Ghislain Samson, Louis Tessier, Guy Vaillancourt* and Leroy Pazdernik
Université du Québec à Trois-Rivières, Département de chimie-biologie
C.P. 500, Trois-Rivières
Québec, Canada, G9A 5H7.

In this study, two aquatic mosses,Fontinalis dalecarlica Schimp ex. B.S.G. and Platyhypnidium riparioides (Hedw) Dix., were exposed to three different aqueous concentrations of Cd (nominal concentrations of 10, 25 and 50 µg•L-¹) in a flow-through system for a period of 28 days. A first order kinetic model has demonstrated that the Cd bioaccumulation by the two species selected can be characterized by a biphasic pattern where a rapid uptake occurs during the first few days of exposure followed by a steady state which represents a saturation of Cd adsorbed onto the cell walls and into the intercellular spaces. In some cases, a very slow accumulation was observed for the rest of the exposure period which may be associated to intracellular uptake of Cd. However, this phenomenon is only significant when the mosses are exposed to high aqueous concentrations of Cd. The difference in the Cd kinetics of the two bryophytes exposed to high Cd concentrations seems to be related to the Cd desorption activity at the extracellular level which is more pronounced for Platyhypnidium riparioides. The use of these two moss species as bioindicators of Cd in an aquatic ecosystem would be more appropriate for long term exposure studies.

It is well known that before undertaking any field studies, more precise information on the heavy metal bioaccumulation kinetics under laboratory conditions is required (Marigomez and Ireland 1989; Mersch et al. 1993; Tessier et al. 1994a). The most important phenomena to be discerned are (1) the effects of aqueous metal concentration on the bioaccumulation processes, (2) the bioconcentration factor extrapolated to steady state conditions, (3) the biological half-life of the metal and (4) the possible differences observed between two species exposed to same experimental conditions. Metal accumulation in mosses is essentially related to cationic exchange on the cell wall surfaces (Brown 1976; Niboer and Richardson 1981; Brown and Wells 1990). Since this exchange is a rapid process, then only a short exposure period is required to obtain significant results (Kelly et al. 1987; Mersch et Pihan 1993). Claveri et al. (1994) stated that the uptake kinetics of metals in mosses can be decribed by non linear equations. The originality of estimating the kinetic parameters of metal accumulation in aquatic organisms with the use of a first order kinetic model, is related to the possibility of predicting the pattern of metal uptake in experimental conditions and to compare more precisely the performance of bioaccumulation among different species.

In the present study two moss species (Fontinalis dalecarlica Schimp ex. B.S.G. and Platyhypnidium riparioides (Hedw) Dix.) have been exposed to three different concentrations of cadmium for a period of 28 days in order to assess the accumulation kinetics for this metal. Using a flow-through system with continuous water and metal input, we have estimated different kinetic parameters based on a first order kinetic model. These parameters were: (1) the concentration at steady state conditions, (2), the desorption (elimination) rate constant, (3), the extracellular adsorption rate constant, (4) the theoretical bioconcentration factor extrapolated to steady state conditions, and (5) the biological half-life of the metal. There were determined for each exposure concentration and for each species studied.

Each moss sample was washed with deionized water as suggested by Wehr et al. (1983). Loss of metal attributed to this procedure is generally about 5 % (Pickering and Puia 1969). Any sub-samples have been submitted independently to the same analytical procedure. The subsample was oven-dried at 110° C for 12 hours. After weighing, the dry moss samples were digested in concentrated nitric acid (HNO3; 70 %) for 7-8 hours until the dissipation of the brown fumes; the reaction was driven to completion with 30 % hydrogen peroxide (H2O2) (Berryman 1991). The digested mosses were analysed by atomic absorption spectrophotometry (Varian model AA1275 equipped with a deuterium lamp) with air acetylene flame. Calibration, using the standard addition method, was applied to avoid matrix effects. Digestion blanks were frequently measurement to verify any possible contamination. About 15 % replicates (Tessier et al. 1994a) were made to check the analysis precision, which was better than 11 %. Certified standards for water (Standard Reference Material 1643 C) and mosses (Certified Reference Material BCR No 61) were used in the quality control scheme. Detection limits were 0.6 ng•g-1 (dry weight). All materials used were rinsed for 24 h with 1 M HNO3 and then rinsed five times with deionized water.

The accumulation of metal in the bryophytes is generally characterized by two distinct patterns; a first rapid metal uptake where a passive ion exchange process is occurring on the cell walls (adsorption) until a plateau is reached (steady state conditions), then followed by a slower linear accumulation. This slow step is considered an active one (absorption) where the metal penetrates into the cells (Pickering and Puia 1969; Breuer and Melzer 1990). A first order kinetic model was used (Moriaty 1983; van Hattum et al. 1989; Tessier et al. 1994a) for analyzing the Cd uptake in the two aquatic mosses.

where

Ct = concentration in the mosses at time t (µg•g-1 dry weight)

Css = concentration in the mosses at steady state conditions (µg•g-1 dry weight)

Cw = concentration in the water (µg•ml-1)

K1 = rate constant for uptake (adsorption) from the water (hour-1)

K2 = rate constant for elimination (desorption) from the mosses (hour-1).

When it was possible, the uptake data were separated in two groups representing the two patterns of accumulation (two compartments). Equation (3) was fitted to the first group (first compartment) for each exposure concentration. A nonlinear iterative least square regression was used to estimate the two parameters of Eq. (3); Css and K2. Using these two values and the actual exposure concentration (Cw), estimation of K1 was determined by fitting the Eq. (1) to the data as described previously. For the data representing the second pattern of uptake (second compartment), the linear regression was applied. The slope of the regression model was used to indicate the rate of accumulation. Significance of the nonlinear and linear regression were assessed with the F-test (ANOVA). This procedure was repeated for each species studied.

The theoretical bioconcentration factor (BCF) for bioaccumulation from the water at steady state conditions was evaluated from the estimated K1 and K2 values (Walker 1987; Tessier et al. 1994a), where:

Biological half-life (T1/2) was estimated from T1/2 = ln 2/K2 (van Straalen and van Meerendonk 1987).

The water chemistry parameters used for the exposure experiments were similar to those measured for the control (Table 1) and did not change significantly during the 28-day exposure (ANOVA; p > 0,05). The actual aqueous Cd concentrations to which the mosses were exposed during the accumulation experiments are presented in Table 2. The actual concentration was lower than the desired nominal exposure concentration, even if the exposed surfaces of the aquaria were saturated before the experiment. This demonstrates that it was difficult to maintain the aqueous cadmium concentration in the media, even with a dynamic flow-system. Most of the concentration decrease occurred during the first few hours of exposure followed by a steady state value. The Cd concentration in each bryophyte was statistically similar for the 10 day-replicate and for the first 10 days at the 50 µg•L-1 exposure test (ANOVA; p>0,05).

The kinetic parameters of cadmium uptake by the mosses in the first compartment are shown in Table 3. These variables were estimated according to the actual Cd concentration measured in water during experimentation. The Cd concentration in the mosses in steady state conditions (Css) increased as the aqueous Cd concentrations increased. For F. dalecarlica the Css values ranged between 85.1 and 265.5 µg•g-1, while P. riparioides was characterized by values ranging from 100.5 to 180.3 µg•g-1 (Table 3). Comparing these results between the two species it seems that F. dalecarlica may accumulate more Cd when exposed to 25 and 50 µg•L-1 than P. riparioides, for the same exposure period.

The values of elimination or desorption constant (K2) are situated between 0.0058 and 0.0088 hour-1 for F. dalecarlica whereas they are slightly higher for P. riparioides with values ranging between 0.0073 and 0.0110 hour-1 (Table 3) with no particular trend relative to the exposure concentration of Cd. The metal desorption from the cell surface seems to be more prominent for P. riparioides. This observation is represented by the slight difference in the biological half-life of Cd on the cell walls of P. riparioides (2.6 to 4.0 days according to the exposure concentration) compared to F. dalecarlica (3.3 to 5.0 days; Table 3).

The calculated accumulation constants (K1) decrease with an increase in the water Cd concentration, where the values are evaluated between 248.8 and 72.0 hour-1 for F. dalecarlica and between 254.8 and 91.5 hour-1 for P. riparioides (Table 3). Comparing the two species, the K1 values are higher for P. riparioides, except for specimens exposed to 25 µg•L-1 (K1 = 109.9 hour-1 for P. riparioides and 129.7 hour-1 for F. dalecarlica). The same decreasing tendency, as observed with the accumulation constants, is also evident for the theoretical bioconcentration factors at steady state conditions (Table 3).

However, the Cd is more bioconcentrated in F. dalecarlica exposed to 25 and 50 µg•L-1 (BCF = 21262 and 12414 respectively) than in P. riparioides (BCF = 15055 and 8318 respectively).Nevertheless, one can notice that P. riparioides has a higher BCF value when exposed to 10 µg•L-1 (Table 3).

As described previously, slow Cd uptake was observed, in certain cases, after a steady state condition has been reached. Table 4 presents the second compartment Cd accumulation rates for both species, which are represented as the slope of the regression line estimated for each Cd exposure concentration. Uptake of cadmium is significant for specimens of F. dalecarlica exposed to 25 and 50 µg•L-1 but the accumulation is weakly linear (R2 values of 0.61 and 0.73 respectively). However, the accumulation is more accentuated for bryophytes exposed to 50 µg•L-1 with an uptake rate of 0.251 µg•g-1•hour-1. For P. riparioides, the only significant uptake of Cd was noted for mosses exposed to 50 µg•L-1 with a high significant linear accumulation rate of 0.451 µg•g-1•hour-1. This last observation clearly indicates that Cd, in the second compartment of P. riparioides is more rapidly accumulated when exposed to high stress of Cd, compared to F. dalecarlica.

The use of a first order kinetic model to assess the potential of the two moss species in accumulating Cd from the aqueous fraction seems to be of great utility for estimating the bioaccumulation process over a long period of time. We have demonstrated that the accumulation of Cd in both species is characterized by a biphasic pattern; a rapid uptake during the first days of experimentation followed by a steady state condition. In some cases, a slower uptake during the rest of the exposure period was also observed which indicates the presence of a second compartment. However, when it was significant the slower uptake in the second compartment was not necessarily linear. The first compartment can be described as the extracellular Cd adsorption (passive uptake) where the metal is bound by ion exchange to ligands located at the cell surface (Brown and Wells 1990). This compartment also represents the intercellular fraction where the metal is found in the available spaces of the cell wall (Claveri, 1995). The major part of the accumulation was accomplished rapidly within the first few hours according to the passive nature of the ionic exchange process for attachment. A steady state was then reached when the available Cd fixation sites became saturated. Thus the term "saturation conditions" would be more appropriate then "steady states conditions" in such circumstances. The second compartment represents the intracellular uptake of Cd or the biologically active uptake (Pickering and Puia 1969; Brown and Wells 1990; Claveri, 1995), which implies the transport of Cd within the plasma membrane by specific carriers (Streit and Stumm 1993). The low intensity of the bioaccumulation in the second compartment is due mainly to the fact that the plasma membrane acts as a barrier, reducing the uptake rate of metal.

Nevertheless, the intracellular bioaccumulation of Cd is only significant when F. dalecarlica is exposed to 25 and 50 µg•L-1 and when P. riparioides is exposed to 50 µg•L-1 of Cd. Also we observe, in the case of F. dalecarlica that the rate of uptake is more accentuated when the Cd water concentration increases. These results clearly show that membrane permeability may increase highly when the bryophytes are exposed to elevated toxic metal ions as stated by Tyler (1989). In other cases, the non significance of the Cd uptake indicates that the saturation of Cd adsorption on the cell walls is nearly complete. Hence, the time needed to reach saturation may follow a gradual response associated with the aqueous Cd concentrations.

By comparing the kinetic parameters of the first compartment (extracellular uptake) for the two bryophytes we have established that F. dalecarlica is characterized by higher concentrations at saturation conditions when exposed to 25 and 50 µg•L-1 of Cd. However, if we analyze the elimination rate constant estimated for both exposure concentrations, it seems that the difference observed between the two bryophytes is related to the fact that the desorption of Cd from the cell walls of P. riparioides is more rapid which reduces the overall bioconcentration capacity of this species. For both mosses exposed to 10 µg•L-1 of Cd, P. riparioides possess a higher accumulation rate and a lower desorption rate constant than F. dalecarlica. This would explain the higher BCF value observed for P. riparioides. Most of the metal accumulated by mosses can be explained by the abundance of leaves containing negatively charged sites such as uronic, glucoronic and other carboxylic acids (Mersch et al. 1993). The quantity of an element bound will depend on the ambiant concentration of the element, its affinity for binding sites, the total number of exchange sites available (Brown and Well 1990), the background concentration present in the bryophytes and on the pH (Gailey and Llyod 1986). Thus we might conclude that the affinity of the binding sites of F. dalecarlica for Cd fixation and their availability are more pronounced at higher ambiant concentrations than P. riparioides. Also, the reversible nature of Cd fixation (desorption) at extracellular level (intercellular and exchangeable fractions) is higher when P. riparioides is exposed to high Cd concentrations. These results are slightly different from those observed by Mouvet (1987) where the Cd concentration in Platyhypnidium ripariodes and Fontinalis antipyretica (a species close to Fontinalis dalecarlica) was not significantly different after 336 hours of exposure at 4, 7.2 and 15 µg•L-1 of Cd.

In order to assess the great potential of the two bryophytes selected in this study, we have compared their theoretical BCF values exhibited at steady state conditions with other aquatic species submitted to the same laboratory conditions and to the same kinetic model. In their study on the bioaccumulation of Cd in freshwater molluscs, Tessier et al. (1994a) have estimated, in the first compartment of different age-classes of the short-lived gastropod Viviparus georgianus and the long-lived pelecypod Elliptio complanata exposed to 10 µg•L-1 of Cd, lower BCF values than those exhibited by the two moss species exposed to 25 µg•L-1 (actual concentration = 10.1 µg•L-1). The highest theoretical BCF value attained by the younger individuals was 3565, for the snail and 1518 for the clam. Hence, the BCF values of both mosses represents an increment of 4.2 to 14 times compared to the freshwater molluscs. Timmermans et al. (1992) have shown that the water mite Limnesia maculata exhibited a BCF value at a steady state condition of 455 when exposed to 94 µg•L-1 (actual concentration) for a period of 32 days. The BCF value for the bryophytes is 18 to 74 times greater than that for the water mites. However this difference among bryophytes, molluscs and the insect larvae is related to the fact that bioaccumulation of metal in molluscs and insects is an active process regulated by metabolic activities which may reduce the uptake rate and/or increase the excretion of Cd. In this sense, the regulated metal concentration in invertebrates may reach a steady state condition with the ambiant environment more rapidly than bryophytes. Hence the use of mosses as a bioindicator of Cd in the aquatic ecosystem would be more appropriated for long term exposure studies due to the passive nature of Cd adsorption on the cell walls.

The authors would like to thank Mr David Berryman from the Quebec Ministry of Environment and Wildlife for his much appreciated assistance during this project and Ms Claudie Gagnon for her comments on the revision of this manuscript.

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