Effects of Age, Fulton's Condition Index (K) and Muscle Fat on Total Mercury Content in Raw, Pre-Canning and Canned Samples of Atlantic Bluefin Tuna (Thunnus thynnus).

A total of 30 samples of Atlantic bluefin tuna were analysed for total mercury concentration. Relationships between total mercury content and age, Fulton's condition index (K) and fat content were statistically evaluated. The effect of muscle status (raw, pre-canning, canned) on mercury content was also investigated. The average total mercury content was: 1.185 ± 0.662 mg/kg in raw, 1.481 ± 0.893 mg/kg in pre-canning and 1.393 ± 0.882 mg/kg in canned samples, respectively. Canning promotes a statistically significant increase in the concentration of mercury. The weight of fish, K and fat content are useful tools to estimate the mercury accumulation in Atlantic bluefin tuna. The results of the present study represent a contribution to the assessment of the EU mercury levels in Atlantic bluefin tuna.

Analytical Approaches in Official Food Safety Control: An LC-Orbitrap-HRMS Screening Method for the Multiresidue Determination of Antibiotics in Cow, Sheep, and Goat Milk.

The presence of unauthorized substances, such as residues of veterinary medicines or chemical contaminants, in food can represent a possible health concern. For this reason, a complete legislative framework has been established in the European Union (EU), which defines the maximum limits allowed in food and carries out surveillance programs to control the presence of these substances. Official food control laboratories, in order to ensure a high level of consumer protection, must respond to the challenge of improving and harmonizing the performance of the analytical methods used for the analysis of residues of authorized, unauthorized, or prohibited pharmacologically active substances. Laboratories must also consider the state of the art of the analytical methodologies and the performance requirements of current legislation. The aim of this work was to develop a multiresidue method for the determination of antibiotics in milk, compliant with the criteria and procedures established by Commission Implementing Regulation (EU) 2021/808. The method uses an LC-Orbitrap-HRMS for the determination of 57 molecules of antibiotic and active antibacterial substances belonging to different chemical classes (beta-lactams, tetracyclines, sulfonamides, quinolones, pleuromutilins, macrolides, and lincosamides) in bovine, ovine, and goat milk samples. It provides a simple and quick sample pretreatment and a subsequent identification phase of analytes, at concentrations equal to or lower than the maximum residual limit (MRL), in compliance with Commission Regulation (EU) 2010/37. The validation parameters: selectivity, stability, applicability, and detection capability (ccß), are in agreement with the requirements of Commission Implementing Regulation (EU) 2021/808 and demonstrated the effectiveness of the method in detecting veterinary drug residues at the target screening concentration (at the MRL level or below), with a false positive rate of less than 5%. This method represents an effective solution for detecting antibiotics in milk, which can be successfully applied in routine analyses for official food control plans.

Colorimetric Analysis and Determination of Histamine in Samples of Yellowfin Tuna (Thunnus albacares) Marketed in Sardinia (Italy) by a Combination of Rapid Screening Methods and LC-MS/MS.

The consumption of fishery products has been steadily increasing in recent decades. Among the quantitatively more important species, the yellowfin tuna (Thunnus albacares), is one of the main at-risk species as regards the possibility to present important levels of histamine and to be associated with the so-called "Scombroid Fish Poisoning". The main aim of the present study was to evaluate the colorimetric parameters, the occurrence, and the quantification of histamine contamination in yellowfin tuna samples marketed in Sardinia (Italy) by a combination of rapid screening and official control methods. A total of 20 samples of yellowfin tuna loins collected from large retailers, fishmongers and local markets were analyzed for the qualitative and quantitative determination of histamine by the lateral flow test HistaSure™ Fish Rapid Test and LC-MS/MS, respectively. Moreover, all the samples were examined to assess the conformity with the EU rules on labelling and subjected to colorimetric analysis according to the CIE-L*a*b* standard. Visual inspection of yellowfin tuna labels highlighted a 30% of non-compliances. A significant (p < 0.05) difference was reported for brightness (L *), redness (a *), and yellowness (b *). The results of histamine occurrence agreed with the food safety criteria (<100 mg/kg) laid down in EC Regulation 2073/2005 in the 95% and in the 90% of the samples with the rapid screening methods and LC-MS/MS, respectively. A highly significant sessional variation (p < 0.00001) was pointed out. Moreover, the two methods showed an agreement rate of 85%. The results of the present study confirmed the utility of lateral flow tests for the fast qualitative determination of histamine in yellowfin tuna. Rapid screening test should be strengthened by comparison with the official method especially in case of uncertain or positive results.

A representative sampling of tuna muscle for mercury control.

The mechanisms of mercury accumulation and distribution in fish tissues are related to its high affinity for sulfhydryl groups in proteins. There is evidence that mercury is distributed unevenly based on the different reactivity of these groups in the various muscle proteins. Tuna fish also shows numerous specialized anatomical features including the structure of the swimming muscles and some form of endothermy, which generates variations in the mercury content between dark and white muscle and between muscle tissues with different lipid content. The aim of the study is to verify, through a suitable sub lot of Thunnus thynnus caught by a static trap in south-western Sardinia, the effective uneven distribution of mercury in the various muscles and also identify the sites representative of the entire carcass. In agreement with other authors, the results show that even in the Bluefin tuna of the Mediterranean, the site "anterior extremity of upper loin (schienale in Italian)" is representative of the mercury average content of muscle tissues as a whole.

Occurrence of hexabromocyclododecanes and tetrabromobisphenol A in fish and seafood from the sea of Sardinia - FAO 37.1.3 area: Their impact on human health within the European Union marine framework strategy directive.

Levels of hexabromocyclododecane isomers α, ß, γ, (HBCDDs) and tetrabromobisphenol A (TBBP-A) were determined in 24 representative samples of different wild fish and seafood species (benthic: N = 16; pelagic: N = 8) and 16 samples of farmed bivalve molluscs from the West Mediterranean Sea (FAO 37, 1.3 sub-area). An LC-MS/MS-based method with limits of quantification (LOQS) in the range of 0.01-0.05 ng g-1 fresh weight (fw) was utilized. While α HBCDD was found in 80% of the 24 wild species samples, ß and γ congeners were found in 33% and 25%, respectively. ΣHBCDD content ranged from 0.03 (Aristeus antennatus) to 0.68 (Sardina pilchardus) ng g-1 fw as Upper Bound values across 2.00-4.46 trophic levels. In farmed molluscs, HBCDD congeners were always present and ranged from 0.22-0.52 ng g-1 fw, with the exception of one farm (1.23-2.06 ng g-1 fw), whose values suggest the presence of a regular emission source. TBBP-A levels always fell below the LOQ of 0.05 ng g-1 fw in all samples. The results are in good agreement with results of previous studies from the Mediterranean Sea. The Environmental Quality Standard for human health from fish and seafood local consumption was set at 165 µg g-1 fw. The Margin of Exposure of 490,020 as the ratio between the considered Health Based Guidance Level of 0.79 mg kg-1 body weight and the geo-referenced HBCDD intake (P95 fish and seafood intake; mean ΣHBCDD contamination) indicates no threat to food safety.

A case study on the development of representative sampling procedure to determine mercury levels in a lot of tuna caught by static trap.

Mercury contamination in fish products, and in large pelagic predatory fish in particular, is a remarkable food safety issue that affects fish industry. A lot made up by Bluefin tunas caught at Porto Paglia - in the south-western coast of Sardinia - was subject to several sessions of fish meat official sampling, as disputable results of mercury level determination suggested a potential non-compliance to maximum allowable limit. Local competent Veterinary Service reviewed the sampling procedure introducing more rigorous details including the identification of a specific muscular portion as sampling site, after having taken into account both statutory provisions laid down by Regulation (EC) No 333/2007 and available scientific evidence on variables affecting uneven distribution of mercury across tuna carcasses. This case-study aims to provide an initial contribution in order to ensure an appropriate and representative field sampling protocol of tuna lots that are to be analysed for mercury content.

Intake estimates of dioxins and dioxin-like polychlorobiphenyls in the Italian general population from the 2013-2016 results of official monitoring plans in food.

The implementation of the European Union strategy for polychlorodibenzo-dioxins and -furans (PCDD/Fs), and dioxin-like polychlorobiphenyls (DL-PCBs) is determining a general reduction of their presence in the environment and in the food chain. The most important route for human exposure to these substances is food consumption and, as a consequence, a progressive decrease of their dietary intake has been observed in the last decades. In this context, it seemed worth updating the PCDD/F and DL-PCB intake estimation for the Italian population. A total of 2659 samples of food of animal and vegetable origin analyzed for PCDD/Fs and DL-PCBs in the period 2013-2016 by accredited official laboratories and the national food consumption database were considered for the dietary intake assessment in different age groups of the Italian general population The median cumulative intake estimates expressed as pg WHO-TEQ/kg body weight per day and computed with a deterministic and a probabilistic approach were 1.40-1.52 for children, 0.82-0.85 for adolescents, and 0.64-0.61 for adults, respectively. Such results confirm the decreasing trend of PCDD/F and DL-PCB dietary intake even though the Tolerable Daily Intake (TDI) value of 2 WHO-TEQ/kg body weight per day is exceeded at the 95th percentile for all age groups, with children as sensitive group. Most contributing food categories to the intake resulted fish, food of vegetable origin, and cheese. A sensitivity analysis was also performed to calculate the target contamination levels able to keep the dietary exposure below the TDI. Computed target levels fall between P50 and P97 of the occurrence distribution of the main food groups, meaning that most of the Italian food production can be considered safe.

Application of a new targeted low density microarray and conventional biomarkers to evaluate the health status of marine mussels: A field study in Sardinian coast, Italy.

In the present study, we investigated the health status of marine mussels (Mytilus galloprovincialis) caged and deployed at three different sites on the Sardinian coastline characterized by different levels of contamination: Fornelli (F, the reference site), Cala Real (CR), and Porto Torres (PT). A new low density oligonucleotide microarray was used to investigate global gene expression in the digestive gland of mussels. Target genes were selected to cover most of the biological processes involved in the stress response in bivalve mollusks (e.g. DNA metabolism, translation, immune response, cytoskeleton organization). A battery of classical biomarkers was also employed to complement the gene expression analyses. Chemical analysis revealed higher loads of heavy metals (Pb and Cu) and total polycyclic aromatic hydrocarbons (PAHs) at PT compared to the other sites. In mussels deployed at CR, functional genomics analysis of the microarray data rendered 78 differentially expressed genes (DEGs) involved in 11 biological processes. Animals exposed at PT had 105 DEGs that were characterized by the regulation of 14 biological processes, including mitochondrial activity, adhesion to substrate, DNA metabolism, translation, metal resistance, and cytoskeleton organization. Biomarker data (lysosomal membrane stability, lysosomal/cytoplasm volume ratio, lipofuscin accumulation, metallothionein content, micronucleus frequency, and cytoskeleton alteration) were in trend with transcriptomic output. Biomarker data were integrated using the Mussel Expert System (MES), allowing defining the area in which the presence of chemicals is toxic for mussels. Our study provides the opportunity to adopt a new approach of integrating transcriptomic (microarray) results with classical biomarkers to assess the impact of pollutants on marine mussels in biomonitoring programs.

[Monitoring plan of environmental contaminants in sheep milk from areas next to an Italian contaminated site named «Aree industriali di Porto Torres e discarica di Calancoi¼ (Industrial areas of Porto Torres and landfill of Calancoi) in Sardinia Region (Southern Italy)]. / Piano di monitoraggio per la ricerca di contaminanti ambientali in latte ovino prodotto in aree prossime al sito di interesse nazionale denominato «Aree industriali di Porto Torres e discarica di Calancoi¼ in Sardegna.

OBJECTIVES: to assess concentration levels of dioxins, PCBs, and chemical elements in sheep milk produced in areas close to the Italian contaminated site (CS) located in the North-West of Sardinia (Southern Italy), named «Aree industriali di Porto Torres e discarica di Calancoi¼ (i.e., industrial areas of Porto Torres and landfill of Calancoi). DESIGN: monitoring plan of environmental contaminants on zootechnical productions based on analysis of dioxins, PCBs, heavy metals (Cd, Hg, Pb), and other elements (As, Be, Cr, Ni, Tl , Zn) in sheep bulk milk. SETTING AND PARTICIPANTS: 60 samples of sheep bulk milk were collected in rural farms where animals were fed mainly with forage and feed produced in the CS: 30 samples from farms located near the industrial area of Porto Torres and 30 taken from farms spread out the landfill area of Calancoi, a former waste dump in the East of Sassari city. MAIN OUTCOME MEASURES: the concentration levels of target substances were determined and compared to legal thresholds fixed on EU regulations. RESULTS: concentrations of target substances in all samples were below the maximum limits set by EU regulations. Dioxins and DL-PCBs (expressed as the sum) were in the range 0.20-1.30 pg WHO-TEQ/g fat, NDL-PCB (sum of the six marker congeners) were in the range 0.50-7.50 ng/g, while concentrations of all heavy metals were not detectable or were near the limit of quantitation of the method. Undetectable concentrations were also observed for the toxic elements beryllium and thallium. CONCLUSIONS: concentration levels of micro-pollutants in milk samples were significantly lower than the legal thresholds. Concentrations and congener profiles showed no relevant differences compared to milk samples from areas with low environmental impact, therefore consumption of milk from the studied area do not produce a higher intake of contaminants respect to the regional milk. The results of this study do not make the resident population safe from health risks caused by critical issue of the CS, but provide information limited to the safety of the milk from these areas, without showing peculiar contamination and hazard for food from local sheep farms.

Influence of metals on rhinosinusal polyposis in Sardinian population (Italy).

Metals have strong toxic effects in humans and can act as immunoregulatory factors. The purpose of our study was to determine whether the concentrations of metals are associated with the clinical course of nasal polyposis (NP). We measured the concentrations of 10 metals and non-metal (Zn, Mn, Se, Fe, Cr, Ni, Pb, Al, Cd, and Cu) in 58 patients with NP, and 29 controls with a healthy nasal mucosa. We used electron microscopy to compare the ultrastructural features of the nasal mucosa between NP patients and healthy controls. Concentrations of metals in nasal polyps and healthy mucosa were determined by mass spectrometry. Transmission electron microscopic (TEM) and scanning electron microscopic (SEM) images of the nasal mucosa were obtained. The mean tissue concentrations of all 10 metals and non-metal were significantly lower in NP patients than in healthy controls (P < 0.05).TEM and SEM revealed changes in the mucosal ultrastructure in NP with progressive fibrosis, devascularisation, and inflammation. Tissue concentrations of metals were lower in NP patients than in healthy controls, and this was particularly evident in massive polyposis.

Arsenic Speciation in Fish Products and Seafood as a Prerequisite for Proper Risk Assessment.

The Boi Cerbus lagoon, facing a mining and industrial site in Sardinia (Italy), is an important fishing area for the local population. Previous studies showed high concentrations of total arsenic (Astot) in fish, molluscs and crustaceans sampled in the lagoon, and a possible exceeding of the provisional tolerable weekly intake set by the Joint FAO/WHO Expert Committee on Food Additives by some local consumer groups. However, the percentage of inorganic As (Asinorg) should be known for a correct assessment of potential risk, as its toxicity is much higher than that of the organic forms. Eighty samples of 14 different species of fish, molluscs and crustaceans, sampled in the Boi Cerbus lagoon in 3 different seasons (winter, spring and summer), were analysed for Astot by inductively coupled plasma mass spectrometry (ICP-MS) and Asinorg by high performance liquid chromatography-ICP-MS. All the data obtained from the analysis were statistically processed to evaluate significant differences based on season, taxon and habitat, in preparation for a subsequent risk assessment.

Microbiological and Chemical Evaluation of Helix Spp. Snails from Local and Non-EU Markets, Utilised as Food in Sardinia.

With this study, 28 pools of snails of the genus Helix, respectively Helix aspersa (n=24) and Helix vermiculata (n=4) were analysed. They were taken from snail farming and stores. The snails were from Sardinia, other regions of Italy, and from abroad. All the samples were examined as pool looking for these microbiological target: Salmonella spp., Listeria monocytogenes, Escherichia coli O157, Clostridium perfringens, Norovirus and Hepatitis A Virus (HAV). In the same pools, the concentration of cadmium and lead by inductively coupled plasma mass spectrometry was also determined. The levels of these heavy metals were quite high, especially for cadmium. Two samples were positive for Salmonella spp., while no sample was positive for Escherichia coli O157, HAV and Norovirus. Two samples were positive for Clostridium perfringens and 8 for Listeria monocytogenes. The microrganisms related to Listeria monocytogenes were identified using biochemical techniques, then serotyped and gene sequenced by multiple loci sequence typing technique. Furthermore, antimicrobial restistence was tested on the same samples.

Individual methylmercury intake estimates from local seafood of the Mediterranean Sea, in Italy.

A Seafood Frequency Questionnaire (SFQ) broken down in more than 42 items with 8-week coverage was interview-administered to 278 adults aged 19-82years (167 women, 98 in the reproductive age 19-45years, and 111 men), resident on the Italian Mediterranean shore and frequent buyer at local fish markets. Methylmercury (MeHg) intake on individual basis was estimated for a selected occurrence equal to the median value+Median Absolute Deviation (MAD) in each seafood species reported (conservative scenario). MeHg occurrence was derived from an extensive seafood database referred to years 2009-2011. Accounting for an average body weight of 62.2kg, 24.6% of women resulted overexposed with respect to the European Food Safety Authority (EFSA) Tolerable Weekly Intake (TWI) for MeHg of 1.3µg/kg bw, with a mean of 0.92µg/kg bw. In the vulnerable group aged 19-45years, 29.6% exceeded the TWI. Rather than the amount of seafood consumed, the seafood choice appears to be the main determinant of the MeHg intake. Risk awareness was reported in the 49% of SFQs. Uncertainties related to such estimates from questionnaires are discussed, in order to give adequate health recommendations without compromising seafood consumption in the Mediterranean region.

Mercury occurrence in Italian seafood from the Mediterranean Sea and possible intake scenarios of the Italian coastal population.

Mercury (Hg) settlements in the Mediterranean Sea determine a potential toxicological relevance of seafood intakes for coastal populations. To assess this possibility, fish, molluscs, and crustaceans of commercial size of 69 different species were sampled and analyzed for total mercury (Hg(TOT)) from georeferenced areas and evaluated for their compliance with the European Union Maximum Residue Limits of 0.5 and 1.0 mg/kg wet weight (ww). Accounting for the weekly estimated seafood intake in the Italian coastal population (mean 469-626 g/person/week) it was then possible to recover threshold contamination values in seafood. Under a Tolerable Weekly Intake of 1.3 µg/kg/bw/week, a threshold seafood contamination <0.10 mg/kg ww has been derived for sensitive groups. A suitable algorithm based on the parallel MeHg and Hg(TOT) analysis on the most consumed species, helped to refine the uncertainties related to the conservative assumption in seafood all the Hg(TOT) present is in form of MeHg. This work aims to improve the link between the risk management and risk assessment strategies, with the identification of those fish and seafood species, that, when regularly consumed, could determine or prevent potential Hg(TOT)/MeHg overintakes in sensitive groups.

Effect of amalgam fillings on the mercury concentration in human amniotic fluid.

BACKGROUND: Methyl mercury (MeHg) and metallic Hg are well known as neurotoxic agents. Dental amalgam contributes significantly to elemental Hg vapour exposure in the general population. There is little information about Hg concentration in human amniotic fluid (AF) of pregnant women and its potential toxic effect on the fetuses. OBJECTIVE: Primary to assess the relationship between the presence of detectable mercury (Hg) concentration in human AF, number and surface areas of amalgam fillings of pregnant women; secondary to analyse their obstetric history and perinatal complications. METHODS: Seventy-two pregnant women took part in this prospective study. One dentist recorded the dental status, presence, number and surface areas of amalgam fillings. Total Hg concentration in AF was determined in digested samples using automatic cold vapour atomic absorption equipment. The detection limit of Hg in AF, determined from blank readings, was 0.08 ng/ml. To estimate the dependence of the explanatory variables (such as number and surface areas of amalgam fillings, fish consumption, presence of liver or neurological diseases and smoking habits) on mercury concentration several linear regression models were built up. Stepwise logistic regression procedures were running on total sample and on patients with at least one amalgam filling (Positive Filling group = PF). Principal component analysis (PCA) provided two factors, which explained for more the 60% of the variance among the variables. RESULTS: The overall mean Hg concentration in AF among all patients was 0.37+/-0.49 ng/ml. Nineteen (26.4%) women had a Hg concentration <0.08 ng/ml (Hg negative group). In 53 (73.6%) patients, with a concentration > or = 0.08 ng/ml (Hg positive group), the mean value of Hg was 0.49+/-0.52 ng/ml. The average number of amalgam fillings was 2.26 +/- 3.19 in the Hg negative group and 5.32+/-3.03 in the Hg positive group (ANOVA one-way p=0.04). A dependence of mercury concentration on number of amalgam fillings (p=0.03), surface area of the amalgam fillings (p=0.04) and fish consumption (p=0.04) was observed but not at a significant level. In stepwise logistic procedure the number of amalgam fillings gave a contribution to the model (p=0.04), although null value was included in the confidence intervals. We observed no statistically significant differences (chi2 test) among the patients with a Hg concentration <0.08 ng/ml (n=19) and those with a concentration > or = 0.08 (n=53) with regard to obstetric history and perinatal complications. CONCLUSIONS: Number and surface areas of amalgam fillings influenced positively Hg concentration in AF but not at a significant level. Moreover Hg levels detected in AF were low and no adverse outcomes were observed through pregnancies and in the newborns.