Evaluation of dicloran phototoxicity using primary cardiomyocyte culture from Crassostrea virginica

Abstract

Dicloran is a commonly used fungicide throughout the Southern and Western United States. Runoff of dicloran from agriculture systems to nearby waterbodies can accumulate in the organisms that inhabit those areas. Although severe damage of dicloran to ecological systems has not been reported, its toxicity has been modified by photodegradation. The objective of this study is to assess the changes of dicloran toxicities during photo exposure using a reliable in vitro biological model. In the present investigation, the photodegradation of dicloran in vitro showed over 90% of dicloran was degraded within 24h of UV exposure in water. Two major intermediate degradation products, 2-chloro-1,4-benzoquinone (CBQ) and 1,4-benzoquinone (BQ), were detected upon UV exposure of dicloran; however, they were rapidly degraded via photolysis. To estimate the impact of the phototoxicity of dicloran to aquatic organisms, we developed an in vitro cell culture system using the C. virginica cardiomyoctes (CvCMs) which were isolated from heart tissues and formed beating cell clusters. The CvCM clusters were treated with irradiated dicloran or the two intermediate standards, CBQ and BQ, and they showed up to 41% decrease in beating rates compared to control cell clusters. Expression levels of selected genes: def, hsp70, and cam, were upregulated in response to stimulations of UV irradiated dicloran and the two standard intermediates. The four-hour irradiated dicloran also resulted in more significant inhibition in the proliferation and small cardioactive peptide β production of CvCMs than other treatment. Tested solutions of photolyzed dicloran showed elevated toxicities opposed to the standard intermediates, CBQ and BQ, suggesting additive toxicity of these dicloran products or toxicity due to other unidentified degradation products. Results of this study supported our hypothesis that the degradation of dicloran caused by photo irradiation results in an elevated toxicity which can be evaluated by the in vitro CvCM model.