Microplastics aggravate the bioaccumulation of three veterinary antibiotics in the thick shell mussel Mytilus coruscus and induce synergistic immunotoxic effects

https://doi.org/10.1016/j.scitotenv.2021.145273Get rights and content

Highlights

  • Immunity of thick shell mussel is hampered by coexposure to OTC, FLO, SMX, and MPs.

  • MP-antibiotic coexposure induces synergic immunotoxic effects on the mussel.

  • Exposure induces alterations in ROS, F-actin disturbance, and cell viability.

  • Exposure downregulates cytoskeleton-, immune-, and detoxification-related genes.

  • Antibiotic accumulation is significantly aggravated by the copresence of MPs.

Abstract

Marine bivalves living in pollution-prone coastal areas may be simultaneously coexposed to veterinary antibiotic residuals and microplastics (MPs). However, the synergistic effects of these two types of emergent pollutants have not been fully elucidated in mussel species. Therefore, the immunotoxic effects of the three representative antibiotics, oxytetracycline (OTC, 270 ng/L), florfenicol (FLO, 42 ng/L), and sulfamethoxazole (SMX, 140 ng/L), with and without the copresence of polystyrene MPs (0.26 mg/L, dimeter: 500 nm), were investigated in the thick shell mussel. Our data showed that the immunity was significantly hampered by exposure to the pollutants and MP-antibiotic coexposure induced synergistic immunotoxicity. For instance, compared to those treated with antibiotics (OTC, FLO, and SMX) alone, mussels coexposed to antibiotic and MPs had significantly lower phagocytic rate (further decline by approximately 28.80%, 34.21%, and 11.22%, respectively) and total hemocyte count (further reduced by approximately 37.45%, 61.67%, and 46.32%, respectively). Exposure to the pollutants tested also led to inductions in intracellular reactive oxygen species (ROS), decreases in the F-actin cytoskeleton, declines in the cell viability of hemocytes, and downregulation of cytoskeleton- and immune-related genes. In addition, mussels coexposed to antibiotic-MP accumulated significantly greater amounts of antibiotics, which may partially explain the synergic immunotoxic effect detected. Exposure to pollutants tested also led to suppression in the activity of glutathione-S-transferase (GST) and downregulation of detoxification-related genes whereas induction in the level of lipid peroxidation (indicated by MDA content) in gills, which may facilitate the entry whereas constrain the exclusion of antibiotics and therefore result in an elevation in accumulation of antibiotics.

Introduction

According to estimates, approximately 4.8 to 12.7 million tons of plastic wastes are released into oceans every year (Thompson et al., 2009; Jambeck et al., 2015; Geyer et al., 2017). Appreciable proportion (approximately 93,000 to 236,000 tons) of these wastes have diameters smaller than 5 mm, which are defined as microplastics (MPs) and are currently ubiquitously detected in aquatic environments (Law and Thompson, 2014; Desforges et al., 2014; van Sebille et al., 2015). Due to their small size, waterborne MPs can be easily ingested by many aquatic species and subsequently induce adverse impacts on a series of physiological processes, such as growth retardation (Besseling et al., 2014), fecundity decline (Besseling et al., 2014; Sussarellu et al., 2016), oxidative damage (Barboza et al., 2018; Zhou et al., 2021), neurotoxicity (Barboza et al., 2018; Lei et al., 2018; Shi et al., 2021), and immunotoxicity (Shi et al., 2020a; Tang et al., 2020a, Tang et al., 2020b).

Meanwhile, both direct application in aquaculture practice and indirect discharge via sewage and surface runoff result in an accumulation of antibiotic residuals in ocean environments, which is regarded as another emergent pollution (Kolpin et al., 2002; Watkinson et al., 2009). For example, according to previous surveys, concentrations of the three most frequently detected veterinary antibiotics, oxytetracycline (OTC), florfenicol (FLO), and sulfamethoxazole (SMX), reach as high as 270 ng/L, 42 ng/L, and 140 ng/L, respectively in the seas of China (Zou et al., 2011; Du et al., 2017). On the one hand, similar to that of MPs, exposure to veterinary antibiotics may exert a series of adverse impacts on marine species, such as causing oxidative stress (Park et al., 2018), constraining metabolism and growth (Martins et al., 2013; Serra-Compte et al., 2019), disrupting behavioral and immune responses (Guardiola et al., 2012; Banni et al., 2015; Bownik et al., 2019), and altering the expression of genes from key molecular pathways (Guardiola et al., 2012; Banni et al., 2015; Zhou et al., 2020a). On the other hand, marine organisms may accumulate waterborne antibiotic residuals into their bodies and therefore aggravate the risk of antibiotic resistance for both the species and for consumers who are exposed to antibiotic-polluted seafood (Yelin and Kishony, 2018; Zhou et al., 2020a).

Owing to their large specific surface areas and high affinity for some pollutants, MPs may act as vectors for some waterborne contaminants, such as heavy metals (Brennecke et al., 2016; Barboza et al., 2018), pharmaceuticals (Shi et al., 2020a), polycyclic aromatic hydrocarbons (Liu et al., 2016; Sun et al., 2020), and veterinary antibiotics (Zhang et al., 2018; Zhou et al., 2020a). In addition, recent findings indicate that exposure of marine organisms to MPs may disrupt the processes of detoxification of these pollutants (Park et al., 2018; Tang et al., 2018; Rocha et al., 2020). Through hitchhiking (or the Trojan horse effect) and inhibiting detoxification, MPs may aggravate the accumulation of these pollutants in marine species and thus affect their toxicites (Paul-Pont et al., 2016; Dawson et al., 2018). Because both MPs and veterinary antibiotics are ubiquitously present in ocean environments, marine species could be simultaneously threatened by these two types of pollutants under realistic scenarios. However, to the best of our knowledge, the synergistic effects of MPs and veterinary antibiotics on marine organisms remain poorly understood to date.

Marine mussels are a family of bivalve species that are widely distributed along coastal areas all over the world (O'Connor, 2002; Li et al., 2016; Shi et al., 2020b; Zhao et al., 2020). By aggregating into beds, mussels create habitats for other marine organisms and are thus recognized as important engineers for marine ecosystems (Borthagaray and Carranza, 2007). In addition to their crucial ecological roles, many marine mussels are also traditional aquaculture species, the production of which reaches more than 85,000 tons according to the Food and Agriculture Organization of the United Nations (FAO, 2019). However, inhabiting pollution-prone coastal areas and possessing a sessile filter feeding lifestyle, marine mussels are more susceptible to waterborne emergent pollutants such as MPs and veterinary antibiotics, highlighting the need to investigate the ecotoxicological impacts of these pollutants (Zhang et al., 2018; Zhou et al., 2021).

Devoid of antigen-antibody mediated immune responses, marine mussels mainly rely on phagocytosis of hemocytes to cope with complicated environmental challenges (Söderhäll, 2010; Wang and Song, 2018). Nevertheless, previous studies suggest that the innate immune response via phagocytosis might be the common target for both MPs and veterinary antibiotics (Guardiola et al., 2012; Zhou et al., 2021). In addition, one case study showed that the accumulation of two veterinary antibiotics, OTC and FLO, was significantly aggravated by MPs in a bivalve species, Tegillarca granosa (Zhou et al., 2020a). Therefore, theoretically, both the add-on effect on common targets and the aggravation of antibiotic accumulation could result in synergic impacts of MPs and veterinary antibiotics on marine mussels. However, this inference still awaits verification with empirical data in mussels.

To fill these knowledge gaps, in the present study, the synergistic effects of MP antibiotics on the phagocytosis and total count (THC) of hemocytes were analyzed in the thick-shell mussel Mytilus coruscus, one of the key aquaculture mussel species in Asian Pacific (Li et al., 2010; Shi et al., 2020b; Zhao et al., 2020). To further reveal the affecting mechanism underpinning the hampered immune response observed, the impacts of MPs and three veterinary antibiotics (OTC, FLO, and SMX) on the intracellular content of reactive oxygen species (ROS), distribution of fibrous actin (F-actin) cytoskeleton, and cell viability were assessed with hemocytes. In addition, the impacts of MPs on the accumulation of the antibiotics tested were examined. The activity of glutathione-S-transferase (GST), which plays a crucial role in detoxification, and the level of lipid peroxidation (an indicator for tissue membrane damage) were estimated as well. Furthermore, the expression of ten genes from the actin skeleton regulation, phagosome, and detoxification pathways was investigated.

Section snippets

Experimental animals and acclimation

Thick shell mussels M. coruscus were collected from Shengsi Island, Zhoushan, China, in July 2019. After collection, mussels were immediately transported to the Qingjiang Research Station of Zhejiang Mariculture Research Institute and acclimated for two weeks in seawater before the experiment. During acclimation, mussels were fed twice daily with live microalgae Platymonas subcordiformis at a rate of approximately 5% of the dry tissue weight, and the seawater was renewed every day. Seawater

Impacts of antibiotics and MPs on phagocytic activity and total count of hemocytes

Exposure of mussel individuals to the three veterinary antibiotics tested led to significant reductions in the phagocytic activity of hemocytes, which were approximately 78.29%, 82.80%, and 76.09% of the control for those exposed to OTC, FLO, and SMX, respectively (Table 3). Similar inhibition of phagocytic activity (approximately declined by 24.90%) was also observed for mussels exposed to MPs (Table 3). In addition, compared to that treated with antibiotics only, mussels coexposed to the

Discussion

Under realistic scenarios, sessile filter feeding bivalve mollusks living in pollution-prone coastal areas may be simultaneously threatened by emergent pollutants such as veterinary antibiotics and MPs (Paul-Pont et al., 2016; Zhou et al., 2020a). However, the synergistic impacts of antibiotics and MPs on the immune responses of marine mussel species remain poorly understood to date. The results obtained in the present study demonstrated that exposure to veterinary antibiotics along with MPs

CRediT authorship contribution statement

Y. Han, W.S Zhou, Y. Tang, and G.X. Liu contributed to experimental design, statistical analysis, and manuscript preparation. Y. Han, W.S Zhou, Y. Tang, and W. Shi carried out the experiments; Y. Han, Y.Q. Shao, P. Ren, J.M. Zhang, and G.Q. Xiao contributed to experiment preparation and data analysis. G.X. Liu, Y. Han, and H.X Sun contributed to substantive discussion of the results and revision of the manuscript. W. Shi, G.X. Liu, Y.Q. Shao, G.Q. Xiao contributed to funding acquisition.

Declaration of competing interest

This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood the journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.

Acknowledgement

This work was funded by the National Key Research & Development Program of China (No. 2018YFD0900603), China Postdoctoral Science Foundation (2020M671743), Science & Technology Project of Wenzhou (Z20170013), Zhejiang Key S&T Project of New Agricultural Varieties (2016C02055-9-2), and Key R&D Program of Zhejiang Province (2018C02039, 2019C02045).

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