Antimony biomethylation by Scopulariopsis brevicaulis: characterization of intermediates and the methyl donor

The filamentous fungus Scopulariopsis brevicaulis biomethylates inorganic antimony(III) compounds to trimethylstibine, that can be detected in culture headspace gases. Dimethylantimony and trimethylantimony species have been detected in the medium of these cultures, but the origin of these species was controversial. We now show that the dimethylantimony species is a true intermediate on the pathway to trimethylstibine (rather than arising from trimethylstibine oxidation or as an analytical artifact) because no dimethylantimony species are formed on trimethylstibine oxidation, as determined by using HG-GC-AAS. Furthermore, the dimethylantimony and trimethylantimony species can be separated, by using anion exchange chromatography, and so the dimethylantimony species is not an analytical artifact, formed during the hydride generation process. The antimony biomethylation mechanism was further probed by measuring incorporation of the methyl group, from 13CD3-L-methionine and CD3-D-methionine, into methylantimony species and, for comparison, into methylarsenic species. The percentage incorporation of the labeled methyl group into methylarsenic and methylantimony species was not significantly different. The incorporation from 13CD3-L-methionine was 54% and 47% for antimony and arsenic, respectively. The incorporation from CD3-D-methionine was 20% and 16% for antimony and arsenic, respectively. It appears that the biomethylation of arsenic and antimony occur by very similar, perhaps identical, mechanisms.     

Bacterial communities and enzyme activities of PAHs polluted soils

Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From the fastest phenanthrene-degrading culture C(B-BT), representative strains were identified as Achromobacter xylosoxidans (100%), Methylobacterium sp. (99%), Rhizobium galegae (99%), Rhodococcus aetherovorans (100%), Stenotrophomonas acidaminiphila (100%), Alcaligenes sp. (99%) and Aquamicrobium defluvium (100%). DGGE-profiles of culture C(B-BT) showed bands attributable to Rhodococcus, Achromobacter, Methylobacterium rhizobium, Alcaligenes and Aquamicrobium. The isolation of Rhodococcus aetherovorans and Methylobacterium sp. can be consistent with the hypothesis that different phenanthrene-degrading strategies, cell surface properties, or the presence of xenobiotic-specific membrane carriers could play a role in the uptake/degradation of solid phenanthrene.     

Rapid biotransformation of arsenic by a model protozoan Tetrahymena pyriformis GL-C. [corrected

Arsenic biomethylation and biovolatilization are thought to be two important metabolic pathways in aquatic and soil environments. Tetrahymena thermophila is a genus of free-living ciliated protozoan that is widely distributed in freshwater environments around the world. In this study, we studied arsenic accumulation, speciation, efflux, methylation and volatilization in this unicellular eukaryote exposed to various concentrations of arsenate. Our results show that T. thermophila accumulated 187 mg.kg⁻¹ dry weight of arsenic when exposed to 40 μM for 48 h, with MMAs(V) (monomethylarsenate) and DMAs(V) (dimethylarsenate) as the dominant species, accounting for 66% of the total arsenic. Meanwhile, arsenate, arsenite, MMAs(V) and DMAs(V) were detected in the culture medium; the last three were released by the cells. The production of volatile arsenic increased with increasing external As(V) concentrations and exposure time. To our knowledge, this is the first study on arsenic metabolism, particularly biomethylation and biovolatilization, in protozoa.     

Headspace solid phase microextraction method for determination of triazine and organophosphorus pesticides in soil

A headspace solid phase microextraction method (HS-SPME) for simultaneous determination of five pesticides belonging to triazine and organophosphorus pesticide groups in soil samples was developed. Microextraction conditions, such as temperature, extraction time and sodium chloride (NaCl) content were investigated and optimized using 100 μ m polydimethyl-siloxane (PDMS) fiber. Detection and quantification were done by gas chromatography-mass spectrometry (GC-MS). Relative standard deviation (RSD) and recovery values for multiple analysis of soil samples fortified at 30 μ g kg^{? 1} of each pesticide were below 13 % and higher than 70 %, respectively. Limits of detection (LOD) for all the compounds studied were less than 3.2 μ g kg^{? 1}. The proposed method was applied in the analysis of some agricultural soil samples.     

Development of a solid phase microextraction-gas chromatography-mass spectrometry method for the determination of pentachlorophenol in human plasma using experimental design

A new method, headspace solid-phase microextraction (HS-SPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS), which was used for the determination of trace amount of pentachlorophenol (PCP) in human plasma was presented. The acetylation derivatization reaction was firstly optimized using a Doehlert design. Then a series of parameters relevant to the headspace SPME procedure, including fiber coating, extraction temperature, extraction time and salt addition, were optimized using a two-level full factorial design expanded further to a central composite design. The validation of method showed that the optimized method had good linearity (R(2)=0.999) within the concentration ranges 0.1-50.0ngml(-1), and was sensitive with the limit of detection of 0.02ngml(-1). Intra- and inter-day precision for pentachlorophenol in human plasma samples were not greater than 11.9% and 12.6%, respectively. The proposed method, to our knowledge, describes the first application of HS-SPME with GC-MS for analysis of PCP in blood plasma sample. Application of the method to real human plasma samples, PCP was successfully detected in some cases at concentration levels 1.2-6.3ngml(-1).     

Alginate immobilized enrichment culture for atrazine degradation in soil and water system

An atrazine degrading enrichment culture, a consortium of bacteria of genus Bacillus along with Pseudomonas and Burkholderia, was immobilized in sodium alginate and was used to study atrazine degradation in mineral salts medium (MSM), soil and wastewater effluent. Sodium alginate immobilized consortium, when stored at room temperature (24 ± 5°C), was effective in degrading atrazine in MSM up to 90 days of storage. The survival of bacteria in alginate beads, based on colony formation unit (CFU) counts, suggested survival up to 90 days and population counts decreased to 1/5^th on 120 days. Comparison of atrazine degrading ability of the freely suspended enrichment culture and immobilized culture suggested that the immobilized culture took longer time for complete degradation of atrazine as a lag phase of 2 days was observed in the MSM inoculated with alginate immobilized culture. The free cells resulted in complete degradation of atrazine within 6 days, while immobilized cells took 10 days for 100% atrazine degradation. Further, immobilized cultures were able to degrade atrazine in soil and wastewater effluent. Alginate beads were stable and effective in degrading atrazine till 3rd transfer and disintegrated thereafter. The study suggested that immobilized enrichment culture, due to its better storage and application, can be used to degrade atrazine in soil water system.     

Stabilization of thioarsenates in iron-rich waters

In recent years, thioarsenates have been shown to be important arsenic species in sulfidic, low-iron waters. Here, we show for the first time that thioarsenates also occur in iron-rich ground waters, and that all methods previously used to preserve arsenic speciation (acidification, flash-freezing, or EDTA addition) fail to preserve thioarsenates in such matrices. Laboratory studies were conducted to identify the best approach for stabilizing thioarsenates by combination and modification of the previously-applied methods. Since acidification was shown to induce conversions between thioarsenates and precipitation of arsenic-sulfide minerals, we first conducted a detailed study of thioarsenate preservation by flash-freezing. In pure water, thioarsenates were stable for 21 d when the samples were flash-frozen and cryo-stored with a minimal and anoxic headspace. Increasing headspace volume and oxygen presence in the headspace were detrimental to thioarsenate stability during cryo-storage. Addition of NaOH (0.1 M) or EtOH (1% V/V) counteracted these effects and stabilized thioarsenates during cryo-storage. Addition of Fe(II) to thioarsenate solutions caused immediate changes in arsenic speciation and a loss of total arsenic from solution during cryo-storage. Both effects were largely eliminated by addition of a neutral EDTA-solution, and thioarsenates were significantly stabilized during cryo-storage by this procedure. Neutralization of EDTA was required to prevent alteration of thioarsenate speciation through pH change. With the modified method (anoxic cryo-preservation by flash-freezing with minimal headspace after addition of neutralized EDTA-solution), the fractions of mono- and dithioarsenate, the two thioarsenates observed in the iron-rich ground waters, remained stable over a cryo-storage period of 11 d. Further modifications are needed for the higher SH-substituted thioarsenates (tri- and tetrathioarsenate), which were not encountered in the studied iron-rich ground waters.     

Antimony accumulation in Achillea ageratum, Plantago lanceolata and Silene vulgaris growing in an old Sb-mining area

Preliminary data of a biogeochemical survey concerning antimony transfer from soil to plants in an abandoned Sb-mining area are presented. Achillea ageratum, Plantago lanceolata and Silene vulgaris can strongly accumulate antimony when its extractable fraction in the soil is high (139-793 mg/kg). A. ageratum accumulates in basal leaves (1367 mg/kg) and inflorescences (1105 mg/kg), P. lanceolata in roots (1150 mg/kg) and S. vulgaris in shoots (1164 mg/kg). In these plant species, the efficiency of antimony accumulation decreases when the antimony availability in the soil is high. In A. ageratum and S. vulgaris, the death of the epigeal target part at the end of the growing season contributes to a reduction of the antimony load in the plant. A study to test the use of these species as bioindicators of antimony availability in soil is suggested by our results.     

Antimony distribution and mobility in topsoils and plants (Cytisus striatus, Cistus ladanifer and Dittrichia viscosa) from polluted Sb-mining areas in Extremadura (Spain)

A study about topsoil antimony distribution and mobility from the soils to the biomass has been afforded in three abandoned Sb mining areas located at Extremadura. Physico-chemical characteristics of the soils and total antimony levels were measured in soils and autochthonous plant species (Cytisus striatus, Cistus ladanifer and Dittrichia viscosa). Comparison with corresponding values in reference areas isolated from the mining activities is discussed. Antimony mobility in the soils was estimated by measuring the water extractable fraction; low results were obtained for the three soil areas, with no statistical differences. Plant ability to accumulate antimony was estimated by use of plant accumulation coefficients (PAC). Seasonal (spring vs. autumn) effects on the antimony content in the plant species. Cytisus striatus from Mari Rosa mine presented antimony excluder characteristics, whereas Dittrichia viscosa specimens growing in San Antonio mine showed a significant antimony bioaccumulation.     

Degradation of atrazine in mineral salts medium and soil using enrichment culture

Atrazine degrading enrichment culture was prepared by its repeated addition to an alluvial soil and its ability to degrade atrazine in mineral salts medium and soil was studied. Enrichment culture utilized atrazine as a sole source of carbon and nitrogen in mineral salts medium and degradation slowed down when sucrose and/or ammonium hydrogen phosphate were supplemented as additional source of carbon and nitrogen, respectively. Biuret was detected as the only metabolite of atrazine while deethylatrazine, deisopropyatrazine, hydroxyatrazine and cyanuric acid were never detected at any stage of degradation. Enrichment culture degraded atrazine in an alkaline alluvial soil while no degradation was observed in the acidic laterite soil. Enrichment culture was able to withstand high concentrations of atrazine (110 μg/g) in the alluvial soil as atrazine was completely degraded. Developed mixed culture has the ability to degrade atrazine and has potential application in decontamination of contaminated water and soil.     

Biodegradation of imidacloprid by an isolated soil microorganism

Imidacloprid (1-[(6-chloro-3-pyridinyl)-methyl]-N-nitro-2-imidazolidinimine), a chloronicotinyl insecticide used to control biting and sucking insects, is very persistent in the soil with a half-life often greater than 100 days. Although a few soil metabolites have been reported in the literature, there are no reports of imidacloprid-degrading soil microorganisms. Our objectives were to discover, isolate, and characterize microorganisms capable of degrading imidacloprid in soil. Two soil-free stable enrichment cultures in N-limited media were obtained that degraded 19 mg L(- 1) (43%) and 11 mg L(- 1) (16%) of the applied imidacloprid, and produced about 19 mg L(- 1) 6-chloronicotinic acid in three weeks. Enrichment media without microorganisms had no loss of imidacloprid. Strain PC-21, obtained from the enrichment cultures, degraded 37% to 58% of 25 mg L(- 1) imidacloprid in tryptic soy broth containing 1 g L(- 1) succinate and D-glucose at 27 degrees C incubation over a period of three weeks. Trace amounts of NO(3)(-)/NO(2)(-)were produced and six metabolites were characterized by high performance liquid chromatography (HPLC) using (14)C-methylene-imidacloprid and liquid chromatograph-electrospray-mass spectrometer (LC-MS). Two of the metabolites were identified as imidacloprid-guanidine and imidacloprid-urea by HPLC standards and LC-MS. During the experiment, 6-chloronicotinic acid was not produced. Less than 1% of the applied (14)C was incorporated into the microbial biomass and no (14)CO(2) was detected. Strain PC-21, identified as a species of Leifsonia by PCR amplification of a 500 bp sequence of 16s rRNA, cometabolized imidacloprid.     

Characterization of chlordecone-tolerant fungal populations isolated from long-term polluted tropical volcanic soil in the French West Indies

The insecticide chlordecone is a contaminant found in most of the banana plantations in the French West Indies. This study aims to search for fungal populations able to grow on it. An Andosol heavily contaminated with chlordecone, perfused for 1 year in a soil–charcoal system, was used to conduct enrichment cultures. A total of 103 fungal strains able to grow on chlordecone-mineral salt medium were isolated, purified, and deposited in the MIAE collection (Microorganismes d'Intérêt Agro-Environnemental, UMR Agroécologie, Institut National de la Recherche Agronomique, Dijon, France). Internal transcribed spacer sequencing revealed that all isolated strains belonged to the Ascomycota phylum and gathered in 11 genera: Metacordyceps, Cordyceps, Pochonia, Acremonium, Fusarium, Paecilomyces, Ophiocordyceps, Purpureocillium, Bionectria, Penicillium, and Aspergillus. Among predominant species, only one isolate, Fusarium oxysporum MIAE01197, was able to grow in a liquid culture medium that contained chlordecone as sole carbon source. Chlordecone increased F. oxysporum MIAE01197 growth rate, attesting for its tolerance to this organochlorine. Moreover, F. oxysporum MIAE01197 exhibited a higher EC₅₀ value than the reference strain F. oxysporum MIAE00047. This further suggests its adaptation to chlordecone tolerance up to 29.2 mg l^?1. Gas chromatography–mass spectrometry (GC-MS) analysis revealed that 40 % of chlordecone was dissipated in F. oxysporum MIAE01197 suspension culture. No chlordecone metabolite was detected by GC-MS. However, weak amount of ¹⁴CO₂ evolved from ¹⁴C₁₀-chlordecone and ¹⁴C₁₀-metabolites were observed. Sorption of ¹⁴C₁₀-chlordecone onto fungal biomass followed a linear relationship (r ²?=?0.99) suggesting that it may also account for chlordecone dissipation in F. oxysporum MIAE01197 culture.     

Uptake and degradation of DDT by hairy root cultures of Cichorium intybus and Brassica juncea

Hairy root cultures of Cichorium intybus and Brassica juncea were used for their ability to uptake and degrade DDT (1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane). After 24 h of 14C DDT treatment, only 12-13% of the total applied radioactivity was detected in the culture media, indicating the efficient uptake of DDT by the hairy roots. The majority of the applied radioactivity was associated with the roots. DDT was degraded to various other products such as DDD, DDE and DDMU, along with some unknown compounds by hairy root cultures, which were detected by thin layer chromatography (TLC) and autoradiography. The rate of in situ degradation was found to be higher during the initial stages of culture and the residual 14C DDT in the roots was found to decrease from 77% to 61% over a period of 10-days. There was no spontaneous degradation of 14C DDT in media lacking hairy root cultures or in media with autoclaved hairy roots. This suggests that endogenous root enzymes play a role in the breakdown of 14C DDT. These results suggest the potential applicability and advantage of using these plant species for phytoremediation of persistent xenobiotics such as DDT in an eco-friendly and efficient manner for environmental clean up.     

Biodegradation of haloacetic acids by bacterial enrichment cultures

Haloacetic acids (HAAs) are toxic organic chemicals that are frequently detected in surface waters and in drinking water distribution systems. The aerobic biodegradation of HAAs was investigated in serum bottles containing a single HAA and inoculated with washed microorganisms obtained from enrichment cultures maintained on either monochloroacetic acid (MCAA) or trichloroacetic acid (TCAA) as the sole carbon and energy source. Biodegradation was observed for each of the HAAs tested at concentrations similar to those found in surface waters and in drinking water distribution systems. The MCAA culture was able to degrade both MCAA and monobromoacetic acid (MBAA) with pseudo-first order rate constants of 1.06 x 10(-2) and 1.13 x 10(-2) l(mg protein)(-1) d(-1), respectively, for concentrations ranging from 10(-5) to 2 mM. The pseudo-first order rate constant for TCAA degradation by the TCAA culture was 6.52 x 10(-3) l(mg protein)(-1) d(-1) for concentrations ranging from 5.33 x 10(-5) to 0.72 mM. The TCAA culture was also able to degrade MCAA with the rate accelerating as incubation time increased. Experiments with radiolabeled HAAs indicated that the 14C was primarily converted to 14CO2 with minor incorporation into cell biomass. The community structure of the enrichment cultures was analyzed by both cultivation-dependent and cultivation-independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragments showed that each of the two enrichment cultures had multiple bacterial populations, none of which corresponded to HAA-degrading bacteria cultivated on HAA-supplemented agar plates. This research indicates that biodegradation is a potential loss mechanism for HAAs in surface waters and in drinking water distribution systems.     

Determination of benzene, toluene, ethylbenzene, xylenes in water at sub-ng l-1 levels by solid-phase microextraction coupled to cryo-trap gas chromatography-mass spectrometry

A trace analytical method of benzene, toluene, ethylbenzene and xylenes (BTEX) in water has been developed by using headspace solid-phase microextraction (HS-SPME) coupled to cryo-trap gas chromatography-mass spectrometry (GC-MS). The chromatographic peak shape for BTEX was improved by using cryo-trap equipment. The HS-SPME experimental procedures to extract BTEX from water were optimized with a 75 microm carboxen/polydimethylsiloxane (CAR/PDMS)-coated fiber at a sodium chloride concentration of 267 g l(-1), extraction for 15 min at 25 degrees C and desorption at 290 degrees C for 2 min. Good linearity was verified in a range of 0.0001-50 microg l(-1) for each analyte (r(2)=0.996-0.999). The limits of detection (LODs) of BTEX in water reached at sub-ng l(-1) levels. LODs of benzene, toluene, ethylbenzene, m/p-xylene and o-xylene were 0.04, 0.02, 0.05, 0.01 and 0.02 ng l(-1), respectively. The proposed analytical method was successfully used for the quantification of trace BTEX in ground water. The results indicate that HS-SPME coupled to cryo-trap GC-MS is an effective tool for analysis of BTEX in water samples at the sub-ng l(-1) level.     

Assessment and distribution of antimony in soils around three coal mines, Anhui, China

Thirty-three soil samples were collected from the Luling, Liuer, and Zhangji coal mines in the Huaibei and Huainan areas of Anhui Province, China. The samples were analyzed for antimony (Sb) by inductively coupled plasma-optical emission spectrometry (ICP-OES) method. The average Sb content in the 33 samples was 4 mg kg(-1), which is lower than in coals from this region (6.2 mg kg(-1)). More than 75% of the soils sampled showed a significant degree of Sb pollution (enrichment factors [EFs] 5-20). The soils collected near the gob pile and coal preparation plant were higher in Sb content than those collected from residential areas near the mines. The gob pile and tailings from the preparation plant were high in mineral matter content and high in Sb. They are the sources of Sb pollution in surface soils in the vicinity of coal mines. The spatial dispersion of Sb in surface soil in the mine region shows that Sb pollution could reach out as far as 350 m into the local environment conditions. Crops in rice paddies may adsorb some Sb and reduce the Sb content in soils from paddyfields. Vertical distribution of Sb in two soil profiles indicates that Sb is normally relatively immobile in soils.     

Treatment of cork boiling wastewater using chemical oxidation and biodegradation

Three cultures were enriched from cork boiling wastewater using tannic acid as the selective carbon substrate, at 25 degrees C and pH 7.2, 25 degrees C and pH 4.7 and 50 degrees C and pH 4.7. The enrichment culture obtained at neutral pH was composed of five culturable isolates, whereas from each acidic enrichment two bacterial strains were isolated. Mesophilic isolates were Gram negative bacteria belonging to the genera Klebsiella, Pseudomonas, Stenotrophomonas and Burkholderia. Thermophilic isolates were members of the genus Bacillus. Despite the capability of the enrichment cultures to use tannic acid as single carbon and energy source, those cultures were unable to reduce the total polyphenols or the total organic carbon content of cork boiling wastewater. In order to increase the bioavailability of the organic carbon in cork boiling wastewater, biodegradation was preceded by Fenton oxidation. It was demonstrated that the combined process, using small amounts of Fenton reagents and biodegradative inoculum added almost simultaneously to cork boiling wastewater, leads to TOC reductions of more than 90%.     

Simultaneous determination of estrogenic odorant alkylphenols,chlorophenols, and their derivatives in water using online headspace solid phase microextraction coupled with gas chromatography-mass spectrometry

A simple online headspace solid-phase microextraction (HS-SPME) coupled with the gas chromatography-mass spectrometry (GC-MS) method was developed for simultaneous determination of trace amounts of nine estrogenic odorant alkylphenols and chlorophenols and their derivatives in water samples. The extraction conditions of HS-SPME were optimized including fiber selection, extraction temperature, extraction time, and salt concentration. Results showed that divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was the most appropriate one among the three selected commercial fibers, and the optimal extraction temperature, time, and salt concentration were 70 °C, 30 min, and 0.25 g/mL, respectively. The developed method was validated and showed good linearity (R ²?>?0.989), low limit of detection (LOD, 0.002–0.5 μg/L), and excellent recoveries (76–126 %) with low relative standard deviation (RSD, 0.7–12.9 %). The developed method was finally applied to two surface water samples and some of these target compounds were detected. All these detected compounds were below their odor thresholds, except for 2,4,6-TCAS and 2,4,6-TBAS wherein their concentrations were near their odor thresholds. However, in the two surface water samples, these detected compounds contributed to a certain amount of estrogenicity, which seemed to suggest that more attention should be paid to the issue of estrogenicity rather than to the odor problem.     

Does malaoxon play a role in the geno- and cytotoxic effects of malathion on human choriocarcinoma cells?

This investigation was undertaken to elucidate whether the active metabolite of malathion, malaoxon, has any role in exerting cyto- and genotoxic effects for human choriocarcinoma (JAR) cell line which is an acceptable model for human placental cells. Gas chromatography-mass spectrometry (GC-MS) analysis were separately performed on the cell compartment and supernatant cell culture medium after subjecting the cell line to different malathion concentrations (10-400 μg/mL) and for various incubation periods (0.5 to 24 hours). GC-MS analysis showed that the sonication performed for the disruption of the cells did not cause the chemical change of malathion. The uptake of malathion by the cells was relatively fast. However, the presence of malaoxon, even in trace amounts, could not be confirmed either in samples originating from disrupted cells or in the cell culture medium. Although the hydrolysis of malaoxon occurred in the culture medium, this degradation process could not be counted as a reason for the absence of malaoxon. Since both malathion and malaoxon standard compounds could be accurately detected and distinguished by the applied liquid-liquid extraction and GC-MS methods, one can conclude that, in the case of JAR cells, the parent compound, (i.e. malathion itself) is responsible for the observed in vitro cyto- and genotoxic effects. Our results indicate that the direct toxicity of malathion contributes to the complications of pregnancy observed for environmental malathion exposure.     

Detection of DNA adducts in declining hop plants grown on fields formerly treated with heptachlor, a persistent insecticide

Hop decline was observed in Alsace, eastern France, in reparcelled sugar beet fields formerly abundantly treated with an insecticide, heptachlor. Leaves were collected from 'declining hops' grown in an heptachlor-contaminated area and from 'healthy hops' grown in a soil not contaminated by heptachlor. These two samples came from hop vines treated with other usual pesticides. 'Control' hop leaves came from soil neither treated with pesticide nor contaminated with heptachlor. Hypermodified nucleotides (DNA adducts) were detected using the (32)P-postlabelling method. No detectable DNA adducts were found in the 'control' specimen, whereas eight adducts were detected in the 'healthy hops' specimen, probably due to the usual pesticide treatment. However, 16 adducts, nine of which were new adducts, could be detected in the 'declining hops' specimen. It may therefore be supposed that the presence of these hypermodified nucleotides perturbs gene expression and so contributes to the hop decline. In addition, to confirm the genotoxicity of heptachlor, it is shown that it induces DNA adducts in bean-cell suspension culture as well. Finally, it is proposed, in the case of alternate cultures scheduled in fields which were formerly treated with pesticides, adapted to other cultures, that particular attention should be given to the history of the soils.