Ana M. Pechen de D'Angelo

Biomarkers of effect in toads and frogs.

Amphibians are good bioindicators of environmental pollution due to their susceptibility to chemicals during their freshwater cycles. The effects of environmental pollution, together with changes in human activity and climate, have contributed to the reduction in the amphibian population over recent decades. However, toxicological research on amphibians has been rather scarce compared with that on other vertebrates. In this article we review the biochemical alterations underlying xenobiotic action and/or the detoxifying responses described for anuran species, with the aim of establishing possible biomarkers of effect. During the embryonic development of anurans, morphological and behavioural alterations are the effects most frequently cited in connection with chemical exposures. However, such biomarkers have a low sensitivity and are unspecific compared with biochemical alterations. Some primary pesticide targets, in particular cholinesterases for organophosphates and carbamates, have been evaluated. Esterases change seasonally and with the stage of development, and their sensitivity to anticholinesterase agents varies between species. Thus their use as biomarkers in anurans must be carefully analysed. Enzymes and endogenous compounds related to oxidative metabolism may also be used as biomarkers of effect. Glutathione pool, glutathione-S-transferases and metallothioneins respond in different ways to pesticides and heavy metals in anuran embryos and tadpoles. Mixed-function oxidases, in turn, are less developed in amphibians, and show a reduced induction in response to pesticide exposures. Endogenous polyamine levels are also proposed as good age-related biomarkers of damage. Finally, molecular biomarkers related to receptor binding, signal transduction and genetic response have gained increasing relevance, as they have been implicated in the fertilisation process and the earliest events in anuran development. The identification of transcription factors associated with the exposure of amphibians to xenobiotics as well as other alterations in hormone signalling appears highly promising. However, these techniques are likely to complement other methods. In conclusion, the use of several biomarkers with multiple endpoints is needed to link exposure to response and to provide better predictive tools for the environmental protection of endangered anuran species.

Thiols and polyamines in the potentiation of malathion toxicity in larval stages of the toad Bufo arenarum.

Treatment with exogenous spermidine enhanced acute malathion toxicity during larval development of the toad Bufo arenarum Hensel. The polyamine was rapidly incorporated in the larvae with a subsequent metabolization to putrescine and spermine, which were excreted to the media. Endogenous polyamine levels were not changed by either spermidine or malathion treatments. However, 0.5-mM spermidine modified malathion uptake and bioavailability increasing the concentration of the xenobiotic in the larvae. The amount of reduced thiols was decreased by both compounds, but the depletion was insufficient to induce cytotoxicity. The oxidative degradation of polyamines competes for the pool of reduced glutathione used in the conjugation of malathion in the larvae, thus leading to the reported potentiation of toxicity. Our results suggest that exposure to thiols-depleting agents may induce alteration of organophosphate degradation in amphibian larvae.

Cholinesterase activities in developing amphibian embryos following exposure to the insecticides dieldrin and malathion

The exposure of toad (Bufo arenarum) embryos to dieldrin2 and malathion3 produced an inhibition of acetyl (AChE) and butyryl (ChE) cholinesterase. Dieldrin treatment induced hyperactivity in the swimming larvae; malathion showed no detectable behavioral effects.In vivo adsorption of dieldrin by the oocyte produced delayed toxicity in the embryo.In vitro inhibition of AChE was not significant; 47.3 mg/L of malathion in the medium interrupted embryogenesis, but 0.47 mg/L permitted the embryo to complete their development without the appearance of any observable malformations.

Toxicological and biochemical response to azinphos-methyl in Cydia pomonella L. (Lepidoptera: Tortricidae) among orchards from the Argentinian Patagonia

BACKGROUND Azinphos-methyl is the main insecticide used to control codling moth on apple and pears in Northern Patagonia. The aim of this study was to evaluate the toxicological and biochemical response of diapausing larvae of codling moth in orchards subjected to different insecticide selection pressure. RESULTS Dose-mortality assays with azinphos-methyl in diapausing larvae of Cydia pomonella L. showed significant differences between the LD(95) from a population collected in one untreated orchard (2.52 microg moth(-1)) compared with that in a laboratory-susceptible population (0.33 microg moth(-1)). Toxicity to azinphos-methyl in field populations of diapausing larvae collected during 2003-2005 was evaluated by topical application of a discriminating dose (2.5 microg moth(-1)) that was obtained from larvae collected in the untreated orchard (field reference strain). Significantly lower mortality (37.71-84.21%) was observed in three out of eight field populations compared with that in the field reference strain. Most of the field populations showed higher esterase activity than that determined in both the laboratory susceptible and the field reference strains. Moreover, there was a high association between esterase activity and mortality (R(2)=0.64) among the field populations. On the other hand, a poor correlation was observed between glutathione S-transferase activity and mortality (R(2)=0.33) among larvae collected from different orchards. CONCLUSIONS All the field populations evaluated exhibited some degree of azinphos-methyl tolerance in relation to the laboratory susceptible strain. Biochemical results demonstrated that esterases are at least one of the principal mechanisms involved in tolerance to this insecticide.

Enhanced esterase activity and resistance to azinphosmethyl in target and nontarget organisms

The organophosphorous compound azinphosmethyl (AzMe) is applied extensively in northern Patagonia (southern Argentina) to manage codling moths (Cydia pomonella). This area is irrigated by fast-flowing channels that provide a favorable habitat for many species, including amphipods (Hyalella curvispina) and a field-mixed population of black flies (Simulium bonaerense, Simulium wolffhuegeli, and Simulium nigristrigatum). In the present study, AzMe susceptibility and carboxylesterase (CarbE) activity from both insecticide-exposed and nonexposed field populations were studied. The median lethal dose determined in codling moths from an insecticide-treated orchard was significantly higher (3.48 microg/insect) than that observed in those from an untreated orchard (0.69 microg/insect). Similarly, the median lethal concentration (LC50) determined in black flies collected from the treated area (0.021 mg/L) was significantly higher than that recorded in those from the untreated site (0.011 mg/L). For amphipods, both a subpopulation susceptible to AzMe (LC50, 1.83 microg/L) and a resistant one (LC50, 390 microg/L) were found in the treated area. Both subpopulations were more resistant to AzMe than the population from the untreated site (LC50, 0.43 microg/L). Significant differences (p < 0.001) in CarbE activities were observed between populations from pesticide-treated and untreated areas. Mean activities +/- standard deviation from treated and untreated sites were 0.21 +/- 0.16 and 0.016 +/- 0.008 micromol/min/mg protein, respectively, for codling moths; 2.17 +/- 1.71 and 0.81 +/- 0.35 micromol/min/mg protein, respectively, for black flies; and 0.27 +/- 0.10 and 0.14 +/- 0.07 micromol/min/mg protein, respectively, for amphipods. The results suggest that enhanced CarbE activity is one of the mechanisms that provide AzMe resistance in H. curvispina, Simulium spp., and C. pomonella populations from the insecticide-treated areas.

Effect of malathion on Bufo arenarum hensel development—I: Esterase inhibition and recovery

Newly fertilized Bufo arenarum Hensel embryos were exposed continuously or for a brief period (72-120 hr) to malathion (44 ppm) and then resuspended in amphibian Ringers solution. Continuous exposure depressed acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) activities. The activities of the three enzymes in embryos treated for 72 hr recovered after a delay of 24 hr, but these enzymes showed different rates of recovery in embryos treated for 120 hr. Acrylamide disc electrophoresis showed several bands of esterase activity in control embryos. Continuous exposure to malathion abolished all esterase activity within 48 hr, but if the exposure continued new bands of esterase activity appeared at 120 hr of exposure. The zymograms of embryos exposed for 72 or 120 hr to malathion and then transferred to uncontaminated medium for 120 hr were similar to that of control embryos.

Risk assessment of Magnacide® H herbicide at Río Colorado irrigation channels (Argentina). tier 3: Studies on native species

We evaluated the potential environmental risk of the herbicide Magnacide (Baker Petrolite, TX, USA) using native species from Argentina, representing the ecosystem at the Irrigation Corporation (CORFO) channels at the Colorado River mouth, Buenos Aires, Argentina. Six species including fish, toads, snails, crustaceans, and insects were selected to perform studies on acute toxicity and repeated exposure effects. Magnacide H susceptibility ranking was Bufo arenarum (lethal concentration 50 [LC50] = 0.023 mg/L), Onchorhynchus mykiss (LC50 = 0.038 mg/L), Heleobia parchappii (LC50 = 0.21 mg/L), Hyalella curvispina (LC50 = 0.24 mg/L), Simulium spp. (LC50 = 0.60 mg/L), and Chironomus spp. (LC50 = 2.83 mg/L). The risk limit of 10th percentile (0.013 mg/L) determined by probit analysis on sensitivity distribution was similar to the one calculated from literature data. Risk assessment based on field application data suggested lethal exposures for more than 70 to 90% of the species up to 20 km downstream from the application point. Repeated exposures to Magnacide H of amphibian larvae at the lowest-observed-effect concentration caused some effects during the first exposure, but without cumulative effects. Amphipods were insensitive to repeated exposures, showing no cumulative effects. Whether short-term exposures may result in long-term sublethal effects on the organisms life history was not addressed by these laboratory tests. In conclusion, tier 3 studies indicate that Magnacide H application schedule is extremely toxic for most native species at CORFO-Rio Colorado channels, representing a high potential risk in the environment. The real environmental impact must be addressed by field studies at tier 4 giving more information on population effects and communities.

DIELDRIN EFFECTS ON PHOSPHOLIPID AND PHOSPHOINOSITIDE METABOLISM IN BUFO ARENARUM OOCYTES

Abstract 1. [ 32 P]Pi incorporation into phosphoinositides was greater in control oocytes than in Dieldrin-treated ones, while in phospholipids [ 32 P]Pi incorporation was similar in both cases. 2. PA and PI were preferentially labelled in Dieldrin-treated oocytes.PC and PE labelling showed that the metabolic flux was directed towards them only in control cells. 3. After 1 min of carbachol stimulation, polyphosphoinositides labelling decreased in both groups. In control but not in Dieldrin-treated cells PIP and PIP 2 radioactivity exceeded the zero time value after 7 min of stimulation. 4. Results suggest that after agonist-dependent hydrolysis of phosphoinositides, PIP and PIP 2 kinases are activated in control but not in Dieldrin-treated oocytes.

Dieldrin modifies the hydrolysis of PIP2 and decreases the fertilization rate in Bufo arenarum oocytes

Abstract Carbachol treatment in Bufo arenarum oocytes decreases the radioactivity in [ 32 P]PIP 2 in the following 20 min after stimulation and increases the [ 3 H]glycerol labeling of 1,2-DAG at 1 min of stimulation. On the contrary, in Dieldrin treated oocytes carbachol stimulation produces an increase in [ 32 P]PIP 2 labeling without changes in [ 3 H]1,2-DAG radioactivity. The sustained hydrolysis of PIP 2 observed in Control oocytes is necessary to generate the intracellular second messengers which initiate the fertilization pathway. The lack of response to muscarinic stimulation in Dieldrin treated oocytes, may be associated with an early activation of PIP 2 -PLC by the insecticide, producing a depletion of the PIP 2 pool previous to the stimulation with carbachol. These changes take place simultaneously with a decrease in the ability of Bufo arenarum oocytes to be fertilized in vitro , suggesting a correlation between impairment in the PIP 2 cascade and a decrease in the fertilization rate.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.