# Morphological and Molecular Alterations Induced by Lead in Embryos and Larvae of _[PERSON]_
## 1 Introduction
It has been recently highlighted that the potential hazard of Pb below a certain threshold dose (i.e., 100 \(\upmu\)g/L) remains largely under-investigated, and even very low Pb concentrations, claimed as harmless, may represent a risk for human health [7; 9]. Much more effort is needed to develop a biological early warning system, especially in aquatic ecosystems [9], also considering both direct Pb effects and its potential of biom magnification through the food chain. In zebrafish embryos and larvae, available studies on Pb toxicity mainly focused on neurotoxicity, neurodevelopmental, and neurobehavior effects [40; 43; 44; 45; 46; 47; 48]. Surprisingly, morpho-functional investigations during the sensitive phase of development are scarce, and only a few studies have furnished an overall morphological evaluation [45; 49; 50; 51; 52; 53]. Moreover, all available information on fish deals with high lead concentrations, and there is a lack of research on the adverse effects of lead at environmentally relevant concentrations [53]. On these bases, here we investigated for the first time the effects induced by two environmentally relevant concentrations of Pb (2.5 \(\upmu\)g/L and 5 \(\upmu\)g/L) in the zebrafish model. Moreover, starting from the assumption that fish embryos are especially susceptible to environmental contaminants during the first 24 h post-fertilization (hpf) [54], we selected for the experiment early development stages (starting from 6 hpf), evaluating the Pb toxic effects after 48, 96, and 144 h. Given the need to identify early, sensitive biomarkers of Pb exposure, we first investigated morphological alterations, recognized as one of the most straightforward tools to investigate the effects of contaminants in fish [55]. To allow an objective comparison of gross morphology deformity after Pb exposure, a morphological grading system was used. Considering that the combination of morphological changes and transcriptional responses is recognized as valid and promising tools for studying ecotoxicological effects [23], in a second step, we also applied molecular biomarkers to have a more comprehensive overview of Pb-induced outcomes. In detail, we evaluated the expression pattern of some genes involved in the early developmental process (_Wnt_ and _\(\beta\)-catenin_) to address the mechanism underlying the putative morphological modifications caused by Pb exposure. Since the mechanisms through which lead may exert its toxicity may be different and stage-specific, we evaluated the expression of several proteins known as valuable biomarkers of metal exposure in adult fish. To protect themselves against heavy metals, living organisms, including fish and mammals, activate several defense systems, including the metallothioneein (MTs) modulation; these are low molecular weight proteins effective for detoxifying non-essential metals, and protecting cells from oxidative stress [35; 56; 57; 58; 59; 60; 61; 62]. Incidence of oxidative stress resulting from exposure to Pb and heavy metal contamination is the most common response in both fish and mammalian models [51]; therefore, we investigated the expression of two markers of oxidative stress induction (superoxide dismutase 1-_sod_1 and lactate dehydrogenase-_ldh_) during zebrafish development. The present study aims to elucidate the role of morphological studies for assessing sub-lethal Pb toxicity and provide a set of sensitive and early informative biomarkers for environmental risk assessment.