Toxicokinetics of Waterborne Trivalent Arsenic in the FreshwaterBivalve Corbicula fluminea

Pedro M. Costa Æ Hugo M. Santos ÆIsabel Peres Æ Maria H. Costa Æ Sheila Alves ÆJose Luıs Capelo-Martinez Æ Mario S. Diniz

Received: 30 May 2008 / Accepted: 12 November 2008 / Published online: 23 November 2008

� Springer Science+Business Media, LLC 2008

Abstract Arsenite (AsIII) uptake and elimination kinetics

were studied in a freshwater bivalve, Corbicula fluminea,

exposed to several nominal concentrations of AsIII (0, 100,

300, 500, and 1000 lg L-1) in a static 28-day assay, fol-

lowed by a depuration stage of 14 days. At the end of each

sampling time (days 0, 7, 28, and 42) whole-body portions

were surveyed for total As concentrations and, compli-

mentarily, surveyed for whole-body metallothionein (MT)

induction to assess its role as a defense mechanism against

exposure to AsIII. Histochemical evaluation of the digestive

gland was performed to verify As deposition and elimina-

tion in the tissue. Results show a significant increase in

whole-body total As after 28 days of exposure for all

treatments, followed by a decrease at the end of the depu-

ration phase. Biodynamic kinetic models for As uptake and

elimination were obtained from bioaccumulation data dur-

ing the exposure phase, for all As treatments, by estimating

uptake and elimination rate constants. Bioconcentration

factors (BCFs) were estimated by the ratio of these

constants. Results revealed that exposure to higher con-

centrations of AsIII causes a decrease in BCFs, suggesting

that C. fluminea triggers effective regulatory mechanisms

when exposed to higher concentrations of the metalloid.

Significant induction of MT was detected during the

exposure phase, followed by a decrease in MT concentra-

tion to control levels after depuration for all treatments. No

significant differences in MT concentrations were observed

between treatments. This finding may confirm the role of

MT as part of the As regulation process, but its indepen-

dence relative to concentrations of AsIII in water suggests

that MT induction is not dose dependent. The histochemical

evaluation provided clear evidence that As was effectively

accumulated in the digestive gland during exposure and

eliminated during depuration. The present work demon-

strated that C. fluminea is capable of regulating As, even at

exposures as high as 1000 lg L-1 of waterborne AsIII.

Arsenic (As) is a nonessential abundant metalloid that may

occur in the environment as a consequence of natural

mobilization and mineralization processes ranging from

erosion to activity of micro-organisms (Duker et al. 2005).

The presence of As in the biota can also result from

anthropogenic contamination since it has been used in

multiple industrial applications, from pesticides to syn-

thetic pigments, and, also, it may be released to the

environment as a consequence of the combustion of fossil

fuels and mining and smelting of metal ores (Mandal and

Suzuki 2002). As a consequence of these processes, As

may be present in trace amounts in organisms, soil, water,

and air, at potentially toxic concentrations (Mandal and

Suzuki 2002).

Arsenic-contaminated water is a serious human health

issue, far from restricted to developing countries. In the

Pedro M. Costa, Hugo M. Santos and Mario S. Diniz contributed

equally to this work.

P. M. Costa (&) � I. Peres � M. H. Costa � M. S. Diniz

IMAR-Instituto do Mar, Departamento de Ciencias e Engenharia

do Ambiente, Faculdade de Ciencias e Tecnologia, Universidade

Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

e-mail: pmcosta@fct.unl.pt

H. M. Santos � J. L. Capelo-Martinez

REQUIMTE, Departamento de Quımica, Faculdade de Ciencias

e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,

2829-516 Caparica, Portugal

S. Alves

Centro de Quımica Estrutural, Instituto Superior Tecnico de

Lisboa, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal

123

Arch Environ Contam Toxicol (2009) 57:338–347

DOI 10.1007/s00244-008-9267-6

United States, for instance, 13 million people are estimated

to drink water with more than 10 lg As L-1 (Bissen and

Frimmel 2003b). In some regions of Asia (e.g., Bangladesh

and India), As contamination of freshwater bodies is a

long-known major public health problem, and it has been

found to be the cause of severe pathologies, including lung,

liver, and skin cancers as well as a variety of cardiovas-

cular disorders (Duker et al. 2005; Mandal and Suzuki

2002). Human health concerns are, however, not limited to

direct exposure to waterborne As but also include con-

sumption resources from polluted areas, such as fish and

mollusks. Hazard quotient models for the consumption of

fish grown in black-foot disease regions (an As-derived

vascular pathology in humans exposed to contaminated

water) have recently been developed, relating carcinogen-

esis risks to As bioaccumulation in meal fish and

reinforcing the importance of understanding toxicokinetics

for such purposes (Liao and Ling 2003).

Arsenic toxicity and carcinogenicity have been found to

be dependent on its chemical and physical forms (species),

as well as exposure concentration and duration (Hossain

and Sivakumar 2006). Trivalent or arsenite (AsIII), and

pentavalent or arsenate (AsV), forms of As are the most

common in natural waters (Feng et al. 2001); however,

trivalent As is in general more toxic than pentavalent

(Cervantes et al. 1994; Duker et al. 2005). Arsenite can

inhibit various enzymatic pathways including glycolysis

and the tricarboxylic acid cycle by binding to sulfydryl

groups of enzymes and arsenate is known to uncouple

mitochondrial oxidative phosphorylation (Hindmarsh

2000). Organic As compounds such as arsenobetaine and

arsenocholine are also less toxic than inorganic forms and

have been found to be rapidly eliminated by mammals

(Kaise et al. 1992; Bissen and Frimmel 2003a).

It is believed that metallothioneins (MTs) play an

important role in the homeostasis of essential metals such

as copper (Cu) and zinc (Zn) and in the detoxification of

nonessential metals such as cadmium (Cd) and mercury

(Hg) (Romero-Isart and Vasak 2002). Bivalve MTs share

biochemical properties with their mammalian counterparts:

low molecular weight (6–7 kDa), high cysteine content,

lack of aromatic amino acids, and heat and acid tolerance

(Romero-Isart and Vasak 2002). As a result of the protein’s

role in metal detoxification, MT induction has been pro-

posed to be a biomarker for the environmental monitoring

of metal contamination (Viarengo et al. 1999). According

to Aposhian and Aposhian (2006) a number of reports have

appeared dealing with the binding of As species to proteins

as a protective mechanism, without, however, providing a

clear relationship between As metabolism and MTs. Still,

other authors have demonstrated that arsenite does bind to

mammalian MTs, including humans’ (Toyama et al. 2002;

Jiang et al. 2003; Ngu and Stillman 2006).

It is recognized that mollusks are capable of concen-

trating metals from their environment and have a high

tolerance to metallic burden (Couillard et al. 1993). Pres-

ently having a worldwide distribution, Corbicula fluminea

is a freshwater bivalve from Asia that was introduced in

Europe in the 1980 s, presenting invasive dynamics of

rivers, channels, and lakes (Mouthon 1981). It has been

considered of great relevance to develop environmental

toxicology studies with C. fluminea due to its distribution,

competitiveness, and tolerance against pollution (Sebesvari

et al. 2005). Still, regardless of the aforesaid characteris-

tics, C. fluminea has been subjected to only a few

ecotoxicological studies regarding As contamination, even

though it is has already been suggested that this species is a

valid biological indicator of contamination by trace metals

and metalloids (Inza et al. 1997; Baudrimont et al. 2003;

Santos et al. 2007).

Although some studies were carried out with respect to

Cd, Cu, and Zn (Baudrimont et al. 1997a; Fraysse et al.

2002), there is still a lack of data on As toxicokinetics and

toxicodynamics of C. fluminea, hence the relevance of the

processes of accumulation and elimination of this metal-

loid. The main objective of the present work was to study

As contamination kinetics by developing biodynamic two-

compartment models and estimating bioconcentration fac-

tors and, secondarily, to study the induction of MT-like

proteins in C. fluminea as a possible defense mechanism

against exposure to trivalent As. A histochemichal

approach is also intended in order to confirm As accumu-

lation in the digestive gland.

Materials and Methods

Experimental Design

The freshwater bivalves, Corbicula fluminea, collected

from the river Minho’s hydrographic basin (northwestern

Portugal) were acclimated in a closed continuous-flow

system with filtered and aerated tap water. Animals were

fed daily with a suspension of minced fish pellets (DibaQ,

Segovia, Spain). The assay was carried out in static mode

and in duplicate, at 20 ± 1�C and pH 7.06 ± 0.07, with a

12:12-h light:dark photoperiod. Bivalves (soft body mass,

1.6 ± 0.3 g; shell length, 2.0 ± 0.3 cm) were randomly

distributed by 20-L-capacity polyvinyl tanks (15 individ-

uals per replica), with constant aeration, and exposed to

different nominal concentrations of AsIII: 0 (control), 100,

300, 500, and 1000 lg L-1. AsIII stock solutions were

prepared from standard inorganic trivalent arsenic (Sigma,

St. Louis, MO, USA). The control test was performed with

clean, dechlorinated, tap water. The assay was divided into

exposure and depuration phases. The exposure phase lasted

Arch Environ Contam Toxicol (2009) 57:338–347 339

123

for 28 days, with three sampling times, at days 0 (T0), 7

(T7), and 28 (T28). After T28 animals were allowed to de-

purate in clean water for a further 14 days (depuration

phase), the assay was terminated at day 42 (T42). At T0 10

animals from acclimation tanks were sacrificed for analy-

sis, and at subsequent sampling times five individuals per

replica were processed for whole-body As and MT con-

centrations and histochemical evaluation. At each sampling

time water samples were collected to determine the real As

concentrations.

Arsenic Determination

Total As in tissue was determined in homogenized whole

soft body samples, dried, and digested in pressurized TFE

cells with HNO3 and H2O2 (Bryan et al. 1985). Total As

concentrations were measured by electrothermal atomic

absorption spectrometry (ET-AAS), with Zeeman correc-

tion and a matrix modifier (Pd[NO3]2 � 2H2O from Fluka,

Buchs, Switzerland), using a graphite tube atomizer

(M6Solaar AAS; Thermo Electron Corp., Waltham, MA,

USA). Analysis of DOLT 3 reference material (National

Research Council, Ottawa, Canada) and blanks followed

the same analytical procedure used for the samples to

validate the procedure and the measured values were found

within the certified range. The calibration curve was

obtained from standard inorganic As for atomic absorption

spectrometry (N206962 standard; Aldrich, Milwaukee, WI,

USA). Concentrations are given as micrograms total As per

gram tissue dry weight (dw). Total As in water was

determined using the same procedure, from water samples

acidified with 1 lL mL-1 Suprapur grade 65% HNO3

(Merck, Darmstadt, Germany).

Uptake and Elimination Kinetics and Bioaccumulation

Factors

First-order two-compartment kinetic models were obtained

to estimate the As uptake and elimination (excretion) rate

constants k. According to the biodynamic models detailed by

De Kock and Kramer (1994) and Luoma and Rainbow

(2005), the bioaccumulation of a contaminant results from

the balance between uptake from water and elimination from

tissues, which depends on the concentration of the bio-

available contaminant in the surrounding medium. Arsenic

bioaccumulation may therefore be given by the model

CAs ¼k1CAs waterð Þ

k2

1� e�k2t� �

þ CAs init:ð Þe�k2t ð1Þ

where CAs is the concentration of total As in tissue at time

t, k1 is the uptake constant, k2 is the elimination constant,

CAs(init.) is the baseline As concentration in tissue (i.e., at

the beginning of the exposure period), and CAs(water) is the

As concentration in the water, assuming that it remains

constant and no decay parameter is necessary in the model.

Since tissue As concentrations are given as micrograms per

gram tissue dw, water As as micrograms per liter, and time

as days, k1 is given as micrograms As per gram tissue dw

per day per microgram As per liter water, which, simpli-

fied, becomes liters per gram per day (L g-1 day-1). The

constant k2 is expressed as days-1, since the elimination

rate over time may be obtained by multiplying this constant

by the As concentration in tissue (Luoma and Rainbow

2005). The constants k1 and k2 are thus random coefficients

related to the removal of the element from the water, as is

its incorporated in the tissues and its elimination from the

organism, respectively.

Under steady-state conditions, the bioconcentration

factor (BCF) can be estimated as the ratio of k constants

(e.g., Bailer et al. 2000):

BCF ¼ k1

k2

ð2Þ

Bioconcentration factors are thus given as liters per

gram (L g-1). Standard errors (SE) for BCFs were

estimated as described by Bailer et al. (2000), according

to the formula

SEðBCFÞ �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffir2

1

k22

� 2k1r1;2

k32

þ k21r

22

k42

s

ð3Þ

where r2 is the variance of each k estimation and r1,2 is the

covariance between both estimations since they were

obtained by the same model. Estimation was done using

bioaccumulation data from the exposure phase. Since water

As concentrations were kept constant during the exposure

phase in each experimental treatment, four different mod-

els (corresponding to the nominal exposures to 100, 300,

500, and 100 lg L-1) were obtained. Rate constants k1 and

k2 were estimated by iterative least squares. Model good-

ness of fit was assessed by the r2 statistic.

Metallothionein Determination

MT was determined by quantification of thiolic (–SH)

groups in heat-treated cytosols from whole-body samples

according to the Brdicka reaction, as described by Diniz

et al. (2007). Whole-body tissue samples were homoge-

nized in the cold (&4�C), in 3 ml of 0.02 M Tris-HCl

buffer (pH 8.6) and centrifuged at 30,000g for 1 h at 4�C.

The supernatant (cytosol) was then separated from the

pellet, heat-treated at 80�C for 10 min, and subsequently

centrifuged at 30,000g for 1 h at 4�C to precipitate residual

high molecular weight and non-heat-stable proteins.

Quantification of MT equivalents in heat-treated cytosols

was performed by means of differential-pulse polarography

with a static mercury drop electrode (DPP-SMDE) using a

340 Arch Environ Contam Toxicol (2009) 57:338–347

123

693 VA processor and a 694 VA stand (Metrohm, Herisau,

Switzerland). The three-electrode system consisted of a

mercury capillary working electrode, a platinum auxiliary

electrode, and an Ag/AgCl reference electrode. The

Brdicka supporting electrolyte was prepared according to

Palecek and Pechan (1971) and contained 1 M NH4Cl, 1 M

NH4OH, and 2 mM (CoCNH3)6Cl3 (hexaminecobaltIII

chloride; Sigma). Triton X-100 (Sigma), 2.5 9 10-2 % (v/

v), was added to the electrolyte prior to use to suppress

baseline noise. The quantification procedure by DPP-

SMDE was adapted from Thompson and Cosson (1984).

MT concentrations were determined using the standard

addition method. In the absence of a standard mollusk MT,

determination of C. fluminea MT was performed using a

rabbit liver MT (forms I and II) standard (Sigma), as val-

idated by Diniz et al. (2007). Results are expressed as

milligrams MT per gram tissue dw.

Histochemistry

Fresh samples of whole-body C. fluminea were placed in

Castel’s fixative, containing 10% (v/v) formaldehyde and

2.5% (m/v) copper sulfate, for 5 days. After washing for

24 h with distilled water, samples were embedded in par-

affin for histological sectioning as described by Martoja

and Martoja (1967). After inclusion in paraffin, 5- to 7-lm

sections were counterstained with Nuclear Fast Red

(Sigma). Castel’s method for arsenic histochemistry was

employed as described by Lillie (1965). Histochemical

evaluation was performed using a DMLB optical micro-

scope (Leica Microsystems, Wetzal, Germany).

Statistical Analysis

The nonparametric statistics Mann–Whitney U and

Kruskall–Wallis H were used to identify differences

between exposure and control treatments and within

groups, respectively, after failure to demonstrate homoge-

neity of variances by Levene’s test. Correlation analysis

was obtained with Spearman rank-order correlations R or

Kendall’s s statistics. Inference on BCF estimates was

obtained with the z-test. The significance level was set at

5%. Statistical analysis and model parameter estimation

were done using the Statistica 6.0 software (StatSoft Inc.,

Tulsa, OK, USA), after Sheskin (2000).

Results

Arsenic Concentrations in Water

During the experimental period no mortality was observed

in any treatment. Total As concentrations determined in

water samples collected from the different treatments were

found to be very close to the nominal concentrations of

AsIII exposure (Table 1). No significant differences were

detected between the beginning and the end of the exper-

imental period (Mann–Whitney U, p [ 0.05), which

confirms that CAs(water) (1) can be regarded as constant.

Arsenic Bioaccumulation and Elimination

The data obtained from clam tissue samples collected at T7

and T28 regarding the different exposures to AsIII (100,

300, 500, and 1000 lg As L-1) are shown in Fig. 1, which

also reports total As concentrations for depurated organ-

isms. Total As concentration in control clams was

10.8 ± 6.4 lg As g-1 dw and did not vary significantly

along the experiment time (p [ 0.05). The levels of As in

organisms sampled at T0 (11.7 ± 4.4 lg g-1 dw) were not

significantly different from controls (p [ 0.05). During the

exposure phase, a very significant increase in accumulated

As was observed for all treatments at T28 (p \ 0.05) but

not at T7. At the end of the exposure term a strong corre-

lation between As in water and As in mollusks’ whole soft

body (Spearman R = 0.75, p \ 0.05) was observed. After

14 days of depuration a significant decrease was observed

(p \ 0.05) in the contents of total As in the whole soft

tissues of the clams in comparison with levels measured at

T28, for all treatments. The decrease in whole-body total As

ranged between 79% and 91%.

Biodynamic Accumulation Model

and Bioconcentration Factors

Bioaccumulation data from the exposure phase were suc-

cessfully fitted to model (1), yielding four different models

that explain 95.5%–98.4% of the total variance (Fig. 2).

Estimated k constants and respective BCFs are presented in

Table 2. The BCF estimated for the 100 lg L-1 exposure

was found to be significantly higher than the other treat-

ments (p \ 0.01). Although no other significant differences

were found between BCFs, these were found to be nega-

tively correlated with As exposures (Kendall’s s = - 1,

p \ 0.05), decreasing with increasing exposures to water-

borne AsIII.

Table 1 Average ± standard deviation values of effective total As

concentrations (lg L-1) measured in water samples in the exposure

assay

Treatment (lg AsIII L-1)

Control 100 300 500 1000

T0 \d.l. 96 ± 2 306 ± 5 540 ± 8 1010 ± 3

T28 \d.l. 92 ± 3 299 ± 3 530 ± 5 991 ± 4

Note: d.l., detection limit

Arch Environ Contam Toxicol (2009) 57:338–347 341

123

Metallothionein Induction

The MT induction in exposed organisms was found to be

significantly different from controls only at T28 (Fig. 3).

Induction was higher in organisms exposed to 100 lg L-1.

The concentrations of MTs in the organisms used as controls

(7.8 ± 0.8 mg g-1 dw) did not vary significantly during the

experimental period (Kruskall–Wallis H, p [ 0.05). The

increase in MT concentrations ranged approximately

between two- and threefold, corresponding to induction rates

ranging between 0.52 and 0.79 mg MT g-1 day-1, respec-

tively, from T0 to T28. After the depuration phase, a decrease

was observed in MT concentrations in all exposed groups,

reaching levels similar to those of controls (p \ 0.05). A

moderate but significant correlation (Spearman R = 0.5,

p \ 0.05) was found between bioaccumulated As and MT

concentrations after 28 days of exposure but not at any other

sampling points.

Histochemistry

The histochemical analysis revealed the presence of As

granules in the tissues of C. fluminea, particularly in the

cells of the digestive gland (Fig. 4) from organisms

exposed to higher concentrations (500 and 1000 lg As

L-1). Histochemical analysis of depurated individuals

depicts a distinctive loss of accumulated As, shown by the

reduction to total or near-total absence of copper arsenite

granules, which is in accordance with the reduction of

tissue As to control levels measured by AAS (Fig. 5).

Discussion

The present work demonstrated that C. fluminea is capable

of bioaccumulating and eliminating As as a result of

Fig. 1 Average total As

accumulation in exposed

organisms. Error bars represent

standard deviation. Significant

differences (*) from control

organisms and (�) from the

other exposed groups,

respectively (Mann–Whitney U,

p \ 0.05)

Fig. 2 Estimated models for all AsIII treatments during the exposure

phase

Table 2 Results of the kinetics study of the exposure phase

Treatment

(lg AsIII L-1)

k1

(L g-1 day-1)

k2

(days-1)

BCF ± SE

(L g-1)

100 -0.019 -0.176 0.110 ± 0.018

300 -0.003 -0.119 0.024 ± 0.015

500 -0.004 -0.173 0.021 ± 0.004

1000 -0.002 -0.162 0.010 ± 0.004

Note: BCF bioconcentation factor; k1 uptake constant; k2 elimination

constant; SE standard error

342 Arch Environ Contam Toxicol (2009) 57:338–347

123

Fig. 3 Average whole-body

MT concentrations in tested

organisms. Error bars represent

standard deviation. Significant

differences (*) from control

organisms and (�) from the

other exposed groups,

respectively (Mann–Whitney U,

p \ 0.05)

Fig. 4 Histochemical analysis of exposed individuals showing an increase in copper arsenite granules (arrows) after 28 days of exposure.

a Digestive gland tubule of a control individual; b digestive gland tubule of a clam exposed to 500 lg AsIII L-1

Fig. 5 Histochemical analysis of digestive glands of depurated individuals showing a decrease in copper arsenite granules (arrows) in digestive

gland tubules of clams exposed to 1000 lg AsIII L-1. a After 28 days of exposure, exhibiting clusters of granules; b after 14 days of depuration

Arch Environ Contam Toxicol (2009) 57:338–347 343

123

exposure to high levels of arsenite in water, which indi-

cates that the species is capable of regulating the metalloid

even following contamination by what is recognized as the

element’s most toxic form.

According to Baudrimont et al. (1997a) C. fluminea is

capable of accumulating high levels of waterborne ele-

ments and is an interesting model organism for

toxicological studies in freshwater ecosystems since it lives

buried in the upper layer of sediments and filters large

quantities of water (10 L clam-1 day-1). Nevertheless,

only limited information is yet available regarding the

exposure of this freshwater bivalve to As, although

Sebesvari et al. (2005) have demonstrated that Corbicula

sp. is capable of accumulating As and stated that the asiatic

clam is suitable for monitoring environmental heavy metals

and metalloids.

A field study carried out by Jonhs and Luoma (1990),

who collected Corbicula sp. from the San Francisco Bay and

the Sacramento/San Joaquin river delta (USA), showed that

mean As concentrations ranged from 5.4 to 11.5 lg g-1

whole tissue dw at all stations (with small temporal fluctu-

ations being detected), generally being consistent with

concentrations found in uncontaminated systems. The con-

trol organisms in the present work show concentrations

within the same range. The results show, however, that As

bioaccumulation is linked to As in water, contradicting

previous findings when bivalves were employed to monitor

waterborne As (e.g., Valette-Silver et al. 1999; Orescanin

et al. 2006). Most of these field studies with bivalves failed

or were unable to relate As concentrations in water with the

metalloid’s levels in typical contaminant storage compart-

ments such as sediments. It is likely that waterborne As

represents a much more bioavailable form of the metalloid,

especially concerning filter-feeding animals such as

bivalves, hence the exponential-type curve of the bioaccu-

mulation models estimated in the present work.

Bioaccumulation of toxic substances by aquatic organ-

isms is a complex phenomenon that results from the

relation between uptake and elimination rates and therefore

from the intrinsic biological variables from which they

derive. From the uptake constants low BCFs (\1) were

obtained. Since accumulation depends on uptake con-

stant 9 As concentration in water (1), low BCFs do not

necessarily mean that the organisms are not bioaccumu-

lating but that the proportion of the metalloid actually

being removed from the water and incorporated into the

tissues is low, at least at the tested concentrations of AsIII.

It is noteworthy that the tested concentrations are realistic

in terms of freshwater highly contaminated with this met-

alloid (reviewed by Mandal and Suzuki 2002).

The results show that As concentrations in soft tissues

decreased in all treatments during the depuration phase.

Furthermore, the BCF estimates, which were found to

decrease with increasing exposures to As, may indicate that

the organisms underwent a regulation process by modulat-

ing the uptake:elimination ratios. Even empirically, it can

be observed that exposure to 1000 lg L-1, for instance,

does not correspond to a 10-fold increase in bioacumulated

As relative to 100 lg L-1, at any sampling time. According

to our results, this can mean that the animals were

responding to exposure especially by restricting uptake,

since the estimated elimination constants failed to show a

clear trend with increasing exposure concentrations. On the

other hand, the results from the depuration phase (confirmed

by histochemistry) indicate that these bivalves have good

mechanisms to eliminate the metalloid at the tested con-

centrations of exposure. These results confirm the strong

metabolic capacity of the species regarding As detoxifica-

tion, as already suggested by Sebesvari et al. (2005). The

phenomenon of decreasing BCFs with increasing metal

exposures has also been observed for other elements

(essential and nonessential) and described as a factor that

may compromise the employment of this guideline as a

biomonitoring tool of aquatic biota (McGeer et al. 2003).

Still, much research is lacking regarding the actual bio-

logical factors that are responsible for this. Nevertheless,

even if BCFs alone are not capable of providing a direct

measure of environmental contamination, our work showed

that BCFs, when associated with uptake and elimination

kinetics, may provide solid insight into the species’ sensi-

tivity and regulatory capacity toward contamination.

The release of contaminants from cells depends on the

different turnover times of the various biochemical com-

partments to which the pollutant is bound (reviewed by

Ravera 2001): the pollutant fraction bound to substances

with a short turnover time is the one initially released (such

as phosphorous in membranes), and only subsequently are

the fractions bound to compounds with a longer turnover

time (like metals precipitated in granules). Regarding As,

these compounds may consist of several detoxifying

mechanisms that are activated to neutralize the toxic

effects and rapidly eliminate the metalloid from tissues

(e.g., formation of metal thiolates by MTs and other che-

lating processes like As methylation [Aposhian 1997]). The

kinetic differences in As elimination in organisms exposed

to different concentrations of contaminants can be attrib-

uted to differences in uptake and depuration rates (Luoma

and Rainbow 2005). Our results show that C. fluminea can

efficiently regulate As by modulating bioconcentration

factors according to exposure concentrations, possibly by

enabling a series of responses of which MT induction and

intracellular storage of arsenic granules may be important

components. The kinetics analysis performed in this work

was effectively based on a two-compartment (water/

organism) partitioning model which is a simplified equa-

tion that reflects the overall processes within the organism.

344 Arch Environ Contam Toxicol (2009) 57:338–347

123

It is not possible at this stage to infer whether uptake is

reduced by specific responses to exposure or if physio-

logical effects, such as a decrease in filtration rates, are

involved, or how elimination is modulated. The low mor-

tality and relative constancy of elimination during uptake

(and the effective depuration that followed) should indi-

cate, however, that exposure to waterborne As did not

considerably impair the animal’s metabolism, suggesting

that direct responses to exposure have occurred. The BCFs,

on the other hand, reflect the combination of all effects and

responses that affect uptake and elimination, a clear neg-

ative correlation with increasing exposures having been

observed.

Even though some inconsistencies and divergences

remain in the scientific community regarding the physio-

logical role of MTs and their use as biomarkers in

environmental monitoring programs (Amiard et al. 2006),

it is generally accepted that these proteins play a role in the

homeostasis of essential metals (e.g., Cu and Zn), as they

can act as essential metal stores ready to fulfill enzymatic

and other metabolic demands (Viarengo and Nott 1993;

Roesijadi 1996). It is also believed that the binding of MTs

to metals has a detoxifying effect, due to their ability to

sequester these elements and consequently decrease their

bioavailability to other cellular ligands (Baudrimont et al.

1999; Ravera 2001). It is noteworthy that, at least con-

cerning mollusks, there are several agents that are also able

to induce MT biosynthesis such as physical stress, tumor

necrosis factor, and reactive oxygen species (Baudrimont

et al. 1997a; Viarengo et al. 1999).

The present study reveals that a significant MT induc-

tion over time of exposure occurs, followed by a decrease

to baseline levels when the organisms are no longer sub-

jected to waterborne As. These results clearly indicate that

C. fluminea MTs play a significant role in As contamina-

tion, confirming the results of previous studies (Santos

et al. 2007; Diniz et al. 2007). The induction of these

proteins was, however, higher in clams exposed to lower

concentrations of AsIII in water (100 and 300 lg L-1),

showing that MT overexpression in this species following

exposure to As is unlikely to be dose dependent, which

may result from other detoxification mechanisms that may

have a more prominent role in As detoxification, a subject

already debated by other authors (e.g., Apeti et al. 2005).

Although many field and laboratory studies were per-

formed with C. fluminea, only a limited number of studies

have been devoted to MT induction in this species and, in

particular, regarding AsIII exposure (Baudrimont et al.

1997b; Santos et al. 2007; Diniz et al. 2007). In fact, studies

focusing on MT induction in freshwater bivalves following

As exposure are scarce. In accordance with the present

findings, Baudrimont et al. (2003) observed that field-con-

taminated C. fluminea MT concentrations rapidly decreased

by 37% following transfer to unpolluted conditions.

According to Viarengo et al. (1987) the elimination of

metals by bivalves seems to be mediated by an active

mechanism involving their accumulation in lysossomes

(with the subsequent excretion of the metal-rich residual

bodies). Since our results show that C. fluminea is able to

regulate this metalloid by accumulating and eliminating the

excess of As in tissues, it is thus possible that MT induction

may partially function as part of a primary defense mech-

anism in favor of bioaccumulation and other biochemical

mechanisms. The presence of intracellular As deposits, as

detected by the histochemical evaluation, may, in fact, be

another primary defense mechanism against contamination,

since precipitated forms of the metalloid should not be

made easily available to affect cellular functions. Arsenic

concretions, observed to form large clusters in tissues of the

digestive gland in the individuals exposed to higher con-

centrations, appear to be linked to the metalloid’s uptake

and elimination and might explain the good potential of this

species to bioaccumulate As; in addition, it may be an

important factor regarding its tolerance to contamination.

From the present results it can be inferred that C. flumi-

nea may be suitable as a monitoring organism of exposure

to AsIII in water, being able to accumulate and effectively

eliminate the metalloid even when exposed to relatively

high concentrations. The present work also shows that

C. fluminea may have a good resistance to this metalloid,

mostly by being able to modulate uptake and elimination

relative to exposure concentrations, from which result low

BCFs. Some caution may thus be required when interpret-

ing As kinetics data on this species. Finally, although it has

been demonstrated that MT induction occurs as a result of

exposure to AsIII, the unclear relationship between MTs and

exposure concentrations may signify that caution should be

implied when considering it as a stand-alone biomarker of

exposure to arsenite in C. fluminea.

Acknowledgment P. M. Costa is supported by a FCT (Portuguese

Foundation for Science and Technology) Ph.D. grant (ref. SFRH/BD/

28465/2006).

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