www.elsevier.com/locate/marpolbul

Marine Pollution Bulletin 48 (2004) 526–532

Mediterranean Talitrus saltator (Crustacea, Amphipoda)as a biomonitor of heavy metals contamination

A. Ugolini a,*, F. Borghini b, P. Calosi a, M. Bazzicalupo a, G. Chelazzi a, S. Focardi b

a Dipartimento di Biologia Animale e Genetica, Universit�a di Firenze, via Romana 17, 50123 Firenze, Italyb Dipartimento di Scienze Ambientali, Universit�a di Siena, via. Mattioli 10, 53100 Siena, Italy

Abstract

The use of sandhoppers and beachfleas as biomonitors of heavy metals contamination is relatively recent. Using adult individuals

of Talitrus saltator from nine localities on the northern Mediterranean Sea, we studied the concentrations of eight trace elements: Al,

Cd, Cr, Fe, Hg, Pb, Cu, Zn, both in the substratum and in the individuals. We also carried out a preliminary investigation of the

correspondence between the sandhoppers’ genetic variability and heavy metal contamination at the sampling sites.

T. saltator accumulated Cd, Cu, Zn and Hg (at higher concentrations than in the sand) and also Al and Fe (at lower concen-

trations than in the sand). It seems that Mediterranean sandhoppers do not accumulate Pb and Cr. An intraspecific comparison

between northern European (Baltic) and Mediterranean populations of T. saltator was made. Finally, we observed a tendency to a

positive correlation between the sandhoppers’ genetic variability and heavy metals contamination.

� 2003 Elsevier Ltd. All rights reserved.

Keywords: Sandhoppers; Talitrus saltator; Mediterranean sea; Heavy metals; Bioaccumulation; Genetic diversity

1. Introduction

Various organisms have been tested as bioindicators

in the submerged and intertidal littoral environment of

rocky and sandy shores. These include various species of

crustaceans, especially decapods, isopods and amphi-

pods (e.g. see Rainbow, 1998). The use of sandhoppersand beachfleas as possible biomonitors in the supralit-

toral band of sandy shores (an ecotonal environment

characterized by the input of substances and material of

both marine and terrestrial origin) has recently received

attention. Studies conducted mainly on northern Euro-

pean sandy shores have shown that many talitrid species

are good bioindicators of contamination by heavy

metals, especially zinc and copper (Moore and Rain-bow, 1987; Moore et al., 1991; Rainbow and Phillips,

1993; Weeks and Rainbow, 1994; Rainbow, 1995, 1998;

Rainbow et al., 1998b; Brown and Depledge, 1998).

Talitrid amphipods constitute one of the largest ani-

mal components (in terms of biomass) in the supralit-

toral band of sandy shore ecosystems and they play an

* Corresponding author. Tel./fax: +39-055-22-88-219.

E-mail address: ugolini_alb@dbag.unifi.it (A. Ugolini).

0025-326X/$ - see front matter � 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2003.10.002

important role in the energy flow among the various

trophic levels (e.g. see Griffiths et al., 1983; Branch and

Branch, 1981; Brown and McLachlan, 1994). Grazers,

detritivores and scavengers, the sandhoppers feed on

plant and animal organic material of both marine and

terrestrial origin (Palluault, 1954; Bergerard, 1989;

Wildish, 1988).The sandhopper Talitrus saltator is widely distributed

in the Mediterranean basin, its distribution area

extending northward to the Swedish and Norwegian

coasts. However, there are several important intra-

specific behavioural differences between sandhoppers

inhabiting the Mediterranean and northern shores. For

example, the latter can use the earth’s magnetic field in

addition to the sun as a chronometrically compensatedorientation reference to maintain the direction of the

beach’s sea-land axis during their movements (Arendse,

1978; Ugolini and Cannicci, 1991); moreover, the feed-

ing excursions are normally performed in the intertidal

zone (Williamson, 1954; Bregazzi and Naylor, 1972;

Williams, 1980).

Along the Italian coast, characterized by modest tidal

excursions, the feeding excursions are carried out alongthe shoreline and, more importantly, extend for sev-

eral tens of meters toward the upper horizons of the

A. Ugolini et al. / Marine Pollution Bulletin 48 (2004) 526–532 527

supralittoral (Geppetti and Tongiorgi, 1967; Ugolini,

1996; Scapini et al., 1992).

In view of the strong differences in spatial use of the

habitat between the northern European and Mediter-ranean Talitrus saltator, we decided to evaluate the

possibility of using the latter as bioindicators for the

monitoring of heavy metals pollution in sandy shore

environments. We also wanted to make a preliminary

assessment of the relationship between genetic diversity

and heavy metals contamination.

2. Study area

The study area partially covers the one considered

during monitoring of the quality of Italian coastal

environments (ICRAM, 2000). Of the nine sampling

sites, mostly situated near river mouths or small streams,

seven are along the Tuscan coast and two in eastern and

southern Corsica (Fig. 1). The area encompassing thevarious localities is of great naturalistic importance since

it includes nine national or regional parks and protected

areas. The sampling localities are

(a) Serchio River mouth (Pisa, Italy)

(b) Livorno Calambrone (Livorno, Italy)

(c) Rosignano Solvay (Livorno, Italy)

(d) Piombino (Livorno, Italy)(e) Ombrone River mouth (Grosseto, Italy)

(f) Albegna (seaside beach) (Grosseto, Italy)

(g) Albegna River mouth (Grosseto, Italy)

(h) Cateraggio d’Aleria (Corsica)

(i) La Piantarella (Bonifacio, Corsica)

Fig. 1. Location of the sampling sites. (A), Serchio River mouth; (B),

Livorno Calambrone; (C), Rosignano Solvay; (D), Piombino; (E),

Ombrone River mouth; (F), Albegna (seaside); (G), Albegna River

mouth; (H), Cateraggio d’Aleria; (I), La Piantarella.

3. Materials and methods

Adult individuals of T. saltator were collected in

March–April and September of 1999 and 2000. Theanimals were transported to the laboratory in plastic

containers with sand and detritus from the sampling site

and then killed by freezing. At each site, a sample of

sand was taken from the zone of wet sand habitually

frequented by the sandhoppers at sunset and dawn and

where they dig temporary refuges in which they spend

the hottest hours of the day.

Samples were dried at 40 �C until constant weight andthen finely ground. Samples of about 150 mg were

mineralised in clean Teflon vessels with 3 ml of HNO3 at

120 �C for 8 h. After digestion, the solution was brought

to 10 ml volume with deionised water. Each digestion

included at least one blank test. Analytical determina-

tions were performed by atomic absorption spectrome-

try (AAS): Al, Fe, and Zn were assayed by inductively

coupled plasma atomic emission spectrometry (Plasma400, Perkin Elmer); Cd, Cr, Cu, Pb were determined by

electrothermal atomic absorption spectrometry with

Zeeman background correction and Hg by flow injec-

tion atomic absorption spectrometry (ASS). Each deter-

mination was carried out three times. The accuracy of

analytical procedures was checked by simultaneous

digestion and analysis of standard reference materials

(SRMs). SRM 1566a ‘‘oyster tissue‘‘ from the NationalInstitute of Standards and Technology (NIST, Gai-

thersburg, USA) was used. Batches with accompanying

SRM outside the certified range were repeated. Element

concentrations (expressed in ppm dry weight basis) were

determined by the addition method. Standard solutions

of inorganic elements were prepared by serial dilution of

stock standard solutions containing 1 g l�1 of the ele-

ment to be determined (Spectrosil, BDH). The recoveryrate ranged between 92% and 105% and the coefficients

of variation calculated through the analysis of five rep-

licates of several samples ranged from 5.5% to 19.4 %,

depending on the element.

For each sampling site and heavy metal, we calcu-

lated the mean of the concentrations determined in each

sample. The relationship between the concentrations in

the sand and in the sandhoppers was analysed by theusual correlation test (e.g. see Zar, 1984). Although the

number of sampling sites is low and hence the degrees of

freedom are not high, the type of representation adopted

allows: (a) a preliminary evaluation of the type of rela-

tionship between the concentrations of a metal in the

substratum and in the individual (there can be bioac-

cumulation also if the concentration in the individual is

less than in the substratum); (b) the existence of bioac-cumulation even when the concentration in the sand-

hoppers appears high but is independent of the quantity

of metal present in the substratum. In fact, the Cartesian

plane is divided in two by the theoretical line for perfect

528 A. Ugolini et al. / Marine Pollution Bulletin 48 (2004) 526–532

correspondence between the concentrations of the ele-

ment in the substratum and in the sandhoppers. If the

data fall in the upper half (i.e. above the line), there is

accumulation by the sandhoppers. If the data fall in thelower half, there is still accumulation by the sandhop-

pers if there is a significant positive correlation; if the

correlation is not significant, the sandhoppers are not

bioaccumulators of the considered element.

3.1. Genetic diversity

DNA preparation: DNA was isolated from frozen orethanol-preserved whole animals with the Quiamp DNA

kit (Quiagen) following the manufacturer’s ‘‘Tissue

Protocol’’. Before extraction, the animals preserved in

ethanol were hydrated at 4 �C in ethanol/water mixtures,

passing through decreasing ethanol concentrations and

ending in pure distilled water. DNA was quantified

spectrophotometrically and diluted to 10 ng/ll in double

distilled sterile water.RAPD amplification and analysis: RAPDs were

performed in a 25 ll total volume containing 10 mM

KCl, 2 mM Tris–HCl (pH 7.5), 0.1 mM DTT, 0.05%

Tween 20 (v/v), 10 lM EDTA, 3 mM MgCl2, 0.2 mM of

each dNTP (Boehringer-Mannheim), 0.7 U of Expand

High Fidelity PCR System (Boehringer-Mannheim),

0.36 lM of primer, 25 ng of template DNA. Reactions

were performed on a Perkin Elmer 9600 thermal cycleras described previously (Paffetti et al., 1996).

An initial screening of 10 RAPD decamer primers

was performed to test amplification profiles for poly-

morphism, readability and reproducibility. After this

screening procedure, three primers were chosen for ana-

lysis: 1247 (50-AAGAGCCCGT), RF2 (50-CGGCCCC-

TGT) and 1290 (50-GTCGATGCGA). Amplification

products were resolved electrophoretically on a 2%agarose gel run with TAE buffer at 100 V for 1 h 30’ and

visualised after ethidium bromide staining. Bands of

equal fragment sizes were interpreted as homologous.

For each site, 20 DNA samples were analysed from

20 individuals, making a total of 180 samples. RAPD

analysis of the samples with the three primers produced

18 different bands (markers). The RAPD phenotype of

each individual was expressed as a vector of 0s (forabsence of the band) or 1s (for its presence) assuming

that RAPD bands represented independent loci. The

analysis was based on Euclidean squared distances

(Excoffier et al., 1992). Genetic diversity was calculated

as the mean number of pairwise differences using Arle-

quin 1.1 software (Schneider et al., 1997).

Classification of the heavy metals contamination at

the various localities was based on the sum of points(from 1 to 8) assigned to the concentration of each ele-

ment in the sand sample. The samples were collected in

the zone with the highest concentration of sandhoppers

during the day (the wet zone of the shoreline). For

each metal, points were assigned to the sampling sites

in increasing order from the highest concentration to

the lowest. In a similar way, the 9 sites were listed

in increasing order on the basis of the index ofgenetic variability. The two lists of ranks were then

compared by the Spearman rank correlation coefficient

test.

4. Results and discussion

At all the sampling sites, the concentrations of Zn,Cu, Hg and Cd are much higher in the sandhoppers than

in the sand (Table 1, Fig. 2). However, considering the

relatively small number of sampling sites, a significant

positive correlation between the concentrations in the

substratum and in the sandhoppers is observed only for

Cu, while it is at the limit of significance for Hg (Fig. 2).

The two concentrations are clearly not correlated for Cd

(Fig. 2), at least in the quantities measured here. Nev-ertheless, it should be noted that, at two localities, there

is a very high Cd concentration in the animals despite a

low concentration in the substratum: Rosignano Solvay

(0.2 ppm in the sand, 3.88 ppm in the animals) and

Cateraggio d’Aleria (0.02 ppm in the sand, 3.32 ppm in

the animals). Although the correlation between Zn

concentrations in the sand and in the sandhoppers does

not reach significance (Fig. 2), there is an inverse ten-dency: as the concentration increases in the sand, it

decreases in the animals.

The concentrations of Al, Fe, Cr, and Pb are lower in

the sandhoppers than in the sand (Table 1, Fig. 2). For

Al and Fe, there is a significant positive correlation

between the two concentrations (Fig. 2). This is also true

for Cr which, however, is not generally accumulated by

the sandhoppers (Table 1, Fig. 2). In fact, the relation-ship corresponds to that known for other species of

crustaceans artificially exposed to high quantities of

contaminants (see Amiard et al., 1987). Moreover, at

one locality (Cateraggio d’Aleria), the high concentra-

tion of the metal in the substratum (201.4 ppm), leads to

a correspondingly high concentration in the animals

(181.5 ppm), supporting the significant correlation. For

Corsica, the high concentration of Cr along somecoastal tracts is not a novelty, as it is related to the

presence of asbestos (e.g. at Canari, see Chiffoleau and

Andral, 2002). The values measured in the sand at

Cateraggio d’Aleria agree quite well with those found at

the stations of Fium’Orbo and Fiume Golo by Chiffo-

leau and Andral (2002); hence the presence of Cr at our

locality can perhaps be considered of natural origin. For

Pb (Fig. 2), the correlation between the concentrationsin the sand and in the sandhoppers is similar to that for

Zn: the concentration in the animals strongly decreases

as the concentration in the substratum increases, al-

though here the correlation is significant.

Table 1

Mean heavy metal concentrations (expressed in ppm) in the sand and in the sandhoppers at each sampling site

Locality Sand Sandhoppers Locality Sand Sandhoppers

Al Cd

A Serchio r.m. 13,976 394 A Serchio r.m. 0.06 0.96

B Livorno 15,619 236 B Livorno 0.07 0.82

C Rosignano 3192 103.2 C Rosignano 0.2 3.88

D Piombino 6392 192 D Piombino 0.03 0.85

E Ombrone r.m. 14,821 329 E Ombrone r.m. 0.09 1.04

F Albegna 19,146 160 F Albegna 0.131 1.3

G Albegna r.m. 26,542 434.5 G Albegna r.m. 0.133 0.82

H C. d’Aleria 25,824 502 H C. d’Aleria 0.02 3.32

I La Piant. 2785 277 I La Piant. 0.2 1.29

Cr Fe

A Serchio r.m. 53.55 19.69 A Serchio r.m. 19,859 335

B Livorno 35.05 2.76 B Livorno 12,792 242

C Rosignano 21.7 1.66 C Rosignano 3934 233

D Piombino 18.5 7.96 D Piombino 6351 259

E Ombrone r.m. 93.5 2.54 E Ombrone r.m. 34,403 540

F Albegna 30.58 0.98 F Albegna 35,152 260

G Albegna r.m. 38.49 1.93 G Albegna r.m. 35,046 280

H C. d’Aleria 201.4 181.5 H C. d’Aleria 40,620 491

I La Piant. 3.26 14 I La Piant. 1243 231

Hg Pb

A Serchio r.m. 0.04 0.21 A Serchio r.m. 17.3 0.95

B Livorno 0.07 0.14 B Livorno 6.72 3.52

C Rosignano 0.16 0.2 C Rosignano 26 1.05

D Piombino 0.05 0.235 D Piombino 8.1 1.66

E Ombrone r.m. 0.07 0.198 E Ombrone r.m. 11.94 0.75

F Albegna 0.135 0.23 F Albegna 15.78 1.33

G Albegna r.m. 0.199 0.203 G Albegna r.m. 10.12 0.53

H C. d’Aleria 0.01 0.12 H C. d’Aleria 12.85 1.3

I La Piant. 0.01 0.16 I La Piant. 3.66 3.31

Cu Zn

A Serchio r.m. 15.1 64 A Serchio r.m. 33.65 134

B Livorno 29.4 87.7 B Livorno 44 152

C Rosignano 8 47 C Rosignano 16.5 146.5

D Piombino 6.7 50.4 D Piombino 27.5 242

E Ombrone r.m. 28.9 54.3 E Ombrone r.m. 46.6 156

F Albegna 26.85 65.2 F Albegna 55.45 155

G Albegna r.m. 28.75 63.4 G Albegna r.m. 58.7 112

H C. d’Aleria 43.6 54.7 H C. d’Aleria 75.5 187

I La Piant. 2.8 32.6 I La Piant. 6.8 236

The letters next to the localities correspond to those reported in Fig. 1.

A. Ugolini et al. / Marine Pollution Bulletin 48 (2004) 526–532 529

Therefore, the elements bioaccumulated by the

sandhoppers are Zn, Cu, Hg, Cd, Fe and Al. It should

also be noted that the graphs for Cu and Zn are mirror

images of each other (Fig. 2). This could depend on theability of Cu to influence the tissue concentration of Zn,

as occurs in some crabs (see Brown and Depledge,

1998), although an interaction between the two metals

has not been clearly demonstrated in supralittoral am-

phipods (Moore et al., 1991; Weeks and Rainbow, 1994;

Casini and Depledge, 1997).

Pb seems not to be accumulated by the sandhoppers;

at concentrations in the sand higher than 3–6 ppm, this

element appears to be subjected to processes of body

burden regulation.

A comparison between our results and the data re-

ported in the literature for the Tuscan and Corsicancoasts is not very indicative because of temporal differ-

ences and/or differences in the choice of localities and

bioindicator organisms (Posidonia, Ferrara et al., 1993;

mussel, ICRAM, 2000; IFREMER, 2003). However, a

rather useful intraspecific comparison can be made by

considering the data for the Gulf of Gdansk reported by

Rainbow et al. (1998a) and Fialkowski et al. (2000)

(Table 2).

Fig. 2. Correlation between the heavy metal concentrations in the sand

and in the sandhoppers. For each graph, we report the equation and

regression line or curve and the coefficient of determination R2. The

theoretical line for perfect correspondence between the concentrations

in the sand and in the sandhoppers (dashed line) is not shown for Al

and Fe for graphical reason (coincidence with the Y axes of the

graphs).

Table 2

Minimum and maximum concentrations (expressed in ppm) of several

heavy metals found in adult individuals of T. saltator in previous

studies (Gulf of Gdansk, Poland) and in the present study

Rainbow et al.

(1998a,b)

Fialkowski

et al. (2000)

Our data

Cd 8.04–35.1 2.41–3.02 0.82–3.88

Cu 49.7–70.4 39.2–57.4 32.6–87.7

Fe 161–492 137–554 108.7–540

Pb 20.9–32.3 25.7–36.0 0.53–3.52

Zn 162–262 94.2–148 112–236

530 A. Ugolini et al. / Marine Pollution Bulletin 48 (2004) 526–532

For Cu, Livorno Calambrone is the locality with the

highest degree of contamination (87.7 ppm).

T. saltator does not show particular efficiency of Zn

assimilation, at least when compared with other supra-

littoral amphipods (Weeks and Rainbow, 1994), and in

some beachflea species the quantity of the assimilated

element does not appear to be positively correlated with

its concentration in the food (Weeks and Rainbow,

1994). Nevertheless, this metal is present at a high

concentration in the individuals of at least two of ourlocalities: Piombino (242 ppm) and La Piantarella (236

ppm).

These results for Cu and Zn are supported by the

data for several localities on the Polish coast (Rainbow

et al., 1998b) considered polluted because of levels of

70.4 ppm (Cu) and 262 ppm (Zn) (Table 2).

The Cd concentrations measured in the sandhoppers

of Rosignano Solvay (3.88 ppm) and Cateraggio d’Aleria(3.32 ppm) are much lower than the minimum found by

Rainbow et al. (1998b) at various sampling sites in the

Gulf of Gdansk (8.04 ppm) (Table 2); however, they are

higher than the maximum found by Fialkowski et al.

(2000) in the Gulf of Gdansk (3.02 ppm) and much

higher than the level considered as the background for

the Tuscan coast (0.5 ppm, ICRAM, 2000).

For T. saltator, there are no reference data regardinglocalities contaminated by Hg, Cr, Fe and Al. Never-

theless, we believe that the following concentrations are

worthy of attention. Al: 502 and 394 ppm measured

at Cateraggio d’Aleria and at Serchio River mouth,

respectively; Cr: 181.5 ppm at Cateraggio d’Aleria, 19.6

ppm at Serchio River mouth and 14 ppm at Piantarella;

Fe: 540 and 491 ppm at Ombrone River mouth and

Cateraggio d’Aleria, respectively. In contrast, the con-tamination by Pb (maximum 3.52 ppm at Livorno

Calambrone) and Hg (maximum 0.23 ppm at Piombino)

can be considered modest or absent.

The Al, Cr, Fe and Pb contents in the sandhoppers

differ greatly between Albegna River mouth and

Albegna seaside. Except for Pb, present to a greater

degree in the animals collected on the seashore, the con-

centrations of the other elements are 2–4 times higher inthe animals collected on the river bank than in those

from the beach. Differences in the concentrations in the

substratum and the effect of salinity do not seem to be

sufficient to explain such a strong discrepancy and fur-

ther investigations are necessary to resolve the problem.

Sandhoppers feed on bacteria and stranded organic

material, and thus absorb trace elements that are mainly

present locally. For this reason, T. saltator represents agood integrator of the two sources of bioavailable trace

elements: those in solution and those present in food. As

indicated by Rainbow (Rainbow and Phillips, 1993;

Rainbow, 1995), once the existence and type of rela-

tionship between the concentrations in the substratum

and in the sandhoppers have been ascertained and

the seasonal variations in bioaccumulation have been

identified (Rainbow and Moore, 1990; Fialkowski et al.,2003), these animals could easily be used in the bio-

monitoring of pollution from several heavy metals

since they exhibit many characteristics of good bioin-

dicators (easily identified, easily found, etc.). The

Table 3

Estimate of genetic diversity within each population and its relation-

ship with heavy metals contamination

Population

(site)

Heavy metals

contamination

(ranks)

Genetic

diversity

Genetic

diversity

(ranks)

C. d’Aleria 1 0.092 1

Piombino 2 0.149 4

Serchio R.

mouth

3 0.139 3

Ombrone R.

mouth

4 0.134 6

La Piantarella 5 0.187 8

Livorno 6 0.191 5

Albegna

seaside

7 0.167 7

Albegna R.

mouth

8 0.221 2

Rosignano 9 0.155 9

A. Ugolini et al. / Marine Pollution Bulletin 48 (2004) 526–532 531

bioaccumulation capacities of conspecific Mediterra-

nean and northern European sandhoppers do not seem

to depend on the well known differences in behaviour,

although a direct comparison between sandhoppersfrom different latitudes would be desirable. In fact, the

Mediterranean T. saltator, like the northern European

conspecifics, are good accumulators of Zn, Cu and Cd,

and also accumulate Hg, Fe and Al.

Genetic diversity was estimated within each popula-

tion (i.e. the samples from a particular site) as described

in Materials and Methods and the values were com-

pared among populations (Table 3). The genetic diver-sity within each population is rather low, ranging from

0.0926 to 0.221, but there are some differences among

the populations. The positive correlation between the

degree of genetic diversity and the degree of heavy

metals contamination at the sampling site (Table 3) is

barely significant (rs ¼ 0:717, N ¼ 9, P < 0:05, Spear-

man rank correlation coefficient test). However, it must

be borne in mind that environmental contamination bytrace elements is not the main factor affecting the genetic

composition of sandhopper populations (Bulnheim and

Schwenzer, 1999; De Matthaeis et al., 2000a,b). There-

fore, this result must be considered as an interesting

premise for further detailed investigations of this topic.

Acknowledgements

Our research was financially supported by local funds

of the Universities of Firenze and Siena respectively

assigned to A. Ugolini and S. Focardi.

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