Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923)

Abstract

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (K_m and V_max) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.     

In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides

In this study, the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations on cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities of the mosquitofish (Gambusia yucatana) were investigated. In a first phase of the study, head and muscle ChE were characterized with different substrates (acetylthiocholine iodide, s-butyrylthiocholine iodide and propionylthiocholine iodide) and the selective inhibitors eserine hemisulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51), and N,N'-diisopropylphosphorodiamic acid (iso-OMPA). The results obtained suggest that the enzyme present in both head and muscle of G. yucatana is mainly acetylcholinesterase (AChE). Acute toxicity was evaluated by exposing fish to several concentrations of single pesticides and of a mixture of chlorpyrifos/glyphosate. LC50 values were determined after 96 h of exposure, except in the case of carbofuran for which LC50 was calculated after 24 h since almost all the fish died within this period. LC50 values were 0.085 mg/l for chlorpyrifos, 17.79 mg/l for glyphosate, 0.636 mg/l for carbofuran and 0.011 mg/l for the chlorpyrifos/glyphosate mixture. A Toxic Unit approach was used to compare the toxicity of chlorpyrifos and glyphosate when occurring in a mixture with their toxicities as single compounds. Synergistic effects of chlorpyrifos and glyphosate when present in a mixture were found. At the end of each bioassay (24 h for carbofuran, 96 for the other substances/mixture), effects on biomarkers were analyzed. Muscle LDH activity was not altered by any of the three pesticides tested. Gill GST activity was significantly inhibited (40%) by carbofuran after 24 h of exposure to concentrations equal or higher than 0.06 mg/l. ChE muscle and head activity were significantly inhibited (50% and 30%, respectively) by carbofuran at concentrations equal or higher than 0.25 mg/l. Chlorpyrifos induced a significant inhibition of both muscle and head ChE (80% and 50%, respectively) after 96 h of exposure to concentrations equal or higher than 0.05 mg/l. Carbofuran did not induce significant alterations of fish ChE. The ChE EC50 determined for chlorpyrifos/glyphosate mixture (0.070 mg/l) was higher than the correspondent value calculated for chlorpyrifos alone (0.011 mg/l) suggesting an antagonistic effect of glyphosate on ChE inhibition by chlorpyrifos. ChE activity of G. yucatana seems to be a good biomarker to diagnose the exposure of wild populations of this species exposed to anticholinesterase pesticides.     

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.     

Influence of temperature and soil type on the toxicity of three pesticides to Eisenia andrei

Expansion of agriculture in the tropics has increased the use of pesticides that may affect the soil ecosystems. Few studies so far determined the effects of pesticides in the tropics and tropical risk assessment therefore often relies on data from temperate conditions. Hence we compared the toxicity of chlorpyrifos, carbofuran and carbendazim to the earthworm Eisenia andrei at two different temperatures reflecting temperate and tropical conditions. The toxicity of the three pesticides in both conditions decreased in the order carbendazim > carbofuran > chlorpyrifos. For chlorpyrifos and carbofuran, but not for carbendazim, survival was more sensitive at the higher temperature, probably due to increased earthworm activity. Sub-lethal effects (reproduction and growth) however, varied inconsistently with temperature and soil types. We conclude that toxicity of pesticides in tropics may not be predicted from data generated under temperate conditions, even within the same species.     

In vitro effects of pesticides and metals on the activity of acetylcholinesterase (AChE) from different tissues of the blue mussel,Mytilus trossulus L.

The objective of this study was to conduct a comprehensive comparison of the effect on acetylcholinesterase (AChE) from various tissues of Mytilus trossulus caused by in vitro exposure to several pesticides and metals, because results available in the literature are inconsistent, difficult to compare, and sometimes contradict each other. For this purpose, fraction S10 extracted from gills, digestive gland, mantle and muscles, and the whole soft tissue of the mussel was exposed to several pesticides (dichlorvos, chlorpyrifos, fenitrothion, carbofuran and carbaryl) and metals (Cu, Zn, Cd, Hg, Pb) at a wide range of concentrations. AChE was inhibited in 50% or more in all the tissues exposed to dichlorvos, Cu, Hg and the mixture of Cu+Cd, and in some tissues exposed to carbaryl and carbofuran. The IC₅₀ was calculated where possible. No inhibition was found in the case of chlorpyrifos, Cd, Pb, and Zn.     

Cholinesterase characterization in Corbicula fluminea and effects of relevant environmental contaminants: A pesticide (chlorfenvinphos) and a detergent (SDS)

This study characterizes the cholinesterasic forms present in the bivalve Corbicula fluminea, through the analysis of differential affinity towards various substrates and inhibitors. This approach also permitted the validation of cholinesterase inhibition in the mentioned species for monitoring purposes by evaluating the inhibitory activity of a detergent (SDS) and a pesticide (chlorfenvinphos), both in vitro and in vivo. The cholinesterasic form present in the tissues of C. fluminea showed intermediate properties in terms of its relation with substrates and inhibitors. Furthermore, it was not inhibited by the detergent SDS under any of the test conditions, but was clearly inhibited by the selected organophosphate. The data show clearly that the main cholinesterasic form in C. fluminea is responsive to the organophosphate chlorfenvinphos, thus supporting the use of this biomarker for the environmental monitoring of specific contaminants such as anticholinesterasic pesticides. Despite not being commonly attained in the wild, the levels of chlorfenvinphos required to elicit a significant ChE inhibition were only one order of magnitude above actual values of aquatic contamination by this pesticide.     

Pesticide effects on crabs: How environmental concentrations of endosulfan and chlorpyrifos affect embryos

The objectives of this work were to evaluate the effects of environmentally relevant chlorpyrifos and endosulfan, concentrations in the incubation period, effective hatching and survival of embryos and neonates of the freshwater burrowing crab, Zilchiopsis collastinensis (Decapoda, Trichodactylidae). Both pesticides were prepared from commercial and technical grade products. The exposure to about 100, 200, and 400 ng endosulfan L^?1, and 48, 240, and 1,200 ng chlorpyrifos L^?1 did not cause differences in the incubation period or in effective hatching but decreased survival of neonates, especially in the concentrations prepared from the technical grade product. Even if these concentrations are below the median lethal concentration (LC₅₀) values for embryos, these caused a significant decrease in the survival of neonates, i.e. when crabs are outside the egg and not protected by chorion. The decrease in the neonate population caused by these concentrations, which could be found in the environment, might impact aquatic communities.     

Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg⁻¹ chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (≤1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field.     

Pesticides residues in the Prochilodus costatus (Valenciennes, 1850) fish caught in the São Francisco River,Brazil

The objective of this study was to determine the levels of pesticides in the fish Prochilodus costatus caught in São Francisco River, one of most important rivers in Brazil. Thirty-six fish were captured in three different areas, and samples of the dorsal muscle and pooled viscera were collected for toxicological analysis. We evaluated the presence of 150 different classes of insecticides, fungicides, herbicides and acaricides by multiresidue analysis technique using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the limit of detection of 5 ppb. In this study, organophosphorus and carbamate pesticides were detected at the highest levels in the caught fish. Among the 41 organophosphorus pesticides surveyed, nine types were detected (chlorpyrifos, diazinon, dichlorvos, disulfoton, ethion, etrimfos, phosalone, phosmet and pyrazophos) in the muscle, viscera pool, or both in 22 (61.1%) fish. Sampled tissues of 20 (55.6%) fish exhibited at least one of the eight evaluated carbamate pesticides and their metabolites: aldicarb, aldicarb sulfoxide, carbaryl, carbofuran, carbosulfan, furathiocarb, methomyl and propoxur. Fungicides (carbendazim, benalaxyl, kresoxim-methyl, trifloxystrobin, pyraclostrobin and its metabolite BF 500 pyraclostrobin), herbicides (pyridate and fluasifop p-butyl), acaricide (propargite) and pyrethroid (flumethrin) were also detected. In conclusion, P. costatus fish caught in the São Francisco River contained residues of 17 different pesticides, in both muscles and the viscera pool, indicating heavy environmental contamination by pesticides in the study area.     

Cholinesterase activities as potential biomarkers: characterization in two freshwater snails, Potamopyrgus antipodarum (Mollusca, Hydrobiidae, Smith 1889) and Valvata piscinalis (Mollusca, Valvatidae, Müller 1774)

Anti-cholinesterase insecticides constitute a major portion of modern synthetic pesticides and the assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure of non-target organisms to these pollutants. However, most studies on this biomarker were developed on vertebrates and among invertebrates, gastropod mollusks are rarely used. Gastropods are important members of aquatic habitats and therefore present a high ecological relevance for freshwater ecosystems. In this context, ChE activities were characterized in two freshwater gastropod mollusks, Potamopyrgus antipodarum and Valvata piscinalis, in order to ascertain their value as sentinel species. Firstly, characterization of ChE activities was performed using different substrates (acetylcholine iodide, butyrylcholine iodide and propionylcholine iodide) and specific inhibitors (eserine, iso-OMPA and BW284c51). Secondly, in vivo effect of a widely used organophosphate insecticide, chlorpyrifos, was tested on ChE activity in both species. Results suggested that P. antipodarum possesses two isoforms of cholinesterases, one isoform which properties are intermediate between an acetyl and a propionyl ChE, and one minor isoform which correspond to a butyryl ChE, while V. piscinalis seems to possess only one isoform which displays typical properties of an acetyl ChE. Chlorpyrifos induced no effect on V. piscinalis ChE. In contrast, P. antipodarum activity was significantly decreased by environmental realistic chlorpyrifos concentrations (2.86 and 14.2 nM) after seven days of contact. The present study suggests that P. antipodarum may be employed as a biological indicator for assessing pesticide contamination.     

Cholinesterase characterization in Corbicula fluminea and effects of relevant environmental contaminants: a pesticide (chlorfenvinphos) and a detergent (SDS)

This study characterizes the cholinesterasic forms present in the bivalve Corbicula fluminea, through the analysis of differential affinity towards various substrates and inhibitors. This approach also permitted the validation of cholinesterase inhibition in the mentioned species for monitoring purposes by evaluating the inhibitory activity of a detergent (SDS) and a pesticide (chlorfenvinphos), both in vitro and in vivo. The cholinesterasic form present in the tissues of C. fluminea showed intermediate properties in terms of its relation with substrates and inhibitors. Furthermore, it was not inhibited by the detergent SDS under any of the test conditions, but was clearly inhibited by the selected organophosphate. The data show clearly that the main cholinesterasic form in C. fluminea is responsive to the organophosphate chlorfenvinphos, thus supporting the use of this biomarker for the environmental monitoring of specific contaminants such as anticholinesterasic pesticides. Despite not being commonly attained in the wild, the levels of chlorfenvinphos required to elicit a significant ChE inhibition were only one order of magnitude above actual values of aquatic contamination by this pesticide.     

Sorption and photodegradation of chlorpyrifos on riparian and aquatic macrophytes

Surface water bodies may become contaminated via spray drift following pesticide application. In this investigation, the photodegradation and sorption of chlorpyrifos was studied in four riparian macrophytes representative of Mediterranean flora (Phragmites australis, Iris pseudacorus, Equisetum pratense and Typha latifolia). The results of experiments with both the active ingredient and the formulation DURSBAN 48® EC confirm the ability of these species to interact with chemicals such as chlorpyrifos. The maximum sorption of chlorpyrifos at equilibrium was observed in Phragmites australis (22%). And, the maximum instantaneous sorption of chlorpyrifos was observed in the dried biomass of Phragmites australis (49%). The epicuticular waxes present on leaves influence photodegradation processes, resulting in a decrease in chlorpyrifos persistence depending on the nature of the extract. The half-life of chlorpyrifos residues in leaf waxes decreased from 34 to 99 minutes when irradiated.     

Leaching of seven pesticides currently used in cotton crop in Mato Grosso State-Brazil

This study aimed to evaluate the leaching of pesticides and the applicability of the Attenuation Factor (AF) Model to predict their leaching. The leaching of carbofuran, carbendazim, diuron, metolachlor, alpha and beta endosulfan and chlorpyrifos was studied in an Oxisol using a field experiment lysimeter located in Dom Aquino-Mato Grosso. The samples of percolated water were collected by rain event and analyzed. Chemical and physical soil attributes were determined before pesticide application to the plots. The results showed that carbofuran was the pesticide that presented a higher leaching rate in the studied soil, so was the one representing the highest contamination potential. From the total carbofuran applied in the soil surface, around 6% leached below 50 cm. The other pesticides showed lower mobility in the studied soil. The calculated values to AF were 7.06E-12 (carbendazim), 5.08E-03 (carbofuran), 3.12E-17 (diuron), 6.66E-345 (alpha-endosulfan), 1.47E-162 (beta-endosulfan), 1.50E-06 (metolachlor), 3.51E-155 (chlorpyrifos). AF Model was useful to classify the pesticides' potential for contamination; however, that model underestimated pesticide leaching.     

Comparative toxicity of chlorpyrifos, diazinon, malathion and their oxon derivatives to larval Rana boylii

Organophosphorus pesticides (OPs) are ubiquitous in the environment and are highly toxic to amphibians. They deactivate cholinesterase, resulting in neurological dysfunction. Most chemicals in this group require oxidative desulfuration to achieve their greatest cholinesterase-inhibiting potencies. Oxon derivatives are formed within liver cells but also by bacterial decay of parental pesticides. This study examines the toxicity of chlorpyrifos, malathion and diazinon and their oxons on the foothill yellow-legged frog (Rana boylii). R. boylii is exposed to agricultural pesticides in the California Central Valley. Median lethal concentrations of the parental forms during a 96 h exposure were 3.00 mg/L (24h) for chlorpyrifos, 2.14 mg/L for malathion and 7.49 mg/L for diazinon. Corresponding oxons were 10 to 100 times more toxic than their parental forms. We conclude that environmental concentrations of these pesticides can be harmful to R. boylii populations.     

A comparison of the persistence in a clay loam of single and repeated annual applications of seven granular insecticides used for corn rootworm control

In May 1983, granular formulations of carbofuran, chlorpyrifos, disulfoton, fonofos, isofenphos, phorate, and terbufos were applied in incorporated bands to duplicate 2 m2 field plots of clay loam. Insecticide concentrations were determined in the bands at 0,1,2,3,4,6,8,10,12,16, and 20 wk. Following spring cultivation, the insecticides were applied to the same plots in 1984 and 1985. In addition, carbofuran was applied to previously untreated plots in 1984 and all 7 materials were applied to previously untreated plots in 1985. Sampling and analysis were carried out as in 1983. Persistence was assessed on the basis of the disappearance rates measured for the 1st 8 wk and of a calculated Effectiveness Potential (the ratio of the average residue in the upper 5 cm of the band at 8, 10 and 12 wk and the published LC95 for western corn rootworm in clay loam soil). Soils treated with carbofuran and isofenphos in 1984 and all soils treated in 1985 were tested for anti-insecticide activity. Soil cores from some carbofuran, chlorpyrifos and terbufos treated plots were sectioned vertically to establish the distribution of the insecticides during 1985. In addition, granular and pure chemical forms of isofenphos and carbofuran were applied at 10 ppm to anti-isofenphos and anti-carbofuran active and control soils (from field plots) maintained at 10 and 20% moisture in the laboratory to assess the effect of formulation and moisture on persistence in active soils. Insecticide concentrations were determined at 0,1,3,7, 10,14,21,28, and 35 days. The persistence of chlorpyrifos, terbufos and phorate was relatively constant over the 3 years and between plots receiving single and multiple treatments. Disulfoton and fonofos behavior was more variable and that of carbofuran and isofenphos was extremely variable. Anti-insecticide activity against carbofuran and isofenphos was detectable 2 wk after an initial application and was still present the following spring. Anti-insecticide activity against fonofos, terbufos sulfoxide, phorate sulfone and disulfoton sulfone was also generated in this soil. Anti-insecticide activity against chlorpyrifos, disulfoton, terbufos and phorate was not present. Carbofuran, chlorpyrifos and terbufos (+ metabolites) present in the upper 5 cm of soil averaged 93, 94 and 94%, respectively, of the total core contents over 12 wk. Significant moisture dependent differences were observed between the behavior of granular carbofuran and granular isofenphos in anti-insecticide active soils.(ABSTRACT TRUNCATED AT 400 WORDS)     

An evaluation of the cytochrome P450 inhibition potential of selected pesticides in human hepatic microsomes

The goal of this work was to study the ability of 18 pesticides to inhibit selective model activities for all major xenobiotic-metabolizing enzymes, namely CYP1A1/2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4. Generally organophosphorus insecticides were the most potent and extensive inhibitors, especially towards CYP1A1/2 (IC₅₀ values of chlorpyrifos, fenitrothion and profenofos ～3 μ M), CYP2B6 (IC₅₀ values of chlorpyrifos and fenitrothion 2.5 μ M), CYP2C8 (fenitrothion 4.3 μ M), CYP2C9 (fenitrothion and malathion 4.8 and 2.5 μ M, respectively), CYP2D6 (chlorpyrifos and phenthoate ～ 3 μ M) and CYP3A4 (chlorpyrifos, fenitrothion and phenthoate 3–4 μ M). Otherwise there were quite considerable differences in potency and extent of inhibition between different organophosphates. Pyrethroids were in general very weak or inactive. Deltamethrin and fenvalerate were potent inhibitors of CYP2D6 (IC₅₀ values of ～ 3 μ M) while lambda-cyhalothrin potently inhibited both CYP2D6 and CYP3A4-mediated activities (IC₅₀'s about 3–4 μ M). Some pesticides caused relatively potent inhibitions sporadically (carbendazim, CYP2D6, IC₅₀ = 12 μ M; atrazine, CYP3A4, IC₅₀ = 2.8 μ M; glyphosate, CYP2C9, IC₅₀ = 3.7 μ M; hexaflumuron, IC₅₀ = 6.0 μ M). With the exceptions of alpha-cypermethrin, cypermethrin, isoproturon, carbaryl and abamectin, most pesticides inhibited relatively potently at least one CYP-selective activity, which may have relevance for potential interactions in occupational exposures and for further studies on the CYP-associated metabolism of respective pesticides.     

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF.     

Cholinesterase activity and effects of its inhibition by neurotoxic drugs in Dictyostelium discoideum

Recently, we found acetylcholinesterase (AChE) activity in the ciliate protozoan Paramecium primaurelia. As in the slime mould Dictyostelium discoideum the presence of a serine esterase was found with strong sequence identity to Torpedo AChE, we extended to D. discoideum the investigation on the characterization and possible functions of cholinesterases (ChEs). In amoeboid cells, histochemical, biochemical, and electrophoresis analyses evidenced both a ChE activity able to hydrolyze the substrate PrTChI, and AChE (E.C. 3.1.1.7.) activity similar to Electrophorus electricus AChE. Conversely, butyrylcholinesterase activity was nearly absent, according to our previous results on P. primaurelia. Moreover, the possibility to utilize D. discoideum in a bioassay for the pre-chemical screening both of moist environments and fresh waters, in relation to the occurrence of the neurotoxic organophosphate drugs, such as "basudin", inhibiting ChE activity, was investigated. Exposure to basudin inhibited propionylcholinesterase (PrChE) activity in a dose-dependent manner in the range 10(-1)-10(-7) M (60% at 10(-4) M), without any significant effect on AChE activity. PrChE activity was inhibited slightly by 10(-5) M eserine, and reduced significantly both by 10(-5) M iso-OMPA and BW284C51, classically used to discriminate the different ChE molecular forms. The effects on cell morphology, cell density, and differentiation were evaluated in cultures exposed to PrTChI 10(-5) M or basudin 10(-4) M for a three-day period. The PrTChI-exposed sample exhibited cell morphology, cell density, ability to aggregate, and to form fruiting bodies similar to the control; whereas, the basudin-exposed sample showed anomalies in cell morphology and lower cell density than the control, together with inability to aggregate.     

N-methylcarbamate pesticides and their phenolic degradation products: hydrolytic decay,sequestration and metal complexation studies

We report on the rates of decomposition of a group of N-methylcarbamate (NMC) pesticides (carbaryl, carbofuran and propoxur) under pre-determined tropical field conditions. Rates of decomposition for three NMCs were determined at pH 7.08 and T?=?20?°C and pH 7.70 and T = 33?°C respectively, as follows: carbaryl (78?days and 69?days); carbofuran (143?days and 83?days) and propoxur (116?days and 79?days). Investigation on methods for removal of NMCs and their phenolic decomposition products shows that activated charcoal outperforms zeolite, alumina, diatomaceous earth, cellulose and montmorillonite clay in the removal of both NMCs and phenols from aqueous solution. Furthermore, metal complexation studies on the NMCs and phenols showed that Fe (III) forms a complex with isopropoxyphenol (IPP) within which the Fe:IPP ratio is 1:3, indicative of the formation of a metal chelate complex with the formula Fe(IPP)₃.     

Comparative sensitivity of Eisenia andrei and Perionyx excavatus in earthworm avoidance tests using two soil types in the tropics

Terrestrial avoidance behavior is proposed as a fast and cost-effective method for assessing effects of pesticides on earthworms. Tropical species however, have rarely been used in avoidance tests. Avoidance tests were performed with Perionyx excavatus, a tropical species, and Eisenia andrei as the standard species, using chlorpyrifos and carbofuran in artificial and natural soil. Earthworms were exposed to concentrations of 1–900 (chlorpyrifos) and 1–32 (carbofuran) mg a.i. kg⁻¹ dry soil in a two-chamber system under tropical conditions (26 ± 2 °C, 48 h). No significant difference was found in the control tests comparing the two soils used, suggesting soil type did not affect the distribution of the worms. The results suggest a higher sensitivity of E. andrei, with EC_50S for the effect on avoidance behavior for both pesticides being a factor of 2–3 lowers than for P. excavatus. Earthworm avoidance tests with local species should therefore be used with caution when applied as a tool for pesticide risk assessment in the tropics. Endpoints generated through avoidance tests in this study are shown to be less sensitive than reproduction and more sensitive than survival. This was further confirmed by literature data available. Earthworm avoidance tests therefore can only replace survival tests as an initial screening tool for risk assessment.