Cytology of pollutant metals in marine invertebrates: a review of microanalytical applications.

X-ray microanalysis (XRMA) is customized for investigations of the metabolic and detoxification strategies of heavy metals taken by marine organisms from polluted environments. Sites of uptake, intracellular accumulation, transport and excretion are visualized, analysed and quantified. Cryopreparation techniques are required to prevent the translocation or loss from specimens of soluble metal species. In marine invertebrates, metals are detoxified by systems of chemical binding and intracellular compartmentalization. XRMA investigations have concentrated on marine molluscs and crustaceans and even within these restricted groups there are marked inter-species differences in the biochemical and cytological processes which reduce metal bioavailability. Some detoxification systems also protect the carnivores which ingest the metal-laden tissues of the prey. This results in the bioreduction of metals along a food chain. These processes are investigated by XRMA which can be tuned to observe the complex interactions which operate at all levels within and between the biota and polluted environments.

Heavy metal pollution induced by a ferro-nickel smelting plant in Greece.

Seven heavy metals, Co, Cr, Cu, Fe, Mn, Ni and Zn, were measured in marine sediments, plants and invertebrates in the vicinity of a ferro-nickel smelting plant in Greece. The concentrations of metals in the sediment were higher than those found in the average unpolluted Greek coastal sediment. High levels of metals were observed in the gastropod molluscs, particularly Cerithium vulgatum, which concentrated metals more than other invertebrates.

Effects of polycyclic aromatic hydrocarbons on molluscan lysosomes and endoplasmic reticulum.

Some effects of two isomeric polycyclic aromatic hydrocarbons, anthracene and phenanthrene, on the fine structure and cytochemistry of digestive cells in the marine mussel Mytilus edulis have been investigated. The cytochemical results show that increasing concentrations of anthracene and phenanthrene have different effects on the acid labilization time for latent beta-glucuronidase which is used to measure the stability of lysosomal membranes. At the ultrastructural level the limiting membranes of secondary lysosomes appear multilayered, with discontinuities and overlaps. Under the conditions of the experiment, only phenanthrene produces changes in this configuration. Both macroautophagic and microautophagic processes occur in the control and hydrocarbon treatments, and complementary data from other studies indicate that autophagic processes are enhanced by polycyclic aromatic hydrocarbons. Phenanthrene also causes proliferation of the smooth endoplasmic reticulum in the digestive cells, although cytochemical measurements of smooth endoplasmic reticulum-associated NADPH-ferrihemoprotein reductase show that anthracene stimulates activity over a greater range of concentrations than phenanthrene. The different effects of the two isomers is taken as evidence that the molecular configuration of the compound determines its reactivity with membranes and its subsequent effect on the physiology of the cells.

Adaptation of a quantitative programme for the X-ray analysis of solubilized tissue as microdroplets in the transmission electron microscope: application to the moult cycle of the shrimp Crangon crangon (L).

A quantitative programme for X-ray microanalysis is used in a non-standard manner on solubilized tissue which has been spiked with cobalt and sprayed as microdroplets on electron microscope grids. During the procedure the count time and the concentration of cobalt is related to the peak integral and, from the relative efficiencies, the concentrations of other elements are computed from the peak integrals. Absorption is taken into account but the X-ray background is not used to estimate the total mass and the beam current is not measured. The method is applied to the hepatopancreas and blood from individual shrimps, Crangon crangon, to give the concentrations of sodium, magnesium, silicon, phosphorus, sulphur, potassium and calcium at different stages of the moult cycle. In the hepatopancreas the absolute and relative quantities of phosphorus, sulphur and other elements change in phase with the moult cycle. This situation must be linked with fluctuations in levels of metabolic activity and may affect the metal-binding capacity of the tissue which is known to fluctuate. The hepatopancreas accumulates lipid and phosphorus during the intermoult period, but the level of phospholipid phosphorus remains as a constant proportion of the tissue wet weight. The gland does not store calcium for hardening the new exoskeleton after ecdysis. Magnesium is a more important and variable component and could be linked with metabolic activity. The blood composition remains more stable. However, sulphur concentration is high and variable and this may, to some extent, reflect changes in the concentration of taurine. The concentration of copper increases towards the end of the moult cycle and decreases during moulting; opposite changes occur in the hepatopancreas.

A rapid, routine technique for the X-ray microanalysis of microincinerated cryosections: an SEM study of inorganic deposits in tissues of the marine gastropod Littorina littorea (L.).

A procedure is described that prepares chemically untreated biological sections for X-ray microanalysis in the scanning electron microscope (SEM). The method aims to retain and localize labile components in tissue sections by a procedure that is both rapid and routine. Large quantities of fresh tissue can be processed for analysis within a single day. Thick cryosections are cut with a steel knife in a conventional cryostat, freeze-dried, and then ashed by either low or high temperature incineration procedures. Controlled microincineration attenuates the organic matrix to reveal sufficient surface relief for effective SEM of some cytological structure and microanalysis of the residual inorganic components. The detectability of various elements is enhanced because the relative concentrations in the residues are increased and the level of nonspecific background in the X-ray spectra is reduced. The technique is applied to different tissues from the visceral complex of the marine prosobranch Littorina littorea. In animals exposed to elevated levels of zinc it can be demonstrated tht the metal is localized both as an insoluble form in granules and as a labile form within the cytoplasm. Other metals, including magnesium, potassium, calcium, manganese, and iron, have been identified and localized. The effectiveness of this technique for retaining labile elements is compared, in outline, with that of conventional fixation procedures.