Introduction
Praziquantel (PZQ) is a synthetic broad-spectrum anthelmintic, widely used in veterinary and human helminthiasis. In farmed fish, PZQ has been demonstrated to be highly effective against a range of internal and external parasites. Thus, PZQ constitutes a good candidate substance based on its proven efficacy against flatworms such as Sparicotyle chrysophrii, a microcotylid blood-sucking monogenean, which is currently the most serious pathogen for gilthead sea bream (Sitjà-Bobadilla et al., 2010, Rigos et al., 2021). PZQ is generally considered safe in treated animals (Norbury et al., 2022). Little work has been done examining the effects of PZQ in the surrounding marine environment, albeit assessment of the effects on non-target organisms is one of the most important components of environmental risk assessment. Therefore, the objective of this study is the evaluation of the toxicity of PZQ on cell culture and selected non-target marine organisms.
Materials and methods
Amphibalanus amphitrite toxicity assay: The barnacle-rearing method was carried out according to Kotsiri et al. (2018). Assays were conducted by adding 10 nauplii into individual wells of a 24-well plate with 2 ml of artificial seawater (25‰) and various concentrations of PZQ. Nauplii have incubated for 48 h in the presence of PZQ at 25°C and the plates were examined after 24 and 48 h. Each animal was inspected under a stereomicroscope and its condition was recorded. The LC50 was determined as the concentration of PZQ that resulted in 50% mortality of the nauplii.
Marine bacteria growth assay: The ability of PZQ to inhibit the growth of the bacteria Vibrio. alginolyticus was assessed by the disk diffusion test. Sterile filter paper discs (4 mm) were loaded with samples of serial dilutions of PZQ, allowed to dry at room temperature and then were placed on agar plates, which were seeded with the strain of bacteria. Plates were incubated for 24 h at 35℃. Marine bacteria growth inhibition was determined by measuring the zone of inhibition in mm around the filter paper disc. As a positive and negative control, standard discs were loaded with 0.5 M penicillin G or solvent, respectively.
Cytotoxicity assays: The cellular toxicity of PZQ was evaluated in epithelial cell line cultures, RTgill-W1. The cytotoxicity of PZQ was evaluated in 96 well plate cultures applying the neutral red (NR) assay to assess the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. In a flat bottom plate, 5×104 cells ml-1 were placed and incubated at 19℃ for 24 h until adherence. Next, the medium was changed and PZQ was added to the culture in different concentrations. After 24 h, NR medium was added, the plate was incubated, washed, the dye was extracted and the absorbance was measured at 540 nm.
Statistical analysis: For statistical comparisons between groups, one-way ANOVA followed by Dunnett’s multiple comparisons test at P<0.05 was used where appropriate using GraphPad Prism v.9. For LC50 all data were fitted to a three-parameter logistic curve according to the following model: (Y=Bottom + (Top – Bottom)/(1+10^((X – LogIC50)).
Results & Discussion
Based on ecotoxicity tests against A. amphitrite (24 h: LC50= 47.5 mg/L), also confirmed by marine bacteria growth assays, PZQ induced no effect on the growth of the tested bacterial strain, the toxicity of PZQ was found to be low (Fig. 1a, c). This conclusion is in agreement with previous findings in acute and long-term experiments (Cioli et al., 2003; Norbury et al., 2022). Also, the results indicated that very low and low concentrations (≤ 1 mg/L) of PZQ induced no effect on RTgill-W1 cells viability after 24 h of exposure, while medium and high concentrations of PZQ exerted cytotoxic effects (Fig. 1b).
While the environmental concentrations are likely dependent upon dose and stocking density, there is no evidence that high concentrations of PZQ are being discharged into the environment after a treatment regimen. Data from a previous study have shown that PZQ was detected in very small concentrations (0.003 mg/L) in water during PZQ administration and was not detected in the sediment (Ido et al., 2019).
In agreement with existing limited knowledge, this trial suggests that PZQ use in aquaculture is relatively safe for the treated animal and the environment. Nevertheless, there is a need to investigate the fate of this anthelminthic in aquaculture systems in order to more accurately assess concerns about ecological impacts.
Acknowledgements
The project is co-funded by Greece and the European Union under the Fisheries and Maritime Operational Program 2014-2020 (75% EMFF contribution, 25% National Contribution). website: https://praziquantel.gr/
References
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