Environmental Research 137 (2015) 246–255

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Review

Effect of heavy metals on fish larvae deformities: A review

D.G. Sfakianakis a,n, E. Renieri b, M. Kentouri a, A.M. Tsatsakis b
a
Biology Department, University of Crete, 71409 Heraklio, Crete, Greece
b
Laboratory of Toxicology, University of Crete, Medical School, Crete 71409, Greece

art ic l e i nf o a b s t r a c t

Article history: Heavy metals have been associated with many fish deformities in natural populations and in laboratory
Received 3 September 2014 produced specimens as well. Deformities in general have devastating effects on fish populations since
Received in revised form they affect the survival, the growth rates, the welfare and their external image. Although the embryonic
5 December 2014
stage in respect to heavy metal exposure has been extensively studied, there is not much information
Accepted 15 December 2014
available as to what happens in fish larvae and adults. In the present article, we present the available
information on the effect of heavy metals on fish larvae deformities. We also address the need for more
Keywords: research towards the effects of metals on the subsequent life stages in order to assess the long-term
Heavy metals consequences of heavy metal poisoning on fish organisms and possibly correlate these consequences
Fish larvae
with the environmental contamination (use as biomarkers).
Deformities
& 2014 Elsevier Inc. All rights reserved.
Environment
Biomarkers

1. Introduction which are equivalent to or worse than those caused by non-

Heavy metals have been associated with many fish deformities
in natural populations as well as in laboratory produced speci-
mens (Bengtsson and Larsson, 1986; Cheng et al., 2000; Jezierska
et al., 2009a, 2009b). The presence of pollutants and especially
heavy metals in the aquatic environments of fish (seas, rivers, la-
goons) leads to severe adverse effects on the organisms and has
been a subject of concern for many decades (Bengtsson et al., 1979;
Muramoto, 1981). Early in the 1970s, many attempts were made to
identify the effects of heavy metals on organisms such as the effect
of cadmium on vertebral deformities of Cyprinus carpio (Mur-
amoto, 1981) and Pimephales promelas (Pickering and Gast, 1972)
or the effect of zinc on the vertebral column of Phoxinus phoxinus
(Bengtsson, 1974).
In general, metals can be categorized as biologically essential
and nonessential. The nonessential metals (e.g., aluminum (Al),
cadmium (Cd), mercury (Hg), tin (Sn) and lead (Pb)) have no
proven biological function (also called xenobiotics or foreign ele-
ments), and their toxicity rises with increasing concentration. Es-
sential metals (e.g., copper (Cu), zinc (Zn), chromium (Cr), nickel
(Ni), cobalt (Co), molybdenum (Mo) and iron (Fe)) on the other
hand, have a known biological role, and toxicity occurs either at
metabolic deficiencies or at high concentrations. The deficiency of
an essential metal can therefore cause an adverse health effect,
whereas it’s high concentration can also result in negative impacts
n
Corresponding author. Fax: þ30 2810394408.
E-mail address: sfak@biology.uoc.gr (D.G. Sfakianakis).
essential metals (Kennedy, 2011). The most commonly found
heavy metals in fish organisms are cadmium, lead, mercury, zinc,
copper, nickel, cobalt, molybdenum, chromium and tin. Amongst
them, the most frequently studied, with respect to fish defor-
mities, include cadmium, copper, lead, zinc, mercury and
chromium.
The most bioavailable form of metals that results in toxicity is
believed to be the dissolved ionic form. Significant metal toxicity
in fish can also derive from the organic forms of several metals
including tin and mercury. Multiple physiological systems are af-
fected by metals (commonly the gills) and toxicity depends on
metal form and speciation, bioavailability, toxicokinetics (absorp-
tion, distribution, biotransformation, and excretion), and tox-
icodynamics (interactions with ligands) (Kennedy, 2011). Heavy
metals accumulate in the tissues of aquatic animals and become
toxic when concentrations reach certain toxicity thresholds, values
which vary considerably among metals, metal species, taxonomic
species and organism life stages. Fish absorb metals mainly
through the gills and the digestive track, and to a lesser extent,
through the skin (Kennedy, 2011). For more information on heavy
metals, environment and fish interactions see Kennedy (2011).
On the other hand, fish deformities have devastating effects on
fish populations since they affect their survival, growth rate,
welfare and external morphology (Boglione et al., 2013; Divanach
et al., 1996). Some of the most common deformities can be located
in the vertebral column (Koumoundouros et al., 2002; Sfakianakis
et al., 2006), the swimbladder (Chatain, 1994; Divanach et al.,
1996), the cephalic region (Georgakopoulou et al., 2007), the fins

http://dx.doi.org/10.1016/j.envres.2014.12.014
0013-9351/& 2014 Elsevier Inc. All rights reserved.
 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255
(Favaloro and Mazzola, 2003; Koumoundouros et al., 2001, 1997;
Sfakianakis et al., 2003) and the lateral line (Sfakianakis et al.,
2013a). The most frequent of them are the ones in the vertebral
column and especially lordosis (V-shaped dorsal–ventral curva-
ture), kyphosis (Λ-shaped dorsal–ventral curvature) and scoliosis
(lateral curvature). Fish deformities (especially skeletal ones) are
quite important since they interfere with the organism's ability to
interact with the environment. A distinctive example is the im-
pairment of the swimming ability (Sfakianakis et al., 2011) which
is the most crucial characteristic in accomplishing life important
actions such as prey hunting, predator avoidance, traveling etc.
Heavy metals may adversely affect various metabolic processes
in developing fish (embryos in particular), resulting in develop-
mental retardation, morphological and functional deformities, or
death of the most sensitive individuals. Additionally, heavy metals
activate energy-consuming detoxification processes; thus, in in-
toxicated fish less energy can be used for growth. Most of the
studies of heavy metals on developing fish (embryos or larvae)
report high incidences of mortality, hatching delay, altered body
shape, and body anomalies (Jezierska et al., 2009a, 2009b).
Fish embryonic and larval stages are generally considered to be
the most sensitive in terms of toxicity during the entire fish life
cycle (Osman et al., 2007; Zhang et al., 2012). On the other hand,
adult exposures are not entirely risk-free; metal exposure of
spawners may result in contamination of eggs and sperm, and
reduce fish fertility and embryonic development. In many cases it
is proven that exposure of adults to heavy metals leads to the
latter being deposited in the testes and ovaries (Jezierska et al.,
2009a). Generally, prenatal or early postnatal life is very vulner-
able and sensitive to any type of xenobiotics (Ankley and Johnson,
2004) and exposure during these critical periods may cause pro-
found effects during the entire lifetime of a fish (Birnbaum, 1994).
Heavy metals also act as endocrine disrupters in fish, e.g.,
cadmium was reported to reduce thyroid hormone levels (Hontela
et al., 1996), inhibit estrogen receptors and disrupt growth hor-
mone expression (Guével et al., 2000), while lead inhibits thyroid
hormone synthesis by affecting iodine metabolism (Chaurasia
et al., 1996). Prooxidative properties of metal ions may result in
oxidative stress in fish and oxidative damage to the cell mem-
branes. Cadmium, copper mercury and lead also exert a genotoxic
effect on fish (Çavaş, 2008; Cavas et al., 2005).
Over the past decades, it has been suggested by scientists and
public organizations that fish deformities can be used as bio-
markers of environmental exposures (Au, 2004). Morphological
deformity assessment is believed to be one of the most straight-
forward methods to study the effects of contamination on fish due
to the ease of recognition and examination when compared with
other types of biomarkers (Sun et al., 2009). In many cases, fish
deformities are easy to distinguish – even macroscopically – and
thus offer a tremendous advantage over other methods especially
for scientists working on the field away from the laboratory
equipment.
Toxicants such as heavy metals in water have posed a serious
risk to aquatic organisms and public safety. These contaminants
are frequently incorporated into food chain via water, micro-
organisms, plants, fish, and then enter human bodies through
drinking water and fishery foods. Many recent studies exhibit the
accumulation of various metals on animals’ livers and kidneys
especially in closed seas such as the Mediterranean and address
the need for close scene monitoring (Storelli et al., 2005).
The embryonic stage has been extensively studied (reviewed by
Jezierska et al. (2009a)) probably due to fact that specimen ma-
nipulation and handling is easier and faster. However, there is still
quite a big gap of knowledge as to what happens in fish larvae and
adults. In the present review we tried to summon the – limited –
relatively recent literature on the matter and address the need for
247
more research, targeted in life stages after the embryonic cycle. In
order to do so, we focused on the studies that test the effect of
metal on a larvae (or adult), but due to the surprisingly small
number of experiments, we also included studies which, although
the exposure to metals was carried out on the embryonic stage,
the assessment of their effects was performed on live hatched
larva.
2. Heavy metal exposure in laboratory fish populations
2.1. The effect of cadmium (Cd)
Cadmium is a naturally occurring non-essential trace element
and its' tendency to bioaccumulate in living organisms often in
hazardous levels, raises environmental concern (Kalman et al.,
2010; Liao et al., 2011). The severity of cadmium toxicity in aquatic
organisms as well as the fact that human activities such as disposal
of industrial wastes and mining, are the primary routes of its re-
lease in the environment, have rendered it a priority pollutant
research wise (Kalman et al., 2010; Maunder et al., 2011). Cad-
mium accumulates in aquatic organisms via dietary or aqueous
exposure (Liao et al., 2011). The toxicity of Cd to aquatic species
depends on speciation, with Cd2 þ being the mainly absorbed
species, following primarily the gill and intestine uptake pathways
and secondarily the branchial one (McGeer et al., 2011). The main
mechanism of toxicity is the antagonistic interaction between the
uptake of Ca2 þ and Cd2 þ , which disrupts Ca2 þ absorption
(McGeer et al., 2011) leading to acute hypocalcaemia and growth
reduction, problematic reproduction, as well as impairments in
development and behavior (Dang and Wang, 2009; Maunder et al.,
2011; McGeer et al., 2011). A concentrated list of cadmium induced
deformities in fish is presented in Table 1.
Witeska et al. (1995) exposed Common carp (C. carpio) eggs at
different concentrations of cadmium (0.001–0.05 ppm) until
hatching. They reported head deformities and spinal curvatures
which ranged from 0% to 47% in the different populations. Inter-
estingly, the higher concentration of cadmium (0.05 ppm) pre-
sented one of the lower deformities incidence (5%). The authors
also used Common carp larvae younger than 10 days, between 10
and 20 days old and older than 20 days for 96-h acute toxicity tests
(0–0.017 ppm) but did not observe any deformities. The results of
this study indicate the protective role of the egg shell, as the newly
hatched larvae proved to be more susceptible than the eggs.
Moreover, the results show that susceptibility decreases with age
since the 96-h LC50 for fish under 10 days, between 10 and 20 days
and over 20 days was 0.002 ppm, 0.005 ppm, 0.007 ppm of cad-
mium respectively.
Williams and Holdway (2000) studied the effects 2 h pulse-
exposure of cadmium on early life stages of Australian crimson
spotted rainbow fish (Melanotaenia fluviatilis). The range of the
concentrations used was 0.033, 0.1, 0.33, 1, and 3.3 mg/L. The age
of the embryos was 3, 46 and 92 h (post fertilization). Cadmium
affected hatching, larval survival and spinal deformities (without
specifying which ones). Higher cadmium concentration and
smaller embryo age resulted in more adverse effects. Deformed
specimens (spinal deformities) reached up to 27% in the popula-
tions. The authors also tested the effect of zinc on the species
larvae without however checking for deformities.
Nguyen and Janssen (2002) studied the African catfish (Clarias
gariepinus) with the effect of metal starting after fertilization and
lasting for 5 days. Concentrations used varied from 0.05 to 5 mg/L
(CdCl2  2.5H2O). The main deformity they reported was reduction
of body pigmentation which reached percentages of up to 100% at
the highest concentration and was significantly higher from the
control (0 mg/L) at concentrations above 0.5 mg/L.
248 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255

Benaduce et al. (2008) studied the effect of waterborne Cd (as

Nguyen and Janssen, (2002)

CdCl2) in eggs and larvae of silver catfish (Rhamdia quelen) at two

Williams and Holdway,

Benaduce et al. (2008)

Benaduce et al. (2008)

Barjhoux et al. (2012)

Barjhoux et al. (2012)
alkalinity levels (63 and 92 mg/ L CaCO3) and at 4 Cd concentra-

Witeska et al. (1995)

Witeska et al. (2014)

tions: 0.5 (control), 4.5, 8, and 18 μg/L from egg fertilization until

Zhang et al. (2012)

Sassi et al. (2010)
Cao et al. (2009)
21 days after hatching. They reported deformities in the barbel and
the spinal column of the larvae (without specifying the types of
the deformities) that reached up to 100% incidence. They also
(2000)
Source

observed that larvae exposed to the lower alkalinity, presented a

higher incidence of barbels and spinal column deformities in the
highest waterborne Cd concentration.
Cao et al. (2009) studied the effect of cadmium on embryonic-
larval development of red sea bream, Pagrus major. They applied
% Deformed in the

concentrations of 0–3.2 mg CD2 þ /L from 3 h post fertilization to

96 h post hatching when larvae opened mouth and were ready to
population

feed (entire embryonic and prelarval stage). Hatchability rate

32.5–52.5
40.7–58.6
22.1–25.6
 5–100

ranged from 100% in the control to 0% in the highest Cd con-

0–91.7
0–100

1–100

15–92
5–27
0–47

 35
centration (3.2) and mortality ranged from 1% to 100% respec-
tively. Abnormalities also ranged from 1% to 100% respectively and
included “cardiac edema, degenerated and hooked tails, fins le-
Spinal (kyphosis, lordosis and scoliosis)
Head deformities and spinal curvatures

Spinal (mainly kyphosis and lordosis)

sions and spinal curvature” with skeletal deformities being the

Skeletal deformities (spine, tail, fins)

most pronounced.
Vertebral curvatures and yolk sac

Sassi et al. (2010) investigated the effect of cadmium (0.4 mg/L

Reduction of pigmentation

CdCl2) in juvenile mosquitofish (Gambusia affinis) in respect to

temperature (24 or 32 °C). The exposure period begun 1 day after
hatching and lasted for 30 days. Deformities reported were at
Spinal curvatures?
Type of deformity

Deformed barbel

32.5–52.5% incidence in the population and included lordosis,

Cardiovascular
Spinal column

kyphosis, scoliosis and 1 case of simultaneous presence of all three

deformities

deformities (LSK syndrome). The authors also concluded that the

higher the water temperature the higher the Cd toxicity and these
Spinal

results should be considered in subsequent studies.

Barjhoux et al. (2012) studied the effect of Cd spiked sediment
on Japanese medaka (Oryzias latipes) during the entire embryonic
From egg fertilization to 21 days after

From egg fertilization to 21 days after

stage (concentrations varied between 0.18 and 19.8 μg/g d.w.).

From egg fertilization to hatching

They observed deformed larvae up to 72% in the populations.

Specimens were examined at hatching and the deformities found
were mostly spinal (mainly kyphosis, lordosis and C-shaped lar-
vae) and cardio-vascular (mainly abnormal positioning and heart
Duration of exposure

looping). They reported LOEC (Lowest Observed Effect Con-

Until first feeding

centration) value for Cd at 1.9 mg/g d.w. sediment.

Until hatching
The effect of cadmium (Cd) exposure on fish deformities (nominal concentrations are used).

Zhang et al. (2012) studied the toxicity of cadmium (0.1–

30,000 μg/L CdCl2) to the embryos and pre-larvae of Soldatov's
hatching

hatching

30 Days
10 Days
10 Days
5 Days

144 h

catfish (Silurus soldatovi) and although they report many adverse

2h

effects (they mention “wavy notochords” but do not specify its

frequency in the populations or whether it is the only deformity
observed in larvae), as they call the various deformities which
Embryonic and prelarval

Embryonic and prelarval

Embryonic and prelarval

reach incidences of up to 92% in the populations, they chose not to

describe them and distinguish their appearance.
3, 46 and 92 h post
Stage of exposure

Witeska et al. (2014) studied the effects of Cd (100 μg/L) on the

1d Post hatching
Embryonic stage

Embryonic stage

Embryonic stage
Embryonic stage
Embryonic stage
Embryonic stage

embryonic, larval or both stages of the ide, Leuciscus idus. Their

fertilization

results showed that metal toxication affected mortality, body size,

various body morphometrics and deformities (vertebral curva-
stage

stage

stage

tures and yolk sac deformities). As far as the deformities are

concerned, they reported that at the end of the embryonic stage,
0.0005–0.018 mg/L

0.0005–0.018 mg/L

0.18–19.8 μg/g d.w.

0.18–19.8 μg/g d.w.
0.0001–30 mg/L

deformed larvae following exposure to Cd were about 35% in the

0.001–0.05 ppm
Melanotaenia fluviatilis 0.033–3.3 mg/L
Concentration

population. Also, they reported that cadmium was more toxic to

0.05–5 mg/L

0–3.2 mg/L

the ide embryos and larvae than copper which was also included
0.4 mg/L

0.1 mg/L

in the same study. The same authors continued assessing the effect
of Cd in the ide larvae until 21 days after hatching but did not
examine the deformities at that stage.

2.2. The effect of copper (Cu)

Clarias gariepinus

Gambusia affinis

Silurus soldatovi
Rhamdia quelen

Rhamdia quelen
Cyprinus carpio

Oryzias latipes
Oryzias latipes

Leuciscus idus
Pagrus major

Copper is an abundant element which occurs as a natural mi-

Species

neral with a widespread use. It is also an essential micronutrient

Table 1

for living organisms on account of being a key constituent of

metabolic enzymes (Monteiro et al., 2009). However it can be toxic
 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255 249

to intracellular mechanisms at concentrations which exceed nor-

mal levels (Hernández et al., 2006). Fish can accumulate copper

Barjhoux et al. (2012)

Barjhoux et al. (2012)
Mochida et al. (2008)
Johnson et al. (2007)

Witeska et al. (2014)

Nguyen and Janssen,
via diet or ambient exposure. A fluctuation of the dietary copper

Kong et al. (2013)

load can lead to altered fish physiology with protein malfunctions
(Grosell, 2011; Hernández et al., 2006). Additionally, it is well
documented in a variety of fish species, that excess waterborne

% Deformed in the population Source

(2002)
copper exposure, will have deleterious effects on the gills, gut and
sensory systems (Johnson et al., 2007). Recently, it has been rea-
lized that extended Cu exposure can evoke an acclimation re-
sponse, allowing fish not only to survive but also endure further
exposure to higher concentrations. However, there are re-
productive impairments, reduced growth and behavioral changes
(Grosell, 2011). Moreover, there is a difference in Cu regulation
among marine and freshwater fish, due to the extra intestinal Cu
Not provided

uptake from seawater in marine fish (osmotic homeostasis).

18.6–35.2
11.5–29.2
 15–70

Freshwater fish seem to accumulate Cu mainly through the dietary

 1–10
0–100

 15
route (Dang et al., 2009). A concentrated list of copper induced
deformities in fish is presented in Table 2.
Lateral line deformities (fewer functional neuromasts)

Nguyen and Janssen (2002) studied the African catfish with the
metal exposure beginning at fertilization and lasting for 5 days.
Vertebral deformities and inflammatory masses

Concentrations used varied from 0.15 to 2.5 mg/L (CuSO4  5H2O).

Vertebral curvatures and yolk sac deformities

The main deformity they reported was the reduction of body

pigmentation which reached percentages of up to 70% and was
Spinal (mainly kyphosis and lordosis)

significantly higher from the control (0 mg/L) at concentrations

over 0.6 mg/L.
Johnson et al. (2007) studied the effect of Cu on zebrafish
From egg fertilization to 24 h after hatching Scoliosis and tail curvatures
Reduction of pigmentation

(Danio rerio) embryos from fertilization up to 120 h. Two copper

concentrations were used (68 and 244 μg Cu L  1) and the results
showed fewer functional neuromasts and larvae’s inability to or-
Type of deformity

ientate in a water current. Moreover, the authors observed mor-

Cardiovascular

tality, hatching inhibition and impairment of larval development.

On the same matter, Hernández et al. (2006) subjected zebrafish
larvae of 2–5 days old to non-lethal concentrations of copper (1–
50 μM CuSO4) for a 2 h pulse exposure and discovered that the
lateral line was impaired (neuromasts cellular damage, apoptosis,
and loss of hair cell markers). They also tested other metals (such
as Zn, Fe, Ag, Mn, Co, Cd and Sn) but did not observe the same
From egg fertilization to hatching

effects. The authors also reported regeneration of the damaged

From egg fertilization for 120 h

area providing that the Cu concentration was below a certain

threshold. The same conclusions for the effect of dissolved copper
The effect of copper (Cu) exposure on fish deformities (nominal concentrations are used).

on zebrafish were reported in Linbo et al. (2006, 2009).

Stage of exposure Duration of exposure

Mochida et al. (2008) used a teleost fish, the mummichog

(Fundulus heteroclitus), to conduct early life-stage toxicity testing
for copper pyrithione (CuPT). Fertilized mummichog eggs were
exposed to CuPT at various concentrations (0.5–4 μg/L) for 50 days
50 Days
10 Days
10 Days
5 Days

under continuous flow-through conditions. Hatchability, survival,

growth, and morphological deformities were measured. Hatch-
ability did not differ significantly between any experimental group
Embryonic stage

stage
stage
stage
stage
stage
stage

and control groups. Survival and growth were significantly re-

duced at 50 days in the groups exposed to 2 or 4 μg/L) CuPT.
Embryonic
Embryonic
Embryonic
Embryonic
Embryonic
Embryonic

During the test, abnormalities, such as vertebral deformity (lor-

dosis, kyphosis and scoliosis) and formation of inflammatory
masses in the lateral muscles, occurred in fish exposed to CuPT.
0.0005–0.004 mg/L
6.95–23.1 μg/g d.w.
6.95–23.1 μg/g d.w.

Light and electron microscopic studies indicated that muscle

0.068–0.244 mg/L

dysfunction played a role in the vertebral deformity and revealed

Concentration

0.15–2.5 mg/L

that the inflammatory masses were composed mainly of macro-

0.1–1 mg/L

phages and necrotic myocytes. The study has great variations in

0.1 mg/L

the deformities incidence but the general trend is that the longer
the exposure (50 days vs. 40 and 30) combined with higher doses
(4 μg/L), the more deformed specimens become (up to 100% in the
Fundulus heteroclitus

population).
Clarias gariepinus

Carassius auratus
Oryzias latipes
Oryzias latipes

Barjhoux et al. (2012) studied the effect of Cu spiked sediment

Leuciscus idus
Danio rerio

on the Japanese medaka during the entire embryonic stage (con-

centrations varied between 6.95 and 23.1 μg/g d.w.). They ob-
Species
Table 2

served deformed larvae up to 52% in the populations. Specimens

were examined at hatching and the deformities found were mostly
250 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255
spinal (mainly kyphosis, lordosis and C-shaped larvae) and cardio-
vascular (mainly abnormal positioning and heart looping). They
reported LOEC (Lowest Observed Effect Concentration) value for
Cu at 8.5 mg/g d.w. sediment.
Kong et al. (2013) studied the effect of various concentrations of
Cu (0.1, 0.4, 0.7 and 1 mg/L) from egg fertilization until 24 h after
hatching on the Goldfish, Carassius auratus. They observed sco-
liosis and tail curvatures (under a microscope) and concluded that
the deformities rate increased gradually with increasing Cu con-
centrations. The highest deformity incidence was about 10% in the
population at Cu concentration of 1 mg/L.
Finally, Witeska et al. (2014) studied the effects of Cu (100 μg/L)
on the embryonic, larval or both stages of the ide, Leuciscus idus.
Their results showed that metal toxication affected mortality, body
size, various body morphometrics (specifically the body
area perimeter and the swimbladder area perimeter) and defor-
mities (vertebral curvatures and yolk sac deformities). As far
as the deformities are concerned, they reported that at the end of
the embryonic stage, deformed larvae following the exposure to
Cu represented about 15% of the population. The same authors
continued studying the effect of Cu in the ide larvae until 21
days after hatching but did not examine the deformities at that
stage.
2.3. The effect of lead (Pb), zinc (Zn) and chromium (Cr)
Those four metals have drawn much less attention compared to
Cd and Cu and the studies on fish deformities are quite scarce. A
concentrated list of lead, zinc and chromium induced deformities
in fish is presented in Table 3.
Lead is a persistent heavy metal which has been characterized
as a priority hazardous substance (EU Directive 1907/2006). Al-
though Pb is a naturally occurring substance, its environmental
concentrations are significantly increased by anthropogenic sour-
ces which include base metal mining, battery manufacturing, Pb-
based paints and leaded gasoline (Mager, 2011; Monteiro et al.,
2011). The aquatic systems are subsequently contaminated via
superficial soil erosion and atmospheric deposition. The con-
centration and bioavailability of Pb is mainly dependent on the
absorption into the sediments and the natural organic matter
content of the water as well as the pH, alkalinity and hardness
(Mager, 2011; Sepe et al., 2003). Aquatic organisms bioaccumulate
Pb from water and diet, although there is evidence that Pb accu-
mulation in fish, is most probably originated from contaminated
water rather than diet (Cretì et al., 2010). The most toxic form in
water is considered to be the free Pb ion (Pb2 þ ) (Mager, 2011;
Monteiro et al., 2011). Toxic effects of Pb in fish include disruption
of Na þ , Cl- and Ca2 þ regulation and development of black tails,
while the principal toxic effects of chronic lead exposure to fish are
hematological and neurological (Mager, 2011).
Table 3
The effect of lead (Pb), zinc (Zn) and chromium (Cr) exposure on fish deformities (nominal concentrations are used).
Species Metal (Concentration) Stage of exposure
Fundulus heteroclitus Pb (0.1– 10mg/L ) Embryonic stage
Clarias gariepinus Pb (0.1–0.5 mg/L ) 6 h Embryos
Clarias gariepinus Pb (0.1–0.5 mg/L ) 6 h Embryos
Clarias gariepinus Pb (0.1–0.5 mg/L ) 6 h Embryos
Clarias gariepinus Pb (0.1–0.5 mg/L ) 6 h Embryos
Acipenser sinensis Pb (0,2–1,6 mg/L) 96 h Embryos
Melanotaenia fluviatilis Zn (0.33–33.3 mg/L) 3, 46 and 92 h post
fertilization
Clarias gariepinus Cr (11–114 mg/L) Embryonic stage
Weis and Weis (1977) studied the effect of mercury, cadmium
and lead exposure on the development of the killifish (F. hetero-
clitus) and observed deformities only as an effect of the lead. They
treated eggs with PbNO3 at 3 different concentrations (0.1, 1 and
10 mg/L) from the blastula stage until hatching where the ex-
amination for skeletal deformities took place. They observed that
at 1 mg/L, only 20% of the larvae were normal, 40% exhibited lor-
dosis and the other 40% were permanently bent (curled up). At the
concentration of 10 mg/L all specimens (100%) were permanently
bent, whereas at 0.1 mg/L, no deformed specimens were observed
(0%).
Osman et al. (2007) studied the effect of lead exposure (100,
300, and 500 μg/L lead nitrate) on embryos of the African catfish.
Larvae were examined at 48 hpf (hours post fertilization), 96 hpf,
144 hpf, and 168 hpf (hatching took place after about 40 h). Ex-
posure to lead nitrate caused a progressively longer delay in
hatching and reduced the percentage of embryos which success-
fully completed hatching from 75% in the control group to 40% in
the group exposed to 500 μg/L lead. Four major deformities were
observed (irregular head shape, pericardial edema, yolk sac ede-
ma, and notochordal defect) and two minor ones (finfold defect
and reduction of pigmentation) while their frequencies increased
significantly with increasing lead concentration in all stages. All
the deformities were recorded in the embryos exposed to 300 and
500 g/L lead. It is worth mentioning that higher frequencies of
deformities were observed at the first sampling point (48 hpf,
right after hatching) proving that most of them were lethal to
larvae.
Hou et al. (2011) monitored the effect of lead on the Chinese
sturgeon, Acipenser sinensis. They used 3 concentrations (0.2,
0.8 and 1.6 mg L  1) for a period of 112 days (from eggs to juve-
niles). They observed deformities in the two higher concentrations
that reached 100% in the populations by the end of the experi-
mental period. The deformities they documented were body
(spinal) curvatures. All deformed specimens recovered completely
after a depuration phase of 5 weeks. The authors also reported
reduced ability of locomotion and foraging by deformed juveniles.
Although it does not represent an actual deformity, it was
found that in Catla catla fingerlings, exposure to 15.5 ppm con-
centration of Pb(NO3)2 for 60 days formed certain changes in the
gills (hypertrophy, hyperplasia etc.), which however recovered
after return to lead free environment (Palaniappan et al., 2008).
Zinc is an essential micronutrient, found almost in every cell
and it is the second most abundant trace element after Fe. Zinc is a
regulatory catalyst of a number of metalloenzymes and is also
required for the metabolism of biological molecules including
proteins and nucleic acids. Additionally, Zn is involved in more
complicated functions, such as the immune system, neuro-
transmission and cell signaling (Celik and Oehlenschläger, 2004;
Hogstrand, 2011). It can become toxic for fish at increased
Duration of exposure Type of deformity % Deformed in the Source
population
From blastula stage Lordosis and curled 0–100 Weis and Weis,
to hatching up body (1977)
48–168 h Irregular head 0–34.3 Osman et al. (2007)
48–168 h Notochord defects 4.3–69.7 Osman et al. (2007)
48–168 h Yolk Sac edema 0–3.3 Osman et al. (2007)
48–168 h Finfold defect 0–35 Osman et al. (2007)
112 Days Spinal curvatures 0–100 Hou et al. (2011)
2h Spinal deformities 3–27 Williams and Hold-
way, (2000)
5 Days Body axis  25–100 Nguyen and Janssen,
(2002)
 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255
waterborne levels (Niyogi and Wood, 2006), while dietary Zn has
mild toxic effects on fish (Hogstrand, 2011). The main target of
waterborne Zn toxicity are the gills (Hogstrand, 2011), where the
Ca2 þ uptake is disrupted, leading to hypocalcemia and eventual
death (Niyogi and Wood, 2006). The other endpoints of toxicity
vary amongst freshwater and marine fish with the most common
being survival, growth, reproduction, and hatching (Hogstrand,
2011). However, chronic exposure to Zn may lead to an acclimation
response for the surviving fish (Hogstrand, 2011; Niyogi and
Wood, 2006).
Williams and Holdway (2000) studied the effect of 2 h pulse-
exposure of zinc on early life stages of Australian crimson spotted
rainbow fish. The zinc concentrations used were 0.33, 1, 3.3, 10,
and 33.33 mg/L. The age of the embryos was 3, 46 and 92 h (post
fertilization) and zinc affected hatching, larval survival and spinal
deformities. The higher the concentration and the younger the
embryo resulted in more adverse effects. Deformed specimens
(considering only spinal deformities) reached up to 27% in the
populations. The authors also tested the effect of zinc on the
species' larvae without however checking for deformities.
Chromium (Cr) is an ubiquitous element which speciates
mainly in two forms in the environment, depending on physico-
chemical characteristics: the most commonly found trivalent Cr
(III) and the most toxic hexavalent Cr (VI) which is primarily
produced from anthropogenic sources such as industrial wastes.
Poor treatment of these effluents can lead to the presence of Cr
(VI) in the surrounding water bodies, where it is commonly found
at potentially harmful levels to fish (Li et al., 2011; Pacheco et al.,
2013). Cr, apart from being classified as a Group I carcinogen (IARC,
1990), has been reported to have a number of adverse effects on
fish organism. Fish assimilate Cr by ingestion or by the gill uptake
tract and accumulation in fish tissues, mainly liver, occurs at
higher concentrations than those found in the environment
(Ahmed et al., 2013; Pacheco et al., 2013). Toxic effects of Cr in fish
include: hematological, histological and morphological alterations,
inhibition/reduction of growth, production of reactive oxygen
species (ROS) and impaired immune function (Reid, 2011; Vera-
Candioti et al., 2011).
The effect of Chromium on the African catfish was studied on
one occasion (Nguyen and Janssen, 2002). The exposure took place
right after fertilization and lasted for 5 days. Concentrations used
varied from 11 to 114 mg/L (K2Cr2O7). The main deformity re-
ported was abnormal body axis which reached percentages of up
to 100% and was significantly higher than the control (0 mg/L) at
concentrations over 36 mg/L.
3. Heavy metal exposure in natural fish populations
The studies of natural fish populations' exposure to heavy
metals have not been conducted under controlled laboratory
conditions and therefore precision in details of important para-
meters (such as the metal's exact concentration, the possible sy-
nergistic effect of other pollutants present in the area etc.) is
lacking. Those studies are based on the metal’s tracing primarily
on fish tissues or secondarily in the water or sediment of the
natural habitat.
As far as fish deformities are concerned, it is well established
today that they mostly appear in cultured populations mainly due
to the environmental conditions prevailing and secondarily due to
other factors such as genetics, general husbandry etc. (Boglione
et al., 2013; Sfakianakis et al., 2006, 2013b). In some cases, the
incidence of deformities in fish populations under aquaculture
conditions has been reported to be over 80% (Sfakianakis et al.,
2006). Wild populations on the other hand, are considered to be
ideal and therefore – in general – free of deformities (Sfakianakis
251
et al., 2004). Consequently, when natural fish populations bearing
deformities are discovered, the first assumption always is that
there must have been – at some level – an exposure to certain
pollutants (usually organic compounds or heavy metals) (Bengts-
son and Larsson, 1986; Bengtsson et al., 1985, 1979).
There are quite a few studies on wild fish deformities but most
of them simply hypothesize that the causative factor is probably
the presence of heavy metals on the natural habitat. Only a limited
number of articles in the recent literature directly correlate the
presence of fish deformities with heavy metals either on fish tis-
sues or on the habitat’s water or sediment.
Kessabi et al. (2009) sampled populations of the Mediterranean
killifish, Alphanius fasciatus, from different unpolluted and polluted
sites off the coast of Tunisia and discovered that the deformed
specimens (originating only from the polluted sampling areas)
presented higher Cd concentrations in their livers and spinal
columns when compared to the normal ones. Moreover, they
reported that the bioaccumulation factors of Cd in the liver in de-
formed fish were also significantly higher than in normal fish, thus
suggesting that the ability of fish to accumulate large amount of Cd
may represent a potential risk to induce spinal deformities. In a
follow up study by the same authors in the same species (Kessabi
et al., 2010), quantitative RNA biomarkers were used and it was
suggested that a combined effect of both heavy metals and organic
pollutants is what actually leads to the presence of deformities.
Sun et al. (2009) studied Tilapia (Oreochromis spp.) populations
collected from different rivers in Taiwan. They discovered the
presence of many different skeletal deformities in the vertebral
column, cranium, operculum, fins and jaws. They correlated these
deformities with the severe pollution of the rivers with heavy
metals (Hg, Zn, Pb, Cu and Cr) and organic compounds. In this
study, there seems to be a slight correlation between certain
deformities and certain pollutants but nothing is definitive. The
authors argue that Tilapia is quite tolerant and presents more
deformities than other species and could therefore serve as a
reliable biomarker of the environment pollution in general.
In a recent study (Kessabi et al., 2013a), skeletal deformities in
the vertebral column of Mediterranean killifish specimens, col-
lected from the Tunisian coast, were linked with high concentra-
tion of heavy metals (Cd, Cu and Zn), various polycyclic aromatic
hydrocarbons (PAHs) and estrogenic compounds discovered in
water and sediment of the sampling areas. The incidence of de-
formed specimens reached up to 18% in a certain polluted sam-
pling area while it did not exceed 5% in the unpolluted reference
area. The authors did not discriminate between the different xe-
nobiotics discovered and therefore the direct effect of only the
heavy metals remains unknown.
Besides sampling fish from their natural habitats, Skinner et al.
(1999) had the idea of exposing hatched eggs of two species (Ja-
panese medaka, O. latipes and Inland Silverside, Menidia beryllina)
to different percentages of storm water sampled in an urban area
and contaminated with various pollutants (including Cd, Cu, Pd,
Zn, Cr and Ni). Their findings showed that the contaminated storm
water induced various deformities in the hatched larvae of both
species with the most prominent ones being the abnormal
swimbladder inflation, the spinal curvatures and the reduction of
pigmentation. The presence of significant adverse effects in this
study did not correlate significantly with any of the individual
pollutants that were traced in the storm water, but did correspond
with total toxic metal pollutants in the samples (especially Cd, Cu,
Pb and Zn).
Messaoudi et al. (2009) studied natural populations of Grass
goby (Zosterisessor ophiocephalus) deriving from one polluted and
one unpolluted area in the Gulf of Gabes in Tunisia. They dis-
covered spinal deformities (mainly kyphosis, scoliosis and lordo-
sis) which were more often in the polluted area specimens
252 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255
(roughly 11% of the population). Liver concentrations of Cd and Zn
were significantly higher in the deformed specimens when com-
pared to the normal ones. The study also included analysis of Cu
(both in water and organisms’ liver) but it did not seem to corre-
late with the observed deformities. Although no other pollutants
were included in this study, it seems that a strong correlation
between accumulation of the two metals (Cd and Zn) and spinal
deformities was revealed.
The general conclusion from the studies on natural fish popu-
lations is that the causes of the occurrence of spinal deformities in
natural populations of fish are complicated and, to a large extent,
unknown. They may be a response to a number of biotic (e.g.,
hereditary defects, parasite infections) and abiotic factors (e.g.,
vitamin deficiencies, electrical shock etc.) which are not related to
pollution (Kessabi et al., 2009).
4. Discussion
The pathway through which heavy metals induce deformities
in fish is not quite clear yet and it seems to be different for each
metal. Sassi et al. (2010) argue that Cd induces disturbance of the
Ca balance in the cellular bone tissue of G. affinis and that in turn
would probably lead to a situation in which hypocalcemia is
compensated by an increased release of Ca from skeletal bone. As a
result, the spinal column would become more fragile and in-
creasingly susceptible to deformities. Cd may also directly induce
bone deformities by negatively affecting the bone matrix or the
bone tissue itself, disturbing bone remodeling and mineralization.
In addition, Cd may interfere with the crystallization of the main
bone mineral hydroxyapatite and osteoblast activity (Blumenthal
et al., 1995; Suzuki et al., 1989). According to Muramoto (1981),
cadmium induced body deformities in fish, were related to a de-
crease in Ca and P content. Developmental deformities may also
originate from genotoxic action of Cd and Cu (Cavas et al., 2005).
The delay of hatching which is very common in heavy metal ex-
posure has been attributed to the effect of the metals on mitotic
divisions (Perry et al., 1988).
Concerning fish larvae, decrease in fish growth caused by
copper or cadmium intoxication may be a result of various meta-
bolic disturbances. Couture and Kumar (2003) reported a direct
inhibition of mitochondrial enzymatic activity and oxidative me-
tabolism in Perca flavescens exposed to copper and cadmium. De-
toxification is a process which requires high metabolic costs and
may also reduce growth of fish exposed to Cd and Cu. According to
Wu and Hwang (2003), Cu and Cd exposures induced me-
tallothionein synthesis in O. mossambicus larvae. These metals also
cause ionoregulatory disorders in fish which may again cause in-
creased metabolic cost of the compensatory osmoregulation and
also lead to growth reduction due to the inevitable energetic de-
ficiency (Witeska et al., 2014).
According to other authors (Kennedy, 2011), the effect of heavy
metal poisoning could also be indirect; vertebral deformities can
be the result of a nutritional deficiency as fish exposed to heavy
metals stop feeding (which results in vitamin C deficiency etc.).
Regarding fish, it is well established that the younger the stage,
the more sensitive the organism is. A recent study on heavy metals
(Zhu et al., 2011) contradicts this general knowledge as it has
shown that the Chinese rare minnow (Gobiocypris rarus) – when
exposed to 3 different heavy metals (Cu, Zn and Cd) – was more
sensitive during the larval stage rather than the embryonic one.
The same conclusion is reached in 2 earlier studies concerning the
effect of cadmium and copper on Common carp (Witeska et al.,
1995; Jezierska et al., 2009b).
In teleost species, it is generally accepted that skeletal defor-
mities can be environmentally induced in two ways: (i) by
alteration of biological processes necessary for maintaining the
biochemical integrity of bone, or (ii) through neuromuscular ef-
fects, which lead to deformities without a chemical change in
vertebral composition (Avyle et al., 1989). Most of the deformities
reported in fish are in the vertebral column or its predecessor – in
fish development –, the notochord. The notochord is the primary
axial structure on which many other tissues depend for their
proper formation and differentiation. Toxicants that disrupt nor-
mal development and differentiation of the notochord may
therefore result in permanent skeletal deformities, muscle ab-
normalities, and neurological dysfunction. Axial structures may
also be malformed by uncontrolled muscle spasms (Nguyen Thi
Hong et al., 1997).
Apart from their negative effects, it is common knowledge that
certain heavy metals (and in certain concentrations) play a posi-
tive role on fish development. Nguyen et al. (2008) tested different
combinations of Artemia enrichment with zinc and manganese
(Mn) – both essential metals for fish – before feeding red sea
bream (P. major) larvae and concluded that the presence of these
metals acts beneficially to growth and skeletal deformities.
Environmental parameters such as water temperature, oxygen
concentration, hardness, salinity, alkalinity and dissolved organic
carbon may affect metal's toxicity to fish (Benaduce et al., 2008;
Linbo et al., 2009; Sassi et al., 2010). Hypoxic conditions, tem-
perature increase and acidification usually render the fish more
susceptible to intoxication, whereas increase in mineral content
(hardness and salinity) reduce metal toxicity (Witeska and Je-
zierska, 2003). Moreover, interactions among various metals pre-
sent in the water may also modify their toxicity and synergistic,
additive or antagonistic effects may also occur (Witeska and Je-
zierska, 2003). Bao et al. (2008) studied the synergistic toxic ef-
fects of zinc pyrithione and copper to three marine species and
concluded that in order to properly protect the marine life, water
quality criteria should be established in such a way that would
take into account the synergistic effects of the studied metals.
Besides heavy metals, fish populations encounter a number of
other hazardous conditions. Murl Rolland (2000) states that there
has been an unprecedented decline in commercial marine fisheries
and in freshwater fish species world-wide, prompting a search for
the causes of these declines and a growing concern for the future
viability of fishery resources. Commercial fishery species have
been subjected to tremendous pressure from overfishing which
has caused rapid declines in some populations. Together with
eutrophication, introduction of exotic species, habitat alterations,
exposure to aquatic pollution, and (potentially) the effects of glo-
bal climate change, it is apparent that fish populations are faced
with a wide variety of stressors generated by human activity. Re-
versal of the current decline in many fish populations and suc-
cessful management in the future depends upon identifying the
risks posed by all factors currently impacting the health and sur-
vival of these populations.
In literature, there are a lot of available studies on other con-
taminants such as selenium (Kennedy et al., 2000) and on nutri-
tional aspects such as vitamins deficiencies (Darias et al., 2011;
Mazurais et al., 2009) but there is little information on the effects
of heavy metal poisoning accompanied by precise monitoring of
the consequences on the fish. Although in the past years quite a
few publications on the subject appeared, the effect of the differ-
ent heavy metals on various fish species still needs to be studied
more thoroughly. Moreover, the precise effect on fish deformities
has not been studied since all studies – or most of them – examine
their samples at hatching or a few days afterwards. Additionally, it
is crucial to have – in a documented form – the exact concentra-
tions (threshold), above which the heavy metal contamination
poses a threat to the organism (reduction of welfare or death), to
the fisheries and aquaculture industry (through fish that are
 D.G. Sfakianakis et al. / Environmental Research 137 (2015) 246–255
unmarketable bearing deformities) and of course to the con-
sumer’s health (through the food chain).
Barbee et al. (2013) state that toxicological research has re-
cently been shifting from the traditional focus on the lethality of
toxicants to organisms, to an increased emphasis on the sublethal
impacts of contaminants. The sublethal effects of environmental
pollutants may be latent, and thus harder to detect, yet can have
significant consequences for the long-term growth, reproduction
and survival of individuals. Therefore, there is a need of future
studies which will focus on using lower exposure concentrations
(non-lethal) thus enabling the fish to reach adulthood (or at least
the juvenile stage), in order to fully understand the long-term
consequences of heavy metals poisoning on fish organisms.
Moreover, as stated many times previously (Au, 2004; Kessabi
et al., 2013b; Sun et al., 2009), the presence of deformities in wild
fish populations could serve as an excellent indicator of water
pollution (in this case, heavy metal contamination). The effects of
sublethal concentrations of metals on fish could offer information
about the contamination level of certain environments and thus
facilitate the use of fish deformities as biomarkers of environ-
mental pollution. In order, however, to achieve this use, tolerant
fish species should be used in addition to the cause-and-effect
relationships between biomarkers and the various pollutants
being well established (Antunes and Lopes Da Cunha, 2002; Sun
et al., 2009).
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