Ecotoxicology and Environmental Safety 212 (2021) 112010

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Elutriate preparation affects embryo development test with Paracentrotus

lividus: An in-depth study on the differences between two protocols and
three different sediment/water mixing times
Davide Sartori a, *, Simona Macchia a, Nicolas Layglon b, Sebastien D’Onofrio b,
Benjamin Misson b, Maria Elena Piccione a, Rosa Maria Bertolotto c, Alice Scuderi a,
Fabiano Pilato a, Silvia Giuliani a, David Pellegrini a, Andrea Gaion d
a
Institute for Environmental Protection and Research, ISPRA, Via del Cedro, 38, 57123 Livorno, Italy
b
Université de Toulon, Aix Marseille Université, CNRS, IRD, Mediterranean Institute of Oceanography (MIO), UM110, France
c
Agenzia Regionale per la Protezione dell’Ambiente Ligure, ARPAL, Via Bombrini 8, 16149 Genova, Italy
d
University Centre South Devon, Vantage Point, Long Road, Paignton TQ4 7EJ, United Kingdom

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

Edited by Dr R Pereira Coastal areas are under continuous and increasing pressure from different human activities. A mixture of con­
taminants (e.g. hydrocarbons, pesticides, persistent organic pollutants (POPs), emerging contaminants, and
Keywords: others), originating mainly from populated, industrialised and agricultural areas, can reach the marine envi­
Sea urchin ronment through different means such as wastewater discharge, soil runoffs, leaching from agriculture, and
Elutriate preparation
volatilisation/deposition. In this context, marine sediments have increasingly been considered repositories for a
Sediment management
variety of pollutants that can accumulate and be stored for long periods, acting as a secondary source of con­
Embryo-development, ecotoxicology
Environmental monitoring taminants during subsequent dredging operation or vessel manoeuvring. Chemical and ecotoxicological analyses
of sediments are routinely conducted to evaluate the potential hazard/risk to the environment, either on bulk
sediment or elutriate. In general, sediment elutriates are commonly prepared according to ASTM Guide even if
alternative protocols are proposed by USACE for the various condition that they have to represent. The goal of
the present study was to determine if the toxicological properties of ASTMprepared elutriates are comparable to
those obtained from the USACE protocol. Sediment coming from 3 harbours (Olbia, Cagliari, and Toulon), as part
of the “Se.D.Ri.Port” Interreg Project, were processed to obtain elutriates according to ASTM Guide and USACE
Dredging Elutriate protocol and tested with the sea urchin Paracentrotus lividus embryo development test.
Moreover, the significance of different stirring times of water/sediment mixture (1 h, 3 h, and 24 h) was tested
with both the ASTM and USACE protocol. In addition to the biological analysis, for each sediment sample, heavy
metals concentration, granulometry, and organic matter were determined. Even if for the ports of Toulon and
Cagliari, the ASTM and USACE elutriates showed comparable results with P. lividus bioassay, for the port of
Olbia the two protocols showed different criticalities. Preliminary results show that for the site Olbia elutriates
prepared with the USACE protocol resulted in higher toxicity than elutriates obtained with ASTM (p < 0.001). In
conclusion, differences in preparation protocols appear to be significant and can lead to different results in
biological testing. To overcome this problem and to obtain more reliable evaluations of risk to the environment,
standardisation and regulation must be the next goals in sediment management procedure.

1. Introduction 2012) and may be transferred through the food web, affecting marine
biota and ultimately human health (Borgå et al., 2001).
Marine sediments have increasingly become repositories for a variety A common approach in sediment risk assessment is to integrate
of pollutants (Huerta Diaz 2006; Sahu et al., 2009) which can accumu­ chemical analysis and toxicological results (Chapman, 2000). Matrices
late and remain in this matrix for long periods (Ruiz-Fernández et al., generally taken into account to evaluate sediment toxicity are: whole

* Corresponding author.
E-mail address: [email protected] (D. Sartori).

https://doi.org/10.1016/j.ecoenv.2021.112010
Received 16 July 2020; Received in revised form 27 January 2021; Accepted 28 January 2021
Available online 4 February 2021
0147-6513/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

sediment, pore water and elutriate. The whole sediment represents the obtained from the USACE protocol, considering the significance of 3
most realistic exposure scenario however, bioassays on this matrix different stirring times of water/sediment mixture (1 h, 3 h, and 24 h).
require a considerable investment of human resources, material and
time (Haring et al., 2010). Pore water can represent a direct exposure 2. Materials and methods
route for many species but, depending on the grain size, it could be
difficult to obtain the right volume to perform bioassays. Even the 2.1. Sediment samples
extraction methodology can lead to chemical-physical alterations in pH
and redox potential of the pore water and in return can affect the real The sediment samples for this study were collected from the harbours
bioavailability of contaminants present (Chapman et al., 2002). of Olbia, Toulon and Cagliari as part of the ’’Se.D.Ri.Port" Interreg
Tests with elutriates simulate exposure to contaminants released in project (See Supplemental Material, Figure OP.1) during the fall-winter
the water column during dredging operation or open water disposal of season of 2019.
dredged material (McDonald, 2005). According to Haring et al. (2012), Sediments were sampled, in each port at three sites along the
elutriate preparation could reduce the toxicity of the sample by diluting contamination gradient. For each Port, sediment collection at the indi­
contaminants and generally only hydrophilic contaminants can be cated site occurred on the same day. In the Toulon Harbour, a sample
extracted in elutriate. Elutriates are commonly prepared according to was collected from presumably few contaminated aquaculture sites
the ASTM E1391 (2014) method, which consists in mixing one part of (Lazaret: 43◦ 05’ 24.088" N, 05◦ 54’ 58.922" E) and the other two-
sediment and four parts (in volume) of water. However, under the samples, in the inner and presumably most polluted part of the port
incentive of several research programmes related to dredging activities (Corsica: 43◦ 06’ 54.546" N, 05◦ 55’ 33.666" E; Suffren: 43◦ 07’ 10.193"
and management of dredged materials, the U.S. Army Corps of Engi­ N, 05◦ 54’ 59.525" E).
neers (USACE) has developed several elutriate preparation protocols In Olbia harbour, sediments were collected from three sites: P1 (40◦
using different water/sediment ratios to best simulate the environ­ 55’ 26.777" N, 09◦ 31’ 30.460" E) is located in the area where ferry boat
mental conditions that occur during handling activities such as sediment daily resuspend sediment with their bow thruster; P2 (40◦ 55’ 09.736"
discharge into confined disposal facilities, offshore spillage, and N, 09◦ 32’ 26.332" E) is at the mouths of the Padrongianus river, and P3
dredging (Di Giano et al., 1995). (40◦ 55’ 23.984" N, 09◦ 33’ 40.082" E) is located in the eastern unpol­
To our knowledge, as of today, there is no universally recognised and luted part of the harbour (reference site). In the Port of Cagliari, sedi­
accepted method in the preparation of elutriates and, in recent years, ment samples were collected in the area of the fishing vessel’s dock
numerous articles have been produced on this issue (Novelli et al., 2006; (Darsena: 39◦ 12’ 39.045" N, 09◦ 06’ 09.910" E), in the eastern part of
Haring et al., 2010, 2012; Vicinie et al., 2011). Similarly, Italian Decree the Port of Cagliari near the mouth of Channel Palma (S. Elmo: 39◦ 12’
173/2016 (MD 173/2016), which established the technical criteria for 03.375" N, 09◦ 07’ 40.431" E; reference site), and in the area of the
the authorisation and management of dredged material, does not pro­ Rinascita Pier (39◦ 12’ 41.807" N, 09◦ 06’ 17.434" E) where the cruise
vide any indication about the method of preparation of elutriate but only terminal is located.
indicates the 1:4 (w/v) water/sediment ratio. Surface sediments were collected with an 8-L van Veen grab, stored
There are reasonable grounds to believe that different elutriate at 4 ◦ C in capped polyethylene vessels and transferred to the laboratory
methods can affect the bioavailability and toxicity of this matrix (Marin for elutriate preparation and chemical analysis to be conducted within 2
et al., 2001; Novelli et al., 2006), and consequently lead to different weeks from sampling. Sediment samples, in order to minimise sources of
management options of sediments. For this reason, as a part of the Se.D. contamination and the variation in the physical, chemical, or biological
Ri.Port Interreg project, for the evaluation of the ecotoxicological effects characteristics, were collected and processed following the indication
related to the resuspension of sediments, the embryo development test reported in the technical manual U.S. EPA (2001).
with sea urchin Paracentrotus lividus was implemented to evaluate the
toxicity of elutriates obtained from sediment sampled in the ports of 2.2. Water Sampling
Olbia (Italy), Cagliari (Italy), and Toulon (France) following a pollution
gradient. In Toulon bay (South East France), surface and deep sediments Elutriate samples were prepared using seawater collected from each
are characterised by a strong anthropogenic contamination in heavy sampling site 1 m above the sediment surface. Water was collected with
metal, (navy base, harbours, etc.); in particular, mercury appears to be a 4.2 L horizontal van Dorn type water sampler (Wildco, model Beta)
the most enriched contaminant with a large pollution dispersion in the decontaminated with a hydrochloric acid solution (10%) and then
whole bay (Tessier et al., 2011). rinsed with Milli Q water the day before its use. On the sampling site, the
Olbia harbour is the most important in Northern Sardinia for ferry water sampler was rinsed with acidified milli-Q (0.1% v/v HCl, Trace­
traffic (https://deims.org/). Previous investigations have shown very Select, Fluka) and then rinsed three times with seawater from the site
low levels of contamination of both metals and organic contaminants (Coclet et al., 2018; Layglon et al., 2020).
(De Luca et al., 2005).
The portual area of Cagliari showed heavy metal contamination, 2.3. Sediment elutriate preparation
especially of As, Cd, Hg, and Pb (Schintu et al., 2016). Sediment coming
from the S. Gilla lagoon, is rich in Hg because of important discharges Sediment elutriate were prepared in 2L capped polyethylene vessel.
from a petro-chemical plant that operated since the 1960s in the pro­ The Dredging Elutriate (Di Giano et al., 1995), developed by USACE
duction of chloride (Contu et al., 1983; Frontalini et al., 2009). To assess to estimate the release of contaminants from sediment due to resus­
the possible influence of sample preparation in the toxicity of sediment, pension during dredging activity, consisted of mixing the sediment and
in the present study elutriates were prepared following both the ASTM site water at a total suspended solid concentration of 10 g L-1 followed by
protocol and Dredging Elutriate method developed by USACE. the aeration of the slurry for 1 h. Aeration was performed in a thermo­
In addition to the biological analysis, for each sediment sample, stated room (4 ± 1 ◦ C) equipped with a compressed air source with
heavy metals concentration, granulometry and organic matter content deionized water trap and tubing for bubble aeration of slurry. Glass
were determined. Furthermore, ammonium and nitrite concentrations tubes were attached to the aeration source and insert the vessel con­
in the elutriate, frequently regarded as confounding factors (Postma taining the sediment suspension. Vessel were covered on the top with a
et al., 2002), have been checked prior to test execution to avoid inter­ lid sealed with a parafilm strip. The flow rate was adjusted to agitate the
ference with the results. mixture vigorously for 60 min. The mixture is allowed to settle for 60
The goal of the present study was to determine if the toxicological min at 4 ◦ C and subsequently the supernatant was centrifuged at 1200 x
properties of ASTM-prepared elutriates are comparable to those g for 20 min using a refrigerated ultracentrifuge (Thermo Scientific,

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D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

Mod. SL16R). According to ASTM protocol (ASTM, 2014), elutriates procedures is regularly assessed by participation in QUASIMEME
were prepared mixing 1 part of sediment with 4 parts of site water (1:4 (Quality Assurance of Information in Marine Environmental in Europe)
v/v ratio), and mixed on a rotary shaker table, located in a thermostaed interlaboratory exercises for trace metal analysis.
room (4 ± 1 ◦ C), for 1 h at a speed of 400 x g. After mixing, the samples The limit of detection of the method and the limit of quantification
were centrifuged at 1200 x g for 20 min to separate the water from the for each metal analysed is given in Table 1.
sediment using a refrigerated ultracentrifuge (Thermo Scientific, Mod. A supplementary analysis was conducted on Olbia sediments to
SL16R). assess the presence of Polycyclic Aromatic Hydrocarbons (PAHs) and
The aqueous fraction (elutriate sample) was stored without filtering Polychlorinated Biphenyls (PCBs) in the matrices.
in 50-mL polyethylene containers at 4 ◦ C until used for sea urchin Polycyclic Aromatic Hydrocarbons (PAHs) were analysed using gas
bioassay. chromatography and mass spectrometry with a Soxhlet sample extrac­
In addition to the procedures described above for both the ASTM and tion technique (US-EPA 3540/8270D) (US-EPA, 2007b; US-EPA,
USACE protocols, additional elutriates were prepared from each sedi­ 1996a). Quantifications of polychlorinated biphenyl (PCBs) were con­
ment sample, by increasing the stirring time of the water-sediment ducted according to the US-EPA 3660B/8082A (US-EPA, 2007a;
mixture to 3 (ASTM 3 h, USACE 3 h) and 24 h (ASTM 24 h, USACE US-EPA, 1996b).
24 h) following the same protocols described before.
2.6. Organic matter
2.4. Ammonium and nitrite determination
The organic matter content was determined using the Loss on Igni­
The Ammonium (NH4) and Nitrite (NO2) content in the elutriate was tion (LOI) methodology (Luckzak et al., 1997). Briefly, the sediment
determined using Hach Lange kit LCK 304 and LCK341 respectively with sample was subjected to three phases of combustion at increasing tem­
a spectrophotometer Hach Lange DR3900. Briefly, the ammonium ions peratures. Approximately 7 g of sediment was dried at 105 ◦ C in
react at pH 12.6 with hypochlorite ions and salicylate ions in the pres­ weighted crucibles, for 24 h. Then, the sample was dried in a muffle, at
ence of sodium nitroprusside as a catalyst to form indophenol blue. For 160 ◦ C and 400 ◦ C, for 6 and 4 h respectively. After each step weight was
nitrite determination, nitrites react with primary aromatic amines in determined. The content of organic matter was expressed by the
acidic solution to form diazonium salts. These combine with aromatic following relation:
compounds that contain an amino group or a hydroxyl group to form
intensively coloured azo dyes. Organic Matter (g/kg) = (Net weight160 ◦ C – Net weight400 ◦ C)*100/(Net
weight105 ◦ C)
2.5. Chemical analyses

At first step, considering the prevalent contamination of the ports

involved in this work linked to inorganic contaminants (De Luca et al., 2.7. Granulometry analysis
2005; Frontalini et al., 2009; Tessier et al., 2011; Schintu et al., 2016), it
was decided to proceed only to the determination of heavy metals Granulometry analysis was conducted on 70 g of sediment. Each
concentrations in sediments. sample was treated with a solution of hydrogen peroxide and distilled
Samples of sediment were dried at 60 ◦ C, homogenised and sifted in a water (1:8 v.v.), for 48 h at room temperature, to facilitate the separa­
mortar, before chemical treatment. Trace metal analyses were per­ tion of the granules. Afterward, moist sediment was separated on a 63
formed with approximately 0.3 g of sample digested with a solution of 5 µm mesh with distilled water; the two fractions obtained were dried at
mL of HNO3, 1 mL of HCl and 2 mL of ultrapure water, using high- 60 ◦ C and subsequently weighed.
pressure Teflon reactors in a microwave oven system (EPA, 3051 A). Sediment fraction > 63 µm was separated with a set of sieves (ASTM
The concentration of As, Cd, Cr, Cu, Ni, Pb, Zn was determined by series) with mesh sizes that decrease downwards from − 1–4 phi with a
inductively coupled plasma optical emission spectroscopy (ICP-OES range of 0.5 phi (½φ) and each fraction obtained was weighed.
720, Agilent). Total mercury content was quantified by cold vapour
atomic absorption spectrometry (CETAC, M-7600). Suitable internal 2.8. Paracentrotus embryo-development test
chemical standards were used to calibrate the instrument and each
reading was replicated 3 times. The analytical and quality assurance was P. lividus adults were collected from an intertidal rocky site along the
performed using certified reference material (LGC6137, accepted % of southern coast of Livorno, Italy (43◦ 25.6020’ N e 10◦ 23.7800’ E) and
recovery between 80% and 120%; APHA, 1992; EMEA, 2006), duplicate immediately transported in an insulated box to the laboratory. The
samples and procedural blanks. The external validation of the analytical embryo toxicity test with P. lividus was performed according to Sartori

Table 1
Heavy Metal Concentration (mg/Kg) and organic matter content (O.M.) (g/kg) in the Study Areas. Concentration are expressed as mean ± SD (n = 6). Limit of detection
(LOD), Limit of quantification (LOQ) and Recovery (%) for each heavy metal analysed are listed in table. Accepted % of recovery between 80% and 120% (APHA, 1992;
EMEA, 2006).
As Cu Ni Cr Cd Pb Zn Hg O.M.

Toulon Lazaret 21,53 ± 1,67 34,58 ± 0,48 15,73 ± 1,55 27,62 ± 0,05 0,05 ± 0,08 48,59 ± 1,65 121,49 ± 1,42 0,71 ± 2,08 10,16 ± 1,15
Corsica 39,23 ± 1,78 462,90 ± 2,97 25,48 ± 0,86 52,23 ± 0,29 0,19 ± 0,29 195,08 ± 3,53 415,38 ± 1,24 9,85 ± 1,15 6,11 ± 0,68
Suffren 16,11 ± 1,41 136,79 ± 1,64 25,47 ± 0,95 42,74 ± 0,95 0,41 ± 0,12 133,27 ± 1,25 396,45 ± 6,81 2,13 ± 1,38 2,81 ± 0,96
Olbia P1 5,55 ± 1,70 26,99 ± 0,82 10,36 ± 0,82 17,43 ± 0,31 0078 ± 0,46 21,73 ± 1,71 119,22 ± 1,42 0058 ± 0,96 4,56 ± 0,38
P2 4,61 ± 1,17 11,11 ± 0,58 5,12 ± 0,58 9,11 ± 0,58 0068 ± 0,41 11,83 ± 2,08 65,95 ± 1,25 0031 ± 1,70 1,78 ± 0,54
P3 6,01 ± 1,00 6,04 ± 0,54 7,50 ± 0,49 13.25 ± 0,49 0031 ± 0,41 10,34 ± 1,15 47,68 ± 1,24 0022 ± 1,86 0,94 ± 0,58
Cagliari Rinascita 9,32 ± 2,99 20,88 ± 2,00 7,74 ± 0,46 15,18 ± 0,86 0,39 ± 0,40 61,87 ± 1,38 94,19 ± 0,92 0,22 ± 0,88 1,69 ± 0,86
Darsena 19,40 ± 0,05 117,18 ± 0,81 20,00 ± 0,47 32,59 ± 0,90 2,20 ± 0,05 304,47 ± 1,88 422,59 ± 1,76 0,26 ± 1,13 4,60 ± 0,96
S. Elmo 5,45 ± 1,27 141,90 ± 1,27 21,11 ± 1,43 30,06 ± 0,95 1084 ± 0,18 120,70 ± 0,96 461,50 ± 3,80 0,64 ± 1,28 14,73 ± 1,25
LOD (mg L-1) 0,0005 0,00004 0,0061 0,0035 0,0001 0,0071 0,0036 0,1672 –
LOQ (mg kg-1) 0,0441 0,0029 0512 0,2951 0,0004 0,5907 0,2998 13,93 –
Recovery (%) 89 93 102 93 97 102 96 98 –

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D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

et al. (2016). Controls were conducted as untreated negative controls (Tuckey for homogeneous variances, Dunnett’s test otherwise). Addi­
(0.22 µm FSW, collected from the sea urchin sampling site) and positive tionally, to compute and measure the associations between the two sets
controls with exposure to CuNO3 * 3H2O (Sigma Aldrich, USA). To of variables “Percentage of malformed plutei” and “Metals”, a Canonical
obtain this reference value, embryos in the positive control group were Correlation Analysis (CCA) has been conducted for each individual
exposed to the following Cu nominal concentration 12, 24, 36, 48, 60 protocol. Statistical significance for all the analytical procedures was set
µg/L. Elutriates were tested both undiluted (100%) and diluted at at values p < 0.05.
dilution values of 25% and 50%. Six replicates were prepared for each
sample using sterile capped polystyrene six-well microplates. Embryos 3. Results
were incubated in a dark room at 18 ◦ C for 72 h; at the end of incubation,
samples were fixed with 1% Lugol solution, rinsed in Phosphate Saline With regards to the grain size composition, sediments of Toulon and
Buffer (PBS) and observed with transmitted light microscopy. Cagliari had a strong silt composition, generally over 45%, and
The percentage of normally developed plutei (P4) in each treatment exceeding the 90% in Corsica and Suffren site in the Port of Toulon and
was determined, following the criteria described in Pagano et al. (1986) S. Elmo site in the Port of Cagliari (Fig. 1). The granulometric compo­
by observation of 100 larvae. Plutei were classified as abnormal when sition of the three sampling sites within each location (Toulon, Olbia and
presents skeletal aberration (apex spicules cross-linked or separated), Cagliari) has been tested with a Chi-square Goodness of fit to assess the
deformation of gastrointestinal apparatus, arms deformation. The deviation of 2 sites (observed) from the remaining reference site (ex­
acceptability of the results was fixed at a percentage of normal plutei > pected). All the individual comparisons resulted statistically different (p
80% in negative control tests (Gaion et al., 2013). < 0.01) with the exception of Corsica vs Suffren in Toulon (p = 0.25)
Data have been expressed as Effective Concentration (EC) values (see Supplementary Table, S4).
using Probit analysis method described by Finney (1971). Except for the P1 site, where silt rises to 42.53% of the grain size
fractions, samples collected in Olbia harbour had a predominant sandy
composition (> 80%).
2.9. Statistical analysis Table 1 shows organic matter content and heavy metal concentra­
tions in the surface sediments of Toulon, Olbia, and Cagliari.
In order to select the appropriate parametric (Anova and T tests) or The highest concentrations of metals were found in the ports of
non-parametric analysis (Kruskall-Wallis and Mann Whitney U test), Toulon and Cagliari. Among the metals analysed, high concentrations of
data have been processed to assess normality and homoscedasticity. mercury were found in Suffren and Corsica sediments (Toulon), where
Data were considered normally distributed if the p value for the Shapiro 2.13 and 9.85 mg/kg of Hg were measured respectively.
Wilk test was ≥ 0.05 (Das and Rahmatullah Imon, 2013), or Skewness The highest Zinc concentration occurred in the Port of Cagliari at S.
and Kurtosis were between − 1.96 and + 1.96 (Cosineau and Chartier, Elmo site, although similar concentrations were recorded, in the same
2010). Homogeneity of variances was tested with Levene’s test (p ≥ harbour, in Darsena site, and for the Port of Toulon in the Corsica
0.05), to select the appropriate post-hoc test in the analysis of variance

Fig. 1. Granulometric composition (%) of surface sediment collected in the Port of Toulon, Cagliari, and Olbia. “CTRL” indicates reference site for each study area.

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D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

sediment. For Lead, the highest concentration has been recorded in the Sediment Quality Guidelines for the Protection of Aquatic Life (CSQG)
Port of Cagliari, in the S. Elmo site, with 461 mg/kg although both in the by the Canadian Environmental Quality Guidelines Canadian Council of
Darsena site, in the port of Cagliari, and in the Corsica site, in the Port of Ministers of the Environment (CCME, 1995).
Toulon, considerable concentrations have been measured (422.59 and In this study, the levels of Cu, Pb, Zn in the ports of Cagliari and
415.38 mg/kg respectively). Particularly critical in the two ports was Toulon were noteworthy and above the L1, L2 for the Italian legislation,
the concentration of copper: in the Suffren and Corsica sites (Toulon), and the PEL values reported in the CSQG (CCME, 1995) derived from
136.79 and 462.90 mg/Kg were measured respectively, whereas in the studies on field-collected sediments that measured chemicals concen­
Port of Cagliari, 141.90 and 117.18 mg/Kg of Cu were recorded in the S. trations and associated biological effects. Samples from these locations
Elmo and Darsena sites, respectively. In Olbia, concentrations of heavy presented fine grain size predominance (Fig. 1) and this fraction was
metals are below the level of potential concern except for the P1 site, strongly correlated with Cu, Pb, and Zn concentrations (Fig. 3), sug­
where Zinc was present with a high concentration of 119.22 mg/Kg. gesting the occurrence of metal complexation processes. The observa­
Chromium was found, in studied areas, with generally low concentra­ tion that sediment grain size has a pronounced effect on heavy metal
tions (Table 1), whereas Nichel ranged, in Cagliari and Toulon harbour, concentrations agreed with other studies, which reported the strong
between 7.74 and 25.48 mg/Kg. affinity of heavy metal with clay fraction (Roussiez et al., 2005; Liu
Regarding the embryo development test with P. lividus, the per­ et al., 2019). The high surface area of fine sediment particle serves as a
centage of well-developed plutei recorded in negative control (0.22 foundation for increased adsorption of heavy metals (Mantie and Sap­
FSW) was 87% while the EC50 value obtained with the reference toxi­ pington, 1994) which can return to the water column, due to dissolution
cant (Cu(NO3)2 * 3H2O) was 24.019 (21,135 − 23.709) µg/L. This value process caused by physical-chemical changes after resuspension (Mad­
falls within the range of laboratory control chart (22.60–68.34 μg/L) dock et al., 2007; Machado et al., 2011). In the port of Toulon consid­
and agrees with the data reported in literature for this species with this erable concentrations have been found also for Hg, whereas in the port
reference toxicant (Arizzi Novelli et al., 2003; Sartori et al., 2016). of Cagliari sediments showed high level of contamination for Cd. Mer­
In the Port of Toulon, as reported in Fig. 2a, in the Corsica site, cury concentration was noteworthy in the port of Toulon; in particular,
elutriate prepared according to ASTM, showed higher toxicity with in Suffren and Corsica sites, the levels of Hg detected were 2.13 and
respect to USACE ones (Fig. 2a), with a percentage of abnormal plutei of 9.85 mg/kg respectively, one order of magnitude higher than the PEL
50% and 20% respectively (p < 0.01). A significant difference value and L1 and L2 values. The contamination found in the two ports is
(p < 0.01), in the percentage of malformed plutei, for ASTM and USACE consistent with the findings of Tessier et al. (2011) for the port of Toulon
method, was observed in the Suffren site as well (33.33% and 64.67% and Schintu et al. (2016) for the port of Cagliari. The Port of Olbia did
respectively). For the samples from the Port of Cagliari, elutriates pre­ not show any particular criticality with regards to heavy metal, except
pared according to the ASTM method, showed the highest criticalities, for Zn which is particularly high in the sediments of the P1 site which
with a percentage of well-developed plutei ranging between 0% and 6%. showed a percentage of silt greater than 40%.
Contrary to what observed in the Port of Cagliari, in Olbia, the samples Considering the effects of metals on the embryonic development of
prepared following the USACE, showed the highest toxicity with a P. lividus reported in the literature, EC50 values vary between 0.02 and
significantly higher percentage of malformed embryos (p < 0.05) in all 0.11 mg/L and 0.5 and 11.24 mg/L for copper and cadmium respec­
three studied areas (Table 2). tively; whereas the EC50 values for zinc ranged from 0.02 to 0.058 mg/L
The percentage of abnormal larvae of P. lividus increased along with (Fernández and Beiras, 2001; Gaion et al., 2013; Manzo et al., 2013;
the stirring time (3 h) in the ASTM method (ASTM 3 h), and values Sartori et al., 2016). The concentrations of these metals found in the
> 50% were reached with the S. Elmo, Rinascita, Darsena (Cagliari) and sediments from the ports of Cagliari and Toulon are well above the EC50
Corsica (Toulon) samples respectively (Fig. 2b). The highest percentages values reported in the literature for this species and this finds a confir­
of abnormal plutei were recorded in the Port of Caglari with values mation in the toxicity of elutriates (Table 4). As a result, possible ad­
rising the 100% in Darsena and S. Elmo elutriates. ditive and synergistic effects should be considered, as reported by Beiras
Samples prepared based on the USACE protocol increasing the stir­ and Fernandez (2001) with regards to different combinations of Hg with
ring time to 3 h (USACE 3 h), showed similar trends of malformed plutei Cu, Pb and Cd.
observed with the unmodified USACE method. An increase in the per­ The total ammonium concentrations detected in the elutriates were
centages of malformed plutei was observed with the USACE 24 h pro­ not significant (see supplementary table, S3) and fall below the EC50
tocol, especially for the port of Olbia where the percentages of well- values reported in the literature for P. lividus and other sea urchin species
developed embryos < 30% in all samples (Table 2). (Arizzi et al., 2003; Saco-Álvarez et al., 2010) and therefore not suffi­
The total ammonium and nitrite concentrations (see Supplementary cient to justify the presence of confounding factor.
Table S3) detected in elutriates were not significant, and therefore not Analysing the results of the embryotoxicity test with P. lividus, it can
sufficient to justify the presence of confounding factors, assuming that be observed that the increase in mixing time in the preparation of elu­
the inhibition is the result of the presence of anthropogenic toxicants. triates contributed to increasing the toxicity of the samples themselves,
Both CCA analyses, resulted in a model significant at p < 0.001; in although with sometimes significant differences between the two pro­
particular, CCA for protocol ASTM showed a total correlation coefficient tocols and between the three ports under investigation. In particular, for
for the two sets of data of 0.935 whereas with regards to USACE protocol the port of Toulon in the Lazaret site, the ASTM 3 h and ASTM 24 h
the model resulted in a correlation coefficient of 0.662. Partial corre­ protocols showed higher toxicity for sediments compared to the USACE
lation coefficients and significances are reported in Table 3. unmodified method, with an EC20 value of 72.95% and 67.38%
respectively. Also, the increase in mixing time between water and
4. Discussion sediment from the Corsica site led to an increase in the toxicity of the
samples analysed, albeit with opposite trends. Indeed, the ASTM 3 h and
The environmental sediment quality was discussed in comparison to USACE 24 h protocol showed higher toxicity for sediments in this area,
the L1 (the lowest chemical level of reference) and L2 (the highest with EC50 value of 77.91% and 78.74% respectively. These results
chemical level of reference), defined by the Italian Ministerial Decree confirm what reported by Arizzi Novelli et al. (2006) which showed that
173/2016 which establishes criteria and methodological procedures for the commonly used 1:4 sediment: water ratio (ASTM) has not always
characterising marine and brackish sediments to be dredged, their been useful in detecting sediment toxicity, generally demonstrating a
classification and identification of appropriate management options and lower discriminatory capability than elutriate prepared with lower
monitoring”. In addition, metal concentration was evaluated consid­ suspended solid concentration (1:20, 1:50, and 1:200 sediment: water
ering the Probable Effect Level – PEL established in the Canadian ratio). However, if for Cu there is a rapid reduction in the amount of

5
D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

Fig. 2. Percentage (%) of well developed plutei (P4) in sediment elutriates prepared according to: standard ASTM and USACE method (a), ASTM and USACE
modified method with 3 h of stirring time (b), and ASTM and USACE modified method with 24 h of stirring time (c). Percentage of normal plutei are expressed as
mean among six replicates (n = 6) ± SD (vertical bars). “CTRL” indicates reference site for each study area, *Indicates statistical significance p < 0.05 (independent
samples t-test, between results obtained from 3 elutriate dilution, six replicate each for ASTM and USACE elutriates), * *Indicates statistical significance p < 0.01
(independent samples t-test, between results obtained from 3 elutriate dilution, six replicate each for ASTM and USACE elutriates). Lack of * indicates no statisti­
cal difference.

6
D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

Table 2
Percentage (%) of normal developed plutei (P4) observed in sediment elutriate (undiluted) prepared according to ASTM and USACE protocol, with three stirring times
(1, 3 and 24 h). Concentration are expressed as mean ± SD (n = 6).
Normal developed P4 (%)

ASTM 1 h USACE 1 h ASTM 3 h USACE 3 h ASTM 24 h USACE 24 h

Toulon Lazaret 74,67 ± 1,53 81,00 ± 1,00 66,00 ± 1,15 81,00 ± 0,58 64,33 ± 1,53 79,33 ± 1,52
Corsica 53,33 ± 2,08 80,33 ± 0,58 31,00 ± 1,00 81,00 ± 2,08 73,67 ± 1,53 35,67 ± 0,58
Suffren 66,67 ± 1,15 35,33 ± 1,53 56,00 ± 2,65 65,00 ± 1,53 33,67 ± 1,53 80,67 ± 0,58
Olbia P1 73,00 ± 1,53 66,00 ± 0,58 77,33 ± 0,58 25,00 ± 1,53 81,00 ± 1,00 28,00 ± 1,00
P2 63,00 ± 1,15 29,00 ± 0,58 58,67 ± 1,53 79,67 ± 1,53 77,67 ± 1,53 10,67 ± 0,58
P3 82,00 ± 1,00 30,00 ± 1,00 54,33 ± 1,53 54,67 ± 1,15 47,33 ± 1,15 4,00 ± 1,73
Cagliari Rinascita 4,00 ± 1,15 67,00 ± 2,08 12,33 ± 2,08 0,00 ± 0,00 7,67 ± 1,53 11,33 ± 1,15
Darsena 6,00 ± 2,65 73,00 ± 1,15 0,33 ± 0,58 32,33 ± 2,51 1,00 ± 1,00 29,67 ± 1,53
Sant’Elmo 0,00 ± 0,00 10,00 ± 0,58 0,00 ± 0,00 19,00 ± 3,60 0,00 ± 0,00 5,67 ± 0,58

dissolved metal as a result of the adsorption and precipitation processes,

Table 3
for Pb there is a continuous release from the sediments, until reaching
Summary of the results from the CCA analysis showing the correlation between
the maximum concentration in solution after 6–9 h. Similarly, in a
heavy metals and sea urchin larvae malformation for ASTM and USEPA protocol.
recent study testing a model combining geochemical and kinetic pro­
Metal ASTM Protocol (p < 0.001; USEPA Protocol (p < 0.001;
cesses, Ciffroy et al. (2019) showed a release of Pb from oxides and
r ¼ 0.935) r ¼ 0.662)
carbonates from sediment during the first 10 h. In the long-term period,
Loadings Corr. Coeff. with % Loadings Corr. Coeff. with % there is a diametrically opposed trend for these two metals, with the
malformed malformed
concentration of dissolved Cu increasing again and those of Pb
As − 0.047 n.s. − 0.649 − 0.430 decreasing (Dang et al., 2020).
Cu − 0.020 n.s. − 0.337 − 0.223
For the port of Cagliari, elutriates prepared with both protocols
Ni 0.280 0.262 − 0.381 − 0.252
Cr 0.151 n.s. − 0.529 − 0.350 showed high toxicity, in line with the high contamination of Pb, Zn, Cu
Cd 0.883 0.825 0.461 0.305 and Cd occurring in these areas. The toxicity of the elutriates remained
Pb 0.610 0.570 − 0.177 n.s. almost unchanged also by increasing the resuspension time of the sed­
Zn 0.515 0.481 − 0.045 n.s. iments (3 and 24 h) with the exception of the Darsena and Rinascita
Hg − 0.095 n.s. − 0.498 − 0.329
samples which did not show any particular criticality, based on the
unmodified USACE protocol. If results obtained in the Rinascita site
were expected, considering the low contamination and the prevalent
sandy composition (more than 50%) of the sediments, the high toxicity

Fig. 3. Correlation between concentration of As, Cu, Ni, Cr, Pb, and Zn (mg/kg) and percentage (%) of silt in sampled sediment. Trace metal concentration are
expressed as mean ± SD (vertical bars). “CTRL” indicates reference site for each study area.

7
D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

Table 4 of metals which positively correlated with malformed plutei. Indeed, in

Correlation between silt abundance (%) in sediment sample, heavy metal of the first model, Ni, Cd, Pb and Zn all correlated importantly with the
potential concern and elutriate protocol which showed the greatest and the least embryotoxicity, with particular emphasis on Cd and Pb whose co­
effect on embryo development test with P. lividus. efficients are 0.825 and 0.570. With regards to the CCA of the USACE
Site Sample Silt Protocol Protocol Heavy metal protocol, only Cd positively correlated with malformed plutei, with a
(%) with with of potential lower coefficient than its counterpart (0.305).
> Effect in < Effect in concern
P. lividus P. lividus
test test 5. Conclusions

Toulon Lazaret 83,7 ASTM 24 h USACE; Zn, Hg

USACE 3 h
The two methodological protocols used in the preparation of the
Corsica 91,95 ASTM USACE; As, Cu, Cr, elutriates provided partially comparable results for the ports of Toulon
USACE 3 h Pb, Zn, Hg and Cagliari, whereas for the port of Olbia the data obtained were
Suffren 95,44 ASTM 3 h USACE 24 h Cu, Pb, Zn, sometimes discordant. In general, the results of the embryo-
Hg
development bioassay with P. lividus showed an increase in toxicity
Olbia P1 42,53 USACE 3 h; ASTM 24 h Zn
USACE 24 h related to the increased mixing time between water and sediment during
P2 13,81 USACE; USACE 3 h; – the preparation of elutriates, both for the ASTM and USACE protocols.
USACE 24 h ASTM 24 h This fact suggests a better extraction capability of hydrophilic com­
P3 4,96 USACE 24 h ASTM – pounds in the aqueous phase as the contact time between water and
Cagliari Rinascita 45,62 USACE 3 h; USACE Pb, Zn
sediment increases. The variation of toxicity as a function of time can be
ASTM
Darsena 81,48 ASTM; ASTM USACE Cd, Cu, Pb, related to the different kinetics of trace metals migrating from sediment
3 h; ASTM Zn to the aqueous phase during the preparation of elutriates (Dang et al.,
24 h 2020), or with the synergistic toxicity of some heavy metals (such as Hg,
S. Elmo 94,01 ASTM; ASTM USACE 3 h Cd, Cu, Pb,
Pb, and Cu)to the larval development of P. lividus (Fernández and Beiras,
3 h; ASTM Zn
24 h 2001).
The results obtained in this paper showed that the elutriate protocols
provide a different biological response depending on the grain size
revealed by P. lividus assay in Darsena elutriate was unexpected. composition, with a higher sensitivity given by the ASTM protocol for
Noteworthy was the result obtained in the port of Olbia where the sample with high percentage of silt.
USACE protocol highlighted the most critical issues with the P. lividus In Italy, dredged sediments can be classified, in five classes (MD
bioassay, despite the low contamination of heavy metals. To better n.173/2016) based on the integration of chemical analyses with the
interpret the ecotoxicological results, PAHs and PCBs analysis on the outcome of toxicological tests (weight of evidence criteria); this classi­
sediments of the port of Olbia (see Supplementary Methods) were con­ fication can be the discriminant factor for the management strategy of
ducted. The analysis of organic contaminants did not detect the presence the sediments, ranging from nourishment of submerged coastal areas to
of important concentrations of PAHs in the sediments of the investigated disposal, and consequent handling precautions, into confined disposal
area (see supplementary Table S1). Similarly, with regards to PCBs, it facilities (CDF). An incorrect ecotoxicological classification of sediment
was found that some congeners were present although with extremely due to inappropriate methodological procedures can result in loss of
low concentrations (see supplementary Table S2) and far below the PEL millions of Euros as well as representing a potentially environmental
levels reported in the CSQG and the L1 values reported in Decree 173/ critical scenario.
2016. The toxicity detected in Olbia sediments could be due to the A correct protocol which can improve the interpretations of the re­
presence of emerging contaminants (such as fragrances, flame re­ sults obtained from bioassay, the uniformity, and the standardisation of
tardants, sunscreens, etc.) which, for the Gulf of Olbia, characterised by the matrices preparation must necessarily be an objective for the future.
a strong increase in human pressure during the spring-summer season, Standardisation will permit to have comparable results obtained from
can considerably increase their concentration in the water column and different areas and different times, allow better management of sedi­
consequently accumulate in the sediment. ments without compromising ecosystems of particular naturalistic value
In Table 4, the percentages of silt in sediment samples and the cor­ and productive activities, such as aquaculture facilities, whose damage
responding elutriate protocol are reported; these factors resulted in the can in turn create a risk to human health.
highest and lowest toxicity in the P. lividus bioassay. In general, for
sediments with a percentage of silt greater than 80%, (Lazaret, Corsica Declaration of Competing Interest
and Suffren in Toulon; Darsena and S. Elmo in Cagliari) the protocol that
highlighted the greatest toxicity is the ASTM protocol, whereas, for The authors declare that they have no known competing financial
sandy samples (P1, P2 and P3 in Olbia), the most suitable protocol to interests or personal relationships that could have appeared to influence
highlight the greatest criticality is the USACE protocol. Furthermore, it the work reported in this paper.
is important to stress that samples with a high level of toxicity in elu­
triates prepared according to the ASTM method are characterised by Acknowledgements
high concentrations of Cu, Pb and Zn. These results show that the ASTM
protocol, although with different intensities linked to the water- The present work has been carried out with funding from the Interreg
sediment mixing time, is the most suitable to reveal toxicity due to the programme Italy-France Marittimo 2014–2020, co-funded by European
presence of high concentrations of heavy metals. Further investigations Regional Development Fund, with the project entitled “Sedimenti,
should be conducted on this issue; however, these preliminary results Dragaggi, Rischi portuali - Se.D.Ri.Port“.
suggest to consider an accurate choice of elutriate methodology for
ecotoxicological bioassays based on the granulometric characteristics of Appendix A. Supporting information
the samples themselves.
From the analysis of CCA of our results, it appears that ASTM pro­ Supplementary data associated with this article can be found in the
tocol is more efficient in extracting metals from the sediment evenly. online version at doi:10.1016/j.ecoenv.2021.112010.
The higher general correlation coefficient compared to USACE method
(0.935 vs 0.662, respectively) is further confirmed by the higher number

8
D. Sartori et al. Ecotoxicology and Environmental Safety 212 (2021) 112010

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