# Morphological and Molecular Alterations Induced by Lead in Embryos and Larvae of _[PERSON]_
## 2 Materials and Methods
### Exposure Conditions  
According to a standard staging guideline, before the experiment started, embryos' developmental stage was determined under a stereomicroscope by distinctive morphological features [63]. Exposure started once embryos were at 6 hpf and was performed in Petri dishes (density: 1 egg/mL) prefilled with the appropriate Pb solution or untreated aged tap water (control). During the experiments, the solutions were renewed daily, according to [PERSON] et al. (2015). The Petri dishes were placed in a temperature- and light-controlled incubator set to the same circadian rhythm used for adult zebrafish. After hatching and the yolk sac regression, larvae were fed ad libitum with brine shrimp nauplii (_Artemia salina_). Two sets of experiments were conducted, and three independent replicates were performed for each. _Mortality and hatching rate_--Embryo mortality and hatching rate were evaluated using a subsample of 30 embryos for each exposed group, including the control. The numbers of survived and hatched embryos were counted every day, and dead embryos were readily removed. Embryos/larvae viability (%) was assessed by observing the following parameters: egg coagulation, heartbeat, and spontaneous motility missing. In each treatment group, hatched larvae were counted, and the hatching rate (%) was determinate as follows: hatched numbers/total exposed numbers \(\times\) 100. _Gross morphology, deformity incidence and quantitative real time PCR_--For morphological analysis, animals (n = 90) were sampled at the selected time points: 48, 96, and 144 h corresponding to 54, 102, and 150 hpf. Prior to the morphological analyses, embryos/larvae were anesthetized in Tricaine methanesulfphone (MS-222) (200 mg/L) to inhibit movement. Observations were conducted using a stereomicroscope Optika SLX-3 equipped with a C-HP Camera (Optika Microscopes ITALY). All individuals were photographed and the obtained images were used to check the morphological malformations. Moreover, to evaluate the severity of morphological defects and allow reproducibility and comparison between the experimental and control groups, a morphological scoring system proposed by [PERSON] (2018) was used with some modifications. Briefly, observing the images acquired for each individual (dorsal and frontal view), the extent of each malformation was quantified by assigning a numerical value to each lesion. The grading system used was: 0 = no effect (\(-\)); 1 = weak (\(+\)); 2 = medium (\(++\)); 4 = strong (\(++\)). The overall severity of deformities is expressed as the sum of the estimated values for each malformation. The obtained numerical scores for all experimental groups (including the control) were statistically compared. Cumulative deformity, defined as individuals with malformations among total observed individuals, and the deformity incidence, defined as the percentage of individuals with a specific malformation among total individuals, were recorded after 48, 96, and 144 h of exposure. All photographed animals were promptly stored at -80 for subsequent real-time PCR analyses. Total RNA of all exposed groups and control group was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. RNAs quantity and quality were determined by spectrophotometry using the NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), and for the cDNA synthesis, we used the High capacity RNA to cDNA Kit (Applied Biosystems, Foster City, CA, USA). The genes analysed were: superoxide dismutase 1 (_sod1_, accession number NM_131294.1), metal-regulatory transcription factor 1 (_mtf1_ accession number NM_152981.1), wingless-type MMTV integration site family, member 3A (_wnt3a_, accession number NM_001007185.1), catenin beta-like 1 (_ctmb1_, accession number NM_200866.1) and lactate dehydrogenase D (_ldhd_, accession number NM_199873.1).