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.     

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.     

The measurement of cholinesterase activities as a biomarker in chub (Leuciscus cephalus): the fish length should not be ignored

Biomarkers are early warning systems of the exposure of aquatic organisms to pollutants. Among them, the measurement of the cholinesterase (ChE) activities in fish muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides. As such it has been used in numerous field studies both in marine and continental waters. Cyprinids (chub, Leuciscus cephalus) were sampled in river sites (France) in relatively clean and polluted areas. We performed the statistical analysis of the ChE activities and we generally observed a statistical relationship between ChE activities and fish length, the larger fish having the lower ChE activities. We concluded that the great majority of the significant differences in ChE activities between sites could be due in fact to differences in fish length between field samples. We stress the importance of taking into account the fish length whenever differences in ChE levels between field sites must be interpreted.     

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.     

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.     

Effects of complex waste mixtures on hepatic monooxygenase activities in brown bullheads (Ictalurus nebulosus)

Hepatic MFO components (cytochrome P-450, cytochrome b(5), and ethoxyresorufin O-deethylase, EROD) were measured in brown bullheads (Ictalurus nebulosus) inhabiting a creek receiving a complex mixture of organics and trace metals. The activities of these same enzymes were also measured in bullheads from an uncontaminated reference site to assess the relative ability of MFO parameters to serve as a biomarker of aquatic pollution. Bullheads analyzed from the polluted site had lower hepatic microsomal P-450 (p < 0.01) concentrations and similar EROD activities per mg protein as compared to bullheads from the reference site. However, analysis of enzyme turnover ratios revealed greater EROD activity per mg cytochrome P-450 (p < 0.05) in the fish from the polluted site. No difference in cytochrome b(5) activities were observed between the two groups. As compared to the reference site, bullheads collected from the polluted creek had an increased occurrence of lip and lower jaw lesions and liver damage, including elevated liver/body weight ratios. Accordingly, the monooxygenase activities measured in this study were not reliable indicators of chemical pollution or contaminant stress in bullheads in the polluted creek. Further research is needed concerning contaminant interactions, particularly among organic pollutants and metals and their effects on monooxygenase activities.     

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.     

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.     

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.     

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.     

Cholinesterase characterization and effects of the environmental contaminants chlorpyrifos and carbofuran on two species of marine crabs,Carcinus maenas and Pachygrapsus marmoratus

Among the most frequent targets for toxic effects of modern pesticides, namely organophosphates and carbamates, one may find cholinesterases (ChEs). ChEs exist in a wide variety of animals and have been used actively to discriminate among the environmental effects of different pollutant groups, including the aforementioned pesticides. This study had three purposes, namely (i) identifying the ChE forms present in tissues (eyes and walking legs muscle) of two crab species, Carcinus maenas and Pachygrapsus marmoratus; to (ii) determine the in vitro toxicological effects, and (iii) compare the sensitivity of such enzymatic forms towards commonly used anti-ChE pesticides, namely the organophosphate chlorpyrifos and the carbamate carbofuran. Our results showed that there was not a clear preference for any of the tested substrates in any of the tissues from both species. Furthermore, the ChE activity was almost completely suppressed following incubation with eserine and with the specific inhibitor BW284C51 in all tissues from both species. In vitro exposure to chlorpyrifos promoted a significant decrease in ChE activity in both species. Furthermore, the ChE activity was completely suppressed following incubation with carbofuran and chlorpyrifos. These results suggest that the major ChE forms present in tissues of both crab species show intermediate structural properties and activity patterns, halfway between classic acetylcholinesterase and pseudocholinesterases. However, the sensitivity of the found forms towards ChE inhibitors was established, and the responsiveness of such forms towards common anti-ChE chemicals was established. Both tested species seem to be promising test organisms to be used in marine and coastal scenarios of putative contaminations by anti-ChE chemicals, considering the here reported patterns of response.

     

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.     

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.     

EROD activity and biliary fluorescence in Schroederichthys chilensis (Guichenot 1848): biomarkers of PAH exposure in coastal environments of the South Pacific Ocean

Schroederichthys chilensis is a common shark that lives in Chilean coastal environments. In this work, the relationship between liver 7-ethoxyresorufin-O-deethylase dealkylation (EROD) activity and Fluorescent Aromatic Compounds (FAC) in bile of S. chilensis sampled in three bays with different degrees of pollution were performed including a reference area. Sixty individuals were collected, 20 for each site; (10 males and 10 females per site) livers and bile samples were obtained and immediately frozen. EROD activity and FAC were measured according to three standard methods. EROD activity and FAC were higher in polluted areas than in the reference area. Synchronous Fluorescence Spectra of the bile from the fish collected at the most polluted area showed a peak at 347nm representing a metabolite corresponding to 1-hydroxypyrene. The low EROD activity in the reference area is likely related to the low level of PAH in sediments. We propose that this species is a good indicator of exposure to FACs, since it presents a series of characteristics that make it suitable for monitoring PAH exposure in coastal zones.     

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.     

Biomarkers of a native fish species (Cnesterodon decemmaculatus) application to the water toxicity assessment of a peri-urban polluted river of Argentina

Environmental monitoring by means of biomarker parameters assessed in different species is a useful tool. It has the advantage of providing a quantitative response as well as valuable information of ecological relevance on the chronic adverse effects caused by water pollution. The aim of this study was to assess the response of biochemical and physiological parameters of Cnesterodon decemmaculatus, a native teleost, simultaneously caught in two sites of Reconquista river, a highly polluted peri-urban river. This study compared the measured parameters with that of specimens of the same species captured in an unpolluted body water, and correlated the detected changes with the physicochemical profile of the water at each site. A comparison was made of selected parameters of gill, brain and liver and of somatic indices of fish collected from polluted and reference sites. The main parameters whose changes allowed to discriminate between sampling sites were gill (Na(+),K(+))-ATPase, brain acetylcholinesterase (AChE) and liver aminotransaminases activities; tissues' protein content and liver somatic index (LSI) were also sensitive biomarkers in brain and liver, respectively. The results showed that the response of the measured biomarkers allowed for the differentiation of sampling sites according to their water quality and confirmed that Cnesterodon decemmaculatus may be a useful test organism for the biomonitoring of freshwater environments. In addition, the simultaneous measurement of the physicochemical parameters of the water samples showed a good correspondence between the biomarkers responses and the environmental chemical stress conditions.     

Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes

The increase of ethoxyresorufin dealkylation (EROD) and the inhibition of acetylcholinesterase (AChE) as biomarkers have been commonly used in vertebrates for the persistent organic pollutants (POPs) biomonitoring of aquatic environments, but very few studies have been performed for invertebrates. Previous researches demonstrated the interference due to some chemicals on EROD and AChE activities of the freshwater bivalve Zebra mussel (Dreissena polymorpha) in laboratory and field studies, showing its possible use for the screening of POP effects. We investigated the contamination of the Italian sub-alpine great lakes (Maggiore, Lugano, Como, Iseo, Garda) by the biomarker approach on Zebra mussel specimens collected at 17 sampling sites with different morphometric characteristics and anthropization levels. Results showed a homogeneous contamination of AChE inhibitors in Lake Garda, Maggiore, Como and Iseo with values ranging from 0.5 to 3 nmol/min/mg proteins and with an average inhibition of about 66% to controls. The planar compounds pollution, able to activate the EROD activity, seems higher in some sampling stations of Lake Garda, Como and Iseo (2-4 pmol/min/mg proteins) than that measured in Lake Lugano (1.5-3 pmol/min/mg proteins). On the contrary, the enzyme activity in Lake Maggiore showed an interesting opposite effect of AhR-binding compounds and trace metals. Finally, the possible use of Zebra mussel specimens maintained at laboratory conditions as controls against the selection of the less polluted sampling site is discussed.     

Morphological and haematological parameters in the Algerian mouse (Mus spretus) inhabiting an area contaminated with heavy metals

In the present study morphological (ratio of body weight to body length, weight of internal organs) and haematological parameters (cells count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration) were used for monitoring potential effects of metal contaminants on the Algerian mouse (Mus spretus). The study also provided the opportunity to evaluate the use of morphological and haematological characteristics as indicators of a metal stress. Data were taken from a riverside population inhabiting a heavy metal-contaminated site and compared with those collected from a reference population of the same species. With the exception of non-reproductive males, all mice weighed less in the metal-polluted site as compared to the reference site. Spleen and kidney masses were also less in contaminated mice but mean haemoglobin concentration was greater in the polluted group versus controls. Responses of mice from the polluted area may indicate physiological stress due to diminished environmental quality. Results also suggest that M. spretus may be used as an indicator in pollution monitoring.     

Comparative study of biochemical and immunological biomarkers in three marine bivalves exposed at a polluted site

A battery of biochemical and immunological biomarkers used for pollution assessment were measured for first time in the clams Venus verrucosa and Callista chione and were compared with those of the mussel Mytilus galloprovincialis, a well-established indicator organism utilized in numerous environmental monitoring programs. Clams and mussel were transplanted at a polluted and a reference site or maintained at the laboratory. Among biochemical biomarkers, acetylcholinesterase did not differ at the polluted site in all species, but there was a significant difference between the mussel and the clams, glutathione S-transferase showed a clear inhibition at the polluted site in all species and a significant difference between the two clams was also indicated, while catalase activities were increased only in V. verrucosa at the polluted site and not in mussel or the other clam. Immunological biomarkers responses were also pronounced at the polluted site. Lysozyme activity was species-dependent whereas respiratory burst activity measured as luminol-dependent chemiluminescence (CL) was site and stimulus dependent, and it was evident in M. galloprovincialis and V. verrucosa and not in C. chione. Further investigation focused on biochemical and immunological biomarkers related with the oxidative mechanisms in clams will strengthen and expand their use as bioindicators for pollution assessment.     

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.