg)	0.3-1.5		<0.3-6
	Range (positive %)	(100%)		(70%)
	Ochratoxin A	n=10	n=10	n=10
	( g/kg)	0.16-0.86	0.16-0.92	0.0-0.64
	Range (positive %)	(100%)	(100%)	(60%)
	Zearalenone	n=10	n=10	n=10
	( g/kg)	20-50	50-200	50-200
	Range (positive %)	(100%)	(100%)	(100%)

Table 2 :

 2 Concentrations of aflatoxin B1, fumonisin B1, ochratoxin A and zearalenone detected in maize samples, (n=10)

	Mycotoxins					Maize samples
		1	2	3	4	5	6	7	8	9	10
	Aflatoxin	1.5	<1.5 1.5	1.5	1.5	<1.5 5	5	1.5	20
	B1 (µg/kg)
	Fumonisin	1.5	0.3	0.3	1.5	1.5	0.3	0.3	1.5	0.3	1.5
	B1 (mg/kg)
	Ochratoxin	0.67	0.29	0.14	0.39	0.15	0.86	0.50	0.32	0.09	0.23
	A (µg/kg)
	Zearalenone
	(µg/kg)

Table 3 :

 3 Concentrations of aflatoxin B1, fumonisin B1, ochratoxin A and zearalenone detected in rice samples, (n=10)

	Mycotoxins					Rice samples
		1	2	3	4	5	6	7	8	9	10
	Aflatoxin	1.5	1.5	5	10	1.5	10	1.5	5	<1.5 <1.5
	B1, (µg/kg)
	Fumonisin					Not detected
	B1, (mg/kg)
	Ochratoxin	0.91	0.68	0.53	0.20	0.92	0.16	0.50	0.20	0.9	0.16
	A (µg/kg)
	Zearalenone,
	(µg/kg)

Table 4 :

 4 Concentrations of aflatoxin B1, fumonisin B1, ochratoxin A and zearalenone detected in peanuts samples, (n=10), nd= not detected

	Mycotoxins					Peanuts samples
		1	2	3	4	5	6	7	8	9	10
	Aflatoxin	1.5	5	5	5	5	1.5	5	5	5	10
	B1, (µg/kg)
	Fumonisin	<0.3 <0.3 <0.3 <0.3 6	6	3	nd	nd	nd
	B1, (mg/kg)
	Ochratoxin	0.26	0.56	0.20	nd	nd	nd	nd	0.64	0.25	0.33
	A (µg/kg)
	Zearalenone,
	(µg/kg)

  Penicillia in Tunisia and in Europe and that in continuously warm regions like Côte d'Ivoire OCHRATOXIN A is produced by Aspergilli mainly

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  /kg and in Thailand 98 µg/kg. It appears that in Côte d'Ivoire, rice is less contaminated than in some other countries including developed countries. However in the present case all samples are contaminated by aflatoxin B 1 and other mycotoxins that could elicit some synergism with AFB1 whereas in other countries aflatoxin B 1 is not necessarily contaminated by some other toxins. So it remains to understand the cause of this multicontamination, if any.

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contamination in rice was about 280 µg/kg, in India, 180 µg/kg,

  (2 µg/kg bw/day) and zearalenone (500 ng/ kg bw/day),[START_REF] Jecfa | Evaluation of certain food additives and contaminants. 49 th Report[END_REF][START_REF] Jecfa | Safety evaluation of certain mycotoxins in food[END_REF].ConclusionsThe combinations of the four mycotoxins assayed in commodities consumed almost daily endanger people's health. This study reveals that mycotoxins multi-contamination occurs in Côte d'Ivoire in most of maize, rice and peanuts samples collected by the end of November 2002, in which ochratoxin A, aflatoxin B 1 , fumonisin B 1 and zearalenone were found in appreciable amounts, some of which exceeded EU regulatory limits such as aflatoxin B 1 that mostly poses problems. This situation is expected to occur in tropical countries and suggests that the crops analysed may have been produced in this geographical area. The calculation of daily intake with the highest concentrations found shows that fumonisin B 1 and zearalenone are also of concern due to eating habits although found at concentrations below regulatory limits. The main question is at present what health hazard could be linked to this cooccurrence of aflatoxin B 1 , ochratoxin A, fumonisin B 1 and zearalenone? Further investigations are under way.

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