Preliminary studies on environmental pollutants in chamois and wild boar from Eastern Piedmont, Italy.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) are synthetic chlorinated compounds classified as POPs whereas only the penta e tetra-brominated polybromodiphenyl ethers (PBDEs) are so defined by the Stockolm Convention (Stockholm Convention, 2005) in order to elimitate or restrict the use of POPs. Organophosphorus insecticides (OCPs) represent important environmental and food contamination sources, widely used in agriculture. Among polyciclic aromatic hydrocarbons (PAHs), benzo[a]pirene is classified by IARC in Group 1, as cancerogen  and Benzo[a]fluoranthene as a Group 2B, as possible cancerogen (IARC, 2012; IARC, 2010).  EFSA (European Food Safety Authority) has released a scientific opinion on the risks to public health related to the presence of brominated flame retardants in food (EFSA, 2011) and in 2014 European commission has asked Member States to monitor the presence of brominated flame retardants (BFRs) in food over the next two years (EC, 2014). Due to their heir n-octanol/water partition coefficient (log Kow), they accumulate in fat tissue, bioconcentrate and biomagnify in the animals at the higher trophic levels, possibly causing, through chronic exposure, endocrine disruption and cancer (Wania et al., 1995; Vallack et al., 1998). The aim of this study is to evaluate the presence of OCPs, PCBs, PBDEs and PAHs in chamois and wild boar from Eastern Piedmont, Italy. A total of 20 chamois and 20 wild boar muscle samples were collected during the hunting season 2017, from Verbania Cusio Ossola (VCO) (Fig 1). The chemical analysis for the detection of OCPs, PCBs, PBDEs, and PAHs   was performed by GC-MS/MS on muscle samples purified and extracted using a QuEChERS technique, validated according to SANTE 2017 (SANTE/11183/2017). These preliminary results show the ubiquitary presence of the studied contaminants. PCBs have been found more in chamois (45%) than in wild boar (35%). No PBDEs were detected in wild boar but in chamois were found with a prevalence of 35%  and  concentration 0.25-1.52 ng g-1. About OCPs, phorate and demeton were found in wild boar (55%-15%) and chamois (32%- 35%) with range concentrations 0.21-20.1 ng g-1. No PAHs were detected, expect antharacene for one samples in wild boar (0.53 ng g-1). Further studies are in progress in order to correlate environmental contamination and game animals

Metals in mussels from Italian mollusc culture plants

The beneficial effects on human health of seafood are well known. However, seafood is a major source of exposition for consumers of most of the contaminants due to human activities such as breeding, industries, mining and agriculture: the overall level in biota, therefore seafood and particularly molluscs, dramatically increased over this last two centuries. This study evaluates the presence of Lead, Mercury, Cadmium, Arsenic, Nickel and Chromium in mussels from the Italian mussel culture plants, and estimate the risk that Italian consumer undergoes eating these molluscs. Mussels where collected at the wholesale fish market of Milan, the most important wholesale Italian fish market. The molluscs belonged to the   37 FAO marine area (corresponding to Mediterranean Sea), particularly from FAO 37.2.1 Ligury, 37.2.2. North Adriatic, middle Adriatic, Puglia, 37.2.3 Lazio and Sardinia, and were collected from July 2016 to February 2017. (FIG1). Analyses were carried out through inductively coupled plasma-mass spectrometry (ICP-MS) according to the Environmental Protection Agency (EPA) 3050B method. The sample concentrations were below the Maximum Levels (ML) given by Commission Regulation (EC) No 1881/2006 for Cadmium, Lead and Mercury, except one sample from south Adriatic sea, that showed Mercury concentration of 0.528 mg kg-1. Arsenic, Nickel and Chromium ML are not stated by EU. Arsenic concentration ranged from 2.05 to 13.35 mg kg-1, with the highest values found in Italian molluscs, Nickel concentration ranged from 0.00 to 3.98 mg kg-1. Chromium was found only in 5 of 30 sample analysed with a maximum concentration of 0.590 g kg-1. The tolerable intakes recommended by EFSA and on EU maximum levels, indicate that Italian mussels do not pose a risk consumers

Preliminary study about the detection of perfluoroalkyl substances in eel samples of Lake Garda by liquid chromatography tandem mass high resolution mass spectrometry (LC-HRMS).

Perfluoroalkyl substances (PFASs) are a large class of fluorinated aliphatic chemical of anthropogenic origin with high chemical stability even at high temperatures and in presence of alkalis, strong acids or oxidizing agents (Lau et al. 2004). All these characteristics make them no biodegradable and very persistent in the environment, associated with adverse health risks (Eriksen et al. 2010). Food, especially fish and other seafood, is considered the main source of exposure to PFASs (EFSA 2012). In this preliminary study we developed and validated a sensitive, selective and specific method by LC-HRMS Orbitrap to monitor the presence of 16 PFASs in eel (Anguilla Anguilla) samples. The clean-up of the lyophilized samples consisted of a previous extraction step with acetonitrile to precipitate also proteins, followed by a purification step through Oasis® WAX SPE (Weak Anionic Exchange Solid Phase Extraction) cartridges. The method applied to 45 farmed eel samples from Lake Garda showed the presence of several PFASs, up to 10 in the same eel, in the order of ng/g (Fig.1). The results provided a representative situation of the  PFASs contamination level of the lake, lower than those of others European countries (Hoff et al. 2005, Kwadijk et al. 2010)

Detection of seven β2-agonists in teeth by LC-MS/MS: preliminary results.

β2-agonists are powerful tocolytic (the only use permitted in cattle) and bronchodilator agents, butmay also be administered as growth promoters to improve the production of lean meat increasing alsothe lipolytic activity. Although the European Union (EU), China and other Asian countries have bannedthe use of β2-agonists for growth promoting purposes (European Union, 2003), the United States ofAmerica (USA) authorised ractopamine as a feed additive for swine, cattle and turkey. These veterinarydrugs, generally show high clearance rates in the conventional biological matrices, as well as urine, liverand muscle, making difficult their detection (Wu, 2014). For this reason, we suggested bovine teeth asa new unconventional matrix of accumulation in a more long-time window, for the detection ofcimaterol, clenbuterol, isoxsuprine, mabuterol, ractopamine, salbutamol and terbutaline. In literature,the few studies on teeth are limited to human (Andra, 2015) and are absent for veterinary medicine.The samples extracted by a simple liquid extraction step with ethyl acetate:tert-butyl methyl ether (4:1,v/v) after washing and pulverization of teeth, through a ball mill, were analysed using a liquidchromatography tandem mass spectrometry (LC-MS/MS) confirmatory method validated according tothe Commission Decision 2002/657/EC criteria (European Union, 2002). Teeth from 8 veal calves,administered per os with 80 mg day-1 oral ractopamine for 32 days, and from seven random bovinesfrom the food chain were collected at the slaughterhouse to test the suitability of this matrix. Theresults demonstrated ractopamine presence in teeth from the treated animals (average concentration8.90 ng g-1). Isoxsuprine was found in a control sample (13.67 ng g-1), demonstrating the effectivenessof this matrix as a powerful tool to ensure illegal treatment

Analyses of organochlorine pesticides residues in eels (Anguilla anguilla) from Lake Garda using Gas chromatography coupled with Tandem Mass Spectrometry (GC-MS/MS).

Lake Garda is located in Insubria region, that is known for being the most populated and industrialized area of Italy (Camusso et al., 2001). Therefore, the Lake water, and also the fish species present, could be affected by environmental contamination.  European eel (Anguilla anguilla) are considered as suitable matrix for biomonitoring environmental contaminants in European water (Belpaire et al., 2007), being widespread in many European waters and highly contaminated by lipophilic compounds, due to the high lipid content (up to 40%) (Larsson et al., 1991). Moreover, eel is an edible species (its farming currently supplies approximately 45,000 tons/year) (Nielsen et al., 2008), so it also represents a public health issue. Based on these considerations, the aim of this study was to evaluate the occurrence of fourteen organochlorine pesticides (OCs) in forty-five eels (Anguilla anguilla) from Lake Garda, using Accelerated Solvent Extraction (ASE) procedure for the analytes extraction and Gas chromatography coupled with Tandem Mass Spectrometry (GC-MS/MS) for the analysis of OCs. GC-MS/MS analysis was developed and validated according to the SANTE/11945/2015 guidelines.  Uncontaminated eel sample (previously checked for the presence of OCs and considered blank with a concentration of compounds < Limit of Detection) were used for all procedure's optimization steps. For all the OCs analysed, satisfactory results were achieved. Regarding eel samples, several pesticides were detected, but DDTs (DDT and its metabolites) were found with the highest prevalence (92 %). The concentration rage was from not detected (n.d.) to 19000 ng g-1. Although DDTs levels in the environment are declining (Albaiges et al., 2011), they continue to bioaccumulate in tissues of human and animal and biomagnify in food chains

Detection of boldenone, its sulfate and glucuronate forms, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone in bovine bile and urine by LC-MS/MS: preliminary results.

The European Union regulations ban or regulate the use of substances with hormonal action. There are, however, some detectable steroids in biological matrices from cattle to which no administration were made. These are the so called “grey-zone” or “pseudoendogenous” substances (endogenously produced under certain circumstances). The administration of boldenone and androstadienedione to cattle is forbidden both for therapeutic or growth promotion purposes, while therapeutic use of prednisolone is permitted. The control of the use of these substances is hampered by their pseudoendogenous nature. We made a comparison between analyses performed on the classical matrix urine with bile (a novel matrix) from the same animals, with the aim to determine the better matrix for the above mentioned steroids. We tested the synthetic corticosteroid dexamethasone, too. The preliminary results do not show any difference between the two matrices, as concern the pseudoendogenous substances. The data about dexamethasone were instead very promising about the use of bile. The detection frequency and concentration levels were, in fact, higher in bile than in urine from the same animals

Are saponins and sapogenins precursors of prednisolone? Preliminary results

The transformation of cortisol into prednisolone in cattle faeces was demonstrated and provided by literature, given the structural similarity of prednisolone with cortisol. In this study, we evaluated a possible neo-formation of prednisolone, as results of a faecal or environmental contamination. A saponine, α-solanine, and a sapogenin, diosgenin, were selected as possible precursors. A simple method without extraction was applied. The analyses were performed by HPLC–MSn to evaluate the possible transformations. The results showed that prednisolone was detected in the faecal suspension spiked with diosgenin at t= 8h, while cortisol was also detected at t = 24h in the sample of faeces spiked with α-solanine. In the feed and in the control samples, no transformations were observed

Determination of thyreostats in bovine urine and thyroid glands by HPLC-MSMS

Thyreostats are orally active substances used in medicine and veterinary work to regulate the production of T3 and T4 hormones by the thyroid gland. They can be illicitly administered to livestock for fattening purposes in order to improve body-weight gain due to an important retention water in edible tissues and in the gastroenteric tract. Their fraudulent utilisation is severely prohibited by the European Union, which requires a precise monitoring. In 2007, European Union of Reference Laboratories in the CRL Guidance paper proposed a recommended concentration of 10 ng mL-1 in urine, which has just been suggested to increase to 30 ng mL-1. In order to facilitate the detection of thyreostats in two bovine matrices, urine and thyroid glands, a method was developed without a previous derivatisation step, frequently used for HPLC analysis of Thiouracil. A salting-out assisted liquid–liquid extraction procedure for preparation was carried out to facilitate the movement of the thyreostats to the tert buthyl methyl ether phase. The HPLC-MS/MS analytical procedure of the method was validated according to the guidelines of Commission Decision 2002/657/EC and has permitted to obtain satisfactory performance parameters and, decision limit and detection capability values for all the thyreostats lower than the recommended values hitherto mentioned

Presence of endogenous prednisolone in human urine

The possibility of an endogenous presence of the glucocorticoid prednisolone has already been demonstrated in bovine and horse urine, with the aim of clarifying its origin in this matrix, which is used by official agencies for the control of illicit treatments. From this point of view, the endogenous nature of prednisolone could be a major topic in doping control of both amateur and professional human athletes. A study was therefore made on 34 human volunteers (13 males and 21 females; aged 22–62) to detect the presence of prednisolone in their urine by HPLC–MS3. One of the volunteers underwent vernal allergy treatment with betamethasone for two subsequent years. An investigation was carried out with the aim of verifying if the suppression, and the circadian rhythm, of cortisol urinary levels could also apply to prednisolone. The results of the study show that prednisolone was present in the urine of all 34 volunteers, with a concentration very close to 100-times lower that of cortisol, with no dependence on gender. The same ratio (1/100) was observed in the prednisolone and cortisol levels detected during the 24 h together with the suppression of prednisolone by betamethasone treatment. These data demonstrate the endogenous nature of low concentrations of prednisolone in human urine, and motivate further studies about the biosynthetic pathways of this corticosteroid and its relationship with stress in humans, as already described in cows

Psychological Reactions during and after a Lockdown: Self-Efficacy as a Protective Factor of Mental Health

The aim of the present study was to investigate the effects of home confinement/social isolation (i.e., lockdown), imposed to reduce large-scale spread of a disease in the population, on the mental health of individuals. Through an online survey during the lockdown (DL) related to COVID-19 (1085 respondents, 627 females, agerange: 18-82) (Italy, 23 April-2 May 2020), we revealed that situational factors, i.e., the presence of children at home and female gender, and psychological factors, i.e., a greater sense of isolation, lower perception of safety outside the home and higher trait anxiety, predicted higher levels of state anxiety (R2 = 0.58). The same factors, but with young age instead of the presence of children, predicted higher levels of perceived stress (R2 = 0.63). Then, these data were compared with those collected after the lockdown (AL) (174 respondents, 128 females, agerange: 19-78) (Italy, 1 July-31 October 2021). The results showed that along with a reduced sense of isolation (DL = 2.90 vs. AL = 2.10) and an increased perception of safety outside the home (DL = 2.63 vs. AL = 3.05), a reduction in state anxiety (DL = 45.76 vs. AL= 40.88) and stress appeared (DL = 18.84 vs. AL = 17.63). However, the situation was better for men than for women. Perceived self-efficacy emerged as a protective factor for mental health (R2range: 0.03-0.27). The results are discussed in light of the evidence on the effects of lockdown on individuals worldwide. These results may be used to make more educated decisions on targeted help for individuals who may be most adversely affected by the adoption of lockdowns in the future