Chemosphere 240 (2020) 124819

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Sea cucumber Holothuria polii (Delle Chiaje, 1823) as new model for
embryo bioassays in ecotoxicological studies
Lorenzo Morroni a, *, 1, Arnold Rakaj b, 1, Luca Grosso b, Alessandra Fianchini b,
David Pellegrini a, Francesco Regoli c
a
Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA), Livorno, Italy
b
Laboratorio di Ecologia Sperimentale ed Acquacoltura, Dipartimento di Biologia, Universita  di Roma “Tor Vergata”, Roma, Italy
c
Dipartimento di Scienze della Vita e dell’Ambiente (DiSVA), Universita Politecnica delle Marche, Ancona, Italy

h i g h l i g h t s

In this study, a standard methodology of Holothuria polii embryo bioassay was defined.

A new integrative toxicity index was developed to refine the sea cucumber bioassay.

Good sensitivity of the test to discriminate toxic samples was verified.

The bioassay proved a useful tool in future assessment of marine pollution.

a r t i c l e i n f o a b s t r a c t

Article history: The sea cucumbers are common members of marine benthic communities, widespread distributed,
Received 20 June 2019 easily available and handled. Nevertheless, no data are available on embryo toxicity assays using sea
Received in revised form cucumbers, despite some of these species could fully meet the requirements for model test organisms.
6 September 2019
Holothuria polii is a key species in soft sediments and seagrass meadows; the aim of the present study
Accepted 7 September 2019
Available online 12 September 2019
was the standardization of a new embryo bioassay with this species, as an ecologically relevant test to
evaluate the effects of environmental stressors. Sequential experiments were carried out, allowing to
Handling Editor: David Volz define the test acceptability, and a minimum sample size of 240 embryos. Temperature of 26  C, salinity
at 36‰ and a density of 60 eggs/ml were identified as optimum experimental conditions for performing
Keywords: the bioassay. The EC50 calculated for Cd2þ and Cu2þ in dose-response experiments indicated a good
Embryo bioassay sensitivity of H. polii, with comparable values with those obtained in embryo toxicity bioassays of other
Holothuria polii marine invertebrates. An Integrative Toxicity Index (ITI) was calculated by integrating the frequency of
Sea cucumber abnormal embryos with the severity of observed abnormalities. The index allows to better discriminate
Ecotoxicology
different levels of toxicity, appearing particularly relevant for validating the usefulness of H. polii in
Water quality
embryo assays and ecotoxicological studies on environmental quality.
Harbor sediments
© 2019 Published by Elsevier Ltd.

1. Introduction consequences (Chapman et al., 1987). For this reason, the chemical
approach is routinely integrated with ecotoxicological analyses for
Human activities introduce significant amounts of pollutants a more realistic estimation of pollution (Long et al., 1996; Bebianno
into marine environment, increasing the background concentra- et al., 2015). The standardization of bioassays implies the compli-
tions of a large range of contaminants. For several decades chemical ance of several requirements of the test species, such as widespread
analyses have been used to define the degree and the nature of distribution, easy availability, ecological relevance and sensitivity to
pollution without providing evidence of potential biological low levels of contaminants (Pe rez et al., 2016). High sensitivity to
toxic substances is typically present during early development of
marine invertebrates (Beiras and His, 1995; Pe rez et al., 2016); in
this respect embryos and larvae belonging to the taxonomical
* Corresponding author.
E-mail address: [email protected] (L. Morroni).
groups, e.g. echinoderms (Pagano et al., 2017; Morroni et al., 2016;
1 
Saco-Alvarez et al., 2010), coelenterates (Faimali et al., 2014),
contributed equally.

https://doi.org/10.1016/j.chemosphere.2019.124819
0045-6535/© 2019 Published by Elsevier Ltd.
2 L. Morroni et al. / Chemosphere 240 (2020) 124819

polychaetes (Oliva et al., 2018), mollusks (His et al., 1997; Galgani 2.2. Sea cucumber embryo test
et al., 2009; Pe rez et al., 2016), crustaceans (Bellas et al., 2005;
Faimali et al., 2006; Buttino et al., 2018), ascidians (Bellas et al., Before spawning induction, adults were kept in substrate-free
2003) have been used increasingly in bioassays in the last decades. aquaria for 48 h in order to void their gut contents. Sea cucum-
The sea urchin embryo-toxicity test is among the most used and bers were then individually transferred to 10 L spawning tanks
standardized routine tools for biomonitoring studies (His et al., containing 0.45 mm filtered and UV-sterilized seawater (FSW), and
1999; ASTM, 2012). However, this bioassay also presents certain spawning induced by thermal shock. In particular, water temper-
disadvantages such as the lack of gamete emission during the ature was rapidly increased by 3e5  C (from 24  C to 27e29  C),
summer which limits the possibility to perform the analysis maintained over 1.5 h, and then returned to the initial temperature
throughout the whole year (Oliva et al., 2018). For this reason, the (Rakaj et al., 2019). When spawning induction was successful, the
availability of complementary embryo-toxicity tests with species sea cucumber showed signs of pre-spawning movements. Eggs of
spawning in different periods, would allow to define batteries of individual females were collected from the bottom of spawning
bioassays which guarantee the possibility to measure develop- tanks and suspended in FSW. Eggs were pooled and examined
mental endpoints in all the seasons. At this time, no data are under a microscope: females releasing non-round eggs, immature
available on similar toxicity assays using sea cucumbers, despite forms or debris were all discarded. Males were identified after the
some of these species could fully meet the requirements for model first spawn: sperm obtained from at least three males was collected
test organisms. Deposit-feeding sea cucumbers are common dry, pooled and stored at 4  C until further use. Sperm motility was
members of marine benthic communities and important ecosystem checked under a microscope and diluted sperm suspension was
engineers that play a key role in sea floor dynamics by processing added to an aliquot of egg suspension to check the success of
and bioturbating the sediment (Purcell et al., 2016; Rakaj et al., fertilization (formation of the fertilization envelope). Males fertil-
2018). These organisms are intensively harvested worldwide, izing less than 95% of the eggs were discarded. Once mobility was
especially in Asia, China and Japan (Purcell et al., 2016), considered checked, a suspension of 1  104 spermatozoa/mL (Rakaj et al.,
an excellent seafood as well as being used in various medical ap- 2019) was diluted in 500 mL of egg suspension (1000 eggs/mL).
plications. To tackle their increasing demand in aquaculture, After fertilization, embryos were washed three times by decanta-
attention has been recently put on fertilization and embryo tion, removing sperms in the supernatant and adding FSW. To avoid
development of sea cucumbers. bacterial overgrowth, an antibiotic solution of Gentamicin 2 mg/L
In this study, we focused on Holothuria polii (Delle Chiaje, 1823), was administered (Fischer, 2013). A period of 20 min was allowed to
a very common Mediterranean species, inhabiting soft sediments pass before starting the incubation with test solutions. One milli-
and seagrass meadows, where it plays a fundamental role in liter of fertilized eggs suspension was added to 9 mL of test solution,
benthic dynamics (Bocagni et al., in press). The spawning period of using 10 mL sterile capped polystyrene six-well microplates. Six
H. polii occurs from June to September; considering its availability, replicates per treatment were carried out. At the end of incubation
maintenance in laboratory conditions and the relative easiness to (72 h) samples were preserved by adding a few drops of 40%
induce fertilization (Rakaj et al., 2019), this species could be pro- buffered formalin and morphologically evaluated under an inverted
posed as an additional model for ecotoxicological studies covering microscope.
the lack of mature sea urchins during the summer period. With this
aim, the present study was intended to develop a standardized and 2.3. Endpoints
reproducible methodology for embryo toxicity bioassays with
H. polii. A set of experiments was set up to measure responsiveness Groups of 100 embryos were analyzed by optical microscopy
of embryos to temperature and salinity, determining the optimal (Zeiss Axio Vert A1) and photographed using a digital camera (Zeiss
conditions for embryogenesis and larval development. Reproduc- Axiocam 105 color) with 25 zoom. Mid-auricularia stage was
ibility of the test, optimum embryo density and minimum sample selected as the developmental endpoint. The degree of toxicity was
size were also investigated. Finally, the embryos were exposed to calculated using the percentage of abnormal embryos and the
cadmium (Cd2þ) and copper (Cu2þ), chosen as model and well- Integrative Toxicity Index (ITI). The standard criteria, routinely
known reference toxic compounds, to assess the applicability and adopted in embryo bioassays, evaluate the percentage of normal
the sensitivity of embryos and larvae of H. polii in ecotoxicological and abnormal embryos without considering different typologies of
tests. malformations nor the phase in which they appear. Embryos are
The percentage of abnormal embryos was chosen as an classified as normal if they satisfy the following morphological
endpoint, and an Integrative Toxicity Index (ITI) was applied, criteria: (1) correct schedule in reaching the developmental
associating different scores to various developmental anomalies, endpoint, (2) left/right and dorso/ventral embryonic axis symme-
further increasing the discrimination capacity of the assay (Morroni try, (3) correct differentiation of axohydrocoel, somatocoele and
et al., 2016). digestive system (mouth, stomach and anus). ITI was slightly
modified from Morroni et al. (2016) and it is based on a more
detailed analysis of development delays. The toxicity was quanti-
2. Material and methods fied by counting the frequency of delayed and/or abnormal embryo
morphology and by quantitatively ranking the severity of effects
2.1. Sampling and acclimatization from 0 (none) to 5 (high). Anomalies were classified as delays in
development with or without further morphological alterations,
Adult sea cucumbers (H. polii) were collected by snorkeling and different stages weighted depending on the severity of
(1e5 m) at Santa Marinella in the central Tyrrhenian Sea, Italy anomalies observed. Size reductions higher than 30% (larval
(42 30 000 N, 11490 900 E) from July to September 2018. Organisms dwarfism) were observed at increasing stressful conditions, and
were transported to the Laboratory of Experimental Ecology and typically associated with alterations of body shape. Consequently,
Aquaculture (University of Rome Tor Vergata) inside 30 L tanks these developmental anomalies were considered as malformations.
equipped with aerators and ice (to maintain the temperature below Lower toxicity values were given to embryos with delay in devel-
28  C), and acclimatized in a recirculating aquaculture system (RAS) opment and absence of defects; higher scores were attributed to
at 24  C for 7 days. embryos showing contemporarily delayed and defected
 L. Morroni et al. / Chemosphere 240 (2020) 124819 3

morphotypes. Details about selected scores and stages of devel- dilutions in filtered seawater (FSW) were prepared to obtain the
opment are given in Table 1. The toxicity score is 0 when normal exposure concentrations of 0.01, 0.02, 0.04, 0.06, 0.1, 0.14, 0.18 mg/L
mid-auricularia (mAu) are observed after 72 h, and 1 for delayed Cu. Nominal concentrations were compared with measured con-
larvae that reach correctly early-auricularia stage (eAu). Toxicity centrations, analyzing metal solutions by inductively coupled
values increase for abnormal mid-auricularia larvae, depending on plasma optical emission spectrometry (ICP-OE, Agilent Technolo-
the defects in size reduction (score 2) or malformations (score 3); gies 7900, Santa Clara, CA, USA).
higher toxicity values are attributed to delayed and malformed
early-auricularia larvae (score 4) or when observing at Gastrula/ 2.6. Data analysis
Blastula/Morula stage (G/Bl/M) (score 5).
The integrative toxicity index (ITI) is calculated as follows: The control results obtained in the experiments were used to
X assess the quality of the biological material. In particular minimum
ITI ¼ ¼ 10ðSi Fi Þ=100 acceptable percentages of abnormal embryos and ITI values were
ni established in order to define test acceptability. Minimum sample
size (n) was also estimated on the basis of tables provided by
where Si is the score associated at each abnormality and Fi is the Burnstein (1971), calculating the proportion of abnormal embryos
frequency observed for that abnormality (i ¼ 10). in the whole population (p) at a given confidence level, assuming
sampling without replacement. The obtained sample is a non-
2.4. Assessment of temperature, salinity and embryo density effects negligible fraction of the total population N (i.e. n >5% N), then n
is over-estimated, and thus the obtained value was corrected for a
H. polii embryos were exposed to FSW with different tempera- finite population.
tures and salinities, in order to investigate the optimal conditions The percentages of abnormal embryos were considered in order
for embryogenesis and larval development. In particular the test to evaluate the toxic effects estimated as EC50 values. The EC values
was performed incubating fertilized eggs at temperature of 22, 24, with 95% confidence limits were calculated by the Trimmed
26, 28, 30  C until 72 h and using FSW with salinity of 30, 33, 36, 39, Spearman-Karber statistical method. Responses in each experi-
42‰. Different eggs suspensions were also prepared obtaining a mental condition were corrected for effects in control tests by
final density of 20, 30, 40, 60, 100, 150, 200 embryos/mL. applying Abbott’s formula (Hamilton et al., 1978). Variations in
embryo development related to temperature, salinity, embryo
2.5. Reference toxicants density and metal exposure were analyzed by one-way ANOVA,
performed considering each parameter as a factor. Homogeneity of
Copper nitrate and Cadmium chloride were used as reference variances was tested by Cochran’s C-tests; Stu-
toxicants. The exposure levels of Cd2þ and Cu2þ were selected after denteNewmaneKeuls (SNK) test was performed to check for a
preliminary experiments (data not shown) based on literature data posteriori comparisons of mean values after significant effects in
on embryo bioassays with the echinoid Paracentrotus lividus ANOVA (Underwood, 1997).
(Morroni et al., 2018) and the bivalve Mytilus galloprovincialis
(Beiras and Albentosa, 2004). Cadmium chloride stock solution was 3. Results
prepared by dissolving reagent grade cadmium nitrate (Sigma
Aldrich srl, Milan, Italy) in bidistilled water (BDW) to obtain a Cd2þ 3.1. Optimal temperature, salinity and embryo density
concentration of 1000 mg/L. Test solutions were obtained by
diluting the stock solution in FSW, obtaining the following The effects of temperature, salinity and embryo density are re-
increasing concentrations: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg/L. A ported in Fig. 1. Temperature significantly affects embryo devel-
10 mg/L Cu2þ solution was prepared in bidistilled water by stock opment (p < 0.01), causing a developmental delay at 22  C and
solution of 1000 g/L (Sigma Aldrich srl, Milan, Italy). Further 24  C (Fig. 1A and B). At these temperatures 100% of larvae were

Table 1
Classification of developmental anomalies of Holothuria polii according to the new Integrative Toxicity Index (ITI) based on developmental delays (delayed embryos) and
malformations (malformed embryos). mAu: mid-auricularia; eAu: eraly-auricularia; G: Gastrula; Bl: Blastula; M: Morula.
4 L. Morroni et al. / Chemosphere 240 (2020) 124819

Fig. 1. The effects of temperature (A, B), salinity (C, D) and embryo density (E, F) on Holothuria polii embryo development. Data are expressed as mean percentage of abnormal
embryos (A, C, E) and ITI (B, D, F) ± standard error. Asterisks indicate significant differences between each treatment and optimum value (temperature of 26  C; salinity of 36‰;
embryo density of 60 embryo/mL). *: p < 0.05, **: p < 0.01.

abnormal (Fig. 1A), mostly delayed at early-auricularia (83% at 22  C the absence of negative effects and the adequate number of em-
and 90% at 24  C), with ITI values from 1.8 to 2 (Fig. 1B). By contrast bryos to be counted at the end of the test.
embryos incubated at 26  C, 28  C and 30  C reached mid-
auricularia stage with a percentage of abnormal embryos from 3.2. Test acceptability and sample size
10% to 22% and ITI from 0.6 to 0.7.26  C was chosen as the optimal
incubation temperature; no significant differences were observed Control data showed a normal distribution expressed both as a
at 28  C and 30  C. percentage of abnormal embryos (Shapiro-Wilk W: 0.95, p ¼ 0.005)
Salinity also significantly affected embryo development and as ITI (Shapiro-Wilk W: 0.92, p ¼ 0.0002). The 10th percentile
(p < 0.01). 34‰e36‰ was identified as the optimal range when of the distribution corresponds to 42.03% (abnormal embryos) and
using percentages of abnormal embryos, with values ranging from 0.89 (ITI); thus, thresholds of acceptability were fixed at 40% and
0% to 9% in the absence of significant differences (Fig. 1C). 0.8, respectively. Concerning minimum sample size, setting an er-
Considering ITI values as the endpoint, the optimum range of ror of 0.05 (confidence level of 95%), values of n for expected p
salinity was 33‰e37‰, with ITI values between 0.8 and 1.5 and values of 1, 0.90, 0.75, 0.60, 0,5, 0.40, 0.25, 0.10 and 0 were,
without significant differences between them (Fig. 1D). Due to the respectively, 58, 200, 334, 400, 400, 400, 334, 200 and 58. After
lowest values, both in terms of abnormal embryos and of ITI score, applying the correction for finite populations, a minimum sample
36‰ salinity was chosen as optimal for this species. size of 240 embryos allows a 95% confidence in the estimate with
Embryo densities lower than 100 embryos/mL showed no effect an error of 5% and was considered as ideal for the test. Each
on development in terms of both abnormal embryos and ITI scores treatment was replicated in 6 wells.
(Fig. 1E). However, adverse and significant (p < 0.01) effects on
embryogenesis became evident at densities 100 embryos/mL. In
3.3. Reference toxicants
fact, highest embryo densities caused 100% of abnormal larvae and
ITI values from 4.7 to 4.9 (Fig. 1EeF). Based on these results, the
Recovery rate of Cd2þ and Cu2þ, measured in each test solution,
density of 60 embryo/mL was proved as optimal one, considering
ranged from 93% to 130% compared to their nominal concentrations
 L. Morroni et al. / Chemosphere 240 (2020) 124819 5

(Table 2), which were thus used for the following calculations. pollutants and the correct assessment of toxicity. Our values are
The incubation of H. polii embryos with the two metal solutions higher than control percentages of mussel and sea urchin, where
induced significant abnormalities (p < 0.01) in a dose-dependent- acceptability criteria are generally between 10 and 30% of abnormal
manner (Fig. 2). Considering the percentage of abnormal embryos embryos (His and Seaman, 2000; Arizzi Novelli et al., 2002), but
the EC50 of Cd2þ (±95% confidence limits) was 2.26 mg/L lower than those obtained in limpet (>60%; Pe rez et al., 2016) and
(1.69e3.31) and the EC50 of Cu2þwas 0.026 mg/L (0.023e0.029) Ascidian (50.2%; Bellas et al., 2003) embryo bioassays. These values
(Table 3). Using ITI as an alternative endpoint the EC50 were lower may depend on different factors as polyspermy and oocyte imma-
than those obtained with the percentages of abnormal embryos. In turity, stating that each batch of eggs contains, inevitably, unripe
fact the EC50 of Cd2þ (±95% confidence limits) was 2.02 mg/L eggs mixed with ripe ones, but also to the reproductive character-
(1.71e3.42) and the EC50 of Cu2þwas 0.012 mg/L (0.009e0.019). rez et al., 2016).
istics and natural variability of the test specie (Pe
This difference between the two endpoints was evident especially We propose to carry out 6 replicates for treatment, by counting
at the highest metal concentrations, where the percentages of 100 embryos per replicate, so exceeding the minimum sample size
abnormal embryos were 100% both Cd-exposed embryos (Fig. 2A) of 240 embryos; the time of incubation is fixed at 72 h before
and Cu-exposed embryos (Fig. 2D). In particular, Cd showed ITI observing the larval development.
values of 2.9, 3.4, 3.7, 3.9 at 3, 3.5, 4, 4.5, 5 mg/L, respectively In toxicity bioassays temperature and salinity are two funda-
(Fig. 2B) and Cu showed ITI values of 3.5, 3.6, 4.3, 4.5, 4.6 at 0.04, mental variables. In this study, temperatures lower than 26  C
0.06, 0.1, 0.14, 0.18 mg/L, respectively (Fig. 2D). This increase of ITI prevented the development to mid-auricularia stage in 72 h, while
values at high metal concentrations reflects a shift from malformed higher values increase the risk of bacterial contamination and
early auricularia larvae to gastrula, blastula and morula embryos. bacteriosis as observed for some outliers at 28  C and 30  C; to
This phenomenon was observed both for Cd2þ and Cu2þ. In addi- better prevent this event, the temperature of 26  C and the use of
tion, embryos exposed to 0.02 mg/L of Cu2þ showed 28% of mid- 2 mg/L of Gentamicin are recommended for the assay.
auricularia stage with a strong size reduction (data not shown). Embryo density is another important variable that needs to be
This morphotype, typically observed in Cu-exposed embryos, in- assessed in order to avoid false positives. An egg density of 60 eggs/
crease the developmental delay and malformations in dose mL was identified as the more appropriate, by combining the
dependent-manner and was partially responsible to the highest absence of negative effects with the quantity required for reaching
sensitivity of ITI. the minimum sample size. Egg size in H. polii is approximately
200 mm (Rakaj et al., 2019), more than two times higher than in sea
urchin P. lividus (Fernandez and Beiras, 2001) and up to five times
4. Discussion
higher than in Crassostrea gigas (Powell et al., 2002): such elevated
diameter has the potential to disturb respiration and available
Crustaceans, echinoderms and mollusks are the most widely
space during embryogenesis, and particular attention has thus been
used marine invertebrates in embryo toxicity bioassay due to their
given to embryo density from the beginning of the test.
widespread distribution, suitability to perform the test, and sensi-
The applicability of the bioassay toward toxic substances was
tivity to environmental stressors (Oliva et al., 2018). The sea cu-
assessed by measuring the sensitivity of embryos to Cd2þ and Cu2þ.
cumber H. polii is a very common, easily maintainable species and,
Using the percentage of abnormal embryos as the endpoint, an EC50
by artificial induction, it spawns large amounts of gametes. Our
of 2.26 mg/L for cadmium and 0.026 mg/L for copper were esti-
study successfully addresses, for the first time, the use of this
mated. These values were comparable with those obtained in em-
species in ecotoxicological bioassays developing a reproducible
bryo toxicity bioassays with P. lividus and M. galloprovincialis, but up
methodology: the endpoint can be measured through a conven-
to ten times higher than those of C. gigas and of the polychaetes
tional approach based on the count of total abnormal embryos, or
Ficopomatus enigmaticus and Hydroides elegans (Table 3). By
through an integrated ITT discriminating both the severity and time
contrast, sea cucumbers were one order of magnitude more sen-
of occurrence of various alterations. Our results estimated an
sitive to Cu2þ than polychaetes, two times more than P. lividus and
acceptability limit for controls up to 40% as abnormal embryos, and
Ciona intestinalis and two times less than bivalve species reported
up to 0.8 for the ITI score: higher levels of developmental anomalies
in Table 3.
in control experiments would reflect a poor quality of gametes,
The sensitivity of H. polii is further increased when alterations
possibly interfering with responsiveness of embryos toward
were scored as ITI, according to the severity of the effects, thus
discriminating between simply delayed larvae and abnormal
Table 2 delayed larvae. Lower EC50 were detected for both Cd2þ and Cu2þ
Nominal and measured concentration of each metal used for toxicity test. n.m. ¼ not compared to those obtained considering the percentage of
measured.
abnormal embryos. In dose-response experiments, ITI values still
Metal Nominal concentration (mg/L) Measured concentration (mg/L) exhibited a gradual increase at 3e5 mg/L Cd2þ and 0.04e0.18 mg/L
Cd 1.0 0.93 ± 0.01 Cu2þ, when percentages of abnormal embryos were already at
1.5 1.60 ± 0.02 100%. These data confirm that differences between the two ap-
2.0 2.09 ± 0.03 proaches are particularly evident in experimental conditions which
2.5 2.59 ± 0.03 induce 100% of abnormal embryos. For instance, at 22  C and 24  C,
3.0 3.03 ± 0.04
3.5 3.51 ± 0.05
100% of the embryos abnormally developed while ITI values
4.0 4.01 ± 0.06 differed, being 1.7 and 2.0, respectively (Fig. 1B); densities of 150
4.5 4.55 ± 0.06 and 200 embyos/mL, induced severe malformations in 100% of the
5.0 5.06 ± 0.1 embryos, showing ITI of 4.7 and 4.9, respectively. These values are
Cu 0.01 0.012 ± 0.001
higher than those observed at 22  C and 24  C, while these differ-
0.02 0.024 ± 0.003
0.04 0.053 ± 0.007 ences could not be evaluated in terms of total abnormal embryos.
0.06 0.065 ± 0.009 This reflects the capability of the index to better discriminate the
0.1 0.100 ± 0.015 samples according to the severity and the developmental stage at
0.14 0.152 ± 0.002 which an effect is observed, considering delays in development as
0.18 0.183 ± 0.027
being less severe than the delays combined with embryo
6 L. Morroni et al. / Chemosphere 240 (2020) 124819

Fig. 2. The effects of Cd 2þ (A, B) and Cu 2þ (C, D) on Holothuria polii embryo development. Data are expressed as mean percentage of abnormal embryos (A, C) and ITI (B,
D) ± standard error and normalized respect to the control by using Abbot’s correction.

Table 3 organisms (e.g. oysters) conditioned for being constantly sexually

EC50 values of Holothuria polii compared with those from literature in Ciona intes- mature.
tinalis, Crassostrea gigas, Ficopomatus enigmaticus, Hydroides elegans, Mytilus gallo-
The sea cucumber H. polii has widespread distribution in Eu-
provincialis, Paracentrotus lividus for Cd2þ and Cu2þ in embryo toxicity assays.
ropean seas and, interestingly, its spawning period is comple-
Test specie Cd2þ EC50 (mg/L) Cu2þ EC50 (mg/L) mentary to that of the sea urchin P. lividus. In fact, H. polii, naturally
Holothuria polii 2.26 (1.69e3.31) 0.026 (0.017e0.029) spawns in summer, with a distinct annual reproductive cycle
Ciona intestinalis e 0.054 a (Aydin and Erkan, 2015), while P. lividus in winter and spring (with
0.212 b 0.013 b
Crassostrea gigas
water temperature at 13,5  Ce18  C) when the photoperiod is short
Ficopomatus enigmaticus 0.79 c 0.2 c
Hydroides elegans 0.18 d 0.12 d
(Byrne, 1990; Lozano et al., 1995; Arizzi Novelli et al., 2002; Shpigel
Mytilus galloprovincialis 1.925 e 0.01 e et al., 2004). Given these characteristics and the comparable
Paracentrotus lividus 1.72 f 0.045 f sensitivity observed toward model toxicants, the H. polii could
a
Bellas et al. (2003). represent a complementary species to P. lividus, allowing to
b
Mai et al. (2012). perform embryo bioassays also during the summer season, when
c
Oliva et al. (2018). gametes of sea urchin are not naturally available. In these pro-
d
Gopalakrishnan et al. (2008).
e spective H. polii embryo bioassay is a promising test for being
Beiras and Albentosa (2004).
f
Morroni et al. (2018). considered among the suitable species for embryo toxicity tests
included in ecotoxicological batteries, guaranteeing an all-year-
round availability of sexually mature species. Additional studies
morphological malformations. Future studies should address the on molecular/cellular responses to pollutants in H. polii would
sensitivity of H. polii toward additional (i.e. organic) pollutants, provide useful insights on mechanisms of action of pollutants,
chemical mixtures and environmental matrices to further expand further reinforcing the suitability of this organism as bioindicator
the potential applications of this bioassay. species in a more integrated WOE approach for ecological risk
The overall results obtained with H. polii embryo bioassay are assessment.
encouraging and might also have important applied consequences.
Standardized toxicity tests are an essential component to evaluate
5. Conclusions
the quality of marine waters and sediments. The weight of evidence
approach (WOE), recently developed for assessing sediment quality
The use of H. polii in embryo bioassays is of ecological relevance,
in harbor areas is based on the combination of chemical and eco-
as the species is widely distributed, easy to handle and to apply in a
toxicological data, which include a battery of bioassays with
bioassay without sophisticated equipment. The present study
different species and biological endpoints (Piva et al., 2011;
provides useful elements for the standardization of the test pro-
Bebianno et al., 2015, Rodríguez-Romero et al., 2016). Among
cedure, describing the optimal conditions of temperature, salinity
these, also embryotoxicity is considered, with sea urchin embryos
and embryo densities for application of the embryo bioassay
mostly applied to check this endpoint. However, due to the sea-
obtaining reliable results. An Integrative Toxicity Index can also be
sonality of reproductive cycle, sea urchin are not always available
applied to better discriminate embryo malformations compared to
for the test, thus forcing the expensive buy of commercial
the count of total abnormal embryos. The sea cucumber embryo
 L. Morroni et al. / Chemosphere 240 (2020) 124819 7

test is a promising tool to complement bioassay test batteries of embryo bioassay. Arch. Environ. Contam. Toxicol. 57, 540e551.
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