|Sea urchin; Fertilization; Embryogenesis; Cytogenetic
abnormalities; Redox alterations
|Sea urchins have proven to be extremely helpful in providing
scientific insight in a number of biological disciplines, including
physiology, embryology, biochemistry and genetics, in studies dating
as far back as the late 19th century to the early 20th century [1-6]. Those
studies provided a body of knowledge that became foundation in
what we know in basic cellular events such as mitosis with the first
reports on chromosomes , fertilization and embryogenesis [3-5],
with implications well beyond echinoderm biology and thereafter
translated into general biology and medicine. Relatively few reports
on the exposures of sea urchin embryos to various chemicals were
available, and focused primarily on the influence of some agents in
modulating normal embryogenesis, rather than on investigating any
adverse effects of chemicals, e.g. lithium-induced “animalization”
|In the wave of the early concern in radiobiology, drug toxicity,
and environmental pollution, sea urchins were first utilized in
pioneering studies evaluating the effects of sperm irradiation ,
marine pollution , pharmaceuticals and pesticides [10-12], and
the action mechanisms of carcinogens [13,14], in bioassays viewed
as complementary to the classical murine models. Those studies
dating up to 1970’s set the grounds for continuing investigations up
|The body of literature of toxicity testing in the sea urchin
model includes reports focused on a number of morphological
and/or molecular endpoints assessing adverse effects. This prevents
attempting extensive comparisons (e.g. scaling EC50’s) in-between
several agents. Though with this limitation, the present review may
both provide an unprecedented archive of sea urchin toxicity testing
reports and a useful contribution in future study design.
|Seawater acidification: As shown in Table 1, pH changes were
initially studied in sea urchin embryos and sperm as a subject
of bioassay standardization, or for their possible implications in
freshwater systems, related to the recognized phenomenon termed
acid rain . Early reports found that developing embryos under
slightly decreased pH conditions, in the order of 0.5 pH units,
underwent damage to embryogenesis and mitotic aberrations, with
stronger effects when treatment was performed before hatching
than in later developmental stages (blastula/gastrula) [16,17]. When
sea urchin sperm were exposed to pH changes, fertilization was
inhibited by either pH decrease (<7) or increase (>8.5). Furthermore,
transmissible damage to the offspring of low pH-exposed sperm was
observed, both in terms of developmental defects and of decreased
mitotic activity [18,19]. It should be noted that slightly decreased pH
(such as 7.5) enhanced fertilization success, as confirmed in a more
recent report . A series of studies by Stumpp et al. [21-24] showed
that elevated seawater pCO2, closely resembling the expected values
by the end of this century, altered the expression of 26 representative
genes important for metabolism, calcification and ion regulation,
along with impacting growth and resulting in developmental delay
of echinoid larvae. Adult sea urchins (P. lividus) were tested by Lewis
et al.  for their sensitivity to micromolar copper levels in ambient
vs. acidified seawater, resulting in DNA damage and oxidative stress
responses, beyond the mere data of metal speciation.
Table 1: Reports on inorganics-associated effects on sea urchin early life stages.
|Altogether, the available literature on the effects of pH decrease in
sea urchin bioassays corroborates the of global concern about ocean
|Other inorganics: A number of inorganics (Cd, As, Cr, Pb, Cl,
Ag, Mn, La, Ce, Gd) were tested for their adverse effects on sea urchin
development, fertilization success, mitotic activity, as well as other effects
as induction of apoptosis, calcification defects, DNA damage and/or
increasing production of reactive oxygen species (ROS) amongst others
[26-59] (Table 1). Groups of inorganics were tested in an extensive body
of studies providing comparative toxicity datasets for various agents
[40,59]. This was the case for early studies [41,42] testing groups of
inorganics as Ag, Cd, Cr, Hg and Pb; other studies reported on the effects
of individual inorganics vs. their mixtures [43,54].
|It has been reported that developmental impairment due to
treatment of fertilized eggs with Cd and Mn [29,38] involved the
action of the physiological messenger nitric oxide . Moreover,
maternal exposure to these metal ions impaired reproduction and
progeny fitness .
|We have reported on the comparative toxicities in P. lividus
embryos and sperm of light rare earth elements (REEs) (Y, La, Ce,
Nd, Sm and Gd) that induced developmental defects and mitotic
abnormalities in exposed embryos, offspring damage following sperm
treatment, and redox anomalies (ROS production, lipid peroxidation,
and nitric oxide release) [58,59]. A current study of heavy REEs (Dy,
Ho, Er, Yb and Lu) suggests more severe toxicity of heavy vs. light
|Nanomaterials: As shown in Table 2, a number of studies have
been focused on the effects of nanomaterials (NM) in sea urchin
early life stages. The growing interest in the multi-fold applications
of NM has led to concerns on their possible health effects in several
biological models, including sea urchins [60-73]. To date, the
reported effects of NM in sea urchin early life stages have included
multiple responses in sea urchin embryos following exposures to
carbon nanoparticles, including developmental defects, abnormal
biomineralization, and altered gene expression [60,61]. Other
adverse effects have been reported in terms of chemosensitization,
increased multidrug resistance, and redox changes following
exposures of sea urchin embryos to Zn and Co oxide nanoparticles
[62-65]. Silica nanoparticles were found to increase developmental
defects while inhibiting fertilization success, and led to the decrease
of cholinesterase activity . TiO2 nanoparticles were found to
enhance phagocyte TLR/p38 MAPK signalling pathway by activating
an internalization mechanism . Altogether, sea urchin bioassays
have been useful in revealing adverse effects after exposure to NM. It
should be recognized, however, that a substantial body of literature,
beyond the scope of this review, points to a number of beneficial effects of NM, especially in medical and agricultural applications. Ad
hoc investigations are warranted to critically re-evaluate beneficial vs.
harmful effects associated with NM exposures.
Table 2: Reports on nanoparticle-associated effects on sea urchin early life stages.
|As shown in Table 3, an extensive body of literature is available
regarding the effects on sea urchin early life stage after exposures
to organic agents. Since early studies [13,14,74] and up to recent
reports [75,76] polycyclic aromatic hydrocarbons (PAHs) and
their hydroxylated derivatives have been investigated for their
bioaccumulation in sea urchin embryos and/or for induction of
developmental defects. Comparisons of the relative toxicities of select
PAHs vs their model mixtures when subjected to fluorescent light
exposure were also investigated in recent literature . Other studies
reported on the relative toxicities of low molecular-weight aromatic
hydrocarbons (benzene and styrene) and their derivatives [77,78].
Table 3: Reports on the effects of organics, complex mixtures and natural products on sea urchin early life stages.
|Extensive studies have focused on the effects of pharmaceuticals
on sea urchin embryogenesis and fertilization since early reports
[10,11,79-82] and up to recent studies [83-94]. The available
database comprehends reports on several classes of agents including
antibiotics and antiseptics [79-82], antineoplastic drugs [84-87,89],
teratogens [10,11,86,88,93], anti-inflammatory drugs, hormones or
hormone-like agents [88,92], anesthetics [94-96], cannabinoids [97-99], and antioxidants [86,100]. Altogether, the currently available
literature on the adverse effects of several pharmaceuticals in sea
urchin bioassays contributes to mechanistic information about druginduced
toxicity to early life stages, warranting further investigations
and risk assessment.
|Pesticides and metal organics: Since a pioneering paper by Bresch
and Arendt , several pesticides have been investigated in sea
urchin early life stages. As shown in Table 3, a number of studies have
reported on halogen, nitrogen, and pyrethroid derivative pesticides
for their effects on sea urchin early development, fertilization, and offspring quality [101-120]. Several studies found embryo- and
spermiotoxicity of chlorinated pesticides [101-109], showing
differences in toxicity as a function of number of chlorine atoms per
molecule [102,103]. Organophosphate pesticides were found to affect
sea urchin metamorphosis , embryo- and spermiotoxicity, along
with induction of offspring damage following sperm exposure .
|In some cases pesticide preparations include organic and
inorganic components, as in the case of R6 fungicide, a mixture of an
acetamide pesticide, cymoxanil (CYM) and cupric oxychloride (Cu-
OCl) . The commercial mixture resulted in toxicity that failed to
appear following exposure to the two mixture components alone. A
mixture prepared from analytical grade CYM and Cu-OCl also failed
to induce toxicity, a result that suggested the occurrence of more toxic
components in the formulation of the commercial mixture . Thus, a critical reappraisal may be suggested in studies where toxicity
is commonly evaluated on pure chemicals, while disregarding the
effects induced by the technical-grade counterpart.
|Either as antifouling agents or as pesticides, a number of
metallorganic compounds have been evaluated for their toxicity
to sea urchin early life stages (Table 3). Most of these reports have
focused on organic tin derivatives used as antifouling agents, while
some studies were aimed at comparing toxicities of individual agents
vs. their mixtures [121-131].
|Complex mixtures: An extensive body of literature refers to the
effects of complex mixtures in sea urchin early life stages, since the
1970’s [132,133]. As shown in Table 3, a number of studies have been
published on the effects of complex mixtures in sea urchin bioassays [132-160], including crude oil and oil fractions; spill-treating agents;
refinery wastewater effluent; pulp mill effluent; bauxite manufacturing
by-products; storm water and parking lot runoff; leather tanning
effluents; landfill leachate; pisciculture effluents, and alum-coagulated
municipal wastewater. This body of literature is not confined to studies
of mixtures directly involved in marine pollution, as in the cases for
industrial sludge and effluents [141-146,148-151,153,158], or landfill
leachate . Thus, one may recognize multiple applications of sea
urchin bioassays in toxicity evaluation of complex mixtures both
impacting on marine and on freshwater or terrestrial environments.
|Another major application of sea urchin bioassays in testing
complex mixture toxicity refers to sediment toxicity evaluations. This
relevant subject is omitted in the present review and will be exposed
in a subsequent paper.
|Natural products: An established body of literature in sea
urchin bioassays has focused on the effects of a number of natural
products (NP) on sea urchin early life stages. The major goals of this
research line have been devoted to either characterizing some natural
products for their mitotic activity and their potential applications as
pharmaceuticals, or as potential stressors to marine ecosystems.
|Sea urchin bioassays were successfully used for assay-guided
isolation of natural products from marine diatoms . A body
of literature has been devoted to these molecules, characterized as
polyunsaturated aldehydes (PUAs), showing antimitotic and proapoptotic
activity inducing malformations in developing embryos
[162-168]. Interestingly, the effect was recorded when PUAs were
added before or soon after fertilization, while they were almost
ineffective when added at 40 minutes post fertilization onwards
. Molecular studies of differential gene expression induced by
PUA treatments revealed that sea urchins activated an orchestrated
defense system involving HSP70 as key stress response mediator
 and showed that this effect is modulated by the messenger
nitric oxide . Analogous to PUA, diatom-derived oxylipins and
hydroxyacids were reported recently to affect sea urchin development
by antimitotic and pro-apoptotic mechanisms [169,170].
|Other studies have reported on mitotoxic or other effects of
natural products on sea urchin early development. Extracts from
several biota, including algae, plants, and sponges were found to
exert mitotic arrest in sea urchin cell division [171-175]. Terpenic
compounds were obtained from sponges and corals providing
evidence for mitotoxicity and embryotoxicity [176,177] and other
effects, such as inhibition of DNA polymerases  and nucleic acid
biosynthesis . Two reports focused on the action mechanisms
of β-amyloid in sea urchin early development [180,181]. A subtle
reproductive impairment through nitric oxide-mediated mechanisms
was reported in sea urchins from a site in the Gulf of Naples affected
by (Ostreopsis cf. ovata) .
|Sea urchin bioassays: Background and perspectives: After
the early laboratory studies of sea urchin bioassays in 1970’s, some
methodology reviews were published in testing developmental
damage, spermiotoxicity and transmissible damage to sperm
offspring, by either treating early life stages or adult sea urchins [183-
|A summarized scenario of assay timing and outcomes that are
tested for in embryo exposure is depicted in Figure 1. The commonly
used embryotoxicity protocol starts at zygote stage (10 min postfertilization)
ending with the observation of developmental defects at pluteus larval stage, 48 to 72 hrs after fertilization, according to
species and culture temperature. In order to elucidate the most
sensitive developmental stage to the action of a given xenobiotic,
embryo exposure may be confined to early, pre-hatching stages,
with prevailing mitotic activity, or to post-hatching stages (blastula
and gastrula), leading to larval differentiation. Thus, for example,
allopurinol, some antioxidants (L-methionine and N-acetylcysteine)
and diatom aldehydes [86,100,168] only induced developmental
defects following early (pre-hatching) exposures. By contrast,
cadmium and chlorobenzenes were only effective when administered
to post-hatching embryos [26,77], while lead exerted significantly
stronger effects following post-hatching compared to pre-hatching
Figure 1: Schematic view of timing and developmental stages in sea urchin
embryos cultured at 18°C (e.g. P. lividus) showing sensitive stages to mitotoxic
agents vs. embryo-selective agents (i.e. affecting later stages in embryogenesis).
Abbreviations: N: Normally developed plutei; P1: Malformed pluteus; P2:
Developmental arrest at abnormal blastula/gastrula stage.
|The major embryological endpoints evaluated in larval cultures
are the frequencies of pluteus malformations (P1), pre-larval arrest
(P2), as shown in Figure 1. The most commonly scored endpoint,
as a function of xenobiotic concentration, is the frequency of
developmental defects (%DD)=(P1+P2). Another scored endpoint
consists of the observation of dead plutei and dead pre-larval (or prehatching)
embryos that point to acute effects; however, these effects
are not usually observed in testing sub-acute developmental toxicity,
confined to %DD.
|In order to perform cytogenetic analysis, evaluating inhibition of
mitotic activity and/or induction of mitotic aberrations in Carnoy’s
fluid, cleaving embryos (approx. 5 hrs post-fertilization) are fixed
and subsequently stained by acetic carmine allowing for observation
of chromosomes, then scoring the frequencies of active mitoses per
embryo, embryos lacking mitotic figures, and the frequencies of
mitotic aberrations. The most commonly observed mitotic aberrations
include anaphase bridges, lagging chromosomes, multipolar spindles,
and scattered chromosomes (Figure 2).
Figure 2: Main cytogenetic endpoints include decreased mitotic activity
(mitoses per embryo) and mitotic aberrations. A. Anaphase bridge; B.Lagging
chromosomes; C. Multipolar spindle; D. Scattered chromosomes.
|Aside embryological and cytogenetic endpoints, a number
of biochemical or other molecular alterations may be detected in
xenobiotic-exposed sea urchin embryos and larvae. This was the case
for the induction of redox anomalies, with changes in ROS production,
oxidative and nitrosative stress endpoints, and oxidative DNA
damage [39,48,49,59,89,90]. Other reported endpoints, evaluated in
testing developmental toxicity in sea urchin embryogenesis, included
calcification defects [23,37-39], induction of multi-drug resistance  and MAPK-mediated signalling pathway , and production
of the endogenous antioxidant, ovothiol .
|Sea urchin sperm are exposed to agents for assigned time
intervals varying upon species, e.g. 60 min for Paracentrotus lividus
or 10 min for Sphaerechinus granularis. The endpoints measured
following sperm exposures to xenobiotics include fertilization
success and offspring damage, expressed as developmental defects
and/or altered redox endpoints (Figure 3). Changes in fertilization
success are mostly evaluated in zygotes by formalin fixation shortly
after fertilization by a number of authors [49,95,96,114,183,184].
We follow - and recommend - a different procedure, by reading
fertilization rate starting from 1 hr post-fertilization on live and
cleaving embryos, which are then allowed to undergo further
development up to pluteus stage. Indeed, reading fertilization rate in
fixed zygotes is confined to the appearance of fertilization membrane,
which may be more or less evident and lead to imprecise evaluation,
or to fixation artifacts. Otherwise, reading fertilization rate in
cleaving embryos is both supported by evidence for fertilization
membrane and by on-going cleavage. Thus, the observation of
any transmissible damage from sperm to offspring is warranted.
The induction of developmental defects in the offspring of sperm
exposed to xenobiotics has been reported in a number of studies
[19,31,33,43,44,54,58,59,89,90,111,143-145,148-151]. The finding of
developmental defects or embryonic larval mortality following sperm
exposures may be attributed to induction of dominant lethals, an
effect referred to since early genetics studies up to date [187,188].
Figure 3: Sperm pretreatment experiments, timing and observation endpoints:
a) Changes in fertilization success; b) Offspring damage, detected as: b1)
Developmental defects; b2) Altered redox endpoints.
|The body of literature on the use of sea urchin in toxicity testing
encompasses an extensive number of xenobiotics and mixtures that are
relevant both in environmental science and in general pharmacology and
toxicology. Well beyond the current database and the on-going research,
the appearance of new pollutants, pharmaceuticals and occupational
agents, along with the need for characterizing widespread complex
mixtures, open the field for further research efforts and future regulation
utilizing sea urchin bioassays in current and forthcoming work.
|Beyond their half-century historical background, sea urchin bioassays represent a thriving tool in future investigations and health
risk assessment, unconfined to marine environment and in prospect
evaluation of novel xenobiotics.
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