C S Tsoutsi
email: htsoutsi@cc.uoi.gr

I K Konstantinou

D G Hela
email: dchela@cc.uoi.gr

Organophosphorus pesticide residues in Greek virgin olive oil: levels, dietary intake and risk assessment

Keywords: Chromatographic analysis, Clean-up -SPME, Exposure assessment, Risk assessment Additives/Contaminants: Pesticides -organophosphorous Olive oil, pesticides, monitoring, dietary intake, risk assessment 21

The occurrence of organophosphorus pesticide (OPs) and their metabolite residues 2 was investigated in 167 samples of Greek virgin olive oil during a two-year (2004-3 2005) sampling campaign. A total of 30.5% of samples contained detectable residues 4 although only one sample contained dimethoate residues above the maximum residue 5 limit. Among the seven detected OPs, fenthion and fenthion sulfoxide residues were 6 detected in 10.8% and 14.4% of the samples respectively at 0.003 to 0.61 mg kg -1 7 followed by dimethoate that was detected in 10.2% of the samples, at 0.003 to 0.057 8 mg kg -1 . The acute dietary risk assessment was undertaken by determining the 9 National Estimated Short-Term Intake (NESTI) while for the chronic dietary risk 10 assessment the National Theoretical Maximum Daily Intake (NTMDI) and National 11 Estimated Daily Intake (NEDI) were calculated. The estimated intakes (NESTI and 12 NEDI) of each pesticide were <7% and <0.86% of the corresponding acute reference 13 doses (ARfDs) and acceptable daily intakes (ADIs), respectively. A cumulative risk 14 assessment was performed using the Hazard Index (HI) and the Toxicity Equivalence 15 Factor (TEF) approaches taking into account that OPs share the same toxicological 16 mechanism. The determined HI and TEFs were found to represent only a small 17 portion of the respective ADIs or ARfDs. These results indicate that there is neither 18 acute nor chronic risk for the Greek population through olive oil consumption. 19 20

Introduction 22

Olive oil production is heavily concentrated in the Mediterranean countries, which 23 have 99 % of acreage and produce 98% of the world's olive oil. The European Union 24 (EU) has the lead role in the world production with a share of about 75%. The 25 consumption of olive oil is also practically concentrated in the producing regions, 26 with 71% is centered in the EU and 92% of this figure corresponds to the three 27 leading countries, Italy, Spain and Greece [START_REF] Luchetti | Geographic Distribution of World Production of Olive Oils in 563 Handbook of Olive Oil[END_REF]. Olive groves occupy more 28 than 60% of cultivated land in Greece. Spread throughout the mainland and the 29 islands, 132 million olive trees produce approximately 350.000 tons of olive oil 30 annually, of which 75% is extra -virgin olive oil (International Olive Oil Council 31 2007a). 32 33The pesticides authorised for the treatment of olives in Greece belong to different 34 chemical families such as organophosphates, pyrethrins, carbamates, benzoylureas 35 and spinosad for use as insecticides, dithiocarbamates as fungicides while substituted 36 ureas, bipyridinium salts, simazine and glyphosate are mainly applied as herbicides. 37

However, organophosphorus pecticides (OPs) are used in the largest quantities in 38 olive groves (Lentza-Rizos et al. 2001[START_REF] Skouras | Organophosphate resistance in olive 583 fruit fly, Bactrocera oleae, populations in Greece and Cyprus[END_REF]) for controlling the olive 39 fruit fly (Dacus oleae), the key insect pest of olives in the Mediterranean area and 40 they are consequently the main target compounds for olive oil residue analysis (Hiskia 41 et al. 1998;Lentza-Rizos et al. 2001;[START_REF] Botitsi | 477 Monitoring of pesticide residues in olive oil samples: results and remarks between 478 1999 and 2002[END_REF]). Among OPs, fenthion and 42 dimethoate are the compounds that are mostly frequently applied [START_REF] Hiskia | Determination of Organophosphorus 525 Pesticide Residues in Greek Virgin Olive Oil by Capillary Gas Chromatography[END_REF]43 Tsatsakis et al. 2003). 44 matrix, which increases the amount of high purity solvents used, the number of 71 manual operations required, the uncertainty in the determination, the cost of the 72 method and can result in the loss of the analyte. Headspace solid phase 73 microextraction (HS-SPME) has proven its ability to analyze trace amounts of 74 pesticides in olive oil [START_REF] Tsoutsi | Optimization of headspace solid-phase 594 microextraction conditions for the determination of organophosphorus insecticides in 595 olive oil[END_REF][START_REF] Tsoutsi | Screening method for 598 organophosphorus insecticides and their metabolites in olive oil samples based on 599 headspace solid-phase microextraction coupled with gas chromatography[END_REF] as well in other matrices 75 [START_REF] Lambropoulou | Recent developments in 547 headspace microextraction techniques for the analysis of environmental contaminants 548 in different matrices[END_REF][START_REF] Lambropoulou | Optimization of headspace solid-phase 543 microextraction conditions for the determination of organophosphorus insecticides in 544 naturals waters[END_REF]. It simplifies the process of 76 sample treatment before quantitative determination by GC or HPLC and avoids the 77 use of organic solvents. 78 79

The assessment of dietary intake can be performed using either deterministic methods 80 that compare point estimates of intake with a toxicologically acceptable level or the 81 probabilistic method (e.g. Monte Carlo) that takes into account the variation in 82 pesticide residue levels, in food consumption and body weight levels of the population 83 addressed [START_REF] Caldas | Probabilistic assessment of the cumulative 492 acute exposure to organophosphorus and carbamate insecticides in the Brazilian diet[END_REF]. Furthermore, the outcomes give information on the 84 likelihood at which a certain exposure level will occur, as well as the uncertainties 85 associated with it. 86 87 The purpose of this study was to apply a HS-SPME method for the determination of 88 OPs in virgin olive oil samples, within the context of a pesticide-monitoring program 89 for the Mediterranean diet in order to estimate the dietary risk exposure to OPs in the 90 Greek population which has not been reported so far in previous studies. Τhe present 91 study has focused on OPs that present a common mechanism of action in order to 92 perform a cumulative risk assessment. Both chronic and acute exposure was 93 considered for the risk assessment that was made based on individual pesticides as 94 well as on cumulative approaches. Two deterministic approaches, the Hazard Index 95 Olive oil samples used to determine the limits of detection (LODs), limits of 133 quantification (LOQs), recovery and precision were "pesticide free" and different 134 from the samples studied. A ''pesticide-free'' virgin olive oil was obtained from 135 organically grown olive tree cultivation with known spray history and environmental 136 background. To certify as residue free, the sample was analyzed for 137 organophosphorus pesticides. The olive oil sample (known as a virgin olive oil blank) 138 did not contain detectable levels of any of the analytes. To obtain spiked olive oil 139 samples, the appropriate volume of standard working solutions was added to 5 g of 140 olive oil blank at spiking levels ranging from 0.025 to 0.50 mg kg -1 . The I.S. solution 141 was also added to each sample at 0.10 mg kg -1 concentration level. 142 Extraction/concentration of pesticides was carried out according to previous HS-143 SPME extraction protocol [START_REF] Tsoutsi | Screening method for 598 organophosphorus insecticides and their metabolites in olive oil samples based on 599 headspace solid-phase microextraction coupled with gas chromatography[END_REF]. Briefly, 5 g of olive oil sample were 144 C. Helium was used as the carrier (1.5 mL min -1 ) and 160 make-up gases (40 mL min -1 ). The detector gases were hydrogen and air, and their 161 flow rates were regulated at 4 mL min -1 and 120 mL min -1 , respectively. After 162 sampling, the SPME fiber was placed into the injection port of the GC system, 163 equipped with an SPME liner from Supelco (Bellefonte, PA), where it was desorbed 164 for 7 min at 250 o C [START_REF] Safe | Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), 577 dibenzofurans (PCDFs), and related compounds: environmental and mechanistic 578 considerations which support the development of toxic equivalency factors (TEFs). 579[END_REF]). The injection port was in splitless mode, the splitter 165 opening after 2 min. 166 167 Confirmatory analysis was performed on selected positive olive oil samples using a 168 During the in-house validation process, the sensitivity, linearity, accuracy and 177 precision were examined in blank matrix samples. Recovery assays and quantification 178 of pesticide residues were accomplished by the internal standard method. The 179 calibration curves constructed were linear over the range of 0.025 to 0.50 mg kg -1 . 180

The correlation coefficient were >0.985. LODs calculated as three times the noise 181 levels ranged between 0.006-0.010 mg kg -1 , thus, the maximum residue limits (MRLs) 182 required by European and international regulations for the selected analytes can be 183 verified without difficulty (Codex Alimentarius Commission 1996). 184 185 Concerning the accuracy of the method, the relative recoveries of the extraction 186 procedure at three fortification levels (0.080, 0.16 and 0.32 mg kg -1 ) ranged between 187 80 % and 106 % for all the analytes studied. The relative recovery R (%) was 188 calculated as follows: 189

R(%) =Cobs/Cspike × 100 190
where Cobs is the mean concentration of the fortified sample and Cspike is the spiked 191 concentration as previously described in Tsoutsi et al. 2006. Repeatability (n=5, intra-192 day precision), expressed as RSD (%) values, were below 5 % while the 193 reproducibility (n=5, inter-day precision) never exceeded 10 %. In all cases the results 194 accuracy data comprised between 70 and 110% with RSD<15% indicating that a 196 method can be considered efficient for analysing the target compounds in a given 197 matrix (EC, 2006). 198 199 Equilibrium methods like SPME are more selective and sampling in the headspace 200 reduces the matrix effects and interferences present in the liquid phase while poses 201 little risks of cross contamination between samples [START_REF] Pawliszyn | Solid phase microextraction: Theory and Practice[END_REF]. Nevertheless 202 relative retention times have been checked for each pesticide and only a maximum 203 deviation of 1% from the expected RRT was considered acceptable in order to prevent 204 misidentification of analytes due to interferences. In addition, confirmation of selected 205 positive olive oil samples was followed using a GC-MS instrument in SIM mode. 206 207

Dietary intake and risk assessment 208

The results of the monitoring program were not only used to check the compliance of 209 olive oil samples with MRLs, but also for the assessment of the consumers' risk 210 resulting from their exposure to pesticide residues via olive oil consumption. The 211 dietary risk was estimated at both acute and chronic level using the determined 212 pesticide residue levels (mean, median and 90 th percentile values) and mean 213 consumption data of olive oil in Greece (69.7 g day -1 ) extracted from the International 214 Olive Oil Council reports (International Olive Oil Council 2007b). An important issue 215 in the exposure assessment was how to treat samples that were reported to contain no 216 residues (<LOD). All intake estimations were performed assuming that non-detected 217 residue concentrations were equal to half of the respective limit of detection (ND=1/2 218 LOD). A person body weight of 60 kg was assumed in the calculation according to 219 This methodology that uses MRL as a parameter of residue concentration in the intake 235 calculation is very conservative as it assumes situations that are unlikely always to 236 occur. The assumptions include a life daily consumption of food items that: 100% of 237 the daily food consumption will be derived from treated crops; 100% of the treated 238 crop will contain pesticide residues at the MRL level; no dissipation or degradation of 239 the pesticide will occur during storage, transport, preparation, commercial processing 240 and cooking. Thus, a refinement of the intake calculation with more realistic residue 241 data is a better assessment of the risks to consumers. (3) 265

In cases where the estimated exposure is at levels below the ARfD, the exposure is 266 considered to be too low for any toxicity to occur, and the risk is deemed to be 267 insignificant or negligible. 268 The HI is the sum of the hazard quotients (HQ) of the individual OPs (Eq. 4) i.e., it is 282 the sum of the exposures to each of the pesticides in the group expressed as a fraction 283 of its respective ARfD (in case of acute hazard index), or its ADI (in case of chronic 284 hazard index) [START_REF] Teuschler | Current and future risk assessment guidelines, 587 policy, and methods development for chemical mixtures[END_REF]. The function of this first profiling step is to 285 identify the potentially critical samples. If a sample has a value of HI that exceeds 1 286 equal to 100% this could indicate an unacceptable risk. The calculations of "acute HI" 287 and "chronic HI" are described below 288 Using the TEF approach OPs with different potencies were normalized to yield a total 292 equivalent exposure related to one of the pesticides, the so-called index compound 293 (IC) (Wilkinson et al 2000). The TEF approach was first developed to estimate the 294 toxicity of mixtures of polychlorinated dibenzo-p-dioxins, dibenzofurans and 295 biphenyls [START_REF] Safe | Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), 577 dibenzofurans (PCDFs), and related compounds: environmental and mechanistic 578 considerations which support the development of toxic equivalency factors (TEFs). 579[END_REF] but since then the TEF approach have also been used in a 296 number of studies for OPs [START_REF] Jensen | Cumulative risk assessment of the intake 535 of organophosphorus and carbamate pesticides in the Danish diet[END_REF][START_REF] Boon | Cumulative exposure to Acetylcholinesterase 473 inhibiting compounds in the Dutch population and young children[END_REF]. As index compounds 297 chlorpyrifos or methamidophos were used and TEFs for nine OPs were extracted from 298 previously published studies (Table 1) [START_REF] Boon | Cumulative exposure to Acetylcholinesterase 473 inhibiting compounds in the Dutch population and young children[END_REF]EPA 2002;Caldas et al. 299 2006). The exposure (mg kg -1 day -1 ) of each pesticide is multiplied by its TEF and the 300 resulting products are summed (Wilkinson et al. 2000) according to equations 5 and 6 301 for the chronic (TEF c ) and acute (TEF a ) effect level respectively. The resulting sum is 302 then compared with the ADI (0.01 and 0.04 mg kg -1 bw day -1 for chlorpyrifos and 303 methamidophos respectively) for the chronic exposure assessment and with the ARfD 304 (0.1 mg kg -1 bw day -1 ) for the acute exposure assessment. If it is lower than the trigger 305 effect values the cumulative risk is assumed to be negligible. 306

( ) i c TEF NEDI TEF ∑ × =
(5) 307

( ) i a TEF NESTI TEF ∑ × = (6) 308 309

Results and Discussion 310

Monitoring data 311

Of the 167 samples analyzed, pesticide residues were detected in 51 samples (≈30 %), 312 while the rest contained no detectable residues. Of the thirteen organophosphorus 313 pesticide residues investigated in this study, only seven, omethoate, dimethoate, 314 malaoxon, fenthion, ethion, fenthion sulfoxide and fenthion sulfone, were found 315 above their detection limits. Fig. 1 shows the pesticides which were detected in the 2. 319 320

The co-occurrence of pesticide residues is shown in Figure 2. A total of 35 samples 321 (20.96 %) had at least one organophosphorus pesticide at detectable levels, 12 322 samples (7.78 %) contained residues of two pesticides and only 4 samples (2.40 %) 323 were contaminated with three or four pesticide residues. The results of our monitoring 324 of organophosphorus pesticides indicate that, among 167 samples of virgin olive oil 325 that were examined, only one sample was found to have dimethoate (0.057 mg kg -1 ) 326 in concentration above the MRL of Codex Alimentarius for olive oil (0.05 mg kg -1 ). 327

Contamination status of OPs varied on the sampling area (Fig. 3). Pesticide residues 328 from different cultivation areas, along with the detection rates are shown in Table 3. 329

The highest percentage of detection (%) was found in samples from the region of 330 Crete (≈55%) one of the most important olive oil producing areas of Greece 331 [START_REF] Tsatsakis | Three-year study of fenthion and dimethoate pesticides in olive 591 oil from organic and conventional cultivation[END_REF]. Olive oil samples from the region of Peloponnese and 332

Chalkidiki followed with ≈15% of pesticide detection. 333

334

The most frequently detected pesticides were fenthion and its metabolite fenthion 335 sulfoxide and dimethoate. Over 27 different formulations for dimethoate and two for 336 fenthion are registered in Greece [START_REF] Tsatsakis | Three-year study of fenthion and dimethoate pesticides in olive 591 oil from organic and conventional cultivation[END_REF]. Fenthion was detected in 18 337 samples (10.8%) at levels ranging from 0.003 to 0.61 mg kg -1 and its metabolite 338 fenthion sulfoxide was found in 24 samples (14.4 %) at levels ranging from 0.005 to 339 0.15 mg kg -1 . Dimethoate was found in 17 samples (10.2%) in the concentration range 340 of 0.003-0.057 mg kg -1 . 341 The occurrence of pesticide residues in olive oil is related also on the fate of 343 pesticides during olive oil extraction that is closely depended to the fat and water 344 solubility of the compounds under consideration. Residues of fat soluble fenthion 345 were generally higher than those of water-soluble insecticide dimethoate. This is 346 probably due to the higher water solubility of dimethoate (25 g L -1 ). Because of this a 347 large fraction of the active ingredient draws away with the water phase during the 348 olive oil production. On the contrary, fenthion is a lipophilic compound, which is only 349 slightly soluble in water and has an octanol-water partition coefficient (log K ow ) of 4.1 350 in contrast to dimethoate which has a log K ow of 0.3. In the study of Cabras et al. 351 (Cabras et al. 1993) it was observed that ca. 70% of fenthion is transferred from the 352 olives to the oil while the remaining part is split between the cake (20%) and 353 vegetation water (10%). Accordingly, the transfer factors of dimethoate and fenthion 354 from olives to olive oil have been as 0.03 and either 3.3 or 5.20 respectively (Farris et 355 al. 1992). Finally, Leone et al. have reached the same conclusion regarding the 356 importance of the fat-or water-solubility of these two insecticides [START_REF] Leone | Research on some phosphorganic and pyrethroid insecticides 559 residues in oil extracted from olives subjected to guided field control trials against 560 Dacus oleae[END_REF]. 357 Furthermore, the persistence of pesticides after their application in olives is also a key 358 factor for the occurrence of their residues. The fate of fenthion and dimethoate in field 359 trials have been studied by several researchers (Cabras et al. 1997a;Cabras et al. 360 1993;[START_REF] Rotunno | Decay of Fenthion in Green Table 574 Olives[END_REF], where low degradation rates have been observed, 361 resulting to the presence of residues in olive oil. The occurrence of fenthion is 362 consistent with the study of [START_REF] Cabras | Persistence and fate 481 of fenthion in olives and olive products[END_REF], that considered a rather slow 363 metabolic breakdown of this insecticide with a half life of ca. 38 days and that there is 364 a good chance to observe their residues in olive oil. On the other hand, dimethoate 365 degrades more rapidly (half-life 4.3 days) (Cabras et al. 1997a) thus, there is a much 366 lower probability to detect residues of dimethoate in higher percentages and levels. 367 368 Earlier pesticide monitoring data conducted in olive oil samples from the Greek 369 territory are similar to the levels and frequencies reported here in. Generally, results 370 from previous studies that indicated a considerable decrease in residue concentrations 371 [START_REF] Hiskia | Determination of Organophosphorus 525 Pesticide Residues in Greek Virgin Olive Oil by Capillary Gas Chromatography[END_REF][START_REF] Tsatsakis | Three-year study of fenthion and dimethoate pesticides in olive 591 oil from organic and conventional cultivation[END_REF][START_REF] Lentza-Rizos | Monitoring of pesticide residues in olive products: 555 organophosphorus insecticides in olives and oil[END_REF] concentrations ranging between 0.01 -0.83 mg kg -1 and 0.016 -0.03 mg kg -1 377 respectively. The insecticides detected, in this study, were mainly fenthion, either as 378 the parent molecule or as its oxidative metabolites (especially fenthion sulfoxide and 379 to a lesser extent fenthion sulfone). Dimethoate was detected less frequently. The 380 detected pesticide concentrations in most samples were bellowing the Maximum 381 Residue Limits. The significant reductions in the use of fenthion for bate spraying and 382 its replacement by spinosad, has resulted in a significant decrease in the OPs residues 383 in olive oil produced in Corfu [START_REF] Kantas | Insecticides residues in olive oil of Corfu, produced under different 539 pesticide management of Bactrocera oleae (Gmel) Evaluation of results. 6th European 540 Pesticide Residue Workshop[END_REF]. 384 385 Contamination by fenthion and dimethoate was observed in more than forty-six virgin 386 olive oil samples analyzed in Greece during 1994-1995 but always below legal limits 387 [START_REF] Hiskia | Determination of Organophosphorus 525 Pesticide Residues in Greek Virgin Olive Oil by Capillary Gas Chromatography[END_REF]. Additionally residues of fenthion and its metabolites fenthion-388 sulfoxide and fenthion sulfone with levels of 0.007-1.57 mg kg -1 have been reported 389 in virgin olive oil samples collected directly from olive mills at various locations on 390 during 1999during -2002during (Botitsi et al. 2004). The analysis of 391 organic and conventional olive oil samples from Crete during 1997-1999 showed the 392 presence of fenthion and dimethoate with mean concentrations ranging between 0.001 393 and 0.0271 mg kg -1 for dimethoate and between 0.0035 and 0.1702 mg kg -1 for 394 fenthion [START_REF] Tsatsakis | Three-year study of fenthion and dimethoate pesticides in olive 591 oil from organic and conventional cultivation[END_REF]. 395 396 Finally, some pesticide monitoring data in olive oil produced in Italy are presented 397 below for comparison purposes. In the Campanian region, it was reported that 398 dimethoate was one of the most important residues of Italian olive oil as it was 399 detected in 44% of the samples with a mean value of 0.061 mg kg -1 and fenthion was 400 detected in 27% of olive oil samples with a mean value of 0.073 mg kg -1 (Rastrelli et 401 al. 2002). In addition, different OPs were also reported like diazinon, methidathion 402 and parathion with mean values 0.083 mg kg -1 , 0.063 mg kg -1 and 0.080 mg kg -1 403 respectively. Finally, only fenthion residues were detected in Sicilian and Apulian 404 samples at concentrations ranging from 0.09 to 0.73 mg kg -1 [START_REF] Dugo | Rapid GC-FPD determination of 499 organophosphorus pesticide residues in Sicilian and Apulian olive oil[END_REF]. 405 406

Assessment of dietary exposure 407

Although the residue levels of these pesticides are below the MRLs, there was no zero 408 risk because there were pesticides present in the olive oil samples. In order to take 409 account of the maximum risk of consumer's exposure in olive oil, the National 410 Theoretical Maximum Daily Intakes (NTMDIs) was calculated. Table 4 shows the 411 NTMDIs of the detected OPs according to European MRLs and the dietary intakes 412 expressed as % of ADI. The NTMDIs for the detected insecticides ranged from 2.86 413 % ADI to 16.3 % ADI for a 60-kg person and from 2.29 % ADI to 13.1 % ADI for a 414 75-kg person. 415 The refinement of the intake values was performed by determining the NEDI using 417 the average or the 90 th percentile residues found in the samples analyzed (Table 5). 418

Refinement of the residue levels resulted in a significant decrease in the % of ADI 419 percentages for all the compounds. Even for the more conservative assumption, i.e. 420 90 th percentile, the calculated NEDIs for all detected pesticides represent only a small 421 portion of the respective ADIs (<0.86% of ADI). The highest NEDI obtained for 422 fenthion and fenthion sulfoxide were 0.78% and 0.86% of the ADI. 423 424

The NESTI is used to estimate acute dietary exposure. All NESTI values (Table 6) of 425 the detected OPs were <7% of the respective ARfDs recommended by the 426 FAO/WHO, indicating minimum acute risk caused by these pollutants. The 427 cumulative dietary risk exposure to OPs of the Greek consumers is shown in Fig 4 and428 Table 7 using the HI and TEFs approaches respectively for the samples with detected 429 multiresidues. As shown in Fig. 4, both cumulative chronic and acute hazard indexes 430 did not exceed 12 and 8% respectively of the risk cup. It should be noted that the 431 chronic hazard index was higher than the acute hazard index in all cases. 432 433

The percentage contribution of the pesticides detected in multi-residue samples is also 434 depicted in Fig. 4. Fenthion, fenthion sulfoxide and dimethoate contributes more to 435 the exposure than the other pesticides. The results for the chronic cumulative 436 exposure based on TEFs are shown in Table 7. The total cumulative intake in 437 chlorpyrifos equivalent for an adult Greek with an average weight of 75 kg varied 438 from 7.7 10 -5 to 1.0 10 -3 mg kg -1 bw day -1 , which corresponds to 0.77-10.3% of the 439 ADI. In methamidophos equivalents, the results obtained were 9.1 10 -7 -2.0 10 -4 mg 440 kg -1 bw day -1 that is <0.01-0.49% of the ADI. The results from the acute exposure 441 risk assessment in chlorpyrifos equivalents were also shown in Table 7. The % ARfD 442 exhaustion for an adult never exceeded 1% for an adult with 60 kg of body weight. 443

From the results of both approaches it can be concluded that the cumulative risk i.e. 444 the acetylcholinesterase inhibiting effect from exposure to OPs though olive oil 445 consumption, was assumed to be very low. Thus, the contribution of olive oil in the 446 cumulative dietary intake of these pesticide residues from the total diet for the Greek 447 adult consumer is expected to be low. 448 449

Conclusions 450

The overall findings of this study demonstrated the occurrence and the dietary risk of 451 organophosphorus pesticide residues in olive oil samples produced in the years 2004-452 2005 in Greece. The results show that the great majority of the pesticide residues 453 detected were below the MRLs established by the EU. Fenthion and dimethoate were 454 the pesticides more frequently detected while their metabolites were also frequently 455 detected. Moreover, the exposure assessment even when using the more conservative 456 assumptions (90 th percentile values) for each pesticide showed that the contribution of 457 olive oil to the dietary intake represents only a small portion of the respective ADIs or 458

ARfDs. The cumulative chronic exposure assessment for an adult Greek was <13% of 459 the ADI in chlorpyrifos equivalents and 0.61% of the ADI in methamidophos 460 equivalents. In conclusion, the study showed that the dietary intake of 461 organophosphorus pesticide residues detected through olive oil consumption for the 462 National Estimated Daily Intakes (NEDIs) (mg kg -1 bw -1 day -1 or % of ADI) of pesticide residues (average and 90 th percentile) by adults in Greece. National Estimated Short-Term Intakes (NESTIs) (mg kg -1 bw -1 day -1 or % of ARfD) of pesticide residues by adults in Greece and ARfDs

Pesticide

Pesticide

  and the Toxicity Equivalence Factor (TEF), were used for the cumulative risk 96 assessment of the intake of OPs and to assess potential health concerns. 97 98Materials and Methods 99Chemicals, reagents and materials 100 Pesticide standards(dimethoate, diazinon, fenitrothion, malathion, fenthion, parathion 101 ethyl, ethyl bromophos, methyl bromophos, methidathion, ethion) and metabolites 102 (omethoate, malaoxon, fenthion sulfoxide and fenthion sulfone) were purchased from 103 Riedel-de Haën (Seelze, Germany). All pesticide standards were 98-99% purity. 104 Bromophos ethyl was used as the internal standard (I.S.) at a concentration of 0.10 mg 105 Kg -1 . Stock standard solutions of 1000 mg L -1 of each compound were prepared in 106 pure acetone and stored at -18 o C. Working standard solutions of analyzed insecticides 107 at concentration levels 50, 100 and 250 mg L -1 were prepared by diluting the stock 108 solutions with acetone. Acetone (Labscan, Ltd., Dublin, Ireland) was pesticide residue 109 analysis grade. SPME holder and fibre assemblies for manual sampling were provided 110 by Supelco (Bellefonte, PA, USA) and used without modification. 111 Polydimethylsiloxane (PDMS, 100 mm) fibre coating was used for the extraction of 112 OPs from oil samples. Before measurements the PDMS fibre was conditioned in the 113 injector of the gas chromatograph (GC) for 3 h at 240 o C, with the split vent open to 114 fully remove any contaminant that might have caused very high baseline noise and 115 large ghost peaks. The fibre was then repeatedly injected into the GC system until One hundred and sixty-seven (167) samples of virgin olive oil samples were collected 120 from some of the major olive oil production areas of Greece, namely Crete (CRE; 121 n=75), Peloponnese (PEL; n=48), Central Greece (CGR; n=21), Chalkidiki (CHA; 122 n=6), Aegean islands (AEI; n=6) and Ionian islands (IOI; n=11), during 2004 and 123 2005. The samples consisted of bulk samples from large volume storing tanks of olive 124 oil merchants thus, representing about 10000 tonnes of olive oil. According to our 125 knowledge this study is amongst those with the highest number of samples and the 126 highest percentage of total production monitored for olive oil in Greece and other 127 Mediterranean countries. Virgin olive oil samples (0.5 L) were collected in dark glass 128 bottles. The samples were transported to the laboratory and kept for a short time at 129 room temperature until analysis. 130 131 Headspace Solid Phase Microextraction procedure 132

  mL crimp-top headspace vials, capped with PTFE-grey butyl-coated 145 septa. The samples were heated at 75 o C ± 1 and after 10 min, the fiber was immersed 146 to the headspace of the sample for 60 min. Magnetic stirring with a PTFE-coated stir 147 bar was used to agitate the sample at 960 rpm. After extraction, the thermal desorption 148 of the analytes was achieved by inserting the fiber into the injection port (held at 250 149 o C) for 7 min. No carry-over effect was detected after that time. 150 151 Analysis of pesticide residues 152 Chromatographic analysis was performed using a Shimadzu 14A capillary gas 153 chromatograph equipped with flame thermionic detector (FTD) operated at 250 o C. 154The DB-1 column, 30 m x 0.32 mm i.d., used contained dimethylpolysiloxane (J & W 155 Scientific, Folsom, CA). The temperature was programmed as follows:

  column i.d., 0.25 mm; film thickness, 0.25 µm) following the previous 170 temperature program. Ηelium with a flow rate of 1.0 ml/min was used as carrier gas. 171The interface was kept at 200 o C and the spectra were obtained at 70 eV in the 172 selected ion monitoring (SIM) mode. For each of the substances analysed, three 173 characteristic ions were monitored during the analysis. 174 175 Method performance and Quality Control 176

  precision fulfilled the criteria of the European guidelines with 195

  adults (WHO 1997) for comparison with other studies while 75kg 220 of adult body weight was also assumed for being closer to the nowadays mean body 221 weight of an adult in developed countries. Acceptable daily intakes (ADI), Maximum 222 Residue Limits (MRLs), and Acute Reference Doses (ARfDs) given by the Food and 223 Agriculture Organization (FAO) are used for the determination of dietary exposure 224 and the estimation of cumulative risk. 225 226 (a) Chronic dietary exposure 227In a first tier, the chronic dietary risk assessment was conducted by comparing the 228 National Theoretical Maximum Daily Intake (NTMDI) of the studied OPs with the 229 ADI (mg kg -1 bw day -1 ). The NTMDI was defined as the Maximum Residue Limits of 230 each pesticide (MRL i , mg kg -1 ) multiplied by the average consumption data (F, kg 231 day -1 ) of olive oil per person in Greece divided by the mean body weight (b.w.

  The refinement of the NTMDI was conducted by estimating the National Estimated 244 Daily Intake (NEDI) that is based on the most realistic estimate of residue levels. The 245 NEDI was calculated replacing the MRL in the calculation for the residue data from 246 the monitoring program (mean or 90 th percentile value of pesticide residue levels; LR i , 247 mg kg -1 ) and the average consumption data (F, kg day -1 ) according to the following values of NTMDI and NEDI are then compared to ADI of each pesticide. The 251 estimate of the total intake is expressed as percentage of the ADI (%ADI). Risk might 252 exist when NTMDI and NEDI exceed ADI. 253254 (b) Acute dietary exposure 255An acute dietary exposure assessment is the exposure resulting from a single eating 256 occasion or short-term (e. g. single day) exposure including the maximum residue 257 level expected in food as consumed. The intake has to be compared with the 258 toxicological effects that are of concern from short-term exposures, i.e. an Acute 259 Reference Dose (ARfD) has to be used instead of the ADI. The National Estimated 260 Short-Term Intake (NESTI) is used to estimate acute dietary exposure. NESTI is 261 calculated as the highest residue (HR, mg kg -1 ) level detected multiplied by the 262 national consumption data (F, kgday -1 ) for the commodity divided by the body weight 263 (kg) as described below:

  Cumulative risk assessment of the intake 270When humans are exposed to multiple pesticides with a common mechanism of 271 toxicological action, the risk associated with exposure can be characterized by a 272 cumulative risk assessment(EPA 2002). Οrganophosphorus insecticides share the 273 same mechanism of toxicological action by inhibition of acetylcholinesterase activity 274 in the nervous system by phosphorylation, thus, in the present work, two deterministic 275 (point estimate) methods was used to perform the cumulative risk assessment of the 276 intake from olive oil. The Hazard Index (HI) and Toxicity Equivalence Factor (TEF) 277 approaches[START_REF] Teuschler | Current and future risk assessment guidelines, 587 policy, and methods development for chemical mixtures[END_REF] Wilkinson et al. 2000; Boon and Van 278 Claveren 2000;[START_REF] Jensen | Cumulative risk assessment of the intake 535 of organophosphorus and carbamate pesticides in the Danish diet[END_REF] were used in both the acute and chronic exposure 279 assessment of the Greek population. 280 281

  > LOD. The percentage of detection, average, median and 90 th 317 percentile concentration as well as the range of organophosphorus residues in olive oil 318 samples are summarized in Table

Figure 1 :

 1 Figure 1: Distribution of OPs residues in the positive olive oil samples

Figure 3 :

 3 Figure 3: Distribution of positive samples (%) in the major olive oil production regions of

Figure 4 :

 4 Figure 4: Acute (A) and Chronic (C) Hazard Indexes (HI) of pesticide residues in olive oil

  a : NEDI calculated for adult 60kg body weight b : NEDI calculated for adult 75kg body weight c :values established by the World Health Organization

FoodFoodFood

  calculated for adult 60kg body weight b : NESTI calculated for adult 75kg body weight c :values established by the World Health Organization d : values established by the Codex Alimentarius Commission (Codex Alimentarius Commission, 1996) /mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk /mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk /mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 1 .

 1 Toxicity equivalence factors (TEFs) used for the calculation of cumulative

	Page 29 of 39	Food Additives and Contaminants
	dietary intake
	Pesticide	Semi-chronic	Acute	Chronic	Chronic
		TEF a (NL)	TEF a (NL)	TEF b (USA)	TEF c
	Omethoate	1.2	4	-	-
	Dimethoate	25	0.5	0.33	0.32
	Diazinon	50	5	0.024	0.01
	Fenitrothion	0.2	0.02	-	0.03
	Malathion	2	0.02	0.0003	0.0003
	Fenthion	0.71	1.43	0.35	0.33
	Methidathion	0.2	5	0.37	0.32
	Ethion	0.6	2	-	1.1
	Chrlorpyrifos	1	1	0.1	0.05
	Methamidophos	66.7	3.33	1	1

a From Boon and van Klaveren 2000

[START_REF] Boon | Cumulative exposure to Acetylcholinesterase 473 inhibiting compounds in the Dutch population and young children[END_REF]

b From EPA 2002 (EPA 2002b) c From

[START_REF] Caldas | Probabilistic assessment of the cumulative 492 acute exposure to organophosphorus and carbamate insecticides in the Brazilian diet[END_REF][START_REF] Caldas | Probabilistic assessment of the cumulative 492 acute exposure to organophosphorus and carbamate insecticides in the Brazilian diet[END_REF]

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Table 2

 2 Occurrence of pesticide residues in olive oil samples during[2004][2005]

				Food Additives and Contaminants		Page 30 of 39
	Pesticide	Minimum	Maximum	Average *	Median *	90 th Percentile *	No of positive	Detection
		(mg kg -1 )	(mg kg -1 )	(mg kg -1 )	(mg kg -1 )	(mgkg -1 )	samples	rate (%)
	Omethoate	<0.005	0.044	0.005	0.005	0.005	2	1.20
	Dimethoate	<0.003	0.057	0.006	0.003	0.012	17	10.2
	Malaoxon	<0.005	0.25	0.006	0.005	0.005	3	1.80
	Fenthion	<0.003	0.61	0.026	0.003	0.048	18	10.8
	Ethion	<0.005	0.093	0.006	0.005	0.005	6	3.59
	Fenthion sulfoxide	<0.005	0.15	0.015	0.005	0.052	24	14.4
	Fenthion sulfone	<0.005	0.13	0.007	0.005	0.005	3	1.80

*

A default value of ½ LOD were used for samples with values below the LOD http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 4

 4 National Theoretical Maximum Daily Intakes (NTMDIs) of pesticide residues by adults in Greece and acceptable daily intake (ADIs)

			Food Additives and Contaminants		Page 32 of 39
		NTMDI a	NTMDI a	NTMDI b	NTMDI b	ADI c	MRL d
	Pesticide	(mg kg -1 b.w. -1 day -1 )	(% of ADI)	(mg kg -1 b.w. -1 day -1 )	(% of ADI)	(mg kg -1 b.w. -1 day -1 )	(mg kg -1 )
	Omethoate	5.7*10 -5	2.86	4.6*10 -5	2.29	0.002	0.05
	Dimethoate	5.7*10 -5	2.86	4.6*10 -5	2.29	0.002	0.05
	Malaoxon	-e	-	-	-	0.300	-
	Fenthion	1.1*10 -3	16.3	0.00	13.1	0.007	1.00
	Ethion	-e	-	-	-	0.002	-
	Fenthion sulfoxide	1.1*10 -3	16.3	0.00	13.1	0.007	1.00
	Fenthion sulfone	1.1*10 -3	16.3	0.00	13.1	0.007	1.00
	a NTMDI calculated for an adult of 60 kg body weight
	b NTMDI calculated for an adult of 75 kg body weight
	c values established by the World Health Organization
	d values established by the Codex Alimentarius Commission (Codex Alimentarius Commission, 1996)
	e NTMDI was not determined since MRL in olive oil was not set for these pesticides

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Table 5

 5

Acknowledgements 465

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Food Additives and Contaminants


Keith Scudamore

E Guy

Brian Kelleher

Susan J Macdonald

Keith ; Scudamore

Mycotoxins Kas

Robin C E Guy

Campden Food

R A Kelleher

Keith A Scudamore

Fate of the fusarium mycotoxins fumonisins B 1 , B 2 and B 3 , deoxynivalenol and zearalenone in maize flour and maize grits during extrusion cooking

Keywords: Mycotoxins -fumonisins, Mycotoxins -fusarium, Mycotoxinstrichothecenes, Mycotoxins -zearalenone Food Types: Cereals and grain, Processed foods, Snack products Fumonisins, deoxynivalenol, zearalenone, fusarium mycotoxins, maize, flour, grits, processing, extrusion

HAL is

Introduction

Extrusion cooking technologies are used to manufacture many forms of foodstuff from cereals and other ingredients. The range of products includes breakfast cereals, snack foods, pregels (modified starches used in food products) and breading crumbs and also animal feeds. Ingredients such as maize flour and grits, wheat flour and other food components are passed through an extrusion cooker under pressure, mechanical shearing stresses and elevated temperature, and expand rapidly as they are forced through the outlet die [START_REF] Riaz | Extruders in Food Applications[END_REF][START_REF] Guy | Extruded snack foods[END_REF]. During this process, mechanical energy is transformed into heat by viscous dissipation in the starch paste. This causes chemical bonds in the polymers to break and free radicals to form. Mycotoxins are naturally occurring contaminants and compounds such as FB 1 , FB 2 and FB 3 , DON and ZON are subjected both to high temperatures and pressures and to chemical reactions by free radical and other mechanisms and might thus be susceptible to breakdown or interaction with other components in the mixture. Temperatures inside Extrudates can be designed with different physical properties and characteristics ranging from highly expanded products, which are used without further processing, to high-density pellets that are subsequently fried in oil for a very short time during which they expand rapidly. Extrudates are used as the basis for a wide range of retail products Results reported in the literature are limited and sometimes conflicting but this may be because all the conditions used are either not reported or not carefully controlled. For example, if chemical breakdown occurs during the extrusion process the extent of any loss will be related to time, so that the reduction in mycotoxin content will depend on the residence time of the material in the extruder, in turn dependant on the material feed rate. The presence of minor ingredients, or additives, may also have a significant effect [START_REF] Accerbi | Utilization of highly deoxynivalenolcontaminated wheat via extrusion processing[END_REF]. When sugars such as fructose and glucose are present in corn-based foods they may react with the amino group of FB 1 during cooking to form the N-(deoxy-D-fructos-yl-) FB 1 (Lu et al. 1997;Howard et al. 1998). These details are often missing from reports.

Extrusion cooking has been shown to have variable effects on mycotoxins and this has been reviewed recently [START_REF] Castells | Fate of mycotoxins in cereals during extrusion cooking: A review[END_REF], Scudamore et al. 2007).

Approximately 15 % of aflatoxins B 1 and B 2 survived extrusion at 150ºC when spiked corn dough was treated [START_REF] Martinez | Aflatoxin occurrence in 1985-86 corn from Venezuela and its destruction by the extrusion process[END_REF]. However, in a study by [START_REF] Cazzaniga | Mycotoxins inactivation by extrusion cooking of corn flour[END_REF] in which the effects of flour moisture content, temperature and addition of sodium metabisulphite were examined, the reduction of aflatoxin B 1 was only between 10 and 25 % for samples of corn flour experimentally contaminated with aflatoxin B 1 at 50 µg kg -1 . Some loss of ochratoxin A, a mycotoxin formed in grain after harvest, was found during pilot scale extrusion, although loss below 150ºC was relatively small [START_REF] Scudamore | The fate of ochratoxin A in the processing of whole wheat grains during extrusion[END_REF]). [START_REF] Saunders | Control of fumonisin: effects of processing[END_REF] showed that extrusion of dry-milled maize products reduced fumonisin concentrations by 30-90 % for mixing type extruders and by 20-50 % for non-mixing extruders. [START_REF] Katta | Screw speed and temperature effects on stability of fumonisin B1 (FB 1 ) in extrusion cooked grits[END_REF] examined the extrusion of corn grits on the stability of FB 1 using different temperatures and screw speeds. Both the barrel temperature and screw speed affected the loss of fumonisin in the extruded product with losses ranging from 34 to 95 %.

However, the loss ranged from 46-76 % in those products with expansion and colour acceptable for commercial use. [START_REF] Ce | Stability of deoxynivalenol in heat-treated foods[END_REF] showed DON to be quite stable in heat-treated foods when the effects of high temperature and high pressure processing of foods spiked with DON were examined.

Reports from other workers have shown that the fusarium mycotoxins can also degrade at high temperatures. The review by [START_REF] Castells | Fate of mycotoxins in cereals during extrusion cooking: A review[END_REF] suggested that FB 1 was degraded with increasing temperatures from 100 o C to give losses of 50 to 92 % at temperatures above 160 o C. The temperatures were only approximate measures of the barrel temperatures in most cases and the effects of moisture were not separated from the frictional effects. FB 2 was degraded in a similar manner to FB 1 with high losses at 160 o C and up to 100 % at 180 o C. Experiments with DON were more extensive, and results complicated by the use of metabisulphite and problems with the analytical procedures.

The current study continues work by the authors examining the fate of fusarium mycotoxins during milling and selected food processing over a wide range of possible extrusion cooking conditions. The fate of DON, NIV and ZON in wheat flour during extrusion showed earlier that temperature and moisture were important variables for degrading mycotoxins [START_REF] Scudamore | Fate of the fusarium mycotoxins deoxynivalenol, nivalenol and zearalenone during extrusion of wholemeal wheat grain[END_REF]). Here we examine the loss of mycotoxins from maize grits when extruded to form directly expanded products and in maize flour either directly expanded or under conditions that produce solid pellets that are then subsequently fried in oil when they rapidly expand. This study concentrates on snack food-like products [START_REF] Guy | Extruded snack foods[END_REF]) made from a basic feedstock of maize grits, and maize flour.

Materials and methods

Maize grits and maize flour containing mycotoxins

Two commercial consignments identified as containing mycotoxins suitable for carrying out the current studies were obtained from UK maize mills. One consisted of 250 kg of maize grits and the other 400 kg of maize flour. The grits had been milled from Argentine maize and consisted of a product with mean particle size between 500 and 600 µm (micron). The maize flour had been milled from French maize giving a product with approximately 70 % of the particles between 150 and 500 µm and about 30% finer particles.

Mycotoxin concentrations found in maize grits are usually low in comparison to those in the whole maize because they are formed in the starchy endosperm of the maize seed that make up the grits on milling. Several consignments of grits from commercial mills were examined before a suitable 1 tonne consignment was found to contain more than 100 µg kg -1 DON and 250 µg kg -1 of fumonisins, but no ZON. In the same way a number of consignments of maize flour were examined before one with a relatively high level of fumonisins (~3000 µg kg -1 of FB 1 + FB 2 ), a low level of ZON and no DON was intercepted. Because the quantity of the naturally contaminated test materials exceeded the capacity of the blender available, each sample was divided into several lots, blended for two hours and placed in a series of polythene bags each containing about 20 kg as described previously for a study on milling and baking [START_REF] Scudamore | The fate of ochratoxin A in the processing of whole wheat grains during milling and bread production[END_REF]. The maize ingredients in these bags were individually mixed again and a series of random samples of about 250 g were taken for mycotoxin analysis for FB 1 , DON and ZON.

The contents of all the individual bags were finally recombined and re-mixed (in several lots) and then stored in batches of 65-70 kg for convenient handling and eventual transfer to the extruder during the study. Individual batches of maize were re-blended and sampled as necessary. After a final blending, samples from each batch of either grits or flour were analysed again for mycotoxin content to determine the values for % standard deviation (SD) of the replicate samples.

The moisture content of the grits was about 13.2 and 13.5 % for maize flour.

Additional mixes were prepared to examine the effect of common food additives on mycotoxin stability. These were 2 % added salt (sodium chloride) and 2 % sugar (sucrose) added to the maize grits and 1 % or 2 % salt and 5 % and 10 % dextrose (glucose) added to the maize flour.

Extruder and processing conditions

A pilot-scale twin-screw extrusion cooker model MPF 50D (supplied by Baker Perkins, Peterborough, UK) was prepared to reproduce conditions simulating commercial manufacturing processes. Within the extruder body, an arrangement of screws comprised of small elements formed sections for conveying cereal, mixing in water to achieve the required moisture conditions, reverse pumping in a shear zone There are a number of possible different combinations of parameters that can be examined during extrusion cooking. To reduce the number of these, the feed rate of the material was standardised to 700 g min -1 . except when examining pellet formation in which case it was 550 g min -1 . Conditions using combinations of up to 3 temperatures and 4 moisture conditions were examined. In an initial trial, the effect of screw speed was determined and this then subsequently standardised to 300 rpm so limiting the number of combinations required for study.

Each extrusion run involved a sequential change in extruder conditions, The extruder was run up to a steady state at each change over and held for 2 minutes to stabilise the extrusion before a sample was collected over 2.5 minutes thus representing about 1.5 to 2 kg of extrudate. This was allowed to cool before packing.

Logging data was taken to confirm the target settings. Data were recorded (but not all shown here) for temperature, ºC, feed rate, g min -1 , SME (specific mechanical energy), kJ kg -1 , pressure, MPa, extrudate moisture content, %, extrudate specific volume, L kg -1 .

Direct extrusion cooking

The arrangement of the screw elements for direct extrusion cooking has been given previously (Scudamore et al. 2007). There is a section of feed screws conveying the maize feedstock into a short mixing section of paddles where water is injected. A second conveying section forces the mixture into a reversing paddles section.

Resistance from the reversing paddles causes the section to fill and generate a powder feed rates and water were adjusted to the computed settings required to run at 700 ±5 g/min and moisture levels from that of the raw material up to 21 ±1 % w/w. In an initial trial the screw speed was adjusted from 200 to 400 ±1 rpm but subsequently standardised at 300 rpm.

Pellet extrusion cooking

A different configuration was used for extrusion cooking with pellet formation. This comprised a conveying section of feed screws leading to a water injection point and a short cooking section with reversing paddles to heat the flour to 110-120 o C and to melt the crystalline structures in the starch. After this heating section, cooling was applied gradually to reduce the mass temperature to 90-95 o C before extrusion through the 6mm dies. The extrudate was cut with a 4-blade knife at 1100 rpm into disc of approximately 1.5mm thickness. After drying at 60 o C for 1-2h, samples (500 g) were ground for analysis. A second portion (500 g) of dried extrudate was fried in vegetable oil at 185 ±5 o C for 20 seconds, drained and ground for analysis. Here, die plates contained two circular dies of 6mm diameter and 4mm land length. At the start of the extrusion cooking run the barrel heaters were set at 20/90/120/50/30 o C and adjusted to heat and then cool the feedstock using the six sections and circulating water set at 10 o C. The exit temperatures were varied from 90 ±2 to 95 ±2 %. The powder and water feed rates were adjusted to the computed settings on the plan to run at 550 ±5 % g/min for 28 ±1 % moisture w/w. The screw speed was held at 250 ±1 rpm for all runs.

Analysis

The methods of analysis used were as described for studies on the extrusion of wholemeal wheat flour (Scudamore et al 2007) seconds, to ensure the sample was mixed with the solvent, and then at high speed for 5 minutes. The sample was filtered and 10 ml of filtrate was transferred into a flask containing 40 ml of phosphate buffered saline solution (PBS). This was mixed well and then filtered through micro fibre filter paper. 10 ml of the filtered aqueous extract was transferred onto a fumonisin immunoaffinity column. The extract was passed completely through the column at a rate of about 1 drop per second under gravity.

The column was washed with 10 ml of PBS and dried to remove all excess PBS by pushing approximately 5 -10 ml of air through it. The fumonisins were eluted from the column with 1.5 ml of methanol at a rate of no more than 1 drop per second and collected in a 4 ml amber vial. The column was washed with 1.5 ml of water and added to the methanol eluate. Automated pre-column derivatisation was carried out using ortho phthaldialdehyde (OPA) derivatisation reagent. This was prepared by dissolving 0.12 g OPA in 3 ml of methanol. 15 ml of 0.1 M disodium tetraborate solution and 150 µl of 2-mercaptoethanol was added and the vial was capped and shaken and kept in the dark. The HPLC system transferred 50 µl sample/standard to a clean vial, added 450 µl OPA reagent, mixed the solutions, and then injected 50 µl of the derivatised solution onto the HPLC column. HPLC used an automatic sample injector (Gilson 231) with dilutor (Gilson 401). A Phenomenex ODS3-Prodigy (15 cm x 4.6 mm i.d.), 5 µm particle size, 250 A pore size HPLC column and guard column, C18 (Anachem, C752), 2.5 cm x 4.6 mm i.d. heated at 32 °C was used with a mobile phase -

DON and other trichothecenes

In summary, a ground sample (20 g) was extracted with 100 ml acetonitrile/water 84:16 by shaking for 2 hours on a wrist action shaker. A 5 ml aliquot of the extract was applied to a pre-washed charcoal/alumina column and 2 ml of clean-ed up extract was transferred to a vial and evaporated to dryness. The residue was derivatised to form the trichothecene -trimethyl silyl (TMS) derivatives and determined by GC/MS operating in selected ion mode, using 4 ions for confirmation. Trichothecenes detected by this method are DON, 3AcDON, 15AcDON, nivalenol, diacetoxyscirpenol, fusarenon-X, T2 toxin and HT2 toxin.

ZON

The method used was that of [START_REF] Macdonald | Determination of Zearalenone in Cereals and Cereal Products by Immunoaffinity Column Cleanup with Liquid Chromatography: Interlaboratory Study[END_REF] and is summarised here. A ground sample (25 g) was extracted with 125 ml of acetonitrile/water 75:25 by blending for 3-5 minutes and the extract was filtered. 15 ml of the extract was diluted with 85 ml of PBS. 50 ml of the diluted extract was passed through an EASI-EXTRACT ZON immunoaffinity column under gravity and the column was washed with 20 ml water. ZON was eluted under gravity with 1.5 ml of acetonitrile. 2 ml of water was added to 1 ml of eluate and mixed well. ZON was detected by HPLC. Liquid chromatography was performed on a Phenomenex ODS3-Prodigy 150 x 4.6 mm, 5 µ particle size, operated at 1 ml min -1 with acetonitrile: water 55:45 as mobile phase. The excitation wavelength of the fluorescence detector was set at 274 nm and the emission wavelength was 440 nm. Sample volumes of 400 µl were injected. LODs were typically 4, 10 and 9 µg kg -1 for FB 1 , FB 2 and FB 3 respectively (based on 3 x signal to noise of the lowest concentration calibration standard of 100/50/50 µg kg -1 ) while LOQ were 14, 34 and 31 µg kg -1 (based on 10 x signal to noise of the lowest concentration calibration standard), 5 µg kg -1 and 10 µg kg -1 respectively for each trichothecene and 3 and 6 µg kg -1 for ZON. For DON and ZON, the limit of detection is defined as 3 times the electronic baseline noise and the limit of determination as 6 times baseline noise. After analysis samples were retained and stored at -20°C.

Results from check trials

Acceptable results

have been achieved in the FAPAS Scheme (www.fapas.co.uk/fapas.cfm) for fumonisins, DON and ZON in cereals during the period August 2000 to April 2007 and were consistently within the limits for Z scores of ± 2 considered acceptable.

Moisture content determination

The moisture content of grits, flour and each extrudate was measured by taking a 5 g sample and heating in an oven at 105ºC for 16 hours.

Results and Discussion

All analytical results were corrected for recovery and all extrudates were corrected for changes in moisture.

Homogeneity of maize grits and maize flour

After initial mixing and blending the % SD between samples from individual bags of maize grits was 17.3 % for FB 1 and 8.3 % for DON using replicate pairs of samples from up to 10 bags. After further mixing prior to extrusion, the bags were re-sampled and the % SD improved to 8.8 % for FB 1 but not for DON (12.1 %). Mean concentrations of mycotoxins in the maize grits were then 274µg kg -1 for FB 1 , 66 µg kg -1 for FB 2 , 22 µg kg -1 for FB 3 and 143 µg kg -1 for DON.

After thorough mixing of the maize flour, 10 pairs of random samples from the bags gave % SD values of 13.5 %, 14.3 %, 14.0 % and 14.1 % for FB 1 , FB 2 , FB 3 and ZON respectively. Mean concentrations were then 2009 µg kg -1 for FB 1 , 409 µg kg -1 for FB 2 , 285 µg kg -1 for FB 3 and 22.0 µg kg -1 for ZON. The precision of the replicate samples which is a combination of a sampling variability factor and analytical precision is quite similar to that for the analytical methods alone, table 1, so that most of the variability is probably due to the analysis and rather than inhomogeneity In addition, in carrying out each study, a sample was taken from the feed hopper for analysis immediately before processing and compared with the corresponding extrudate sample after extrusion. This was carried out by timing the flow of material through the extruder. The result for every set of conditions examined in this work was the combined result from separate runs thus based on two replicates. The mean % SD for all the replicate pairs of results is given at the foot of each table of results. This represents the combined variability due to sample in-homogeneity and to the analysis. The values for grits and flour are mostly between 1% and 4% that compares very favourably with values usually expected from trace analysis alone at these levels, table 1. A wide range of conditions have been examined, not all of which may be used in commercial operations. Thus, in assessing the likely behaviour of mycotoxins in a particular process the manufacturer should ensure that the appropriate conditions in this study are referred to.

Maize Grits

Effect of extruder screw speed

An initial study was carried out to determine whether the screw speed of the extruder feed shaft affected the results obtained. To do this the barrel temperature was set to 160ºC and moisture contents of 17 % and 21 % were used. The results obtained for FB 1 and DON are shown in table 2. Once each extrudate condition had stabilised, a sample of grits was taken from the feed hopper and the corresponding sample of extrudate as described earlier. The mean values from two separate runs are given.

Extrusion of maize grits of 17 % moisture content showed that there was a loss of FB 1 that increased as screw speed increased from 200 to 400 rpm. A similar higher loss occurred at 21 % moisture content as the screw speed increased from 300 to 400 rpm.

Results show that losses of the fumonisins are consistently greater at lower moistures.

More energy is applied to the extrusion system under drier conditions and this may be the reason for an increased breakdown of the thermally labile fumonisins. However, there was no significant effect on DON concentrations as the screw speed was changed. These findings are consistent with the relative thermal stability of the two compounds. As a result of this initial study, it was decided thereafter to standardise screw speed at 300 rpm and thus reduce the number of variables.

Effect of extruder temperature and maize moisture content

Table 3 shows the loss of FB 1 during direct extrusion of maize grits at different temperatures and extruder moistures. Results for FB 2 and FB 3 (not shown here) were very similar but subject to greater variability probably because their concentrations were lower. The loss of fumonisins was greater at 17 % moisture content than at 21 %. The loss also increased slightly as the temperature was raised as might be expected with a thermally labile compound although the moisture of the extruded product had a more significant effect than temperature. In contrast, temperature had little or no effect on the survival of DON suggesting it is heat stable under the conditions used.

However there was a small loss under all conditions that might be due to reduced extractability during analysis or to some binding with the extrudate material. The % SD for each pair of results was between 3 and 4 % that showed that the variability in analysis and sampling was very small and that the effects shown are significant. The Specific Volume of the products (s.v) are given and this indicates how greatly the density of the products formed can change depending on the conditions of extrusion.

The presence of small amounts of certain other ingredients added to the cereal extruder feed has been shown previously to affect the stability of some mycotoxins during processing. The effect of two commonly used additives were examined here, 2 % w/w sodium chloride (salt) and 2 % w/w sucrose. These additives were each added to a separate batches of the maize grit feed and examined in separate extruder runs. Table 4 shows the result on concentrations of FB 1 and DON of adding 2 % NaCl to the maize grits. The loss of fumonisin B 1 is less in the presence of sodium chloride at both temperatures although the overall loss is less at the higher moisture content. The reason for this is unclear but higher extraction efficiency is sometimes obtained when salt is present in analytical extraction solvent mixtures and suggests that it may be an extractability effect. Less pronounced results were obtained for DON which is less susceptible to breakdown. .

Salt

Sucrose

Table 5 shows that the addition of 2 % sucrose has no effect on the levels of FB 1 or DON when compared with samples without sucrose processed under similar conditions although losses were consistently greater under the drier conditions as discussed earlier..

Maize Flour

The flour fraction from maize milling is composed of smaller particles than those comprising the grits and has a higher oil content of about 2-3 %. There are commercial processes that use both direct expansion in which the extruder operates with lower moisture content material and higher temperatures and pellet formation that is used with higher moisture extrudate (28-30 %) and lower temperatures (~100°C). The dense pellets that are formed are then fried in oil at high temperature for about 20 seconds and as a result they expand rapidly to form the end product. Both processes were examined here. The mycotoxin concentrations in the maize flour were 2009 µg kg -1 for FB 1 , 409 µg kg -1 for FB 2 , 285 µg kg -1 for FB 3 , 22 µg kg -1 for ZON. The DON concentration was too low to obtain quantitative results.

Direct extrusion

The losses of FB 1 , FB 2 and FB 3 shown in table 6 were all >39 % under all conditions studied. Loss also increased as temperature was raised but this effect was much less than that caused by changes in moisture content. However, the effect of temperature became more marked at lower moisture contents. Each fumonisin was affected in the same way. The loss of ZON was unexpected as it is usually regarded as a very thermally stable mycotoxin and moisture content had the opposite effect when compared to the fumonisins. This is difficult to explain although the concentration of ZON was low (22 µg kg -1 ) and this might play a role.

Unfortunately no study of the effect on ZON in maize grits was possible in the absence of this mycotoxin from that ingredient. However, in a previous study of ZON in wholemeal wheat, a much higher concentration was present (300 µg kg -1 ). In that study, there was a tendency for more than 100 % of ZON to be measured although this was less marked at the higher moisture contents. Limited studies of the effects of extrusion have been carried out but some loss of ZON has been reported previously [START_REF] Ryu | Stability of zearalenone during extrusion of corn grits[END_REF]).

Our study with maize grits showed that the addition of sodium chloride produced higher recovery of fumonisins. Here in table 7, the addition of 1 % sodium chloride also similarly reduced the apparent loss of fumonisin under each of the conditions examined. We reported a similar result with wholemeal wheat in which the presence of added salt also reduced the levels of mycotoxin lost. Food manufacturers are under pressure to reduce salt levels in food so it is important that the effect of salt and other ingredients is noted. It was not possible to decide whether this is an analytical artefact due to better extractability or whether thermal breakdown or binding to the maize structure is reduced. This study was repeated with maize flour and the results in table 7 confirm the effect of salt. Higher values for mycotoxins are found consistently in the presence of salt.

Studies with maize grits showed that the addition of sucrose had no effect on either fumonisins or DON so that it was decided to examine whether reducing sugars affect concentrations of some mycotoxins. Table 8 shows the effect of adding 5 % and 10 % of dextrose (glucose) to maize flour under the same conditions as tested for salt.

Levels of FB 1 recovered were much reduced under all conditions by the addition of only 5 % of dextrose. The higher 10 % addition further increased the loss but clearly even a small addition of reducing sugar has a significant effect. However, fumonisins are known to bind with glucose.

It was observed that extrudate expansion of grits was similar to normal snack products at 17 % moisture but denser and more like breakfast cereals at 21 % moisture.

The results for ZON, Table 8 shows that its loss does not appear to be affected by the presence of dextrose at either moisture at 180ºC although there was some loss under all concentrations. However, the loss appears to be reduced by the presence of dextrose at 160ºC. ZON is much less polar than DON and this may be a factor. It is difficult to explain some of the results for both of these fusarium mycotoxins.

However, they are both relatively resistant to loss during extrusion compared with the fumonisins. The apparent increase under some conditions requires further confirmatory studies. The trend for residual ZON values to increase going from 140ºC The answer to these changes may lie with the efficiency of the extraction solvent used for removing mycotoxins in the extruded materials or that bound mycotoxins are being released by the extrusion conditions-or a combination of both.

It has already been established that DON is very stable under the conditions studied but the presence of 2 % sodium chloride seems to result in an even higher survival. This may be that sodium chloride assists in the extraction, as has been noted

and used in some analytical extraction procedures for several mycotoxins. This effect also seems to occur for FB 1 although the overall survival is less than for DON. There appears to be no such effect with sucrose, where the presence of 2 % does not appear to cause any significant difference under any conditions tested.

Reduction of salt content by processors in line with current requirements may apparently reduce fumonisins concentrations because recovery is affected. The extent of this will much depend on the physical properties of the extrudate. This factor needs to be examined in detail. On the other hand, the addition of sucrose under these conditions did not significantly lower fumonisin concentrations.

Extruded pellets

Conditions used to produce extruded pellets are very different from those needed for direct expansion. The moisture of the material in the extruder is about 30 % and only a little lower at the point when forced through the die. Barrel temperature was set to 110ºC or 120ºC but this fell to 90ºC and 95ºC respectively at the die face. The pellets were then dried to about 9-10 % and stored until required. In the final frying stage the pellets are dropped into hot cooking oil for a short time during which they expand 9.

The results in table 9 show that there appears to be an increase in FB 1 level during pellet formation after correction for the change in moisture content. This was the same for pellets formed at both 110ºC and 120ºC. However in the same samples there was more loss for ZON at the lower temperature. Throughout this study each value is the mean of 2 samples taken from different runs. The variability is small for the fumonisin results but rather larger for the lower levels of ZON. During the subsequent frying, there was a substantial reduction in FB 1 , particularly in the higher temperature pellets. In contrast the loss of ZON was much less. Changes over the whole process are very similar for FB 1 , FB 2 and FB 3 and this further suggests that these differences are real and not artefacts or analytical problems. The degree of expansion of the pellets from the 110ºC and 120ºC material is quite different giving products of specific volume of 2.1 and 3.2 respectively and this suggests that the composition of the pellets plays a significant part in determining the loss of mycotoxins.

The loss of each fumonisin from the pellet to the fried product might be due to dissolution of the mycotoxin in the frying oil. However this seems unlikely as little loss of ZON occurred during the frying. This compound is less polar and would be expected to dissolve more readily in the oil. It thus suggests that the fumonisins, but not ZON, were partially degraded in the oil.

The effect of salt was examined again in this process by adding 1 % and 2 % to the maize feed recipe. In contrast to studies on direct expansion, salt did not produce any clear effect during pellet formation although the high loss of fumonisins from the 120ºC pellets was subsequently reduced during frying and for the overall

Conclusions

Extrusion cooking technology is complex and can affect mycotoxin concentrations in different ways. Some factors can produce apparently opposite effects in different mycotoxins. Moisture content has been found to be a more important factor than temperature. Operation of the extruder with low moisture ingredients requires a higher energy input and tends to result in higher losses of fumonisins at all temperatures although the opposite effect has been found for ZON. Temperature alone does not appear to significantly degrade any of the fumonisins, DON or ZON under the extrusion conditions studied.

Because loss is less affected by temperature than by moisture content it seems likely that binding to cereal components and/or solvent extractability are also significant factors. Current EC legislation (EC, 2007) sets limits for FB 1 + FB 2 concentrations of 2000 µg kg -1 in flour (milling fraction with particles ≤500 µ) or 1400 µg kg -1 (particles >500 µ) and 1000 µg kg -1 in maize-based foods for direct human consumption or 800 µg kg -1 in breakfast cereals and 200 µg kg -1 in processed maize-based foods and baby foods for infants and young children. Similar catagories apply to limits for DON and ZON (EC, 206). Thus processes using extrusion need to be carefully considered to ensure that mycotoxins in the appropriate consumer end product will meet legislative limits.

Acknowledgement

F

  an extruder can range from 100ºC to 200ºC and moisture content can be used from 13 % to 30 % depending on the characteristics of the end product required.

  pressure pumping. The extruder was calibrated for flow rate and moisture prior to setting up each condition required for extrusion cooking.

  twin start screws. This in turn generates heat and melts the crystalline structures in the starch granule so that a melt fluid forms. In the final screw section the hot fluid is sealed under pressure and the water superheated to > 140 o C and as it forced through a die it returns to atmospheric pressure outside the barrel when the water vaporises rapidly with the immediate expansion of the extrudate. In this work, the die plate contained two circular dies of 4 mm diameter and 4 mm land length. At the start of the extrusion cooking run the barrel heaters were set at 0/50/70/120/140 o C and adjusted to obtain a mass temperature of 140 ±5 o C in the shear zone and final pumping section. Subsequently the barrel temperature was raised in the last two sections to obtain mass temperatures of 160 o C and then 180 ±5 o C. The

  with the addition of a method for the determination of fumonisins. The methods used are UKAS (ISO 17025) accredited, inhouse validated, published in AOAC, being processed as CEN standards and follow the UK Food Standards Agency Requirements of External Contracts (Thompson and Wood, 1995). The methods for DON and ZON are summarised below. All reagents were purchased from commercial sources and were of analytical grade. Solvents were of HPLC grade. FUMONIPREP® columns, TRICHOTHECENE P columns and EASI-EXTRACT® Zearalenone columns were supplied by R-Biopharm Rhone (Glasgow, Scotland). Mycotoxin standards of DON and ZON were purchased from Sigma-Aldrich Company Ltd., Gillingham, Dorset, UK. DON and ZON were quantified prior to use by UV spectrometry, prepared 3-monthly. FB 1 , FB 2 and FB 3 were obtained from PROMEC Unit, MRC Cape Town, PO Box 19070, Tygerberg 7505, Cape Town. Prior to analysis, all samples were ground to 1µm particle size in a laboratory mill and mixed for 1 hour on a Winkworth cone blender and Winkworth paddle mixer.

  the ground sample were extracted with 125 ml of acetonitrile:methanol:water 25:25:50 (v/v/v) by homogenising using an Ultra Turrax on slow speed for 30

  1 M sodium dihydrogen phosphate solution (76:24, v/v) adjusted to pH 3.35 by addition of phosphoric acid. Detection was by fluorescence (Jasco FP-1520) set at excitation 335 nm and emission 440 nm.

  and Limit of Detection of mycotoxinsSamples were analysed in batches and each batch included a blank maize flour sample spiked with a known amount of mycotoxin to allow assessment of recovery, with 70-110 % being the acceptable range. The spiking levels were 250 µg kg -1 FB 1 , 125 µg kg -1 FB 2 and FB 3 , 200 µg kg -1 DON and 50 µg kg -1 ZON. The recovery and % SD for 10 replicate determinations are given intable 1

  the same time the moisture content falls to between 2 and 4 % depending on the pellet, table

  Recoveries of ZON were also improved overall in the expanded product in the presence of salt.

  to 180ºC in table7is contrary what would be expected from chemical degradation.

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Table 2 .

 2 These studies were supported by the UK DEFRA Food Safety LINK Programme and UK Food Standards Agency. Maize flour and maize grits were supplied by UK maize millers Initial mixing and blending was carried by RHM Technology, High Wycombe and analysis of fusarium mycotoxins at the Central Science Laboratory, York, UK. The effect of screw speed on FB 1 (mean concentration 274 µg kg -1 ) and DON (mean concentration 143 µg kg -1 ) in maize grits at constant feed rate and two

	Extrusion cooking was carried out at the Campden and Chorleywood Food Research
	Association, Chipping Campden, UK.
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Table 3 .

 3 Effect of extruder temperature and maize moisture content on FB 1 (mean

	concentration 274 µg kg -1 ) and DON (mean concentration 143 µg kg -1 )during
	extrusion of maize grits
		mc, %	sp. v	FB 1		DON
	MC, % T,ºC grits ext	SV L kg -1 value	mean loss, % values	DON, µg/kg mean loss, %
				s
	140	17		219		128
			7.88	7.60	203	26.1	130	9.6
				186		131
	160	17		*		128
			7.78	9.12	189	31.2	128	10.7
				189		128
	180	17		177		129
			7.70	8.21	174	36.5	124	13.4
				172		119
	140	21	12.4	246		146
				2.51	243	11.2	139	3.3
	140	21	3	240		132
	160	21	12.1	234		141
				2.24	235	14.4	136	4.8
	160	21	8	235		132
	180	21	11.9	230		134
				3.06	233	15.1	139	2.9
	180	21	6	236		144
		% SD for the sample pairs	4.13		3.94

Table 4 .

 4 Effect on FB 1 (mean concentration 274 µg kg -1 ) and DON (mean concentration 143 µg kg -1 )concentrations of adding NaCl to maize grits

	NaCl T, ºC	mc, %	FB 1 , µg kg -1		DON, µg kg -1
		grits Ext. value mean loss,	value	mean loss, %
	180	17		1767		129
	0		7.70	174	36.5		124	13.4
	180	17		172		119
	180	17		210		137
	+ 2 %		7.80	205	25.2		140	2.0
	180	17		200		144
	180	21	11.9	230		134
	0			233	15.1		139	2.9
	180	21	6	236		144
	180	21	12.1	231		143
	+ 2 %			244	10.9		141	1.7
	180	21	3	257		139
	% SD for the sample pairs	3.67			4.14

Table 5 .

 5 Effect on FB 1 (mean concentration 274 µg kg -1 ) and DON (mean concentration 143 µg kg -1 )concentrations of adding sucrose to maize grits

	sucrose T, ºC	mc, %	FB 1 , µg kg -1	DON, µg kg -1
		grits	ext.	value mean loss,	value mean loss, %
	180	17		177		129
	0		7.70	174	36.5	124	13.4
	180	17		172		119
	180	17		177		129
	+ 2 %		7.26	177	35.5	122	15.0
	180	17		177		115
	180	21		230		134
	0		11.96	233	15.1	139	2.9
	180	21		236		144
	180	21		217		131
	+ 2 %		11.61	221	19.2	135	5.8
	180	21		226		139
	% SD for the sample pairs	1.63		5.73

Table 6 :

 6 Reduction in FB 1 , FB 2 and FB 3 (mean concentrations 2009, 409 and 285 µg kg -1 respectively) and ZON (22 µg kg -1 ) content during direct extrusion of maize flour at constant screw speed and feed rate

		mc, %	sp.v.	barrel		loss of mycotoxin, %
	flour	extrudate	L kg -1	T., ºC	FB 1	FB 2	FB 3

Table 7 .

 7 Effect of NaCl on the loss of FB 1 (2009 µg kg -1 ) and ZON (22 µg kg -1 ) content during direct extrusion of maize flour.

	Page 31 of 34		Food Additives and Contaminants
		mc, %		NaCl, %
	flour	extrudate	Barrel T,ºC	0	2
		FB 1		loss, %
	17	6.20/5.25	160	51	41
	17	5.60/6.33	180	58	49
	15	5.80/4.93	160	53	48
	15	4.40/5.79	180	68	40
		% SD for the sample pairs		2.03
		ZON		loss, %
	17	6.20/5.25	160	40	24
	17	5.60/6.33	180	23	10
	15	5.80/4.93	160	34	15
	15	4.40/5.79	180	16	16
		% SD for the sample pairs		1.27

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Table 8 .

 8 Change in FB 1 (2009 µg kg -1 ) and ZON (22 µg kg -1 ) content during direct extrusion of maize flour, effect of dextrose

			Food Additives and Contaminants		Page 32 of 34
		mc, %		Dextrose, %
	flour	extrudate*	barrel T,ºC	0	5	10
		FB 1		loss, %
	17	6.20/4.57	160	51	80	88
	17	5.60/4.62	180	58	84	88
	15	5.80/4.80	160	53	87	93
	15	4.40/4.59	180	68	90	93
		% SD for the sample pairs		1.45
		ZON		loss, %
	17	6.20/4.57	160	40	23	18
	17	5.60/4.62	180	23	19	26
	15	5.80/4.80	160	34	20	18
	15	4.40/4.59	180	16	12	20
		% SD for the sample pairs		3.71
	* = 0/10 % dextrose

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Table 9 .

 9 Change in FB 1 , FB 2 and FB 3 (mean concentrations 2009, 409 and 285 µg kg -1 respectively) and ZON (22 µg kg -1 ) during pellet production and frying of maize flour and effect of NaCl.

			Food Additives and Contaminants
				loss of mycotoxin, %
		NaCl,	flour:	pellet:	flour			mc, %,
	T, ºC	%	pellet	fried	:fried	flour :fried	after
								frying,
				FB 1		FB 2	FB 3
	110	0	13 +	22	12	11	12	4.00
	120	0	13 +	60	46	46	50	2.41
	110	1	7 +	20	14	20	12	3.73
	120	1	8 +	33	28	34	24	2.53
	110	2	11 +	25	17	31	21	3.12
	120	2	0	21	21	31	25	2.89
		% SD		4.43	3.93
				ZON
	110	0	31	5	34
	120	0	8	13	18
	110	1	14	10	9
	120	1	5	+1	5
	110	2	12	+3	10
	120	2	14	4	18
		% SD	8.84	6.01

+ Apparent increase

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