Experimental study of mercury transfer between artificially contaminated sediment (CH3HgCl) and macrophytes (Elodea densa)

Aquatic Toxicology, 12 (1988) 213-228 Elsevier

213

AQT 00268

Experimental study of mercury transfer between artificially contaminated sediment (CH3HgC1) and macrophytes (Elodea densa) R. Maury, A. Boudou, F. Ribeyre and P. E n g r a n d Laboratoire d'Ecologie Fondamentale et Ecotoxicologie, Universitd de Bordeaux I, Talence, France (Received 4 April 1987; revision received 2 September 1987; accepted 8 September 1987)

The quantification of metal transfer routes from a natural sediment enriched with methylmercury (4 mg • Hg kg -1 fresh weight) to Elodea densa, shows a high mercury accumulation in the plant organs (leaves, stems and roots). The experimental approach developed shows that, in the long term (28 days), root absorption of the organic compound (direct route) represents the dominating vector of metal accumulation in the plant, the leaves being the principal organ for storage. Two mechanisms, far less important from a quantitative point of view, are superimposed in this direct transfer: contamination by the water, linked to the releasing phenomenon at the interface 'water-sediment', during the initial exposure phase (4 days), and inter-plant transfers resulting from decontamination processes, acting together with direct metal accumulation in the E. densa. Key words: Methylmercury; Experimental ecosystem; Sediment; Freshwater; Macrophytes (Elodea densa); Bioaccumulation and transfer

INTRODUCTION

We are developing a research programme in experimental ecotoxicology. It has as its main objective the study of the processes of bioaccumulation and transfer of mercury compounds in freshwater systems. It is based on the consideration of the three fundamental components of ecotoxicology: abiotic factors (physicochemistry of the medium); biotic factors, e.g. biological diversity, inter-individual and interspecies relationships; and contamination factors, e.g. sources of pollution, concentrations in the biotope and chemical speciation.

Correspondence to: R. Maury, Laboratoire d'Ecologie Fondamentale et Ecotoxicologie, Universit~ de Bordeaux I, 33405 Talence Cedex, France. 0166-445X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

214

After setting up, perfecting and experimenting on a linear model of trophic transfer (experimental food chain) (Ribeyre et al., 1979; Boudou and Ribeyre, 1983), we elaborated a new, more complex methodology, based on the concept of experimental ecosystems (interactive models), enabling us to quantify the actions and interactions of the various parameters considered upon the processes studied (Ribeyre, 1985; Ribeyre and Boudou, 1985). The first step in carrying out this new approach is based on a system of three compartments: water, natural sediment and Elodea densa; the initial source of contamination from mercury compounds (HgCI2 and CH3HgCI) being the 'sediment' source. The actions and interactions of two abiotic factors, i.e., temperature and photoperiod, were studied using factorial experimental designs. After perfecting the ecotoxicological model, experiments were undertaken to analyse the bioaccumulation of mercury in the macrophytes, at organism and organ levels. This was related to the chemical form of metal introduced into the substratum, and to the different abiotic conditions, determined by the two controlled factors (for example, 24°C/16 h; 18°C/16 h; 18°C/8 h; etc.). It is clear from our results that there is a definite inequality in the transfer of mercury compounds between the sediment and E. densa (CH3HgC1 -> HgC12). The influence of the two abiotic factors (temperature and photoperiod) is high, both at the level of biotic criteria (weight, length and growth factor) and of contamination criteria (concentration and content of mercury in whole plant and organs) (Engrand et al., 1985; Maury and Engrand, 1986). Many questions were raised during the interpretative analysis of the results, but the objective of the present study is to assess the qualitative and quantitative importance of the different ways of mercury transfer between the sediment and the rooted macrophytes. The information collected in the bibliography indicates the controversy which exists regarding the importance of direct routes, via the sediment (root absorption), and indirect routes, via the intermediary of releasing processes (sediment --, water ~ plant). This controversy concerns both the processes of contamination (Dollar et al., 1971; Bristow, 1975; Baker, 1979) and the absorption mechanisms of nutrients by the rooted macrophytes, depending on the nature of the sediment, on the species of plant studied, on the physicochemical characteristics of the medium, etc. (Denny, 1980; Taylor and Crowder, 1983; Campbell et al., 1985). The various results obtained during previous experiments, especially those relating to the bioaccumulation of methylmercury in E. densa, show a very clear exponential tendency for the evolution of metal contents in plants (chronological analysis on a 28-day exposure time) (Fig. 1). The concentrations measured in the plant as a whole are high, especially in the leaves (for example, concentrations greater than 1 ppm wet weight for the experimental condition: CH3HgC1, 1 ppm Hg in the sediment, 24°C/16 h, 28 days; Maury and Engrand, 1986). In our experimental system, given that the initial source of contamination is the sediment, which transfer routes are at the origin of the bioaccumulation of mercury in E. densa?

215

T ~ 24°C1/6h

1500.

== 1000 /

.2

18°C/8h

/ /

500

duration (days) Fig. 1. Speed of bioaccumulation of mercury in E. densa, originating in the source sediment (initial contamination condition; CH3HgCl 1 ppm) and in two abiotic conditions: 24°C/16 h and 18°C/8 h.

In this paper, we present the main results obtained, after describing the methodological steps elaborated to analyse mercury transfer routes between the 'sediment' source and E. densa. Only the methylmercury was retained to contaminate the natural substratum, at an initial concentration of 4 mg H g. kg -1 (wet weight of sediment). According to the bibliography consulted, the concentrations o f CH3HgCI found in sediments in the natural medium vary enormously: from the order of only a few ppb (Batti et al., 1975; Matsunaga et al., 1978; Jackson, 1986) to several hundred ppb (Akagi et al., 1979; Jackson et al., 1982). The concentration of mercury in the sediment selected for our experiments, of the order of a few ppm, is no greater than that found in certain parts of the natural environment. These values were chosen with the following considerations in mind: - the amount of mercury transferred into the plant should be large enough to improve understanding of the contamination process; - errors during contamination of sediments and measuring of mercury in the plants should be as small as possible; - to amplify processes linked to the presence of mercury in the systems, particularly the inhibition of plant growth and the desorption phenomenon of mercury 'sediment ~ water'. Similarly, only one combination of the two controlled abiotic factors was chosen, i.e. 'temperature: 24°C and photoperiod: 16 h of light per day', as this corresponds to the optimal condition for accumulation of mercury in plants observed during a comparative study of 9 different sets of experimental conditions (Maury and Engrand, 1986).

216 EXPERIMENTAL PROTOCOL AND METHODS OF ANALYSIS

In order to meet the main aims described in the introduction, we developed an experimental protocol which depended on E. densa cuttings, planted in natural sediment (flask T) or in contaminated sediment (flask C: CH3HgC1, 4 mg Hg. kg-1), being present in the same experimental units (see Fig. 2). Each flask (glass, diameter 4.5 cm, height 7 cm) containing contaminated sediment is covered with a layer of substratum not enriched with mercury, thus minimizing for each plant 'C', the processes of indirect metal transfer. In order to investigate the phenomenon of mercury release (contaminated sediment --, water) in relation to the bioaccumulation of metal in E. densa, two experimental conditions were analysed simultaneously. These

TEMPErlURE

PHOIOPER I OD

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24°C

t

water

sediment

condition ]

condition

2

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O 0 o

o

o

O

0

0

o

0

0

0

0

0

o

0

0

o

Q

Arrangement o f Elodea densa i n t h e e x p e r l m e n ~nits. T = n a t u r a l sediment, C = contaminated sediment.

tXPIRININIAL UNIIS Duration condition condition (adys) I 4

I

2

3

4

28

5

6

7

8

Fig. 2. Experimental protocol undertaken to quantify the various pathways of mercury transfer between the 'sediment source' and E. densa.

217

differed in the level of contamination of sediment in the flasks (condition 1, sediment 'T'; condition 2, sediment 'C', CH3HgCI 4 ppm). The surface of this sediment represented 75070 of the area of the base of the experimental units, and 68°70 of the total volume of the substratum introduced into each tank. The three compartments of the experimental units (glass aquarium 25 × 25 × 30 cm) correspond to those used for experiments on the ecotoxicological model:

"Sediment" compartment The sediment comes from a site on the banks of the river Garonne, upstream from Bordeaux. Its geochemical and granulometrical characteristics are periodically monitored by the 'Institut de G6ologie du Bassin d'Aquitaine'; it is a very homogeneous sediment (silt), rich in clay (75-80°7o) and has a particularly low concentration of organic carbon (2°7o on average) (Donard, 1983). The natural level of total mercury in this substratum is 0.124 + 0.012 ppm (wet weight). The 'T' and 'C' sediments are prepared from an initial batch of substratum homogenized by mechanical mixing. The level of contamination desired is obtained by adding methylmercury solution to a known quantity of sediment (e.g. 400 ml of methylmercury solution at 100 ppm for 10 kg of sediment, fresh weight). After a second mixing, samples are taken (10 on average) permitting, after dosage, verification of the mercury concentrations and also the homogeneity of the distribution of metal added to the contaminated sediment.

"Water" compartment Tap water is used after preliminary dechloridation by aeration. The source is groundwater; its physicochemical characteristics are relatively standard throughout the year and are periodically checked by Bordeaux Municipal Laboratory. The main parameters are: NO3=0.14 mg.1-1, PO4 = undetected, Resistance: 2100 ohms/cm-2/cm -1, organic matter =0.64 mg. 1-1; heavy metals (Pb, Se, Hg etc.) undetected. After introducing the sediment, the experimental units are filled with water, taking the maximum amount of precautions to minimize the mixing at the sediment-water interface. The volume of water in each unit is 11.5 1. The pH, at time zero, is 7.5 _+0.2. The medium is not renewed during the experiment, but water from the original source is added to compensate for evaporation, taking of water samples, etc., thus maintaining constant levels in the tanks.

"Macrophytes" compartment A room for cultures has been installed at the laboratory. The production capacity is about 3500 cuttings from E. densa supplied by a local importer (Aqua Decor) and

218 TABLE 1 Measurement of mercury concentration in water (ppb; confidence interval a = 0.05) after the introduction of contaminated sediment (4 p p m of CH3HgCI) into experimental unit. (ND = not detected, 0* = day of planting of E. densa.) Days

Condition 1

Conditon 2

l0 7 3 0*

0.03 (0.04) ND ND ND

2.00 (0.30) 0.32 (0.03) 0.12 (0.08) ND

cultivated in a natural sediment identical to that used during the experiments. The cuttings taken, correspond to the apical part of the lateral branches; average length = 12 ___1 cm; average weight = 0.610+_0.025 g (fresh weight). One cutting is placed in each flask with many precautions. Temperature regulation is ensured by using thermostated tanks (200 1, 24+_0.2°C). The amount of light per day (16 h/24 h) is adjusted thanks to a programmed clock: the average light intensity at the level of the flasks is 1600 lux (neon tubes: 33 RS, 40 W). A periodic check of abiotic factors - temperature, pH, dissolved oxygen - is undertaken, simultaneously with measurements of mercury concentrations in the aquatic phase. Between filling the experimental units with water and planting the cuttings, a period of 8-10 days is allowed for the medium to stabilize, especially when mercury is present in the water. In fact, the evolution of metal concentration in the aquatic phase, based on the releasing phenomenon at the water-sediment interface during the filling of the tanks or afterwards, shows a rapid decrease. On the day of planting E. densa, the concentration of mercury in the water - notably condition 2 - is less than the detection limit of our measuring technique (0.1 ppb) (Table I; no values are given after planting because we are below the detection limit). Two exposure durations were noted: 4 days (corresponding to the average period before the appearance of adventive roots in the water column) and 28 days (condition fixed after several preliminary experiments while perfecting the system). Replicate experiments are carried out by subjecting two experimental units to each exposure time and each experimental condition. At 4 and 28 days, E. densa samples are taken by sectioning the main stem at the sediment surface level. Surface water is removed with absorbant paper, and then each plant is weighed (total fresh weight; establishment of growth factor: ['Final weight-Initial weight']/'Initial weight') and the overall morphological characteristics are noted (the length of the main stem and the lateral branches, number of adventive roots, etc.). Dissection then enables the separation of leaves, stems and adventive roots. The samples, thus collected, are weighed and placed in a deep-freeze. To be precise, the fresh weight of the leaves, closely dependent on the processes of desiccation during dissection, is obtained from the difference be-

219

tween the total weight, measured as soon as the plant is removed, and that of the stem, much less sensitive to this phenomenon. Lastly, the roots of flasks 'T' and 'C' are collected by filtering the substratum, rinsed to eliminate the sedimentary particules adsorbed, weighed and measured (mercury analysis). Because the weight of this organ is so small, it is very difficult to analyse the roots of each plant separately; we, therefore, regrouped them for each experimental condition studied (conditions 1 and 2, 'T' and 'C'), still keeping two replications. Measurement of the mercury is carried out by atomic absorption without flame (spectrophotometer Varian-AA 475). The detection limit is 5 ng of mercury. The sediment or plant samples are first mineralized by a nitric acid treatment (HNO3 pure), in a pressurised medium (Pyrex glass tubes) at 95°C for 3 h. For the water samples, a treatment with bromine salts is applied before the addition of stannous chloride (Farey et al., 1978). The technique used makes it possible to dose total mercury without distinguishing between organic and mineral forms. The results of mercury accumulation in plants ('organism' and 'organ' levels) are expressed as concentration (ng Hg. g-~ fresh weight or ppb) and content (ng Hg/plant or organ). Confidence intervals of average values are calculated for a = 0.05. Statistical analysis relative to average values, is carried out using Student's t-test and variance analysis. RESULTS AND DISCUSSION

We decided to present the results in two main stages: (a) analysis of mercury accumulation in E. densa, taking the plant as a whole, after 4 and 28 days' contamination, and morphometrical characteristics (growth factor, length of the main stem and lateral branches); and (b) study of metal accumulation at 'organ' level (leaves, stems and roots) after 28 days' contamination. Analysis of the processes at "whole plant" level After a 4-day exposure, the levels of contamination measured in E. densa are all significant compared to background levels of mercury in plants (natural level = NL = 5 + 0.5 ppb, fresh weight). Observation of 'whole plant' contents (Fig. 3A) and concentrations (Fig. 3B) shows a similarity between these two criteria, related to the conditions studied: T1, C1, T2 and C2. This similarity is due to a very low increase in weight of E. densa during this short period of time, resulting in no significant differences between the average weights of plants in the various conditions studied (average weight of all the E. densa at 4 days = .0.652___0.038 g). The comparable conditions - T~ and T2 on the one hand and C~ and C2 on the other - show much higher metal accumulation levels in E. densa for experimental units of condition 2:T2 > T~/C2 > C1 (significance level of differences between the average contents: 99.9°7o).

220

4 DAYS

CONCENTRATION (ppb Hg) lO0

CONTENT ~ - ~ (ng Hg) 100

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Condition I Condition

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I 28 DAYS 1 CONTENT ~ ' ~ (ng Hg)

CONCENTRATION ppb Hg) 3 000

5000

2 000

I

,T! Cl,

,,

000 ,~~,

iT2 C21

iT,] C,,],

Condition I Condition 2

Condition I

T..2 C2~ Condition 2

Fig. 3. Average total contents (A and C) and concentrations (B and D) of mercury in E. densa, after 4 a n d 28 days' contamination, for all experimental conditions (N.L. = natural level; confidence interval = 0.05).

221

For each of the two experimental conditions studied (1 and 2), 'C' plants show more contamination than 'T' plants, but the differences observed are not statistically very significant.

After 28-day contamination, the total contents of mercury measured in E. densa (Fig. 3C) are very close for conditions C1 and C2 (no significant difference) and correspond to average concentrations of 2096 + 140 and 2580_+ 690 ppb (Fig. 3D). For c o n d i t i o n s T1 and T2, the differences noticed between the total contents (T2 > T 1 ) are significant (99.9%), the contamination levels, expressed in concentration, being 190_+60 and 280_+ 10 ppb. Calculation of the ponderal growth factors of E. densa after 28 days' contamination (Fig. 4A) shows no significant differences between Tz and T2 plants on the one hand and Cz and C2 on the other. Despite having no strict control units during this experiment, comparison of this ponderal criterion between the 'T' and 'C' E. densa reveals a very large growth difference, exceeding 40%, due to the methylmercury originally introduced into the sediment.

GROWTH FACTOR

PRINCIPAL STEM LENGTH

LATERAL BRANCH .ENGTH

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(cm)

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2

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Condition [

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Condition

I 2

TI C1

Condition

iT2 C2 I [

Condition

2

Fig. 4. Growth factor (A, = (P-Po)/Po) principal stem length ( B ) a n d lateral branch length (C) of E. densa after 28 days' contamination (confidence interval c~= 0.05).

222 The inversion observed above between the mercury contents in E. densa (condition C2 > C1, Fig. 3C) and that of the concentrations (condition C~ > C2, Fig. 3D), can be linked to the growth differences between the two lots of plants (growth factors C1 < C2, Fig. 4A). Study of the two morphometric criteria - principal stem length (Fig. 4B) and 'lateral branch length' (Fig. 4C) - shows that the inhibition of ponderal growth of E. densa, corresponding to C~ and C2 conditions, is essentially due to a different development of the lateral branches, the growth of the main stem not being significantly affected.

Analysis of accumulation processes at the "organ" level After 4 days" contamination, the mercury content in E. densa leaves 'T' and 'C' corresponds to more than 90% of the quantity of metal accumulated in the whole plant (leaves + stems); no significant difference was noted between the experimental conditions studied. After 28 days" exposure, the distribution of mercury in the three macrophyte organs is similar for the two conditions TI and T2: relative mercury contents (To~gans/Tglobal × 100)= 83 and 86% and for the leaves, 12% for the stems, 5 and 2% for the roots, respectively and for the other two conditions, C1 and C2, relative contents are 57 and 69% for the leaves, 32 and 22% for the stems, 11 and 9% for the roots, respectively (Fig. 5). On the other hand, comparison between T and C conditions shows a different typology: the E. densa grown on sediment initially contaminated by methylmercury show high relative contents of metal in the roots and in the stems; conversely, more than 80% of the metal is located in the leaves of plants in condition T. In view of the very large difference in weights of the organs (average weight of the roots = 5% of the total weight), the quantification of mercury accumulation by the intermediary of 'concentration' criterion shows different contamination levels from those observed using the 'contents'. Thus, for the two experimental conditions C1 and C2, the average concentrations after 28 days' exposure are: 3400 and 3200 ppb for the leaves, 2200 and 1400 ppb for the stems and 8300 and 5100 ppb for the roots, respectively. It is, however, important to remember that the mercury measurements in the roots correspond to one value per lot of plants (T and C), for each experimental unit; in addition, because of the low biomasses, the estimation of metal concentrations in the roots (content/weight) is more sensitive than in the other organs. The 'concentration' criterion, from a toxicological point of view, is associated with a notion of risk of cellular or molecular damage (e.g. enzymatic inhibitions) and the various physiological functions they maintain. Thus, the level of contamination in the E. densa organs, measured after an exposure of 28 days, can explain the

223

CONTENT (ng Hg) 3 000

2 000

1 000

,R I

S

Li

iR

T1

S C1

Condition 1

Lj

i R S I

I

Lj

,R

T2

S L I C2

I

Condition 2

Fig. 5. Comparative study of mercury contents in the 3 organs of E. densa: roots (R), stems (S), leaves

(L), after 28 days' contamination (confidenceinterval c~=0.05).

inhibition in growth of 'C' plants in comparison to 'T' plants (Fig. 4A). Mercury concentrations greater than 5000 ppb (fresh weight) in the roots may affect the absorption of the nutrients and/or their transport towards the plant's other organs.

Interpretative analysis o f the results The overall results obtained on methylmercury transfers between the 'sediment' source and E. densa, reveal two distinct processes, characterizing the two periods of observation studied.

After 4 days" exposure, experimental condition 1 results in a very low level of plant contamination, almost all the metal being located in the leaves. Although no

224

mercury was detected in the medium at the moment of planting (concentration 0.1 ppb), these results show contamination of E. densa by an 'indirect' route. The metal present in the water (micro-traces) either came from residual quantities, after filling the experimental units for example, or from a very low rate of releasing from the contaminated sediment, during the period before a n d / o r after planting. The quantities of mercury accumulated in the plants in condition C1 are greater than those in condition T~ plants. This difference is caused either by: (i) a metal concentration gradient in the water, the level being higher near the flasks with the contaminated sediment (CO, in spite of the natural sediment on the surface; or (ii) by the onset o f mercury transfer between the substratum and the plant, directly via the b o t t o m of the stem in the sediment or through the 'underground' roots, the appearance and growth of which is very rapid after planting the cuttings. In accordance with the anatomical and histological structures of E. densa, characterized by absence of vascular elements (Feuillade, 1961), a transport of organic mercury at the level of symplasmic and vacuolar structures of the stem can be considered. For condition 2, if one takes into account the presence in the experimental units o f a large quantity of contaminated sediment, and no 'protection' from the releasing p h e n o m e n o n 'sediment -+ water' by the intermediary o f a substratum layer on the surface, the hypothesis of an indirect route o f plant contamination is reinforced. This would result in an increase in mercury concentrations and contents compared to condition 1. We can also note that the difference observed between the mean values Ca and T2 (Ca > TE) tends to sustain the hypothesis of the beginning of root transfer in E. densa in CE flasks. The existence o f a metal concentration gradient between C and T plants is much less likely here than in condition 1.

After 28 days" exposure the very great difference (a factor of 10) separating the contamination levels of C1 and C2 plants on the one hand, and T1 and T2 on the other, discloses transfer mechanisms distinct from those considered for the 4-day period. Indeed, only a 'direct' contamination of E. densa C1 and C2, via the intermediary o f their root system, can explain these differences. This is supported by the similarity in the content and concentration values for conditions C1 and C2. Other arguments can be used to confirm this interpretation, notably the importance of the relative levels o f mercury contamination in root and stem, the two organs which ensure transport of mercury from the 'sediment source' to the leaves. The results of mercury fixation in conditions T1 and T2 are very much reduced compared to C1 and C2 plants. They do, nevertheless, correspond to an important increase in comparison to the 4-day period. These E. densa have been grown on noncontaminated sediment; the mercury transfers are, therefore, via the water, unless there is a possibility o f metal fixation on the sediments of T flasks, especially in condition 2 where the mercury concentrations in the water, at least in the first phase, are liable to play an important part. Although such a mechanism could be sug-

225 gested, it would only explain a very small proportion of the accumulation mechanism observed. If the T2 plants are more contaminated than T1 plants, the differences between the average global contents are nevertheless slight: 711 and 435 ng. The presence of mercury in the medium coming from the transfer 'sediment --, water' cannot explain such results. In fact, the curves revealing the evolution of metal concentrations in the water show a rapid decrease (Table I); in addition, the quantities of mercury in the sediments and the exchange surfaces of the 'natural' or 'contaminated' sediments with water are very different in conditions 1 and 2. In an attempt to explain the mercury accumulation in the 'control' elodea, T~ and T2, one might consider a hypothesis of decontamination, on plant level C1 and C2. This would ensure a mercury transfer between the macrophyte organs, especially the leaves, and the water. The metal thus 'excreted' could attach to various accessible sites in the experimental system (suspended matter, walls of the tanks, sediments, etc.), and on the other plants - T1 or T2. Such a hypothesis is based on two essential arguments: (a) The decontamination processes, at the level of organism, organ and even at the cell level, are coupled with physiological and biochemical activities (transmembrane exchanges, secretions, cellular turnover, etc.) and can be accentuated when the toxic products modify certain structures or certain functions. In aquatic organisms, especially in fish, they are also dependent on contamination levels (contents and concentrations), penetration routes in the organism (direct or trophic), and abiotic factors (temperature, etc.) (Boudou, 1982; Ribeyre and Boudou, 1983) As far as we are aware, only the work of Czuba and Mortimer (1980) has shown decontamination in E. densa, after contamination of the macrophytes from the water column, together with 'migration' of mercury compounds, young tissues (CH3HgC1) and older tissues (HgCl2). (b) The quantities of mercury accumulated in E. densa for both conditions C~ and C2, are comparable for the 28 day period. Thus the decontamination processes may be at the origin of a similar metal fixation by 'T' plants. However, the difference between the global contents, T2 > T1, can be related to mercury transfers between the contaminated sediment and the water, higher inside the experimental units of condition 2. CONCLUSION The overall results obtained during this experimental approach and the different mechanisms they revealed enable us to submit a synthesis relative to transfer processes between the sediment initially contaminated with methylmercury and E. densa. The synthesis diagrams shown in Fig. 6 correspond to the two exposure times studied (4 and 28 days). The methylmercury is considered to be uniformly spread

226

N 4 DAYS

28 DAYS

Fig. 6. Schematicrepresentation of the main mercury transfer routes and their importance, between the 'sediment' source (CH3HgC1)and E. densa. (A) Release of mercurypresent in the sediments and indirect contamination of E. densa. (B) Mercury vaporization at the water-atmosphere interface. (C) Adsorption of mercury to the experimental unit walls. (D) Direct contamination of E. densa via the stem and the root system. (E) Mercury transfer between the plants (decontamination processes).

t h r o u g h o u t the sediment compartment, by analogy with the structure of our ecotoxicological model (Maury and Engrand, 1986), and in order to show in a more general way the mercury movements likely to appear between the three compartments 'water-sediment-macrophytes'. In the short-term (Fig. 6A), mercury accumulation by E. d e n s a comes from the superposition of two contamination routes: indirect route (A), i.e. release of metal from the sediment to the plant (essentially the leaves), and direct route (D), via the stem base of the cuttings and the subterranean roots which are starting to grow. Within the experimental system, other exchanges may take place from the metal first added to the sediment: mercury vaporization at the interface 'water-atmosphere' (B) or mercury adsorption on the aquarium walls (C). In the longest term (Fig. 6B), direct contamination (D) of E. d e n s a by the conductive routes (roots and stems) becomes clearly predominant in comparison to indirect contamination (A). In addition, the results obtained reveal a new pathway of metal transfer, related to the processes of decontamination of the plants (E).

227 ACKNOWLEDGEMENTS

We would like to thank Mrs. V. Serre, J. Jouvenel and D.W. Geffre, as well as Mr. H. Bouillard for their help in this work. This research was financed by the French Ministry of the Environment, the Commission of the European Communities, the Regional Public Establishment of Aquitaine and the National Scientific Research Centre (CNRS).

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