The effect of acute exposure of four disinfectants commonly used in aquacultural practice (formalin, potassium permanganate, benzalkonium chloride and malachite green) was studied on the histological structure of adult zebrafish (Danio rerio) gills. Groups of 8 individuals were exposed to a dose of each disinfectant corresponding to the therapeutic dose (TD) and five folds of the therapeutic dose (5xTD). Gills of all exposed zebrafish showed a higher occurrence of histopathological changes. These alterations included a slightly focal proliferation of interlamellar cells with obliteration of interlamellar spaces, mild infiammatory reaction with leucocyte infiltration and lifting of the epithelial layer from gill lamellae. Fish exposed to potassium permanganate showed more severe histopathological changes consisting of necrotic change of lamellar cells, distorsion and apical necrosis of secondary lamellae

Abramo, Francesca

Braca, G

Gregori, M

Intorre, Luigi

Pretti, Carlo

English

Gregori M

PRETTI, CARLO

INTORRE, LUIGI

Braca G

ABRAMO, FRANCESCA

Archivio della Ricerca - Università di Pisa

Bull. Eur. Ass. Fish Pathol., 27(5) 2007,  185Gill histopathology in zebrafish modelfollowing exposure to aquaculturaldisinfectantsM. Gregori1*, C. Pretti1, L. Intorre2, G. Braca1 and F. Abramo11 Department of Animal Pathology, University of Pisa, Pisa, Italy; 2 Section of Pharmacology andToxicology, Department of Veterinary Clinics, University of Pisa, Pisa, Italy.AbstractThe effect of acute exposure of four disinfectants commonly used in aquacultural practice(formalin, potassium permanganate, benzalkonium chloride and malachite green) was studiedon the histological structure of adult zebrafish (Danio rerio) gills. Groups of 8 individuals wereexposed to a dose of each disinfectant corresponding to the therapeutic dose (TD) and five foldsof the therapeutic dose (5xTD). Gills of all exposed zebrafish showed a higher occurrence ofhistopathological changes. These alterations included a slightly focal proliferation of interlamellarcells with obliteration of interlamellar spaces, mild infiammatory reaction with leucocyteinfiltration and lifting of the epithelial layer from gill lamellae. Fish exposed to potassiumpermanganate showed more severe histopathological changes consisting of necrotic change oflamellar cells, distorsion and apical necrosis of secondary lamellae.* Corresponding author’s E-mail: mgregori@vet.unipi.itIntroductionThe use of pharmaceutical substances islimited in fish, in contrast to mammaliantherapeutics. It is basically restricted toanesthetic and anti-infective agents forparasitic and microbial diseases. Disinfectantsare compounds that reduce pathogenicorganisms on biological surfaces. Idealcharacteristics of a disinfectant include abroad spectrum, fast action and low toxicity(Heit & Riviere, 1995). The most widely useddisinfectants are iodophores, salts (potassiumpermanganate), organic chlorocompounds,aldheydes (formalin), hydrogen peroxide,quaternary ammonium compounds (benzal-konium chloride) and antiseptic dyes(malachite green) (Burka et al., 1997). Ingeneral, disinfectants are non-selective in theirsite of action, and lack selective toxicity.Furthermore, doses currently used to treat petfish are, in most cases, based on empirical andanecdotal information, rather than onpharmacological and clinical data, with theresult that fish toxicity may occur even atrecommended dosages, particularly whenfish of different species are treated at the sametime (Stoskopf, 1988). Toxicity tests may beuseful in order to evaluate the effects ofxenobiotics at the organism level (Bernet etal., 1999) and histopathology could be usedas an instrument or endpoint in toxicitystudies on fish (Wester et al., 2002).In this study we have investigated the gilltoxicity of four disinfectants which are widelyused in aquaculture, using zebrafish (DanioBull. Eur. Ass. Fish Pathol., 27(5) 2007, 186rerio) as test species. The four disinfectantsexamined in this study include: formalin (F),potassium permanganate (PM), benzal-konium chloride (BC) and malachite green(MG).Materials and methodsFishAdult male zebrafish, 3.5±0.6 cm lenght,0.60±0.2 g body-weight were purchased fromErreCI (Pisa, Italy). They were acclimated in50 L glass aquaria containing dechlorinatedtap water (temperature 23±1°C; pH 7.8) for atleast 1 week before the experiment and fedon commercial pellets. Fish were kept undernormal laboratory illumination with a 12hlight/dark cycle. Procedures for the care andmanagement of animals were performed inaccordance with the provisions of the ECCouncil Directive 86/609 EEC, recognised andadopted by the Italian Government (DL27.01.1992, n° 116).ChemicalsF, BC, MG and PM were purchased (SigmaAldrich, Milan, Italy).Toxicity tests, exposure and mortalityrecordsThe tolerance of fish to each disinfectant wasassessed after fish were exposed to therecommended therapeutic dose (TD) and fivetimes the recommended dose (5xTD). Thechoice of each disinfectant TD and theduration of exposure were derived from theanalysis of literature (Burka et al., 1997;Southgate, 1993). The exposure times wereperformed as follows: BC 1h, F 1h, MG 0.5h,PM 1h. Experimental groups also includeduntreated control fish, maintained in rearingwater up to five folds of each disinfectant timeexposure (BC 5h, F 5h, MG 2.5h, PM 5h) (table1).Fish were divided into nine groups with eightfish each (8 test groups and one untreatedcontrol) and kept in 20 L recirculating systemaquaria. Then they were transferred into testaquarium (aerated to restore theconcentration of dissolved oxygen to at least90% of its air saturation value) where theywere subjected to static bath exposure of eachof the 4 disinfectants at the TD and 5xTD. Acalculated volume of each disinfectant wasadded to each test aquarium based on itsvolume of water. Before addition, eachdisinfectant was diluted in 1 L of waterremoved from the test aquarium and thenmixed throughout the test aquarium. Thecontrol group was exposed to untreated water.At the end of treatment, fish were thenreturned to untreated aquaria and monitoredfor up to 96 hours.Table 1. Testing concentrations of the disinfectants employed in the test.* TD = Therapeutic dose.Group Benzalkonium chloride Formalin Malachite green Potassium permanganate  Dose (ppm) Time of  exposure Dose (ppm) Time of  exposure Dose (ppm) Time of exposure  Dose (ppm) Time of exposure TD 2.0 1 h 250 1 h 2.0 0.5 h 5 1 h 5 x TD* 10.0 1 h 1.250 1 h 10.0 0.5 h 25 1 h Bull. Eur. Ass. Fish Pathol., 27(5) 2007,  187Histopathological procedures andassessmentAll exposed fish were collected immediatelyafter death.  The other fish were sacrificied atthe end of the 96 hours. All fish were fixed ina 10% buffered formalin solution (pH 7.4) for24 hours and routinely processed. Wholebody para-sagittal sections were prepared.Gill morphological alterations were classifiedinto one of these main categories: proliferative(increase of the number of specific cell types),degenerative (breakdown of tissue and/orcells), inflammatory (increased presence ofcells in tissue repair; response to damagedFigure 1. Gill histological sections of adult zebrafish; (A) Fish exposed to 1250 ppm F for 1 h. Note a slightproliferation of lamellar and interlamellar epithelium and epithelial lifting; (B) Fish exposed to 25 ppm PMfor 1h. Severe degeneration and necrosis of the epithelial cells; (C) Fish exposed to 10 ppm MG for 0.5 h.Separation of secondary lamellar epithelium from the pillar cell (lamellar edema); (D) Fish exposed to 10ppm BC for 1 h. Infiltration of inflammatory cells between the layers of the lamellar epithelium and epitheliallifting; (E) Normal gill structure from control zebrafish (H&E stain, scale bar = 50 μm).Bull. Eur. Ass. Fish Pathol., 27(5) 2007, 188tissue) and structural (changes in tissuearchitecture), each representing a generaltissue response to a particular stressor.The degree of histopathological alterationswas qualitatively and semi-quantitativelyevaluated for each individual fish, byassessing a score to tissue lesion severity andcomparing it to control sections. Ranking wasas follows: - = no pathological alterations; + =mild to moderate focal changes; ++ = severepathological alterations. The cumulative effectof disinfectants exposure to histopathologicalgill alterations was then assessed.Figure 2. Semiquantitative evaluation (score assessment) of histopathological alterations in the gills ofzebrafish exposed to each disinfectant, at TD and 5xTD.Bull. Eur. Ass. Fish Pathol., 27(5) 2007,  189ResultsHistopathological cumulative findingsControlsIn histological sections obtained from allzebrafish from the control group each gill wasmade up of filaments of primary lamellae.Secondary gill lamellae, originating from thefilaments in double rows, were disposedperpendicularly to each filament and werecovered by a single layer of normotipicflattened respiratory epithelium (figure 1E).F- exposed fishHistopathological changes in the gills werecharacterized primarily by epithelial cellproliferation at the base of the secondarylamellae resulting in obliteration of theinterlamellar space and, in some cases, infusion of secondary lamellae. Therefore themajor point of interest in F-exposed fish wasthe hyperplasia of the respiratory epithelium(figure 1A). Inflammatory alterations, such aslamellar edema, appeared as a separation ofthe respiratory epithelium from the pilar cellsin the secondary lamellae.PM-exposed fishThe predominant findings consisted ofdegenerative and necrotic changes ofrespiratory epithelium and pilar cells (figure1B) and focal obliteration of the interlamellarspaces by an inflammatory exudatecontaining necrotic material. The majority ofexposed fish showed numerous gill archfilaments that were disorganized and in mostcases secondary lamellae exhibited distorsion,apical necrosis and fusion. Inflammatoryalterations consisted mainly of a lifting of thegill epithelium (epithelial lifting), and aninfiltration of leukocytes and lymphocytes inthe secondary lamellae.MG- and BC- exposed fishThe lesions observed in the gills of the MG-and BC- exposed animals exibithed similarhistopathological features. Hyperplasia ofinterlamellar cells could be observed in a fewanimals. Multifocal degenerative and necroticareas, which involved the epithelial cells bothat the tip and along the edge of secondarylamellae, were also observed. The mostcommon change, which was observed inalmost all cases, was the lifting of theepithelium layer from gill lamellae due tolamellar oedema (figure 1C). Inflammatoryinfiltrate, which was represented by cellslocated in the region between the pilar cellsand the outer layer of lamellar epithelium,was a finding in both groups (figure 1D).The overall score assessment to tissue lesionseverity for TD and 5xTD exposure is reportedin Figure 2. Fish exposed to the 5xTD showeda higher level of histopathological alterationwhen compared to the TD exposed ones.DiscussionZebrafish, a cyprinid, is a relevant fish speciesfor ornamental aquaculture and a model fortoxicity tests (Spitsbergen & Kent, 2003). Inthe present study gill alterations followingexposure to F, BC, PM and MG werehistologically evaluated on test-zebrafish. Thetolerance of F, PM, MG and BC exposure forzebrafish have been previously evaluated byMeucci et al., (2005). The authors graded thetoxicity of disinfectants for zebrafish as MG <BC < F< PM.The range of the structural alterations of thegill lamellae observed included lifting oflamellar epithelium, swelling and fusion ofsecondary lamellae, hyperplasia of lamellarBull. Eur. Ass. Fish Pathol., 27(5) 2007, 190and interlamellar cells, necrosis andinflammation. All the categories of alterationswere present, with different scores, in all deadand killed fish after disinfectant exposureboth at the TD and 5xTD. The higher severityof histopathological alterations detected inthe group of fish exposed to the 5xTDcorrelated well with the mortality records.Moreover, the presence of histopathologicalalterations in the TD exposed fish which didnot show mortality in a 96 h period underlinethe relevance of histopathological assessmentto detect sublethal alterations.F-exposed fish showed predominantlyproliferative alterations, while PM-exposedfish exhibited necrotic changes. Gillpathological features such as inflammatoryalterations and hyperplasia of interlamellarepithelium were similar in MG and BCexposed fish.The observed proliferative changes mightrepresent a physiological adaptative responsewhich reduces the vulnerable surface area ofthe gills rather than a direct effect of toxicants.In addition to this, hyperplasia of the lamellarepithelium, as well as epithelial lifting,increases the distance across whichwaterborne irritants must diffuse to reach thebloodstream. These changes may beadvantageous for fish by reducing toxicantentry, but the efficiency of gas exchange willbe severely impacted (Hemalatha & Banerjee,1997). Despite the widespread use of F, fishfarmers and fish health professionals areconcerned about its potential to damagebranchial architecture (Speare et al., 1997).Previous reports (Rucker, 1962) recorded thateven a single F treatment of juvenile rainbowtrout, Onchorhynchus mykiss, at recommendeddose levels, was seriously damaging to the gillof some fish. Our study in zebrafish exposedto F demonstrates gill alterations, whetherthese are non-specific response pattern ordirectly related to the toxicant exposure is stillto be investigated.Necrotic changes were the predominantlesions observed in the PM exposed fish. Thesefindings are in accordance with data reportingthat PM was the least “safe” disinfectant forzebrafish (Meucci et al., 2005). Necrotic lesionsare believed to reflect the direct deleteriouseffects of toxicant, they are generallyirreversible, and their persistence andprogression may lead to a partial or total lossof organ function (Nero et al., 2005). Since thepermanganate ion MnO42¯ is a strong oxidant(Tucker & Boyd, 1977), the mechanism oftoxicity may be mediated through oxidativestress.Different patterns of histopathologicalalterations, including cell necrosis, weredetected in BC- and MG-groups, howeverthese were less severe. Gill lesions havepreviously been described in healthy rainbowtrout after BC-exposure at a dose minimallyabove recommended treatment levels (Byrneet al., 1989) and following repeated exposuresto MG (Gerundo et al., 1991), but histologicallesions have not been documented.Inflammatory alterations, such as lamellaredema and inflammatory infiltrate, werefound in almost all exposed fish and the liftingof the secondary lamellar epithelium likelycontributes to changes in gill interlamellardistance. The amount of inflammatoryinfiltrate mainly represented by neutrophilswas generally mild and focal. InfiltratingBull. Eur. Ass. Fish Pathol., 27(5) 2007,  191leukocytes may release cytokines which inturn can activate mononuclear phagocytes anddirectly induce synthesis of the enzymes thatmediate the respiratory burst.In conclusion, our study demonstrates gillhistopathological alterations in fish exposedto F, BC, PM and MG, with distinct responsepattern identified in the F and PM groups andwith different level of severity based on thedosage of the disinfectant used. Furtherinvestigations are needed to understand therole of each disinfectant in the pathogenesisof both specific and aspecific responsepatterns.ReferencesBurka JF, Hammell KL, Horsberg TE, JohnsonGR, Rainnie DJ and Speare DJ (1997). Drugsin salmonid aquaculture- A review. Journal ofveterinary pharmacology and therapeutics 20, 333-349.Byrne P, Speare DJ and Ferguson HW (1989).The effects of a cationic detergent on gills andblood chemistry of rainbow trout (Salmogairdneri L). Diseases of aquatic organisms 6,185-196.Gerundo N, Alderman DJ, Clifton-Hadley RSand Feist SW (1991). Pathological effects ofrepeated doses of malachite green: apreliminary study. Journal of fish diseases 14,521-532.Heit MC and Riviere JE (1995). Antiseptic anddisinfectants. In “Veterinary Pharmacologyand Therapeutics” (H.R. Adams Ed.), pp. 741-752. Iowa State University Press, Ames, IA.Hemalatha S and Banerjee TK (1997).Histopathological analysis of sublethaltoxicity of zinc chloride to the respiratoryorgans of the airbraeathing catfishHeteropneuster fossilis. Biological research 30,11-21.Meucci V, Pretti C, Di Bello D, CognettiVarriale A, Soldani G and Intorre L (2005).Acute toxicity of disinfectants to ornamentalfish. Toxicology letters 158, 254-255.Nero V, Farwell A, Lee LEJ, Van Meer T,MacKinnon MD and Dixon DG (2005). Theeffects of salinity on naphtenic acid toxicityto yellow perch: gill and liver histopathology.Ecotoxicology and environmental safety 63, 365-377.Rucker RR (1962). Progress in sport fisheryresearch. US Bureau of Sport Fisheries andWildlife Circular 160, 13.Southgate P (1993). Disease in aquaculture.In “Aquaculture for Veterinarians: FishHusbandry and Medicine” (L. Brown, Ed.),pp 91-129. Pergamon, New York.Speare DJ, Arsenault G, MacNair N andPowell MD (1997). Branchial lesionsassociated with intermittent formalin bathtreatment of Atlantic salmon, Salmo salar L.,and rainbow trout, Onchorynchus Mykiss(walbaum). Journal of fish diseases 20, 27-33.Spitsbergen JM and Kent ML (2003). The stateof the art of the zebrafish model for toxicologyand toxicologic pathology research.Advantages and current limitations.Toxicologic pathology 31, 62-87.Stoskopf MK (1988). Fish Chemoterapeutics.The Veterinary clinics of North America. Smallanimal practice 18, 329-347.Tucker CS and Boyd CE (1981). Relationshipbetween potassium permanganate treatmentand water quality. Transactions of the americanfisheries society 106, 481-488.Wester PW, van der Ven LTM, Vethaak AD,Grinwis GCM and Vos JV (2002). Aquatictoxicology: opportunities for enhancementthrough histopathology. Environmentaltoxicology and pharmacology 11, 289-295.

Gill histopathology in zebrafish model following exposure to aquacultural disinfectants

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Gill histopathology in zebrafish model following exposure to aquacultural disinfectants

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