Clinical effects and cholinesterase activity changes in workers exposed to phorate (Thimet)

Abstract

Phorate (Thimet), an aliphatic derivative of phospnorus is a hignly toxic insecticide. In order to implement the safety measures, the clinical manifestations and cholinesterase (ChE) activity were evaluated before and after 2 weeks of exposure to this insecticide in 40 male tormulators.

The 2 week's exposure reveal signs and symptoms of toxicity in 60% of the formulators. Gastrointestinal symptoms and lowering of heart rate (bradycardia) were more prominent as compared to the neurological symptoms. A significant depression in plasma ChE activity was observed at the end of 1st week (55%) and 2nd week (71%) as compared to the respective pre‐exposure values. A recovery up to 79% of the pre‐exposure activity of this enzyme was noticed 10 days after cessation of the above exposure.     

Seasonal variations in cholinesterase activity, nerve conduction velocity and lung function among sprayers exposed to mixture of pesticides

Pesticide spraying operation is associated with the increased risk of adverse health effects among sprayers who do not follow safe farm work practices. A study was conducted among pesticide sprayers in North India to evaluate the clinical and subclinical variations in their vital health parameters before and after the pesticide spraying season. Blood cholinesterase levels, pulmonary function test, nerve conduction velocity and self-reported symptoms were studied among 18 eligible and consenting male sprayers. Mean acetylcholinesterase activity was reduced by 55 % in the post-exposure assessment (P?<?0.001) as compared to pre-exposure levels. Mean forced expiratory volume in 1 s was 20 % lower in the post-exposure assessment as compared to the pre-exposure level (P?<?0.05). No significant change was observed in the motor and sensory nerve conduction velocity in the median nerve of sprayers before and after the spraying activity. Also, no significant variation was observed with respect to self-reported symptoms except weakness in arms and legs (P?<?0.05). The significant decline in lung function and acetylcholinesterase level after pesticide exposure reflects the strongly negative effect of exposure to pesticides during spraying activity. More longitudinal studies among pesticide sprayers must be undertaken to further substantiate the cause–effect relationship between pesticide exposure and its subclinical effects. There is a strong necessity to minimise the exposure through the use of personal protective equipment in pesticide sprayers.     

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.     

Effects of the organophosphate insecticide azinphos-methyl on the reproduction and cholinesterase activity of Biomphalaria glabrata

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The snail Biomphalaria glabrata is a freshwater gastropod widely distributed in South America, Central America and Africa. The aim of the present work was to investigate whether azinphos-methyl causes alterations in the reproduction of B. glabrata. To this end, gastropod pigmented specimens were exposed to various concentrations of the insecticide (0.021, 0.5, 2.5, and 5 mg L⁻¹) for either 2 or 14 d. Along 14 d, several reproduction parameters and cholinesterase (ChE) activity were evaluated. In each group, the number of egg masses, the number of eggs per mass, the number of hatchings, the time to hatching, and the survival of the offspring after one month of treatment was evaluated. The results showed that, depending on the concentration and time of exposure, azinphos-methyl induced alterations in the reproduction of B. glabrata. These alterations were mainly represented by a decrease in the number of egg masses, and, in some cases, by a lower number or even the total absence of hatchings. Thus, the gastropods exposed to 2.5 and 5 mg L⁻¹ of azinphos-methyl for 14 d showed ChE inhibitions higher than 35% along time and completely lost their ability to reproduce. On the other hand, exposure to high acute concentrations or exposure to low concentrations for 14 d resulted in ChE inhibition equal to or lower than 35% between 7 and 14 d of treatment and similar alterations in reproduction. These were represented by a decrease in the number of egg masses. At low pestice levels, the number of egg masses and the number of offspring resulted to be more sensitive biomarkers than ChE inhibition. It is concluded that the insecticide azinphos-methyl can cause a decline in the reproductive performance of B. glabrata.     

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.     

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.     

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.     

Response of thermal stressed bobwhite to organophosphorus exposure

Constant and intermittent cold stressed juvenile northern bobwhite (Colinus virginianus) were exposed to chlorpyrifos, an organophosphorus insecticide, following avian dietary LC(50) test procedures. Chlorpyrifos concentrations ranged from 100 to 1000 ppm; ambient temperatures (T(a)) included 35.0 (control), 32.5, 30.0 and 27.5 degrees C. Survival decreased significantly under constant cold conditions. Non-survivors averaged 33% weight loss, but survivor weight dropped with increasing dose and decreasing T(a). Control quail at all temperatures maintained a constant body temperature (T(b)); however, T(b) of dosed quail fell with increasing dose and decreasing T(a). Non-surviving cold stressed quail had higher cholinesterase (ChE) activity that non-survivors at 35 degrees C; in addition, dead quail failed to average the 50% inhibition of ChE used to implicate a ChE depressant in death. Daily exposure of quail to 12 h of warmth increased survival. Survivors of intermittent cold stress experienced less weight loss than constant cold survivors due to reduced metabolic costs. Synergistic effects were evident in juvenile northern bobwhite exposed simultaneously to chlorpyrifos and cold stress; however, mortality and certain sublethal effects were mitigated when cold exposure was intermittent.     

Impact of insecticides on the American robin (Turdus migratorius) in a suburban environment

The purpose of this study was to examine the effects of diazinon and acephate applications to ornamental trees on the breeding behaviour and productivity of the American robin and to measure the effect of repeated chlorpyrifos spraying on robin productivity. No cases of adult or juvenile mortality were recorded, and nestling production was not affected. Plasma cholinesterase (ChE) activity in females exposed to diazinon or acephate was significantly lower than that in controls. However, there were no pronounced changes in parental care. The absence of a significant impact on behaviour despite the marked change in plasma ChE levels may have been due to one of several factors, i.e. (1) the insecticide had not reached the brain 18 to 24 h after spraying, and (2) exposure was insufficient to produce behavioural changes. The use of diazinon and acephate on ornamental trees should not be harmful to robins. There was a negative correlation between nest productivity and the number of lawns surrounding the nest treated with chlorpyrifos once or more in the preceding 2 years. The decline in earthworm numbers and their reduced biomass due to repeated chlorpyrifos use may account for the lower productivity recorded during the nesting season.     

Neurotoxic effect and metabolic responses induced by a mixture of six pesticides on the earthworm Aporrectodea caliginosa nocturna

The effects of a mixture of insecticides and/or fungicides at different environmental concentrations were investigated on a Aporrectodea caliginosa nocturna population. This laboratory experiment was carried out in order to reproduce Gaillac (France) vineyard conditions. Neurotoxicity (cholinesterase), metabolisation (glutathione-S-transferase) and oxidative stress (catalase) enzymes were studied as biomarkers in earthworms after short-term exposure in terraria. The aim was to observe the global effects of pesticide exposure, as in a vineyard, rather than focus on each isolated biomarker variation, or on each compound's impact. ChE activity was inhibited after a few days of insecticide and/or fungicide exposure, indicative of a neurotoxic effect in earthworms. The significant increase in GST and CAT activities revealed the metabolisation of these products resulting in the production of reactive oxygen species. After a long period of exposure or high concentrations, earthworms were physiologically damaged: they could not cope with the high toxicity (cellular dysfunction, protein catabolism...). Chemical analysis showed that pesticide bioaccumulation in earthworm tissues, even in those exposed to the highest concentrations and for the longest periods, was very low (under LOD) or absent. However, the study of pesticide residues in terraria after 34 days in a climate chamber suggested that earthworms participate in soil pesticide breakdown.     

Effects of azinphos-methyl exposure on enzymatic and non-enzymatic antioxidant defenses in Biomphalaria glabrata and Lumbriculus variegatus

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The oligochaete Lumbriculus variegatus and pigmented and non-pigmented specimens of the gastropod Biomphalaria glabrata are freshwater invertebrates that have been recommended for contamination studies. Recently, it has been shown that L. variegatus worms exhibit a higher cholinesterase (ChE) activity and a greater sensitivity to in vivo ChE inhibition by azinphos-methyl than pigmented B. glabrata snails. The aims of the present study were (1) to investigate if, in addition to its anticholinesterase action, azinphos-methyl has also pro-oxidant activity in L. variegatus and B. glabrata, and (2) to examine if species that are highly susceptible to the neurotoxic effects of organophosphates also suffer a greater degree of oxidative stress. Therefore, total glutathione (t-GSH) levels and activities of cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PDH) were measured in the whole body soft tissue of organisms exposed for 48 and 96 h to a level of azinphos-methyl that produces 50% of inhibition on ChE. Results showed different patterns of antioxidant responses between the gastropods and the oligochaetes, and even between the two phenotypes of gastropods: (1) in exposed L. variegatus t-GSH levels increased and CAT and SOD activities decreased with respect to control organisms, (2) in pigmented gastropods, SOD decreased while CAT transiently diminished, and (3) in non-pigmented gastropods, SOD activity showed a biphasic response. GST and G6PDH were not altered by azinphos-methyl exposure. Of note, t-GSH levels were 4-fold times higher in L. variegatus than in both phenotypes of B. glabrata. This may suggest that GSH could play a more important role in antioxidant defense in L. variegatus than in B. glabrata.     

Controlled exposures of volunteers to respirable carbon and sulfuric acid aerosols.

Respirable carbon or fly ash particles are suspected to increase the respiratory toxicity of coexisting acidic air pollutants, by concentrating acid on their surfaces and so delivering it efficiently to the lower respiratory tract. To investigate this issue, we exposed 15 healthy and 15 asthmatic volunteers in a controlled-environment chamber (21 degrees C, 50 percent relative humidity) to four test atmospheres: (i) clean air; (ii) 0.5-microns H2SO4 aerosol at approximately 100 micrograms/m3, generated from water solution; (iii) 0.5-microns carbon aerosol at approximately 250 micrograms/m3, generated from highly pure carbon black with specific surface area comparable to ambient pollution particles; and (iv) carbon as in (iii) plus approximately 100 micrograms/m3 of ultrafine H2SO4 aerosol generated from fuming sulfuric acid. Electron microscopy showed that nearly all acid in (iv) became attached to carbon particle surfaces, and that most particles remained in the sub-micron size range. Exposures were performed double-blind, 1 week apart. They lasted 1 hr each, with alternate 10-min periods of heavy exercise (ventilation approximately 50 L/min) and rest. Subjects gargled citrus juice before exposure to suppress airway ammonia. Lung function and symptoms were measured pre-exposure, after initial exercise, and at end-exposure. Bronchial reactivity to methacholine was measured after exposure. Statistical analyses tested for effects of H2SO4 or carbon, separate or interactive, on health measures. Group data showed no more than small equivocal effects of any exposure on any health measure.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of widely used pharmaceuticals and a detergent on oxidative stress biomarkers of the crustacean Artemia parthenogenetica

Pharmaceuticals are continuously dispersed into the environment as a result of human and veterinary use, posing relevant environmental concerns. The present paper reports the acute toxic effects of three therapeutic agents (diazepam, clofibrate and clofibric acid) and a detergent, sodium dodecylsulphate (SDS), to the hypersaline crustacean Artemia parthenogenetica. This study specially focused on oxidative stress parameters, namely (1) total and selenium-dependent glutathione-peroxidase (GPx), (2) glutathione reductase (GRed), (3) total superoxide dismutase (SOD), and (4) glutathione-S-transferases (GSTs). The effects of tested substances on lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and soluble cholinesterases (ChE) were also investigated. Diazepam caused a significant inhibition of ChE (LOEC = 7.04 mg/l) and total GPx activities. SDS was responsible for a decrease in the activity of both ChE (LOEC = 8.46 mg/l) and GRed (LOEC = 4.08 mg/l). Both fibrates (clofibrate and clofibric acid) were responsible for significant decreases in Se-dependent GPx, with LOEC values of 176.34 and 3.09 mg/l, respectively. Clofibrate also caused a slight increase of TBARS content of A. parthenogenetica homogenates. These results indicate that the exposure to all the tested compounds induced alterations on the cellular redox status in A. parthenogenetica. In addition, diazepam was shown to have the capability of interfering with A. parthenogenetica neurotransmission, through the inhibition of ChE.     

Enzymatic biomarkers in the crab Carcinus maenas from the Minho River estuary (NW Portugal) exposed to zinc and mercury

Zinc and mercury are common contaminants of estuaries and coastal ecosystems where they may induce adverse effects on the biota. Carcinus maenas is a key-species in several European estuaries, living in close association with the sediment where a considerable number of environmental contaminants, including zinc and mercury may accumulate. In the present study, the acute effects of zinc and mercury on C. maenas from the Minho River Estuary (NW Portugal) were investigated by using the activity of the enzymes cholinesterase (ChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GST) as effects' criteria. Crabs were exposed for 96h to several concentrations of Zn(2+) (0, 1.84, 3.71, 7.39 and 14.79mg/l) or Hg(2+) (0, 0.09, 0.19, 0.37 and 0.74mg/l). The choice of Zn(2+) test concentrations was based on the LC50 value (14.86mg/l) determined in a first part of the study, while the choice of Hg(2+) concentrations was based on values from the literature. At the end of the bioassays, eye, muscle and hepatopancreas tissues were isolated and used for ChE, LDH and GST determinations, respectively. Zinc significantly inhibited ChE activity (p<0.05, EC50=14.68mg/l), caused significant alterations of GST activity (p<0.05) and induced LDH activity (p<0.05) at the exposure of 14.79mg/l. Mercury significantly inhibited ChE activity (p<0.001, LOEC=0.09mg/l, EC50=0.235mg/l) and increased both GST (p<0.05, LOEC=0.774mg/l) and LDH activities (p<0.05, LOEC=0.119mg/l). These results suggest that both metals interfere with cholinergic neurotransmission in C. maenas by inhibiting ChE activity. In addition, they also interfere with GST activity and this may reduce the capacity of detoxification of some chemicals and/or to increase the probability of oxidative stress to occur. Furthermore, both metals increase LDH activity, suggesting an interference with energy production pathways. Therefore, the presence of zinc and mercury in estuaries at concentrations in the mg/l or mug/l range, respectively, may have a negative impact on C. maenas.     

Biochemical and locomotor responses of Carcinus maenas exposed to the serotonin reuptake inhibitor fluoxetine

The aim of this study was to assess the effects of the widely used anti-depressant fluoxetine on behaviour (locomotion), moulting, neuromuscular transmission, energy production and anti-oxidant defences’ efficiency of the epibenthic crab Carcinus maenas. Crabs were individually exposed to fluoxetine concentrations for 7 d. Effects on locomotion were assessed at the end of the exposure using an open field test adapted to C. maenas in the present study. Tissue samples were later collected to evaluate fluoxetine effects on physiological functions using the activity of key enzymes and other parameters as biomarkers, namely: N-acetyl-β-glucosaminidase (NAGase) in the epidermis (moulting) and the hepatopancreas; cholinesterases (ChE) in muscle (neuromuscular cholinergic transmission); NADP⁺-dependent isocitrate dehydrogenase (IDH) and lactate dehydrogenease (LDH) in muscle (energy production); glutathione S-transferases (GST) in hepatopancreas (biotransformation and oxidative stress system); glutathione reductase (GR), and glutathione peroxidade (GPx), total glutathione levels (TG) and lipid peroxidation levels in the hepatopancreas (anti-oxidant defences and oxidative damage). Because no information on C. maenas NAGase activity was previously available, its variation during the moult cycle was also investigated. The results showed that locomotion was significantly increased at fluoxetine concentrations equal or above 120 μg L⁻¹, with animals spending more time moving, walking longer distances than controls. Levels of NAGase activity were found to vary in relation to C. maenas moult cycle, but no alterations were observed after exposure to fluoxetine. Significant increases in the activity of ChE, GST and GR enzymes, and the levels of TG were found, with a lowest observed effect concentration (LOEC) of 120 μg L⁻¹. Effects on locomotion were significantly and positively correlated to those induced on ChE activity. The results raise concern when hypothesising conditions of chronic exposure in the wild.     

Assessing dimethoate contamination in temperate and tropical climates: potential use of biomarkers in bioassays with two chironomid species

This study was conducted to investigate the potential use of biomarkers in bioassays with chironomids to assess contamination by pesticides in temperate and tropical climates. Two species of midge were studied, the widespread Chironomus riparius and the tropical Kiefferulus calligaster (Kieffer, 1911). Preliminary studies included investigations of the effects of temperature on larval development and the influence of larval age on normal variability of cholinesterase (ChE) and glutathione S-transferase (GST) activities and protein content. In the second phase, the influence of two abiotic factors particularly important in tropical conditions (temperature and oxygen concentration) and of the organophosphorous (OP) insecticide dimethoate on biochemical and conventional endpoints was investigated. Results showed that K. calligaster is morphologically and physiologically similar to C. riparius and for both, the time of larval development decreases with the increase of temperature. Moreover, 3rd and 4th instars appeared to be the most suitable for biomarkers determinations. ChE activity seems to be valuable biomarker regarding temperature and dissolved oxygen (DO) variations, while some caution should be taken when using GST as an environmental biomarker, since it shows some dependence of these parameters. C. riparius was more sensitive to dimethoate than K. calligaster suggesting that the use of bioassays with the former species in tropical conditions may overestimate the toxicity of OP pesticides to autochthonous species. When testing sub-lethal effects of dimethoate to C. riparius, ChE activity showed to be a very sensitive parameter detecting significant effects at the lowest concentration that caused emergence delay of larvae, suggesting that it is an ecologically relevant parameter.     

Effects of organophosphate compounds on a soil protist, Colpoda inflata (Ciliophora, Colpodidae)

Many investigations on protists indicate that they play an important role in agricultural soils. We have tested the effects of three organophosphate (OP) pesticides, basudin, cidial, and fenix, on the soil ciliate Colpoda inflata, and examined its viability, fission rate, ability to excyst and extrude macronuclear chromatin into cytoplasm. Exposure to these OPs caused a dose-dependent effect on cell viability, and significantly reduced the mean fission rate at a concentration of 1/10(5) v/v. After exposure of resting cysts to 1/10(5) v/v or 1/10(6) v/v concentrations of basudin or cidial, the number of excysted cells was significantly lower than that of the controls. Conversely, exposure to a 1/10(5) v/v fenix concentration did not affect excystment and exposure to 1/10(6) v/v was found to promote excystment. Moreover, exposure to these OPs (1/10(4) v/v or 1/10(5) v/v) interferred with the ability to extrude macronuclear chromatin. The median lethal concentration in 60 min for each OPs tested was at least a hundred times lower than the doses recommended by the manufacturer. Finally, as the inhibition of cholinesterase (ChE) activity is the first target of OPs, the presence of ChE activity was checked in C. inflata. Three ChE activities were found, hydrolyzing the substrates acetyl-beta-methyl thiocholine iodide, propionyl thiocholine iodide and butyryl thiocholine iodide, that appeared to be very low and not inhibited by OP-exposure.     

Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.     

Effects of nitrogen dioxide on leaf chlorophyll and nitrogen content of soybean

One-month-old soybean (Glycine max [L.] Merrill), cultivar 'Williams', plants were exposed to nitrogen dioxide (0.1, 0.2, 0.3 and 0.5 ppm) and carbon filtered air (control), 7 h per day, for 5 days, under a controlled environment. Leaf chlorophyll content (Ch a, Ch b, and total Ch content) and foliar nitrogen content (%N) were determined before and after the exposure. The influence of NO(2) treatments up to 0.3 ppm on leaf chlorophyll content was negligible although a stimulatory effect was evident in Ch a and total Ch content with 0.2 ppm NO(2). Marked decline in Ch content was observed with 0.5 ppm treatment; the reductions in Ch a and total Ch were 45% and 47%, respectively. Foliar-N contents of plants treated with 0.2 and 0.3 ppm NO(2) were higher than the control; plants exposed to 0.5 ppm NO(2) showed a 41% reduction in foliar-N compared to pre-exposure values.