Tissue distribution of inorganic mercury, methylmercury and cadmium in the Asiatic clam (Corbicula fluminea) in relation to the contamination levels of the water column and sediment

~ )

Pergamon

Chemosphere, Vol. 35, No. 12, pp. 2817-2836, 1997 © 1997 Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain

PII: S0045-6535(97)00342-1 TISSUE DISTRIBUTION AND CADMIUM

OF INORGANIC

IN THE ASIATIC CLAM

THE CONTAMINATION

MERCURY,

0045-6535/97 $17.00+0.00

METHYLMERCURY

(Corbiculafluminea)

IN RELATION

TO

LEVELS OF THE WATER COLUMN AND SEDIMENT

Inza B., Ribeyre F., Maury-Brachet R. and Boudou A.

L a b o r a t o i r e d ' E c o t o x i c o l o g i e - Universite B o r d e a u x I / C N R S A v e n u e des Facultes - 33405 Talence Cedex, France (Received in Germany 26 May 1997; accepted 30 June 1997)

ABSTRACT The comparative experimental study of inorganic mercury (HglI), methylmercury (MeHg) and cadmium (Cd) bioaccumulation in the Asiatic clam Corbiculaflumineawas based on a 14 days' exposure to the water column or sediment compartments, as initial contamination sources. For each contaminant and exposure source, a five-point concentration range was set up in order to quantify the relationships between the contamination pressure and bioaccumulation capacity, at the whole soft body level and in five organs: gills, mantle, visceral mass, kidney and foot. Hg and Cd bioaccumulation at the whole organism level was proportional to the metal concentrations in the water column or sediment. For similar exposure conditions, the average ratios between the metal concentrations in the bivalves - [MeHg]/[HglI] and [MeHg]/[Cd] were close to 10 and 5 for the sediment source and 8 and 15 for the water column source. Metal distribution in the five organs revealed strong specificities, according to the different contamination modalities studied: kidney and gills were clearly associated with Cd exposure, mantle and foot with MeHg exposure and the visceral mass with inorganic Hg exposure. 01997 Published by Elsevier Scien,zeLtd

Keywords: Asiatic clam,

Corbicula, cadmium,

inorganic mercury, methylmercury, bioaccumulation, organotropism,

indoor microcosms, sediment, metal bioavailability.

1. INTRODUCTION

The current state of knowledge of the chemical fate of trace metals in freshwater systems clearly demonstrates the fundamental role of sediments as storage reservoirs, as sites for the transformation of metal chemical forms and species, and as potential endogenous contamination sources for the pelagic food 2817

2818 webs, via release processes and trophic transfers. Benthic species therefore occupy a key position within the metal biogeochemical cycles in aquatic systems, closely linked to their structural and functional properties and also their microhabitats: detritivorous burrowing species, filter feeders at the sediment/water interface, etc. [1, 2]. The Asiatic clam, Corbicula flummea, was selected in order to study mercury and cadmium bioaccumulation both at the laboratory scale, using indoor microcosms, and at the field level, at different polluted sites along the Lot and Garonne rivers, in South-West France [3]. There are many ecological and biological arguments to justify this choice of species: - C. fluminea, originating from China, was introduced into North America some time prior to 1938; it has become a major component of the benthic communities in lotic and lentic habitats south of latitude 40°N [4] . It is currently presenting very strong, invasive dynamics in rivers, channels and lakes in South-West France [5]. Its biotic potential and its development cycle, combined with a very limited degree of predator pressure are producing densities of several hundred individuals per m2, even several thousand in some cases [4, 6, 7] ; -

this bivalve has high adaptation capacities towards natural or anthropogenic variations in

the principal ecological factors. For example, this species is able to live within wide ranges of temperature (8 to 27 °C) and pH (5.9 to 8.2) in the Nina river (Portugal) [5]. Hence, large batches of molluscs are easily maintained in the laboratory for several months or transplanted into field conditions using caging procedure; -

this filter-feeding mollusc lives buried in the superficial sediment layers. Its estimated

filtration capacity is between 0.5 and 20 liters per day, with abiotic factors having a marked effect on this rate [6, 8]. Several field studies and experiments on artificial indoor streams have shown that C. fluminea is a good bioindicator of metal contamination [9, 10] ; -

the size of the adult individuals, shell length of around 2 cm (maximum anteroposterior

dimension), makes it possible to dissect the principal organs and analyze metal organotropism after different exposure conditions. This paper presents a comparative experimental study of inorganic mercury, methylmercury and cadmium bioaccumulation in C. fluminea, after a 14 days' exposure to the water column or sediment as initial contamination sources. For each contaminant and exposure source, a five-point concentration range, including controls, was studied, in order to quantify the relationships between the contamination pressure and bioaccumulation capacity, at the whole organism level (soft body) and in five organs (gills, mantle, visceral mass, kidney, foot).

2. MATERIAL AND METHODS

2.1. Structure

of the Experimental

Units (EUs)

2819 3 liters of dechlorinated tap water were first introduced into glass tanks (12x12x30 cm), which had been lined with plastic film (Plastiluz bags, alimentary standard). The general chemistry of the tap water was: resistivity 2,470 ohm.cm-1; HCO3, 231.8 mg.L-1; CI, 16.0 mg.L-l; SO4, 37.5 mg.L-1; Ca, 53.5 mg.L-1; Mg, 12.2 mg.L-1; NH4 <0.01 mg.L-1; NO2, 0.09 mgL-l; NO3, 1.8 rag.L-l; PO4 <0.05 mg.L -1. One kilogram (ww) of a 50/50 "natural sediment + pure sand" mixture was introduced into each EU using plastic containers (Monoplast, alimentary standard: 1lxl lx5 cm). The sand was 98% silica, with a granulometry of between 0.8 and 1.4 mm (SILAQ, France). Sediment was collected from the banks of the Garonne river, upstream from Bordeaux: it was a very homogeneous silt, rich in clays (75-80 %), with a low total organic carbon content (2 % on average). Wet weight (ww)/dry weight (dw) = 2.1 ± 0.2 (dw after 48 h desiccation at 60°C) and ww/volume = 1.96. Background Hg and Cd concentrations were 85 ± 8 and 240 ± 42 ~tg.kg-1 (ww) respectively. Seven days after the setting up of the water column and sediment compartments, delay required for the stabilization of the physico-chemical conditions, 6 Corbicula were added to each EU. Molluscs were collected from the Canal du Mid±, about 15 km from where it joins the Garonne river, and were then maintained in the laboratory on a sand substrate, with 3 phytoplanktonic algae additions per week from dense cultures of Scenedesmus acutus. The batches of molluscs were selected in order to obtain a homogeneous distribution of individuals throughout the EUs: 6 classes were defined using the shell length criterion (extreme values: 1.2 and 1.8 cm). One individual from each class was then randomly allocated to each EU. It is better to use the shell length criterion, rather than the weight, given that there are sometimes considerable variations in the amounts of water contained in the pallial cavity [4] . Oxygen saturation was maintained throughout experiment by permanent air bubbling (Pump RENA 301), with diffusers placed 5 cm below the water column surface. The EUs were placed in larger tanks (140x65x30 cm), which were themselves in enclosed containers, with thermoregulation equipment (heating and cooling systems). Temperature was fixed at 21 ± 0.2 °C. Artificial light was produced from 2 neon tubes (Sylvania F36W/GRO) in each tank, positioned 45 cm above the surface of the EUs and operated by timer switches. The daily period of light was fixed at 12h/24h; the average light intensity at the EU surface was 35 laE.cm-2.s-1. Periodic measurements of pH (pHmeter Hanna Inst., H18424) and turbidity (turbid±meter ESD 800) were carried out in the water column. No external food supply was added during the experiment.

2.2. Contamination of the EUs from the water column or sediment source

In close association with the objectives of this experiment, metal concentration ranges in the water column or in the sediment were defined according to progressions adapted to the data treatment procedure (Table 1). Thus, for each metal, ten experimental conditions were studied: 2 sources x 5 contamination

2820 levels, including the control conditions. Two replicates were made for each condition, giving 60 EUs set up simultaneously. CO

C1

C2

C3

Cd

control

0.8

2.3

5

MeHg

control

0.3

0.6

1

HglI

control

0.6

1.5

2.8

Cd

control

1

3.6

8.8

MeHg

control

0.3

0.6

1

Hgll

control

0.6

1.5

2.8

Table 1: Nominal concentrations selected for the two chemical forms of mercury - inorganic Hg (HglI) and methylmercury (MeHg) - and for cadmium (Cd) added to the water column or sediment as initial contamination sources. Concentration ranges were based on the progression log(Cj+ 1) = aj.

The contamination of the sediment was based on initial metal additions from concentrated aqueous solutions of 0.5 gHg.L-1 for methylmercury (MeHg: CH3HgCI, Merck) and 1 g.L -1 for inorganic mercury (HglI: HgC12, Merck) and cadmium (Cd: CdC12, Merck). After a mechanical mixing stage and before the introduction of sediments in the EUs, samples were collected from the different batches in order to control contamination levels and the homogeneity of the metal distribution. Water column samples were also analysed during the experiment, in order to quantify the metal release from the sediment. For the water column source, metals were added daily to the EUs (8 a.m) from aqueous solutions of 6 mg.L-1 for Cd and 3 mg.L-1 for HglI; additions were constant throughout the experiment but equivalent to half of the first additions. There were two daily additions of MeHg (at 8 a.m and 6 p.m.) from an aqueous solution of 1 mgHg.L-1 identical to that of the initial contamination step. The volumes added were defined according to metal determinations on water samples collected after 1, 2, 3, 6, 7, 8, 10 and 13 days' exposure, at the end of the contamination cycles (24 h for HglI and Cd; 14 h for MeHg). This procedure was adopted in order to take into account the complex processes which give rise to the decrease in metal concentrations in the water column after each addition, due to adsorption on the tank walls, transfers at the sediment interface, volatilization, bioaccumulation in the molluscs. Marked differences emerged in the evolution of metal concentrations in the water column: the average decrease of [Cd] and [HglI] during the 24 h cycles was around 40 % and 48 % respectively; for MeHg, it was close to 90 % after 14 h. These differences are in agreement with previous data obtained in similar experimental conditions; for MeHg, the permanent aeration of the water column played an important role [11, 12]. The contamination pressure was estimated for each EU contaminated via the water source using a global index called "Concentration.Days" Equivalent (CDE) [13] . This index is based on the integration of

2821 the different Hg and Cd concentrations measured in the water column (Ct), according to the length of time between sampling points (tj - ti): CDE (Ixg.L-1.days) = [(Co + C1)/2](tl - tO) + [(C1 + C2)/2](t2 - t l ) + ..- [(Ci + Cj)/2](tj - ti) (Cj = metal concentration measured at tj; Ci = average concentration between the measured value at ti and the following theoretical concentration after metal addition). As metal transfers to the sediment Compartment could represent a secondary contamination source for the benthic molluscs, Hg and Cd determinations were made on 4 sediment cores (glass tubes, inner diameter: 5 mm) collected from each EU at the end of the experiment and stored at -20 °C. In order to assess the vertical distribution of the two metals in the sediment, the cores were sliced into 3 strata: the uppermost layer, in direct contact with the water (0-0.5 cm), and two underlying strata (0.5-1 cm and 1-5 cm). Each sediment sample was dried (60°C, 24 h) before weight measurement and metal dosage. This drying stage was particularly necessary for the surface layers which contained varying amounts of water; preliminary experiments have shown that no significant metal loss occurred during this treatment. The method used to estimate Hg and Cd stratification in the sediment was based on the establishment for each core of the relationship between metal cumulated burdens (MBc) and the corresponding cumulated dry weights (Wd), from the uppermost layer down to the two underlying strata [14]. For the majority of cores, the relationship between MBc and Wd (3 measurements/core) could be satisfactorily estimated with a simple linear regression model (MBc = b0 + blWd), where bl corresponds to the background concentration in the sediment and b0 to the quantity of metal accumulated at the sediment surface. However, for the highest [Cd] in the water, the relationship between the three measured values was not linear, indicating metal transfers beyond the first sediment layer; in this case, the regression models were based on the two underlying strata.

2.3. Sampling and measurements of the clams (C fluminea) The 6 molluscs from each EU were collected after a 14 days' exposure and stored at -20°C. 4 organisms were used for metal determinations in five organs: mantle, visceral mass, foot, gills and kidney, the other 2 individuals being retained in the event of further checks being required. For analytical reasons, and despite the fact that it represented about 87 % of the total weight of the bivalves, the shell was not taken into account in this study. The soft bodies were dissected while still frozen. The four samples of each organ were pooled and weighed (ww, after drying on absorbant paper sheets) in glass tubes used for the digestion stage, before Cd or Hg determination. The relationship between the fresh weight (fw) and dry weight (dw) of the soft body of

C fluminea is well established: dw = 0.182 x fw (r = 0.99). For each EU, average metal concentrations in the soft bodies were obtained from the sum of the burdens in the five organs divided by the sum of the corresponding weights. Metal burdens associated with liquid lost during dissection were negligible.

2822 For metal determination in the biological matrices and sediment, samples were first digested by nitric acid (pure HNO3 - 3 mL) in a pressurized medium (borosilicate glass tubes), at 95°C for 3 h. Digestates were then diluted up to 20 mL with ultra-pure water (MilliQ plus). After mixing, the samples were left to stand, in order to facilitate redeposition of solid materials, especially for sediment samples. Metal determinations on the water samples were carried out without filtration. Total Hg determination was carried out by Flameless Atomic Absorption Spectrometry (Varian AA 475). A bromine salt treatment was applied to water samples and diluted digestates before the addition of stannous chloride [15] . The detection limit was 0.2 ~tg.L-1. Cd determination was carried out with a Varian AA 20 spectrophotometer equipped with a model GTA 96 graphite tube atomizer and autosampler. Samples of 10 ~tL were taken for the metal determination and mixed before atomization with 4 BL of a mixture "50 % Pd + 50 % Mg(NO3)2", to facilitate removal of the matrix. The detection limit was 0.1 ~tg.L-1 . The accuracy of the two analytical procedures was monitored by periodic analyses of standard reference materials from BCR (Brussels, Belgium), KFA (Jtilich, Germany) or IEAE (Monaco), together with biological samples series. Values for total Hg and Cd were consistently within the certified ranges for each element. Background concentrations in the control molluscs collected at the end of the experiment are shown in Table 2.

G

VM

M

F

SB

Hg (ng.gl, fw)

27.5±4.4

34.1±6.4

16.1±3.1

30.44-1,9

28.3±4.1

Cd (ng.g-t, fw)

308.7± 79.8

61.9 ± 10.5

30.4 ± 4.9

20.3 ± 10.0

85.5 ± 16.2

Table 2: Background concentrations of total mercury and cadmium in Corbicula fluminea, in the four organs (gills: G, visceral mass: VM, mantle: M, foot: F) and at the whole organism level (SB: soft body).

2.4. Data treatment

In order to quantify the isolated actions and the combined interactions between the different factors taken into account within the complete factorial design, the multiple linear regression technique was used. Due to the definition of the metal concentration ranges and to the regressor coding, orthogonal polynomials were used, which simplified the interpretation of the effects of each factor because the regression coefficients were independent. Regressor coding was based on tables [16] : for example, Co = -2, C1 = -1, C2 = 0, C3 = 1, C4 = 2 for the first level. Depending on the variance/average relationships for each set of data, different types of transformation of the explained variables were used (logY, I/Y, ~Y, ...). The alpha risk adopted for the statistical significance of the effects observed was equal to 0.01 or 0.05; F values were calculated with reference to the inter-replicate variance. Multiple regressions and 2D plots were made using Excel and softwares developed in our Laboratory.

2823 A global approach to the distribution of inorganic Hg, MeHg and Cd in the five organs of C

fluminea

after exposure to the two contamination sources was established using multivariate analysis

(factorial correspondence analysis). Data treatment based on the relative metal burdens in the organs was carried out using the Statlab 2.1 software.

3. RESULTS

3.1. Exposure conditions from the water column and sediment sources

For the water source, the average metal concentrations in the water column obtained from the CDE values (Cm=CDE/13 days) were very close to the nominal concentrations (Cn): the ratios Cm/Cn were 1.23 for Cd, 1.21 for MeHg and 1.08 for HglI. Mercury and cadmium accumulation in the sediment, via transfers from the water column, showed a marked proportionality between the b0 coefficient values from the regression models set up for the 4 sediment cores per EU, and the corresponding nominal Hg and Cd concentrations in the water column (Figure 1).

~

200.

4o

150

30

1oo

M ~

120

2O

.g 10

50

|

|

i

|

2.5

5

7.5

10

[Cd] ht the water (ml~L-I)

0

.~ 4o

/. i 0.5

| 1

i 1.5

[Mettgl m the water (~.L-I)

0 1

2

3

4

5

[Hgll] inthe water (rag.L-l)

Figure 1: Relationships between the nominal concentrations of the metals in the water column - cadmium (Cd), methylmercury (MeHg), inorganic mercury (I-IglI) - and the average metal burdens accumulated in the sediment cores (bo coefficient) at the end of the experiment. Regressionmodels: Cd: b0 = 20.0[Cd] (R = 0.97) MeHg: b0 = 24.5[MeHg] (R = 0.88) - HglI: bO = 19.7[HglI] (R = 0.98)

Vertical Hg distribution in the sediments clearly showed that most of the transfers are limited to the first layer (0-0.5 cm). For the highest [Cd] in the water (C3 and C4 levels), relationships between the cumulative burdens (Be) and the corresponding cumulative dry weights (We) were non-linear, indicating a significant metal diffusion towards the underlying sediment (data not shown). This metal distribution could lead to very high concentrations in the superficial sediment layer: for example, [Cd] were close to 5 ~tg.g-1 (dw) when the nominal concentration in the water column was 10 gg.L-1; for HglI (C4 = 5 lag,L-l) and MeHg (C4 = 1.5 lag.L-l), they were 2.3 and 1.5 pg.g-1 (dw) respectively. The amounts of the metals transferred to the

2824 sediment at the end of the experiment were estimated from the average burdens measured in the cores; average values were around 40 % of the total amount of Cd added to the water column, 26 % for inorganic Hg and only 14 % for MeHg For the sediment source, metal concentrations measured in samples collected before the sediment was introduced into the EUs were very close to the nominal values selected for the three concentration ranges (data not shown). Estimates of the quantities of Hg or Cd transferred to the water column during the experiment were based on metal determinations on water samples collected at time zero (7 days after the setting up of the EUs) and after 8 and 13 days. Only Cd determinations gave significant results higher than the detection limits of the analytical methods. At the end of the experiment, [Cd] in the water were close to 0.3 lag.L-1 for the C3 level (8.8 ~tg.g-1) and 0.6 lag.L-1 for the C4 level (20 ttg.g-1). Estimated contamination pressures from this secondary source, based on the CDE indexes calculated from the metal concentrations measured in the water column, showed a clear relationship with the initial metal concentrations in the sediment.

3.2. Weight of the clams at the end of the experiment No mortality was observed during the experiment. Cd concentrations in the water column were lower than the 55 and 100 ~tgCd.L-1 used by Graney et al. [9] and Doherty el al. [10] respectively, and which produced mortality rates of 15 % and 60 % after 30 days of exposure in artificial streams. To our knowledge, no data were available in the literature on the acute and chronic toxicity of Hg compounds to this benthic species. Multiple regression analysis of the soft body weights after 14 days of exposure shows that the different factors taken into account - contamination sources, metal concentration levels - have no significant effects in comparison with the control organisms (ct = 0.01 - data not shown). In relation to the study of metal distribution within the organisms, a comparative analysis of the relative fresh weights of the five organs was realized: no significant differences were observed between the exposure conditions (data not shown). The average relative weights of the visceral mass, mantle, foot, gills and kidney were 49.1%, 19.3 %, 18.8 %, 12.5 % and 0.3 % respectively.

3.3. Mercury and cadmium bioaccumulation in the clam (C. fluminea) 3.3.1. Whole organism level The average concentrations of the metals measured in the soft body, function of the different contamination levels of the water column or sediment source, are presented in Figure 2. Bioaccumulation was proportional to the metal concentrations in the water column or in the sediment compartment, though with an asymptotic tendency for the inorganic Hg.

2825 ~

900.

d

A

MeHg

CA

v,m

6OOO

tX

0.~

.t

600 9

,,

- .s

O

O

40O0

#

MeHg

°. J

O

O

s-"

N

CA

Itgn

e~

2000

300 °m

/l

].,.;

[] °

°.--A

8 0

.

• ~'

i S*'

A A

-0

s~

~..._tr'" w

0

!

w

i

2.5 5 7.5 [metal] in the water (pg.La) Water source

I

10

!

i

i

|

i

0 5 10 15 20 [metal] in the sediment (mg.kg-1, ww) Sediment source

1 /

Figure 2: Bioaccumulation of cadmium (Cd), methylmercury (MeHg) and inorganic mercury (HglI) in the soft body of Corbiculafluminea after a 14 days' exposure, function of the nominal concentrations of the metals in the water column or in the sediment, as initial contamination sources. Symbolscorrespond to the measuredvalues(2 replicatesper condition)and the curvesto the multipleregressionmodels(~ = 0.01).

In our experimental conditions, bioaccumulation in the soft body was greater when the metals were added to the water column: for the C4 contamination levels, Hg concentrations were about 10 times higher; for Cd, this ratio was only 2.5. Nevertheless, comparison of the two contamination sources has little meaning, given the wide difference in their contamination levels (factors of 1,000 for the Hg and 2,000 for the Cd, in favour of the sediment source) and the differences in the metal bioavailability to the benthic organisms. The comparison between the two chemical forms of Hg and Cd, for similar nominal concentrations, clearly shows the preponderance of the MeHg bioaccumulation, leading to concentrations higher than 6,000 ng.g-1 (fw), after 14 days of exposure from the water column source (C4 level: 1.5 ~g.L-1). The average ratios between the metal concentrations in the bivalves - [MeHg]/[HglI] and [MeHg]/[Cd] -, are close to 10 and 5 for the sediment source, and to 8 and 15 for the water source. The estimated bioconcentration factors from the water column source (BCF = [organism]fw/[water]) are close to 4,000 for MeHg, between 300 and 500 for HglI and around 150 for Cd. This average BCF value for Cd was lower than those obtained by Graney et al. [9] after 28 days of exposure in artificial streams: 630 for a [Cd] of 23 ~g.L -1 and 300 for a

2 8 2 6

[Cd] of 55 ~tg.L-1, maximal concentrations in the molluscs being observed after 11 days. Similarly, an average BCF close to 500 was found by Doherty et al. [10] after a 22 days' exposure to 100 ~tgCd.L-1. Differences in bioaccumulation capacities between Cd and HglI were inverted for the two contamination sources (Figure 2). The higher values found with cadmium after contamination of the EUs by the sediment source may be explained in part by the release processes in the water column, which induce a secondary contamination source for the molluscs. If the estimated contamination pressure from this secondary source is taken into account, via the CDEs calculated from the measured [Cd] in the water column, and with reference to the data obtained for the water column source, 40 % of the Cd burdens accumulated in the soft body of C. fluminea

could result from metal transfers between the sediment

compartment and the underlying water column. For the two Hg compounds, metal determinations on water samples were always under the detection limit (0.2 ~tg.L-l): it is thus impossible to establish links between bioaccumulation and Hg transfers from the sediment to the water column compartment.

3.3.2. Organ level Analysis of Hg and Cd distribution in the organs of C. fluminea reveals the specificities of the different biological compartments (Figure 3) : -

relationships between metal bioaccumulation in the organs and the corresponding nominal

concentrations in the water column or in the sediment are generally of a linear type or else they revealed greater accumulation values for the lower exposure levels within the concentration ranges studied: note, for example, a plateau tendency for the gills after contamination via the "water column/MeHg" source and for the visceral mass after exposure to the "water column/HglI" source; - for the Cd, the lowest concentrations were observed in the foot; Hg accumulation in this organ were similar to those observed in the mantle, except for the C4-MeHg level, where accumulation in the foot is greater; -

the highest concentrations were observed in the gills after contamination of the EUs with

MeHg and Cd; differences from other organs were amplified for the water source and, on the contrary, markedly reduced for the sediment source, notably for MeHg. Thus, concentrations higher than 10,000 ng.g -1 (fw) were measured in the gills after a 14 days' exposure to MeHg, via the water column source (BCF > 7,000); similarly, concentrations close to 3,000 ng.g"1 (fw) were measured for the condition "Cd/water source/10 ~tg.L-1. (BCF = 300). For the cadmium and "MeHg-sediment source", concentrations in the visceral mass occupied an intermediary position. When the microcosms were contaminated by inorganic Hg, the visceral mass showed the highest concentrations, [Hg] in the gills being close to those measured in the foot and mantle; for the sediment source, the ratio between [Hg] in the visceral mass and gills was close to 3, metal concentrations in the gills, foot and mantle being very low, close to the background levels.

2827 4000

12000 ~ o

.+

.+--8

4000.

G

+.

G

3000

"7 ~

.' o

¢

.+" o

.7 ~

o+

+

VM

.

/

+~o M

0. 5

7.5

1o

F

,,-7, M

~

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~,:

+

2000

o

[]

1000

o

o/'

..-"

+ ' i/ r

o

i

o M

-~"

V

. - ' / x.O ++'+

.

t,

~

1

2

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[ H g I I ] in t h e w a t e r 0 t g . L -~)

800

300

o

o

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1500

~"

600

:'°0

.." o VM ~- ,,..,,~

e~ S

o

/

."

1000

~

+"

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• °•"

0 0.5 1 1.5 [MeHg I in the water (pg.U ~)

[Cd] in t h e w a t e r ( r a g . L - l )

2000

~'

7

...... 2 2.5

o

:'

4000

=_

1000

o VM o

:'o

2000

3000.

+-

8000

7

:

r*"

..,~

400

/./. /

0 VM

.

,%

. VM

,_~

dr ~~

200

•

F

o

+

o z i

o:"

~"

""

500

."o.,'

-+

.,"

M

'o..0+~+

F

200

5

10

15

.~

100

o ro o+ s

s

,, "'

--

20

~

OG

-0 . . . . . ..--

0FT/-+, 0

[Cd] i n t h e s e d i m e n t ( m g . k g - l , ww)

13

oF

.0:.-.+.....~ . . . . ,*m o

i

0

/ /

~' M

0.5

1

1.5

[ M e H g l in t h e s e d i m e n t (mg.k g-l,ww)

0

1

2

3

.

.

4

5

[ H g l l l in t h e s e d i m e n t ( m g . k g - 1 , ww)

Figure 3: Cadmium (Cd), methylmercury (MeHg) and inorganic mercury (HglI) and bioaccumulation in the different organs of

Corbiculafluminea

-

gills: G; visceral mass: VM; mantle: M; foot: F -, function of the

different levels of contamination of the water column and of the sediment, as initial contamination sources. Symbols correspond to the measured values (2 replicates per condition) and the curves to the multiple regression models ((x = 0.01). When considered in relation to the major differences in weight between some of the organs studied, the relative metal burdens provide complementary information on Cd and Hg organotropism. Results shown in Figure 4 correspond to average values for the two Hg compounds and Cd, without differentiating between contamination levels of the water column and sediment sources. Contamination of the EUs with inorganic Hg produced very high relative burdens in the visceral mass (>70%), the values obtained for MeHg and Cd being between 45 and 50%. After exposure to MeHg, the relative burdens in the foot were 12 and 16%, much higher than the corresponding burdens for the two inorganic Hg contamination conditions (3 and 8%). After exposure to cadmium, two organs were characterized by high metal relative burdens in comparison to results for the two Hg compounds: the gills

2828 (26 and 29 % against 9-8% for HglI and 21-17% for MeHg) and especially the kidney (12 and 6% against I-2% for HglI and 3% for MeHg).

K 12%

M 6% 15%

M 11%

G 26%

VM

VM

45%

45% G 29%

F 6%

K 3%

M

M

14%

16%

GIi

F 5%

K 3%

G

VM

17%

50%

21%

F 16%

F 12%

M K G 5% 1% 9%

11%

3%

2%

8% F

8% VM 71%

VM

82%

Water source

[

I

Sedimentsource

]

Figure 4: Average relative burdens of cadmium (Cd), methylmercury (MeHg) and inorganic mercury (HglI) in five organs - gills: G; visceral mass: VM; mantle: M; foot: F; kidney: K - of

Corbiculafluminea, after

days of exposure from the water column or sediment as initial contamination sources.

14

2829 A global and comparative approach to Cd, inorganic Hg and MeHg distribution in the five organs of

C. flurainea, based on the relative metal burdens, was realized using the factorial correspondence analysis (Figure 5).

axe 2

ID

" C1

Cd -W

\\

\

[]

~1

callS

\\\\ ,\ I I

i MeHg-W

i000

Hgn-W

axe 1

.

II

Figure 5: Plan 1-2 (% cumul = 81,3) from the factorial analysis based on the relative metal burdens in the five organs of Corbiculafluminea (gills: G; visceral mass: VM; mantle: M; foot: F; kidney: K), after 14 days of exposure to the water column (W) or sediment (S) contamination sources. ---->: increasing metal concentration ranges in the water column or in the sediment.

Analysis of the plan 1-2 clearly confirms the specificity of the organotropism: the clusters of points corresponding to the three contaminants are well differenciated. They are very clearly associated with the organs already mentioned, namely the kidney (K) and the gills (G) after exposure to Cd, the mantle (M) and the foot (F) after exposure to MeHg and the visceral mass (VM) after exposure to inorganic Hg. Some complementary information can be deduced: - for the inorganic Hg, metal distribution in the organs was markedly influenced by the initial contamination source of the microcosms and by the contamination levels. The importance of the visceral mass increased after exposure via the water column source but decreased when HglI concentration in the water column moved from the C1 to the C4 level; conversely, Hg relative burdens increased in the visceral

2830 mass when the contamination levels of the sediment increased, leading to a similar organotropism for the highest contamination levels of the two sources. - for methylmercury, when the metal concentrations increased in the water column or in the sediment compartment, the relative burdens in the foot and in the mantle predominate in the detriment of the visceral mass after exposure to the sediment source, and to the gills for the water source. - for the other contamination conditions, notably after exposure to the water column or sediment contaminated with Cd, it was not possible to establish any relationship between the Cd concentration range and the metal organotropism in the so~ body of the molluscs.

4. DISCUSSION AND CONCLUSION

This comparative study of inorganic Hg, methylmercury and cadmium bioaccumulation in the Asiatic clam, in a wide range of metal concentrations in the water column or in the sediment compartment as initial contamination sources, reveals some very marked similarities and differences, both in respect of metal transfer capacities between biotopes and organisms and metal distribution in the main organs, after 14 days of exposure. In conjunction with the physico-chemical properties of the two mercury compounds and cadmium, many other ecotoxicological mechanisms can act on the metal bioaccumulation processes in filtering molluscs: metal chemical fate in the biotopes, bioavailability, interactions with the biological barriers at the interface between the soft body and the surrounding medium, absorption efficiency, distribution in the different internal organs, and excretion. Among the important processes relative to the chemical fate of the two metals in the biotopes and their bioavallability, complexation reactions with ligands present in the particulate and dissolved phases play an important role. Within the microcosms, the bioturbation activity of the molluscs is weak: turbidity measurements on water samples revealed very small concentrations of suspended particles (< 5 NTU on average). According to pH and pC1 values, thermodynamic calculations with the "MINEQL+" programme [17] show that inorganic Hg and MeHg are almost exclusively represented by neutral chemical species in solution: Hg(OH)2, HgOHCI, HgCI2 - CH3HgCI, CH3HgOH. For Cd, the two species Cd 2+ and CdCO3 are predominant. When the EUs are contaminated by the sediment source, the affinity of the two metals for the sedimentary particles is very important, as it contributes to a considerable imbalance in partitioning between the porewater and the particulate phase [1]. Metal transfers between the two biotopes, depending on the initial contamination source, can also play an important part in the contamination of the bivalves: - releasing processes from the sediment were detected only for Cd but were probably also present for the two chemical forms of mercury. They may explain a large proportion of metal bioaccumulated at the end of the experiment;

2831 -

metal accumulation in the sediments when the EUs were contaminated by the water source

is restricted mainly to the superficial layer (< 0.5 cm), and may be the cause of the contamination of the bivalves by direct transfers from the sediments, but more especially through the intermediary of microorganisms living at the sediment surface: algae, bacteria, protozoans. In our experimental conditions, these represent the greater part of the trophic support for these filter-feeders, which, in certain circumstances, can become deposit feeders [8, 18]. Transformations of inorganic Hg and MeHg, via the methylation and demethylation reactions, can modify mercury transfers from the water column and sediment compartments. In any case, given the wide differences in bioaccumulation capacities between the two Hg chemical forms, demethylation can only minimise transfers between the water column or the sediment and the organisms. On the other hand, the methylation of inorganic Hg must contribute to increasing bioaccumulation if the methylated form produced is bioavaitable to the benthic molluscs. If methylation reactions were able to take place in our experimental conditions, especially when the microcosms were contaminated via the sediment source, the contamination procedure used for the water source, based on daily additions of inorganic Hg or MeHg in the EUs, should minimize their importance. Exchanges between Corbiculafluminea and the water column are based on currents via the inhalant and exhalant siphons, for respiratory and nutritive purposes. It should be noted that the presence of toxic products in the external environment may cause the valves to close or to slow down their opening and closing rhythm. Studies by Doherty et al. [19]

showed that when populations of Asiatic clams were

exposed to concentrations of between 100 and 400 ~tg.L-1 of Cd they rapidly closed their valves; no information is available for Hg compounds. Metal concentrations measured at the whole body level revealed the very high predominance of MeHg bioaccumulation over that of inorganic Hg and cadmium; also the preferential transfer capacity of both metals from the water source, despite initial contamination levels in the sediment being one or two thousand times greater. For most aquatic organisms, biological barriers are easily crossed by MeHg, often with very high transfer rates [20, 21] . The liposolubility of MeHg is not the direct cause of its transmembrane transport: in fact, the octanol/water partition coefficients are very low, between 0.7 and 1.7, depending on the chemical species (CH3HgOH or CH3HgC1), whereas they reach 3.3 for HgCI2 [22, 23] . "Rapid diffusion across membranes rather than lipoid affinity is responsible for the transport of MeHg" [24]. The organotropism of mercury after contamination of the molluscs by MeHg indicates a large accumulation of the metal in all the organs, and a marked affinity for the foot, similar to the bioaccumulation capacity in the adductor muscle of the oyster or the skeletal muscle of the rainbow trout. Moreover, this tissue acts as a receiver compartment during the depuration periods, the Hg burden increasing via Hg transfers from donor organs (gills, liver, etc...) [11, 25] . The bioaccumulation of inorganic Hg clearly reveals the predominance of the visceral mass. The surface of the visceral mass is densely covered in cilia and is in direct contact with the water inside the

2832 mantle cavity. If direct metal transfers from this internal medium are probable, via adsorption or absorption mechanisms, the trophic route, based on contaminated food particles and/or ingested water, may also play an important part. In our experimental conditions, no external food was added during the 14 days of exposure. However, recent studies on periphyton communities in similar indoor microcosms including rooted macrophytes have shown that a large number of diatom species (130 taxa) are present and able to colonize artificial substrates in the water column [26] . It is also important to note that the Asiatic clam is one of the rare species of suspension feeders able to capture small-size particles, such as bacterial cells with an average size of about 0.5 lam [27] . Studies on contamination via the trophic route of different aquatic species show that the intestinal barrier is not particularly permeable to inorganic Hg (transfer rates were less than 10%); and yet this barrier is able to fix large quantities of metal, probably via adsorption reactions at the level of the enterocytes' microvillosities and their abundant cell-coat [11, 12, 20]. Cd bioaccumulation from the water source was characterized by concentrations in the whole soft body which are about half those observed after contamination by inorganic Hg. For the sediment source, Cd bioaccumulation can be explained in part by the metal release in the water column. Although there are wide differences in the concentrations of Cd in the molluscs, according to the inital contamination source, organotropism is very similar. In both cases, gills have characteristically high concentrations, with their relative burdens being 26 and 29% respectively. There were also very large amounts of Cd measured in the kidneys: 12 and 6%. After exposure of C. fluminea via the water column in artificial streams or via Cdcontaminated algae, metal distribution in the visceral mass and in the "gill+mantle+adductor muscle" compartment was comparable with our results: the ratios between average Cd concentrations were, in both cases, close to 1.2, in favour of the "gill+mantle+adductor muscle" compartment [10] . Concentrations and relative Cd burdens in the foot of C. fluminea are comparatively low: these results are in agreement with the very limited accumulation capacitiy of Cd in the muscle tissue, especially in fish [28]. Among the cellular processes which may play an important role in metal storage in the different organs of the freshwater molluscs, metallothioneins (MTs) and their induction by essential and nonessential metals have the ability to sequester Cd and possibly Hg, and to protect against their toxicity [29] . Doherty

et al. [10] have shown a significant increase in MT-like concentrations in C. fluminea depending on the duration and mode of exposure to cadmium. Recent experimental studies based on indoor microcosms showed a significant induction of MTs in the gills and visceral mass (including the kidneys) of C. fluminea after direct exposure to cadmium. On the other hand, no significant increase of the MT concentrations was observed after exposure to inorganic Hg, despite very high bioaccumulation in the different organs [30]. No data are actually available on the sequestration capacity of MeHg by these cytosolic proteins; saturation techniques used for MT determination in biological samples reveal a very small binding capacity of this organic compound to the MT thiol clusters [30]. Several studies support also the view that the cellular lysosomal system is involved in the incorporation of Cd and Hg via granule formation, especially in the molluscan digestive gland and kidney [29]. Experimental approaches are currently underway to further our

2833 investigations into mercury and cadmium bioaccumulation in C. fluminea. A detailed analysis of metal distribution at the organ and cell levels, based on autoradiography after exposure via the water column or sediment sources enriched with radioisotopes (l°gCd, 2°3Hg) should enable us to consolidate the interpretative analysis of the role of the biological barriers in the fixing and uptake of metals; these studies will also enable us to investigate the bioaccumulation mechanisms under lower exposure levels, especially for MeHg

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