3.1. Speciation of metals in aquatic systems
In aquatic systems, the concentrations of metals vary according to their geographical localization. For example, Fig. 8 shows the range of total concentrations of the majors cations and trace metals that can be found in the open sea or freshwaters (Buffle and De Vitre, 1994).
The concentration of cadmium varies between 10-12 M and 10-9 M in open sea water and between 10-10 M - 10-8 M in freshwater systems.
Figure 8. Trace and major metal concentrations in open sea and freshwaters (Buffle and De Vitre, 1994).
Metals may be partitioned among a number of different forms or species. Indeed, in most natural systems, metals are rarely found as free ion (e.g. Cd2+) but more often in the form of trace metal complexes (CdCl-, CdCl2, Cd(CO3
-). Metals may also form stable complexes by interacting with inorganic (e.g. CO3
2-) and organic ligands (e.g. proteins, amino acids2-), particles (e.g. colloids, phytoplankton, bacteria). The complexation of metals is dependant on
physicochemical conditions such as pH, temperature, ionic strength redox potential, presence and concentration of complexing agents. Each of the complex species may behave differently with respect to their mobility, lability and bioavailability and thus natural systems are under constant change and practically never reach true equilibrium. Therefore, the study of metal speciation in natural environments is complicated and the elucidation of the relationship(s) between the different physicochemical forms of the metals and their reactivity, mobility and bioavailability is often difficult. In the laboratory, under well controlled conditions, we can predict the equilibrium concentrations of most metal species by using equilibrium modelling software (e.g. MINTEQA2; (Allison et al., 1999)). Nonetheless, accurate model predictions are difficult to do for natural waters due to the fact that it is very difficult to have the precise chemical composition (all inorganic and organic components) of natural waters, and the stability constants for all metal complexes.
3.2. Physicochemical conditions that favors metal uptake
As mentioned previously, the determination of trace metal speciation in a specific environment is important because not all metallic species are bioavailable to the organism to the same extent. The physicochemistry of the interactions of trace metals with a microorganism can be schematized in Fig. 9. In order to understand the biological effects that are induced by trace metals, it is important to understand the role of physicochemical processes on the biouptake or metal adsorption to the cell wall or biological membrane since this is the first step of any toxicological impact.
Diffusion
The metal generally diffuses through bulk solution to the biological membrane where it can be adsorbed prior to desorption or transport into the microorganism. The process of adsorption and internalization describes the metal bioaccumulation step. Inside the diffusion layer the metal can form complexes with hydrophilic ligands (Lh-M) or lipophilic ligands (LL -M). These ligands include organic ligands such as fulvic substances, humic substances, proteins or organic acids. The metal can also form complexes with strongly binding biological ligands, M-Lbio, the best known of which are the siderophores. As a result, the metal can attain the cell membrane as a complex (e.g. Lh-M, LL-M, Lbio-M) or as the free aquo ion (M). At the
level of the cell membrane, the metal might react with a sensitive site by adsorption which may or may not be followed by biological transport (internalization).
Figure 9. Interaction of metals with microorganism at the cellular level. Diffusion, complexation, adsorption and desorption are generally accepted as the main steps leading to metal internalization. Upon internalization, the metal can interact with ligands inside the cell (M-Lbio), initiate signals at the molecular level, be expulsed by efflux or secretion or be stored into vacuoles (Worms et al., 2006).
Transport
The membrane is composed of phospholipids, proteins, and polysaccharides; some of them containing ionisable groups that confer a (often negative) charge on the biological surface.
The metal can bind to both unspecific sites (e.g. carboxyl groups of the cell wall) and specific transport sites. The cell wall and cell membrane contain proteins, some of which are transporters. Metals can be internalized by carrier mediated transport, through protein channels and by passive diffusion. Metal speciation will have an important influence on the type of transport, i.e. lipophilic species such as HgCH3Cl species likely cross the membrane by passive diffusion while most other complexes of toxic metal (e.g. Cd, Ni, Pb, Cr) must dissociate to release the free metal which use the essential metal (Ca, Mg, Zn) transporters, to
cross the membrane via carrier mediated transport. It is thought that Cd enters the cell through Zn and Ca transporters (Gitan et al., 2003).
Internalization
The overall biouptake process depends on the rate-limiting step that is encountered during bioaccumulation. Internalization (Jint) is often assumed to be the slowest step in the overall reaction, i.e. Jint <<Jdiff,Jkin, Jkin'. Jint however depends on the internalization rate constants and the density of transport sites on the membrane. Several steps of the overall process are potentially rate-limiting:
- Diffusion of trace metal complexes to the biological surface (Jdiff). Diffusion is slower when the metal is complexed to a macromolecule or colloidal ligand. Diffusion limitation (Jint > Jdiff) is more frequently observed in marine rather than freshwater systems (Wilkinson and Buffle, 2004). It has been shown that in natural waters (contrary to laboratory conditions), the density of transport sites may be large and Jdiff may become rate limiting. This situation arises when the transport system is stimulated in response to metal depletion, most likely for essential metals only (Hassler et al. 2003).
- Chemical reactivity (Jkin) of the trace metal in the bulk solution. The Jkin can become limiting (Jint > Jkin) in situations where complex (metal-ligand) lability is very low and the free metal concentration is too low. Some complexes are inert and dissociate very slowly: the metal is then “trapped” in the external solution.
- Chemical reactivity (Jkin') of the trace metal with transport sites at the cell membrane surface.
In either of these limiting cases (either Jdiff or Jkin are limiting), bioavailability will be best predicted by a ponderated sum of free and labile species. Nonetheless, even though natural systems are practically never at equilibrium in the vicinity of the cell, it is often possible to assume a rate-limiting internalization that leads to steady-state conditions that can be predicted on the basis of equilibrium considerations.
In all cases, the physicochemistry of the bulk media surrounding the cell will have an important influence on the biouptake process. Furthermore, certain biological responses to the metal (e.g. efflux, pH changes at the cell surface, modification of surface charge) can also influence the physicochemistry of the surface, thus potentially modifying the internalization flux.
Not all organisms react in the same manner to trace metal exposures. In addition to the chemistry, the process of biouptake and metal sensitivity are dependent on biological factors such as the nutrient status and tolerance mechanisms. For example, for a decreased nutrient supply, the cell is more likely to be susceptible to metal toxicity because of a tendency to take up non essential trace metals at the same time as essential metals. In addition, differences may be observed between prokaryotes and eukaryotes since prokaryotes possess numerous systems that are designed to exclude bioaccumulated metal (Nies, 2003; Nies and Silver, 1995).
Eukaryotes generally reduce metal bioaccessibility by complexing the internalized metal, and then redistributing it among different compartments or vacuoles (Zenk, 1996; Zhou and Qiu, 2004).
3.3 Models describing biouptake
Most studies that examine the bioaccumulation of trace metals suggest that trace metal uptake is best related to the concentration of free metal in the bulk solution which has been interpreted to suggest that the metals in solution are in equilibrium with those at the surface of the biological organism and that equilibrium models based upon measurements of the free ion are likely the best predictors of trace metal bioaccumulation (Morell, 1983; Slaveykova and Wilkinson, 2005). Indeed, the FIAM (Campbell, 1995b; Morell, 1983) and BLM (Paquin et al., 2002) models have been developed to explain correlations between the concentration of free metal ions in solution and observed biological effects. For both models, metal uptake fluxes can be predicted from any of the metal species at equilibrium. In the case of the BLM and the FIAM, competitive effects are taken into account by predicting quantities of metal bound to site of action using equilibrium constants that have been determined from toxicity experiments.