Quantitative determination of tetracyclines in medicated feed for food-producing animals by HPLC-DAD.

Tetracyclines are a group of antibiotic substances largely administered through medicated feed to control diseases in food-producing animals. Fine dosing of antibiotics contained in medicated feed is crucial for the success of the treatment as well as minimising potential threats such as the spread of antimicrobial resistance and the transfer of antibiotic residues in food. A rapid analytical method based on HPLC with diode array detection (HPLC-DAD) was developed to quantify oxytetracycline, chlortetracycline and doxycycline in medicated feed. The reported method underwent in-house validation and was found to be suitable for the quantification of three target tetracyclines within the concentration range of 40-1000 mg kg-1 in official routine analysis. The method was applied to 103 official samples in the framework of the Italian National Plan on animal feed during the years 2021-2023 and nine non-compliant concentrations were identified in swine and fish feed samples.

Occurrence of Glyphosate and Other Polar Pesticides in Honey from Lombardy and Emilia-Romagna Regions in Italy: Three-Year Monitoring Results.

Intensive agricultural practices, such as pesticides use, may negatively affect bee health and hive products. Glyphosate is one of the most widely used polar pesticides applied in crops for weed control. In this study, honey samples, collected from beekeeping farms located in the Lombardy and Emilia-Romagna regions in Italy in the framework of regional monitoring plans activated from 2020 to 2022, were analyzed for the presence of residues of polar pesticides. The analytical method based on ion chromatography coupled to high-resolution mass spectrometry was applied to quantify glyphosate, glufosinate, ethephon, fosetyl aluminum, and their related metabolites. Residues of glyphosate were detected in around 28% of analyzed honey samples. Observations on the distribution of the honey-production-site locations suggest that honey samples originating from the provinces within the Lombardy region, where the agricultural sector is highly developed, were more affected by glyphosate contamination than the samples collected from the areas with low agricultural activity, where no glyphosate residues were detected over the three years of the monitoring program.

Safety Evaluation and Probabilistic Health Risk Assessment of Cow Milk Produced in Northern Italy According to Dioxins and PCBs Contamination Levels.

Contamination levels of dioxins and polychlorinated biphenyls (PCBs) were monitored over 2018-2021 in 214 bovine milk samples from farms located in two regions in northern Italy (Lombardy and Emilia-Romagna). The average concentrations of the sum of dioxins and dioxin-like PCBs (0.78 ± 0.55 pg TEQ/g fat) and six non-dioxin-like PCBs (6.55 ± 2.24 ng/g fat) were largely below the maximum, and action limits established at European level, confirming a decreasing trend observed both locally and across Europe in recent years. The impact of contamination levels on chronic dietary exposure of the Italian population to dioxins and PCBs was found to be highly variable based on the type of cow milk (skimmed, semi-skimmed, or whole-fat milk) and the population age group considered. Indeed, a first-tier screening of the potential exposure via determinist methods allowed for the identification of the youngest population as the group with the worst risk profile. The refinement of exposure assessment via Monte Carlo probabilistic methods suggested that, at the less pessimistic middle-bound simulation scenario, infants, toddlers, and children consuming whole cow milk may be exposed to dioxins and PCBs levels above the toxicological reference values with a probability of 76, 56, and 22%, respectively.

Prenatal Environmental Stressors and DNA Methylation Levels in Placenta and Peripheral Tissues of Mothers and Neonates Evaluated by Applying Artificial Neural Networks.

Exposure to environmental stressors during pregnancy plays an important role in influencing subsequent susceptibility to certain chronic diseases through the modulation of epigenetic mechanisms, including DNA methylation. Our aim was to explore the connections between environmental exposures during gestation with DNA methylation of placental cells, maternal and neonatal buccal cells by applying artificial neural networks (ANNs). A total of 28 mother-infant pairs were enrolled. Data on gestational exposure to adverse environmental factors and on mother health status were collected through the administration of a questionnaire. DNA methylation analyses at both gene-specific and global level were analyzed in placentas, maternal and neonatal buccal cells. In the placenta, the concentrations of various metals and dioxins were also analyzed. Analysis of ANNs revealed that suboptimal birth weight is associated with placental H19 methylation, maternal stress during pregnancy with methylation levels of NR3C1 and BDNF in placentas and mother's buccal DNA, respectively, and exposure to air pollutants with maternal MGMT methylation. Associations were also observed between placental concentrations of lead, chromium, cadmium and mercury with methylation levels of OXTR in placentas, HSD11B2 in maternal buccal cells and placentas, MECP2 in neonatal buccal cells, and MTHFR in maternal buccal cells. Furthermore, dioxin concentrations were associated with placental RELN, neonatal HSD11B2 and maternal H19 gene methylation levels. Current results suggest that exposure of pregnant women to environmental stressors during pregnancy could induce aberrant methylation levels in genes linked to several pathways important for embryogenesis in both the placenta, potentially affecting foetal development, and in the peripheral tissues of mothers and infants, potentially providing peripheral biomarkers of environmental exposure.

A New Method for Ivermectin Detection and Quantification through HPLC in Organic Matter (Feed, Soil, and Water).

Ivermectin is a macrocyclic lactone widely used in veterinary medicine for its broad-spectrum antiparasitic properties. It has been proven to be effective and safe. The purpose of this study was to develop a high-performance liquid chromatography method with a diode array detector for ivermectin screening in feed and water for animal consumption. Furthermore, the objective was to quantify ivermectin levels that were higher than 0.5 mg/kg in solid matrixes and 0.1 mg/kg in water. Doramectin was used as process standard. Samples were extracted using solid phase extraction with silica and C-18 columns. The method involved the use of high-performance liquid chromatography (HPLC) with a diode array detector (DAD). The results were interpreted using a calibration curve built with ivermectin standards at multiple concentrations (0.5, 1, 2, 5, and 12.5 mg/kg). Statistical evaluation of data was done using ANOVA. The data analysis showed that the linear regression was highly significant (P < 0.001), the intercept values were not significantly different from zero, and the correlation coefficient values (>0.999) indicated excellent linearity. Further tests demonstrated that this method is also useful when studying soil matrixes. The soil was dried and analyzed in the same way as feed; the same recoveries were realized on the spiked samples. The method is easy, inexpensive, precise, and repeatable; it requires very small amounts of sample.

Iron Content, Iron Speciation and Phycocyanin in Commercial Samples of Arthrospira spp.

Cyanobacteria are characterized by high iron content. In this research, we collected ten commercial samples of Arthrospira spp. sold as food supplement to determine iron content and assess whether iron speciation showed variability among samples and changed respect to A. platensis grown in controlled conditions. Particular attention was also paid to phycocyanin, as an iron-binding protein. In six of the ten samples, 14 essential and non-essential trace elements were analysed using ICP-MS. Iron content measured in samples using atomic absorption spectrometry (AAS) varied from 353 (sample S5) to 1459 (sample S7) µg g-1 dry weight and was in the range of those reported by other authors in commercial supplements. Iron speciation was studied using size exclusion chromatography followed by the analysis of the collected fraction for the determination of iron by AAS and for protein separation using SDS-PAGE. Overlapping chromatographic profiles were obtained for total proteins, phycocyanin and iron, although quantitative differences were evidenced among the samples analysed. In most samples, iron was mainly bound to ligands with high molecular mass; however, in four samples iron was also bound to ligands with low molecular mass. In fractions containing the most relevant iron burden, the principal protein was phycocyanin, confirming its role as an iron-binding protein in commercial samples.

The influence of different production systems on dioxin and PCB levels in chicken eggs from Emilia-Romagna and Lombardy regions (Italy) over 2017-2019 and consequent dietary exposure assessment.

Hen eggs from farms with different production systems (organic, free range, barn, and in cage) sited in two regions of Italy (Emilia-Romagna and Lombardy) were collected from 2017 to 2019 to monitor the levels of 17 polychlorinated dibenzo-p-dioxin/dibenzofurans (PCDD/Fs), 12 dioxin-like polychlorinated biphenyls (dl-PCBs) and the 6 non-dioxin-like polychlorinated biphenyl indicators (ndl-PCBs). Average concentrations of PCDD/Fs (0.21 pg WHO(2005)-TEQ/g fat), PCDD/Fs + dl-PCBs (0.43 pg WHO(2005)-TEQ/g fat) and ndl-PCBs (6.41 ng/g fat) were below the maximum limits established at European level in eggs, but significantly higher amounts of PCDD/Fs and PCDD/Fs + dl-PCBs were found in eggs from free-range housing system compared to barn, cage, and organic ones. The potential dietary intake of the monitored contaminants by Italian population age groups through the consumption of locally produced eggs was also evaluated. Exposure levels to PCDD/Fs plus dl-PCBs were below the tolerable weekly intake (TWI) of 2 pg WHO(2005)-TEQ/kg bw/week recently set by the European Food Safety Authority (EFSA). However, the younger population was found to be more vulnerable than other age groups to exposure to these contaminants. Specifically, the preferential consumption of free-range eggs by infants, toddlers, and children contributed more than 30% to the TWI. The results confirm the need to continuously monitor the levels of chemical contaminants in the environment and provide a reminder of the importance of targeted intervention measures aimed to reduce their occurrence in food and feed, firstly by improving the management practices on egg-producing farms.

New Trends in the Occurrence of Yessotoxins in the Northwestern Adriatic Sea.

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers' revenues and the availability of local fish, causing a major economic loss in Italy's main shellfish sector. METHODS: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. RESULTS: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. CONCLUSION: In this regard, it is essential to develop and activate, as soon as possible, an "early warning" system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.

Heavy Metal Assessment in Feathers of Eurasian Magpies (Pica pica): A Possible Strategy for Monitoring Environmental Contamination?

In the present study, the Eurasian magpie (Pica pica), was evaluated as a possible bioindicator of environmental pollution by heavy metals (HMs). Levels of Ni, Pb, Cd, and Hg in feathers of 64 magpies (31 males and 33 females) were measured by ICP-MS technique. Plasmatic biomarkers of oxidative stress (OS) were also assessed. The birds were captured in the province of Parma (Italy), in different capture sites within 1 km from urban area (UZ), and farther than 5 km from urban area (RZ). Median HM levels were 0.68 mg/kg (0.18-2.27), 2.80 mg/kg (0.41-17.7),

Lead Levels in Wild Boar Meat Sauce (Ragù) Sold on the Italian Market.

Game meat is endowed with excellent nutritional value, but it may also be a possible source of harmful substances, such as mycotoxins and heavy metals. In particular, several studies showed that lead fragments from hunting ammunition are able to represent a residual contaminant in the meat of wild boars or deer, representing a possible source of lead absorption. Even though wild boar meat consumption in Italy is rather limited, this meat could also be present in very popular Italian recipes, such as the typical meat sauce called ragù. We evaluated the lead levels in 48 samples (three different batches for each of the 16 brands) of ready-to-eat wild boar meat ragù sold on the Italian market in food stores and online distribution with the inductively coupled plasma-mass spectrometry (ICP-MS) technique. A high variability was found in the lead levels detected in the samples, with a median lead level of 0.10 mg/kg (0.01-18.3 mg/kg) and some of the samples showing very high lead concentrations. Since no intake level of lead is considered completely safe, and maximum levels for game meat have so far not been established, a greater attention on the risks to consumers' health related to the presence of this heavy metal in game meat is recommended.

Heavy metals and potential risks in edible seaweed on the market in Italy.

The seaweed food has always been important in Asia, but recently increased in the Western diet. Superfood known for health benefits and rich in essential elements, can also accumulate high contents of heavy metals and iodine from the environment, becoming a health hazard. In particular for iodine, an appropriate labelling of seaweed is needed to warn the consumer of the potential risks. The aim of the study was to analyze the content of 20 heavy metals in seaweeds, distributed in Italy, by ICP-MS, also determining iodine and arsenic (total and inorganic fraction). A total of 72 samples of European and Asian seaweed of 8 genera were analyzed and the results correlated the content of heavy metals to genus, geographical origin and type of sample; 8.33% of the products lacked in the label of the indications of allergens, while 9.72% had irregularities in the label language. The highest concentration of elements was found in the Rhodophyta. The Aluminum level was the highest in the mixed seaweed (165.39 mg/kg) and for the Cadmium in the Asian seaweed (1.16 mg/kg). The amounts of Iron, Zinc and Magnesium, was highest in the Asian seaweed. The values of Arsenic (total and inorganic contents) were compared with the limits: 2.78% exceeds France and USA limits for inorganic, while higher content of total was found in Phaeophyta, which also showed the highest Iodine content (6770.80 mg/kg) that can be dangerous if not reported correctly in the label.

Trace Elements in Home-Processed Food Obtained from Unconventional Animals.

Wild animals have been used as food since ancient times and, currently, the consumption of unconventional animals is increasing worldwide. The process of cooking meat using traditional recipes includes a variety of ingredients, which can influence the total metal intake from the diet. In this study, the concentrations of eight essential (Fe, Zn, Cu, Mn, Se, Ni, Mo, and Co) and six non-essential (Pb, Cd, Hg, Al, As, and Cr) trace elements were determined in home-processed food obtained from snails and from three common species of game animals (woodcock, pheasant, and hare), seasoned with anchovies, mushrooms, and different vegetables using inductively coupled plasma mass spectrometry (ICP-MS). In general, Fe was the most abundant trace element, ranging from 18 ± 8 µg/g in pheasant to 99 ± 76 µg/g in snail, and Co was the least abundant, ranging from 0.007 ± 0.003 µg/g in hare to 0.093 ± 0.048 µg/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 µg/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 µg/g. These alternative food sources can offer an important contribution to the human nutritional requirements of essential trace elements, in particular of Fe. The high concentrations of Pb and Cd present in some samples should be considered as potentially dangerous for the consumers.

Environmental exposure to non-essential trace elements in two bat species from urbanised (Tadarida teniotis) and open land (Miniopterus schreibersii) areas in Italy.

Bats are particularly suited as bioindicators of trace element pollution due to their longevity and their position in the trophic chain. In this study, the concentrations of ten non-essential trace elements (Al, As, Ba, Cd, Hg, Pb, Sb, Sr, Th, Tl) were determined in the tissues (whole body, skin-fur, skinned body, liver, kidney and bone) of lactant Tadarida teniotis from a nursery colony in Rome. A large number of bats from this nursery died before fledging and had bone deformities and fractures. The concentrations of non-essential trace elements in bone and whole body were also analysed in adult specimens of Miniopterus schreibersii from a colony located in a natural park in Northern Italy. In lactant T. teniotis, the Pb concentration decreased in the following order: bone>liver>skinned body>whole body>skin-fur>kidney, and exceeded the toxic threshold associated with negative effects reported for different mammalian species. The levels of the other non-essential trace elements were within a range indicative of low environmental contamination in both species. Significant interspecies differences (P < 0.05) were observed for concentrations of Pb and Ba, higher in the bones of T. teniotis, and of Cd, Hg and Sr, higher in the bones of M. schreibersii. In lactant T. teniotis, the different sources of Pb exposure, through inhalation and/or food, may represent a potential threat to the colony of this synanthropic European bat.

Lead and copper in hunted wild boars and radiographic evaluation of bullet fragmentation between ammunitions.

The purpose of the present study was to evaluate the content of lead in carcasses of wild boars shot with lead bullets, in comparison with that of copper caused by lead-free ammunitions. Radiographic images of hunted boars were obtained in order to assess the degree of bullet fragmentation in the carcasses. Samples of meat were collected from different body areas at increasing distance from bullet trajectory, to be analysed by ICP-MS for lead and copper levels. In wild boars shot with lead ammunitions, a massive dispersion of bullet fragments and very high lead levels were detected. By contrast, in wild boars killed with copper ammunitions no radiographic signs of bullet fragmentation were observed. Copper ammunitions seem therefore a safer alternative to standard lead-core ones, due to their minimal fragmentation and the relatively low toxicity of this metal.

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.

Survey on Urinary Levels of Aflatoxins in Professionally Exposed Workers.

Feed mill workers may handle or process maize contaminated with aflatoxins (AFs). This condition may lead to an unacceptable intake of toxins deriving from occupational exposure. This study assessed the serological and urinary levels of AFs in workers exposed to potentially contaminated dusts in two mills. From March to April 2014, blood and urine samples were collected, on Monday and Friday morning of the same working week from 29 exposed workers and 30 non-exposed controls. AFs (M1, G2, G1, B1, B2) and aflatoxicol (AFOH) A were analyzed. Each subject filled in a questionnaire to evaluate potential food-borne exposures to mycotoxins. AFs contamination in environmental dust was measured in both plants. No serum sample was found to be positive. Seventy four percent of urine samples (73.7%) revealed AFM1 presence. AFM1 mean concentration was 0.035 and 0.027 ng/mL in exposed and non-exposed workers, respectively (p = 0.432); the concentration was slightly higher in Friday's than in Monday's samples, in exposed workers, 0.040 versus (vs.) 0.031 and non-exposed controls (0.030 vs. 0.024, p = 0.437). Environmental AFs contamination ranged from 7.2 to 125.4 µg/kg. The findings of this study reveal the presence of higher AFs concentration in exposed workers than in non-exposed controls, although these differences are to be considered consistent with random fluctuations.