Bull Environ Contam Toxicol (2013) 91:565–570
DOI 10.1007/s00128-013-1098-0
Arsenic Exposure Affects Embryo Development of Sea Urchin,
Paracentrotus lividus (Lamarck, 1816)
Andrea Gaion • Alice Scuderi • David Pellegrini •
Davide Sartori
Received: 10 January 2013 / Accepted: 5 September 2013 / Published online: 28 September 2013
 Springer Science+Business Media New York 2013
Abstract Toxicity tests were performed with embryos of
Paracentrotus lividus to investigate the toxicological effect
of two arsenic species: arsenate (AsV), expected to be more
toxic, and dimethyl-arsinate (DMA) expected to be less
toxic. Exposures to toxicants were performed at different
developmental stages in order to identify the most sensitive
phase of embryological development. Statistical analysis
revealed a high significance of each factor (Molecule,
Concentration and Time of exposure) and their interaction
for the dependent variable ‘‘Percentage of normal-shaped
plutei’’. In particular, the 8 cell stage was the most sensitive
to arsenic; at a concentration of 50 lg L-1 DMA proved to
be more toxic than AsV, resulting in nearly 50 % of nor-
mal-shaped plutei against the 74 % recorded for AsV.
Starting the administration of arsenic at the morula stage,
arsenate proved to be significantly more toxic when com-
pared to DMA.
Keywords Embryotoxicity  Sea urchin 
Larval stages  Arsenate  Dimethyl-arsinate
A. Gaion (&)  D. Pellegrini
Istituto Superiore per la Protezione e la Ricerca Ambientale
(ISPRA), STS Livorno, Piazzale dei Marmi, 57123 Livorno,
Italy
e-mail: andrea.gaion@isprambiente.it
A. Scuderi
Dipartimento di Scienze della Vita e dell’Ambiente, Università
Politecnica delle Marche, Edificio 3, Via Brecce Bianche,
60131 Ancona, Italy
D. Sartori
Centro interdipartimentale di ricerche per la gestione delle
risorse idrobiologiche e per l’acquacoltura (CRIAcq), Parco
Gussone-Edif. 77, Via Università 100, 80055 Portici, NA, Italy
Environmental contaminants, like metal and metalloid, can
be accumulated in larval stages of sea species and can
affect the proper development during the beginning of life
cycle (Durkina and Evtushenko 1991). However, these
observed relationships followed different patterns depend-
ing on the metal considered. Arsenic (As) is one of the
principal environmental contaminants which shows its
toxicity according to the chemical speciation: generically
organic forms are less toxic than inorganic ones (Fattorini
et al. 2004; Sharma and Sohn 2009).
In this context, many authors confirmed that ecotoxi-
cological assays are the best approach to integrate the
effects of contaminants on biota with values measured by
chemical analyses in different environmental matrices
(Persoone and Gillelt 1990; Macken et al. 2009; Palma
et al. 2010). The usefulness of toxicity tests, combining fast
response with high sensitivity and ecological importance,
oriented the international scientific community towards the
development of assays employing the most critical and
sensitive stages of the life cycle. These bioassays can
involve fertilization and embryological development in
model animals (Dinnel et al. 1988; Nipper et al. 1997).
The reliability of the Mediterranean Sea urchin Para-
centrotus lividus as a bioindicator species was recognized
in different studies (Marin et al. 2001; Cunha et al. 2005;
Álvarez et al. 2010; Sanchez-Marı̀n et al. 2010; Fabbrocini
and D’Adamo 2011) and its employment is a common
approach to monitor marine pollution. The two main
applications of this bioassay include both fertilization
toxicity and embryotoxicity. In the first bioassay, exposure
of sperm to a toxicant is performed in order to evaluate the
success of the consequent fertilization (Lera and Pellegrini
2006; Chapman 1995). Whereas for the second bioassay,
exposure commences at the embryo stage and continues for
72 h until pluteus achievement (Losso et al. 2007).
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Embryotoxicity assays are to be considered much more
sensitive than sperm toxicity assays, as they evaluate a
more complex endpoint, distinguishing normal larvae from
those with skeletal, digestive tract or arm malformations
(Losso et al. 2004).
This study focused on two different species of arsenic:
arsenate (AsV) as the most abundant form in the sediments
and water, expected to be more toxic, and the less toxic
dimethyl-arsinate (DMA). In this study, the administration
of the molecules at different concentrations was performed
at distinct stages of embryological development. The most
critical and sensitive phase in the life cycle of the sea
urchin P. lividus may be identified within the first 72 h,
when the zygote reaches the stage of pluteus through dif-
ferent stages (Gilbert 2000; Buznikov et al. 2003). How-
ever, it is essential to understand which stage of
development is the most sensitive towards these chemicals.
The aim of this study was to preliminary investigate the
toxicity of these two species of arsenic at different con-
centrations, and to identify the most sensitive develop-
mental stage of the larval cycle of the sea urchin.
Materials and Methods
Sea urchin adults of the species P. lividus were collected in
May 2012 from an intertidal rocky site along the coast of
Livorno (Italy) [43250 3200 N, 10230 3800 E] and transported
to the laboratory within 1 h from the collection.
Organisms, in a minimum of 3 individuals per sex, were
used to obtain gametes by injecting 1 mL of 0.5 M KCl
solution into the coelom through the peristome. Sperm
from each male was collected and preserved at 4C, while
oocytes from each female were shed into 50-mL beakers
previously filled with 0.22 lm Filtered Sea Water (FSW).
Spawned oocytes were allowed to settle and washed three
times with FSW; cell solutions were then diluted to a final
concentration of 1,000 oocytes*mL-1. Once sperm
mobility had been checked, sperm density was determined
by means of a hemocytometer (Thoma chamber). Sperm
was then diluted into filtered seawater in order to reach a
sperm: eggs ratio of 15,000:1 (Lera and Pellegrini 2006).
Fertilization was executed by gently mixing the sperm and
egg suspension.
Embryos were kept at 20C in a controlled temperature
chamber, a salinity of 38 ± 1 % and 9 h daylight. Arsenic
was administrated in two different species: arsenate (AsV)
and dimethyl-arsinate (DMA) (Sigma-Aldrich, USA).To
appraise the toxicity of arsenic each chemical species was
tested with three different concentrations (50, 100, and
200 lg L-1) at different times of early larval development,
following a hierarchical experimental pattern with crossed
factors.
In order to check the arsenic concentration in tested
solutions, chemical analysis of each solution was per-
formed with Agilent 4100 Microwave Plasma-Atomic
Emission Spectrometer with Multi-mode Sample Intro-
duction System.
Arsenic molecules were administered to untreated plutei
at the attainment of different developmental stages, as
shown in Table 1, independently of the time from fecun-
dation. Exposure ended for all the treatments after 72 h
from fecundation, when pluteus stage was achieved.
Six replicates were fixed for each concentration and the
final volume of the well plate was 10 mL. Controls were
conducted as untreated negative controls (0.22 lm FSW,
collected from the sampling site) and positive controls with
an exposure to CuNO3*3H2O (Sigma Aldrich, USA). To
obtain this reference value, embryos in positive control
group were exposed to copper solution of 24, 48, 60, 72,
96 lg L-1. Normal plutei were discriminated from
incomplete developed plutei, the ones with skeletal

Table 1 Development stage of P. lividus pluteus corresponding to each time of arsenic molecules administration (T0…T7)
Administration Corresponding development stage of Paracentrotus lividus pluteus
time

T0 20 min Post Fertilization (PF), when the increase in thickness of the extracellular envelope (vitelline layer) is definite; before
the first mitotic division
T1 First mitotic division; two blastomers are formed (about 1 h PF)
T2 8 blastomers; first horizontal division is executed (about 3 h PF)
T3 Morula stage reached (about 4 h PF)
T4 Blastula; the cluster of primary mesenchyme cells near the flattened vegetal side. of the blastocoels (about 6–7 h PF)
T5 Gastrula; contact of archenteron with the larval wall in the region of future mouth (oral contact); first signs of bilateral
symmetry (about 15 h PF)
T6 Prism; larva assumes the characteristic angular form; digestive tract consists of weakly delineated esophagus, stomach, and
intestine; developed larval skeleton (about 24 h PF)
T7 Early pluteus; small, non-divided rudiment of second pair of arms appears; the color of larval suspension changes sharply
because of chromatophore (about 48 h PF)

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aberrations (apex spicules cross-linked or separated) and
the ones with deformation of gastrointestinal apparatus.
The acceptability of the results was fixed at a percentage
of normal plutei C80 % in negative control tests. Regard-
ing the positive control, EC50 value with copper had to be
comprised within the acceptability range of 51–87 lg L-1
(Arizzi Novelli et al. 2002, 2003). Development was
stopped by adding 1 mL of 40 % formalin (Carlo Erba
Reagents, Milan). Fixed samples were then rinsed in
Phosphate Saline Buffer (PBS) and observed with trans-
mitted light microscopy.
In order to evaluate the contribution of individual factors
(Molecule, Concentration and Time of exposure) and their
interaction (level of significance p \ 0.05), Permanova
analysis was performed with statistical software PRIMER
v6 with the add-on package PERMANOVA? (Anderson
2001). Pair-wise test was performed as a posteriori com-
parison of levels for single factors, including within indi-
vidual levels of other factors in the case of significant
interactions.
Results and Discussion
Measured arsenic in tested solutions was respectively
53 ± 4, 98 ± 8 and 210 ± 6 lg L-1, confirming the
acceptability of the preparation process.
The percentage of normal plutei in control groups ranged
from 80 to 85 % in all the experiments, whereas EC50 mean
value ± SD for the positive control (54.23 ± 2.13 lg L-1)
was consistent with value reported in literature confirming
the good health of gametes (Anderson et al. 2004; His et al.
1999; Fernández and Beiras 2001; Arizzi Novelli et al.
2003). Arsenic concentration of 200 lg L-1, regardless of
the chemical form, proved to be lethal for the totality of the
development stages investigated, with the exception of
administration of DMA at the pluteus stage (28.83 ±
4.71 % of normal-shaped plutei, Table 2). For this reason,
this concentration was not included in the statistical analysis.
Permanova analysis revealed a high significance of each
factor and their interaction for the dependent variable
‘‘Percentage of normal-shaped plutei’’ (p = 0.001; data not
shown).
As shown in Table 2, T2 (the 8 cell stage) was the most
sensitive to arsenic; at a concentration of 50 lg L-1 DMA
proves to be more toxic than AsV, resulting in nearly 50 %
of normal-shaped plutei against the 74 % recorded for AsV
(Fig. 1).
As reported by Cameron et al. (1987), in the early sea
urchin embryos, the cell lineages provide a means for
generating spatial patterns of cell function that follow from
the process of founder cell specification during cleavage.
The evidence indicated that P. lividus embryos interacted
with the two arsenic molecules in different ways, and their
developmental fate can be influenced by the factors studied
in this experiment (Molecules, Concentration and Time of
administration) and their interaction. Based on statistical
comparison between the toxicity of arsenicals registered in
T2 stage and the other stages, this crucial phase in the larval
development of sea urchins proved to be significantly dif-
ferent from all the others, except for the T0 and T1
administration at 100 lg L-1 (Table 3).
As can be seen in Fig. 1, the overall trend of toxicity
increased until the 8-cell stage (T2), and after this step
started to decrease. In particular, all the exposures to
100 lg L-1 of arsenate resulted to be statistically different
from the control group. At the same concentration, DMA
exerted its toxicity until the T5 administration time. At an
exposure of 50 lg L-1 of arsenic, independently by the
chemical form tested in this experiment, no toxic effect in
respect with control group was measured for administration
after the blastula stage.

Table 2 Mean percentages of normal-shaped plutei after each administration (T0…T7)

Time of admin. 50 lg L-1 100 lg L-1 200 lg L-1
AsV SD DMA SD AsV SD DMA SD AsV SD DMA SD

Control 85.50 4.18 85.50 4.18 81.00 3.58 81.00 3.58 80.67 2.66 80.67 2.66
T0 70.67 2.16 68.83 5.04 44.83 10.1 54.67 4.18 0.00 0.00 0.00 0.00
T1 74.50 3.62 71.50 3.08 50.67 3.33 52.17 11.1 0.00 0.00 0.00 0.00
T2 73.50 8.26 50.17 4.88 41.50 8.46 48.00 7.92 0.00 0.00 0.00 0.00
T3 77.67 3.33 80.00 2.61 47.50 3.27 66.50 4.23 0.00 0.00 0.00 0.00
T4 81.00 5.22 80.00 4.90 57.83 4.71 67.67 3.83 0.00 0.00 0.00 0.00
T5 78.83 6.55 78.67 4.18 60.17 4.26 69.00 3.79 0.00 0.00 0.00 0.00
T6 80.00 4.20 79.17 2.48 69.17 2.64 77.67 3.78 0.00 0.00 0.00 0.00
T7 79.33 5.43 81.50 2.43 68.50 3.94 79.00 2.37 0.00 0.00 28.83 4.71
-1 V
Molecules concentrations are expressed as nominal concentration of As (lg L ). As Arsenate, DMA dimethyl-arsinate, SD standard deviation,
n=6

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Fig. 1 Percentages of normal

shaped plutei after arsenic
administration treatments (T0,
T1, T2, T3, T4, T5, T6, T7)
compared with control group
(Cont). AsV Arsenate, DMA
dimethyl-arsinate. *Indicates
statistical significance p \ 0.05,
**Indicates statistical
significance p \ 0.01

Table 3 Permanova results of

Statistical differences between T2 stage and the other stages
statistical differences between
T2 stage and the other stages 50 lg L-1 100 lg L-1
referred to each concentration
Groups t p (perm) Groups t p (perm)

T2 versus T0 3.5099 0.0025 T2 versus T0 1.5371 0.1421

T2 versus T1 5.1087 0.0002 T2 versus T1 1.9968 0.0605
T2 versus T3 7.9431 0.0001 T2 versus T3 4.702 0.0001
T2 versus T4 7.6401 0.0001 T2 versus T4 6.7408 0.0001
T2 versus T5 6.711 0.0001 T2 versus T5 7.5217 0.0001
T2 versus T6 8.0796 0.0001 T2 versus T6 11.261 0.0001
T2 versus T7 8.0647 0.0001 T2 versus T7 11.397 0.0001
Pairwise test within levels of factor ‘Time of administration’
DMA versus AsV DMA versus AsV

Pair-wise test for the factors Level T2 5.9566 0.0001 Level T3 8.7025 0.004
‘‘Molecule’’, ‘‘Concentration’’ Level T4 3.9688 0.009
and the factor ‘‘Time of Level T5 3.7915 0.012
administration’’, including their
levels. Statistical significance Level T6 4.5184 0.003
(p \ 0.05) is marked as Italic Level T7 5.5992 0.005

Deepening the analysis within the factor ‘‘Time of
administration’’, DMA was more toxic only at T2 stage for
the 50 lg L-1 concentration in respect with arsenate,
whereas for the others administration no differences were
registered in toxicity of the two species.
Regarding the exposure to 100 lg L-1 solution, arse-
nate was more toxic after the T2 administration (Table 3;
Fig. 1) up until the end of the experiment. In fact micro-
meres appeared and the fate of each blastomere proved to
be irreversible after this limit (Tufaro and Brandhorst
1979). In the 100 lg L-1 exposure, the effect of arsenic
supplied at previous larval stages showed no statistical
difference related to chemical form (p [ 0.05); besides,
starting the administration at the morula stage (T3), arse-
nate proved to be significantly more toxic when compared
to DMA.
The findings of our trials confirmed that the 8-cell stage
is a milestone in the proper development of sea urchin
(Table 3; Fig. 1). Furthermore, the 8-cell stage is the result
of the first horizontal division: after this stage the formation
of micromeres begins. The importance of micromeres as
pacemakers of cell divisions during cleavage for proper
development, has been fully studied (Yamazaki et al.
2012). Recent studies confirmed that this stage is particu-
larly sensitive also for other species, apart from sea urch-
ins. In fact when cells were removed from the 8-cell
tunicate embryo, the consequent larva lacked those struc-
tures normally produced by the missing cells. Moreover,
the isolated cells produced these structures separately from
the embryo. Whittaker (1973) provided dramatic bio-
chemical confirmation of the cytoplasmic segregation of
the morphogenetic determinants responsible for this

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pattern. Arceci and Gross (1980) studied histone synthesis
in three different cell types which resulted in the 16-cell
stage embryo. Authors discovered that the divergence of
the histone synthesis pattern among the three cell types was
due to a divergence of cell cycle parameters at this stage.
Even if less toxic, DMA seemed to exert its adverse
effect for a longer time during larval development.
To our knowledge, toxicological data for arsenic for
embryo of P. lividus have been reported only in two pre-
vious works, although with some differences and without
differentiating embryological steps (Pagano et al. 1982;
Arizzi Novelli et al. 2003). Both of these studies, however,
confirmed the importance of monitoring as toxicity to P.
lividus as a useful tool for environmental protection stud-
ies. Pagano et al. (1982) highlighted the high toxicity of
AsV to embryos of P. lividus. Although the exposure was
shorter (48 h) and the damage was measured as disruption
with normal mitotic activity (Mitoses Per Embryo, MPE),
this molecule proved to be more toxic than AsIII, as it
increased MPE and caused morphological abnormalities.
In Arizzi Novelli et al. (2003) toxicity of arsenic was
measured after an exposure of embryos to AsIII for 72 h. As
a consequence, the results from this work can be consid-
ered as a comparison between the effect of the two inor-
ganic forms of this metalloid.
Although arsenic in the marine environment is stored in
sediments (Neff 1997; Bennett et al. 2012), many reactions
can cause changes in chemical speciation of this element
(Pantsar-Kallio and Manninen 1997; Turpeinen et al. 2002).
It is important to understand the biological damage that these
different compounds can cause to the most sensitive stages of
nektonic larvae (Sharma and Sohn 2009).
In conclusion, this study drew attention to different
toxicological patterns for two arsenic species on P. lividus
pluteus development. Overall, our findings suggested that
further research is required to relate toxicity to cell speci-
fication and for a better understanding of the biochemical
fate of this element in sea urchin larval stages.
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