Development of Rapid Cleanup Method Using New Cleanup Agents for Analysis of Pesticide Residues in Tea.

Cleanup using two types of agents (S-NH2 and S-Si) developed by the authors was investigated with the aim of removing interfering substances such as catechin and caffeine to enable analysis of pesticide residues in tea. S-NH2 and S-Si removed approximately 100% of catechin and caffeine in 6 species of tea. Recoveries of 61 pesticides in tea were tested at the level of 0.1 µg/g, and 44 pesticides showed recovery within the range from 70 to 120%, with RSD of less than 10%. With cleanup using S-NH2 and S-Si, pesticide residues in tea could be analyzed within two hours.

Evaluation of New Cleanup Agents for Analysis of Pesticide Residues in Tea.

We evaluated the effectiveness of new cleanup agents (S-NH2 and S-Si) compared with other previously reported cleanup agents (octadecylsilane, graphitized carbon, aminopropyl and silica gel) for removal of interfering substances such as catechin and caffeine prior to analysis of pesticide residues in tea. S-NH2 and S-Si were highly efficient in removal of catechin and caffeine, respectively. Recoveries of 80 pesticides using S-NH2 and S-Si were tested, and more than 70% of pesticides showed recovery greater than 70%. These results indicate that S-NH2 and S-Si agents will be useful for analysis of pesticide residues in tea.

Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface.

The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to ∼70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments.

Concentrations of organochlorine pesticides (OCPs) residues in foodstuffs collected from traditional markets in Indonesia.

A total 23 of organochlorine pesticides (OCPs) residues were determined in five groups of foodstuffs, i.e.: vegetables (carrot, potato, cucumber, corn, and onion), rice, pulses (green bean and soybean), nuts (peanut), and fish (milkfish), which collected from traditional markets in three big cities of Indonesia; Jakarta, Bogor, and Yogyakarta. OCPs were only detected in fatty foodstuffs, such as soybean, peanut, and milkfish. The concentration of HCB (expressed as ng g(-1) on a whole basis), ΣDrins, ΣDDTs, ΣHeptachlors, and ΣHCHs were in the range of <0.3-0.74 ng g(-1), <0.03-0.42 ng g(-1), <0.02-0.41 ng g(-1), <0.03-0.14 ng g(-1), and <0.03-0.06 ng g(-1), respectively, which were far below the maximum residue limits (MRLs) as established by FAO/WHO. These very low concentrations of OCPs residues in foodstuffs indicated that OCPs were used only in past time and no recent input into the environment. Furthermore, the estimated daily intake (EDI) of HCB, ΣDDTs, ΣDrins, ΣHeptachlors, and ΣHCHs in five group foodstuffs, which were 60% of total daily diet of Indonesian, were 0.09 ng kg(-1) bw d(-1), 0.04 ng kg(-1) bw d(-1), 0.01 ng kg(-1) bw d(-1), 0.003 ng kg(-1) bw d(-1), and 0.002 ng kg(-1) bw d(-1), respectively. These results were far below the acceptable daily intake (ADI) as established by FAO/WHO, which indicated that consumption of foodstuffs from Indonesia were at little risk to human health in term of OCPs at present.

Suppressive effect of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls transfer from feed to eggs of laying hens by activated carbon as feed additive.

In this study, we investigated the suppressive effect of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) transfer from the feed to the eggs of laying hens by using activated carbon as a feed additive. Four groups of six hens (White Leghorn egg-layers; age, 11weeks) were housed as two control groups and two exposure groups for a period of 20weeks. Two control groups were fed with either the basal feed "Control" or basal feed additing activated carbon "Control+C". Another two exposure groups were fed with feed contaminated (about 6ng TEQ kg(-1) feed) by standard solutions of PCDDs/PCDFs and DL-PCBs "Exposure" alone and contaminated feed adding activated carbon "Exposure+C". There was no significant effect on each groups for the growth rate, biochemical blood components, and egg production: these were around the standard levels for poultry in general. Moreover the results in this study showed the availability of activated carbon as a feed additive owing to the reduction in the risk of food pollution by PCDDs/PCDFs and DL-PCBs. The concentration in the eggs of the Exposure group gradually increased following the start of egg-laying but reached a steady state after about 1month. In contrast, the concentration for the Exposure+C group was stationary and below the maximum EU level (6pgTEQg(-1)fat). In comparison to the Exposure group, the Exposure+C group showed a significant decline in the percentage of bioaccumulation into the egg. This reduction due to activated carbon was also observed in the muscle and abdominal fat. The reductions were compound- and congener-dependent for DL-PCBs as follows: PCDDs/PCDFs, non-ortho-PCBs, and mono-ortho-PCBs were more than 90%, 80%, and 50%, respectively, irrespective of the type of tissues. Fat soluble vitamin concentrations in the eggs of the Exposure+C group showed lower trends than the Exposure group. The γ-tocopherol and α-tocopherol concentrations in eggs of Exposure+C group showed a significant reduction of about 40%. However, the addition of activated carbon into animal feed could obviate the remote potential for accidents causing unintentional food pollution with PCDDs/PCDFs and DL-PCBs.

Development of the automated cleanup system for the analysis of PCDDs, PCDFs and DL-PCBs.

A new automated cleanup system for the analysis of dioxins (PCDDs, PCDFs and DL-PCBs) has been developed. It was controlled by PLC through the touch-panel. This automated cleanup system can simultaneously treat six samples in 2h, using only about 30 mL of solvent. In this study, the recovery rates of the internal standard added as cleanup spiked were between 70% and 120% in the fly ash sample. The RSDs (relative standard deviations) were below 15%. The shortest analysis time from cleanup to calculation of concentration was approximately 6h. Moreover, this automated cleanup system eliminates personal error in sample preparation and training time for the analyst, and improves the accuracy of the experiment. Additionally, this automated cleanup system allowed rapid analysis and less consumption of organic solvent.

Physicochemical characteristics of carbonaceous adsorbent for dioxin-like polychlorinated biphenyl adsorption.

It has been known that dioxin-like polychlorinated biphenyls (DL-PCBs) are present in almost all types of environments worldwide. Activated carbon treatment has been expected for the removal of DL-PCBs because it is a simple and low-cost removal technology. In the present study, the physicochemical properties of activated carbon were investigated to identify the characteristics of 16 different types of activated carbon on adsorption properties for DL-PCBs. To accomplish this, micropore volume, and pore diameter were calculated by t-plot analysis and the mesopore volume was analyzed by the Barrett-Joyner-Halenda (BJH) method. In addition, the Brunauer-Emmett-Teller (BET) surface area, pH, metal elements, and surface acid functional groups were analyzed. Then, adsorption experiments using DL-PCB in hexane solution were conducted, and the relationship between adsorption and physicochemical properties of activated carbon was investigated. The results showed that activated carbons having a surface area of 700-1200 m(2)g(-1) and micropores with diameters of about 0.7-0.8 nm exhibited high activity for the adsorption of PCBs. The results also clearly showed that the mesopore volume of activated carbon influenced the adsorption rate and the equilibrium adsorption.

Dioxin-like polychlorinated biphenyl adsorbent obtained from enzymatic saccharification residue of lignocellulose.

In this study, the effective utilization of lignocellulose residue as an adsorbent was investigated. Japanese cypress wood flour subjected to hydrothermal pretreatment and ball-mill grinding was saccharified with an enzyme. The residual wood flour was carbonized and activated by physical and chemical activation to produce adsorbents for persistent organic pollutant removal. The adsorption properties were investigated by pore analysis using the N(2) adsorption/desorption isotherm and adsorption tests for dioxin-like polychlorinated biphenyls in a hexane solution. The obtained adsorbents showed high production yields and adsorption properties for dioxin-like polychlorinated biphenyls.

Removal of dioxins and dioxin-like PCBs from fish oil by countercurrent supercritical CO2 extraction and activated carbon treatment.

It has been known that fish oils are prone to contamination by polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs). In this study, the removal of contaminants from fish oil by countercurrent supercritical CO(2) extraction (CC-SCE) and activated carbon treatment was investigated. Fish oil was treated by CC-SCE at 70 degrees C and 30MPa and with a CO(2)/oil ratio of 72; this resulted in a 93% reduction in the sum of PCDDs, PCDFs and DL-PCBs concentration level by and 85% reduction in toxic equivalency (TEQ). CC-SCE uses 40% less CO(2) and yields 30% more refined oil than semi-batch-type processes. Subsequent treatment by activated carbon reduced the concentration level by 94% and TEQ by 93%. CC-SCE is effective for the removal of DL-PCBs, whereas activated carbon treatment is effective for the removal of PCDD/Fs. These results reveal that the combination of CC-SCE and activated carbon treatment is applicable to the removal of PCDD/Fs and DL-PCBs from fish oil.

Acceleration of catalytic activity of calcium oxide for biodiesel production.

This research was aimed at studying the acceleration of the catalytic activity of calcium oxide (CaO) for developing an effective heterogeneous catalyst for biodiesel production by the transesterification of plant oil with methanol. CaO was activated by pretreatment with methanol and was used for the transesterification reaction. The activation and transesterification reaction conditions were examined. The obtained optimal reaction conditions were 0.1-g CaO, 3.9-g methanol, 15-g rapeseed oil, and 1.5-h activation time at room temperature that provided methyl ester in approximately 90% yield within a reaction time of 3h at 60 degrees C. The activation mechanism was also investigated, and the proposed mechanism is as follows. By pretreatment with methanol, a small amount of CaO gets converted into Ca(OCH(3))(2) that acts as an initiating reagent for the transesterification reaction and produces glycerin as a by-product. Subsequently, a calcium-glycerin complex, formed due to the reaction of CaO with glycerin, functions as the main catalyst and accelerates the transesterification reaction.

Validation of high-throughput measurement system with microwave-assisted extraction, fully automated sample preparation device, and gas chromatography-electron capture detector for determination of polychlorinated biphenyls in whale blubber.

Validation of a high-throughput measurement system with microwave-assisted extraction (MAE), fully automated sample preparation device (SPD), and gas chromatography-electron capture detector (GC-ECD) for the determination of polychlorinated biphenyls (PCBs) in minke whale blubber was performed. PCB congeners accounting for > 95% of the total PCBs burden in blubber were efficiently extracted with a small volume (20 mL) of n-hexane using MAE due to simultaneous saponification and extraction. Further, the crude extract obtained by MAE was rapidly purified and automatically substituted to a small volume (1 mL) of toluene using SPD without using concentrators. Furthermore, the concentration of PCBs in the purified and concentrated solution was accurately determined by GC-ECD. Moreover, the result of accuracy test using a certified material (SRM 1588b; Cod liver oil) showed good agreement with the NIST certified concentration values. In addition, the method quantification limit of total-PCB in whale blubbers was 41 ng g(-1). This new measurement system for PCBs takes only four hours. Consequently, it indicated this method is the most suitable for the monitoring and screening of PCBs in the conservation of the marine ecosystem and safe distribution of foods.

Development of supercritical carbon dioxide extraction with a solid phase trap for dioxins in soils and sediments.

A method involving supercritical fluid extraction (SFE) with a solid phase trap containing activated alumina was investigated for the rapid analysis of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin like polychlorinated biphenyls (DL-PCBs) in soils and sediments. The samples were extracted by using supercritical carbon dioxide with water (2% versus CO(2) flow velocity) being used as an entrainer at a pressure of 30 MPa and a temperature of 130 degrees C for 50 min. The extracts were adsorbed on an activated alumina trap that was maintained at a temperature of 150 degrees C, and then, PCDD/DFs and DL-PCBs were eluted with 20 ml of hexane at 60 degrees C. After concentration, they were measured with a high-resolution gas chromatograph interfaced to a high-resolution mass spectrometric detector. The average concentrations of PCDD/DFs and DL-PCBs corresponded to the results obtained by the conventional method, and the reproducibility of this SFE method was below 21% of the relative standard deviations for all samples. The total time required for the analysis of the pretreatment of this method was only 2 h.

Development of heterogeneous base catalysts for biodiesel production.

Investigations were conducted on heterogeneous base catalysts for the transesterification of oil aimed at effective production of biodiesel. Thirteen different kinds of metal oxides containing calcium, barium, magnesium, or lanthanum were prepared as catalysts. Their catalytic activities were tested for transesterification at 60 degrees C with a 6:1 molar ratio of methanol to oil and a reaction time of 10h. The calcium-containing catalysts - CaTiO3, CaMnO3, Ca2Fe2O5, CaZrO3, and CaO-CeO2 - showed high activities and approximately 90% yields of methyl ester. Furthermore, catalytic durability tests were performed by repeating the transesterification reaction several times with the calcium-containing catalysts recovered from the previous reaction mixture. It was found that CaZrO3 and CaO-CeO2 show high durability and have the potential to be used in biodiesel production processes as heterogeneous base catalysts.

Experimental study on the removal of dioxins and coplanar polychlorinated biphenyls (PCBS) from fish oil.

Recently, it has been found that fish oils contain a high proportion of contaminants, namely, polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (cPCBs). In this study, the removal of contaminants from fish oil by supercritical CO2 extraction (SCE) and by using adsorbents (0.13 wt % of oil) was investigated. Dioxins and cPCBs were extracted from fish oil by SCE at a temperature of 60 degrees C and a pressure of 28 MPa, and the removal efficiencies for PCDDs and PCDFs were in the range of 15-90% and those for cPCBs were in the range of 70-90%. However, 40% of the oil was extracted simultaneously with contaminants. On the adsorbent treatment, activated carbon showed high efficiency, and the removal efficiencies were >90% for PCDDs and PCDFs, but below 30% for cPCBs. A combination of both of these methods is more effective, and almost 100% of the total toxicity equivalence quantity value could be reduced.

Influence of the characteristics of soil and fly ash on the supercritical carbon dioxide extraction of dioxins.

Several investigations on the extraction of dioxins from soil and fly ash with supercritical fluid have been reported; however, few of them describe the influence of components on the extraction. We extracted dioxins from eight samples with different values of organic carbon content and surface area with supercritical CO(2) at a temperature of 463 K, a pressure of 40 MPa, and using 10% toluene as an entrainer. We researched the influence of the characteristics of soil and fly ash on supercritical CO(2) extraction of dioxins. The results revealed that the extraction efficiencies of PCDD/DFs and PCBs were high for all soil samples, while that of fly ash samples decreased with the increase in organic carbon content and surface area. The extraction efficiencies of dioxins from four standard samples, activated carbon, humic acid, alumina, and florisil, were also examined. The results revealed that the extraction efficiencies were strongly influenced by activated carbon like components present in the samples.

Relationships between the abnormalities of the male reproductive organs and the accumulation of phenol compounds in the striped field mouse, Apodemus agrarius, inhabiting Korea.

The relationships between the abnormalities of the male reproductive organs in striped field mice and the accumulation of endocrine-disrupting chemicals were assessed. Most mice collected at three areas were contaminated with phenol or organotin compounds. Fourteen to 42% of the mice at each area had abnormally shrunken reproductive organs, and some of them were contaminated with high levels of phenol compounds. Moreover, all the shrunken reproductive organs were damaged in the histological structure. The damage was observed also from several mice accumulating a high level of phenol compounds even though they had normally developed reproductive organs, although no damage was found in the mice accumulating a high level of organotin compounds. Collectively, the abnormalities of the reproductive organs in the mice seemed to be related to the accumulation of phenol compounds rather than of organotin compounds.

Bisphenol a accelerates terminal differentiation of 3T3-L1 cells into adipocytes through the phosphatidylinositol 3-kinase pathway.

In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein lipase (LPL) and adipocyte-specific fatty acid binding protein (aP2) mRNAs. These findings indicate that BPA was able to accelerate terminal differentiation of 3T3-L1 cells into adipocytes. LY294002, a chemical inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), blocked completely the increasing effect of BPA on TG accumulation and expression of LPL and aP2 mRNAs. Western blot analysis revealed that BPA increased the level of phosphorylated Akt kinase. Based on these findings, we concluded that BPA acted through the PI 3-kinase and Akt kinase pathway, resulting in increased TG accumulation and expression of adipocyte genes. The structure-activity relationship for BPA-related chemicals was examined. Eight derivatives of BPA (three diphenylalkanes with different substituents at the central carbon atom, three diphenylalkanes with ester bonds on hydroxyl groups in the phenolic rings, one bisphenol consisting of a sulphur atom at the central position, one chemical with cyanic groups, instead of hydroxyl groups, in the phenolic rings) accelerated terminal adipocyte differentiation and their potencies to increase TG accumulation were 73-97% of that of BPA. Two diphenylalkanes with ether bonds on hydroxyl groups and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) did not have the ability to accelerate terminal adipocyte differentiation.

Effect of 4-nonylphenol on cell proliferation and adipocyte formation in cultures of fully differentiated 3T3-L1 cells.

The effect of 4-nonylphenol (NP) on cell proliferation and adipocyte formation was examined in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for 8 days with or without NP. NP at 5 and 10 microg/ml increased the DNA content by 32% and 68%, respectively, compared with that of the untreated cultures, in which NP was absent during the treatment period. There were many more bromodeoxyuridine (BrdU)-positive cells in the NP-treated cultures, in which NP was present at a concentration of 10 microg/ml during the treatment period, compared to the untreated cultures. These results indicate that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. NP at 5 and 10 microg/ml decreased the triacylglycerol (TG) content by 26% and 58%, respectively, and decreased the lipoprotein lipase (LPL) activity by 51% and 71%, respectively. The lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures. The mRNA levels of LPL and adipocyte-specific fatty acid binding protein (aP2) were considerably lower in the NP-treated cultures than in the untreated cultures. Thus, NP also had the ability to inhibit adipocyte formation in cultures of fully differentiated 3T3-L1 cells. A study using an antiestrogen ICI 182,780 showed that the NP-stimulated cell proliferation was mediated partly by the estrogen receptor, while the NP-induced inhibition of adipocyte formation was mediated by a mechanism other than the estrogen receptor.

Bisphenol A in combination with insulin can accelerate the conversion of 3T3-L1 fibroblasts to adipocytes.

The confluent cultures of 3T3-L1 fibroblasts were treated with or without bisphenol A (BPA) for 2 days and subsequently treated with insulin (INS) alone for 9 days. When BPA was absent during the first 2-day treatment period, the cultures contained 1.6 microg/microg DNA of triacylglycerol (TG), 202 mU/mg DNA of lipoprotein lipase (LPL) activity, and 462 nmol/min/mg DNA of glycerol-3-phosphate dehydrogenase (GPDH) activity. The presence of BPA during the same period caused a 150% increase in the TG content, a 60% increase in the LPL activity, and a 500% increase in the GPDH activity. Thus, BPA by itself can trigger 3T3-L1 fibroblasts to differentiate into adipocytes. Next, the confluent cultures were treated with BPA for 2 days and subsequently treated with a combination of INS and BPA for 9 days. The simultaneous presence of BPA with INS caused a 370% increase in the TG content, a 200% increase in the LPL activity, and a 225% increase in the GPDH activity compared with the cultures treated with INS alone. The amount of [(3)H]thymidine incorporated into DNA was lower in the cultures treated with INS in the presence of BPA than in those treated with INS alone, indicating that BPA has an anti-proliferative activity on 3T3-L1 cells. Taken together, our results indicate that BPA in combination with INS can accelerate the adipocyte conversion.