Environmental Research 137 (2015) 246–255

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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 b

Biology Department, University of Crete, 71409 Heraklio, Crete, Greece 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: Received 3 September 2014 Received in revised form 5 December 2014 Accepted 15 December 2014

Heavy metals have been associated with many fish deformities in natural populations and in laboratory produced specimens as well. Deformities in general have devastating effects on fish populations since they affect the survival, the growth rates, the welfare and their external image. Although the embryonic stage in respect to heavy metal exposure has been extensively studied, there is not much information 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 research towards the effects of metals on the subsequent life stages in order to assess the long-term consequences of heavy metal poisoning on fish organisms and possibly correlate these consequences with the environmental contamination (use as biomarkers). & 2014 Elsevier Inc. All rights reserved.

Keywords: Heavy metals Fish larvae Deformities Environment Biomarkers

1. Introduction Heavy metals have been associated with many fish deformities in natural populations as well as in laboratory produced specimens (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, lagoons) 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 (Muramoto, 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 elements), and their toxicity rises with increasing concentration. Essential 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: [email protected] (D.G. Sfakianakis).

http://dx.doi.org/10.1016/j.envres.2014.12.014 0013-9351/& 2014 Elsevier Inc. All rights reserved.

which are equivalent to or worse than those caused by nonessential 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 deformities, 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 affected by metals (commonly the gills) and toxicity depends on metal form and speciation, bioavailability, toxicokinetics (absorption, distribution, biotransformation, and excretion), and toxicodynamics (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

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(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 curvature), 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 impairment 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 developmental retardation, morphological and functional deformities, or death of the most sensitive individuals. Additionally, heavy metals activate energy-consuming detoxification processes; thus, in intoxicated 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 vulnerable and sensitive to any type of xenobiotics (Ankley and Johnson, 2004) and exposure during these critical periods may cause profound 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 hormone 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 membranes. 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 biomarkers of environmental exposures (Au, 2004). Morphological deformity assessment is believed to be one of the most straightforward 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, microorganisms, 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 manipulation 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

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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 release in the environment, have rendered it a priority pollutant research wise (Kalman et al., 2010; Maunder et al., 2011). Cadmium 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. Interestingly, the higher concentration of cadmium (0.05 ppm) presented 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 cadmium respectively. Williams and Holdway (2000) studied the effects 2 h pulseexposure 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 populations. 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.

Sassi et al. (2010) Barjhoux et al. (2012) Barjhoux et al. (2012) Zhang et al. (2012) Witeska et al. (2014) 32.5–52.5 40.7–58.6 22.1–25.6 15–92  35 Spinal (kyphosis, lordosis and scoliosis) Spinal (mainly kyphosis and lordosis) Cardiovascular Spinal curvatures? Vertebral curvatures and yolk sac deformities 30 Days 10 Days 10 Days 144 h From egg fertilization to hatching

Cao et al. (2009) 1–100 Skeletal deformities (spine, tail, fins)

Benaduce et al. (2008) 0–91.7 Spinal column

 5–100 0–100 Reduction of pigmentation Deformed barbel

5 Days From egg fertilization to 21 days after hatching From egg fertilization to 21 days after hatching Until first feeding

0–47 5–27 Head deformities and spinal curvatures Spinal Until hatching 2h

0.4 mg/L 0.18–19.8 μg/g d.w. 0.18–19.8 μg/g d.w. 0.0001–30 mg/L 0.1 mg/L Gambusia affinis Oryzias latipes Oryzias latipes Silurus soldatovi Leuciscus idus

0–3.2 mg/L Pagrus major

0.0005–0.018 mg/L Rhamdia quelen

Clarias gariepinus Rhamdia quelen

0.05–5 mg/L 0.0005–0.018 mg/L

Embryonic stage 3, 46 and 92 h post fertilization Embryonic stage Embryonic and prelarval stage Embryonic and prelarval stage Embryonic and prelarval stage 1d Post hatching Embryonic stage Embryonic stage Embryonic stage Embryonic stage Cyprinus carpio 0.001–0.05 ppm Melanotaenia fluviatilis 0.033–3.3 mg/L

% Deformed in the population Type of deformity Duration of exposure Stage of exposure Concentration Species

Table 1 The effect of cadmium (Cd) exposure on fish deformities (nominal concentrations are used).

Witeska et al. (1995) Williams and Holdway, (2000) Nguyen and Janssen, (2002) Benaduce et al. (2008)

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Benaduce et al. (2008) studied the effect of waterborne Cd (as CdCl2) in eggs and larvae of silver catfish (Rhamdia quelen) at two alkalinity levels (63 and 92 mg/ L CaCO3) and at 4 Cd concentrations: 0.5 (control), 4.5, 8, and 18 μg/L from egg fertilization until 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 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 embryoniclarval development of red sea bream, Pagrus major. They applied 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 feed (entire embryonic and prelarval stage). Hatchability rate ranged from 100% in the control to 0% in the highest Cd concentration (3.2) and mortality ranged from 1% to 100% respectively. Abnormalities also ranged from 1% to 100% respectively and included “cardiac edema, degenerated and hooked tails, fins lesions and spinal curvature” with skeletal deformities being the most pronounced. Sassi et al. (2010) investigated the effect of cadmium (0.4 mg/L 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 32.5–52.5% incidence in the population and included lordosis, kyphosis, scoliosis and 1 case of simultaneous presence of all three deformities (LSK syndrome). The authors also concluded that the higher the water temperature the higher the Cd toxicity and these 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 stage (concentrations varied between 0.18 and 19.8 μg/g d.w.). 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 larvae) and cardio-vascular (mainly abnormal positioning and heart looping). They reported LOEC (Lowest Observed Effect Concentration) value for Cd at 1.9 mg/g d.w. sediment. 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 catfish (Silurus soldatovi) and although they report many adverse 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 reach incidences of up to 92% in the populations, they chose not to describe them and distinguish their appearance. Witeska et al. (2014) studied the effects of Cd (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 and deformities (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 exposure to Cd were about 35% in the population. Also, they reported that cadmium was more toxic to the ide embryos and larvae than copper which was also included 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) Copper is an abundant element which occurs as a natural mineral with a widespread use. It is also an essential micronutrient for living organisms on account of being a key constituent of metabolic enzymes (Monteiro et al., 2009). However it can be toxic

0.068–0.244 mg/L 0.0005–0.004 mg/L 6.95–23.1 μg/g d.w. 6.95–23.1 μg/g d.w. 0.1–1 mg/L 0.1 mg/L Danio rerio Fundulus heteroclitus Oryzias latipes Oryzias latipes Carassius auratus Leuciscus idus

Embryonic Embryonic Embryonic Embryonic Embryonic Embryonic

stage stage stage stage stage stage

From egg fertilization for 120 h Lateral line deformities (fewer functional neuromasts) 50 Days Vertebral deformities and inflammatory masses 10 Days Spinal (mainly kyphosis and lordosis) 10 Days Cardiovascular From egg fertilization to 24 h after hatching Scoliosis and tail curvatures From egg fertilization to hatching Vertebral curvatures and yolk sac deformities

Not provided 0–100 18.6–35.2 11.5–29.2  1–10  15

Nguyen and Janssen, (2002) Johnson et al. (2007) Mochida et al. (2008) Barjhoux et al. (2012) Barjhoux et al. (2012) Kong et al. (2013) Witeska et al. (2014)  15–70 Reduction of pigmentation 5 Days Embryonic stage 0.15–2.5 mg/L Clarias gariepinus

Stage of exposure Duration of exposure Concentration Species

Table 2 The effect of copper (Cu) exposure on fish deformities (nominal concentrations are used).

Type of deformity

% Deformed in the population Source

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to intracellular mechanisms at concentrations which exceed normal levels (Hernández et al., 2006). Fish can accumulate copper via diet or ambient exposure. A fluctuation of the dietary copper 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 copper exposure, will have deleterious effects on the gills, gut and sensory systems (Johnson et al., 2007). Recently, it has been realized that extended Cu exposure can evoke an acclimation response, allowing fish not only to survive but also endure further exposure to higher concentrations. However, there are reproductive 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 uptake from seawater in marine fish (osmotic homeostasis). Freshwater fish seem to accumulate Cu mainly through the dietary route (Dang et al., 2009). A concentrated list of copper induced deformities in fish is presented in Table 2. Nguyen and Janssen (2002) studied the African catfish with the metal exposure beginning at fertilization and lasting for 5 days. Concentrations used varied from 0.15 to 2.5 mg/L (CuSO4  5H2O). The main deformity they reported was the reduction of body pigmentation which reached percentages of up to 70% and was 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 (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 orientate in a water current. Moreover, the authors observed mortality, 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 effects. The authors also reported regeneration of the damaged area providing that the Cu concentration was below a certain threshold. The same conclusions for the effect of dissolved copper on zebrafish were reported in Linbo et al. (2006, 2009). 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 under continuous flow-through conditions. Hatchability, survival, growth, and morphological deformities were measured. Hatchability did not differ significantly between any experimental group and control groups. Survival and growth were significantly reduced at 50 days in the groups exposed to 2 or 4 μg/L) CuPT. During the test, abnormalities, such as vertebral deformity (lordosis, kyphosis and scoliosis) and formation of inflammatory masses in the lateral muscles, occurred in fish exposed to CuPT. Light and electron microscopic studies indicated that muscle dysfunction played a role in the vertebral deformity and revealed that the inflammatory masses were composed mainly of macrophages and necrotic myocytes. The study has great variations in 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 population). Barjhoux et al. (2012) studied the effect of Cu spiked sediment on the Japanese medaka during the entire embryonic stage (concentrations varied between 6.95 and 23.1 μg/g d.w.). They observed deformed larvae up to 52% in the populations. Specimens were examined at hatching and the deformities found were mostly

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spinal (mainly kyphosis, lordosis and C-shaped larvae) and cardiovascular (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 scoliosis and tail curvatures (under a microscope) and concluded that the deformities rate increased gradually with increasing Cu concentrations. 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 deformities (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). Although Pb is a naturally occurring substance, its environmental concentrations are significantly increased by anthropogenic sources which include base metal mining, battery manufacturing, Pbbased paints and leaded gasoline (Mager, 2011; Monteiro et al., 2011). The aquatic systems are subsequently contaminated via superficial soil erosion and atmospheric deposition. The concentration 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 accumulation 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).

Weis and Weis (1977) studied the effect of mercury, cadmium and lead exposure on the development of the killifish (F. heteroclitus) 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 examination for skeletal deformities took place. They observed that at 1 mg/L, only 20% of the larvae were normal, 40% exhibited lordosis 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). Exposure to lead nitrate caused a progressively longer delay in hatching and reduced the percentage of embryos which successfully 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 edema, 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 juveniles). They observed deformities in the two higher concentrations that reached 100% in the populations by the end of the experimental 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 concentration 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, neurotransmission and cell signaling (Celik and Oehlenschläger, 2004; Hogstrand, 2011). It can become toxic for fish at increased

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

Duration of exposure

Type of deformity

% Deformed in the population

Source

Fundulus heteroclitus

Pb (0.1– 10mg/L )

Embryonic stage

Pb Pb Pb Pb Pb Zn

0–34.3 4.3–69.7 0–3.3 0–35 0–100 3–27

Clarias gariepinus

Cr (11–114 mg/L)

6 h Embryos 6 h Embryos 6 h Embryos 6 h Embryos 96 h Embryos 3, 46 and 92 h post fertilization Embryonic stage

Lordosis and curled up body Irregular head Notochord defects Yolk Sac edema Finfold defect Spinal curvatures Spinal deformities

0–100

Clarias gariepinus Clarias gariepinus Clarias gariepinus Clarias gariepinus Acipenser sinensis Melanotaenia fluviatilis

From blastula stage to hatching 48–168 h 48–168 h 48–168 h 48–168 h 112 Days 2h 5 Days

Body axis

 25–100

Weis and Weis, (1977) Osman et al. (2007) Osman et al. (2007) Osman et al. (2007) Osman et al. (2007) Hou et al. (2011) Williams and Holdway, (2000) Nguyen and Janssen, (2002)

(0.1–0.5 mg/L ) (0.1–0.5 mg/L ) (0.1–0.5 mg/L ) (0.1–0.5 mg/L ) (0,2–1,6 mg/L) (0.33–33.3 mg/L)

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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 pulseexposure 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 physicochemical 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; VeraCandioti 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 reported 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 parameters (such as the metal's exact concentration, the possible synergistic 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

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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) (Bengtsson 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 tissues 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 deformed 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, collected from the Tunisian coast, were linked with high concentration 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 deformed specimens reached up to 18% in a certain polluted sampling area while it did not exceed 5% in the unpolluted reference area. The authors did not discriminate between the different xenobiotics 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 (Japanese 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 discovered spinal deformities (mainly kyphosis, scoliosis and lordosis) which were more often in the polluted area specimens

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(roughly 11% of the population). Liver concentrations of Cd and Zn were significantly higher in the deformed specimens when compared to the normal ones. The study also included analysis of Cu (both in water and organisms’ liver) but it did not seem to correlate 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 populations 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 increasingly 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 decrease 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 exposure 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 metabolic disturbances. Couture and Kumar (2003) reported a direct inhibition of mitochondrial enzymatic activity and oxidative metabolism in Perca flavescens exposed to copper and cadmium. Detoxification 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 metallothionein synthesis in O. mossambicus larvae. These metals also cause ionoregulatory disorders in fish which may again cause increased metabolic cost of the compensatory osmoregulation and also lead to growth reduction due to the inevitable energetic deficiency (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 deformities 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 effects, 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 normal development and differentiation of the notochord may therefore result in permanent skeletal deformities, muscle abnormalities, 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 positive 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, temperature increase and acidification usually render the fish more susceptible to intoxication, whereas increase in mineral content (hardness and salinity) reduce metal toxicity (Witeska and Jezierska, 2003). Moreover, interactions among various metals present in the water may also modify their toxicity and synergistic, additive or antagonistic effects may also occur (Witeska and Jezierska, 2003). Bao et al. (2008) studied the synergistic toxic effects 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 global climate change, it is apparent that fish populations are faced with a wide variety of stressors generated by human activity. Reversal of the current decline in many fish populations and successful management in the future depends upon identifying the risks posed by all factors currently impacting the health and survival of these populations. In literature, there are a lot of available studies on other contaminants such as selenium (Kennedy et al., 2000) and on nutritional 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 different 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 concentrations (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

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unmarketable bearing deformities) and of course to the consumer’s health (through the food chain). Barbee et al. (2013) state that toxicological research has recently 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 environmental 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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