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.

Distribution of rare earth elements (REEs) in the feathers of gentoo penguins (Pygoscelis papua) from different geographical locations of the Antarctic peninsula area.

Antarctica is the most remote and coldest regions of the planet, but the presence of REEs there has received little attention. This study assessed REE-contents in the feathers of adult gentoo penguins from Ardley Island, Kopaitic Island and Base O'Higgins. Field work was accomplished during 2011 (austral summer), and determination of elements was performed with ICP-MS. In general, REE-levels showed descending relations as follows: Ce > La > Y > Nd > Sc > Pr > Gd > Sm > Dy >Er > Yb > Eu > Ho > Tb > Tm > Lu. The data showed an increase of the levels of REEs from the lower part of the feather to the tip. This finding seems to be spatially dependent, but geochemical, anthropogenic conditions, feeding habits, sex, or even health status of birds should also be considered. It is a subject that requires deeper attention in future studies.

Passive surveillance of wild fauna in northwestern Italy: a possible cognitive approach through geographical district study and fur elementary analysis.

The analysis of animal hair is a useful and non-invasive investigation method for monitoring metal content, whose beginning dates to a few decades ago. This study addresses the issue of wildlife mortality in Piedmont and Aosta Valley by linking the evidence to the characteristics of the territories and to hair elemental profile. The considered animal species were badger, fox, marten, and wolf. The quantitative data for 11 trace metals and 16 lanthanides were evaluated considering animal location and death causes regarding impacts, whose relevant number was confined to the Turin province and to the badger and fox species. The evaluation of the analytical results was performed after the out of bounds sample identification by mean of statistics. For trace metals, some areas have thus been identified, mainly in Turin province, in which the large excess of As, Cd, and Pb was related to district pollution. Moreover, the lower contents of Fe, Mn, and Al in Aosta Valley badgers' hair seem influenced by the different characteristics of the subsoil, in comparison to Piedmont, and a relationship with the living environment and the habits was suggested. Regarding lanthanides, Biella province represents an area in which environmental contamination and abundance of rare-earth elements was recorded.

Burden of rare earth elements and trace elements in feathers of magellanic penguins (Spheniscus magellanicus) from the southern Chilean Patagonia.

Patagonia is one of the last pristine regions on the southern hemisphere. The impact of rare earth element (REEs) and trace elements (TEs) in this region have received little attention. The main goal was to assess REEs burden in feathers of adult magellanic penguins (Spheniscus magellanicus). Sampling was performed at Magdalena Island of the Chilean Patagonia during the austral summer of 2011. Multi-elemental determination of 16 REEs and 24 TEs was performed with ICP-MS. The levels of REEs, TEs, and stable isotopes (δ15N, δ13C, and δ34S) were measured to assess the factors that condition the avian exposure to environmental contaminants. The results showed an increase of the levels of REEs and TEs from the calamus to the feather's tip. In the whole feather, the highest levels corresponded to Ce, which exhibited more than two order of magnitude than Lu and Tm levels. Similar to other penguin species, magellanic penguins can be vectors of REEs and metals in remote regions. Stable isotopes revealed that trophic ecology may influence some of the element concentrations in feathers of magellanic penguins, an issue that requires deeper attention.

Phthalate diester occurrence in marine feed and food (Mediterranean Sea).

Organic contaminants such as diesters of phthalic acid (PAEs) can be conveyed by microplastics in aquatic environment and constitute a relevant risk to marine organisms and humans that consume them. A method was developed for the identification and quantitative detection of 6 dimethyl phthalate (DMP), di-ethyl phthalate (DEP), di-n-butyl phthalate (DNBP), butyl benzyl phthalate (BBP), di-2-ethylesyl phthalate (DHEP), and di-n-octyl phthalate (DnOP). PAEs were then quantified in mesozooplankton, mollusk bivalves, and fish from the north-western Mediterranean Sea. Among all PAEs, DEHP was found in all zooplankton samples, in 30% of fish samples, and in 10% of bivalve samples. DBP was instead recovered in only 4% of samples (plankton and fish).

Identification of Illicit Conservation Treatments in Fresh Fish by Micro-Raman Spectroscopy and Chemometric Methods.

In the field of food control for fresh products, the identification of foods subjected to illicit conservation treatments to extend their shelf life is fundamental. Fresh fish products are particularly subjected to this type of fraud due to their high commercial value and the fact that they often have to be transported over a long distance, keeping their organoleptic characteristics unaltered. Treatments of this type involve, e.g., the bleaching of the meat and/or the momentary abatement of the microbial load, while the degradation process continues. It is therefore important to find rapid methods that allow the identification of illicit treatments. The study presented here was performed on 24 sea bass samples divided into four groups: 12 controls (stored on ice in the fridge for 3 or 24 h), and 12 treated with a Cafodos-like solution for 3 or 24 h. Muscle and skin samples were then characterized using micro-Raman spectroscopy. The data were pre-processed by smoothing and taking the first derivative and then PLS-DA models were built to identify short- and long- term effects on the fish's muscle and skin. All the models provided the perfect classification of the samples both in fitting and cross-validation and an analysis of the bands responsible for the effects was also reported. To the best of the authors' knowledge, this is the first time Raman spectroscopy has been applied for the identification of a Cafodos-like illicit treatment, focusing on both fish muscle and skin evaluation. The procedure could pave the way for a future application directly on the market through the use of a portable device.

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.

Multi-Level System to Assess Toxicity in Water Distribution Plants.

The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

Trace and rare earth element bioaccumulation in the spotted dogfish (Scyliorhinus stellaris).

Metals (trace elements and rare earth elements, REEs) were analysed by inductively coupled plasma-mass spectrometry in blood, the liver, the kidney and muscle of ex situ spotted dogfish (Scyliorhinus stellaris). The controlled environment in which these elasmobranchs were hosted allowed to assess a baseline level of metals in the different organs since exposure via water and food can be easily monitored. The highest arsenic, chromium, copper, and iron values were found in the liver, cobalt in the kidney, and cadmium and rubidium in muscle. The highest total trace elements content was found in the trend liver (75 mg kg-1) > blood (33 mg kg-1) > muscle (31 mg kg-1) > kidney (10 mg kg-1), while the ΣREEs was the liver (30 µg kg-1) > muscle (15 µg kg-1) > kidney (13 µg kg-1) > blood (4.1 µg kg-1). Between REEs, the most represented element was scandium. Significant differences in the concentration of metals among organs were observed for almost all elements. Nonessential elements were generally lower and essential elements higher in the examined specimens compared to wild elasmobranchs, suggesting a close relationship between a balanced diet and animal welfare.

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.

Processed animal proteins (PAPs) in animal nutrition: Assessment of the chemical risk of essential and non-essential elements.

BACKGROUND: Processed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. METHODS: PAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). RESULTS: Essential elements were found with the following decreasing order iron>zinc>copper>manganese>chromium>nickel>cobalt (mg kg-1). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. CONCLUSIONS: This study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.

Occurrence of trace elements in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

The present study aimed to determine trace elements in Mediterranean mussels (Mytilus galloprovincialis) from an experimental pilot farm of the Calich Lagoon, a typical Sardinian brackish area (Italy). Two sampling sessions were scheduled in February and May 2019 and the occurrence of 24 metals (Hg, Ag, Al, As, Be, Bi, Cd, Co, Cr, Cu, Fe, Ga, In, Mg, Mn, Mo, Ni, Pb, Rb, Se, Sn, Ti, V, Zn) in bivalves was considered. Environmental conditions of water (temperature, salinity, pH, dissolved oxygen, and chlorophyll a) were also measured in situ. A high significant (P<0.001) difference was reported for temperature, pH, and dissolved oxygen. Our results showed a significant sessional variation of Mo (P<0.001); Cd, V (P<0.01); Ni, Pb and Co (P<0.05) in examined M. galloprovincialis samples; as all values were higher in February than those for May session samples, meanwhile the highest levels were reported for Mg (mean±s.d. 1151±263 mg kg-1 wet weight), Al (mean±s.d. 341±192 mg kg-1 w.w.), and Fe (mean±s.d. 212 ±75 mg kg-1 w.w.) in February samples. The European Union uppermost values (EC Reg. 1881/2006) for Cd, Hg, and Pb were never overpassed. The results confirmed the role of M. galloprovincialis as one of the most appropriate biological indexes to track the presence of trace elements in brackish environments. It could be concluded that the current ecology of the Calich Lagoon suggests that compatibly with the transitional ecosystem, the classification as a bivalves' production area and the implementation of extensive shellfish farming can improve its production capacities. The knowledge of the lagoon ecology is an essential tool for its sustainable exploitation, preserving biodiversity, and mitigating the effects of anthropogenic activities on public health.

Wildlife hair as bioindicators of metal exposure.

Animal hair is a useful biomonitoring tool for assessing the occurrence of trace elements in ecosystems. Essential (chromium, copper, iron, manganese, nickel, and zinc) and nonessential (aluminum, arsenic, cadmium, mercury, lead, palladium, platinum, rubidium, tin, and vanadium) elements were investigated in hair of badger (Meles meles), wild boar (Sus scrofa), marmot (Marmota marmota), wolf (Canis lupus), fox (Vulpes vulpes) and deer (Cervus elaphus) from Northwestern Italy. Badger was found to be the highest bioaccumulator of metals, while wolf, fox and deer recorded the lowest values. Essential elements contribute in higher degree to the sum of metals for all species except for wild boar. Results have shown that animals with omnivorous diet such as badger, marmot and wild boar have metal content (especially Al, As, Cr, Cu, Fe, Ni, V) higher than carnivores (wolf and fox) and herbivores (deer) and could represent an effective sentinel of environmental exposure to metals.

Microplastics as vectors of metals contamination in Mediterranean Sea.

Microplastics are contaminants of great concern all over the world. Microplastics constitute pollutants themselves; moreover, other contaminants such as metals are easily absorbed on their plastic surface, becoming bioavailable to marine biota such as zooplankton.We collected marine zooplankton from Mediterranean Sea to investigate trace elements associated with microplastics. Samples were subjected to visual sorting by a stereomicroscope, collected with sterile tweezers, pooled and subjected to sonication, filtration, and drying before being subjected to acid extraction. An ICP-MS was utilized for multi-elemental determination.Aluminum, iron, chromium, zinc, nickel, molybdenum, manganese, lead cobalt, and copper were found at concentrations of mg/kg while arsenic, vanadium, rubidium, and cadmium at level of µg kg-1. Other elements such as silver, beryllium, bismuth, selenium, tin, and thallium were under the limit of quantitation. Lower levels of iron and manganese in samples from Italy were found in comparison to England and Brazil, while aluminum, copper, and zinc registered comparable values. The presence of metals in marine waters is strictly related to sediment lithology and anthropogenic inputs, but plastic plays a key role as vectors for metal ions in the marine system, being able to concentrate metals several order of magnitude higher than in surrounding waters and exerting potential toxicity for living beings after chronic exposure.

Aluminium occurrence in plant feed from Northwestern Italy.

BACKGROUND: Aluminium (Al) is widespread in nature, but the main route of exposure for humans and animals is through food. Cereals and vegetables give and important contribute to dietary intake. This investigation focused on the occurrence aluminium in raw materials intended for animal consumption. In fact, Al is not included in the official monitoring programs, and little or no data are still available about its presence in feed or plant material. METHODS: Rice, wheat, corn, barley, rye, triticale, oats, and soy samples were collected in Northwestern Italy in an area characterized by clayey soil rich in aluminium silicates. Samples were subjected to homogenization and microwave acid digestion and Al was quantified by Inductively Coupled Plasma-Mass Spectrometer (ICP-MS). RESULTS: Different aluminium concentrations were found in the analyzed raw materials: soy (89 mg kg-1) > rye > rice > oat > triticale > barley > wheat > corn (2.9 mg kg-1). In this study, Al concentrations were found higher than those reported for same matrices in other UE and non-EU countries, especially in rice and soy. CONCLUSIONS: Al concentrations were related to the soil geological feature and to the physiological characteristics of the plant species. These two factors must be carefully considered for a reliable evaluation of aluminium exposure through feed and food.

Poaching of endangered species in Mediterranean Sea: Alopias vulpinus.

A thresher shark poached by fishermen in the stretch of sea between the island of Ponza and the coast of Terracina (Lazio, Central Italy, Mediterranean Sea) and confiscated by the Italian health authorities was evaluated for the presence of chemical contaminants. Hg mean concentration of 2.1 mg kg-1 was found in muscle; speciation analysis confirmed it was almost in the toxic organic form of MeHg (2.0 mg kg-1). The Se:Hg molar ratio was <1 not protective against the toxic effect of Hg. 137Cs was detected at a mean concentration of 0.53 Bq kg-1, and 40K, radioisotope of natural origin was found at 91 Bq kg-1. Dioxins and PCBs were found in the common thresher shark at mean concentrations of 0.012 and 0.088 pg TEQ-WHO g-1 w.w. while NDL PCBs at 4.5 ng g-1 w.w., lower than limits set by European Regulation. The major concern is constituted by the presence of Hg twice the maximum limit set by EU Regulation, then the consumption of thresher shark flesh should be strongly discouraged.

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.

Metal load and oxidative stress driven by organotin compounds on rainbow trout.

Tributyltin-based (TBT) antifouling paints, widely used for the treatment of flooded surfaces, have been banned in 2008 for their high environmental persistence and bioaccumulation in aquatic organisms. Although it is still present in aquatic ecosystems, oxidative stress driven by TBT has been still poorly investigated in fish. The aim of the study was to examine the time-course stress responses in liver of rainbow trout that received a single intraperitoneal injection of tributyltin chloride (TBTC) or tributyltin ethoxide (TBTE), both at a dose of 0.05 and 0.5 mg/kg. Levels of metallothioneins, total glutathione, malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were evaluated at 3 and 6 days post-injection. Tin load was measured in the muscle of the same fish. Differences were observed in the time-course accumulation of tin with a clear dose-response relationship. Although individual oxidative stress biomarkers varied, the biomarker profile indicated different stress mechanisms caused by both TBTC and TBTE. The weak induction of metal-trapping metallothioneins and the changes of oxidative stress biomarkers suggested a stress-pressure in both TBT-treated trout, advising for an ecotoxicological risk for freshwater ecosystems.