Marine Pollution Bulletin 141 (2019) 16–23

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Marine Pollution Bulletin

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Baseline

Baseline evaluation of metal contamination in teleost fishes of the Gulf of T

Tigullio (north-western Italy): Histopathology and chemical analysis
Anna Reboa, Alberta Mandich, Laura Cutroneo , Cristina Carbone, Arianna Malatesta,
⁎

Marco Capello
DISTAV, University of Genoa, Corso Europa 26, I-16132 Genoa, Italy

ARTICLE INFO ABSTRACT

Keywords: Metals, whether essential (Cu, Zn, Cr, Fe, Mn) or non-essential (Al, As, Cd, Ni, Pb, Hg) for organism metabolism,
Metal contamination occur naturally in the marine environment and their abundance can increase due to the presence of human
Bioaccumulation activities. In this study, fish were used as bio-indicators, to determine a correlation between the bio-accumu-
Histopathology lation of metals in muscle and gill tissues and the health status of fish. The study area was the Gulf of Tigullio
Fish
(north-western Italy), which is impacted by various sources of metal contamination. Histopathology served as a
Gills
significant tool to investigate possible alterations in gills, one of the main organs involved in fish physiology.
Results highlighted some correlations between certain metals (e.g. Pb, Ni) and gill alterations (e.g. epithelial
hyperplasia, epithelial lifting), providing baseline data from a pool of different fish species, which can be used
for comparison purposes in further studies.

Biological monitoring is becoming more and more important in
investigating ecosystem health status, as it is applied without modifying
the natural environmental conditions and aims to relate occurrence of a
chemical or stressor with ecological impacts on living organisms (Gupta
and Singh, 2011). Bio-monitoring is based on the use of bio-indicators,
which are organisms particularly representative in a certain habitat and
which can accumulate pollutants; the exposure to and interaction be-
tween selected bio-indicators and environmental contaminants causes
physiological modifications, called biomarkers, which can be analysed
and quantified at different biological levels (van der Oost et al., 2003;
Zhou et al., 2008; Walker and MacAskill, 2014). Hence, it is possible to
use this method with the purpose of coupling the compromised health
status of a bio-indicator with the degree of pollution of the environment
they represent (Gupta and Singh, 2011). Furthermore, it is also neces-
sary to perform chemical analyses, in a way that establishes which
chemicals an organism has been in contact with. Thus, it is possible to
assume that the presence of the chemicals can be related to the sub-
lethal alteration of the biomarker, treating this last as a ‘warning signal’
of a deleterious exposure, even when the stressor is no longer detectable
in the external compartment (Pretti and Cognetti-Varriale, 2001;
Ravera, 2001). Marine environments are strongly affected by pollution,
being the final reservoir of a range of substances introduced by human
activities (Pan and Wang, 2012). In this context, metals are one of the
main pollutants, coming from different natural and anthropogenic
sources, after which they can remain in the water column, adsorbed
into the bottom sediments or accumulated in the tissues of organisms
such as fish and mussels (Montaser et al., 2010; Gupta and Singh, 2011;
Walker et al., 2013; Walker and MacAskill, 2014; Vikas and Dwarakish,
2015; Walker and Grant, 2015). This last possibility is critical because
metal concentration can increase when passing through the food chain
due to the bio-magnification process; this means that higher levels of
contaminants can be found in top predator tissues, representing a risk
for human consumption (Gupta and Singh, 2011). Fish can work as bio-
indicators in monitoring marine metal pollution, since they show most
of the required characteristics, being part of the upper levels of trophic
webs; additionally, they have key ecological and commercial roles,
being very important in human diets (Murtala et al., 2012; El-Moselhy
et al., 2014).
In this study, metal concentrations were investigated in two fish
tissues, muscles and gills, and the selected biomarker was the histo-
pathology of fish gills. Although muscle tissue is not very active in
metal accumulation, it is frequently analysed because it is the main
edible part of fish, so its contamination can potentially lead to serious
disorders in human health (Bat et al., 2017). In contrast, gills are very
important in fish physiology, regarding respiration, osmoregulation,
acid–base balance, nitrogenous waste excretion, thermal exchange and
mucous production (Au, 2004). This is the reason why pathological
modifications of this organ indicate a reduced possibility of fish survival

⁎
Corresponding author.
E-mail address: laura.cutroneo@dipteris.unige.it (L. Cutroneo).

https://doi.org/10.1016/j.marpolbul.2019.02.024
Received 25 December 2018; Received in revised form 6 February 2019; Accepted 12 February 2019
Available online 16 February 2019
0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
A. Reboa, et al.
Fig. 1. Sampling area between Chiavari and Sestri Levante (Gulf of Tigullio, north-western Italy) with the three different sites established for fish sampling (black
circles). Site C: Chiavari; site L: Lavagna; site S: Sestri Levante. Sampling points are shown with red points. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
(Abdel-Moneim et al., 2012). In addition, gills are highly sensitive to
xenobiotic exposure because they are continuously in direct contact
with the external compartment through a very extensive surface
(Figueiredo-Fernandes et al., 2007). The aim of the study was to in-
vestigate the gill conditions of several fish species from the Gulf of
Tigullio (north-western Italy), to relate the alterations in this tissue with
the metals accumulated in the tissue itself.
The study area was the eastern part of the Gulf of Tigullio (Fig. 1),
which is located along the Italian coast of the Ligurian Sea. Various
human activities occur in this area and inland: harbours, industrial
plants, discharges of sewage and wastewater, fish farms, quarries and
mines (such as the abandoned copper mine of Libiola; Fig. 1), crops,
tourism facilities and ports, the main one being the port of Chiavari. All
these activities lead to a huge input of organic substances and metals,
both through direct discharges into marine coastal waters, and in-
directly by the supply of the two most important torrents of the area
(Entella and Gromolo torrents, Fig. 1), where a torrent is a small river
characterised by a periodical stream of fast-moving water (Bertolotto
et al., 2005; Capello et al., 2016; Capello et al., 2017; Consani et al.,
2019a).
We chose three different sampling sites in the study area (Fig.1): site
C – Chiavari, at the mouth of the Entella Torrent; site L – Lavagna, off
the Lavagna beaches; and site S – Sestri Levante, at the mouth of the
Gromolo Torrent. Sampling areas were set up along the shoreline
(5–30 m depth) to investigate the health status of fishes that live and/or
feed near the coast. Different species were line-caught: Chelidonichthys
lucerna, Trachinus draco, Serranus cabrilla, Coris julis, Mullus barbatus,
Pagrus pagrus, Pagellus erythrinus, Pagellus acarne, Trachurus mediterra-
neus, Bothus podas and Scomber scombrus. Sampling at Chiavari was
performed at three different periods: November 2016, July 2017 and
January 2018. Two samplings during the winter season were selected
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Marine Pollution Bulletin 141 (2019) 16–23
for the site at Sestri Levante: November 2016 and January 2018. A
single sampling event was conducted at Lavagna: December 2016.
Fish were immediately euthanised with a quick blow to the head,
then photographed and length and weight measured. Gills were im-
mediately dissected and the second gill arch on the left side of each fish
was fixed in Bouin solution (Bouin 20:1 acetic acid). After 24 h, fixed
tissues were transferred to ethanol 70% and conserved at room tem-
perature. The remaining gills and a sample of muscle tissue from each
fish were immediately frozen with dry ice and stored at −18 °C for
chemical analysis. Twelve metals (Sb, As, Al, Cd, Cr, Fe, Mn, Hg, Ni, Pb,
Cu, Zn) were analysed using inductively coupled plasma optical emis-
sion spectroscopy (USEPA Method 6010D, Optima 8300) of muscle and
gill tissues, after prior digestion of samples with aqua regia (USEPA
Method 3050B corrected with protocol UNI EN 13657:2004 for organic
samples) at Eurochem Italia (Genoa, Italy). These 12 metals were se-
lected due to their wide use in the literature, since they can be found in
all environmental compartments; furthermore, it is interesting to
compare the concentration of non-essential metals, such as Pb, Hg and
Cd, with others that are essential for organism metabolism, such as Fe,
Zn and Mn (Uysal et al., 2008; Vicente-Martorell et al., 2009; Kaoud
and El-Dahshan, 2010; El-Moselhy et al., 2014; Authman et al., 2015;
Bat et al., 2017; Yilmaz et al., 2018). Finally, we selected these metals
because they have already been considered in several recent studies
that involved the marine and terrestrial environment of the Gulf of
Tigullio (Capello et al., 2016, 2017; Consani et al., 2017; Consani et al.,
2019a, 2019b; Tolotti et al., 2019) Data for muscle and gill metal
content from the first sampling period were correlated using a scatter
plot with linear trend-line with R-squared value (R2).
Fixed gill samples for histology were dehydrated through a graded
series of ethanol, infiltrated with bioclear (Bio-Optica) and then em-
bedded in paraffin. Samples were then serially sectioned at 4 μm using a
A. Reboa, et al. Marine Pollution Bulletin 141 (2019) 16–23

Table 1
Ecological characteristics and morphometric measures of examined fish species. S: Sestri Levante; C: Chiavari; L: Lavagna; 1st, 2nd, 3rd: period of sampling; B:
benthic fish; B/N: bento-nektonic fish; B/P: benthic fishes that show pelagic habits during winter; N: nektonic fish.
Fish species Common name Sampling area Habitat Number of samples Range of total Range of body Picture
for species length (cm) weight (g)

Chelidonichthys lucerna Tub gurnard S-1st B 2 18.6–23.2 51–121

C-1st

Trachinus draco Greater weever S-1st B/P 5 11.8–28.2 14–143

L-1st

Serranus cabrilla Comber S-1st B 8 11.2–17.8 16–60

C-2nd
L-1st

Coris julis Mediterranean rainbow S-1st B 6 14.0–17.5 25–57

wrasse

Mullus barbatus Red mullet S-2nd B 6 13.5–17.8 32–77

C-2nd
L-1st

Pagrus pagrus Red porgy S-2nd B/N 3 12.8–14.0 37–48

L-1st

Pagellus erythrinus Common Pandora S-2nd B/N 16 13.2–19.4 23–110

C-1st; C-2nd;
C-3rd
L-1st

Pagellus acarne Axillary Seabream C-1st; C-3rd B/N 9 12.0–16.0 25–63

L-1st

Trachurus mediterraneus Mediterranean horse C-1st; C-3rd N 8 14.0–22.7 20–118

mackerel L-1st

Bothus podas Wide-eyed flounder C-2nd B 4 13.2–16.8 12–52

L-1st

Scomber scombrus Atlantic mackerel C-3rd N 1 17.5 57

microtome (Leica RM 2135), and stained with haematoxylin and eosin
in between hydration and dehydration in ethanol series. One slide for
each fish was analysed, using microphotographs (20× or 40× mag-
nifications; Leica microscope) of nine fields with 10 secondary lamellae
each. Evaluation of several pathological modifications of the tissue was
conducted for each field of every sample. A semi-quantitative scoring
system was developed, containing index and ancillary criteria, where
the score for index criteria was based both on their presence and extent
(Mitchell et al., 2012), while ancillary criteria scores were based only
on their presence/absence. Index criteria were: primary lamellae (PL)
hyperplasia and PL haemorrhage; secondary lamellae (SL) hypertrophy
and/or hyperplasia, SL fusion, and SL epithelial lifting. Ancillary cri-
teria were: SL shortening, SL blood vessel congestion, and aneurysms in
SL blood vessels. Alterations of gill tissue were classified into two
progressive stages, assigning importance factors (w) to the different
stages of change (w = 1 for the first stage; w = 10 for the second stage)
(Poleksić and Mitrović-Tutundžić, 1994). Using score values and im-
portance factors, an organ index was calculated for each sample (Bernet
et al., 1999):
Iorg = (aalt · walt )
where org is organ, alt is alteration, a is score value, and w is the im-
portance factor (Table S1, Supplementary materials). A scale of nu-
merical values of I was developed, in order to evaluate the degree of
damage in each gill examined (Poleksić and Mitrović-Tutundžić, 1994).
The I-values were associated with the following effects on gills: 0–10,
functionally normal; 10–20, slightly to moderately damaged; 20–50,
moderately to heavily damaged; > 50, irreparably damaged. Gill tissue

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A. Reboa, et al. Marine Pollution Bulletin 141 (2019) 16–23

Table 2 2008).
Concentrations (in mg kg−1) of metals which present international legislation In our study, many muscle samples showed a detectable con-
limits (Cd, Hg, Pb; European Commission, 2008) in muscle tissue of fishes centration of Cd and all were above the legislation limits (Table 2) and
sampled at Sestri Levante, Chiavari and Lavagna during all the different sam- also higher than those observed in other studies (Signa et al., 2017;
pling periods. Censored data were substituted with 0.5DL, where DL are:
Yilmaz et al., 2018). Detectable concentrations of Hg were always
0.25 mg kg−1 for Hg; 0.5 mg kg−1 for Cd and Pb.
higher than legislation limits, as already found by Keskin et al. (2007)
Sampling area Sampling Fish species Cd Hg Pb and Llull et al. (2017). For Pb concentrations, most muscle samples
were below the detection limit, but all samples that showed detectable
Sestri Levante 1st sampling C. lucerna 0.25 0.13 0.9
T. draco 0.25 0.13 2.4
values of Pb were above the legislation limit; in addition, some species
S. cabrilla 0.25 0.13 2.6 (T. draco, C. lucerna and S. cabrilla) presented a Pb concentration higher
S. cabrilla 0.26 0.13 2.0 than others in the literature (Yilmaz et al., 2018). The detection limit of
S. cabrilla 0.25 0.53 0.25 the method adopted, which ‘reads’ as a minimum value a concentration
C. julis 0.25 0.54 0.25
of < 0.5 mg kg−1 for Cd and Pb, prevented detection of any additional
C. julis 0.53 0.13 0.25
C. julis 0.83 0.59 0.25 samples above the limit range for these two metals.
2nd sampling M. barbatus 0.71 0.13 0.25 Concentrations of metals in gill tissue of fish collected during the
P. pagrus 0.57 0.61 0.25 first sampling period are shown in Table 3. No legislation limits exist for
P. erythrinus 11 0.13 0.8 metal concentration in gill tissue. Many samples were below the de-
P. erythrinus 0.33 0.13 0.25
P. erythrinus 0.4 0.13 0.25
tection limit, especially Cd, Hg, Pb, Sb, Cr, Ni and Cu. Some samples
Chiavari 1st sampling T. mediterraneus 0.56 0.13 0.25 showed values of certain metals that were considerable higher than in
T. mediterraneus 0.25 0.79 0.25 other samples: 23 mg kg−1 of Pb; 3.5 and 3.6 mg kg−1 of Hg, and 420
T. mediterraneus 0.51 0.13 0.25 and 460 mg kg−1 of Ni. The metal that showed the highest values for
2nd sampling S. cabrilla 0.25 0.7 1.0
almost all samples was Fe, where the maximum reached was
P. erythrinus 0.25 4.4 1.9
P. erythrinus 0.25 0.13 0.5 210 mg kg−1. Furthermore, Al concentration was very high in many
3rd sampling S. scombrus 1.0 0.13 0.25 samples, showing a highest value of 380 mg kg−1. Two other metals
T. mediterraneus 1.1 0.76 0.25 presented values that were clearly higher than those recorded in other
P. erythrinus 0.47 0.13 0.25 samples: 12 and 20 mg kg−1 of As and 26 mg kg−1 of Cr.
Lavagna 1st sampling T. mediterraneus 0.69 0.13 0.25
P. pagrus 0.62 0.13 0.25
Comparing the metal concentrations in muscle and gill tissue, they
P. pagrus 0.7 0.13 0.25 showed the following order, which is similar to data from the literature
B. podas 0.26 0.13 0.25 (Vicente-Martorell et al., 2009; Yilmaz, 2009):
T. draco 0.54 0.13 0.25
M. barbatus 1.4 0.13 0.25 Muscle:Fe > Al > Zn As > Mn Cu > Cr Ni Sb Pb Cd
M. barbatus 1.7 0.13 0.25
Hg
M. barbatus 1.6 0.13 0.25
M. barbatus 0.68 0.13 0.5
Gills:Fe > Zn > Al > Mn > As > Ni Cr Cu Sb Hg > Pb Cd,

where essential metals usually presented higher concentrations than

modifications cannot be compared to each other when they are sub-
mitted to different ranges of score value [(i.e. 0–6, 0–3, 0 or 1, (Table
S1)]; this valuation is not homogenous and the risk is that overlooked
lesions occur due to their more extended range of score (Mallatt, 1985).
To avoid this type of error, all gill tissue modifications were also scored
according only on their presence/absence. The standardisation of the
protocol made possible to do between-lesion comparisons.
Sixty-eight fish and 11 different fish species were collected during
the period November 2016–January 2018, as reported in Table 1.
Metal concentrations in muscle tissue were measured in each fish
from the three sites and reported in Supplementary materials (Tables
S2–S4). Some samples fell above the detection limits, which were:
0.25 mg kg−1 for Hg; 0.5 mg kg−1 for Cd, Pb, Sb and Cr; 1 mg kg−1 for
Cu; 2 mg kg−1 for Mn; 10 mg kg−1 for Al and Fe. Two of the main as-
pects involved in metal bio-accumulation are fish feeding and living
habits (Uysal et al., 2008). It is suggested that benthic fishes are likely
to have higher metal concentrations compared to fishes in the upper
water column, because the first ones live and feed directly in contact
with the sediments. In the present study, this strong correlation did not
occur, as already reported from other studies (El-Moselhy et al., 2014).
For Cd, Hg and Pb, international legislation limits are available (FAO,
1983; European Commission, 2008; Government of Canada, 2018):
0.3 mg kg−1 (Pb) for all sampled fishes; 0.5 mg kg−1 Hg for all sampled
fishes except Mullus sp. and Pagellus spp. (1 mg kg−1) (European
Commission, 2008), 0.05 mg kg−1 Cd for all sampled fishes except
Trachurus sp and Scomber sp. (0.1 mg kg−1) (European Commission,
non-essential ones, an exception being that of Al, which is a non-es-
sential metal but is one of the most abundant on Earth (Fernandes et al.,
2007; Uysal et al., 2008; Yilmaz et al., 2018).
It is also possible to compare metal concentrations in muscle and gill
tissue from the first sampling interval in Sestri Levante, Chiavari and
Lavagna. The comparison is shown in Fig. 2, where concentrations
falling below the detection limit (DL) were considered as 0.5 times the
relative limits (0.5DL). Data that were known to exceed or to be less
than a threshold value were named censored data; the worst practice
when dealing with nondetects is to exclude or delete them (Helsel,
2006). The most common procedure to avoid a strong bias is the sub-
stitution of censored data with a fraction of the DL. In this study,
substitution with 0.5DL values was chosen, this being a method fre-
quently used for chemical analysis (MacAskill et al., 2016).
Muscle is described in the literature as a tissue with low metal ac-
cumulation activity (Murtala et al., 2012; El-Moselhy et al., 2014), but
in our samples As concentration in muscle tissue was always higher
than in gills. Other metals showed variable concentrations in muscle
and gill tissue. However, there were some outliers (not shown) for
which gill concentrations of certain metals were considerably higher
than in other fish and also higher than the muscle concentration of
those metals in the same fish sample: 23 mg kg−1 of Pb in a sample of S.
cabrilla; 26 mg kg−1 of Cr in a sample of C.s julis; 420 mg kg−1 of Ni in a
sample of M. barbatus. These values are also higher than those observed
in the literature (Uysal et al., 2008; Murtala et al., 2012; Yilmaz et al.,
2018). Statistical analysis showed no significant correlation between

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A. Reboa, et al. Marine Pollution Bulletin 141 (2019) 16–23

Table 3
Concentrations (mg kg−1) of all measured metals in gills tissue of fishes sampled at the three sites (Sestri Levante, Chiavari and Lavagna) during the 1st sampling
periods. Censored data were substituted with 0.5DL, where DL are: 0.25 mg kg−1 for Hg; 0.5 mg kg−1 for Cd, Pb, Sb, As, Cr, and Ni; 1 mg kg−1 for Cu; 2 mg kg−1 for
Mn and Zn; 10 mg kg−1 for Al and Fe.
Sampling area Fish species Cd Hg Pb Sb As Al Cr Fe Mn Ni Cu Zn

Sestri Levante C. lucerna 0.25 0.13 0.25 0.25 0.25 2.9 0.25 32 15 0.25 0.5 18
T. draco 0.25 0.64 0.25 0.25 0.25 30 0.25 44 3.1 0.25 0.5 26
S. cabrilla 0.25 0.13 0.25 0.25 0.25 5.0 0.25 58 2.4 0.25 0.5 14
S. cabrilla 0.25 0.13 0.25 0.25 0.25 12 0.25 57 3.3 0.25 0.5 14
S. cabrilla 0.25 0.13 23 0.25 1.9 170 5.5 0.3 1.0 0.25 0.5 9.8
S. cabrilla 0.25 0.13 0.25 0.25 0.25 16 0.25 56 3.8 0.25 0.5 21
S. cabrilla 0.25 0.27 0.25 0.25 0.25 5.0 0.25 44 2.5 0.25 0.5 15
S. cabrilla 0.25 0.13 0.25 0.25 0.25 5.0 0.25 47 3.4 0.25 0.5 20
C. julis 0.25 3.50 0.25 0.25 0.25 67 26 5 1.0 0.25 0.5 40
C. julis 0.25 0.44 0.25 0.25 3.2 44 0.25 80 20 0.25 0.5 37
C. julis 0.25 0.46 0.25 1.40 2.9 42 0.25 160 18 0.25 17 26
C. julis 0.25 0.13 0.25 0.25 3.1 23 0.61 77 14 0.25 0.5 25
C. julis 0.25 0.13 0.25 0.25 0.25 15 0.25 72 16 0.25 0.5 28
C. julis 0.25 0.45 0.25 1.30 3.6 27 0.25 150 13 0.25 0.5 25
Chiavari C. lucerna 0.25 0.13 0.25 0.25 0.25 4.8 0.25 23 22 0.25 0.5 18
P. erythrinus 0.25 0.13 0.25 0.25 1.0 25 0.25 95 11 0.25 0.5 12
S. scombrus 0.25 3.60 0.25 0.25 0.25 92 0.25 5 1.0 0.25 0.5 34
T. mediterraneus 0.25 0.49 0.25 0.25 0.25 18 0.25 55 3.6 0.25 0.5 12
T. mediterraneus 0.25 0.29 0.25 0.25 1.1 13 0.25 85 4.9 0.25 0.5 16
T. mediterraneus 0.25 0.31 0.25 0.25 1.3 14 0.25 61 2.3 0.25 0.5 16
T. mediterraneus 0.25 0.13 0.25 0.25 1.4 4.0 0.25 51 2.4 0.25 0.5 20
T. mediterraneus 0.25 0.13 0.25 0.25 0.81 8.3 0.25 64 2.2 0.25 0.5 20
T. mediterraneus 0.25 0.13 0.25 0.25 1.0 13 0.25 72 3.0 0.25 0.5 21
Lavagna T. mediterraneus 0.25 0.13 0.25 0.25 2.3 6.2 0.25 97 3.6 0.25 0.5 24
P. erythrinus 0.25 0.13 0.25 0.25 1.5 5.0 1.1 72 7.4 0.72 1.0 15
S. cabrilla 0.25 0.13 0.25 0.25 1.6 52 0.93 71 3.7 2.70 1.0 20
P. pagrus 0.25 0.13 0.25 0.25 1.5 5.0 0.25 100 8.9 0.25 0.5 20
P. pagrus 0.25 0.13 0.33 0.25 3.5 380 0.25 210 7.3 0.89 3.1 22
B. podas 0.25 0.13 0.25 0.53 1.3 140 2.2 110 3.2 2.20 1.8 16
B. podas 0.25 0.13 0.86 0.25 0.25 5.0 0.25 80 3.6 0.25 0.5 21
B. podas 0.25 0.13 0.25 0.25 0.25 2.9 0.25 41 3.2 0.25 0.5 18
T. draco 0.25 0.13 0.25 0.25 0.25 3.2 0.25 60 3.0 0.25 0.5 28
T. draco 0.25 0.13 0.25 0.25 0.75 35 0.78 52 1.1 0.25 0.5 23
T. draco 0.25 0.13 0.25 0.25 0.67 18 0.54 44 1.7 1.50 0.52 25
T. draco 0.25 0.13 0.25 0.25 0.83 62 0.73 75 1.2 0.25 1.2 30
M. barbatus 0.25 0.13 0.25 0.25 2.9 16 0.25 130 5.5 0.25 1.4 20
M. barbatus 1.7 0.13 0.7 0.25 12 53 1.3 150 36 420 2.5 33
M. barbatus 0.25 0.13 0.25 0.25 4.8 40 0.25 69 3.5 0.25 1.0 14
M. barbatus 3.3 0.13 0.8 0.25 20 100 1.0 160 42 460 3.0 19
M. barbatus 0.58 0.13 0.25 0.25 5.5 57 0.99 96 6.3 36 7.8 16

muscle and gill concentration, with R2 = 0.1967 (Fig. 3).
From histopathology investigation, indices were calculated for
every fish to represent the degree of damage to gill tissue (Table S5,
Supplementary materials). The frequency of the four categories of gill
damage within the sampling population was calculated: the first cate-
gory shows the highest frequency, after that the frequency decreases
progressively in the second and third categories, down to the fourth
category, which is totally absent (Fig. 4).
This decrease could be due to the exponential nature of the devel-
opment of gill lesions from a normal structure to a more severe con-
dition. If exposure ceases, lesions can be repaired, but, if it persists,
modifications can turn into irreversible damage, decreasing chances of
fish survival (Poleksić and Mitrović-Tutundžić, 1994; Monteiro et al.,
2008). Indices were calculated based on gill tissue modifications,
among which some were present in a large number of samples: SL
shortening; SL epithelial hyperplasia; PL epithelial hyperplasia, some-
times with occurrence of lamellar fusion; SL epithelial lifting; and blood
vessels congestion (Fig. 5). Although histopathology is not a specific
biomarker, it is interesting to compare the metal concentrations of some
gill tissue samples showing high concentrations with the histological
alterations shown in the same tissue. A sample of S. cabrilla from Sestri
Levante was characterised by the accumulation of 23 mg kg−1 of Pb
that could be related to the occurrence of PL epithelial hyperplasia
leading to lamellar fusion, as supported by the exposure study of Olojo
et al. (2005); a concentration of 170 mg kg−1 of Al had been found in
the same sample, and this metal is proved to determine structural al-
terations in tissues (Authman et al., 2015). A high concentration of Al
(92 mg kg−1) occurred even in a sample of S. scombrus from Chiavari;
this sample also accumulated 3.6 mg kg−1 of Hg and showed SL epi-
thelial hyperplasia and lifting, as was already found in an exposure
study on Dicentrarchus labrax (Giari et al., 2008). Finally, two samples
of M. barbatus were characterised by an intense SL epithelial lifting, and
one of the causes of this alteration could be the really high con-
centration of Ni (420 and 460 mg kg−1) found in their gill tissue, as
observed in the literature (Poleksić et al., 2010). Furthermore, these
samples presented a high concentration of Fe (150 and 160 mg kg−1)
and As (12 and 20 mg kg−1), which could exacerbate histological al-
terations (Abalaka, 2015; Authman et al., 2015).
Gill indices of our samples were mostly represented in the lowest
category, indicating no serious alteration of the tissue. On the other

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A. Reboa, et al.
Fig. 2. Metal concentrations comparing muscle and gill tissues of all fishes
sampled during the 1st interval sampling in Sestri Levante, Chiavari, and
Lavagna. Plots use three different scales, dividing metals for which very low or
even undetectable concentrations have been measured from those that present
higher concentrations (mg kg−1): A) Cd, Hg, Pb, Sb; B) Cr, Ni, Cu, As; C) Al, Fe,
Mn, Zn; Censored data were substituted with 0.5DL.
hand, several fish from our study indicated metal contamination in
muscle tissue, where many samples showed Cd, Hg and Pb concentra-
tions above legislation limits. This is obviously to be considered posing
a risk for human consumption.
Histopathology is confirmed to be a valid biomarker to assess the
health status of bio-indicators. However, it is necessary to consider that
21
Marine Pollution Bulletin 141 (2019) 16–23
Fig. 3. Correlation between muscle and gill concentration (mg kg−1) for sam-
ples from the 1st sampling at Sestri Levante, Chiavari, and Lavagna (scatter plot
with linear trend-line with R2 value). Censored data were substituted with
0.5DL.
Fig. 4. Frequency of four categories of Index of gill damage in sampling po-
pulation.
it is absolutely non-specific, because tissue modifications can be caused
by different pollutants and stressors, even if they can also obviously be
related to metal contamination when it occurs. Random sampling is
proved important to obtain actual and realistic data on different fish
species and to compare them, but the sampling effort should be in-
creased, focusing on only one or two species. Despite bio-monitoring
being affected by several confounding factors, it is the most realistic
approach to assess the actual health status of an ecosystem, and our
study confirms the benefits of using it to obtain data for a preliminary
evaluation.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.marpolbul.2019.02.024.
Acknowledgements
This work was self-funded and did not receive any specific grant
from funding agencies in the public, commercial, or not-for-profit sec-
tors.
A. Reboa, et al.
Fig. 5. Images of 4 μm gill sections stained with Haematoxylin and Eosin at 20× magnification, showing tissue alterations: A) Secondary Lamellae Shortening
(double arrow); B) Secondary Lamellae Epithelial Hyperplasia (blank arrows); C) Primary Lamellae Epithelial Hyperplasia resulting in lamellar fusion (stars); D)
Secondary Lamellae Epithelial Lifting (arrows) with oedema (*) and blood vessels congestion (thin arrows); Abbreviations: primary lamellae (PL).
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