Impacts of heavy metals on early development, growth and reproduction of fish – A review

Heavy metals pollution causes a threat to the aquatic environment and to its inhabitants when their concentrations exceed safe limits. Heavy metals cause toxicity in fish due to their non-biodegradable properties and their long persistence in the environment. This review investigated the effects of heavy metals on early development, growth and reproduction of fish. Fish embryos/larvae and each developmental stage of embryo respond differently to the intoxication and vary from species to species, types of metals and their mode of actions, concentration of heavy metals and their exposure time. Many of the heavy metals are considered as essential nutrient elements that positively improve the growth and feed utilization of fishes but upon crossing the maximum tolerable limit these metals cause not only a hazard to fish health but also to human consumers and the disruption of ecological systems. Reduced gonadosomatic index (GSI), fecundity, hatching rate, fertilization success, abnormal shape of reproductive organs, and finally failure of reproduction in fish have been attributed to heavy metal toxicity. In summary, this review sheds light on the manipulation of fish physiology by heavy metals and seeks to raise sensitivity to the prevention and control of aquatic environmental contamination, particularly from heavy metals.


Introduction
Heavy metals pollution is a great concern to aquatic environments because they impart a wide range of toxicities with serious impacts to the aquatic faunal communities [1,2]. Most of the heavy metals accumulated in aquatic water bodies are originate from anthropogenic activities such as agricultural cultivation, erosions of landfills, docking and embarking activities, sewage from industrial and domestic wastewater and some natural processes [1,3]. The uncontrolled population growth, intensive agricultural activities and heavy industrialization result in a wide range of pollutants which eventually inflict serious consequences on aquatic ecosystems as well as associated faunal and floral communities [4][5][6]. Commonly, trace amount of heavy metals (non-degradable) cause serious difficulties in aquatic systems as a result of their assimilation, deposition and even incorporation at a specific concentration in abiotic substances and ultimately, accumulated into the body of associated aquatic organisms [7]. Heavy metals accumulate into the tissues of aquatic organisms throughout different aquatic food chains where they can be concentrated; bioaccumulated metals can result in substantial human health hazards upon consumption of these contaminated aquatic foods [8]. The rapid growth of industrialization across the cities results in the release of effluents contaminated with toxic metals including chromium (Cr), nickel (Ni), copper (Cu), lead (Pb), iron (Fe), and zinc (Zn). In broad, metals can be classified as biologically essential and nonessential. Metals like aluminum (Al), cadmium (Cd), mercury (Hg), tin (Sn) and lead (Pb) have no records of specific biological functions and therefore their toxicities rise with high concentration. On the other hand, essential metals (Cr, Zn, Ni, Cu, Co, Fe) have established biological functions and toxicities occur in response to either their deficiencies or excessive concentrations. Essential metals positively improved the growth and feed utilization of several species [9][10][11][12][13][14][15] but when maximum allowable/tolerable limit these metals are exceeded, they hamper the normal physiological and ecological systems in the aquatic environment [16,17], causing toxicity within the organisms and ultimately causing a substantial threat to human health [1,8]. Most of these heavy metals are highly carcinogenic in nature and in addition they can cause serious health complexities like liver disorders, cardiovascular difficulties, kidney dysfunctions and in extreme cases death. Heavy metal pollution severely disrupts the physiology of several aquatic organisms, especially fish [4,18,19]. Heavy metal contamination greatly changed the hemato-biochemical scenario of fish and also resulted several deformities (cellular and nuclear) in different blood cells [19][20][21]. Genetic damages as a result of heavy metal toxicities have also been recorded by several studies [18]. Heavy metals contamination significantly hampers the reproductive performances of fish [22][23][24]. Investigations have reported several reproductive compromises including reduced GSI, fecundity, hatching rate, fertilization success, abnormal shape of reproductive organs, and finally overall reproductive success in response to a variety of heavy metals [25][26][27][28][29][30]. Moreover, heavy metals severely affected the embryonic and larval development of fish through resulting number of complexities such as increased heart rate, reduced cardiac activity, increased mortality rate, deformed shape, vertebral column deformities etc. in different developmental stages of embryo [11,[31][32][33][34][35]. Despite the destructive impacts of several heavy metals on fish physiology and reproductive performance in fishes, few if any generalized or comprehensive patterns of these responses are available. The current review focuses on the aggregation of up-to-date information about the impacts of heavy metals on embryonic and larval development, growth, reproductive performance with an emphasis of the most commercially important aquaculture species.

Heavy metals effects on embryonic and larval development of fish
Early developmental stages of fish, specifically embryos and larvae, are more susceptible to pollutants such as heavy metals than juvenile and adult fish are, and are widely used as bio-indicators to determine the toxicity of such chemicals to the aquatic organisms [36,37]. Various endpoints such as developmental malformations (teratogenicity), physiological and biochemical alterations, behavioural and functional deformities are used to assess and predict the toxicity of heavy metals to fish population [35]. Fish embryos/larvae at each developmental stage of embryo (blastula, gastrula, segmentation, hatching etc.) respond differently to the intoxication and vary from species to species, types of metals and their mode of actions, concentration of heavy metals and their exposure time etc. [38,39]. For instance, hatching and embryo survival of African catfish (Clarias gariepinus) were unaffected by Cd exposure at a concentration ranging from 0.05-5 mg/L. Another study reported that embryo and larvae survival, hatching of Ide (Leuciscus idus) were significantly affected by Cd exposure (100 μg/L; [35,40]. The types of deformities in different fish species due to expose to different heavy metals are summarized in Table 1. Most of the literature reported reduced embryonic and larval survival, reduced and delayed hatching, stunted growth rate and morphological abnormalities such as skeletal deformities, vascular system abnormalities, reduction in pigmentation, eye anomalies etc. among different fish species exposed to lethal and sub-lethal doses of essential (Cu, Zn) and non-essential (Cd, Cr, Hg and Pb) heavy metals [32,38,[40][41][42]. Cardiovascular endpoints such as hyper or hypo dystrophia, positioning abnormality, incomplete or abnormal heart looping, tubular heart, oedemata, megalocardia etc. are important parameters to assess the toxicity of heavy metals in embryos and larvae, revealing species-dependent differences in the responses to various heavy metals. For example, Cu exposure significantly increased heart rate in zebrafish embryo [31], whereas cardiac activity is reduced in red sea bream [32] and zebrafish [43] embryos exposed to Cd. Larvae are less tolerant to heavy metals than the embryo since embryos have protective hard chorion layers and perivitelline fluid that can impede the entry of heavy metals [44,45]. Catalase (CAT, the enzyme which converts relatively toxic hydrogen peroxide to oxygen activity is significantly reduced in Table 1 Effect of heavy metals on embryonic and larval development of fish. the larvae compared to embryos, which might contribute to the resistance of embryos to heavy metals. Toxicity levels of heavy metals in embryos and larvae of freshwater fish are different from marine fish because of salinity differences. At higher salinity levels, the bioavailability of the toxic forms of heavy  MN; micronucleus, NB; nuclear bud, BN; bi-nucleated metals in water decreases. Information is limited about the toxic effects of heavy metals on marine fish embryos and larvae. Low hatchability, high mortality, morphological abnormalities etc. are reported in embryos and larvae of marine fish exposed to different heavy metals [11,32]. Environmental cues especially high temperature is known to cause developmental deformities in fishes and it has been reported that combined application of high temperature (24-32⁰C) and heavy metal such as Cd causes intense increase in skeletal deformities in juvenile mosquito fish (Gambusia affinis) than Cd or temperature alone [46]. High temperature increases the metabolic activity of fish, increasing the potentiality of metal ion action (Cd in this case) on cellular enzyme and cell membrane.
The mode of action (especially changes in enzyme and DNA) of each heavy metal exposure in embryo and larvae are at early stage of investigation and gaining importance among the researchers investigating molecular mechanisms of their effects in fish. Superoxide dismutase (SOD) and catalase (CAT) enzymes are known to convert reactive oxygen species to non-toxic oxygen in the liver. It has been found that in embryos and larvae of goldfish (Carassius auratus), these enzymatic activities were significantly inhibited due after exposure of high Cu concentration (1.0 mg/L), causing oxidative stress responsible for lipid peroxidation [44]. Moreover, Cd and Cu exposure to 2 dph larvae of Japanese medaka (Oryzias latipes) induced significant DNA damage [47] determined by Comet assay (a reliable method to assess genotoxicity in all stages of fish).
There are numerous reports on the effect of single heavy metal on the ontogenic development embryos and larvae. Because most of the open water environment is contaminated with mixtures of heavy metals (from anthropogenic and geogenic sources), it is important to evaluate the combined effects of those heavy metals on embryonic and larval development. The combined effect of Cu-Zn and Cd-Zn has been investigated in Rainbow trout Oncorhynchus mykiss [48] and common carp Cyprinus carpio [49] embryos respectively, revealing increased embryonic mortality and physical deformities (e.g. vertebral column deformities). Hg and Pb toxicity resulted defects of important organs of fish such as abnormal and irregular fins, head, tails and several spinal difficulties [38,42]. Moreover, Zn contamination negatively affected the hatching success and survival of several fish species as well as hampered the normal formation and pigmentation of several organs [33,35,41,48].
Supplementation of vitamin C with the dry feed to the embryo and larvae of common carp (Cyprinus carpio) exposed to mixture of Zn and Cd increased the ontogenic development and quality and quantity of the larvae through the improvement of immune system [49]. It has been reported that Cd exposure under conditions of high alkalinity can significantly increase the hatching, survival rate and growth of larvae of Silver catfish Rhamdia quelen [50].

Impact of heavy metals on growth performance of fish
Nutritional adequacy is prerequisite sustainable aquaculture. The overall growth, health status and reproductive performances of various aquaculture species especially fish are dependant on appropriate nutrition [63][64][65]. Among the various candidates that contribute nutritional demand of various aquaculture species, heavy metals play important roles in this regard. Various types of trace metals significantly contribute to different physiological processes including growth of fish (Table 2). Several trace metals such as Mn, Fe, Co, Cu, Cr and Zn are known to be important minerals with positively influences on the physiology and metabolism of fish [9,10]. Cr has been regarded as very important trace element that improved the health status of several animals through upgrading the physiology as well as their metabolism [66,67]. Cr directly involved in nutrient (protein, lipid and carbohydrates) metabolism significantly influences the growth and feed utilization of several fish species [68,69]. Moreover, Cr also altered the fatty acid profile in blood through participating in fatty acid metabolism in various animals [70,71]. It has been found that Cr supplementation lowered the   Salmo salar 50, 180 180 Increased SGR at higher [144] (continued on next page) cholesterol, triglycerides level in blood and increased the high density lipoprotein (HDL) cholesterol level [72,73]. Dietary Cr significantly influenced the expression of several genes related to glucose metabolism, lipogenesis, apparently playing a key role in growth enhancement [74]. Cr supplementation in diet significantly improved the growth and feed utility of striped catfish (Pangasianodon hypophthalmus) upto 4 mg/kg but greater concentrations resulted in lower growth with higher micronucleus frequencies (Fig. 1) [10]. On the contrary, presence of Cr in excess level led to several toxicities and therefore, reduced the growth and feed palatability of several species [75][76][77]. Zn is an essential trace element that plays a significant role in the life processes of animals including fish [78][79][80]. Zn acts as a co-factor of several metallo-enzymes (carbonic anhydrase, alkaline phosphatase, alcohol dehydrogenase etc.) ensuring the availability and activities of those important enzymes to stimulate digestion and metabolism of nutrients [81][82][83]. Zn also regulates the nucleic acid metabolism, protein synthesis and anti-oxidative enzymes functionalities of fish [84]. The anti-oxidative roles of Zn were well demonstrated in several studies [85,86]. Dietary Zn supplementation considerably improved the growth of fish through upgrading muscle morphology [9]. Dietary Zn provisions also influence the whole body composition of fish muscle. Zn significantly enhanced the lipid content and lowered the moisture and ash level of fish carcass [87]. However, Zn deficiency hampers the nucleic acid and protein biosynthetic pathways [66,88], impairment of bone development [87] and various other pathological effects [89]. On the other hand, excess amount of Zn resulted various negative impacts such as growth and reproductive performance reduction [90], oxidative stress [91] and poor feed utilization [92][93][94]. Moreover, Zn toxicity resulted in delayed hatching, malformations in bone calcification and growth defects [95]. Cu is an essential element that plays a pivotal role in various physiological as well as biological systems such as hemoglobin and bone formation, control the activities of myelin in the nervous system and finally acts as an activists of many important enzymatic action including cytrochrome oxidase, lysyl oxidase, dopamine hydroxylase ferroxidase, tyrosinase and Cu-Zn superoxidase dismutase [93,96]. Various studies revealed that dietary Cu supplementation significantly improve the growth, oxidative status and immune system of several aquatic species [96][97][98][99].
In the very recent years, aquaculture nutritionists find out the outstanding role of Cu particles has caught the attention aquaculture personnel as potentially interesting feed supplement [100,101]. On the contrary, dietary Cu toxicity exhibited several adverse effects including reduced growth, greater FCR, lower feed efficiency [102,103]. Fe, an essential element that helps to maintain the normal activities of different organs and tissues of animals including fish because of its active role in physiological processes like oxygen gas transportation, cellular respiratory activities, and lipid peroxidation processes. Fe modulated the immune system of animals and thus protects against various infectious agents and also actively participates in the synthesis of steroid and DNA, drug metabolism and electron transportation [104].

Heavy metals effect on reproduction of fish
Reproduction is essential to all animals and successful reproductive performance among the most important determinants of survival at the species level [147][148][149]. Heavy metals pollution negatively affects the reproductive performance of fish resulting low quality gametes that may influence not only success rate of fertilization but also hatching as well as survival rate of the offspring (Table 3) [150]. Various types of heavy metals accumulated into the fish body from the environment and their continuous accumulation disrupt the formation and activities of various tissues and organs including reproductive organs [62]. Heavy metals caused anomalies in reproductive cell/organ development. Arsenic (As) pollution seriously affected the reproductive performances of fish through inhibition of spermatogenesis and oogenesis including reduced egg and sperm quality and quantity, hatching and fertilization rate [22][23][24]. Cd is a potent hazardous metal that resulted several dysfunctions of reproductive process of fish. Various studies demonstrated several difficulties in reproductive performance of fish such as abnormal oocytes structure, empty follicle and loosing follicular line, retraction as well as condensation of cytoplasm, total GSI reduced and so on [27]. Moreover, Cd toxicities cause shrinkage of spermatic lobules and fibrosis in testis, lower sperm motility and viability as well as reduced fertilization rate [26,[150][151][152][153]. Cr has been regarded as one of the most biologically potent heavy metals due to its summative destructive effects on living organisms [154]. Long term exposure to Cr drastically reduced (continued on next page) the spawning success [155], fibrotic and pyknotic testis [26], significantly reduced the GSI, fecundity, lowered number of oocytes and matured spermatozoa [156], hampered the motility of sperm [150] and finally gradual decrease of vitellogenic oocytes [124]. Various studies revealed that reduced GSI, fecundity, hatching rate, fertilization success, abnormal shape of reproductive organs, and finally overall reproductive success resulted from the toxicities created by Cu and Hg [28,29,151,[157][158][159][160]. Pd and Zn resulted similar deformities as well as negative impacts in Carassius gibelio [30], Odontesthes bonariensis [26]; Oryzias melastigma [27] and Clarias magur [25].

Conclusion and future perspectives
Heavy metals contamination is a serious threat to entire aquatic ecosystems including associated flora and fauna. The devastating impacts of heavy metals on aquatic organisms specifically fish result an irreparable loss in aquaculture industry. In this review, destructive effects of heavy metals on fish focusing the embryonic and larval development, growth and reproduction of commercially important species are discussed very concisely with a view to using it as a tool for further genotoxicity related experiments by the researchers of the associated areas. Heavy metals resulted in severe deformities in several aquatic organisms that will ultimately pose a substantial threat to associated consumers. To enlarge the sustainability of the aquaculture sector and to produce safe fish for human consumption, regular monitoring of the fish and associated environment should be done by the appropriate authorities at the local government, state, and national levels. A wellestablished framework should be developed as soon as possible to mitigate this great problem.

CRediT authorship contribution statement
Khanam Taslima: preparation of the first draft of the manuscript. Md Al-Emran, Mohammad Shadiqur Rahman, Jabed Hasan, Zannatul Ferdous and Md Fazle Rohani: data collection and preparation of the Tables. Md Shahjahan: conceptualization, edited the manuscript and final approval. All authors have read the final version and approved the manuscript.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Sharing of data is not permissible for this article. The data that support the outcomes of this study are available on request from the corresponding author [M Shahjahan]. GSI; gonad-somatic index,