Introduction
The aquaculture sector is susceptible to the contamination from microplastics (MP) which mainly derive from environmental pollution, the use of plastic tools, and fish feeds, whether in freshwater or marine farming. Although fish possess the ability to differentiate suspended MP from edible particles, the inherent contamination of aquafeeds presents a less detectable exposure route (Siddique et al., 2023). This results in increased MP ingestion by farmed fish, thereby posing potential health risks to the fish themselves and raising concerns for consumer safety. In fact, dietary MP smaller than 20 µm can be absorbed in the fish intestine, leading to a consequent translocation to other tissues, among which liver acts as a retaining organ (Cattaneo et al., 2024). This limits the accumulation in other sites but causes the triggering of oxidative stress. In this context, the dietary supplementation of natural antioxidants, like astaxanthin (AX), could strengthen the intrinsic defence mechanisms of fish, contributing to mitigate the MP-induced oxidative stress. Particularly, natural AX has a higher antioxidant efficacy compared to its synthetic counterpart. However, natural AX is characterized by a rapid degradation due to its low stability, particularly under industrial processing conditions. To address this limitation, microencapsulation techniques have emerged as promising strategies to enhance the stability of the AX molecule and improve its bioavailability and delivery through aquafeeds. Additionally, the microcapsules’ wall matrix can have a crucial effect in preventing the MP absorption at fish intestinal level. In fact, starch, one of the most frequently preferred components for microcapsules preparation, has been demonstrated to aggregate MP in both suspended water and inside fish gut. Within this context, the present study aimed to: (i) implement fish feeds with natural AX included in microcapsules made of an organic starch-based wall matrix able to preserve it and, at the same time, to exert a crucial role in limiting the MP absorption at fish intestinal level; (ii) testing the experimental diets on European seabass (Dicentrarchus labrax) juveniles over a 60-day feeding trial; (iii) investigating the MP assimilation and accumulation in different fish tissues along with their overall health status.
Materials and Methods
Starting from a control fluorescent MP-free diet (named C), a MP diet, was prepared by adding purchased fluorescent microbeads (range size 1-5 µm) to the control mixture (50 mg/kg feed). Then, the C+AX and MP+AX diets were prepared by adding 7 g/kg feed of AX microcapsules (corresponding to 175 mg/kg of AX) to the C or MP diets, respectively. Five hundred and forty European seabass juveniles were then divided in six experimental group (in triplicate) and were fed as follows: (i) C group: C diet for 60 days; (ii) C/C+AX group: C diet for the first 30 days and C+AX diet for the following 30 days; (iii) C+AX group: C+AX diet for 60 days; (iv) MP group: MP diet for 60 days; (v) MP/MP+AX group: MP diet for the first 30 days and MP+AX diet for the following 30 days; (vi) MP+AX group: MP+AX diet for 60 days. Growth indexes were calculated by weighting all the fish at the end of the trial. MP were localized with confocal microscopy in fish intestine, liver, blood, muscle and adipose tissue, whilst they were quantified on filters through epifluorescence microscopy after digesting the above-mentioned tissues with a KOH solution. Fish welfare was assessed with histological and molecular (real-time PCR on markers involved in immune and oxidative stress response) analysis on liver and intestine samples. Finally, to assess their potential role in MP aggregation, whole microcapsules (empty or filled with AX), and starch (as main microcapsules’ component) were tested in an in vitro experiment.
Results and Discussion
The different dietary treatments did not affect fish survival rate and growth performance and did not cause pathological alterations or signs of inflammation in the intestine as evidenced by both the histological indexes and the relative expression of immune response markers (il1b, il10, and tnfa). However, the dietary fluorescent MP microbeads were absorbed at intestinal level, leading to a consequent translocation and accumulation in the liver which caused, in fish from the MP group, a sod1, sod2, and cat upregulation. Interestingly, the dietary implementation of AX microcapsules led to a mitigation in the expression of oxidative stress markers, particularly in the MP+AX group where it reached levels comparable to those observed in all the controls. Additionally, the microcapsules used in the present study played a significant role in limiting the intestinal MP absorption. In fact, fish from MP+AX group revealed a significantly lower intestinal absorption of fluorescent microbeads, leading to a consequently reduced accumulation in all the organs analysed compared to those from MP group. Of particular interest was the absence in the muscular tissue. These results were supported by: (i) an in vitro coagulation test which demonstrated that the microcapsules (empty or filled with AX) and the starch, tested at the same concentration used in the experimental diets, effectively trapped the fluorescent microbeads, forming MP aggregates too large to be absorbed at the intestinal level; (ii) the number of fluorescent microbeads detected in the fish faeces samples which was significantly higher in both the MP/MP+AX and MP+AX groups compared to the MP one. These results can be of great interest for the aquaculture sector in light to promote fish welfare and quality product. Further studies are necessary to depict a more comprehensive overview of the effectiveness of this technology in relation to the wide variability of conditions within the aquaculture sector and the types of polymers that can be ingested by the fish.
References
Cattaneo, N.; Zarantoniello, M.; Conti, F.; Tavano, A.; Frontini, A.; Sener, I.; Cardinaletti, G.; Olivotto, I. Natural-based solutions to mitigate dietary microplastics side effects in fish. Chemosphere 2024, 367, 143587.
Siddique, M.A.M.; Tahsin, T.; Hossain, I.; Hossain, M.S.; Shazada, N.E. Microplastic contamination in commercial fish feeds: A major concern for sustainable aquaculture from a developing country. Ecotoxicol. Environ. Saf. 2023, 267, 115659.