Subchronic oral exposure to polystyrene microplastics affects hepatic lipid metabolism, inflammation, and oxidative balance in gilthead seabream (Sparus aurata).

Microplastics (MPs) pose a clear threat to aquatic organisms affecting their health. Their impact on liver homeostasis, as well as on the potential onset of nonalcoholic fatty liver disease (NAFLD), is still poorly investigated and remains almost unknown. The aim of this study was to evaluate the outcomes of subchronic exposure to polystyrene MPs (PS-MPs; 1-20 µm; 0, 25, or 250 mg/kg b.w./day) on lipid metabolism, inflammation, and oxidative balance in the liver of gilthead seabreams (Sparus aurata Linnaeus, 1758) exposed for 21 days via contaminated food. PS-MPs induced an up-regulation of mRNA levels of crucial genes associated with lipid synthesis and storage (i.e., PPARy, Srebp1, Fasn) without modifications of genes involved in lipid catabolism (i.e., PPARα, HL, Pla2) or transport and metabolism (Fabp1) in the liver. The increase of CSF1R and pro-inflammatory cytokines gene expression (i.e., TNF-α and IL-1ß) was also observed in exposed fish in a dose-dependent manner. These findings were confirmed by hepatic histological evaluations reporting evidence of lipid accumulation, inflammation, and necrosis. Moreover, PS-MPs caused the impairment of the hepatic antioxidant defense system through the alteration of its enzymatic (catalase, superoxide dismutase, and glutathione reductase) and non-enzymatic (glutathione) components, resulting in the increased production of reactive oxygen species (ROS) and malondialdehyde (MDA), as biomarkers of oxidative damage. The alteration of detoxifying enzymes was inferred by the decreased Ethoxyresorufin-O-deethylase (EROD) activity and the increased activity of glutathione-S-transferase (GST) at the highest PS-MP dose. The study suggests that PS-MPs affect the liver health of gilthead seabream. The liver dysfunction and damage caused by exposure to PS-MPs result from a detrimental interplay of inflammation, oxidative damage, and antioxidant and detoxifying enzymatic systems modifications, altering the gut-liver axis homeostasis. This scenario is suggestive of the involvement of MP-induced effects in the onset and progression of hepatic lipid dysfunction in gilthead seabream.

Fish welfare during slaughter: the European Council Regulation 1099/09 application.

The Treaty of Lisbon states that animals are sentient beings. Fish species show physiological differences from terrestrial animals and are slaughtered and killed in a very different context. Many existing commercial killing methods expose fish to extensive suffering over a prolonged period of time, and some of the slaughtering practices they experience can cause pain and distress. This study highlights the limited feasibility of European Council Regulation 1099/09 requirements on welfare when killing cephalopods and crustaceans. Sentience is the animal's capacity to have positive (comfort, excitement) and negative (pain, anxiety, distress, or harm) feelings. Considerable evidence is now showing that the major commercial fish species, including cephalopods and crustaceans, possess complex neurological substrates supporting pain sensitivity and conscious experiences. In the legislation applied to scientific procedures, the concept of sentience in these species is important. Therefore, it would be appropriate to acknowledge current scientific evidence and establish reference criteria for fish welfare. For the welfare of fish species during slaughter, European Council Regulation 1099/09 applicability is limited. Fish welfare during slaughter is more than just an ethical problem. According to the One-Health approach, food safety should also include the concept of sentience for fish welfare. Pending studies that dispel all doubt, the precautionary principle of European Council Regulation 178/04 remains valid and should be applied to fish welfare.

Do Organochlorine Contaminants Modulate the Parasitic Infection Degree in Mediterranean Trout (Salmo trutta)?

We investigated the occurrence of organochlorine pollutants (OCs) in the muscle of brown trout and evaluated their potential modulation of parasite infection. The toxicological risk for consumer health was assessed, too. Trout were collected from the Sila National Park (Calabria region, South of Italy). The highest concentrations emerged for the sum of the 6 non-dioxin-like (ndl) indicator polychlorinated biphenyls (Σ6ndl-PCBs), followed by the 1,1,1-trichloro-2,2-di(4-chlorophenyl)-ethane (DDT), dioxin-like PCBs, hexachlorobenzene (HCB), and dieldrin. Measured on lipid weight (LW), the mean value of Σ6ndl-PCBs amounted to 201.9 ng g-1, that of ΣDDTs (the sum of DDT-related compounds) to 100.2 ng g-1, with the major contribution of the DDT-metabolite p,p'-DDE which was detected in all sample units (97.6 ng g-1 on average). Among dioxin-like congeners, PCB 118 showed the highest mean concentration (21.96 ng g-1 LW) and was detected in all sample units. Regression analysis of intestinal parasites on OC concentration was performed, controlling for two potential confounding factors, namely sex and sexual stage. The results evidenced the existence of interactions between the dual stressors in the host-parasite system in the wild. A negative and statistically significant correlation was estimated, suggesting that OCs may decrease parasite infection degree. Regarding the toxicological risk evaluation, OC concentrations were consistently below the current European Maximum Residue Limits.

Research and characterization of fibrous microplastics and natural microfibers in pelagic and benthic fish species of commercial interest.

The ingestion of synthetic microfibers, the most prevalent type of microplastics in marine environments, and natural fibers was assessed in Engraulis engrasicolus and Mullus barbatus, two commercially important fish species in the Mediterranean Sea. Microfibers were isolated from the fish gastrointestinal tract using a 10% potassium hydroxide solution. For the microfiber characterization, the evaluation of specific morphological features using a light microscope, coupled with the Fourier-transform infrared (FTIR) analysis of a subsample of isolated particles, was applied. The preliminary results showed the occurrence of microfibers in 53 and 60% of European anchovy and Red mullet, respectively. A mean of 6.9 microfibers/individual was detected in anchovies, while on average Red mullet samples contained 9.2 microfibers/individual. The most common colors of fibers in both species were black, blue, and transparent. Visual characterization of fibers allowed the classification of 40% of the items as synthetic microfibers. FTIR spectroscopy confirmed the visual classification by fiber morphology. Microfibers were made of different typologies of polymers, represented by cellulose, cotton, and polyester. These findings confirm as the wide distribution of fibrous microplastics, and natural microfibers may impact both pelagic and deep-sea trophic webs. Despite the presence of microfibers in fish species poses a potential risk to human health, the literature is scarce regarding studies on the uptake by commercial marine fish mostly due to methodological issues. The visual characterization, corroborated by spectroscopic techniques, may be useful to differentiate synthetic and natural fibers, representing a fast and easy method to assess fibrous microplastic pollution in commercially important fish species.

Focus on histamine production during cheese manufacture and processing: A review.

Histamine (HIS) intoxication is a poisoning caused by histamine in food. Cheese is one of the most common dairy products associated with histamine levels which vary depending on the processing methods. The final content of histamine in cheese is influenced by intrinsic and extrinsic factors, their interactions, and contamination stemming from food processing. The application of control measures may be useful to inhibit/reduce production during cheese manufacture and processing but have a limited effect. To reduce histamine intoxication outbreaks from cheese consumption the introduction of quality control programs and appropriate risk mitigation options should be applied along the dairy chain from an overall perspective of food safety based on individual susceptibility and consumer sensitivity. As key food safety, this topic should be considered in future regulations in dairy products because the lack of a clear law on HIS limits in cheese may result in a significant potential deviation from the EU food safety strategy.

Subchronic Exposure to Polystyrene Microplastic Differently Affects Redox Balance in the Anterior and Posterior Intestine of Sparus aurata.

Microplastics (MPs) are pollutants widely distributed in aquatic ecosystems. MPs are introduced mainly by ingestion acting locally or in organs far from the gastroenteric tract. MPs-induced health consequences for fish species still need to be fully understood. We aimed to investigate the effects of the subchronic oral exposure to polystyrene microplastics (PS-MPs) (1-20 µm) in the gilthead seabreams (Sparus aurata) used as the experimental model. We studied the detrimental impact of PS-MPs (25 and 250 mg/kg b.w./day) on the redox balance and antioxidant status in the intestine using histological analysis and molecular techniques. The research goal was to examine the anterior (AI) and posterior intestine (PI) tracts, characterized by morphological and functional differences. PS-MPs caused an increase of reactive oxygen species and nitrosylated proteins in both tracts, as well as augmented malondialdehyde production in the PI. PS-MPs also differently affected gene expression of antioxidant enzymes (i.e., superoxide dismutase, catalase, glutathione reductase). Moreover, an increased up-regulation of protective heat shock proteins (HSPs) (i.e., hsp70 and hsp90) was observed in PI. Our findings demonstrate that PS-MPs are responsible for oxidative/nitrosative stress and alterations of detoxifying defense system responses with differences in AI and PI of gilthead seabreams.

Pathologic characterization of white striping myopathy in broiler chickens.

White striping (WS) is an emerging myopathy of broiler chickens characterized by white striation of muscle. Despite the recent advances, the pathomechanism underlying the WS remains elusive. The aim of this study was to characterize morphological and molecular features of WS in broiler chickens. 50 pectoralis muscles were collected from 55 days old ROSS 308 broiler chickens with a mean weight of 3.5 kg. Samples were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Real-time-PCR was used to evaluate the expression of different cytokines. Histological lesions were observed in all examined animals, both with and without macroscopic evidence of WS. WS muscles showed endomysial and perivascular inflammatory infiltrates of macrophages and cluster of differentiation (CD)8-positive T lymphocytes with severe myofiber atrophy, necrosis, fibrosis and replacement by adipose tissue. There was diffuse sarcoplasmic and sarcolemmal overexpression of the major histocompatibility complex class I (MHC I). The severity of the histologic lesions was positively correlated with the macroscopic degree of white striations. IL-6, IL-17 and lipopolysaccharide-induced TNF-α factor (LITAF) were overexpressed in severe lesions of WS. The presence of the CD8/MHC I complexes, together with the higher expression of IL-6, IL-17 and LITAF in severe degree of WS, suggest that the immune response may be involved in the progression of this myopathy and can be consistent with a hypoxia-induced inflammatory myopathy. These results help to understand the pathomechanism of WS contributing to the reduction of economic losses and improving poultry welfare.

Extraction and identification of microplastics from mussels: Method development and preliminary results.

Microplastics (MPs) are an emerging threat to marine ecosystems. One of the primary environmental risks is their bioavailability for aquatic organisms. Some fish and bivalves are of particular interest because their feeding strategies expose them to particles present in the water column. The aim of the study was to assess an extraction method in order to isolate and quantify MPs from fish gastrointestinal tract (n.8) and muscle (n.4), and bivalves (n.8) samples. The accuracy of the method was assessed through the calculation of the recovery percentage in samples spiked with a known number of MPs using microscopic observation. Successively, the extraction was preliminarily applied on n.20 mussels collected from mariculture plants of the Tyrrhenian and the Adriatic Sea. The results of the digestion protocol showed an average extraction yield of 80% in fish gastrointestinal tracts, 90% in fish muscle samples, and 95% in mussels. Preliminary analysis carried out on farmed mussels showed an average abundance of 3.8 items/individual, and 0.5 items/g of tissue, among those black, was the most represented color.

Occurrence of bisphenol A in the milk chain: A monitoring model for risk assessment at a dairy company.

Bisphenol A (BPA) as a chemical hazard may enter the milk chain during primary production at the farm and, successively, during milk processing at a dairy company. To identify the potential pathways that play a role in the occurrence of BPA, a monitoring model for risk assessment based on the identification of the hazards at each stage of milk processing was investigated. Milk samples were analyzed using liquid chromatography with fluorescence detection. Quantifiable levels were detected in samples obtained from the raw milk storage tank, pasteurized milk from the storage tank, and packaged milk. The highest BPA contamination levels were detected in raw milk from the storage tank (mean 0.265 µg/L). Despite the fact that dietary exposure levels were below the temporary daily intake, BPA may have adverse effects, particularly for vulnerable population groups. New monitoring programs involving each stage of milk processing should therefore be applied.

Occurrence of potential plastic microfibers in mussels and anchovies sold for human consumption: Preliminary results.

There is a global concern over the impact of microplastics on marine species and trophic webs. Microfibers commonly represent the greater portion of microplastics in the aquatic environment, but little is known about fiber uptake and accumulation by marine biota. The aim of the study was to investigate the potential plastic microfiber contamination in mussels (Mytilus galloprovincialis) and anchovies (Engraulis encrasicolus) from the Tyrrhenian Sea sold for human consumption. Anthropogenic debris was extracted from the digestive tracts of fish and the whole shellfish using a 10% KOH solution and quantified under a light microscope. The preliminary results showed the occurrence of potential plastic and natural microfibers in 73% of the samples. On average mussels contained 1.33 microfibers/g w.w. and 7.66 items/individual, while anchovies contained 9.06 microfibers/individual. Considering that mussels are consumed as a whole, and small pelagic fish, as anchovy, may be eaten without removing the gastrointestinal tract, microfiber contamination may lead to human exposure. More research is required to adequately assess the risk that microplastics, including microfibers, may pose for food safety and human health.

Development of a high-performance liquid chromatography method to assess bisphenol F levels in milk.

Bisphenol F (BPF) is a bisphenol A (BPA) analogue. As an endocrine disruptor, BPF shows a similar BPA hormonal activity and greater endocrine effects. To assess BPF levels in milk a selective method based on solvent extraction with acetonitrile, solid-phase extraction (SPE), high-performance liquid chromatography with fluorescence detection (HPLC-FD) system, was developed. The method showed high recovery values (from 97.60 to 107.16%), and good detection and quantification limits (LOD=0.03 µg/L; LOQ=0.1 µg/L). To validate the analytical method, quantitative analyses of n.20 milk samples of whole milk were preliminarily carried out applying a monitoring system based on the control of different stages of pasteurized whole milk processing at a dairy company. The proposed method is simple, sensitive, and might be suitable to detect BPF residues in milk processing. At the dairy company, the occurrence of BPF levels ranging from

Occurrence of Microplastics in Commercial Seafood under the Perspective of the Human Food Chain. A Review.

The occurrence of microplastics in the marine ecosystem and aquatic organisms, their trophic transfer along the food web, and the identification of seafood species as suitable indicators have become a research priority. Despite the high quantity of research in this field, a comparison between the available data and an appropriate risk assessment remains difficult. In this perspective, as an innovative approach, the association of the feeding strategies of commercial seafood and the microplastic level was considered. Further research to assess the occurrence of microplastics in the marine food web, the long-term effects on animals and humans, and the health implications is needed.

Hot topic: Bisphenol A in cow milk and dietary exposure at the farm level.

Chemical hazards may enter the milk chain during primary production. The study, for the first time, investigated the occurrence of bisphenol A (BPA) levels in cow milk samples collected on the farm following manual or mechanical milking and from the cooling tank. We applied a new monitoring model based on the identification of the hazards at each stage of the milk chain to identify potential pathways for contamination along the milk chain. We evaluated exposure to BPA through milk consumption based on detected contamination levels and the temporary tolerable daily intake established by the European Food Safety Authority (EFSA). Milk samples (n = 72) were analyzed using liquid chromatography with fluorescence detection. The mean BPA concentrations were 0.757 µg/L in manually milked samples, 0.580 µg/L in mechanically milked samples, and 0.797 µg/L in milk from the cooling tank. Bisphenol A occurred in the milk chain as a result of different stages of milking, and reached the highest levels at the end of the milk chain. Although the dietary intake of BPA was below the EFSA's temporary tolerable daily intake, exposure to BPA, even at low doses, through milk consumption represents a public health concern. Therefore, to ensure milk safety, new monitoring plans should be applied based on the identification of hazards at each stage of the milk chain.

Study on endocrine disruptors levels in raw milk from cow's farms: Risk assessment.

Diet represents the primary route for human exposure to bisphenol A (BPA). As endocrine disruptor (ED), BPA has raised concerns about its adverse effects on human health. Therefore, EFSA recommended a tolerable daily intake (t-TDI) of 4 µg/kg bw/day and the EU Regulation n. 2018/213 fixed a specific migration limit (SML) of 0.05 mg/kg for BPA in food from plastic materials intended to come in contact with food. BPA could be present in milk due to environmental contamination, and also as a result of the migration from contact materials used during milking and storage. Considering the widespread consumption of milk and milk products, the contamination of dairy products is a matter of public health concern. The aim of the study was to investigate the BPA contamination levels of raw cow's milk from two farms located in Campania region, Italy. The milk samples (n=22), weekly collected from the cooling tank, were analyzed using liquid chromatography with fluorescence detection. In raw milk from both farms, preliminary results showed the occurrence of BPA levels lower than the SML limit, ranging from not detected to 2.34 µg/L. The consumer exposure, calculated considering a hypothetical raw milk consumption and three possible scenarios, was below the t-TDI. Despite the low levels of exposure through milk consumption, low doses can have lasting effects during human development. Thus, new approaches, methods, and plans should be applied to monitor ED contamination, such as BPA and other pollutants, and to assure milk safety.

Investigation on bisphenol A levels in human milk and dairy supply chain: A review.

Bisphenol A (BPA), widely used as additive in food packaging, is an environmental and food contaminant that shows a weak estrogenic activity in general population and toxicity in the infant population. A temporary tolerable daily intake (t-TDI) of 4 µg/kg bw/day and a migration limit of 0.6 mg/kg in food from plastic materials, intended to come in contact with food, were fixed. Dietary milk is important in the human diet. The review investigated the contamination levels in human milk and along the dairy supply chain. Despite the reported levels are generally below the fixed limits, breast milk is considered a continuous low-level exposure to endocrine-active compounds for infants. In addition, BPA residues are detected in milk and dairy products posing a risk to human health. BPA enters into milk chain via multiple pathways at various points during milk production (e.g., PVC tubing used during the milking process, transfer from bulk milk to storage tanks, during milk processing). To prevent or mitigate this hazard, a specific TDI for infants is recommended and evaluation of risk factors at each phase of the dairy supply chain, in the quality systems, is recommended.

Screening of Oxalate Degrading Lactic Acid Bacteria of Food Origin.

A screening for oxalate degrading abilities was initially carried on within Lactic Acid Bacteria cultures of different food origin. Seventy-nine strains were drop-inoculated onto MRS agar plates containing calcium oxalate. By comparing colonies diameters, 31 strains were used to inoculate, in parallel, MRS and MRS modified by sodium oxalate addition. Differences in the strains' growth were assessed by colony forming unit counts. For two strains, the growth in oxalate enriched medium was significantly higher; while, for eleven strains an opposite behaviour was recorded. Two strains - probiotic Lactobacillus rhamnosus LbGG and Enterococcus faecalis 59 - were chosen. The first strain appeared to be able to metabolize oxalate more efficiently than the other tested cultures, while strain 59 appeared unable to gather advantage by oxalates and, indeed, appeared to be inhibited by the salt presence in the medium. Outcomes revealed that higher glucose concentrations may favour oxalates utilization. In MRS with oxalate, but without glucose, citrate was completely metabolized. Evaluation along time confirmed that the oxalate degradation is more significant in presence of glucose. Outcomes may represent a good start for the development of a safe and even probiotic culture able to lower the oxalates content of food.

Preliminary Study on Physicochemical and Biochemical Stress Markers at Poultry Slaughterhouse.

Pre-slaughter stress can result in variations in the glycogen storage and metabolic changes of muscle, responsible for quality poultry meat. Aim of this study was to investigate, as pre-slaughter stress markers and quality meat, physicochemical (pH), biochemical (muscle glycogen content), and chemical (super oxides free radicals) parameters. The carcass quality, as incidence of individual carcass defects, was also evaluated. Twenty broilers were processed with two different electrical stunning: high (250 Hz; 640 mA; 60V) (Lot C or control) and low (150 Hz; 360 mA; 60 V) (Lot A) frequency and intensity, using sinusoidal alternating current. As preliminary results, the use of low frequency and intensity induced faster pH decline post mortem and adequate acidification of pH at 3 hours (6.49 Lot C; 6.37 Lot A), better muscle glycogen reserve (0.770 µL/50 mL Lot C; 1.497 µL/50mL Lot A), and lightly more rapid muscle oxidation (IDF: 0.109 Lot C; 0.122 Lot A), (FOX: 0.131 MeqO2/kg Lot C; 0.140 MeqO2/kg Lot A). The incidence of individual carcass defects sufficient to cause downgrading or rejection, both in Lot C and Lot A, was generally low. In a multidisciplinary approach, to assess animal welfare and quality poultry meat, additional and feasible parameters should be implemented. Monitoring of pH, muscle glycogen reserve and superoxide free radical production measurements might be markers easier to use, routinely, in practice at abattoir. Further studies are needed to evaluate the usefulness of these parameters.

Study on the occurrence of polycyclic aromatic hydrocarbons in milk and meat/fish based baby food available in Italy.

The study compared the profile of 14 polycyclic aromatic hydrocarbons, frequently occurred in food, in milk (N = 22) and meat/fish based (N = 18) baby foods available on the Italian market. PAH total levels, markers (Regulation EC/835/2011) and carcinogenic PAHs were determined by high- performance liquid chromatography with fluorescence detector (HPLC-FD). The average of total PAHs was 52.25 µg kg-1 in milk and 11.82 µg kg-1 in meat/fish based baby foods. The levels of PAH markers were higher than the permissible EU limits of 1 µg kg-1 in 18.2% and 77.7% milk, and 5.6% and 44.4% meat/fish based baby foods. Milk based samples showed significant higher values (P < 0.05) of carcinogenic and possible carcinogenic hydrocarbons than meat/fish based products. The Margins of Exposure (MOE) value of milk based baby food samples indicated a potential concern for consumer health. Monitoring programs, and good agriculture and manufacture practices should be recommended.

Evaluation of virulence genes in Yersinia enterocolitica strains using SYBR Green real-time PCR.

Yersinia enterocolitica comprises six biotypes 1A, 1B, 2, 3, 4, and 5. The virulence of the strains belonging to biotypes 1B and 2-5 depends on the presence of both chromosomal and plasmid-borne genes. Strains belonging to biotype 1A do not carry the virulence plasmid pYV. However, they carry other virulence genes, such as ystB and hreP. The aim of this study was to investigate the distribution of yadA, virF, inv, ystA, ystB, myfA, hreP and ymoA virulence genes in Y. enterocolitica strains in order to select the target genes that could be used for the development of a probe-specific real-time PCR to determine the presence of Y. enterocolitica in food samples. A total of 161 Y. enterocolitica strains isolated in eight countries and grouped into biotypes 1A, 2 (serotypes O3, O5 and O9), 3 (serotypes O3 and O9) and 4 (serotype O3) were examined for virulence genes. The most common virulence-associated gene in pathogenic Y. enterocolitica proved to be ystA, which can therefore be considered the best target gene to be amplified in order to evaluate the presence of pathogenic biotypes. By contrast, to identify Y. enterocolitica 1A strains, ystB, which codes for the enterotoxin YstB, can be proposed. This has been found in all non-pathogenic biotypes studied, but never in pathogenic biotypes.

Comparative study on the occurrence of polycyclic aromatic hydrocarbons in breast milk and infant formula and risk assessment.

The study compared the polycyclic aromatic hydrocarbons (PAH) profile of human milk collected from Italian mothers and different brands of infant formula available on Italian market. Levels of 14 PAHs most frequently occurred in food, PAH markers listed by Commission Regulation (EC) No. 1881/2006, and carcinogenic PAHs classified by the International Agency for Research on Cancer, were determined by high-pressure liquid chromatography with fluorescence detector. The average concentrations of total PAHs were 114.93 in breast milk and 53.68 µg kg-1 in infant formula. Furthermore, Benzo(a)pyrene (BaP) and the sum of ∑PAH4 markers (BaP, Chrysene, Benzo(a,h)anthracene and Benzo(b)fluoranthene) were higher than the permissible limit of 1 µg kg-1 in 43% and 86% for breast milk and in 10% and 76% for infant formula samples, respectively. Breast milk showed higher levels (P < 0.05) of carcinogenic, and possible carcinogenic hydrocarbons than infant formula samples. Both in human and commercial milk, data showed the occurrence of low and high molecular weight PAHs, respectively from petrogenic and pyrolytic environmental sources, characterizing the infant and mother exposure. Particularly, waste incineration could have represented an important exposure source for infants during breastfeeding, through exposition of mothers resident in some areas of Southern Italy. High PAH levels detected in infant formula enriched with LC-PUFA might be related to the contamination of the vegetable oils added as ingredients. Results showed a high percentage of samples of both breast milk and infant formulas with margin of exposure (MOE) value indicating a potential concern for consumer health.