MARINE

ENVIRONMENTAL
RESEARCH
Marine Environmental Research 58 (2004) 845850
www.elsevier.com/locate/marenvrev

Chemical speciation of arsenic in dierent

marine organisms: Importance in
monitoring studies
Daniele Fattorini a, Carlos M. Alonso-Hernandez b,
Misael Diaz-Asencio b, Alain Munoz-Caravaca b,
Federica G. Pannacciulli c, Michele Tangherlini c,
Francesco Regoli a,*
a

Istituto di Biologia e Genetica, Universita Politecnica delle Marche, Via Ranieri Monte DAgo, 60100
Ancona, Italy
b
Centro de Estudios Ambientales de Cienfuegos, Cuba
c
ENEA, Centro Ricerche Ambiente Marino, La Spezia, Italy

Abstract
Arsenic is a widely distributed element in the marine environment. Inorganic and organic
compounds have extremely dierent toxicological eects, and their characterization is thus of
great utility when monitoring and assessing the impact of arsenic pollution. In this study both
the levels of total arsenic and its chemical speciation were analyzed in several marine organisms collected from Cienfuegos Bay (Cuba) following an episode of acute As-contamination. Fish from the more impacted site were characterized by elevated concentrations of
arsenic (up to 500 lg/g d.w.) and inorganic species represented the predominant forms in
muscle tissues of these organisms.
2004 Published by Elsevier Ltd.
Keywords: Arsenic; Chemical speciation; Monitoring; Risk assessment

Arsenic toxicity is related to its chemical form: inorganic arsenic (arsenate

and arsenite, (i)As) is most toxic, while methylated molecules, such as methylarsonate (MMA), dimethylarsinate (DMA), trimethylarsine oxide (TMAO) and
*

Corresponding author. Tel.: +39-071-2204613; fax: +39-071-2204609.

E-mail address: regoli@univpm.it (F. Regoli).

0141-1136/$ - see front matter 2004 Published by Elsevier Ltd.

doi:10.1016/j.marenvres.2004.03.103

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D. Fattorini et al. / Marine Environmental Research 58 (2004) 845850

tetramethylarsonium (TETRA) are considered moderately toxic. These compounds represent precursors of more complex organic and non-toxic forms, like
arsenobetaine (AsB), arsenocholine (AsC) and arsenosugars (AsS) (Gebel, 2001).
In the marine environment, inorganic arsenic predominates in seawater and
sediments, whilst the organisms bioaccumulate the element generally as organic
non-toxic compounds; these forms have been suggested to be nal products of
detoxication processes (Francesconi, Goessler, Panutrakul, & Irgolic, 1998;
Phillips, 1990). Noticeable variation can be found in the basal concentrations and
distribution of chemical forms of arsenic in marine organisms, often reecting
their trophic position, the inherent capability of biotransformation or other
species-specic traits in As metabolism. Anthropogenic contamination can modify
the environmental bioavailability of this element, and potentially inuence the
relative presence of toxic forms in tissues of exposed organisms.
The aim of this study was a preliminary investigation on the levels and chemical
speciation of arsenic in various marine organisms (algae, crustaceans and shes)
collected from Cienfuegos Bay (Cuba); this area was recently subjected to an episode
of acute contamination due to the accidental release of 3.7 tons of arsenic (as arsenate oxides) from a local nitro-fertilizer factory situated in Cienfuegos city. This
investigation was expected to highlight the importance of analyzing the chemical
speciation of arsenic compounds in order to assess both bioavailability and detrimental risk associated with its release in the marine environment.
Samples and sites: Cienfuegos is situated on the south coast of Cuba and it is
characterized by a closed bay with a shoreline of approximately 90 Km. Release of
arsenical compounds into the Bay occurred in December 2001 and representative
samples of biota were collected 15 days after the contamination episode. Several
species were analysed to provide a preliminary assessment of the environmental
impact in dierent sites of the Bay (Bahia, Calicito, Corona, Cuatro Boyas, La Milpa
and Marina Puerto Sol) and of potential risk associated to human consumption of
these organisms. Analyzed species included algae (Dictyota sp., Hypnea spinella,
Laurencia papillosa), crustaceans (Callinectes sapidus, Farfantepenaeus notialis, Litopenaeus schmitti) and shes (Albula vulpes, Caranx sp., Haemulon sp., Lutjanus
synagris, Micropogonias furnieri).
For determination of arsenic compounds,approximately 500 mg of lyophilized
tissues of algae, crustaceans or sh (muscle) were extracted with 5 ml of methanol
using microwave digestion (10 min at 150 W, Gomez-Ariza, Morales, Giraldez,
Sanchez-Rodas, & Velasco, 2001). Samples were centrifuged at 5000 rpm for
5 min to remove insoluble residues. Separation of arsenic compounds was per
formed using ion exchange HPLC, following the method described by Slejcovec
(1999). Fractions (0.5 ml) were collected at 30 second intervals and 0.5 ml of
nitric acid added. Total arsenic in these fractions was determined by graphite
furnace AAS with Zeeman background correction. Arsenic compounds were
identied by comparing their retention times with those reported for reference

standard materials (Slejkovec,
Van Elteren, & Byrne, 1999). Tissues were also
directly digested in nitric acid to quantify the total content of arsenic (Regoli &
Orlando, 1994). The standard reference material DORM-2 (lyophilized dogsh

D. Fattorini et al. / Marine Environmental Research 58 (2004) 845850

847

muscle, by NRC of Canada) provides certied values also for some As-compounds and it was used to check the quality of both speciation procedure and of
chemical analyses; concentrations measured for dierent forms and total arsenic
were always within the 95% condence interval of reported levels. Levels of total
arsenic and distribution of chemical forms analyzed in various species is reported
in Table 1.
Algae. The total content of As did not exhibit marked variations in dierent
species, with values ranging from about 5 to 10 lg/g (dry weight) (Table 1).
Similar levels appear typical for algae and comparable to those already reported
by other authors: Rhodophyceae (including Laurencia sp. and Hypnea sp.) ranged
from about 1 to 10 lg/g, whilst concentrations up to 100 lg/g were observed in
Phaeophyceae (including Dictyota sp., Tukai, Maher, McNaught, & Ellwood,
2002).
Crustaceans. Concentrations of As measured in tissues of crabs and shrimps
(between 13 and 25 lg/g) were very similar to basal levels described in other crustaceans. Chemical speciation of As revealed that organic non-toxic compounds are
the most common forms, with about 95% occurring as AsB or AsC (Table 1). These
data conrm the capacity of crustaceans to bioaccumulate moderate quantities of
organoarsenic compounds (Larsem, Pritzl, & Hansen, 1993).
Fish. Interesting results were obtained for the concentrations and chemical
speciation of As in sh collected from dierent locations of Cienfuegos Bay, although a direct comparison between sites was limited to a few species. Lutjanus
synagris from Corona exhibited concentrations of As (
12 lg/g) similar to those
reported in some Caribbean shes (Lewis, Scott, Bearden, Quarlesa, Moore,
Strozier, et al., 2002) mainly consisting of AsC (90%). In contrast, the same species
from the site of Bahia exhibited about 500 lg/g of As in the muscle tissue, mainly
as inorganic forms (
98%) (Fig. 1). Similar results for total As were obtained of
comparing Caranx sp. from Corona and Bahia (Table 1); organisms from the latter
site showed much greater concentrations of As (close to 500 lg/g d.w.) and a
chemical speciation characterized by the predominance of inorganic forms. Interestingly, concentrations of As measured in tissues of Haemulon sp. from Bahia,
were only slightly higher than reference values measured in the other sh species,
i.e., Albula vulpes and Micropogonias furnieri respectively from the sites of Corona
and Cuatro Boyas (Table 1).
An overall analysis of obtained results conrm that As in marine organisms is
mainly present as organic non-toxic forms. The accidental contamination in Cienfuegos Bay resulted in a marked accumulation of inorganic As only in two sh
species from the site of Bahia, while no variation from normal values (Larsem et al.,
1993; Lewis et al., 2002; Tukai et al., 2002) was observed in organisms from the other
locations. Although this work was not aimed to provide an extensive monitoring of
the impacted area, nonetheless the elevated levels of inorganic arsenic measured in
sh muscle tissues might be of toxicological relevance. These preliminary results
highlight the importance of measuring chemical speciation of As to better assess the
environmental impact caused by anthropogenic contamination and to evaluate the
potential risks on public health.

848

Species

Site

1
2
3

Dictyota sp.
Laurencia papillosa
Hypnea spinella

Algae
Algae
Algae

4
5

Callinectes sapidus
Farfantepenaeus
notialis
Litopenaeus
schmitti
Caranx latus
Haemulon sp.
Lutjanus synagris
Albula vulpes
Caranx hippos
Lutjanus synagris
Micropogonias
furnieri

Crab
Shrimp

6
7
8
9
10
11
12
13

Shrimp
Fish
Fish
Fish
Fish
Fish
Fish
Fish

La Milpa
La Milpa
Marina
Puerto Sol
Calicito
Calicito
Cuatro
Boyas
Bahia
Bahia
Bahia
Corona
Corona
Corona
Cuatro
Boyas

(i)As
(%)

MMA
(%)

DMA
(%)

TMAO
(%)

TETRA
(%)

AsB
(%)

AsC
(%)

Unknown
(%)

Total As

8.57  0.75
10.14  0.68
4.98  0.51
1.16

95.20
7.13

1.70
92.87

1.94

24.99  0.97
16.26  0.85
13.25  0.91

98.67
0.54
97.70

3.77

0.61

2.06

0.04
1.76
0.05

1.29
0.42

2.97

2.06

Results expressed in lg/g d.w. (mean values  standard deviations, n 4).

Chemical speciation and relative distribution of As-compounds were characterized in species n 4, 5, 7, 8, 9, 12.

96.67
2.25

89.14

488.99  18.58
26.72  0.96
497.71  21.52
4.40  1.05
2.17  0.78
11.91  0.03
10.11  1.31

D. Fattorini et al. / Marine Environmental Research 58 (2004) 845850

Table 1
Total content of arsenic in tissues of several species of algae, crustaceans and shes collected from Cienfuegos Bay (December 2001) after an episode of acute
contamination

D. Fattorini et al. / Marine Environmental Research 58 (2004) 845850

(a)

849

(b)

AsC
(i)As

DMA
(i)As

TETRA

AsB

AsC

12

15

Time (minutes)
inorganic-As

TETRA

12

15

Time (minutes)
methylated-As

organic-As

Lutjanus synagris
1000,0

100,0

10,0

1,0

0,1

Corona

Bahia

Fig. 1. Example of chromatograms and relative distribution of arsenic compounds (lg/g d.w.) in muscle
tissues of Lutjanus synagris collected from Corona (a) and Bahia (b).

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D. Fattorini et al. / Marine Environmental Research 58 (2004) 845850

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