Abstract
Dichlofluanid has long been employed as a fungicide in agriculture and has been massively introduced in antifouling paints for boat hulls over the last two decades. One of the most important toxic effects of antifoulants is represented by immunosuppression in marine invertebrates, which can be analysed in vitro with a number of short-term toxicity assays on haemocytes. Among bioindicators, the colonial ascidian Botryllus schlosseri is a useful candidate; it is a filter-feeding organism living in the water-sediment interface that is found worldwide and is sensitive to antifouling xenobiotics. Dichlofluanid adversely affects both immunocyte lines (phagocyte and cytotoxic lines) after exposure to sublethal concentrations. At 0.05 μM (16.65 μg/L), dichlofluanid induced haemocyte apoptosis and cell shrinkage with a decrease in both motility and phagocytosis. At the lowest concentration (0.01 μM, 3.33 μg/L), inhibition of pivotal enzymatic activities of phagocytes and cytotoxic cells occurred. At the highest concentration (0.1 μM, 33.3 μg/L), dichlofluanid increased glutathione oxidation, leading to stress conditions. The effects of dichlofluanid on immune defence responses are similar to those of organometal-based antifoulants (i.e., organotin compounds and zinc pyrithione), and its use in coastal areas requires attention.
Highlights
Fouling consists of a community of organisms that have settled and grown on natural and artificial hard surfaces submerged for a long period of time in aquatic environments [1,2,3,4]
After adhesion to the coverslips, the haemocyte monolayers were incubated with dichlofluanid, and after washing thoroughly with filtered seawater (FSW), were immediately observed with a light microscope (LM) to determine the number of living haemocytes able to adhere firmly even after exposure to the contaminant and compared with the cells counted in absence of contaminant and adhering on poly-l-lysine-coated (50 μg/mL, Sigma, Hong Kong, China) slides used as reference controls (100% adhesion)
The haemocytes were fixed in a solution of 1% glutaraldehyde plus 1% sucrose in FSW at 4 ◦C for 30 min, washed for 10 min with 0.1 M phosphate-buffered saline (PBS: NaCl 8 g/L, KCl 0.2 g/L, KH2PO4 0.2 g/L, Na2HPO4 1.15 g/L, pH 7.2), stained with a 10% aqueous solution of Giemsa dye (Fluka) for 5 min and mounted with Acquovitrex (Carlo Erba) on glass slides for observation with a LM to count the percentage of cells with an amoeboid shape on the total number of cells
Summary
Fouling consists of a community of organisms that have settled and grown on natural and artificial hard surfaces submerged for a long period of time in aquatic environments [1,2,3,4]. Anti-mould and anti-dandruff compounds from the polymer (chlorothalonil), leather (TCMS pyridine) and cosmetic-pharmaceutical (zinc pyrithione) industries, respectively [8] These antifouling agents are less effective in comparison to organotin compounds—the average duration of these antifouling paints on boats is 2 years compared to 5 for the TBT-based paints—many of these compounds are of environmental concern for their potential widespread toxicity from different sources. Its estimated bioconcentration factor (BCF) of 72 (±14) in fish (Lepomis macrochirus) and its high octanol/water partition coefficient (3.7 at pH 7.0) suggest that dichlofluanid tends to associate with particulate matter [18], forming strong bonds with clay sediments rather than sandy sediments [19], and the adsorption increases as the pH increases with a releasing factor from the polluted sediments corresponding to less than 1% [20] For these reasons, it may moderately bioconcentrate in aquatic organisms [21]. Effects on two immunocyte cell lines present in the haemolymph, represented by cytotoxic cells and phagocytes [41], were described through the analysis of results (toxicity indexes) from a series of functional assays considering the lowest observed effect concentration (LOEC) values as endpoints, i.e., the lowest concentrations of a substance that had a statistically significant adverse effect
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