Résumés
Résumé
Des études microanalytiques ont été menées sur des truites Salmo trutta fario, âgées de deux ans, récoltées dans une rivière des Vosges soumise aux retombées des pluies acides et sur des truites témoins récoltées dans une rivière d'Auvergne, non soumise aux pluies acides. La rivière des Vosges est caractérisée par un pH de 5,42 et par une concentration en aluminium de 200 µg/L-1. Notre but étant de déterminer les tissus, cellules et organites cibles de bioaccumulation éventuelle de l'aluminium, nous avons analysé rein, foie, branchie et tractus digestif. Deux méthodes microanalytiques ont été utilisées pour localiser l'aluminium à l'échelle cellulaire et subcellulaire et connaître les éléments avec lesquels il peut être associé; ce sont la spectrométrie de masse par émission ionique secondaire (microscope ionique associé à un système informatisé de traitement d'images) et la spectrométrie des rayons X (microsonde électronique de Castaing associée à un microscope électronique à transmission).
La microanalyse des rein, foie, branchie et tractus digestif montre l'existence de deux processus conduisant à la bioaccumulation de l'aluminium. Le premier, classiquement connu pour d'autres métaux, met en évidence une insolubilisation de l'aluminium sous forme de phosphate, dans des organites limités par une membrane : les lysosomes et les granules pigmentaires des mélanocytes. Le second, démontre la formation de volumineux dépots extra-cellulaires, atteignant 100 µm de long et entraînant la destruction du tissu. Aucune bioaccumulation significative d'aluminium n'a été observée chez des truites témoins, récoltées dans le centre de la France, où l'eau à pH 7.9 est dépourvue d'aluminium.
Mots-clés:
- truite,
- pluies acides,
- aluminium,
- microanalyse (microscopie ionique, spectrométrie des rayons X),
- ultrastructures,
- lysosomes
Abstract
The major harm caused by acidic precipitation is shown by a disappearance of fish. Other factors besides acidity such as aluminium levels are significantly harmful and many studies have shown that aluminium ions are toxic to fish. The only sensible course of action is to investigate the basic mechanisms by which each of the metal pollutants enters and attacks living systems. For this, one needs to be a combination of physicist, chemist, biochemist, physiologist and toxicologist. Investigations on metal bioaccumulation require very sensitive analytical instrumentation. Total analytical methods commonly used are inadequate : absorbed and adsorbed elements cannot be distinguished.
Therefore, interesting information can be obtained by using physical methods of chemical microanalysis : two available microanalytical techniques are particularly suitable, X-ray spectrometry and secondary ion mass spectrometry.
X-ray spectrometry, also called Electron Probe X-ray Microanalysis or Electron Microprobe (EMP) provides a means for studying the local chemical composition and structure of biological specimens. EMP can be used in association with a photon microscope or with a transmission electron microscope allowing the detection of elements at subcellular level.
The Secondary Ion Mass Spectrometry (SIMS), also called Ion Microscopy, allows to visualize, analyse and photograph the microscopical distribution of the stable or radioactive isotopes of the elements present in a histological section. The sensitivity of the method is very high, ranging from 0.1 to 1 ppm. In association with SIMS, a processing of secondary ion images is used.
Two years-old samples of Salmo trutta fario, from wild populations living in acidified waters of Eastern France (near Cornimont in the Vosges moutains) were studied for aluminium detection at cellular and subcellular levels. The acidified waters were characterized by a low pH (5,42) and high aluminium level reaching 200 µg/L-1. Control trouts living in non acidified waters (pH : 7.9 and aluminium free) of central France (near Clermont-Ferrand) were used for comparison. In order top determine the tissues, cells and organelles of a possible aluminium concentration, the following organs were investigated : kidney, liver, gill and digestive tractus, using both microanalytical techniques described above.
In the kidney ion images showed aluminium emission from tubule lysosomes with a ring-shaped localization along the apical border of the epithetial cells; aluminium emissions from the tubule lumen and from the pigment granules of the melanocytes were also observed. Using the electron microprobe, X-ray emission spectra of aluminium associated with phosphorus were obtained from lysosomes and pigment granules. In the liver and in the gills, ion images showed a high aluminium emission from the same organelles and X-ray spectra of aluminium and phosphorus were also obtained. Moreover, in the pyloric caeca, large extracellular deposits of aluminium were detected : they measured about 100 µm in length and were located in places where tissues had been destroyed.
The same structural, ultrastructural and microanalytical investigations were performed on the control trouts from which non aluminium detection was obtained.
In conclusion, two processes appear to be involved in the aluminium accumulation in the brown trout. The first one corresponds to a well known insolubilisation of aluminium phosphate inside the lysosomes, due to an acidic phosphatase enzymatic activity; aluminium is also trapped inside pigment granules. Both of these mechanisms of storage inside membrane-limited organelles, prevent cells from any interior damage. The second one corresponds to the formation of large extracellular deposits which are likely to provoke injuries leading to the tissue destruction. Such data demonstrating basic mechanisms of aluminium accumulation in a fish, could not have been obtained using total analytical methods.
Keywords:
- Trout,
- acid rain,
- aluminium,
- microanalysis (ion microscopy and X ray spectrometry) ultrastructure,
- lysosome
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