Marine Environmental Research 159 (2020) 104967

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Marine Environmental Research

journal homepage: http://www.elsevier.com/locate/marenvrev

Sea urchin chronicles. The effect of oxygen super-saturation and marine

polluted sediments from Bagnoli-Coroglio Bay on different life stages of the
sea urchin Paracentrotus lividus
Antonia Chiarore a, *, Luigi Musco b, Iacopo Bertocci b, c, Alessandra Gallo d,
Antonio Cannavacciuolo a, Mirko Mutalipassi e, Davide Caramiello f, Folco Giomi g, Marco Fusi h,
Roberto Danovaro i, Marco Munari a, **
a
Department of Integrative Marine Ecology, Ischia Marine Centre, Stazione Zoologica Anton Dohrn, Punta San Pietro, 80077, Ischia, (Naples), Italy
b
Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
c
Department of Biology, University of Pisa, CoNISMa, Via Derna 1, 56126, Pisa, Italy
d
Department of Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
e
Department of Marine Biotechnology, Ischia Marine Centre, Stazione Zoologica Anton Dohrn, Punta San Pietro, 80077, Ischia, (Naples), Italy
f
Unit Marine Resources for Research, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
g
Padova, via Maniciati 6, 35129, Italy
h
King Abdullah University of Science and Technology (KAUST), Red Sea Research Center (RSRC), Thuwal 23955-6900, Saudi Arabia
i
Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: In marinas and harbours, the accumulation of pollutants in sediments, combined with poor exchange of water
Development with the open sea, poses a major environmental threat. The presence of photosynthetic organisms and the related
Marine pollution oxygen production, however, may alleviate the negative effects of environmental contamination on heterotro­
Sediment
phic organisms, enhancing their physiological defences. Furthermore, possible transgenerational buffer effects
Anthropogenic impact
Oxygenation
may increase the ability of natural populations to face environmental stress. Here we tested the occurrence of
Water turbulence transgenerational effects on larvae of the sea urchin Paracentrotus lividus, whose parents were exposed, during the
gametogenesis, to contaminated sediments subject to two temporal patterns of water re-suspension events and
normal- (90%) vs. super-saturated (200%) levels of O2. The study site was Bagnoli-Coroglio (Gulf of Naples,
southern Tyrrhenian Sea), a historically polluted brownfield and Site of National Interest for which environ­
mental restoration options are currently under exploration. Larvae from different adult populations were
significantly, although not linearly, affected by the interaction of all factors to which parents were exposed, at
both 24h and 48h post fertilization. Specifically, the exposure of larvae to elutriates from contaminated sedi­
ments determined a developmental delay, a reduction in size and an increased percentage of abnormalities in all
larval populations independently of their parental exposure. On the contrary, larvae from parents exposed to
contaminated sediments, when reared in clean filtered sea water, succeeded in developing until the echino­
pluteus stage after 48h, with size and abundance comparable to those of larvae from control parents. Pre-
exposure of parents to contaminated sediments did not successfully buffer the negative effects of elutriates on
their offspring, and no positive effects of ‘super-saturated’ levels of O2 in response to contaminants were
observed, suggesting that the Bagnoli-Coroglio area is currently not suitable for the re-stocking or re-introduction
of this species.

* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (A. Chiarore), [email protected] (M. Munari).

https://doi.org/10.1016/j.marenvres.2020.104967
Received 11 December 2019; Received in revised form 19 March 2020; Accepted 21 March 2020
Available online 27 March 2020
0141-1136/© 2020 Elsevier Ltd. All rights reserved.
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

1. Introduction Zhang et al., 2012). While the maintenance of homeostasis allows

acclimatization, it may result in a reallocation of resources away from
Human activities have heavily modified marine environments, growth and reproduction. Furthermore, it is widely recognized that
especially in coastal areas where increasing urbanization and associated early life stages of many organisms are more sensitive than adults to
activities are responsible for the release of a range of contaminants that environmental stress (Byrne, 2012; Moran, 1994), representing a
ultimately accumulate in marine sediments and contribute to habitat possible bottle-neck for natural populations. However, negative effects
alteration (Agardy et al., 2005; Arizzi Novelli et al., 2006; Bertocci et al., on early life stages imposed by environmental perturbations can be
2019; Vitousek et al., 1997). Disused industrial plants are often sources mitigated by anticipatory parental effects (Agrawal et al., 1999;
of a wide range of chemicals that crucially threaten both environmental Marshall and Uller, 2007; Thor and Dupont, 2015). This type of heri­
and human health. Furthermore, inshore areas are particularly vulner­ table plasticity, being a source of phenotypic individual variation within
able to ongoing climate change, including the current and predicted populations, has important ecological and evolutionary consequences
increase in the intensity and frequency of severe storms and associated (Uller, 2008), and may allow populations to adapt to new environmental
water turbulence, which can promote the resuspension of polluted conditions (Miller et al., 2012; Parker et al., 2012; Reed et al., 2011).
sediments and availability of toxic chemicals in the water column and In this study, we focused on the purple sea urchin Paracentrotus liv­
nearby habitats (Neumann et al., 2015; Spalding et al., 2014). The idus (Lamarck, 1816) since it is widely used as a model species in many
Bagnoli-Coroglio brownfield (Southern Tyrrhenian Sea, Italy) is an ecotoxicological studies, being considered a good bioindicator of envi­
infamous example of a heavily polluted marine area. All industrial ac­ ronmental change (Bellas, 2008; Bonaventura et al., 2011; Bo�snjak et al.,
tivities at the site ceased in the mid 1990s, after almost a century of 2011; Dinnel et al., 1988; Kobayashi and Okamura, 2004; Matranga
concrete, steel, chemical and asbestos production resulting in heavy et al., 2000; Morale et al., 1998; Pinsino et al., 2010; Sconzo et al., 1995;
contamination of both the soil and marine sediments. In particular, high Zito et al., 2005). Furthermore, the species has a key role in shaping
concentrations of heavy metals, polycyclic aromatic hydrocarbons benthic marine communities in the Mediterranean Sea and Atlantic
(PAHs) and polychlorinated biphenyls (PCBs) occur in this area (Alba­ Ocean (Guidetti et al., 2003; Guidetti, 2004; Sala and Zabala, 1996),
nese et al., 2010; Arienzo et al., 2017; De Vivo and Lima, 2008; Trifuoggi where it also represents an important commercial resource (Bertocci
et al., 2017). In 2001 Bagnoli-Coroglio bay was designated as a Site of et al., 2014). Paracentrotus lividus is a gonochoric species lacking sexual
National Interest (SNI) requiring environmental rehabilitation. dimorphism (Gianguzza et al., 2009). The reproductive cycle of P. lividus
Several studies reported that the exposure to polluted sediments can is annual, with three main phases: the growing phase (late autumn and
have severe consequences on marine organisms, including changes in winter), when gonads accumulate reserve material; the maturation
biodiversity and ecosystem functioning (Bertocci et al., 2019; phase (spring and early summer), in which gametogenesis and spawning
Martínez-Llado � et al., 2007; Ryu et al., 2011). These effects may be take place; and the spent/regenerating phase, in which relict gametes
exacerbated by those of global climate change, such as warming and are reabsorbed by phagocytes (Spirlet et al., 1998). Here, we first
ocean acidification, which can further contribute to dramatically alter exposed adult sea urchins, during their gametogenesis phase, to exper­
the development of marine organisms (Bressan et al., 2014; Matozzo imental combinations of two water turbulence patterns (‘aggregated’ vs.
et al., 2012, 2013; Munari et al., 2016, 2018; 2019; Range et al., 2014). ‘spaced’ events of re-suspension of contaminated sediments) and two
Additional concerns arise from the current and predicted modification of levels of dissolved oxygen (‘normal’ vs. ‘super-saturated’). Oxygen
patterns of intensity and temporal occurrence of extreme weather con­ super-saturation was suited to simulate oxygen production by photo­
ditions (Aumann et al., 2008; Burge et al., 2014; Easterling et al., 2000; synthetic organisms (Viaroli and Christian, 2003). Larval populations
Trapp et al., 2007; Wolff et al., 2016), which can result in more severe were obtained from adults exposed to each of these combinations of
and frequent exposure of marine organisms to the re-suspension of factors and reared either in clean filtered sea water (FSW) or in the
chemical substances accumulated in sediments. Thus, it is pivotal to presence of elutriates from contaminated sediments. The aims were (i)
assess the combined effects of sediment pollution and changing patterns to assess the simultaneous and potential interactive effects of patterns of
of water turbulence on marine organisms, to better understand and turbulence associated with the resuspension of contaminated sediments
predict wide anthropogenic impacts on marine systems and to effec­ and oxygen availability on larval development after the exposure of
tively direct environmental restoration plans. adults during gametogenesis; (ii) to quantify the parental effect on
The effects of environmental chemical contamination can be poten­ offspring performance after the pre-exposure of adults to stressful
tially buffered by oxygen release in coastal habitats, due to the activity conditions.
of photosynthetic organisms that may enhance animal aerobic perfor­
mance under stressful conditions, as reported for several marine taxa in 2. Materials & methods
response to ocean warming (Giomi et al., 2019). It must also be
considered that, under natural conditions, marine organisms are typi­ 2.1. Study site
cally adapted to respond to environmental changes within a tolerance
range set by internal physiological regulation (Po €rtner et al., 2004; The study site was the coastal area of Bagnoli-Coroglio, an

Fig. 1. Map of the Bagnoli-Coroglio study area.

2
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

Fig. 2. Adult experimental design. See M&M section for details. POP 1 and POP 2: control adults without sediment, 90% oxygen saturation, aggregated and spaced
pattern of turbulence respectively; POP 3 and POP 4: adults with sediment, 90% oxygen saturation, aggregated and spaced pattern of turbulence respectively; POP 5
and POP 6: adults with sediment, 200% oxygen saturation, aggregated and spaced pattern of turbulence respectively.

industrially contaminated region located in the south-eastern part of the Paracentrotus lividus is not protected or endangered and all experimental
Pozzuoli Bay (Gulf of Naples – southern Tyrrhenian Sea, Fig. 1), char­ procedures on animals were in compliance with the guidelines of the
acterized by sandy and clay substrates protected by artificial rocky reefs European Union (Directive 609/86).
(Bertocci et al., 2019; Cocco et al., 1988; De Pippo et al., 1988). In­
dustrial activities were developed in this area from 1905 to 1990, pre­ 2.4. Adult sea urchin rearing mesocosms
dominantly in the form of steel production, asbestos manufacturing,
concrete and chemicals production. In 1994, the Italian Government ‘Ad hoc’ mesocosms comprising twelve independent, closed recir­
implemented a remediation plan for this brownfield, which was classi­ culating tanks (~50 L each), filled with natural sea water, were set up in
fied as a SNI in 2001. Previous studies on the chemical composition of the facility for the maintenance of marine organisms of Stazione Zoo­
coastal marine sediments in the Bagnoli area highlighted heavy logica Anton Dohrn in Naples. Each tank had an internal filter divided
contamination by metals, PAHs and PCBs (Albanese et al., 2010; Arienzo into three sequential compartments. The first compartment was filled
et al., 2017; Trifuoggi et al., 2017). High sediment concentrations of with filtering wool and foam sponges, ensuring mechanical filtration by
heavy metals such as Cu, Fe, Hg, Mn, Ni, Pb and Zn, were reported by collecting particulate matter and debris. The second compartment
Romano et al. (2018), although they could also be related to local hy­ housed SUBSTRATpro (Ehiem GmbH & Co. KG, Deizisau, Germany), a
drothermal activity and other anthropogenic sources (De Vivo and Lima, pearl-shaped sintered glass specifically designed for the colonization of
2008). aerobic chemoautotrophic bacteria belonging to genus Nitrosomonas,
Environmental data including temperature, conductivity/salinity, Nitrosococcus and Nitrospira (Schreier et al., 2010) as well as several
dissolved oxygen and fluorescence profiles were collected during five ammonia-oxidizing archaea (Hatzenpichler, 2012), to ensure biological
cruises from February to October 2019 in seven stations within the filtration. The third compartment contained a Syncra Silent recirculat­
Pozzuoli Bay, and results are shown in Margiotta et al. (2020). ing pump (pump flow rate 700 L/h, pump wattage 8 Watt; SICCE S.r.l,
Analyses of historical data on the benthic diversity in the Bagnoli- Pozzoleone, Italy) to ensure water recirculation in the internal filter. The
Coroglio site reported the occurrence of P. lividus in this area in the sea water level was checked on a daily basis and manually adjusted to
past (Gaglioti et al., 2020), although further studies are needed to assess balance evaporation. Chemical and physical variables of both sea water
the current state of the local P. lividus populations. and sediments are reported by Ruocco et al. (2020).

2.2. Field oxygen data collection 2.5. Adult sea urchin experimental design

To evaluate natural fluctuations in water oxygenation, dissolved The bottom of eight tanks, randomly chosen out of the total twelve,
oxygen was monitored by mooring an oxygen probe equipped with was covered with approximately 5 centimeters of sediment collected
calibrated dissolved oxygen concentration (optical) sensors (MiniDOT from a sampling station located within the Bagnoli-Coroglio area
sensors, PME, Vista, Ca, USA). The probe, with a logging frequency of 5 (40� 480 25.9’’N, 014� 090 33.2’’E; water depth: 3.8 m). This sediment
min, was deployed within a seagrass (Posidonia oceanica) meadow in an came from the same sampling core and was uniformly mixed before
uncontaminated area in the Gulf of Naples (40� 430 58"N 13� 570 59"E; being used. Data regarding the composition and quantification of the
temperature 15.00 � 0.50 � C, salinity 38.00 � 0.03 psμ), in order to contaminants within the sediment are described in Morroni et al.
reflect the expected conditions at the study site once the local ecosystem (2020).
was possibly restored. The remaining four tanks were maintained with sea water without
sediment. Two of these tanks without sediment, and four of the tanks
2.3. Animal collection with sediment were randomly assigned to exposure to the ‘spaced’ tur­
bulence pattern (two events, 48h duration, separated by two weeks
Total 200 adult P. lividus individuals were collected in March 2019 at within one month of exposure). The remaining tanks were assigned to
San Pancrazio (40� 420 15.0"N 013� 570 22.3"E), Ischia Island (Gulf of the exposure to the ‘aggregated’ turbulence pattern (two events, 48h
Naples), at a maximum depth of 5 m with the authorization of the Ma­ duration, separated by three days within one month of exposure). This
rine Protected Area ‘Regno di Nettuno’. Animals were kept in a cool box allowed the manipulation of the temporal variance of turbulence events,
after collection and in the laboratory prior to experimental procedures. while maintaining the same the total frequency and intensity of events

3
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

established over the course of the experiment (Benedetti-Cecchi, 2003;

Benedetti-Cecchi et al., 2006; Bertocci et al., 2005). The application of
water turbulence events in tanks without sediment allowed us to test for
potential effects of hydrodynamics, independently of the presence of
sediments and associated chemical and physical effects. A detailed
illustration of the experimental setting and design was provided by
Ruocco et al. (2020). To produce water turbulence, with the associated
re-suspension of sediment, an electric pump (flow rate 4500L/h, power:
10 W) was placed in each experimental tank. The duration and fre­
quency of experimental re-suspension events were established based on
the records of natural storm events in the study area. Data on wind
Fig. 3. P. lividus developmental stages. From left: arrested embryos, gastrulae,
speed, wave height and period were collected daily from an oceano­
prism, early pluteus and echinopluteus.
graphic buoy within a monitoring program of Stazione Zoologica Anton
Dohrn, as illustrated in detail by Ruocco et al. (2020). Water and sus­
pended particles were collected on the second day following each event
of turbulence and analyzed for heavy metals and PAHs content. Chem­
ical analyses were performed by Bioscience Research Center SRL
(Orbetello, Italy). Details of the analyses are reported in Ruocco et al.
(2020).
Oxygen was provided by bubbling it into four tanks randomly chosen
out of the eight tanks with sediment using an ACQ140 automatic control
system (Aquatronica, Reggio Emilia, Italy) connected to submersed
dissolved oxygen probes (Aquatronica, model ACQ310N–O2) via digital
oxygen interfaces (Aquatronica, model ACQ210N–O2). In addition, in
Fig. 4. Examples of anomalous 4-arm pluteus at 48 hpf. A: crossed spicules, B
order to ensure a constant dissolved oxygen concentration, the auto­ and C: asymmetrical or completely degenerated arms.
matic control system digitally controlled eight solenoid valves (Aqua­
tronica, model ACQ421) connected to a 40-liter oxygen tank.
Specifically, a value of 90% of oxygen saturation served as reference
while a value of 200% was chosen as representative of the daily super-
saturation (Fig. 2). Ten adult sea urchins per tank (seven females and
three males) were then exposed to each experimental condition for a
month, during gonad development, under a 12h:12h light: dark photo­
period setting. Adults were daily checked for mortality and fed with ‘sea
lettuce’, Ulva lactuca Linnaeus, ad libitum.

2.6. Larval experimental design

At the end of the exposure period, adults from each tank were
weighed and the test diameter of each individual was measured. A
minimum of three females and three males from each experimental
condition (n ¼ 6) were separated to constitute pools of gametes in order
to establish six unique laboratory populations of embryos. Sperms were
collected with a micropipette and kept dry on ice in a plastic tube until
use. Eggs were collected in 500 mL beakers filled with 0.22 μm FSW.
Prior to use, egg and sperm quality and quantity were evaluated. Pools
of eggs and sperms were established using the same number of gametes Fig. 5. Somatic (SS) and oral (OS) spicules at 48 hpf.
from each individual, in order to have the same contribution from every
parent during fertilization. A constant sperm:egg ratio (1250:1) was
the potential to highlight: (a) specific effects of parental exposure on
used (Moschino and Marin, 2002), and fertilization was checked after
embryonic and larval development through comparing with larval
30 min by observing the elevation of the jelly coat. Embryo populations
populations reared in FSW; (b) selective pressure acting during game­
were then maintained in 25 ml vials, three replicates for each experi­
togenesis and resulting into a better response to stress of offspring from
mental condition, for 48h of exposure at 22 � C. To test parental effects in
adults reared in harsh conditions (in this case adults with the highest
response to the experimental factors, offspring from each parental
phenotypic plasticity are most likely to spawn and pass down their ge­
population were exposed to the conditions that parents experienced
notypes to the next generation).
during gametogenesis. Embryos coming from parents exposed to sedi­
ments were reared in elutriates. Elutriates were prepared as reported by
Gallo et al. (2020) from the same sediment core used in the tank where 2.7. Larval developmental traits
adults were kept. Specifically, POP 1 and POP 2 embryos were exposed
to FSW at O2 90%-saturation level (FSW_90% O2), POP 3 and POP 4 to The effects of the three combined factors on larval growth were
elutriate at O2 90%-saturation level (ELU_90% O2), and POP 5 and POP examined at two developmental stages (24h and 48h post-fertilization).
6 to elutriate at O2 200%-saturation level (ELU_200% O2). Furthermore, Embryos at 24h and 48h post-fertilization (hereafter as hpf) were
POP 1 and POP 2 embryos were transplanted into FSW at ‘super-satu­ observed with an optical macroscope (Leica Z16 APO), equipped with a
rated’ O2 level (FSW_200% O2), into elutriates at ‘normal-saturated’ O2 Leica DFC 300FX camera connected to a computer with the Leica LAS
level (ELU_90% O2) and elutriates at ‘‘super-saturated’’ O2 level program (Leica Application Suite, Version 4.5). A minimum of 100
(ELU_200% O2), while POP 3, 4, 5 and 6 embryos were transplanted into embryos per replicate were photographed. Different developmental
FSW at ‘normal-saturated’ O2 level (FSW_90% O2). This approach has stages were distinguished to evaluate any delay after 24 and 48 h of

4
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

F(1,17) ¼ 429.680

F(1,17) ¼ 472.850

F(1,17) ¼ 112.050
F(5,17) ¼ 246.920

F(1,17) ¼ 50.987

F(1,17) ¼ 205.34
p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001
PC - rearing
PERMANOVA results. Pseudo–F values (indicated as F) and Monte Carlo p-values for all the parameters measured for larvae reared in both filtered sea water FSW and under parental conditions (PC).
Oral Spicules (OS) 48h

F(1,17) ¼ 137.52

F(1,17) ¼ 19,239
F(5,17) ¼ 33.995

F(1,17) ¼ 4.654

F(1,17) ¼ 5.433
p(MC) ¼ 0.001

p(MC) ¼ 0.049

p(MC) ¼ 0.035
F(1,17) ¼ 4.328
p(MC) ¼ 0.057
FSW - rearing

p(MC)<0.001
p(MC)<0.001

F(1,17) ¼ 2389.7

F(1,17) ¼ 1653.2

F(1,17) ¼ 364.79

F(1,17) ¼ 776.57
F(5,17) ¼ 1043.8

F(1,17) ¼ 0.421
p(MC) ¼ 0.529

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001
PC - rearing
Fig. 6. Diel seawater dissolved oxygen fluctuations (5 min logging frequency).

Somatic Spicules (SS) 48h

exposure, and their frequencies were calculated. Five main classes were
considered: 1) arrested, corresponding to undeveloped embryos; 2)

F(1,17) ¼ 34.023

F(1,17) ¼ 16.886

F(1,17) ¼ 11.190
F(5,17) ¼ 11.260

p(MC) ¼ 0.001

p(MC) ¼ 0.007
F(1,17) ¼ 1.865

F(1,17) ¼ 1.321
gastrulae, distinguished by the onset of invagination; 3) prism, charac­

p(MC) ¼ 0.196

p(MC) ¼ 0.272
FSW - rearing

p(MC)<0.001
p(MC)<0.001
terized by the early development of calcareous rods and the typical
cuboid shape of the embryos; 4) early plutei, distinguished by the
beginning development of the four appendixes; 5) echinoplutei, i.e., the
48 hpf stage characterized by the total development of oral and aboral
rods (Fig. 3).

F(1,17) ¼ 14.360

F(1,17) ¼ 726.17

F(1,17) ¼ 182.86

F(1,17) ¼ 19.840

F(1,17) ¼ 14.588
F(5,17) ¼ 190.4

p(MC) ¼ 0.749
For echinoplutei, the frequency of abnormal larvae (Fig. 4) was

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001
evaluated and the length of somatic (SS) and oral (OS) spicules (Fig. 5) PC - rearing
was assessed using image analysis software (IMAQ™ Vision, National
Instrument™, Version 6.0).

F(1,17) ¼ 31.554

F(1,17) ¼ 19.808
2.8. Statistical analyses
F(5,17) ¼ 11.417
Anomalies 48h

F(1,17) ¼ 2.655

F(1,17) ¼ 3.928

F(1,17) ¼ 0.170
p(MC) ¼ 0.116

p(MC) ¼ 0.063

p(MC) ¼ 0.749
FSW - rearing

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001

A non-parametric PERMutational multivariate ANalysis Of Variance,

PERMANOVA (Anderson, 2001a) applied on the Euclidean distance
matrix of raw data was chosen to test differences on larval growth
among experimental conditions. A one-way PERMANOVA model was
F(1,17) ¼ 88.150

F(1,17) ¼ 84.888

F(1,17) ¼ 17.435

F(1,17) ¼ 8.8705
used to test for differences among larval populations reared in FSW_90%
F(5,17) ¼ 41.511

p(MC) ¼ 0.004
F(1,17) ¼ 3.405
p(MC) ¼ 0.067

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

O2 only, as well as to test differences among original populations. In

p(MC)<0.001
PC - rearing

cases where results were significant, PERMANOVA was used to test for
the interactive effect of turbulence pattern, sediment presence and
Developmental stages 48h

oxygenation, to which parents had been exposed on larval development.

Furthermore, PERMANOVA was used to run relevant pair-wise com­
F(1,17) ¼ 15.787

F(1,17) ¼ 23.206

F(1,17) ¼ 26.779

parisons among and within larval populations. In cases where the

F(5,17) ¼ 11.999

F(1,17) ¼ 2.651

F(1,17) ¼ 1.595
p(MC) ¼ 0.121

p(MC) ¼ 0.229
FSW - rearing

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001

number of unique values from permutations was too low, the

Monte-Carlo procedure was used to calculate p values. The software
package PRIMER 6 PERMANOVA Plus (PRIMER-E Ltd, Plymouth, UK)
was used for all statistical analyses.
F(1,17) ¼ 15.245

F(1,17) ¼ 26.045

F(1,17) ¼ 24.294
F(5,17) ¼ 17.248

3. Results
F(1,17) ¼ 8.133

F(1,17) ¼ 7.864
p(MC) ¼ 0.011

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001
PC - rearing

3.1. Natural variation of seawater oxygenation

Developmental stages 24h

Oxygen levels within the examined P. oceanica meadow fluctuated

during the day. In particular, peaks of oxygen production occurred at
mid-day causing local conditions of oxygen super-saturation. Diel (i.e. a
F(1,17) ¼ 19.303

F(1,17) ¼ 31.161

F(1,17) ¼ 40.853

F(1,17) ¼ 10.559
p(MC) ¼ 0.0014

p(MC) ¼ 0.0271
F(5,17) ¼ 22.551

F(1,17) ¼ 4.086

24-h period) seawater oxygen fluctuations are shown in Fig. 6.

p(MC)<0.001

p(MC)<0.001

p(MC)<0.001
p(MC)<0.001
FSW rearing

3.2. Larval developmental stages

3.2.1. Effects of parental exposure on larval developmental stage in FSW

Turbulence, T
population

Sediment, S

After 24 and 48 hpf, a significant effect of parental pre-exposure on

TxO2 (S)

larval development was evident on larvae reared in FSW (Table 1). At 24

Table 1

O2 (S)
Larval

hpf, all the interactions between parental factors were significant

TxS

(Table 1). Overall, the adults that had experienced a spaced pattern of

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A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

Fig. 7. Percentage of developmental stages at 24 hpf. On the X axis the parental populations (POP1-6) and larval exposure conditions are shown. Significant dif­
ferences (p < 0.05) among larval populations reared in FSW for each experimental parental condition are represented with lower-case letters (a–d), while capital
letters (A–E) represent the significant differences among the larval populations reared under similar parental conditions. Asterisks represent the intra-group
(FSW_90% O2 vs. all other larval conditions) significant differences (*: p < 0.05, **: p < 0.01, ***: p < 0.001), n.s. ¼ not significant (p > 0.05).

turbulence gave birth to a higher percentage of early plutei than those almost all (99%) larvae from POP 3 and POP 4 succeeded to develop up
having experienced an aggregated turbulence pattern. In general, an to the echinopluteus stage, while the average amount of echinoplutei
average 65% of early plutei from parents exposed to a spaced pattern of between POP 5 and POP 6 was around 87% (Fig. 8). These results,
turbulence was recorded, while an average 41% of early plutei from however, take into account the total percentage of echinoplutei without
parents exposed to an aggregated pattern was observed (Fig. 7). distinguishing among normal and abnormal ones.
Oxygen super-saturation had a negative effect on the percentage of
early plutei as well. In particular, an average 78% of early plutei be­ 3.2.2. Transgenerational effects of parental exposure on larval
tween POP 3 and POP 4 was observed, while the average between POP 5 developmental stages
and POP 6 was around 41% (Fig. 7). In general, after 24 and 48 hpf, a buffer effect of the adults’ pre-
At 48 hpf, the interaction between the pattern of turbulence and the exposure to contaminated sediments was not observed in larvae
presence of sediment were significant (Table 1). Overall, the averages of reared in the presence of elutriates. Larvae grew better in FSW inde­
4-arms echinoplutei from both parents exposed to the spaced and the pendently of the origin of their parents. For each experimental larval
aggregated pattern of turbulence were comparable (89% and 94%, population, the percentage of developmental stages of larvae reared in
respectively) (Fig. 8). At 24 hpf, the oxygen super-saturation had a FSW and of those reared in the presence of elutriates was always
negative effect on the average percentage of echinoplutei. In particular, significantly different (Table 1). At 24 hpf, all the interaction between

6
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

Fig. 8. Percentage of developmental stages at 48 hpf. On the X axis the parental populations (POP1-6) and larval exposure conditions are shown. Significant dif­
ferences (p < 0.05) among larval populations reared in FSW for each experimental parental condition are represented with lower-case letters (a–b), while capital
letters (A–F) represent the significant differences among the larval populations reared under similar parental condition. Asterisks represent the intra-group (FSW_90%
O2 vs. all the other larval conditions) significant differences (*: p < 0.05, **: p < 0.01, ***: p < 0.001), n.s. ¼ not-significant (p > 0.05).

parental factors were significant (Table 1). Overall, as observed in all (Table 1).
larval populations reared in FSW, at 24 hpf a negative effect of the pre- The interaction between turbulence pattern and the presence of
exposure of adults to the aggregated pattern of turbulence was recorded sediment affected the normal formation of the calcareous structures in
in the larvae reared in the presence of elutriates (Fig. 7). Oxygen super- larvae maintained in FSW (Table 1). Overall, the percentage of normally
saturation had a negative effect on the average percentage of early plutei developed echinoplutei from parents who experienced a spaced vs. an
as well in all larval populations exposed to elutriates (Fig. 7). aggregated pattern of turbulence was comparable (77%) (Fig. 9). Oxy­
At 48 hpf, all the interaction between parental factors were signifi­ gen super-saturation had a negative effect on the proportion of normally
cant (Table 1). The same trend described for larvae at 24 hpf was developed echinoplutei. In particular, an average 88% of normal echi­
recorded for the larval development at 48 hpf, i.e., a negative effect of noplutei between POP 3 and POP 4 was observed, while the average
adults’ pre-exposure to both aggregated pattern of turbulence and ox­ between POP 5 and POP 6 was around 65% (Fig. 9).
ygen super-saturation (Fig. 8). The length of both SS (Fig. 10 A) and OS (Fig. 10 B) was significantly
affected by the parental population of origin in larvae reared in FSW
(Table 1). In general, larvae reared in FSW had longer SS and OS
3.3. Larval skeletal abnormalities and growth
compared to their siblings reared in elutriates (Fig. 10A–B), regardless of
the parent of origin.
3.3.1. Effects of parental exposure on larval skeletal development in FSW
After 48 hpf, the percentage of normally developed echinoplutei was
dependent on the parental pre-exposure for larvae reared in FSW

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A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

Fig. 9. Percentage � SD (n ¼ 3) of normal echinoplutei at 48 hpf. On the X axis parental populations (POP1-6) are shown. Larval exposure conditions are shown in
the legend. Significant differences (p < 0.05) among larval populations reared in FSW for each experimental parental condition are represented with lower-case
letters (a–d), while the capital letters represent the significant differences among the larval populations reared at the same parental condition. Asterisks repre­
sent the intra-group significant differences (*: p < 0.05, ***: p < 0.001, n.s. not-significant; n ¼ 3).

3.3.2. Transgenerational effects of parental exposure on larval skeletal Carballeira et al., 2012; Corinaldesi et al., 2017; Morroni et al., 2016,
development 2018). Marine sediments close to marinas, harbors and industrial plants
After 48 hpf, the percentage of normally developed echinoplutei was are likely to be enriched, in particular, with heavy metals and PAHs, as
dependent on the parental pre-exposure for larvae reared in the presence recently documented in the Bagnoli-Coroglio area (Morroni et al., 2020;
of elutriates (Table 1). Ruocco et al., 2020). Exposure to these substances can jeopardize the
In general, it is possible to observe a negative effect of both aggre­ normal development of early life stages of echinoderms, and make their
gated pattern of turbulence and super-saturated oxygen level to which populations more sensitive to environmental perturbations (Byrne,
parents were exposed (Fig. 9). Furthermore, no normal echinoplutei 2012). Despite the large number of studies that have addressed the ef­
were observed in both POP 2 and POP 5 when exposed to elutriates fects of single stressors on benthic organisms, those that experimentally
(Fig. 9). examined the interaction between different types of physical, chemical
The length of both types of spicules was affected by parental expo­ and mechanical variables are scarce and often focused on a single life
sure as well (Table 1). Larvae maintained in elutriates had shorter stage.
spicules compared to their siblings reared in FSW, regardless of the Furthermore, in natural habitats, increased water oxygenation due to
parent of origin, with a mean length reduction of 40% for both somatic the photosynthetic activity of algae and macrophytes can have a positive
and oral spicules (Fig. 10A–B). effect on the physiological performance of associated or nearby organ­
isms. Giomi et al. (2019) found that ‘super-saturated’ (140 � 3%) oxy­
4. Discussion gen conditions increased the LT50 of six Red Sea species (one decapod,
one echinoderm, one holothurian, one bivalve, two teleost fishes) by 2
In this study, the combined effect of contaminated sediment and its
�
C compared to those maintained at normal oxygen saturation levels (97
re-suspension associated with different turbulence patterns at both � 2%).
normal and ‘super-saturated’ levels of oxygen were studied for the first Migliaccio et al. (2015) found that the exposure of P. lividus adults for
time. A generally negative effect of the pre-exposure of adults to the nine days to cadmium (Cd) and manganese (Mn) during gametogenesis
examined experimental factors on larvae was observed. Furthermore, resulted in a higher percentage of abnormal larvae and delayed larval
larvae derived from parents maintained in the presence of sediments did development when compared with larvae from control parents,
not perform better when exposed to elutriates, even when reared at a although all larval populations were reared in control sea water.
‘super-saturated’ level of oxygen. Conversely, in our study, when all larval populations were exposed to
Echinoderms, including the sea urchin P. lividus, have been widely elutriates to mimic the effect of re-suspended sediments, there was a
used as marine model organisms to study the effect of different significant difference in larval development when compared to their
anthropogenic stressors such as heavy metals, personal hygiene prod­ siblings reared in FSW_90% O2. These findings are in accordance with
ucts, PAHs, as well as environmental variability (Byrne, 2012; several studies that found the exposure to a mix of heavy metals present

8
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

Fig. 10. A–B: Mean length � SD (n ¼ 3) of somatic (A) and oral (B) spicules at 48 hpf. On the X axis parental populations (POP1-6) are shown. Larval exposure
conditions are shown in the legend. Significant differences (p < 0.05) among larval populations reared in FSW for each experimental parental condition are rep­
resented with lower-case letters (a–b), while capital letters (A–E) represent the significant differences among the larval populations reared under the same parental
conditions. Asterisks represent the intra-group (FSW_N–O2 vs. all other larval conditions) significant differences
(*: p < 0.05, **: p < 0.01, ***: p < 0.001), n.s. ¼ not-significant.

in marine sediments to cause a negative effect on larval development in which could have led to oxidative damage by causing the accumulation
echinoderms, including the sea urchins P. lividus, Psammechinus miliaris of reactive oxygen species (ROS) in larval tissues (Hassoun and Stohs,
and Strongylocentrotus intermedius, and the sea star Asterias rubens 1996), and the subsequent intensification of the sensitivity of cells to
(Coteur et al., 2003; Marin et al., 2001; Xu et al., 2011). It is widely other co-occurring toxicants (Brzo�ska et al., 2008; Chowdhury et al.,
known that the combination of different heavy metals is often more 1987).
toxic than one alone, due to additive and/or synergistic effects Furthermore, abnormal larval development can be due to the
(Kobayashi and Fujinaga, 1976; Pagano et al., 1996). Xu et al. (2011), reduction of acetylcholinesterase (AChE) activity in plutei, as observed
for example, highlighted that the combination of heavy metals, such as by Maisano et al. (2015) after larval exposure to increasing concentra­
Cd, Cu, Pb, Zn, had a negative synergistic effect on the larval develop­ tions of CuO nanoparticles for 24h. AChE plays a pivotal role in the
ment of S. intermedius. This can be explained with the presence of Cd, migration of primary mesenchyme cells (PMCs) and spicule elongation

9
A. Chiarore et al. Marine Environmental Research 159 (2020) 104967

(Ohta et al. (2009). PMCs are responsible for the formation of skeletal draft, Writing - review & editing, Project administration. Alessandra
spicules, which migrate into the blastocoel and produce these calcareous Gallo: Conceptualization, Resources. Antonio Cannavacciuolo:
structures. Moreover, PAHs can interfere with echinoderm larval Formal analysis, Data curation, Writing - original draft. Mirko Muta­
development, as observed in sea urchin Hemicentrotus pulcherrimus lipassi: Resources, Writing - original draft. Davide Caramiello: Re­
larvae exposed to two PAHs, namely benz[a]anthracene (BaA) and sources. Folco Giomi: Resources, Writing - original draft. Marco Fusi:
4-hydroxybenz[a]anthracene (4-OHBaA), for 53h post-fertilization Resources, Writing - original draft. Roberto Danovaro: Writing - orig­
(Suzuki et al., 2015). This was due to a reduction of the expression of inal draft, Funding acquisition. Marco Munari: Conceptualization,
several genes involved in the formation of spicules, which the authors Formal analysis, Investigation, Resources, Data curation, Writing -
attributed to the suppression of the transcription factor for spicule for­ original draft, Writing - review & editing, Visualization, Project
mation by 4-OHBaA. Furthermore, Pillai et al. (2003) suggested that administration.
several PAHs (phenanthrene, fluorene, and pyrene) can cause exogas­
trulation in exposed embryos, causing a vegetative state and death. This Acknowledgement
is corroborated by the high percentage of dead embryos in larval pop­
ulations exposed to elutriates at both 24h and 48h post fertilization in This study was supported by the project ABBaCo funded by the
our study. Italian Ministry for Education, University and Research (grant number
Contrary to the expectation that adults experiencing more stressful C62F16000170001). Dr. Antonia Chiarore was supported by a fellow­
conditions would produce more resistant offspring (Thor and Dupont, ship funded by ABBaCo. We thank Dr. Elisabetta Tosti for coordinating
2015; Weston et al., 2013), larvae from parents reared with sediments at the work package for the experimental research in this project. We thank
both O2 levels and turbulence patterns were less resistant to elutriates Marco Cannavacciuolo, Giovanni De Martino and Francesco Terlizzi for
than those from control parents, as highlighted by the total absence of assistance in the field during sea urchins sampling.
echinoplutei in POP 5. Nevertheless, it may be possible that one month
of exposure during the gametogenesis was too short to trigger a trans­
Appendix A. Supplementary data
generational buffer effect against sea water pollution.
Furthermore, no positive effects of higher oxygen levels were
Supplementary data to this article can be found online at https://doi.
observed, since larvae exposed to oxygen super-saturation showed an
org/10.1016/j.marenvres.2020.104967.
even higher percentage of abnormal larvae and echinoplutei compared
to larval populations reared in FSW_90% O2. Consistently, it was re­
ported that the accumulation and toxicity of Ag, As, Co and Cr in the References
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