The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF-I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF-I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.     

The effect of bee pollen on secretion activity, markers of proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The general objective of this in vitro study was to examine the effect of bee pollen on the release of insulin-like growth factor I (IGF-I) and steroid hormone progesterone, and expression of markers of proliferation (PCNA) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA method and expression of PCNA and caspase-3 by immunocytochemistry. Bee pollen addition at the dose of 10 ng/mL significantly (P<0.05) inhibited IGF-I release by porcine ovarian granulosa cells. This growth factor was not influenced by 100 and 1000 ng/mL doses of bee pollen. Progesterone release by cells was not influenced by bee pollen addition at the doses of 10, 100 and 1000 ng/mL as used in our study. Similarly expression of PCNA and caspase-3 was not affected by bee pollen addition. The present study shows dose-dependent regulation of IGF-I by experimental bee pollen addition in vitro. Progesterone release, expression of PCNA and caspase-3 in porcine ovarian granulosa cells was not induced by pollen. Our results contribute to new insights regarding the possible effect of bee pollen on IGF-I release, which is important for regulation of porcine ovarian functions.     

The influence of deoxynivalenol and zearalenone on steroid hormone production by porcine ovarian granulosa cells in vitro

Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) are frequently occurring in feed of pigs together. The aim of this study was to evaluate the possible in vitro effects of DON and ZEA, alone or their combination on steroid secretion of porcine ovarian granulosa cells (GCs). A species-specific model with porcine ovarian GCs was used to study the potential endocrine disrupting effects of DON and ZEA alone and in co-exposure. Progesterone (P4) and estradiol (E2) were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The results of this study demonstrate that DON alone at the higher concentrations may act to stimulate P4 (at 1,000, 2,000, 3,000 and 5,000 ng mL^?1 but not 10 and 100 ng mL^?1) and E2 (at 2,000, 3,000 and 5,000 ng mL^?1 but not 10, 100 and 1000 ng mL^?1) secretion. The effects of ZEA on P4 and E2 secretion were not confirmed. DON in combination with the other fusariotoxin ZEA may impair steroidogenesis. Results aslo demonstrate different toxicological effects of fusariotoxins on follicle stimulating hormone-induced secretion of P4 and E2. All these results taken together suggest that fusariotoxin and their interactions can impact ovarian steroidogenesis, thereby demonstrating their potential reproductive effects in pigs.     

Assessment of a potential preventive ability of amygdalin in mycotoxin-induced ovarian toxicity

The possible effects of a natural substance amygdalin and its combination with the mycotoxin deoxynivalenol (DON) on the steroid hormone secretion (progesterone and 17-β-estradiol) by porcine ovarian granulosa cells (GCs) were examined in this in vitro study. Ovarian GCs were incubated without (control group) and with amygdalin (1, 10, 100, 1,000 and 10,000 μg mL¹), or its combination with DON (1 μg mL¹) for 24 h. The release of steroid hormones was determined by ELISA. The progesterone secretion by porcine ovarian GCs was not affected by amygdalin in comparison to the control. However, the highest amygdalin dose (10,000 μg mL¹) caused a significant stimulation of the 17-β-estradiol release. A combination of amygdalin with DON significantly (P < 0.05) increased the progesterone release at all concentrations. Similarly, a stimulatory effect of amygdalin co-administered with DON was detected with respect to the 17-β-estradiol secretion at the highest dose (10,000 μg mL¹) of amygdalin and 1 μg mL¹ of DON. Noticeable differences between the effects of amygdalin alone and its combination with DON on the progesterone release were detected. In contrast, no differences between the stimulatory effects of amygdalin and its combination with DON on the 17-β-estradiol synthesis by porcine GCs were observed. Findings from this in vitro study did not confirm the expected protective effect of amygdalin on mycotoxin induced reprotoxicity. Our results indicate that the stimulatory effect of amygdalin combined with DON on the progesterone release was clearly caused by the DON addition, not by the presence amygdalin per se. On the other hand, the stimulation of 17-β-estradiol production was solely caused by the presence of amygdalin addition. These findings suggest a possible involvement of both natural substances into the processes of steroidogenesis and appear to be endocrine modulators of porcine ovaries.     

Dietary bioflavonoid quercetin modulates porcine ovarian granulosa cell functions in vitro

Quercetin is a dietary bioflavonoid used widely as a food supplement and is generally recognized as safe. The aim of this in vitro study was to examine the steroid hormone (progesterone and 17- β estradiol) release, proliferation (PCNA and cyclin B1) and apoptosis (caspase 3 and p53) of porcine ovarian granulosa cells after the addition of quercetin at concentrations 0.01, 0.1, 1, 10 and 100?μmol?L^?1. Progesterone release was stimulated at the concentration 10?μmol?L^?1. Quercetin neither had any impact on 17-β estradiol secretion nor on the presence of PCNA. However, a significant enhancement of the occurrence of cyclin B1 was noted except for the lowest concentration 0.01?μmol?L^?1. Quercetin did not have any influence on the number of granulosa cells containing caspase 3, but at the concentration 10?μmol?L^?1 it inhibited p53 occurrence. Results confirm the safety of quercetin in porcine ovarian granulosa cell model and further suggest its possible concentration-dependent influence on ovarian functions through pathway that may involve progesterone, cyclin B1 and p53.     

Cobalt-induced hormonal and intracellular alterations in rat ovarian fragments in vitro

The objective of this in vitro study was to examine dose-dependent changes in the secretion activity (progesterone, 17β-estradiol and insulin-like growth factor-I) of rat ovarian fragments after experimental cobalt (Co) administration including the apoptotic potential of Co on rat ovarian fragments by evaluating the expression of apoptotic markers Bax and caspase-3. Ovarian fragments were incubated with cobalt sulphate (CoSO₄.7H₂O) at the doses 90, 170, 330 and 500 μg.mL^?1 for 24 h and compared with control group without Co addition. Release of progesterone (P₄) 17β-estradiol and insulin-like growth factor-I (IGF-I) by ovarian fragments was assessed by RIA, expression of Bax and caspase-3 by SDS-PAGE and Western blotting. Observations show that P₄ release by ovarian fragments was significantly (P < 0.05) inhibited after cobalt sulphate addition at higher doses 170–500 μg.mL^?1 used in the study in comparison to control. However, cobalt sulphate addition did not cause any significant change in the release of 17β-estradiol by ovarian fragments at all the doses used in the study (90–500 μg.mL^?1) in comparison to control. On the contrary, IGF-I release by ovarian fragments was significantly (P < 0.05) stimulated after cobalt sulphate addition at the lowest dose 90 μg.mL^?1 in comparison to control, while other doses did not cause any significant change. Also, addition of cobalt sulphate decreased the expression of both the apoptotic peptides Bax and caspase-3 at the higher doses 170, 330 and 500 μg.mL^?1, but not at the lowest dose 90 μg.mL^?1 used in the study. Obtained results suggest Co induced (1) inhibition in secretion of steroid hormone progesterone, (2) dose-dependent increase in the release of growth factor IGF-I, and (3) decrease in the expression of markers of apoptosis (Bax and caspase-3) of rat ovarian fragments.     

Estrogenic and antiestrogenic effect of in vitro treatment of follicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin

OBJECTIVE: Two types of follicular cells from preovulatory ovary were cultured in vitro separately and in co-culture to test difference in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) action on particular cell types. METHODS: The accumulation of TCDD in follicular wall was analysed using coupled capillary gas chromatography mass spectrometry. Whole preovulatory follicles were isolated from ovary and incubated with prolonged exposure to 0.1 nM TCDD or single exposure to 10 nM TCDD for four days. In the second part of experiments direct effects of TCDD on steroidogenesis were investigated in porcine theca cells (Tc) and granulosa cells (Gc) cultured alone and in co-culture (GT). The media were collected after four days for steroid analysis. RESULTS: 59.3% and 81.2% of TCDD added to the culture medium was accumulated after 0.1 and 10 nM, respectively. TCDD in a dose-dependent manner increased estradiol secretion with concomitant progesterone secretion by theca interna cells. On the other hand decrease of both progesterone and estradiol secretion by granulosa cells cultured alone and in co-culture with theca cells was noted. CONCLUSION: Different cell-specific estrogenic or antiestrogenic effect of TCDD were found in ovarian follicles.     

Nontransmission of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows

The absorption of deoxynivalenol (DON; vomitoxin), a trichothecene mycotoxin produced by Fusarium species, was studied in the dairy cow. Serum and milk DON levels were quantitated following a single oral dose of 920 mg DON to each of two lactating cows of similar weight. Maximum blood levels for the two animals following DON administration were 200 and 90 ng/ml serum, occurring at times 4.7 and 3.5 hr, respectively. By 24 hr after dosing only trace levels (less than 2 ng/ml) were still detectable. DON in its conjugated form accounted for 24-46% of the total levels present in serum. Free and conjugated DON were also present in cow's milk, but only extremely low amounts (less than 4 ng/ml) were detected. Detection of DON was carried out utilizing Sep-Pak C18 extraction cartridges for isolation, with additional purification of the sample achieved by passing the extract through a short charcoal/alumina column. The extract was then reacted with N-heptafluorobutyrylimidazole prior to quantitation of the resulting DON-tris-heptafluorobutyrate derivative by combined gas chromatography-quadrupole mass spectrometry, using multiple selected ion monitoring. Detection limits were as low as 1 ng/ml (1 ppb).     

Development of enzyme immunoassay for detection of DDT

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p′-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42–10.53% for intra-assay and 6.04–7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p′-DDT, p, p′-DDD, o, p′-DDD, p, p′-DDE, o, p′-DDE, p, p′-dichlorobenzophenone (DCBP), o, p′-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p′-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p′-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.     

Fast determination of phenoxy acid herbicides in carrots and apples using liquid chromatography coupled triple quadrupole mass spectrometry

A fast, simple and inexpensive method has been developed for the analysis of phenoxy acid herbicides: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), 2-(4-chloro-o-tolyloxy)propionic acid (MCPP), 2-(4-aryloxyphenoxy)propionic acid (Fluazifop) and 2-(4-aryloxyphenoxy)propionic acid (Haloxyfop) in carrots and apples by liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS). The compounds were analyzed by QuEChERS (quick, easy, cheap, effective, rugged, safe) methodology without cleanup.

The recoveries were performed at two spiked levels (0.05 and 0.5 mg/kg) for both matrices with six replicates for each level. The mean recoveries ranged from 70–92% for both apples and carrots. The precision of the method expressed as relative standard deviation (RSD%) was found to be in the range 3–15%. For all compounds, good linearity (r² > 0.99) was obtained over the range of concentration from 0.05 μ g/mL to 0.5 μ g/mL, corresponding to the pesticide concentrations of 0.05 mg/kg and 0.5 mg/kg, respectively. The determination limits (LOQs) ranged from 0.01 ng/mL to 1.3 ng/mL in solvent, whereas, the LOQs calculated in matrix ranged from 0.05 ng/g to 21.0 ng/g for apples and from 0.06 ng/g to 10.2 ng/g for carrots. The developed methodology combines the advantages of both QuEChERS and LC/MS/MS producing a very rapid, sensitive and cheap method useful for the routine analytical laboratories.     

Effect of the appetite stimulant cyproheptadine on deoxynivalenol ‐ induced reductions in feed consumption and weight gain in the mouse 1

Abstract

Deoxynivalenol (DON, vomitoxin), a Fusarium mycotoxin, is suspected of inducing its anorectic/feed refusal activity through a serotoninergic (5HT) mechanism, possible via 5HT₂‐receptors. In this study the efficiency of cyproheptadine (CYP), a serotonin antagonist and known appetite stimulant, to attenuate the adverse effect of DON was investigated in mice. CYP was administered in the feed for two days before animals began receiving the DON, which was also added to the feed. Both agents were administered concurrently thereafter for a 12‐day period. Dosing levels included various combinations of the two compounds, ranging from 0–16 ppm DON and 0–20 ppm CYP.

Results showed that CYP could effectively offset the reduction in feed intake caused by dietary DON, but only when dose levels were optimized (CYP appeared to have a narrow effective dose range, which was also dependent on the DON concentration). In fact, optimum CYP doses were able to enhance feed intake in DON‐fed mice above control levels, indicating that more than just a direct block of the toxin's effect was occurring. Conversely though, the effect of CYP on weight gain (WG) was ambiguous. At the lower CYP doses (≤ 5 ppm), alone or in combination with the lowest DON level tested (4 ppm), a modest positive effect was noted, but at the higher DON concentrations (≥ 8 ppm) the overall WG generally remained depressed, and tended to be reduced further by increasing doses of CYP.

These results provide additional support that serotoninergic mechanisms probably play a role in mediating DON‐induced reduction in feed intake. Where and how DON actually initiates its anorectic effect, however, has not been resolved.     

The role of tryptophan in don‐induced feed rejection

Abstract

Deoxynivalenol (DON, vomitoxin) commonly produced by Fusarium fungi can alter feeding behaviour of pigs and other farm animals. The effects of dietary tryptophan (TRP, precursor of brain amine serotonin) in combination with DON were examined in mice to determine if TRP can modulate DON toxicity. Results indicated that brain TRP can be influenced by dietary TRP, but no evidence of TRP potentiating DON toxicity was observed. Higher TRP levels likely induced amino acid imbalance leading to weight gain suppression.     

Distribution of deoxynivalenol in cerebral spinal fluid following administration to swine and sheep 1

Abstract

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi. In evaluating DON as a potent CNS (emetic, anorexic) agent, its cerebral spinal fluid (CSF) and plasma pharmacokinetics were studied in pigs, a species very sensitive to the effects of DON, and sheep, a more tolerant animal. After intravenous administration, DON was detected very rapidly (<2.5 min) in the CSF of both species, but whereas peak levels (t‐max) occurred at 5–10 min in sheep, in swine it was 30–60 min. It would appear that the very rapid and extensive tissue distribution of DON in swine (Vdγ = 1.13 1 kg^‐1) may be slowing the rate of diffusion of the toxin into the CSF compared to sheep (Vd_β = 0.19 1 kg^‐1) where the toxin is confined essentially to the extracellular compartment. Area under curve calculations indicate approximately 2 1/2 times the amount of toxin eventually reaches the pig CSF compared to sheep CSF.

A good relationship between blood‐CSF DON levels was apparent in both species, although limitations in detection methods made it impossible to resolve a slow terminal phase (γ) in swine CSF which was evident in the plasma profile after iv administration.

Following oral administration of DON to pigs, a close correlation between plasma and CSF DON levels was observed. The toxin could be detected in CSF for up to 20 hr post‐dosing.     

A study on the effect of deoxynivalenol on serotonin receptor binding in fig brain membranes

Abstract

Central serotoninergic (5‐hydroxytryptamine, 5HT) pathways are believed to be involved in the mechanisms of anorexia and/or emesis evoked by the trichothecene mycotoxin deoxynivalenol (DON). Using an in vitro membrane receptor binding assay, the competitive potency of DON was investigated against several radioactive ligands that have a high affinity for selective 5HT‐receptor subgroups. Receptor site densities and displacement profiles in twelve selected regions of pig brain were investigated. Overall, DON possessed only minimal efficacy to competently block any of the 5HT‐ligands tested. IC₅₀ values (50% inhibitory concentration) of at least 5 mM DON were required to inhibit binding, and in certain regions concentrations of 100 mM were ineffective. In comparison, several standard 5HT‐antagonists showed 10³‐10⁵ times greater capability than DON to displace binding of these ligands. Because these results indicated DON possesses only weak affinity for the 5HT‐receptor subtypes investigated here, this suggested that in vivo, unless relatively high concentrations of the toxin are present, its pharmacological effects may be mediated by mechanisms other than a functional interaction with serotoninergic receptors at the central level.     

Comparison between solid-phase extraction methods for the chromatographic determination of organophosphorus pesticides in water

A solid-phase microextraction (SPME) procedure has been developed to extract eight organophosphorus pesticides (OPs) in water and the method was compared with a conventional solid phase extraction (SPE) technique. The extracted OPs were analyzed by gas chromatography using thermionic specific detection. Both extraction methods presented linear calibration at least over the concentration range investigated (100 to 1000 ng x mL(-1) for SPE and 1 to 100 ng x mL(-1) for SPME). SPME method presented higher sensitivity than SPE. The quantitation limits were between 0.1 to 1.0 ng x mL(-1) for SPME depending upon the analyte, and 100 ng x mL(-1) for SPE. The precision, as measured by the standard deviations (RSD), were in the range 3.6% to 5.8% for SPME and 2.4% to 9.2% for SPE. Along with the feature of being a solvent - free sampling technique, SPME offers additional benefits due to its high sensitivity, simplicity, and small size sample required (typically: SPE - 500 mL, SPME - 5 mL).     

Development of enzyme immunoassay for detection of DDT

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.     

Occurrence of mycotoxins in wheat grains exposed to fungicides on fusarium head blight control in southern Brazil

Mycotoxins occurrence in wheat grains impose risks to human and animal health. The southern Brazil has favorable weather conditions for Fusarium graminearum infections and consequently for mycotoxins accumulation on grains. The goal of this study was to evaluate the behavior of new wheat commercial genotypes to Fusarium Head Blight (FHB), to control performance of new fungicide formulations and their relationship with mycotoxins concentration in grains. The manly mycotoxin occurrence on wheat grains in southern Brazil was deoxynivalenol (DON). Two cultivars showed high DON concentration above the tolerance limits (>3000 μg kg^?1). Many other mycotoxins monitored presented concentrations below method detection limit. Satisfactory levels of fungicide effectiveness were achieved against F. graminearum. Some fungicides promoted a satisfactory decrease on DON accumulation in grains. The best results were obtained when prothioconazole was present. SDHI (Succinate dehydrogenase inhibitors) + QoI (Quinone outside inhibitors) fungicides showed benefic effects at FHB control at field, but it did not promote satisfactory reduction on DON contamination. Fungicides can be used satisfactory for FHB control and reduce DON contamination in grains in southern Brazil. The presence of prothioconazole should be recommended. Some genotypes showed high DON concentration and it was not directly related with FHB severity at field.     

Development of a sensitive,competitive, indirect ELISA for the detection of fumonisin B1 in corn originating from Anhui province,China

Fumonisin B₁ (FB₁) is a secondary metabolite produced by Fusarium verticillioides or Fusarium proliferatum, which present in food and feed. It causes hazardous effects on human and animal health. A monoclonal antibody (mAb) against FB₁ was produced and a simple, reliable and sensitive, competitive, indirect enzyme-linked immunosorbent assay (ci-ELISA) for detection of FB₁ was developed and the experiment conditions were optimized. The coating concentration of FB₁-ovalbumin (FB₁-OVA) was 500 ng mL^?1, the action concentrations of anti-FB₁ mAb and goat anti-mouse IgG were 1.28 × 10⁴ and 1:5000, respectively. The 50% inhibitory concentration (IC₅₀) was 11 ng mL^?1, with a detectable range of 1.25–250 ng mL^?1, and a limit of determination (LOD) of 1.15 ng mL^?1. The cross-reactivity (CR) of the antibody against fumonisin B₂ (FB₂) was 60.4, and <1% against deoxynivalenol (DON), aflatoxin B₁ (AFB₁), ochratoxin A (OTA) or zearalenone (ZEN). In spiked samples (250 ng g^?1, 500 ng g^?1, 1000 ng g^?1), the mean recoveries ranged from 86.7 ± 5% to 102 ± 4%, and the coefficient of variation (CV) ranged from 3% to 10%. A survey of 96 corn samples from Bozhou, Fuyang, Bengbu, and Hefei, in Anhui province, China, was performed. Frequencies of FB₁ contamination were 83.3%, 95.8%, 20.8% and 91.7%, and the mean concentrations of positive samples were 0.702 μg kg^?1, 0.883 μg kg^?1, 0.074 μg kg^?1, and 0.276 μg kg^?1, respectively. The results of this study suggest that the ci-ELISA developed in this study can be used to identify FB₁ in corn, furthermore, further study is needed to investigate FB₁ contamination in food and feed to prevent its harmful health effects.     

Monoclonal antibody produced by heterologous indirect enzyme-linked immunosorbent assay and its application for parathion residue determination in agricultural and environmental samples

A heterologous indirect enzyme-linked immunosorbent assay (ELISA) was developed, which was based on monoclonal antibody (Mab) to determine parathion residue in agricultural and environmental samples. Eight Mabs were produced by introducing the heterologous indirect ELISA into the screening procedure. It was shown that these Mabs had more broad-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening). So it became much easier using these new Mabs to develop heterologous immunoassays. In addition, all the developed heterologous ELISAs could be used to determine parathion residue in semiquantitative or quantitative level, and their detection limitation (LOD) was around 2 ng/mL. Compared with the previous heterologous ELISA (hapten 11/5H7) with IC₅₀ of 13.3 ng/mL, a better heterologous ELISA (hapten 11/1E1) was obtained with IC₅₀ of 3.8 ng/mL, which improved the sensitivity 3.5 times. Finally, the latter was applied to parathion residue determination in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, Chinese cabbage and carrot were between 80.4 % and 111.8 %. The LOD for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.     

COMPARISON BETWEEN SOLID–PHASE EXTRACTION METHODS FOR THE CHROMATOGRAPHIC DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES IN WATER

A solid-phase microextraction (SPME) procedure has been developed to ex tract eight organophosphorus pesticides (OPs) in water and the method was compared with a conventional solid phase extraction (SPE) technique. The extracted OPs were analyzed by gas chromatography using thermionic specific detection. Both extraction methods presented linear calibration at least over the concentration range investigated (100 to 1000 ng.mL^?1 for SPE and 1 to 100 ng.mL^?1 for SPME). SPME method presented higher sensitivity than SPE. The quantitation limits were between 0.1 to 1.0 ng.mL^?1 for SPME depending upon the analyte, and 100 ng.mL^?1 for SPE. The precision, as measured by the standard deviations (RSD), were in the range 3.6 % to 5.8 % for SPME and 2.4 % to 9.2 % for SPE.

Along with the feature of being a solvent – free sampling technique, SPME offers additional benefits due to its high sensitivity, simplicity, and small size sample required (typically: SPE – 500 mL, SPME – 5 mL).