influence of static and fluctuating salinity on cadmium uptake and

Influence of static and fluctuating salinity on

cadmium uptake and metallothionein expression by

the dogwhelk Nucella lapillus (L.)

Kenneth M.Y. Leung a, Jorundur Svavarsson b, Mark Crane c,David Morritt c,*

aThe Swire Institute of Marine Science and Department of Ecology and Biodiversity,

The University of Hong Kong, Hong Kong, ChinabInstitute of Biology, University of Iceland, Grensasvegur 12, 108 Reykjavik, Iceland

cSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK

Received 21 November 2001; received in revised form 8 February 2002; accepted 22 May 2002

Abstract

The aim of this study was to investigate the effect of salinity on cadmium (Cd) accumulation and

metallothionein (MT) expression in the dogwhelk Nucella lapillus (L.). Adult dogwhelks (shell

length: 23.4F1.3 mm) were acclimated to salinity of 33 psu (control), 22 or 11 psu under controlled

laboratory conditions (9.5 jC; pH 7.9) for 10 days in a stepwise manner by reducing the salinity by 5.5

psu day�1. Ten treatment groups were used and comprised five salinity regimes (three fixed salinity

[33, 22 or 11 psu] and two fluctuating salinity [varied daily between 33 and 22 psu or 33 and 11 psu in

a cyclic manner]) at each of two Cd concentrations (control: <0.001 Ag Cd l�1 or treatment: 500 Ag Cdl�1). After acclimation, groups of 20 dogwhelks were exposed to each of the 10 Cd/salinity

combinations. All the control and Cd-exposed dogwhelks exposed to 11 psu were dead within 5 days

of exposure due to hypo-osmotic stress. Twenty days after exposure to all other treatments,

concentrations of Cd and MTs in the tissues of surviving dogwhelks were quantified using atomic

absorption spectrophotometry and the silver saturation method, respectively. Both Cd accumulation

and MT induction in control or Cd-exposed N. lapillus were significantly influenced by changes in

salinity, especially at a prolonged and fixed low salinity (22 psu), although such influences of salinity

on the concentration of MTs were dependent on the tissue type. The study highlights that salinity

should be considered when monitoring trace metals and/or MTs in intertidal molluscs, particularly in

estuarine or transplanted biomonitors. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Biomonitor; Biomonitoring; Cadmium; Metallothioneins; Nucella lapillus; Salinity

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -0981 (02 )00209 -5

* Corresponding author. Tel.: +44-1784-443971; fax: +44-1784-470756.

E-mail address: [email protected] (D. Morritt).

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

274 (2002) 175–189

1. Introduction

Anthropogenic metal contamination, associated with sewage discharge and dredging

activity, is a prime concern in the conservation of marine ecosystems because of the high

persistence of metals in the environment and their toxicity to wildlife (Phillips and

Rainbow, 1993). In order to assess the potential threat to natural resources and human

health, the United Nation and many other countries have organised routine monitoring

programmes for trace metals in biomonitor species, e.g. mussels, barnacles and gastro-

pods, one example being the global ‘‘Mussel Watch’’ programme (Goldberg et al., 1978;

O’Connor, 2002). Although quantification of metal concentrations in biomonitors may

reflect metal bioavailability, this may not accurately indicate the toxic effect of the metal to

marine animals. For example, a high concentration of trace metals (e.g. zinc) can be

accumulated by barnacles and gastropods, being sequestered as pyrophosphate-based

granules, but these are not toxic to the organism (Nott and Langston, 1989; Pullen and

Rainbow, 1991; Nott and Nicolaidou, 1990, 1993). Theoretically, any detrimental effects

of metal exposure will initially manifest themselves at the cellular level, which suggests

that measurements made at this level will be most sensitive. Sub-cellular or biochemical

responses (i.e. biomarkers) have the advantage that they can be specific to a particular

group of contaminants, it is possible to relate effects to essential cellular processes, and

they may provide an early warning signal of harmful effects (Depledge and Fossi, 1994).

In view of this, there are increasing calls to incorporate measurements of biomarkers into

biomonitoring programmes (Ringwood et al., 1999; Cajaraville et al., 2000; De Lafontaine

et al., 2000; Viarengo et al., 2000; Downs et al., 2001; Wells et al., 2001).

It has been widely suggested that metallothioneins (MTs) can serve as biomarkers for

metal exposure and toxicity in marine biomonitors, because of their important and unique

roles in homeostasis of essential metals (e.g. zinc and copper) and in the detoxification of

toxic metals (e.g. cadmium and mercury) (Roesijadi, 1992, 1996; Langston et al., 1998;

Viarengo et al., 1999). MT expression generally increases with elevation of tissue

concentrations of MT-inducing metals (Zn, Cu, Cd, Hg and Ag) (Roesijadi, 1992;

Carpene, 1993; Viarengo et al., 1999). Cellular toxicity may result if the rate of metal

influx into the cell exceeds the rate of MTs synthesis and/or the maximum level of MTs

produced by the cell (Di Giulio et al., 1995). MT expression may also be influenced by

various natural (e.g. temperature, salinity, reproductive state, size/age) and anthropogenic

factors (e.g. exposure to pollutants other than metals). If the measurement of MTs is to be

incorporated into biomonitoring programmes, factors such as these should be carefully

considered.

Monitoring programmes for trace metals often use organisms from saltwater environ-

ments, such as estuaries and intertidal zones, where salinity can fluctuate on hourly, daily,

weekly and seasonal time scales. Changes in salinity can affect not only the bioavailability

of trace metals, but may also cause physiological and behavioural changes in the

biomonitor (Phillips and Rainbow, 1993; Mouneyrac et al., 1998; Legras et al., 2000).

Consequently, these salinity-mediated changes may directly influence the uptake rate of

metals and MT expression in the biomonitor. Most toxicity studies, for convenience, use a

fixed salinity regime. In nature, however, the salinity of marine coastal waters, especially

estuaries, fluctuates randomly or systematically depending on climate, surface runoff and

K.M.Y. Leung et al. / J. Exp. Mar. Biol. Ecol. 274 (2002) 175–189176

precipitation. To respond to such short-term changes in salinity, animals have different

strategies to counteract the resultant osmotic stress compared to long-term adaptation at a

fixed salinity. Nevertheless, the influence of fluctuating salinity on MT induction in marine

gastropod molluscs is still largely unknown.

The dogwhelk Nucella lapillus (L.) is one of the most commonly used biomonitor

species, especially in relation to tributyltin contamination (Bailey and Davies, 1991; Gibbs

et al., 1991; Evans et al., 1996). It has been demonstrated that MTs can be induced in this

gastropod species by waterborne Cd and/or hydrogen peroxide (Leung and Furness, 1999,

2001a). Recently, Leung et al. (2001) conducted a field study and showed that concen-

tration of MTs in the Leiblein gland is significantly correlated with the concentration of Cd

or Cu in the digestive gland/gonad complex of N. lapillus. However, the concentration of

MTs in tissues of N. lapillus can also be influenced by temperature, animal size, growth

rate, nutritional state and prey type (Leung et al., 2000; Leung and Furness, 2001b).

The aim of this study was to determine the concentrations of Cd and MTs in the tissues

of N. lapillus exposed to a sublethal concentration of waterborne Cd while subject to

various salinity regimes, under controlled laboratory conditions.

2. Materials and methods

2.1. Experimental animals and design

Adult dogwhelks were collected from Strandakirkja, southwest coast of Iceland (Grid

reference: 63j50VN, 21j43VW) and transported back to the Sandgerdi Marine Centre

where exposure experiments were performed within 7 days of collection. Two hundred

dogwhelks within a specified size-range (23.4F1.3-mm shell length, meanFSD) were

randomly divided into 10 groups of 20 snails. Each group was caged in a plastic net cage

(volume 500 cm3), which was then submerged in a 1-l plastic tank with a flow through

seawater system (Fig. 1). The water was pumped at a constant rate (ca. 4.2 ml min�1)

using a programmable peristaltic pump equipped with multiple-channels (Ismatec, IPC-

N-24, Supplier: Ismatec, Labortechnik-Analytik, Switzerland; Fig. 1). All tanks were

submerged in a water bath to maintain a constant water temperature at 9.5F0.3 jCthroughout the entire experiment. Six groups were acclimated at a salinity of 33 psu (the

salinity at the sampling site), while two each of the four remaining groups were

acclimated to either 22 or 11 psu for 10 days in a stepwise manner by reducing the

salinity by 5.5 psu day�1 (Fig. 2a). The clean seawater used in the present study was

taken from a 50-m deep drilling hole, and free from any contaminants (Svavarsson,

unpublished data). De-chlorinated and filtered drinking tap water was utilised as dilution

water throughout the entire experiment. The levels of dissolved oxygen and pH in the

seawater were monitored using a portable dissolved oxygen meter (OxyGuard Handy

Gamma, OxyGuard International, Denmark) and pH meter (Hanna Instruments, pHep 3,

Portugal), respectively. The overall dissolved oxygen was >80% saturation while the

mean pH was 7.91F0.07 (meanFSD). Salinity and temperature were measured using a

salinity and temperature recorder with accuracy at F0.01 psu or jC (Seamon CT,

Hugrun, Iceland).

K.M.Y. Leung et al. / J. Exp. Mar. Biol. Ecol. 274 (2002) 175–189 177

Ten treatment groups were used and comprised two Cd concentrations (i.e. one control

and one treatment) and five salinity regimes. Ten days after acclimation, 5 out of the 10

cages were exposed to a nominal Cd concentration of <0.001 Ag Cd l�1 (clean seawater as

a control) or 500 Ag Cd l�1 [as CdCl2�5H2O, equivalent to 2.2% of 96 h LC50 (Leung and

Furness, 1999)] under the five salinity treatments, which included three fixed salinity (33,

22 or 11 psu) and two fluctuating salinity regimes. For the latter, the salinity was varied

daily between 33 and 22 psu or 33 and 11 psu in a cyclic manner: for example, 12 h at 33

psu; 4 h changing from 33 to 22 psu; 4 h at 22 psu and 4 h changing from 22 to 33 psu

(Fig. 2b). The rationale for choosing these fluctuating salinity regimes was to mimic heavy

surface runoff during summer months associated with rainfall and ice-melting. The change

of salinity was initiated manually by switching the valves on or off and allowing an inflow

with the appropriate salinity (Fig. 1). The dogwhelks were starved during the exposure

period of 20 days. Test water was freshly prepared every day. Mortality was monitored

daily: death was defined as a failure to respond to probing with forceps; any dead animals

were removed from the tanks to avoid cannibalism. At the end of the exposure, the viable

dogwhelks were collected and stored at �20 jC.

2.2. Metallothionein and metal determinations

The soft body was removed from the dogwhelks after carefully breaking open the shell

with a vice. The soft-tissue was blotted dry using an absorbent tissue and the wet weight

determined to the nearest 10 mg (Sartorius, MCI electronic balance, Laboratory LC 2200

Fig. 1. A schematic diagram illustrating the experimental set-up and the operation system for manipulating

salinity level. For example, for fluctuating salinity between 22 and 33 psu, the valve for seawater at 22 psu is

closed manually during 00.00–12.00 when full strength seawater (33 psu) is required. At 1200, the valve for

seawater at 33 psu is closed and the other valve opened, allowing an inflow with low salinity. The flow rate is

adjusted so that the salinity of the tank is altered to 22 psu after 4 h. From 16.00 to 20.00, salinity in the tank is

constant at 22 psu. At 20.00, the inflow is then switched back to 33 psu. By 24.00, the salinity returns to 33 psu

(see Fig. 2 for the daily cycle).


P). Afterwards, eight individuals were selected randomly from each treatment group and

used for Cd and MT analyses, except that there were only seven dogwhelks for the group

exposed to Cd at 22 psu due to high mortality in this exposure regime. The whole Leiblein

gland and the digestive gland/gonad complex were dissected from the soft-tissue and

weighed. The gland of Leiblein of N. lapillus is the most important and sensitive tissue for

Cd accumulation and Cd–MT induction (Leung and Furness, 1999). However, the amount

of tissues from an individual Leiblein gland was only enough for the MT assay. Both

Leiblein gland and the digestive gland/gonad complex were used for quantification of MT

concentration while Cd concentrations were also determined for the digestive gland/gonad

complex. The weighed Leiblein gland was homogenised with 0.5 ml of 0.25 M sucrose at

4 jC using an Ultraturax homogeniser (T25 Janke & Kunkel, IKA Labortechnik). The

homogenate was centrifuged at 20,000�g for 20 min at 4 jC. The supernatant was

Fig. 2. Salinity regimes used in the present study: (a), fixed salinity 11, 22 and 33 psu; and (b) two fluctuating

salinity profiles.


collected and weighed and 300 Al aliquots of supernatant was analysed for MT content

using the silver saturation method (Scheuhammer and Cherian, 1991; as modified by

Leung and Furness, 1999). Briefly, samples were incubated with 0.4 ml glycine buffer (0.5

M, pH 8.5) and 0.5 ml of 20 mg l�1 Ag solution for 20 min at 20 jC to saturate the binding

sites of MTs. Excess Ag ions were removed by the addition of 100 Al ovine red blood cell

haemolysate (Oxoid, Hampshire, England) to the assay tubes followed by heat treatment in

a water bath (100 jC for 5 min). The heat treatment caused precipitation of Ag-bound

haemoglobin and other proteins, except for MTs, which are heat-stable. The denatured

proteins were removed by centrifugation at 1200�g for 10 min. The haemolysate addition,

heat treatment and centrifugation were repeated three times for each sample. Finally, the

supernatant was centrifuged at 20,000�g for 10 min. The amount of Ag in the final

supernatant fraction, which was proportional to the amount of MTs present, was

determined using an atomic absorption spectrophotometer (UNICAM 929 AAS, Analyt-

ical Technology, TJA solutions, UK) with deuterium background correction. Calibration in

the concentration range 2–20 Ag was achieved using purified horse kidney MT standards

(Sigma) (i.e. Ag Ag ml�1 vs. Ag MT ml�1) for MT quantification. The results were

expressed as Ag MT g�1 wet tissue wt.

For the digestive gland/gonad complex, the weighed tissues for each dogwhelk were

homogenized in 1.0 ml of 0.25 M sucrose at 4 jC. Then 0.5 ml of the homogenate was

centrifuged at 20,000�g for 20 min at 4 jC, and 300 Al supernatant was analysed for MT

content following the same protocol as described above. The remaining homogenate was

dried at 60 jC for at least 96 h until constant mass was achieved. Dry weight of the tissue

was obtained by the difference between the total dry weight and the amount of sucrose

added. Homogenates were then digested in concentrated nitric acid for 24 h at room

temperature followed by boiling for at least 2 h until a clear solution was obtained. The

concentrations of Cd were determined using the UNICAM 929 AAS. Accuracy was

regularly checked by including a standard reference material (dogfish muscle, DORM-1,

from the National Research Council, Canada). The concentration of Cd was expressed as

Ag Cd g�1 dry tissue wt.

2.3. Data analysis

Normality of the data was checked using normal probability plots and Kolmogrov–

Smirnov tests while homogeneity of variances was checked with Bartlett’s test. For the

mortality data, Kaplan–Meier (KM) survival analysis was utilised to compare the

survivorship between the Cd-exposed and control groups at each salinity regime

(Tabachnick and Fidell, 2001). In the present study, the main null hypothesis was that

there would be no significant difference among the concentrations of Cd or MTs in the

tissues of N. lapillus exposed to the four salinity regimes at either Cd level of <0.001 Ag

Fig. 3. Cumulative survivorship of control (dashed line) and Cd-exposed (solid line) N. lapillus at the five

different salinity regimes throughout the exposure period. The value and significance of Kaplan–Meier (KM)

survival analyses for comparison of the survivorship between the control and Cd-exposed animals are also

presented.


Cd l�1 (control) or 500 Ag Cd l�1. Therefore, for each Cd level, one-way analysis of

variance (ANOVA) was used to test any significant differences among the concentrations

of Cd or MTs in the Leiblein gland or digestive gland/gonad complex of N. lapillus

exposed to the four salinity regimes, with subsequent comparison between individual

Fig. 4. Mean concentrations of cadmium in the digestive glands/gonad complex of (a) control and (b) Cd-exposed

N. lapillus at various salinity regimes. Values are meanF1 SD. Significantly different means are indicated by

asterisks (**P<0.01; ***P<0.001).


means using Tukey’s multiple comparison test. Because of using multiple tests, statistical

significance was defined as P<0.01 in order to minimise the chance of committing a Type I

Error (Zar, 1999).

Fig. 5. Mean concentrations of metallothioneins (MTs) in the digestive glands/gonad complex of (a) control and

(b) Cd-exposed N. lapillus at various salinity regimes. Values are meanF1 SD. Significantly different means are

indicated by asterisks (**P<0.01).


3. Results

All the control and Cd-exposed N. lapillus constantly exposed to a fixed salinity at 11

psu swelled during the acclimation period, and died within 5 days of exposure due to

Fig. 6. Mean concentrations of metallothioneins (MTs) in the gland of Leiblein of control and Cd-exposed N.

lapillus at various salinity regimes. Values are meanF1 SD. Bars with same letter are not significantly different

( P<0.01).


hypo-osmotic stress (Fig. 3a). The dogwhelks exposed to Cd and a fixed higher salinity

(22 or 33 psu) exhibited a significantly higher mortality than the control group (Fig. 3).

However, there was no significant difference in survivorship between the control and Cd-

exposed groups exposed to both fluctuating salinity regimes.

The concentrations of Cd in the digestive glands/gonad complex of both control and

Cd-exposed N. lapillus were significantly affected by salinity (Fig. 4; one-way ANOVA:

Control, F3, 28=25.21, P<0.0001; Cd-exposed, F3, 27=6.985, P=0.0013). Among the

controls, the dogwhelks at fluctuating salinities 33 X 22 psu had a significantly lower

concentration of Cd in the tissues than at the other salinity regimes (Fig. 4a). The Cd-

exposed N. lapillus accumulated significantly higher concentrations of Cd in these tissues

compared to that of control animals (Fig. 4). At 22 psu, Cd-exposed dogwhelks had a

significantly lower Cd concentration in these tissues compared with those exposed to both

fluctuating salinity regimes (Fig. 4b).

The concentrations of MTs in the digestive glands/gonad complex and Leiblein gland

were significantly affected by salinity (Figs. 5 and 6). For the digestive glands/gonad

complex, control N. lapillus exposed to a fixed salinity of 22 psu had a significantly higher

MTconcentration whereas there was a significantly higher MTconcentration in Cd-exposed

dogwhelks at 33 psu (Fig. 5; Control, F3, 28=16.5, P<0.0001; Cd-exposed, F3, 27=6.259,

P=0.0023). In the gland of Leiblein, control N. lapillus showed significantly higher levels of

MTs at fixed salinity (22 or 33 psu) compared with those exposed to either of the fluctuating

salinity regimes (P<0.001), while the control dogwhelks at fluctuating salinities 33 X 22 psu

had a significantly lower level of MTs than those at fluctuating salinities 33 X 11 psu (Fig.

6a, F3, 28=46.30, P<0.0001). Cd-exposed dogwhelks at 22 psu also showed a significantly

lower MT concentration in the Leiblein gland (Fig. 6b, F3, 27=5.348, P=0.0051).

4. Discussion

In the present study, high mortality in Cd-exposed N. lapillus at fixed salinity (22 or 33

psu) implies that these intertidal gastropods have a higher tolerance to Cd toxicity under

fluctuating salinities in natural habitats, especially in the estuarine environment.

Our results also indicate that salinity has significant effects on Cd accumulation and

MT induction in the control or Cd-exposed dogwhelks. Among the controls, high Cd

accumulation and MT expression were observed in N. lapillus exposed to the lowest

salinity (22 psu). An increase in Cd uptake is likely attributable to (1) the increased

bioavailability of Cd ions at low salinity due to lower chlorocomplex formation (Rainbow,

1997) and (2) the decrease in soft-body weight or tissue wastage (Leung and Furness,

2001b). In addition to starvation, physiological responses to low salinity stress, which

would require an extra-energy demand, might further reduce the body weight (Cheung,

1997). Nevertheless, it is not clear why control N. lapillus at the fluctuating salinity

33 X 22 psu showed a significantly lower Cd concentration in their tissues. Although this

might be explained by reduced weight loss in this particular group due to a better

adaptation, we cannot support this hypothesis using the current data set. A further study is

needed to confirm whether salinity-mediated change in body weight would have effects on

residual Cd concentration in the tissues of both starved and fed animals.


On the contrary, N. lapillus exposed to the high sublethal concentration of Cd at 22 psu,

exhibited a significantly lower Cd concentration in the digestive glands/gonad complex, and

lower induction of MTs in the Leiblein gland, suggesting that dogwhelks may have a

reduced infiltration rate under such continuous Cd and hypo-osmotic stresses, thereby

reducing uptake of Cd and thus minimising the toxicity (Leung et al., 2000). However, at

both fluctuating salinity regimes, Cd-exposed N. lapillus accumulated high concentrations

of Cd, similar to those at full-strength seawater (33 psu; Fig. 4b). This is possibly a

consequence of a combination of periods of reduced infiltration in reduced salinity followed

by periods of ‘‘catch-up’’ filtration at elevated rates when the environmental salinity

increases again. Intertidal molluscs, including N. lapillus, temporarily react to hypo-osmotic

stress by initiating a ‘‘shell-closing’’ mechanism, which in the case of N. lapillus leads to

withdrawl of the body into the shell and occlusion of the aperture with the operculum

(Hoyaux et al., 1976). The effect of such ‘‘shell closure’’, as evidenced by measures of

mantle fluid concentration, was reduced by gradual acclimation to lower salinity in steps

(Hoyaux et al., 1976). Based on the results presented here, intertidal gastropod species,

which experience fluctuating salinity, may have a similar uptake rate of trace elements when

compared with those living in environments with full strength seawater.

In order to detoxify Cd, N. lapillus, like other marine molluscs, can produce MT to bind

the toxic Cd and transform it into a less toxic compound (Cd–MT) (Roesijadi, 1996),

although they can also store Cd in phosphate granules and excrete these Cd-containing

granules through the renal system (Nott and Nicolaidou, 1990). In the present study, the

tissues of N. lapillus exposed to Cd and fluctuating salinities not only showed high levels

of MT induction in the Leiblein gland but the animals also exhibited high survivorship

even with a very high Cd concentration in their tissues. Our results support the theory that

MTs play a vital role in protection and detoxification of toxic metals in the dogwhelk.

Notwithstanding, it might be argued why the Cd-exposed N. lapillus having a high level of

MTs at the fixed salinity 33 psu also exhibited a high mortality. Leung and Furness (1999)

observed that the concentration and distribution of MTs in different tissues of Cd-exposed

N. lapillus changed with time of exposure. From the present results, the highest MT

expression in the digestive gland/gonad complex of Cd-exposed N. lapillus at 33 psu

might imply a faster rate of transportation of Cd–MTs from other tissues to the digestive

gland for excretion. This is likely to be associated with a high Cd-toxicity occurring in this

group as indicated by their high mortality.

It has been shown that Cd and Cu concentrations in the gills and hepatopancreas of

shore crabs Pachygrapsus marmoratus and Carcinus maenas are inversely related to

salinity under field conditions, but their MT concentrations are associated more with

changes in general protein metabolism than with changes in metal accumulation (Legras et

al., 2000). However, estuarine and marine molluscs are generally osmoconfomers (e.g.

Hoyaux et al., 1976) unlike the majority of estuarine and intertidal crustaceans which are

generally osmoregulators (e.g. Pequeux, 1995) and may therefore respond differently to

the combined effects of salinity and trace metal exposure.

Viarengo et al. (1988) demonstrated that changing the salinity from 22 to 36 psu could

significantly increase the cellular concentration of Cu-induced MTs in the digestive gland

of the bivalve mollusc Mytilus galloprovincialis. Recent field studies on natural popula-

tions of bivalves Crassostrea gigas and Macoma balthica further support the idea that


salinity can indirectly affect the MT concentration in these biomonitor species (Mouneyrac

et al., 1998, 2000). Hypothetically, salinity-mediated change in MT expression could be

attributed to readjustment of physiology (and behaviour) in the organism, as well as

changes in metal speciation and cellular metal distribution. While testing these hypotheses,

there is also a need for research into study on the influence of salinity on MT turnover in

molluscs so that the underlying mechanism of the effect of salinity on MT expression can

be elucidated.

5. Conclusion

Cadmium accumulation and MT induction in N. lapillus can be significantly influenced

by changes in salinity, especially at a prolonged and fixed low salinity, although such

influences of salinity on the concentration of MT are dependent on the tissue types. It is

therefore possible that sampling sites with different salinity profiles may result in variable

endpoint measures, causing poor agreement between the results of MT concentrations in

biomonitor species between different monitoring programmes and/or locations with

different salinity profiles. In conclusion, salinity should be considered when monitoring

trace metals and/or MTs in intertidal molluscs, particularly in estuarine or transplanted

biomonitors.

Acknowledgements

The authors are grateful to the two anonymous referees for providing their useful and

valuable comments, and to Mrs. Margaret Onwu (Royal Holloway, University of London)

for helping with the metal analysis. This work was funded by a Large Scale Facility TMR

(European Commission) research grant, which allowed K.M.Y. Leung to use the facilities

and work at the Sandgerdi Marine Centre, Iceland. K.M.Y. Leung was supported by The

Croucher Foundation, Hong Kong. [SS]

References

Bailey, S.K., Davies, I.M., 1991. Continuing impact of TBT, previously used in mariculture, on dogwhelk

(Nucella lapillus L.) population in a Scottish Sea Loch. Mar. Environ. Res. 32, 187–199.

Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. The use of biomarkers

to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci.

Total Environ. 247, 295–311.

Carpene, E., 1993. Metallothionein in marine molluscs. In: Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of

Metals in Invertebrates. Lewis Publishers, London, pp. 55–72.

Cheung, S.G., 1997. Physiological and behavioural responses of the intertidal scavenging gastropod Nassarius

festivus to salinity changes. Mar. Biol. 129, 301–307.

De Lafontaine, Y., Gagne, F., Blaise, C., Costan, G., Gagnon, P., Chan, H.M., 2000. Biomarkers in zebra mussels

(Dreissena polymorpha) for the assessment and monitoring of water quality of the St. Lawrence River

(Canada). Aquat. Toxicol. 50, 51–71.


Depledge, M.H., Fossi, M.C., 1994. The role of biomarkers in environmental assessment (2). Invertebr. Ecotox-

icol. 3, 161–172.

Di Giulio, R.T., Benson, W.H., Sanders, B.M., Van Veld, P.A., 1995. Biochemical mechanisms: metabolism,

adaptation, and toxicity. In: Rand, G.M. (Ed.), Fundamentals of Aquatic Toxicology, 2nd edn. Taylor &

Francis, London, pp. 523–562.

Downs, C.A., Dillon Jr., R.T., Fauth, J.E., Woodley, C.M., 2001. A molecular biomarker system for assessing the

health of gastropods (Ilyanassa obsolete) exposed to natural and anthropogenic stressors. J. Exp. Mar. Biol.

Ecol. 259, 189–214.

Evans, S.M., Evans, P.M., Leksono, T., 1996. Widespread recovery of dogwhelk, Nucella lapillus (L.), from

tributyltin contamination in the North Sea and Clyde Sea. Mar. Pollut. Bull. 32, 263–269.

Gibbs, P.E., Bryan, G.W., Pascoe, P.L., 1991. TBT-induced imposex in the dogwhelk, Nucella lapillus—geo-

graphical uniformity of the response and effects. Mar. Environ. Res. 32, 79–87.

Goldberg, E.D., Bowen, V.T., Farrington, J.W., Havey, G., Martin, J.H., Parker, P.L., Risebrough, R.W., Rob-

ertson, W., Schneider, E., Gamble, E., 1978. Mussel watch. Environ. Conserv. 5, 101–125.

Hoyaux, J., Gilles, R., Jeuniaux, C., 1976. Osmoregulation in molluscs of the intertidal zone. Comp. Biochem.

Physiol. 53A, 361–365.

Langston, W.J., Bebianno, M.J., Burt, G.R., 1998. Metal handling strategies in molluscs. In: Langston, W.J.,

Bebianno, M.J. (Eds.), Metal Metabolism in Aquatic Environments. Chapman & Hall, London, pp. 219–283.

Legras, S., Mouneyrac, C., Amiard, J.C., Amiard-Triquet, C., Rainbow, P.S., 2000. Changes in metallothionein

concentrations in response to variation in natural factors (salinity, sex, weight) and metal contamination in

crabs from a metal-rich estuary. J. Exp. Mar. Biol. Ecol. 246, 259–279.

Leung, K.M.Y., Furness, R.W., 1999. Induction of metallothionein in dogwhelk Nucella lapillus during and after

exposure to cadmium. Ecotoxicol. Environ. Saf. 43, 156–164.

Leung, K.M.Y., Furness, R.W., 2001a. Metallothionein induction and condition index of dogwhelks Nucella

lapillus (L.) exposed to cadmium and hydrogen peroxide. Chemosphere 44, 321–325.

Leung, K.M.Y., Furness, R.W., 2001b. Survival, growth, metallothionein and glycogen levels of Nucella lapillus

(L.) exposed to sub-chronic cadmium stress: the influence of nutritional state and prey type. Mar. Environ.

Res. 52, 173–194.

Leung, K.M.Y., Taylor, A.C., Furness, R.W., 2000. Temperature-dependent physiological responses of the dog-

whelk Nucella lapillus to cadmium exposure. J. Mar. Biol. Assoc. UK 80, 647–660.

Leung, K.M.Y., Morgan, I.J., Wu, R.S.S., Lau, T.C., Svavarsson, J., Furness, R.W., 2001. Growth rate as a factor

confounding the use of dogwhelks Nucella lapillus (L.) as biomonitors of heavy metal contamination. Mar.

Ecol., Prog. Ser. 221, 145–159.

Mouneyrac, C., Amiard, J.C., Amiard-Triquet, C., 1998. Effects of natural factors (salinity and body weight) on

cadmium, copper, zinc and metallothionein-like protein levels in resident populations of oysters Crassostrea

gigas from a polluted estuary. Mar. Ecol., Prog. Ser. 162, 125–135.

Mouneyrac, C., Geffard, A., Amiard, J.C., Amiard-Triquet, C., 2000. Metallothionein-like proteins in Macoma

balthica: effects of metal exposure and natural factors. Can. J. Fish. Aquat. Sci. 57, 34–42.

Nott, J.A., Langston, W.J., 1989. Cadmium and the phosphate granules in Littorina littorea. J. Mar. Biol. Ass. UK

69, 219–227.

Nott, J.A., Nicolaidou, A., 1990. Transfer of metal detoxification along marine food chains. J. Mar. Biol. Ass. UK

70, 905–912.

Nott, J.A., Nicolaidou, A., 1993. Bioreduction of zinc and manganese along a molluscan food chain. Comp.

Biochem. Physiol. 104A, 235–238.

O’Connor, T.P., 2002. National distribution of chemical concentrations in mussels and oysters in the USA. Mar.

Environ. Res. 53, 117–143.

Pequeux, A., 1995. Osmotic regulation in crustaceans. J. Crust. Biol. 15, 1–60.

Phillips, D.J.H., Rainbow, P.S., 1993. Biomonitoring of Trace Aquatic Contaminants. Elsevier, London, p. 371.

Pullen, J.S.H., Rainbow, P.S., 1991. The composition of pyrophosphate heavy metal detoxification granules in

barnacles. J. Exp. Mar. Biol. Ecol. 150, 249–266.

Rainbow, P.S., 1997. Trace metal accumulation in marine invertebrates: marine biology or marine chemistry? J.

Mar. Biol. Ass. UK 77, 195–210.

Ringwood, A.H., Hameedi, M.J., Lee, R.E., Brouwer, M., Peters, E.C., Scott, G.I., Luoma, S.N., Digiulio,


A.H., 1999. Bivalve biomarker workshop: overview and discussion group summaries. Biomarkers 4,

391–399.

Roesijadi, G., 1992. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22, 81–

114.

Roesijadi, G., 1996. Metallothionein and its role in toxic metal regulation. Comp. Biochem. Physiol. C 113, 117–

123.

Scheuhammer, A.M., Cherian, M.G., 1991. Quantification of metallothionein by silver saturation. Methods

Enzymol. 205, 78–83.

Tabachnick, B.G., Fidell, L.S., 2001. Using Multivariate Statistics, 4th edn. Allyn and Bacon, Boston, p. 966.

Viarengo, A., Mancinelli, G., Orunesu, M., Martino, G., Faranda, F., Mazzuccotelli, A., 1988. Effects of sublethal

copper concentrations, temperature, salinity and oxygen levels on calcium content and on cellular distribution

of copper in the gills of Mytilus galloprovincialis Lam.: a multifactorial experiment. Mar. Environ. Res. 24,

227–231.

Viarengo, A., Burlando, B., Dondero, F., Marro, A., Fabbri, R., 1999. Metallothionein as a tool in biomonitoring

programmes. Biomarkers 6, 455–466.

Viarengo, A., Lafaurie, M., Gabrielides, G.P., Fabbri, R., Marro, A., Romeo, M., 2000. Critical evaluation of an

intercalibration execise undertaken in the framework of the MED POL biomonitoring program. Mar. Environ.

Res. 49, 1–18.

Wells, P.G., Depledge, M.H., Butler, J.N., Manock, J.J., Knap, A.H., 2001. Rapid toxicity assessment and

biomonitoring of marine contaminants—exploiting the potential of rapid biomarker assays and microscale

toxicity tests. Mar. Pollut. Bull. 42, 799–804.

Zar, J.H., 1999. Biostatistical Analysis, 4th edn. Prentice Hall, New Jersey, p. 663.


influence of static and fluctuating salinity on cadmium uptake and

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