Fast and Sensitive Analysis of Short- and Long-Chain Perfluoroalkyl Substances in Foods of Animal Origin.

The availability of sensitive analytical methods to detect per- and polyfluoroalkyl substances (PFASs) in food of animal origin is fundamental for monitoring programs to collect data useful for improving risk assessment strategies. The present study aimed to develop and validate a fast and sensitive method for determining short and long-chain PFASs in meat (bovine, fish, and swine muscle), bovine liver, hen eggs, and cow's milk to be easily applicable in routine analysis of food. A QuEChERS extraction and clean-up method in combination with liquid chromatography coupled to mass spectrometry (LC-MSMS) were used. The method resulted in good linearity (Pearson's R > 0.99), low limits of detection (7.78−16.35 ng/kg, 8.26−34.01 ng/kg, 6.70−33.65 ng/kg, and 5.92−19.07 ng/kg for milk, liver, egg, and muscle, respectively), and appropriate limits of quantification (50 ng/kg for all compounds except for GenX and C6O4, where the limits of quantification were 100 ng/kg). Trueness and precision for all the tested levels met the acceptability criteria of 80−120% and ≤20%, respectively, regardless of the analyzed matrix. As to measurement uncertainty, it was <50% for all compound/matrix combinations. These results demonstrate the selectivity and sensitivity of the method for simultaneous trace detection and quantification of 14 PFASs in foods of animal origin, verified through the analysis of 63 food samples.

TMT-Based Proteomics Profiling of Bovine Liver Underscores Protein Markers of Anabolic Treatments.

Illegal use of growth promoter compounds in food production exposes consumers to health risk. Surveillance of such practices is based on direct detection of drugs or related metabolites by HPLC-MS/MS. Screening strategies focusing on indirect biological responses are considered promising tools to improve surveillance. In this study, an untargeted shotgun proteomics approach based on tandem mass tags (TMTs) is carried out to identify proteins altered in bovine liver after different anabolic treatments. Three controlled pharmacological treatments with dexamethasone, a combination of dexamethasone and clenbuterol, or a combination of sexual steroids (trenbolone and estradiol) are analyzed. Untargeted TMT analysis of liver digests by high resolution MS allowed for the relative quantification of proteins. Thanks to partial least squarediscriminant analysis, a set of proteins capable to classify animals treated with dexamethasone alone (11 proteins), or in combination with clenbuterol (13 proteins) are identified. No significant difference is found upon administration of sexual steroids. After relative quantification of candidate markers by parallel reaction monitoring (PRM), two predictive models are trained to validate protein markers. Finally, an independent animal set of control bulls and bulls treated with dexamethasone is analyzed by PRM to further validate a predictive model giving an accuracy of 100%.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Targeted proteomics for the indirect detection of dexamethasone treatment in bovines.

The illegal use of pharmacologically active compounds for growth promotion in food-producing species poses risks for consumer health and animal welfare. Surveillance relies on the quantification of drug residues in animal fluids or tissues, but the efficacy can be negatively affected due to undetectable residual concentrations in biological matrices. Consequently, techniques focusing on the indirect biological effects of exogenous compound administration have been proposed as more sensitive detection methods. The purpose of the present study is to develop a tandem mass spectrometry analytical method based on low-energy collision-induced dissociation (CID-MS/MS) using multiple reaction monitoring (MRM) for the quantification of 12 potential protein markers of skeletal muscle to detect anabolic treatments with dexamethasone. Protein markers identified in a previous study applying a 2D-DIGE proteomics approach have been quantified using the signature peptide method. A group of proteins were confirmed as reliable markers. Quantitative results enabled a predictive model to be defined based on logistic regression for the detection of treated animals. The developed model was finally cross-validated in an independent animal set. Graphical abstract Analytical workflow used for the quantification of indirect protein markers of dexamethasone treatment.

Proteomics for the detection of indirect markers of steroids treatment in bovine muscle.

Despite the ban by the European Union, anabolic steroids might still be illicitly employed in bovine meat production. The surveillance of misuse of such potentially harmful molecules is necessary to guarantee consumers' health. Analytical methods for drug residue control are based on LC-MS/MS, but their efficacy can be hindered due to undetectable residual concentrations as a result of low-dosage treatments. Screening methods based on the recognition of indirect biological effects of growth promoters' administration, such as the alteration of protein expression, can improve the efficacy of surveillance. The present study was aimed at identifying modifications in the muscle protein expression pattern between bulls treated with an ear implant (Revalor-XS®) containing trenbolone acetate (200 mg) and estradiol (40 mg), and untreated animals. The analysis of skeletal muscle was carried out using a tandem mass tags shotgun proteomics approach. We defined 28 candidate protein markers with a significantly altered expression induced by steroids administration. A subset of 18 candidate markers was validated by SRM and allowed to build a predictive model based on partial least square discriminant analysis. Our findings confirm the effectiveness of the proteomics approach as potential tool to overcome analytical limitations of drug residue monitoring.

Confirmation of protein biomarkers of corticosteroids treatment in veal calves sampled under field conditions.

In veal calf production, growth promoters are still illicitly used. Surveillance of misuse of such molecules is necessary to preserve human health. Methods currently adopted for their analysis are based on liquid chromatography-tandem mass spectrometry, but their efficacy can be affected by undetectable residual concentrations in biological matrices due to treatments at low-dosage or based on unknown anabolic compounds. The development of screening methods to identify the indirect biological effects of administration of growth promoters can improve the efficiency of drug residue monitoring. To this purpose, an integrated approach has been used to further validate the set of protein biomarkers defined in a previous controlled study to detect the use of corticosteroids through the changes caused in muscle protein expression. The thymus morphology of 48 samples collected under field conditions was evaluated to assess the presence of potential corticosteroids treatment. Animals were divided on the basis of their thymus characteristics in negative or suspected for illegal corticosteroids treatment. Drug residue analyses were performed on the liver, giving a satisfactory correlation with the histological examination (∼85%). Finally, the proteomics analysis of muscle protein extracts was carried out by 2D differential in gel electrophoresis, and proteins that were differentially expressed between the two animal groups (p value <0.01) were selected for MALDI-MS/MS analysis. This approach allowed us to identify 29 different proteins, and our findings indicate that the altered protein expression pattern can be used as an indirect method for the detection of illicit corticosteroids administration. A subset of the identified proteins was already reported in a previous controlled study, proving that these biomarkers can be used to develop a screening assay to improve the tools currently available for the detection of corticosteroids abuse in bovine meat production.

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast.

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 µg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 µg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 µg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 µg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions.

Effect of TiO2 Nanoparticle on Bioaccumulation of ndl-PCBs in Mediterranean Mussels (Mitilus galloprovincialis).

The interaction of nanomaterials with pollutants in the marine environment might alter bioavailability, as well as toxicity, of both nanomaterials and pollutants, representing a risk, not only for marine organisms, but also for consumers through the marine food chain.The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO2NPs) in terms of bioaccumulation and toxicity on Mediterranean mussels (Mytilus galloprovincialis) exposed to six-indicator non-dioxin-like polychlorinated biphenyls (ndl-PCBs). Mussels were exposed to ndl-PCBs (20 µg/mL) (groups 3-4) or to a combination of ndl-PCBs (20 µg/mL) and TiO2NPs (100 µg/mL) (groups 5-6) for four consecutive days. TiO2NPs was detected in groups 5-6 (3247 ± 567 and 1620 ± 223 µg/kg respectively), but their presence did not affect ndl-PCBs bioaccumulation in mussels. In fact, in groups 3-4, the concentration of ndl-PCBs (ranging from 3818.4 ± 166.0-10,176 ± 664.3 µg/kg and 2712.7 ± 36.1-9498.0 ± 794.1 µg/kg respectively) was not statistically different from that of groups 5-6 (3048.6 ± 24.0-14,635.9 ± 1029.3 and 5726.0 ± 571.0-9931.2 ± 700.3 µg/kg respectively). Histological analyses showed alterations to the structure of the gill tissue with respect to the control groups, with more severe and diffuse dilatation of the central hemolymphatic vessels of the gill lamellae in groups 5-6 (treated with TiO2NPs and ndl-PCBs concurrently) compared to groups 3-4 (ndl-PCBs only). Finally, in mussels submitted to a seven-day depuration process, most TiO2NPs were eliminated, and NPs had a synergistic effect on ndl-PCBs elimination; as a matter of fact, in groups 5-6, the percentage of concentration was statically inferior to the one observed in groups 3-4. In any case, consumers might be exposed to TiO2NPs and ndl-PCBs (both concurrently and separately) if edible mussels, harvested in a contaminated environment, are consumed without a proper depuration process.

Proteins from Tenebrio molitor: An interesting functional ingredient and a source of ACE inhibitory peptides.

The angiotensin-converting enzyme (ACE) inhibitory potential of the main protein fractions from Tenebrio molitor larvae (TML) was examined to evaluate their use as a novel antihypertensive functional food. Both fractions contained YAN tripeptide, previously reported as responsible for ACE inhibition. Although YAN has been synthesized and was used as a standard for LC-MS/MS quantification and IC50 against ACE was determined, low yields of YAN from TML did not explain adequately the activity of the whole protein fraction. LC-HRMS/MS investigation led to the identification of other three peptides, which were evaluated in silico, synthesized and tested against ACE. Among them, tetrapeptide NIKY showed the most promising activity (52 µM), highlighting once more the potential of TML and paving the way for exploitation in novel foods.

Investigation of levels of perfluoroalkyl substances in freshwater fishes collected in a contaminated area of Veneto Region, Italy.

The bioaccumulation of 12 perfluoroalkyl substances (PFASs) in 107 freshwater fishes collected during 2017 in waterbodies of a contaminated area in Veneto Region (Italy) was evaluated. The contamination had been previously ascribed to a fluorochemical manufacturing plant that discharged mainly perfluorooctanoic acid (PFOA), among other PFASs, into the surrounding environment. Perfluorooctane sulfonate (PFOS) was the most abundant compound, detected in almost 99% of the fish with an average concentration of 9.23 µg/kg wet weight (w/w). Other detected compounds were perfluoroundecanoic acid (PFUnA) (98%, 0.55 µg/kg w/w), perfluorodecanoic acid (PFDA) (98%, 2.87 µg/kg w/w), perfluorododecanoic acid (PFDoA) (93%, 1.51 µg/kg w/w), and PFOA (79%, 0.33 µg/kg w/w). Bioaccumulation of PFASs was species related, with Italian barbel being the most contaminated, followed by chub, wels catfish, and carp, reflecting animals' habitat use and feeding behavior. A significant negative linear relation between PFAS concentration and fish weight was observed no matter the considered species, with smaller fish having proportionally higher bioaccumulation. PFOS concentrations were strongly correlated with the concentrations of other PFASs, suggesting a similar source of contamination or a contamination from ubiquitous sources. Correlation analysis showed PFOA likely originated from a separated source, unlinked to other PFASs. Although the fishes studied are not usually consumed by local people, with the likely exception of freshwater anglers (and relatives), their consumption has been banned by Veneto Authority since the time this study was conducted. In fact, the study suggests that a medium/high consumption frequency (superior to 1 portion per month) of fish from the investigated area might result in a high exposure to PFASs.

Rapid, novel screening of toxicants in poison baits, and autopsy specimens by ambient mass spectrometry.

Animal poisoning and dissemination of baits in the environment have public health and ethological implications, which can be followed by criminal sanctions for those responsible. The reference methods for the analysis of suspect baits and autopsy specimens are founded on chromatographic-based techniques. They are extremely robust and sensitive, but also very expensive and laborious. For this reason, we developed an ambient mass spectrometry (AMS) method able to screen for 40 toxicants including carbamates, organophosphate and chlorinated pesticides, coumarins, metaldehyde, and strychnine. Spiked samples were firstly purified and extracted by dispersive solid phase extraction (QuEChERS) and then analyzed by direct analysis in real time high-resolution mass spectrometry (DART-HRMS). To verify the performance of this new approach, 115 authentic baits (n = 59) and necropsy specimens (gastrointestinal content and liver, n = 56) were assessed by the official reference methods and combined QuEChERS-DART-HRMS. The agreement between the results allowed evaluation of the performances of the new screening method for a variety of analytes and calculation of the resultant statistical indicators (the new method had overall accuracy 89.57%, sensitivity of 88.24%, and a specificity of 91.49%). Taking into account only the baits, 96.61% of overall accuracy was achieved with 57/59 samples correctly identified (statistical sensitivity 97.50%, statistical specificity 94.74%). Successful identification of the bitter compound, denatonium benzoate, in all the samples that contained rodenticides (28/28) was also achieved. We believe initial screening of suspect poison baits could guide the choice of reference confirmatory methods, reduce the load in official laboratories, and help the early stages of investigations into cases of animal poisoning.

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle.

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and ß(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.

A novel tool to screen for treatments with clenbuterol in bovine: Identification of two hepatic markers by metabolomics investigation.

Surveillance of illegal use of growth promoters such as ß2-agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5'-deoxy-5'-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (ß2-agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity.

Fast and simultaneous analysis of carbamate pesticides and anticoagulant rodenticides used in suspected cases of animal poisoning.

Carbamate pesticides (CBs) are reported as one of the main causes of intentional or accidental poisoning of animals. Anticoagulant rodenticides (ARs) form the main class of poisons implicated in analyzed poisoned baits. These two groups of pesticide compounds include multiple substances, and thus, the development of a simple and rapid multiclass/multiresidue analytical method for simultaneous identification of both toxicant classes should be a useful strategy for analytical laboratories to reduce analysis time and cost. The present study aimed to elaborate and validate a rapid method to simultaneously determine 11 CBs and 8 ARs in samples of real matrices (bait, stomach content, and liver) from suspected animal poisoning cases. QuEChERS sample treatment and liquid chromatography coupled to hybrid high resolution mass spectrometry were used. The method resulted in good linearity (R2 ≥ 0.98) for all compounds, recovery was between 70% and 120% for CBs and 40-90% for ARs, and precision was ≤ 20% for all compounds. The method was successfully applied to the analysis of 871 real samples originating from suspected cases of animal poisoning, collected from April 2019 to October 2020. Furthermore, full scan dependent data acquisition allowed qualitative retrospective data analysis of an additional 15 compounds outside the scope of the method to be performed; these compounds could potentially be involved in unresolved poisoning cases.

Canine nephrotoxicosis induced by melamine-contaminated pet food in Italy.

Two young dogs belonging to the same kennel placed nearby Treviso (north-eastern Italy) died at the end of 2008 with clinical signs of renal failure. They were subjected to necropsy and were evaluated for histopathological and toxicological changes. Both the animals had same clinical signs and laboratory evidence of uremia. Post mortem investigations revealed severe nephrotoxicosis, associated with uroliths deposition within renal tubules and pelvis. The predominant crystal type was identical to those observed in the kidneys of animals involved in the 2004 and 2007 melamine-associated renal failure epidemic in Asia and US, providing evidence that they share the same causative agent. High doses of melamine were detected in the pet food administered to the dogs, likewise melamine was identified in renal tissue from one dead dog and in urine samples from both the animals. Therefore, a diagnosis of melamine-related nephrotoxicosis was made. To the author's knowledge this is the first report about melamine contamination of pet food from EU.

LC-HRMS/MS for the simultaneous determination of four allergens in fish and swine food products.

The inclusion on the label of packed foods of any ingredient or technological adjuvant causing allergies is required by EU food legislation. In this study a targeted proteomics method for detecting four allergens in animal-derived food matrices was developed. Liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) was used to select marker peptides from four allergens and develop a quantitative method able to simultaneously detect the presence of milk, egg, crustaceans and soy. The method was validated on fish or swine processed food products contaminated at 5 µg g-1 for milk and egg and 10 µg g-1 for soy and crustaceans. The method was tested by analyzing commercial food products with high protein content and was compared to the ELISA technique. Our results indicated the presence of soy not reported on the food label of some products, pointing out the need for efficient controls to protect allergic consumers.

Abuse of anabolic agents in beef cattle: Could bile be a possible alternative matrix?

European Union prohibited the use of anabolic agents in food producing animals since 1988. An efficient control of abuses is guaranteed not only by highly performing analytical methods, but also by knowledge of metabolic pathways, kinetics of elimination and tissue distribution. To obtain data concerning metabolites production and accumulation in bile, two typical growth promoting treatments are carried out in cattle. In the first study, sixteen beef cattle were implanted with trenbolone acetate and estradiol. In the second one, three animals were implanted with zeranol and three were fed a diet containing zearalenone. Methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) were developed and validated to quantify the analytes of interest. The results evidenced that the biliary concentrations of the marker residues were always higher than those determined at the same time in urine and liver which are the matrices generally collected within the official monitoring programmes.

First report of a fish kill episode caused by pyrethroids in Italian freshwater.

INTRODUCTION: Fish kills are events of strong emotional impact on the population because of the frequent suspicion that they can be the result of serious pollution accidents. As a matter of fact, they are often due to natural occurrences, such as low levels of dissolved oxygen in the water, but in many cases the causes remain unknown. Fish are particularly sensitive to pesticides and pyrethroids are reported to be the most ecotoxicologically active in the aquatic environment. Nevertheless, the reported cases of massive wild fish mortalities due to these toxicants are very few. This paper describes a fish kill episode occurred in the Padua Province (Veneto Region - North Eastern Italy) which involved several fish species and for which it was possible to identify the cause in the presence of pyrethroids in the water. CASE PRESENTATION: When a whitish liquid coming from the rainwater drain of an industrial area was seen to be spilling into a drainage channel, a fish massive mortality was noticed and investigated. The collected water samples showed the presence of relevant concentrations of cypermethrin, permethrin, deltamethrin and tetramethrin. Analyses on the fish tissues revealed the presence of cypermethrin and permethrin at a concentration range of 476-2834µg/kg and 346-2826µg/kg on a lipid basis, respectively. DISCUSSION: According to the results of the performed analyses, we can reasonably state that the described episode had been caused by the exposure of biota to high concentrations of pyrethroids. The present case report significantly contributes to the limited literature available on pesticides-related fish kills. Moreover, it highlights the importance of sharing protocols for fish kill management at a national level, as this would help to better define the roles of the different institutions involved and to improve the investigation and the reporting of these events.

Urinary Concentrations of Steroids in Bulls under Anabolic Treatment by Revalor-XS® Implant.

Despite the European ban of using anabolics in food-producing animals, growth promoters might still be illegally used in the European Union. To control the food chain and guarantee consumers' health, there is a need of highly sensitive analytical methods for the identification of marker residues of such treatments. In the present study, a group of bulls (n = 16) received trenbolone acetate (200 mg) and estradiol (40 mg) by a commercial ear implant during a time range of 71 days, and a second group (n = 16) was kept for control. The aim of the research was to measure the residual urinary concentrations of the administered drugs (ß-trenbolone and ß-estradiol), their main metabolites (α-trenbolone and α-estradiol), and possible alterations of the urinary profile of other endogenous hormones metabolically related. The analytical method was based on liquid chromatography-tandem mass spectrometry. Results showed average urinary concentrations of α-trenbolone and α-estradiol during treatment in the range of (0.81 ÷ 2.1) ng mL-1 and (0.96 ÷ 4.4) ng mL-1, respectively, whereas ß-trenbolone and ß-estradiol exhibit urinary concentrations lower than 0.22 ng mL-1 in both cases. Data obtained from the urinary profiles of endogenous steroids indicate that they could be useful to indirectly detect the ongoing treatment.

Epidemiology of animal poisoning: An overview on the features and spatio-temporal distribution of the phenomenon in the north-eastern Italian regions.

In the present paper we analyze and discuss about the records referring to animal poisonings and poisoned baits cases covering the period between 2007 and 2013 and submitted for diagnostic investigations to the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), which is the public veterinary health institute competent for the north eastern Italian regions. All data were gathered by a passive surveillance system based on voluntary reporting, which became mandatory in 2009 after a decree of the Italian Ministry of Health had come into force. This prohibited the use and detention of poisoned baits and ordered to selected institutions and professionals to carry out standardized surveys to assess suspect and/or confirmed reported cases; all the necessary anatomopathological and toxicological investigations to confirm the reported cases were then performed for free by public veterinary health institutes whenever a veterinarian diagnosis or clinical suspicion were provided. Totally, 1831 suspected animals poisoning and 698 cases of supposed poisoned baits recovery episodes were registered. 642/1831 (35.1%) animal poisoning cases were confirmed and the presence of toxic agents was verified in 292/698 baits (41.8%). The most severely affected territories were the ones with the highest level of urbanization and those most densely populated in the study area. Dogs and cats seemed to be greatly affected by poisoning cases and a characteristic seasonal trend was noticed, with an increase of episodes in late Winter/early Spring and in Autumn. Carbamate insecticides resulted to be the main cause for animal poisoning, while anticoagulants rodenticides played a primary role among toxicants found in poisoned baits. The presented results emphasize that malicious animal poisoning is a widespread problem in north-eastern Italy. The still relevant number of reported poisoning events caused by some banned pesticides poses the problem of identifying where these substances come from and brings to light the popular knowledge about the high toxicity of these compounds. Moreover, the noticeable increase of the number of episodes registered in 2009 pointed out how the above mentioned decree may have contributed to reveal a number of hidden cases which had not been investigated before, probably due to economic reasons related to the costs of toxicological analyses.