Domestic dogs as environmental sentinel in comparative toxicologic pathology: Assessment of metals and rare earth elements concentrations in healthy and neoplastic mammary glands.

Quantification of trace element concentrations in human and animal tissues has acquired great importance in the last few years, considering the pivotal role of these elements in several physiological and pathological processes. Variations in their concentrations appear to have a role in the development and advancement of diseases in both humans and animals, for example, cancer. The purpose of this study was to investigate the concentration of rare earth elements and metals in healthy and neoplastic Formalin-Fixed Paraffin-Embedded (FFPE) mammary gland tissue of dogs. All samples were processed to have a quantitative determination of inorganic elements including metals of known toxicological interest such as Pb, Cd, Tl, As, Hg, the trace elements Mn, Fe, Co, Cu, Zn, Se, and other elements including Cr, V, Mo, Ni, Sb, W, Sn. Moreover, rare earth elements (REEs) (Sc, Y, Lu, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb) were also investigated. Cu and Mo concentrations in mammary cancerous tissue were greater than those in normal mammary glands (p < 0.05). In non-neoplastic tissue increased concentrations of Cd, Co, Ni, Tl, and V were also reported (p < 0.05). The mammary tissue of healthy individuals had greater concentrations of REEs than the neoplastic mammary glands (p < 0.05). The results of our study confirmed differences in mammary inorganic element concentrations between healthy and neoplastic groups, highlighting the potential relevance of these fluctuations in toxicologic pathology.

Comparative analysis of rare earth elements concentrations in domestic dogs and Apennine wolves of Central Italy: Influence of biological, nutritional, and lifestyle factors.

Rare Earth Elements (REEs) are strategical elements playing a crucial role in the industry, especially in producing high-tech materials. Therefore, REEs are new contaminants of emerging concerns. However, due to the lack of exposure data on REE occurrence in environmental matrices, especially in European countries, it is still tricky to establish environmental background levels to assess the ecotoxicological risk related to REEs exposure. The present study aimed to evaluate the liver concentrations of REEs in domestic dogs (Canis lupus familiaris) and Apennine wolves (Canis lupus italicus) living in the Abruzzo region, Italy. Moreover, for the scope of the present study, the dog's group was divided according to their sex, age, lifestyle, and diet. Wolves were categorized concerning their sex and genetic characteristics. Liver samples from dogs and wolves were collected during diagnostic necropsies from carcasses, sample mineralization was performed by a microwave digestion system with a single reaction chamber, and simultaneous determination of the presence of REEs was performed by Inductively Coupled Plasma Mass Spectrometer (Q-ICP-MS) using standard mode for all rare earth elements except scandium (Sc) which was acquired in kinetic energy discrimination (KED) mode. Hepatic concentrations of REEs were statistically significantly higher in wolves compared to dogs. Moreover, significant differences in REEs concentrations arose also from the genetic type of wolf, since "pure wolves" had higher liver concentrations of REEs compared to wolf-dog hybrids. Female and adult dogs also showed elevated REEs compared to male and juvenile dogs, while no significant differences were demonstrated for dogs' diet and lifestyle. The results of the present study confirm the exposure of domestic and wild carnivores to REEs, showing also the ability of REEs to accumulate in carnivore livers, suggesting the potential role of this species as an alternative bioindicator.

Rare earths and other elements content in hen eggs sold in Italy: comparison among main types of farming methods and consumer's health risk assessment.

Recently, in Italy, consumers are choosing hen eggs from farming systems with higher ethical value, due to their perception of a related higher quality and safety. The purposes of this study were to evaluate the existence of differences in elemental content in Italian eggs from organic, barn, and caged hen farming methods and to determine the related potential consumer exposure risk to inorganic contaminants due to the consumption of eggs. One hundred seventy-six egg samples were collected and analyzed using Q-ICP-MS to investigate the content of 14 elements (Pb, As, Hg, Cd, Tl, Fe, Zn, Mn, Cu, Se, Co, Ni, V, and Cr) and 13 rare earth elements (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb). The scenarios of exposure to rare earth and other elements from eggs were estimated for three age groups of consumers. The daily intake values were always lower than the respective safety reference values. In conclusion, Italian hen eggs contain low levels of rare earth and other elements, and therefore, their consumption does not represent a risk of exposure. Finally, no significant differences in contaminants between conventional and organic farming methods were found.

Official controls for the determination of lipophilic marine biotoxins in mussels farmed along the Adriatic coast of Central Italy.

In the present study, 334 samples of mussels (Mytilus galloprovincialis) harvested along the coasts of the Central Adriatic Sea during the years 2020-2021 were analyzed for the presence of lipophilic marine biotoxins according to the European Harmonized Standard Operating Procedure. The results showed that 74 (22%) and 84 (25%) samples were positive to okadaic acid and yessotoxin groups, respectively. Among them, only 11 (3.3%) samples resulted as non-compliant, as they exceeded the maximum limits (160 µg okadaic acid equivalent/kg) established by the Regulation (EC) 853/2004. The method applied in this study was able to detect and quantify lipophilic marine biotoxins concentrations, in order to monitor their presence in molluscs and avoid the risk of consumer exposure.

A new method for determining PFASs by UHPLC-HRMS (Q-Orbitrap): Application to PFAS analysis of organic and conventional eggs sold in Italy.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous environmental pollutants with the ability to uptake to food and feed. Among food, fish, fruits and eggs are considered as major contributors to human dietary exposure. A new method was developed and validated for the simultaneous determination of 18 PFASs in eggs using isotope dilution followed by ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry. The analysis of 132 samples (organic, barn and caged eggs) was performed. Levels were always close to the detection limits and no significant difference emerged among the 3 groups. The highest PFAS concentration in eggs was used to estimate the dietary exposure of different Italian population groups. As expected, children were more highly exposed than adults due to lower body weight. This data suggests that the recent tolerable weekly intake of 4.4 ng kg-1b.w. could be exceeded when the cumulative intake arising from other food products is considered.

Chlorinated Persistent Organic Pollutants (PCDD/Fs and PCBs) in Loggerhead Sea Turtles Stranded along the Central Adriatic Coast.

Persistent organic pollutants are widespread in the marine environment. They can bioaccumulate and biomagnify in marine organisms through the food web with a potentially toxic effect on living organisms. The sea turtle Caretta caretta is a carnivorous animal with opportunistic feeding behavior. These turtles tend to bioaccumulate pollutants through food, and hence they can be considered an indicator of chemical pollutants in the marine ecosystem. In this study, 44 loggerhead sea turtles were considered, and liver and fat tissue were sampled from each of them to investigate the levels of dioxins (PCDD/Fs) and polychlorinated biphenyls (PCBs) in sea turtles and their potential correlation with sex and size in terms of curved carapace length (CCL). Results suggested that these contaminants were easily bioaccumulated, and PCBs were predominant compared to dioxins in both liver and fat tissue. The congener patterns were similar to those found in sea fish. Moreover, there were no differences in the contamination levels between females and males, nor was there a correlation with the size. There is a need to harmonize the methodological approaches to better evaluate the results and trends over time and to monitor the species and indirectly the health status of the marine environment.

Determination of hair cortisol in horses: comparison of immunoassay vs LC-HRMS/MS.

The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depending on the detection technique. The methods were validated according to EMA guideline for bioanalytical method validation, in the range 2-50 pg mg-1 (ELISA) and 1-100 pg mg-1 (LC-HRMS/MS). Satisfactory quantitative performances were obtained for both of the approaches, but this latter demonstrated better precision. The detected concentrations in real samples were encompassing the range 1.3-8.8 pg mg-1 and 2.0-17.9 pg mg-1 by means of LC-HRMS/MS and ELISA, respectively. Overall, HCCs measured with ELISA technique were 1.6 times higher. The overestimation of immunoassay results might be caused by cross-reactivity phenomena of laboratory reagents and other structurally similar hormones present in the mane.

Brominated and chlorinated contaminants in food (PCDD/Fs, PCBs, PBDD/Fs PBDEs): Simultaneous determination and occurrence in Italian produce.

Validated methodology for the simultaneous determination of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) and polybrominated diphenyl ethers (PBDEs) in foods of animal origin is presented. Method performance indicators were equivalent or better than those required for the control of EU regulated (EU, 2017/644) PCDD/F and PCB congeners in these foods, and for risk assessment through dietary intake. The method uses a high (>90%) proportion of 13Carbon-labelled surrogates for internal standardisation combined with high resolution mass spectrometry that allow accurate quantitation, and this was confirmed by multiple successful participations in proficiency testing for PCDD/Fs, PCBs and PBDEs in food. The same validation and method performance requirements as used for PCDD/Fs were followed for PBDD/Fs. The analysis of a range of food samples (eggs, milk, fish, shellfish, pork, beef and poultry), showed the occurrence of all four classes of contaminants at varying concentration ranges. In general, PCBs were the most prominent contaminant, both, in terms of dioxin-like toxicity, as well as in the occurrence of non-dioxin-like congeners, an observation that concurs with those made in other studies on Italian foods. The levels of PCDD/F and PCB occurrence are consistent with a gradual decline in contamination as reported by some other similar studies. Although all the determined contaminants were detected in the sampled foods, there was poor correlation between the occurrences of the brominated and chlorinated contaminants, and between PBDEs and PBDD/Fs, but better associations were observed between the occurrences of the chlorinated contaminants.

Heavy Metals Contamination in Shellfish: Benefit-Risk Evaluation in Central Italy.

Seafood is a source of nutrients in human diet but also of environmental contaminants and its consumption could pose a risk to consumers' health. A survey regarding the exposure to cadmium, lead and mercury through the consumption of bivalve mollusks, gastropods and sea urchins collected on Italian coasts was carried out among central Italian population over a period of three years. A limited number of samples exceeds the threshold set by legislation (6 samples) and the average level of contamination was low in all the species considered. The contribution Acceptable Daily Intake (ADI) was higher for cadmium (9.17%) than lead (1.44%) and mercury (0.20%). The benefit-risk evaluation suggests that the bivalve mollusks and sea urchins consumption (Benefit Risk Quotient < 1) could be increased without health detrimental effects.

Investigation of phenylbutazone and its metabolite oxyphenbutazone in horse meat products during years 2013-2017.

This study showed the results of a monitoring program aimed at detecting the fraudulent use of horse meat in samples collected in the framework of official controls during the years 2013-2017 after the so-called horse meat scandal that caused a decreasing consumer confidence in the food industry, particularly in meat products. A total of 125 samples diversely distributed in the 5 years of monitoring were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to Commission Decision 657/2002/EC with regard to the following parameters: linearity, specificity, decision limit (CCα), detection capability (CCß), recovery, and precision (repeatability and within-laboratory reproducibility). Even if the results of this study revealed no positive sample, the need for constant assurance of food safety and consumers' health protection requires the implementation of preventive as well as corrective actions in the management of risks linked to the food chain in a more global context.

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.

Investigation of Nonionophoric Coccidiostat Residues in Feed as a Consequence of Carryover.

Residues of nonionophoric coccidiostats at carryover concentrations in feedstuffs collected from feed mills or animal farms in central Italy were detected as part of the official controls carried out from 2011 through 2016. The 118 samples were collected on the production line or during feed distribution and storage to determine the sampling sites at major risk of cross-contamination. For determination of nonionophoric coccidiostats, a fast, easy, and cheap method was developed and validated. Feed samples were extracted with acetonitrile-methanol and directly injected for liquid chromatography with tandem mass spectrometry. A total of 24 samples (20.3%) were positive, but only 5 (4.2%) of these samples exceeded the maximum limit set by European legislation. Most of the positive samples were collected from a batch of feed produced immediately following processing of another batch to which the coccidiostat robenidine had been added.

Occurrence of Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans and Polychlorinated Biphenyls in Fruit and Vegetables from the "Land of Fires" Area of Southern Italy.

The concentrations of polychlorinated dibenzo-p-concentrations dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) (PCDD/Fs), and polychlorinated biphenyls (PCBs) were determined in fruit and vegetables collected in farms located in the well-known "Land of Fires" area of Southern Italy, in an effort to learn more about the environmental pollution of this high-risk area due to illegal waste dumping and uncontrolled burning near cultivated fields. Concentrations were in the range 0.011-2.26 ng g-1 for the six "indicator" non-dioxin-like PCBs (NDL-PCBs), and 0.0009-0.096 pg WHO toxic equivalent (TEQ) g-1 for the sum of dioxin-like PCBs (DL-PCBs) and PCDD/Fs. Lacking maximum limits for these contaminants in fruit and vegetables, the concentration values found were compared with the action levels set out in the EU Recommendations. These levels were never exceeded in the examined samples. In the present study, the highest mean value for PCDD/Fs + DL-PCB corresponded to apricots, olives, and nuts, while the lowest values were observed in endive and green beans. The results showed also that NDL-PCB levels in apricots were much higher than in any other food, suggesting that they can accumulate PCBs: this fruit might be proposed as a "sentinel" of the presence of these contaminants in the environment.

Determination of regulatory ionophore coccidiostat residues in feedstuffs at carry-over levels by liquid chromatography-mass spectrometry.

In this study samples of feedstuffs were collected from different feed mills and animal farms located in central Italy and analyzed for ionophore coccidiostat residues at carry-over levels by liquid chromatography-mass spectrometry. Since unavoidable cross-contamination of feedstuffs may occur during their production as well as distribution and storage, the collection of samples covered all these different stages. Residues of lasalocid, monensin, salinomycin and maduramicin were detected in 32.4% of samples, both at production and storage level. The maximum content for unavoidable carry-over set by Regulation (EU) No 574/2011 was exceeded in 11.3% of samples. The variability of the results highlighted the different approach of each investigated feed business operator to avoid any cross-contamination in non-target feed. The method developed in this study can be able to detect ionophore coccidiostats at low concentrations consequent to carry-over.

Simultaneous determination of aflatoxins, T-2 and HT-2 toxins, and fumonisins in cereal-derived products by QuEChERS extraction coupled with LC-MS/MS.

A fast, easy, and cheap method for the simultaneous determination and quantification of aflatoxins (B1, B2, G1, G2), T-2 and HT-2 toxins, and fumonisins (B1, B2) in cereal-derived products was developed. This method involved a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction coupled with liquid chromatography-tandem mass spectrometry. The method validation was performed by analyzing samples spiked at four levels, and the recoveries ranged from 83.6 to 102.9%, whereas the maximum values of repeatability and within-laboratory reproducibility were 14.3 and 15.7% following the performance criteria set by the European legislation. The method was then applied for the analysis of 21 cereal-derived products purchased on the Italian market, which were correctly packaged and labeled as intended for human consumption. The co-occurrence of more than one mycotoxin in the analyzed samples could represent a risk for consumers, and the described method could be a valid alternative for their simultaneous detection in the framework of official control. Graphical Abstract ᅟ.

Comparison of ochratoxin A levels in edible pig tissues and in biological fluids after exposure to a contaminated diet.

The aim of this study was to compare ochratoxin A (OTA) levels in pig tissues and biological fluids after animal exposure to contaminated diet (250 µg OTA/kg of feed) during 4 weeks of fattening. OTA concentrations were quantified using a validated immunoassay method (ELISA) and high-performance liquid chromatography with fluorescence detector (HPLC-FD). The highest mean OTA concentration in pig tissues was determined in kidneys of exposed animals (13.87 ± 1.41 µg/kg), followed by lungs (10.47 ± 1.97 µg/kg), liver (7.28 ± 1.75 µg/kg), spleen (4.81 ± 0.99 µg/kg), muscle tissue (4.72 ± 0.86 µg/kg), fat tissue (4.11 ± 0.88 µg/kg), heart (3.71 ± 1.09 µg/kg), and brain (3.01 ± 0.25 µg/kg). Furthermore, on the last day of exposure (day 28), significantly higher mean OTA levels were determined in urine (16.06 ± 3.09 µg/L) in comparison to serum (4.77 ± 1.57 µg/L) showing that OTA urine analysis could be a good marker to identify elevated levels of this contaminant in porcine tissues used for human consumption. This study gave guidelines for the most efficient OTA control in pig-derived biological materials that can be exercised at slaughterhouses.

Occurrence of polybrominated diphenyl ethers (PBDEs) in foodstuffs in Italy and implications for human exposure.

Polybrominated diphenyl ethers (PBDEs) were determined in various foodstuffs in Italy and the dietary intake was estimated. PBDEs were detected in all analysed samples at concentrations that spanned over five orders of magnitude. The most abundant congeners were the BDE-209, followed by BDE-47 and BDE-99. Fish oil and milk samples showed the highest PBDE concentrations among all samples. The daily dietary intake values were found to be in good agreement or higher to literature values, impacted mainly from the contribution of the analysed dairy products. The cancer risk values estimated for BDE-209 indicated that this specific risk associated with the studied foodstuffs is limited. Italy is one of the world-leading countries in the production of furniture and clothes and has extremely developed medium enterprise industrial sector, where PBDEs were historically used suggesting that their occurrence may be linked to these activities.

Micro-solid-phase extraction (µ-SPE) of organophosphorous pesticides from wheat followed by LC-MS/MS determination.

A rapid, selective and effective method of extraction, clean-up and concentration of organophosphorous pesticides from wheat followed by electrospray (ESI) LC-MS/MS analysis was developed. The µ-SPE (micro-solid-phase extraction) procedure resulted in good analytical performance and reduced at the same time matrix effects, analysis time and solvent consumption. Limits of detection (LODs) and quantification (LOQs) were in the range of 0.3-10 and 1-30 µg kg(-1), respectively, with good reproducibility (RSD ≤ 13.8) and recoveries between 75% and 109%. Coefficients of determination (r(2)) were greater than 0.996 for the studied pesticides. Despite the reduced sorbent bed mass of µ-SPE tips (4.2 mg), the analytical data showed that no saturation phenomena occurs in the tested range of concentration both for single compounds and mixtures. Several real samples were analysed and the concentrations of the selected pesticides were found to be below the respective maximum residue limit (MRLs).

Concentrations of Contaminants with Regulatory Limits in Samples of Clam (Chamelea gallina) Collected along the Abruzzi Region Coast in Central Italy.

Concentrations of pollutants with regulatory limits were determined in specimens of Chamelea gallina, a species of clam collected along the Abruzzi coastal region of the central Adriatic Sea. Nine sampling sites were selected to evaluate the distribution of contaminants in the environment and the health risk for consumers. The concentrations of all the examined compounds were lower than the maximums set by European legislation. Polycyclic aromatic hydrocarbons and total mercury were below the detection limit (0.18 µg/kg for benzo[a]anthracene, 0.30 µg/kg for chrysene, 0.12 µg/kg for benzo[b]fluoranthene, 0.08 µg/kg for benzo[a]pyrene, and 0.0050 mg/kg for total mercury) in all the analyzed samples. Mean concentrations of lead and cadmium were 0.104 and 0.110 mg/kg, respectively. Of the non-dioxin-like polychlorinated biphenyls, PCB-153, PCB-180, and PCB-138 were the most abundant at all sampling sites (1a to 9a) at 0.25 mi (ca. 0.4 km) and at some sampling sites (1b, 2b, 3b, 5b and 7b) at 0.35 mi (ca. 0.56 km). Principal component analysis revealed that the concentrations of dioxin-like polychlorinated biphenyls were similar at the majority of sampling sites, and O8CDD and 2,3,7,8-T4CDF were the predominant dioxin congeners.

Data on milk dioxin contamination linked with the location of fodder croplands allow to hypothesize the origin of the pollution source in an Italian valley.

Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) have similar toxic, endocrine-disrupting, and carcinogenic activity. They are classified as persistent organic pollutants accumulating in the environment and the tissues of living organisms. High concentrations of PCDD/F and dl-PCB have been detected in bovine milk collected in a Piedmont valley (Northwestern Italy) since 2004. This geographic study describes the local distribution of pollution from PCDD/Fs and dl-PCBs. Since their presence in animal products could be traced back to the ingestion of contaminated fodder, dioxin levels in cow milk were related to the distribution of fodder cropland parcels. Specifically, the aim of the study was to determine, through an exploratory approach, whether the contamination was consistent with one common point source of contamination or different scattered sources. Data for PCDD/F and dl-PCB concentrations in the bulk milk from 27 herds, sampled over a 4-year period (2004-2007), were matched to the georeferenced land parcels the dairy farmers used for growing fodder. Isopleth maps of dioxin concentrations were estimated with ordinary kriging. The highest level of pollution for both PCDD/Fs and dl-PCBs was geographically juxtaposed: in both instances, the location of the local steel plant was within this extremely highly polluted area. The study results support the hypothesis for one common point source of contamination in the valley. The exploratory spatial analysis applied in this research may provide a valuable, novel approach to straightforward identification of a highly likely source of dioxin contamination of dairy products (even in the absence of top soil contamination data).