Approximate Bayesian computation analysis of EST-associated microsatellites indicates that the broadleaved evergreen tree Castanopsis sieboldii survived the Last Glacial Maximum in multiple refugia in Japan.

Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.

Investigation of SO3 absorption line for in situ gas detection inside combustion plants using a 4-µm-band laser source.

We have investigated 4-µm-band SO3 absorption lines for in situSO3 detection using a mid-infrared laser source based on difference frequency generation in a quasi-phase-matched LiNbO3 waveguide. In the wavelength range of 4.09400-4.10600 µm, there were strong SO3 absorption lines. The maximum absorption coefficient at a concentration of 170 ppmv was estimated to be about 3.2×10-5 cm-1 at a gas temperature of 190°C. In coexistence with H2O, the reduction of the SO3 absorption peak height was observed, which was caused by sulfuric acid formation. We discuss a method of using an SO3 equilibrium curve to derive the total SO3 molecule concentration.

The population demography of Betula maximowicziana, a cool-temperate tree species in Japan, in relation to the last glacial period: its admixture-like genetic structure is the result of simple population splitting not admixing.

Conservation of the local genetic variation and evolutionary integrity of economically and ecologically important trees is a key aspect of studies involving forest genetics, and a population demographic history of the target species provides valuable information for this purpose. Here, the genetic structure of 48 populations of Betula maximowicziana was assessed using 12 expressed sequence tag-simple sequence repeat (EST-SSR) markers. Genetic diversity was lower in northern populations than southern ones and structure analysis revealed three groups: northern and southern clusters and an admixed group. Eleven more genomic-SSR loci were added and the demographic history of these three groups was inferred by approximate Bayesian computation (ABC). The ABC revealed that a simple split scenario was much more likely than isolation with admixture, suggesting that the admixture-like structure detected in this species was due to ancestral polymorphisms. The ABC analysis suggested that the population growth and divergence of the three groups occurred 96 800 (95% CI, 20 500-599 000) and 28 300 (95% CI, 8700-98 400) years ago, respectively. We need to be aware of several sources of uncertainty in the inference such as assumptions about the generation time, overlapping of generations, confidence intervals of the estimated parameters and the assumed model in the ABC. However, the results of the ABC together with the model-based maps of reconstructed past species distribution and palaeoecological data suggested that the modern genetic structure of B. maximowicziana originated prior to the last glacial maximum (LGM) and that some populations survived in the northern range even during the LGM.

Total serum IgE level influences oral food challenge tests for IgE-mediated food allergies.

Probability curves predicting oral food challenge test (OFC) results based on specific IgE levels are widely used to prevent serious allergic reactions. Although several confounding factors are known to affect probability curves, the main factors that affect OFC outcomes are currently unclear. We hypothesized that an increased total IgE level would reduce allergic reactivity. Medical records of 337 and 266 patients who underwent OFCs for 3.5 g boiled hen's egg white and 3.1 ml raw cow's milk, respectively, were examined retrospectively. We subdivided the patients into three groups based on total IgE levels and age by percentile (<25th, 25-75th, and >75th percentiles), and logistic regression analyses were performed on each group. Patients with higher total IgE levels were significantly less responsive. In addition, age did not significantly affect the OFC results. Therefore, total IgE levels should be taken into account when predicting OFC results based on food-specific IgE levels.

Genome scanning for detecting adaptive genes along environmental gradients in the Japanese conifer, Cryptomeria japonica.

Local adaptation is important in evolutionary processes and speciation. We used multiple tests to identify several candidate genes that may be involved in local adaptation from 1026 loci in 14 natural populations of Cryptomeria japonica, the most economically important forestry tree in Japan. We also studied the relationships between genotypes and environmental variables to obtain information on the selective pressures acting on individual populations. Outlier loci were mapped onto a linkage map, and the positions of loci associated with specific environmental variables are considered. The outlier loci were not randomly distributed on the linkage map; linkage group 11 was identified as a genomic island of divergence. Three loci in this region were also associated with environmental variables such as mean annual temperature, daily maximum temperature, maximum snow depth, and so on. Outlier loci identified with high significance levels will be essential for conservation purposes and for future work on molecular breeding.

Impact of negative frequency-dependent selection on mating pattern and genetic structure: a comparative analysis of the S-locus and nuclear SSR loci in Prunus lannesiana var. speciosa.

Mating processes of local demes and spatial genetic structure of island populations at the self-incompatibility (S-) locus under negative frequency-dependent selection (NFDS) were evaluated in Prunus lannesiana var. speciosa in comparison with nuclear simple sequence repeat (SSR) loci that seemed to be evolutionarily neutral. Our observations of local mating patterns indicated that male-female pair fecundity was influenced by not only self-incompatibility, but also various factors, such as kinship, pollen production and flowering synchrony. In spite of the mating bias caused by these factors, the NFDS effect on changes in allele frequencies from potential mates to mating pollen was detected at the S-locus but not at the SSR loci, although the changes from adult to juvenile cohorts were not apparent at any loci. Genetic differentiation and isolation-by-distance over various spatial scales were smaller at the S-locus than at the SSR loci, as expected under the NFDS. Allele-sharing distributions among the populations also had a unimodal pattern at the S-locus, indicating the NFDS effect except for alleles unique to individual populations probably due to isolation among islands, although this pattern was not exhibited by the SSR loci. Our results suggest that the NFDS at the S-locus has an impact on both the mating patterns and the genetic structure in the P. lannesiana populations studied.

Range shifts of Potentilla fruticosa on the Qinghai-Tibetan Plateau during glacial and interglacial periods revealed by chloroplast DNA sequence variation.

The Qinghai-Tibetan Plateau is one of the most extensive habitats for alpine plants in the world. Therefore, the patterns of genetic variation in populations on the Plateau can reveal the detailed demographic history of alpine plants. We analysed the geographical structure of chloroplast matK sequence variation in Potentilla fruticosa L. (Rosaceae), a shrub currently found across the entire Plateau. We obtained sequence data from 508 individuals from 23 populations at sites ranging from the high-altitude interior to the relatively low-altitude northeastern Plateau. In the interior region, genetic diversity was high and included ancestral haplotypes. In contrast, northeastern populations were characterized by relatively low genetic diversity and recently derived haplotypes. The estimated expansion time in the interior population was 17 times that in the northeastern population. These data suggest that P. fruticosa expanded its range on the Plateau during periods of climatic cooling and contracted to the interior region during warmer periods. Thus, the interior region acted as a refugium and greatly contributed to the diversification of P. fruticosa.

Effects of kin-structured seed dispersal on the genetic structure of the clonal dioecious shrub ilex leucoclada.

Nonrandom patterns of gene dispersal have been identified as possible causes of genetic structuring within populations. Attempts to model these patterns have generally focused solely on the effects of isolation by distance, but the processes involved are more complex than such modeling suggests. Here, we extend considerations of gene dispersal processes beyond simple isolation by distance effects by directly evaluating the effects of kin-structured gene dispersal mediated by the group dispersal of related seeds within fruits (i.e., kin-structured seed dispersal) by birds on genetic structure in Ilex leucoclada, a clonal dioecious shrub. To examine the genetic structure patterns, we established two 30x30 m plots (one with immature soils in old-growth forest and one in secondary forest, designated IM and SC, respectively) with different I. leucoclada stem densities. In these two plots 145 and 510 stems were found, representing 78 and 85 genets, respectively, identified by analyzing their genotypes at eight microsatellite loci. The clonal structure was stronger in the SC plot than in the IM plot. Correlograms of coancestry for genets in both plots exhibited significant, positive, high values in the shortest distance class, indicating the presence of strong genetic structure. However, Sp statistics revealed that the pattern of the genetic structure differed between the plots. In addition, to estimate the family structure within fruits, we sampled forty fruits, in total, from 15 randomly selected plants in the area around the IM and SC plots, and found that 80% of the fruits were multiseeded and 42-100% of the multiseeded fruits contained at least one pair of full sibs. Simulations based on these estimates demonstrated that the group dispersal of related seeds produced through correlated mating both within and across fruits, but not unstructured half-sib dispersal, could generate the observed magnitude and trends of genetic structure found in the IM plot. Furthermore, in addition to kin-structured seed dispersal, isolation by distance processes is also likely to promote genetic substructuring in the SC plot. After discussing possible ecological factors that may have contributed to the observed genetic structure, we contrast our results with those predicted by general isolation by distance models, and propose that kin-structured seed dispersal should promote some evolutionary phenomena, and thus should be incorporated, where appropriate, in models of gene dispersal in natural plant populations.

Genetic diversity and the genetic structure of natural populations of Chamaecyparis obtusa: implications for management and conservation.

We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H(e)) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G(ST)=0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F(ST)) and H(e) values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.

Half-sib family structure of Fagus crenata saplings in an old-growth beech-dwarf bamboo forest.

Half-sib family structure of Fagus crenata saplings was examined in an old-growth beech-dwarf bamboo (Sasa spp.) forest using microsatellite genotypes in parentage analysis to identify the half-sib families in two 50 x 50 m plots: one with 36 adults, 641 saplings and no Sasa cover, the other with 21 adults, 61 saplings and Sasa cover. For large proportions of the saplings (44.6% and 75.4%, respectively) both of their parents were found within the same plot, indicating that pollination events frequently involved short-range pollen dispersal, probably because of the high density of adults in the study population. Although almost all of the adults had half-sib families, the number of offspring in the families was highly variable. In the plot with no Sasa cover, the variation in the number of offspring was significantly explained by the size of parents, i.e. the reproductive success is higher for large adults than for small adults. The half-sibs were aggregately distributed around their parents and the distribution overlapped among different half-sib families, which may be due to the limited seed dispersal and overlapping seed shadows of this species. As expected, there was weak genetic structure in the plot. By contrast, in the plot with Sasa cover, the half-sibs were distributed sparsely with a low density, and the degree of genetic structure was very weak. The difference in the half-sib family structure and genetic structure among saplings presumably reflects the difference in density that should be affected by regeneration dynamics associated with environmental conditions.

Spatial genetic structure among and within populations of Primula sieboldii growing beside separate streams.

We investigated the hierarchical genetic structure of SSR (simple sequence repeats) and cpDNA (chloroplast DNA) polymorphisms among and within populations of Primula sieboldii, a heterostylous clonal herb. Seven out of eight populations at the study site, located in a mountainous region of Nagano Prefecture, had each developed alongside a different stream, and the other occurred on a flat area 70 m from the nearest stream. The magnitude of genetic differentiation among streamside populations in maternally inherited cpDNA (Phi = 0.341) was much higher than that in biparentally inherited SSRs (Phi = 0.011). This result suggests that seed dispersal among streams was restricted, and pollen was the primary agent of gene flow among streamside populations. In contrast, genetic differentiation among subpopulations within streams were low at both markers (Phi = 0.053 for cpDNA, Phi = 0.025 for SSR). This low differentiation among subpopulations in cpDNA compared with that among streamside populations suggest that seed dispersal occur along the stream probably during flooding. This hypothesis was supported by the fact that in cpDNA haplotypes, no clear genetic structure was detected within the streamside population, while a significant genetic structure was found within 20 m in the nonstreamside population. Furthermore, within the streamside populations, two pairs of ramets with identical multilocus genotypes for eight SSR loci were distantly (> 50 m) distributed along the same streamside, suggesting dispersal of clonal propagule. Our study showed that the heterogeneity of the landscape can influence gene flow and hence spatial genetic structure.

Evaluation of cleaved amplified polymorphic sequence markers for Chamaecyparis obtusa based on expressed sequence tag information from Cryptomeria japonica.

We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (approximately 30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (P<0.01) deviation from the expected segregation ratio. In total, 136 polymorphic markers were developed using C. japonica-based STS primers without any sequence modification. In addition, the applicability of STS-based markers developed in one species to other species was found to closely reflect the evolutionary distance between the species, which is roughly concordant with the difference between their rbcL sequences. We plan to use these markers for genetic studies in C. obtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.

Heterogeneous genetic structure in a Fagus crenata population in an old-growth beech forest revealed by microsatellite markers.

The within-population genetic structure of Fagus crenata in a 4-ha plot (200 x 200 m) of an old-growth beech forest was analysed using microsatellite markers. To assess the genetic structure, Moran's I spatial autocorrelation coefficient was calculated. Correlograms of Moran's I showed significant positive values less than 0.100 for short-distance classes, indicating weak genetic structure. The genetic structure within the population is created by limited seed dispersal, and is probably weakened by overlapping seed shadow, secondary seed dispersal, extensive pollen flow and the thinning process. Genetic structure was detected in a western subplot of 50 x 200 m with immature soils and almost no dwarf bamboos (Sasa spp.), where small and intermediate-sized individuals were distributed in aggregations with high density because of successful regeneration. By contrast, genetic structure was not found in an eastern subplot of the same size with mature soils and Sasa cover, where successful regeneration was prevented, and the density of the small and intermediate-sized individuals was low. Moreover, genetic structure of individuals in a small-size class (diameter at breast height < 12 cm) was more obvious than in a large-size class (diameter at breast height >/= 12 cm). The apparent genetic structure detected in the 4-ha plot was therefore probably the result of the structure in the western portion of the plot and in small and intermediate-sized individuals that successfully regenerated under the favourable environment. The heterogeneity in genetic structure presumably reflects variation in the density that should be affected by differences in regeneration dynamics associated with heterogeneity in environmental conditions.

Comparison of the fine-scale genetic structure of three dipterocarp species.

We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.

Genetic diversity of nuclear and mitochondrial genomes in Pinus parviflora Sieb. & Zucc. (Pinaceae) populations.

Genetic diversities of the nuclear and mitochondrial genomes in Pinus parviflora were studied in 16 populations, which were distributed across most of the species' range in Japan. Six mitochondrial DNA haplotypes were identified among the 16 populations. The intrapopulation diversity of allozymes was similar to that of other endemic woody species (H(S)=0.259). Although P. parviflora is distributed in discrete populations, differentiation between these was very low (G(ST)=0.044). In addition, the extent of genetic differentiation between two varieties (var. pentaphylla and var. parviflora) was extremely low (G(VT)=0.001). Intrapopulation diversity of mitochondrial DNA was also very low (H(S)=0.098), but population differentiation was high (G(ST)=0.863). Moreover, the distribution of haplotypes reflected the taxonomic differences between P. parviflora var. pentaphylla and var. parviflora. The populations of var. pentaphylla and var. parviflora contained different haplotypes. Differing modes of inheritance may account for the differences in nuclear and mitochondrial genetic diversity.

Estimated daily intake of plasticizers in 1-week duplicate diet samples following regulation of DEHP-containing PVC gloves in Japan.

Duplicate hospital diet samples obtained over 1 week in 2001 were analysed to estimate the daily intake of plasticizers and the results were compared with those obtained in 1999. The plasticizers quantified in this study were: dibutyl phthalate, butylbenzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), di(2-ethylhexyl) adipate (DEHA), diisononyl adipate (DINA) and O-acetyl tributyl citrate (ATBC). Dipropyl, dipentyl, dihexyl and dicyclohexyl phthalate were also analysed but not detected. The analytical procedure for this follow-up study was essentially the same as in the previous one. Detection limits were 0.1-15.6 ng g(-1) for each plasticizer. One-week duplicate diet samples provided by three hospitals in three remote prefectures of Japan were analysed as individual meals. DEHP was detected at 6-675 ng g(-1) in 62 of 63 meals, significantly lower levels compared with those detected in 1999. Levels of DEHA and DINP also decreased. The mean intake of plasticizers estimated from all samples was 160 microg DEHP day(-1), 12.5 microg DEHA day(-1), 4.7 microg DINP day(-1) and 3.4 microg BBP day(-1). Levels of DINA were relatively high in meals from one hospital: in those meals, the average daily intake was 1338 microg day(-1). Those of ATBC were also higher in meals from another hospital: the average daily intake was 1228 microg day(-1). The sources of DINA and ATBC can be cling-film or sausage packaging.

Development and characterization of microsatellite markers for Cryptomeria japonica D.Don.

Thirty four microsatellite markers for Cryptomeria japonica D. Don were developed by searching three types of library: a database of C. japonica cDNA sequences, a standard non-enriched genomic DNA library and a microsatellite-enriched library using magnetic particles. The enrichment of microsatellite sequences using magnetic particles is very efficient compared to the other two methods both in terms of the numbers of markers generated, and in the polymorphism they detect. The microsatellites developed from the genomic DNA library generally have longer repeat sequences and are more polymorphic than those from cDNA. All the developed microsatellite markers in this study showed polymorphism among 28 plus trees selected from locations scattered throughout Japan. The mean number of alleles per locus (MNA) detected in the 28 plus trees ranged from 2 to 21 with an average of 7.5. The Polymorphism Information Content (PIC) ranged from 0.160 to 0.936 with an average of 0.666. Co-dominant segregation of alleles in a three-generation pedigree of C. japonica was demonstrated at 34 microsatellite loci, and the segregation was not distorted from Mendelian expectation for all loci. In 12 out of 34 loci, a null allele was detected. Key relationships between polymorphic parameters, such as MNA and PIC, and the characteristics of microsatellite sequences, such as the longest repeat number, total repeat number and total number of nucleotides, were investigated using rank correlation coefficients, Kendall's tau. A positive correlation was found between repeat lengths and polymorphisms. The markers provide sufficient resolution for investigating gene flow within forests and seed orchards, and for genome mapping.

Determination of clethodim and its oxidation metabolites in crops by liquid chromatography with confirmation by LC/MS.

A method was developed for determination of the herbicide clethodim (C0) and its oxidation metabolites clethodim sulfoxide (C1) and clethodim sulfone (C2) in agricultural products. Upon extraction, both C0 and C1 were oxidized to C2 by m-chloroperoxybenzoic acid, and C2 was determined by liquid chromatography (LC). The C2 peak was confirmed by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI). Recoveries of C0 from radish, tomato, onion, sweet potato, kidney bean, carrot, cabbage, and lettuce ranged from 91 to 118% following fortification at 0.05-1.0 ppm. The detection limit of C2 in crops was 0.01 ppm (S/N > 3). The fortified samples of onion, sweet potato, kidney bean, and carrot were confirmed by LC/MS (ESI), and the peak of C2 was detected.

[Contents of eleven phthalates and di(2-ethylhexyl) adipate in retail packed lunches after prohibition of DEHP-containing PVC gloves for cooking purposes].

Ten samples of retail packed lunches purchased from convenience stores were determined for 11 phthalates and di(2-ethylhexyl) adipate (DEHA) in August 2000, 2 months after the prohibition of DEHP-containing PVC gloves in Japan. Each homogenized sample was extracted with acetonitrile, partitioned with n-hexane, and cleaned up using Florisil and PSA columns. Phthalates in the extract were determined by GC/MS (SIM). The limits of detection were 14.9 ng/g for di(2-ethylhexyl) phthalate (DEHP) and 18.6 ng/g for dibutyl phthalate (DBP). Levels of phthalates in packed lunch samples were 45 to 517 ng DEHP/g (198 ng/g, average), ND to 90 ng DEHA/g, and ND to 10.0 ng BBP/g. Diisononyl phthalate (DINP) was detected in one sample at 76 ng/g. Average DEHP level in ten samples was 4% of that in 1999. The contents of other phthalates were also reduced. DBP was not detected in any sample. Recovery of deuterated isomers added as surrogates was 27.9% for DNP-d4, and 40.6 to 101.5% for the other phthalates.

Di(2-ethylhexyl) phthalate contamination of retail packed lunches caused by PVC gloves used in the preparation of foods.

Plasticizer contamination of foods sold in retail packed lunches and set lunches in restaurants was determined by GC/MS. The phthalate esters were as follows: diethyl, dipropyl, dibutyl, dipentyl, dihexyl, butylbenzyl, dicyclohexyl, di(2-ethylhexyl), dioctyl, diisooctyl (mixture of isomers) and diisononyl (mixture). Di(2-ethylhexyl) adipate was also determined. Sixteen packed lunches and ten set lunches were analysed, and in all samples the concentration of di(2-ethylhexyl) phthalate (DEHP) was the highest, at 0.80-11.8 mg/kg in packed lunches and 0.012-0.30 mg/kg in set lunches. The DEHP content of five packed lunches exceeded 1.85 mg, which is the EU tolerable daily intake (TDI) for a person of 50 kg body weight. Foodstuffs that were components of the packed lunches were taken from the factory at each step of preparation and phthalates were determined. For example, chicken contained 0.08 mg/kg DEHP when uncooked, 13.1 mg/kg after frying and 16.9 mg/kg after packing. Disposable PVC gloves used in the preparation of foods were apparently the source of high DEHP concentrations. The gloves used during cooking or packaging were sprayed with 68% (w/w) ethanol to sterilize them. PVC gloves from the factory contained 22 or 41% by weight of DEHP. To confirm the link with the contamination problem, samples of boiled rice, croquette and boiled dry radish were handled in the laboratory with PVC gloves containing 30% (w/w) DEHP. DEHP migration levels of 0.05 mg/kg in rice or 0.33 mg/kg in croquette, and 11.1 mg/kg in radish were found. The alcohol sprayed onto the gloves increased the migration of DEHP to 2.03 mg/kg in rice, 2.45 mg, kg in croquette, and 18.4 mg/kg in radish.