Investigation Of The Effect Of Typical Biocides On The Ocular Development Of Zebrafish

Biocides are one of the members of emerging pollutants in recent years.Because of the incomplete removal in wastewater treatment plants,various biocides are directly discharged into the receiving water after applications,thereby posing risks to aquatic organisms such as fish.Most of the existing research focuses on developmental toxicity,neurotoxicity,and reproductive toxicity;however,the study regarding ocular toxicity is quite limited.As a commonly used model animal,zebrafish has a similar eye structure to humans,and its eye development is rapid,making zebrafish become the best choice for studying the effects of environmental pollutants on the visual system.Two biocides,triclocarban and triclosan,are considered endocrine disruptors with thyroid receptor antagonistic activities.In addition,methyl triclosan,as the metabolite of triclosan,has been reported to have the potential to affect thyroid hormone signaling.Given the reports about the regulation of thyroid hormone signaling on retinal cone subtype specifications,we hypothesize that these pollutants may impair visual function,which is critical to wildlife.Therefore,this study took zebrafish as an aquatic biological model and investigated the effects of triclocarban,triclosan,and methyl triclosan on the visual function of zebrafish from phenotype to mechanism.After144 hours of exposure,changes in the retinal structure(e.g.,retinal layer thickness and cell density),visually mediated behavior,opsin gene expression,and protein immunostaining of zebrafish larvae exposed to the target pollutants at the environmentally relevant concentrations and the effective concentrations.The main conclusions of this study are as follows:(1)Triclocarban,triclosan,and methyl triclosan decreased eye size of larval zebrafish at environmentally relevant concentrations and reduced retinal ganglion cell(RGC)density in ganglion cell layer(GCL)based on histopathological analysis.Triclocarban exposure led to a drop in thickness of the outer nuclear layer(ONL),lens,and retinal pigment epithelial layer(RPE)of zebrafish larvae in the high concentration group.Besides,exposure to all concentration groups of triclosan and methyl triclosan resulted in thinner thickness of the ONL and GCL.These results show that exposure to typical biocides disrupted the normal retinal development of zebrafish larvae,including GCL involved in signal transmission and ONL and RPE involved in light sensitivity.(2)The optokinetic response(OKR)and phototactic behavior(PTR)of zebrafish larvae exposed to the target biocides were significantly reduced.After exposure to triclocarban and triclosan,larval zebrafish showed unresponsive to green,red,and white light stimuli and performed normally on blue light.In contrast,a reduction in the visual behavioral response of zebrafish exposed to methyl triclosan under the stimulation of all color light was observed.Combined with histopathological analysis,decreased sensitivity of visually mediated behavior in zebrafish larvae could be related to a decrease in outer nuclear layer thickness.And at the same time,the difference in the sensitivity of the target biocides to different colors light stimuli indicates that changes in the differentiation of cone cells sensitive to different colors of light lead to abnormal responses to green and red light stimuli in zebrafish exposed to triclocarban and triclosan.decreased sensitivity of visually mediated behavior in zebrafish larvae could be related to decreased RGC density.(3)The target biocides downregulated the transcriptional levels of some key opsin genes.Triclocarban downregulated the expression of opn1sw2,opn1mw1,opn1mw3,opn1lw1,opn1lw2 and rho genes,and decreased the immunofluorescence intensity of rhodopsin in the high concentration group.Triclosan-exposed zebrafish larvae showed a downregulation of the transcriptional levels of opn1mw1,opn1mw4,opn1lw1,opn1lw2 and rho genes,and the fluorescence intensity of rhodopsin.Exposure to methyl triclosan at the high concentrations caused a decrease in the expression of opn1sw2,opn1mw1,opn1mw3,opn1lw1 and rho genes,and the fluorescence intensity of rhodopsin was reduced in larval zebrafish exposed to methyl triclosan at the environmentally relevant concentrations.Based on the behavioral and opsin gene expression analyses,the target biocides both affect the rhodopsin expression in zebrafish's rod cells,probably leading to poor performance in the phototactic behavior.It should be noted that triclocarban and triclosan may tend to impact the green-and red-sensitive opsin genes,while exposure to methyl triclosan disrupted all opsin gene expression in zebrafish larvae.Downregulated essential opsin genes and rhodopsin expression levels could be related to the thinner thickness of ONL.These findings provide evidence to support our hypothesis that triclocarban,triclosan,and methyl triclosan impair the visual system of zebrafish.Therefore,we urgently call for more in-depth exploration of the possibility of other biocides-induced ocular toxicity to aquatic organisms and even humans.We also highlight the pollutant-triggered visual lesion,in which actions should be taken to fill the data gaps for existing environmental pollutants.Lastly,the visual toxic effects of triclocarban and triclosan are a warning to human vision because they can be readily in contact with humans as the active ingredient in personal care products.