BDE-47 exposure modulates cellular responses, oxidative stress and biotransformation related-genes in Mytilus galloprovincialis.

Polybrominated diphenyl ethers (PBDEs) are flame retardants, characterized by elevated stability in the marine environment, where are accumulated by organisms, inducing a wide panel of negative effects. In this study, some biochemical patterns related to toxicity, biotransformation and oxidative stress, were studied in the marine model system, Mytilus galloprovincialis, exposed to BDE-47. Mussels were fed with microalgae, previously treated with increasing concentrations of PBDEs (maximum dose 100 ng L-1 of BDE-47 per day). After 15 days of treatment, mussels were fed with the same diet without BDE-47, for additional 15 days. Gills and digestive glands were analyzed at T 0, at 15 and 30 days. Histopathological lesions were assessed in digestive glands of contaminated mussels, while expression of genes, related to cell cycle, multidrug resistance, oxidative stress and detoxification was evaluated on both gills and digestive glands. After 15 days, BDE-47 exposure significantly affected the cell activity in digestive gland and, at 30 days, only mussels exposed to the lower doses showed a certain recovery. Regarding the gene expression, both gills and digestive glands showed a significant down-regulation of the target genes at 15 days, although most of them were up-regulated at 30 days in digestive gland. The results on BDE-47 accumulation in mussels revealed a dose-dependent concentration in tissues, which remained elevated after further 15 days of depuration. This trend supports the responses of the biomarkers, indicating that exposure, at environmentally realistic concentrations of BDE-47, strongly modulates oxidative stress and related patterns of gene expression, suggesting concerns for long-term effect in the biota.

Heavy metals concentrations in some commercially key species from Sicilian coasts (Mediterranean Sea): Potential human health risk estimation.

Trace elements were measured in the edible tissues of target marine species (Sardina pilchardus, Mullus barbatus, Mullus surmuletus, Merluccius merluccius and Parapenaeus longirostris) collected in some of the main ports of the Sicilian coast (southern Italy) in order to evaluate the potential human health risk associated to their consumption. Cadmium, Pb and Hg were below the tolerable limits reported by the European Union in foodstuffs (Commission Regulation (EC) 1881/2006) except for Hg in P. longirostris from one sampling site. The health risk for consumers was assessed both for adults and children calculating the estimated weekly intake (EWI), the target hazard quotient (THQ) and the lifetime cancer risk (CR) for each element. The EWI did not exceed the provisional tolerable weekly intake (PTWI) recommended by the European Food Safety Authority and THQs were < 1 in most cases. Further, the probabilistic distributions of health hazard were evaluated through Monte-Carlo simulation which confirmed a non-carcinogenic risk. With some exceptions related to As exposure, the results obtained definitively confirm the goodness of the fishery commercial resources in one of the most important seafood market of the central Mediterranean Sea and represent a positive data for the assessment of the good environmental status for descriptor 9 of the Marine Strategy Framework Directive in this area of the basin.

Bioaccumulation of heavy metals in fish, crustaceans, molluscs and echinoderms from the Tuscany coast.

The concentration of As, Cd, Cr, Cu, Hg, Ni, Pb and Zn were analyzed in the edible part of several species of fish, crustaceans, molluscs and echinoderms collected in sensitive areas of the Tuscany coast (northern Italy). The concentration of As (0.39-78.1 µg g-1) and Hg (0.01-1.56 µg g-1) resulted in most cases higher than reference thresholds. Target hazard quotient (THQ) and lifetime cancer risk (TR) indexes were calculated to assess cancer and non-cancer risk due to oral exposure; the highest THQ values referred to As and Hg, with values ≥ 1 in 39% and 48% of cases, respectively. Total target hazard quotients (TTHQ) values suggested that the local population could experience adverse health effects due to consumption of local seafood, mainly of demersal and benthic species. Cancer risk was mainly associated with As exposure, and with Cd intake, especially through molluscs consumption. The NMDS model highlighted species specific bioaccumulation processes and specific sensitivity of species to different bioavailable heavy metals. Specifically, Mullus spp. and Scorpaena porcus preferentially accumulate Hg and Cr, Octopus vulgaris specimens were discriminated by the presence of Pb and Zn, while an evident preference for Cd and Cu was recorded in Squilla mantis. In addition, the distribution of heavy metals in organisms revealed sound differences between Follonica and Livorno sampling sites, demonstrating a highly heterogeneous anthropogenic impact in terms of heavy metals input from the industrial activity resting on land.

Hg and Se exposure in brain tissues of striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus) from the Tyrrhenian and Adriatic Seas.

In this study we analyzed Hg and Se concentrations in dolphin brain tissues of fifteen specimens of striped dolphin (Stenella coeruleoalba) and eight specimens of bottlenose dolphin (Tursiops truncatus) stranded in the Tyrrhenian and Adriatic Seas, in order to assess the toxicological risks associated with Hg exposure. High Hg concentrations were found in brain tissues of both analyzed specie (1.86-243 mg/kg dw for striped dolphin and 2.1-98.7 mg/kg dw for bottlenose dolphin), exceeding levels associated with marine mammals neurotoxicity. Althougth the results clearly suggest that the protective effects of Se against Hg toxicity occur in cetaceans' brain tissues, a molar excess of mercury with respect to selenium was found, particularly in adult specimens of Stenella coeruleoalba. On contrary, negligible neurotoxicological risks were found for Tursiops truncatus specimens, due to detoxification processes. Data obtained allowed to prove a more marked neurotoxicological risk for adult specimens of Stenella coeruleoalba in both Tyrrhenian and Adriatic Seas.

Assessment of mercury exposure in human populations: A status report from Augusta Bay (southern Italy).

Here we investigate mercury concentrations in the blood (HgB), urine (HgU) and human hair (HgH) of 224 individuals from a coastal area (Eastern Sicily, SE Italy) strongly affected by Hg contamination from one of the largest chlor-alkali plants in Europe. The factors affecting the distribution of Hg and the extent of the exposure of individuals have been explored with a multidisciplinary approach. Multiple regression analyses, together with evidence of high levels of HgB (exceeding the HBMI recommended levels in 50% of cases) and HgH (exceeding the EPA reference dose in 70% of cases), primarily suggest that the consumption of local fish is the main source of Hg for humans. no. significant exposure to inorganic mercury was identified. Toxicokinetic calculations produced a provisional tolerable weekly intake (PTWI) level that, in most cases, exceeds international recommendations, particularly for residents in the studied area.

[Evaluation of mercury exposure in populations living near the industrial Augusta Bay (Sicily Region, Southern Italy)]. / Valutazione dell'esposizione al mercurio nelle popolazioni residenti in prossimità dell'area industriale della Rada di Augusta (Siracusa).

OBIETTIVI: valutare e quantificare l'esposizione umana al mercurio (Hg) ed esplorare i fattori chiave che influenzano la contaminazione nelle popolazioni residenti in prossimità della Rada di Augusta, un'area a elevato rischio ambientale a causa degli effetti degli sversamenti incontrollati di Hg da parte di uno dei più grandi impianti cloro-soda d'Europa. DISEGNO: un campione di residenti nei comuni di Augusta, Priolo e Melilli (Sicilia orientale) è stato selezionato per lo studio di biomonitoraggio umano. Un questionario dettagliato è stato somministrato a ogni partecipante per raccogliere informazioni anagrafiche sulle abitudini di vita e alimentari. I livelli di Hg in sangue e capelli, usati come traccianti di esposizione a metilmercurio, sono stati messi in relazione al consumo di pesce locale. Il contenuto di Hg nelle urine è stato utilizzato per esplorare eventuali esposizioni atmosferiche a Hg inorganico. Una regressione lineare multivariata è stata applicata con l'obiettivo di esplorare i fattori che influenzano l'esposizione umana al Hg. L'approccio tossicocinetico è stato impiegato al fine di calcolare la dose settimanale provvisoria tollerabile ( provisional tolerable weekly intake, PTWI) e di predire le concentrazioni di Hg attese nel sangue degli individui con una dieta alimentare basata esclusivamente su pesce locale. SETTING E PARTECIPANTI: 224 individui residenti nei comuni di Augusta, Melilli e Priolo. RISULTATI: eccessi di Hg sono stati misurati nel sangue e nei capelli del campione considerato. I più alti livelli sono stati rilevati ad Augusta, dove anche il consumo di pesce locale risulta maggiore. I valori di dose settimanale tollerabile provvisoria (PTWI) calcolati eccedono, nella maggior parte dei casi, le raccomandazioni internazionali, specialmente nei residenti ad Augusta. CONCLUSIONI: gli elevati livelli di Hg registrati nei campioni di sangue e capelli derivano, con ogni probabilità, dal consumo di pesce locale. Rispetto ad Augusta, gli individui di Priolo e Melilli subiscono un impatto più limitato. L'esposizione a Hg inorganico può essere considerata trascurabile.

Synthesis of water suitable as the MEPC.174(58) G8 influent water for testing ballast water management systems.

Here, we describe the methodologies adopted to ensure that natural seawater, used as "influent water" for the land test, complies with the requirement that should be fulfilled to show the efficacy of the new ballast water treatment system (BWTS). The new BWTS was located on the coast of SW Sicily (Italy), and the sampled seawater showed that bacteria and plankton were two orders of magnitude lower than requested. Integrated approaches for preparation of massive cultures of bacteria (Alcanivorax borkumensis and Marinobacter hydrocarbonoclasticus), algae (Tetraselmis suecica), rotifers (Brachionus plicatilis), and crustaceans (Artemia salina) suitable to ensure that 200 m(3) of water fulfilled the international guidelines of MEPC.174(58)G8 are here described. These methodologies allowed us to prepare the "influent water" in good agreement with guidelines and without specific problems arising from natural conditions (seasons, weather, etc.) which significantly affect the concentrations of organisms at sea. This approach also offered the chance to reliably run land tests once every two weeks.

Electrochemical remediation of synthetic and real marine sediments contaminated by PAHs, Hg and As under low electric field values.

To date, remediation, protection, and restoration of contaminated sites is a global concern. The current technologies to restore sediments characterized by heterogeneous characteristics, several pollutants, fine grains, and low hydraulic permeability are poorly effective; hence their remediation is still challenging. A promising approach for the sediment's remediation could be the electrochemical route since it is a not-expensive, effective and noninvasive in situ technology. Electrochemical remediation (ER) is commonly studied under relatively high electric fields (E ≥ 1 V cm-1) and using costly processing fluids in a three compartments cell aiming to desorb and transport the contaminants into the processing fluids (secondary dangerous effluent). In this work, contaminated marine sediments were electrochemically treated focusing on the insertion of electrodes directly in the sediments and adopting, for the first time for real sediments, low E values (≤ 0.25 V cm-1) for 4-days period. It was observed that PAHs can be simultaneously transported and degraded in situ preventing the production of a secondary dangerous effluent and reducing the energy consumption. Firstly, clay marine sediments dragged from Capo Granitola Coast (Trapani, Italy) spiked with five PAHs congeners (5PAHs), Hg and As were used as a simplified model matrix and treated to simulate a real case study. A total PAHs removal efficiency of 57% was reached after 96 h of treatment under 0.05 V cm-1. Then, real polluted marine sediments from Augusta Bay (Syracuse) and Bagnoli-Coroglio Bay (Naples) in the southern Italy were treated as real contaminated sediments to be restored, to validate the proposed approach for real cases. A quite good removal efficiency of PAHs was reached after 96 h of electrochemical treatment coupled with a low energetic consumption due to the rather E values adopted. In addition, it was observed that this approach, under the adopted conditions, is unsuitable for the remediation of Hg and As.

Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy).

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

Hydroclimate variability in the central Mediterranean during MIS 17 interglacial (Middle Pleistocene) highlights timing offset with monsoon activity.

Mediterranean climates are characterized by warm, dry summers and mild, rainy winters. Previous studies suggest that over the last 1.36 Myr, Mediterranean winter rainfalls were in phase with the African monsoon. Here we present a high-resolution terrestrial and marine dataset for the Marine Isotope Stage 17 interglacial (Middle Pleistocene) from Southern Italy, showing that precipitation rates and regimes in the central Mediterranean varied independently of the monsoon system. Specifically, events of extreme summer precipitation were promoted by increased regional insolation rates and/or extratropical cyclones, and their magnitude was further enhanced by the advection of cool and humid North Atlantic air during stadials. Our findings provide new information on the short- to mid-term natural hydroclimatic variability of the Mediterranean basin, and offer new critical insights on land-ocean interactions at the regional scale by complementing previous analyses on the displacement of storm tracks toward southern Europe.

Atmospheric Deposition around the Industrial Areas of Milazzo and Priolo Gargallo (Sicily-Italy)-Part A: Major Ions.

The chemical composition of rainwater was studied in two highly-industrialised areas in Sicily (southern Italy), between June 2018 and July 2019. The study areas were characterised by large oil refining plants and other industrial hubs whose processes contribute to the release of large amounts of gaseous species that can affect the chemical composition of atmospheric deposition As in most of the Mediterranean area, rainwater acidity (ranging in the study area between 3.9 and 8.3) was buffered by the dissolution of abundant geogenic carbonate aerosol. In particular, calcium and magnesium cations showed the highest pH-neutralizing factor, with ~92% of the acidity brought by SO42- and NO3- neutralized by alkaline dust. The lowest pH values were observed in samples collected after abundant rain periods, characterised by a less significant dry deposition of alkaline materials. Electrical Conductivity (ranging between 7 µS cm-1 and 396 µS cm-1) was inversely correlated with the amount of rainfall measured in the two areas. Concentrations of major ionic species followed the sequence Cl- > Na+ > SO42- ≃ HCO3- > ≃ Ca2+ > NO3- > Mg2+ > K+ > F-. High loads of Na+ and Cl- (with a calculated R2 = 0.99) reflected proximity to the sea. Calcium, potassium, and non-sea-salt magnesium had a prevalent crustal origin. Non-sea salt sulphate, nitrate, and fluoride can be attributed mainly to anthropogenic sources. Mt. Etna, during eruptive periods, may be also considered, on a regional scale, a significant source for fluoride, non-sea salt sulphate, and even chloride.

A chemical remediation technique for a nearly-total removal of arsenic and mercury from contaminated marine sediments.

After decades of industrial exploitation of the coast and consequent contamination of the sites and marine sediments, it became essential to recover the marine ecosystem by remediation methods to remove toxic contaminants. In this work, a remediation method was developed to clean marine sediments contaminated by arsenic (As) and mercury (Hg). The method can be applied to mobile platforms and is based on an environmentally friendly approach designed to minimise further contamination. The method was tested on two artificially contaminated sediments and two real samples collected from two highly contaminated sites in southern Italy, Augusta Bay and Bagnoli Gulf, characterised by high Hg and As concentrations, respectively. The method consists of four steps: washing with sodium hydroxide (NaOH) to remove metals associated with humic acid; Fenton-reaction using α-CycloDextrin (aCD) to stabilise Fe(II) at natural pH and oxidise As (III) and Hg (0 or I); complexation reaction with aCD; and complexation with sodium sulfide (Na2S) to remove Hg as soluble Hg-polysulfides. Compared to other remediation experiences in literature, this technique provides the best removal efficiency for As and Hg (ranging between 26 -71 % and 57-95 %, respectively). Considering the residual concentrations of As and Hg and the contamination threshold fixed by European Regulation for re-use, the treated sediment can be used in several civil and industrial contexts. The presented method operates in line with the principles of the circular economy to preserve natural resources, prevent secondary pollution, and promote the effective re-use of clean environmental matrices (soils, sediments and aqueous solutions), thus minimising landfill waste.

Exposure profiles in pregnant women from a birth cohort in a highly contaminated area of southern Italy.

Protecting the health of pregnant women from environmental stressors is crucial for reducing the burden of non-communicable diseases. In industrially contaminated sites, this action is particularly challenging due to the heterogeneous pollutant mixtures in environmental matrices. The aim of this study was to evaluate distribution patterns of mercury, hexachlorobenzene and polychlorobiphenyls in the serum of 161 pregnant women recruited in the framework of the Neonatal Environment and Health Outcomes (NEHO) cohort and living both inside and outside the National Priority Contaminated Site (NPCS) of Priolo. Food macro-categories were determined, and serum levels of contaminants were used to perform k-means cluster analysis and identify the role of food in pollutant transfer from the environment. Two groups of mothers with high and low measured pollutant levels were distinguished. Concentrations in mothers in the high-exposure cluster were at least twofold for all the evaluated pollutants (p < 0.0001) and included mothers living inside and outside NPCS, with a predominance of individuals from the NPCS (p = 0.045). Fish consumption was higher in the high-exposure cluster (p = 0.019). These findings suggest a link between contamination of environmental matrices such as sediment with maternal exposure, through the intake of local food. Such consideration appears poorly investigated in the context of contaminated sites.

Distribution of Cd and As in organs and tissues of four marine mammal species stranded along the Italian coasts.

Concentrations of Cd and As were determined in organs and tissues (muscle, heart, kidney, lung and liver) of four cetacean species (Stenella coeruleoalba, Truncatus truncatus, Grampus griseus and Ziphius cavirostris) stranded along the Italian coasts during the period 2000-2009. Significant differences were found between Cd concentrations in the different analysed tissues. Particularly, the kidney shows the highest concentrations of Cd in all analysed specimens, followed by the liver. No systematic or statistically significant difference in As concentrations was found in the analysed tissues. Considerable differences of Cd and As values in samples from different geographical areas (Sicily Channel, Adriatic Sea and Tyrrhenian Sea) were found, with the highest values measured in samples from the Sicily Channel. This basically suggests that anthropogenic and natural sources significantly affect Cd and As content in cetaceans.

A multipollutant low-grade exposure regulates the expression of miR-30b, Let-7a and miR-223 in maternal sera: Evidence from the NEHO cohort.

There is growing evidence that environmental pollutants can induce epigenetic modifications altering the balance of miRNAs and inducing the onset of pathological conditions in animals. In this study, we measured the serum concentration of a suite of inorganic and organic pollutants (Cu, Zn, Se, Hg, HCB, p,p'-DDE, PCBs) and their association to serum miR-30b, miR-223 and Let-7a microRNA expression in 68 healthy pregnant women from the NEHO birth cohort sited in a highly industrialized area. The effects of the pollutants on the modulation of circulating miRNAs' expression were first investigated using linear continuous regression models with a single-compound approach showing that miR-223 expression was significantly associated with serum concentration of Se and Zn (pSe = 0.0336; pZn = 0.0225) and miR-30b was associated with Hg levels (pHg = 0.019). Furthermore, when contaminants were categorized into tertiles, miR-223 and miR-30b showed a positive association with higher tertiles of Zn, p,p'-DDE (pZn = 0.023; pDDE = 0.041) and Hg (pHg = 0.008), respectively. Moreover, Let-7a expression was exclusively influenced by medium tertiles levels of Se (low vs medium tertiles, p = 0.001). Simultaneous exposure to multi-pollutant mixture was approached by WQS regression model. Statistical analysis shows a driving effect of Zn, Se, Cu, Hg and HCB on significant increased expression of Let-7a (p = 0.045). Mercury and Se significantly amplified the expression for miR-30b (p = 0.038). Differently, the combined effect of p,p'-DDE, Zn and Se decreased miR-223 expression (p = 0.0001). The documented modified expression of circulating miRNAs in the serum of pregnant women, exposed to low-medium dose contaminants mixtures offers innovative early-warning approaches to human health risk assessment.

Large-Scale Mercury Dispersion at Sea: Modelling a Multi-Hazard Case Study from Augusta Bay (Central Mediterranean Sea).

This contribution discusses an example of potential multi-hazard effects resulting from an earthquake in a highly seismogenic area of the Mediterranean Sea, the Augusta Bay, which presents high levels of contamination in sediments and seawater, due particularly to high-concentrations of mercury as a result of a long-term industrial exploitation. In particular, a high-resolution hydrodynamic and transport model is used to calculate the effects of enhanced mercury spreading in the open sea after significant damage and collapse of the artificial damming system confining the embayment where a very high concentration of Hg occurs in seafloor sediments and seawater. Coupling high-resolution 3D dynamic circulation modelling and sediment-seawater Hg fluxes calculated using the HR3DHG diffusion-reaction model for both inorganic and organic Hg species offers a valuable approach to simulating and estimating the effects of spatial dispersion of this contaminant due to unpredictable hazard events in coastal systems, with the potential attendant enhanced effects on the marine ecosystem. The simulated scenario definitely suggests that a combination of natural and anthropogenic multi-hazards calls for a thorough re-thinking of risk management in marine areas characterised by significant levels of contamination and where a deep understanding of the biogeochemical dynamics of pollutants does not cover all the aspects of danger for the environment.

10-year time course of Hg and organic compounds in Augusta Bay: Bioavailability and biological effects in marine organisms.

In the last century, many Mediterranean coastal areas have been subjected to anthropogenic disturbances from industrial activities, uncontrolled landfills, shipyards, and high maritime traffic. The Augusta Bay (eastern Sicily, Italy) represents an example of a strongly impacted coastal environment with an elevated level of sediments contamination due to the presence of one of the largest European petrochemical plants, combined with an extensive commercial and military harbor. The most significant contaminants were represented by mercury (Hg) and hexachlorobenzene (HCB), derived from a former chlor-alkali plant, and other organic compounds like polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). Since the 1970s, Augusta Bay has become internationally recognized as a contaminated marine environment, although very little information is available regarding the temporal trend of contaminants bioavailability and biological impacts on aquatic organisms. In this study, the Hg and HCB concentrations were investigated over 10 years (from 2003 to 2013) in sediments and invertebrate and vertebrate organisms; these two contaminants' ecotoxicity was further evaluated at a biochemical and cellular level by analyzing the induction of organic biotransformation processes and DNA damages. The results showed high concentrations of Hg and HCB in sediments and their strong bioaccumulation in different species with significantly higher values than those measured in reference sites. This trend was paralleled by increased micronuclei frequency (DNA damage biomarker) and activity of the biotransformation system. While levels of chemicals in sediments remained elevated during the time course, their bioavailability and biological effects showed a gradual decrease after 2003, when the chlor-alkali plant was closed. Environmental persistence of Hg and HCB availability facilitates their bioaccumulation and affects the health status of marine organisms, with possible implications for environmental risk, pollutants transfer, and human health.

COVID-19 lockdowns reveal the resilience of Adriatic Sea fisheries to forced fishing effort reduction.

The COVID-19 pandemic provides a major opportunity to study fishing effort dynamics and to assess the response of the industry to standard and remedial actions. Knowing a fishing fleet's capacity to compensate for effort reduction (i.e., its resilience) allows differentiating governmental regulations by fleet, i.e., imposing stronger restrictions on the more resilient and weaker restrictions on the less resilient. In the present research, the response of the main fishing fleets of the Adriatic Sea to fishing hour reduction from 2015 to 2020 was measured. Fleet activity per gear type was inferred from monthly Automatic Identification System data. Pattern recognition techniques were applied to study the fishing effort trends and barycentres by gear. The beneficial effects of the lockdowns on Adriatic endangered, threatened and protected (ETP) species were also estimated. Finally, fleet effort series were examined through a stock assessment model to demonstrate that every Adriatic fishing fleet generally behaves like a stock subject to significant stress, which was particularly highlighted by the pandemic. Our findings lend support to the notion that the Adriatic fleets can be compared to predators with medium-high resilience and a generally strong impact on ETP species.

Corrigendum: 10-year time course of Hg and organic compounds in Augusta Bay: Bioavailability and biological effects in marine organisms.

[This corrects the article DOI: 10.3389/fpubh.2022.968296.].