Seasonal variability of trace elements bioaccumulation in Pacific Oysters (Crassostrea gigas) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

BACKGROUND: Metals pollution is a worldwide environmental issue due to their persistence in the ecosystems, non-degradability, and bioaccumulation in marine biota. Pacific Oysters (Crassostrea gigas) are highly nutritious bivalve representing an important dietary constituent but may accumulate metals through feeding on suspended sediments from surrounding water, then represent a suitable tool for biomonitoring. MATERIALS AND METHODS: The occurrence of trace elements (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Rb, Se, Sn, V, Zn) was investigated in Pacific Oysters (Cassostrea gigas) collected from Calich Lagoon in each season of 2019. Samples were homogenized and subjected to microwave acid digestion before being analyzed by inductively coupled plasma-mass spectrometer (ICP-MS). RESULTS: The results showed a significant seasonal variation for temperature, dissolved oxygen, chlorophyll, and pH. Moreover, high significant seasonal variation in concentrations of Cd, Mn, Ni, and V was recorded. The highest values were found for Fe (128 mg kg⁻1 w.w.), and Al (112 mg kg⁻1 w.w.) in October, for Zn (113 mg kg⁻1 w.w.) in March and May. CONCLUSIONS: Pacific Oysters were confirmed as suitable bioindicators of the health status of coastal lagoons; trace elements concentrations were highly affected by season of collection, and according to literature the highest values were recorded in autumn and summer. The EU legal limits for Cd and Pb were not exceeded, then the farmed oysters were safe to consumers.

Trace and rare earth elements in phytoplankton from the Tyrrhenian Sea (Italy).

Plankton plays a very crucial role in bioaccumulation and transfer of metals in the marine food web and represents a suitable bioindicator of the occurrence of trace and rare earth elements in the ecosystem. Trace elements and REEs were analyzed by ICP-MS in phytoplankton samples from the northwestern Mediterranean Sea. Metal concentrations in phytoplankton were found strongly influenced by seasons and depth of collection (- 30 m, - 50 m). Principal component analysis (PCA) has shown that Al, As, Cr, Cu, Ga, and Sn concentrations were related to summer and autumn in samples collected at 30 m depth, while Fe, Mn, Ni, V, and Zn levels related strongly with summer and spring at 50 m depth. Fe, Al, and Zn were the most represented elements in all samples (mean values respectively in the ranges 4.2-8.2, 9.6-13, and 1.0-4.4 mg kg-1) according to their widespread presence in the environment and in the earth crust. Principal component analysis (PCA) performed on REEs showed that mostly all lanthanides' concentrations strongly correlate with summer and autumn seasons (- 30 m depth); the highest ∑REE concentration (75 µg kg-1) was found in winter. Phytoplankton REE normalized profile was comparable to those of other marine biota collected in the same area according to the suitability of lanthanides as geological tracers.

Optimization and validation of a quick and responsive LC-ESI-MS/MS method to evaluate tetracycline residues in processed animal proteins (PAPs).

Following the bovine spongiform encephalopathy (BSE) in 2001, processed animal proteins (PAPs) reintroduction is envisaged in non-ruminant feed thanks to their high protein content, easy availability and cost-effective characteristics. PAPs must be submitted to rendering practices, providing sterilization of products, under standardized conditions of temperature and pressure, according to Regulation (EC) No 142/2011. However, the chemical risk associated to these raw materials has been never evaluated. The aim of this study was to develop and validate a reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of tetracycline residues in PAPs at µg kg-1 level. The LC-MS/MS method performances were evaluated in terms of specificity, linearity (25-500 µg kg-1), limit of quantitation (LOQ) (25 µg kg-1), accuracy and precision (CV% < 25%), uncertainty, recovery (80-120%) and ruggedness. All the evaluated parameters fulfilled the analytical performance criteria, and the validated LC-MS/MS method fits for purpose as confirmatory method on the occurrence of residues (µg kg-1) of tetracyclines in PAPs. PAPs are a powerful product which could be used both as raw materials in feed and in organic fertilizer production in a circular economy context. Therefore, the lack of regulation and control over antibiotic occurrence should be implemented to avoid a misuse and an increment of antibiotic resistance pressure over the environment and to ensure safety of the feed and food chain.

Preliminary Data on Essential and Non-essential Element Occurrence in Processed Animal Proteins from Insects.

Insects represent a valuable and environmentally friendly protein alternative in food and feed. The Farm to Fork strategy encouraged the reintroduction of animal by-products in feed production to optimise recycling and to valorise under-used resources. In order to grant safe and valuable feed products, this study investigated the black soldier fly (BSF) (Hermetia illucens) chemical risk. Samples collected in different steps of production (8 samples of substrate for culturing, 7 samples of larvae, 15 samples of protein meal, 18 samples of spent substrate) were analysed for microessential elements (chromium, copper, iron, nickel, selenium and zinc) and inorganic contaminants (aluminium, arsenic, cadmium, lead, tin and vanadium) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Microessential elements were found in the following order: Fe > Zn > Cu > Ni > Se > Cr (mg kg-1). Non-essential element concentrations were found lower than the set limits according to the European Union Regulations. The growing demand for alternative protein sources for feed production could be partially compensated by black soldier fly (BSF) (Hermetia illucens) meal, as it appears a good source for high-quality proteins and microessential elements which play a pivotal role in animal growth. In the foreseeable future the current legislation and the official monitoring plans may be implemented and broaden, to focus and assess limits for upcoming matrices, and to ensure feed and food safety.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse.

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg-1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety.

Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity.

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg-1). Among samples containing more than 25 µg kg-1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg-1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Ecology of oxidative stress in the Danube barbel (Barbus balcanicus) from a winegrowing district: Effects of water parameters, trace and rare earth elements on biochemical biomarkers.

Fish can be highly vulnerable to environmental pressures because they are exposed to oxidative stressors in the aquatic environment. Such stressors can affect the levels of antioxidant biomarkers against reactive oxygen species (ROS). With this study we investigated the oxidative stress ecology in Danube barbel (Barbus balcanicus) from the Barbucina creek (northeast Italy), a watercourse in the Collio winegrowing district. To do this, superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activity was measured in gills, liver, and muscle, while metallothioneins (MT) and trace and rare earth elements (REEs) levels were determined in muscle. The effect of environmental factors (physicochemical parameters of water, trace elements and REEs) on oxidative stress biomarkers was thus assessed. High concentrations were determined for cerium (Ce), scandium (Sc), neodymium (Nd), lanthanum (La), yttrium (Y), and praseodymium (Pr) among the REEs. Among the trace elements, arsenic (As), copper (Cu), and mercury (Hg) levels were higher compared to published data, suggesting their role as stressors. The multiple linear regression (MLR) model showed a statistically significant association (R2 = 0.858; F = 10.07; p = 0.015) between As, Cu, Hg, and Pr and SOD activity in the gills, indicating a functional relationship between them. Differently, CAT activity was significantly higher in the liver, probably in response to long-term Cu contamination of the watercourse. This was confirmed by the MLR model that showed a significant association (R2 = 0.638; F = 8.152; p = 0.02) between the concentration of MT and of Cu. Our data show a biochemical defensive response by Danube barbel to the disturbances in the aquatic ecosystem of the Barbucina creek. These insights advance our understanding of the role and the effects of environmental factors as trace elements and REEs on oxidative stress in fish.

Local context and environment as risk factors for acute poisoning in animals in northwest Italy.

Animal and bait poisoning data for northwest Italy collected between 2012 and 2017 were described and analyzed to estimate the risk of exposure to hazardous substances by animals. In about 4% of animals necropsied (n = 356/9512), the cause of death was poisoning and domestic pets (9.5%) and synanthropic animals (12.2%) appear to be the most involved. Furthermore, 294 out of 728 baits (40.4%) were positive for toxic substances and/or inert hazardous material. Application of a mixed-effects Poisson regression model and local cluster analysis evidenced increased risk of exposure to poisoning with altimetry (>600 m.a.s.l.) and municipality (PR = 1.6, 95%CI 1.2-2.1 for poisoning, PR = 2.2, 95%CI 1.2-4.2 for poisoning by insecticides and PR = 2.9, 95%CI 1.4-6.2 for poisoning by metaldehyde). Since the mountainous areas in the region are mostly devoted to pasture and extensive farming, the high frequency of animal and bait poisoning events may be related to farmers' need to protect their livestock and crops against foxes, wolves, rodents or wild boars. Summarizing, the type of land use and context may influence the frequency and type of toxin chosen to kill animals considered a nuisance for hunting, farming, agriculture and apiculture. Despite bans and limitations, the use of harmful substances is not perceived as an environmental threat but rather as routine pest control. Animal and bait poisoning constitute a public health concern because it is potentially harmful to humans and the environment. Our findings may inform risk communication strategies, as well as prevention and control measures for the reduction of illegal and non-targeted species poisoning.