## 2 Materials and Methods
### Sampling and Analysis  
Starting in 2010, the Environmental Protection Agency of the Friuli Venezia Giulia Region (ARPA FVG) planned numerous monitoring activities in the MGL according to the Water Framework Directive [47]. The final goal was to classify the quality of biological elements in seventeen water bodies (hereafter referred to as WBs) that were defined following the Italian Legislative Decrees [63] (Figure 1).  
Surface sediments (n = 17) were collected using a van Veen grab (A = 0.047 m\({}^{2}\)), transferred into pre-cleaned bottles, frozen (T = \(-\)25 \({}^{\circ}\)C), and, before analysis, air-dried, sieved, and homogenised. Total Hg (THg) was determined with a Direct Mercury Analyser (DMA-80, Milestone, Sorisole (BG), Italy) following the EPA method 7473 [64]. The limit of quantitation (LOQ) was <0.06 mg kg\({}^{-1}\). The accuracy was checked via periodic proficiency tests conducted on certified samples (UNICHIM). The speciation of Hg was conducted following the selective sequential extraction (SSE) proposed by [PERSON] et al. [48]. Additionally, the obtained fractions were analysed with atomic fluorescence spectrometry (EPA method 1631e, CVAFS, Brooks Rand MERK-T, Seattle, WA, USA; LOQ = 4.0 ng L\({}^{-1}\)) [65]. The recovery percentage was checked using reference standard materials (NIST-1646a = 0.04 mg kg\({}^{-1}\) and BCR-580 = 132 \(\pm\) 3 mg kg\({}^{-1}\); 77.9 and 104%, respectively) as well as real samples (95 \(\pm\) 19%).  
Surface waters were collected with a telescopic bar coupled to pre-cleaned borosilicate bottles, filtered through 0.45 \(\upmu\)m pore-size membrane filters, and added to 5 mL L\({}^{-1}\) of BrCl solution [65]. The total dissolved Hg (THgD) was finally determined with the same instrumentation and method employed for [PERSON]'s fraction.  
Fish samples of numerous species were initially collected for total Hg (THg) analysis during 2010 and 2011, simultaneously with the monitoring of the fish fauna for the biological quality element (BQE) according to [47]. During spring 2012, targeted monitoring of specimens of the big-scale sand smelt _A. boyeri_ was conducted at twenty-two sampling stations. For each station, 100 individuals were randomly selected and 40 g of fish were homogenised for THg analysis. Finally, the golden grey mullet, _C. auratus_, was selected as a target species for monitoring priority pollutants in biota _sensu_ WFD. Thus, a total of three specimens in each WB were caught during 2021. The total length (TL, cm) was measured in all specimens, and fish fillets for THg analysis were selected from the largest individuals,  
Figure 1: The Marano and Grado Lagoon with the monitored water bodies (WBs) defined _sensu_ WFD/2000/60/CE [47].  
whereas juveniles and species characterised by small size (e.g., _A. boyeri_) and mullets were analysed _in toto_. All catches were conducted using the most appropriate and sustainable method depending on the targeted species. THg analyses were conducted by EPA 7473 [64] directly on fresh samples. Quality assurance was performed by blank control, analysis of replicates, and the use of certified reference material (DORM-2).  
Statistical analysis was performed using EXCEL(tm) and PAST (Paleontological Statistics, Version 4.08) statistical programmes [66]. The non-parametric Kruskal-Wallis H test assesses significant differences between two or more groups of an independent variable [67]. The Mann-Kendall trend test was employed to test for the presence of significant temporal increase/decrease in the selected parameters [68]. The distribution maps were realised using the QGis Version 3.16.3 \"Hannover\" program.