Abstract

Triclosan (TCS) is an antimicrobial and antimycotic agent widely used in personal care products. In aquatic environments, both TCS and its biomethylated more persistent form, methyl-triclosan (MeTCS), are usually detected in wastewater effluents and rivers, where are commonly adsorbed to suspended solids and sediments. The aim of this study was to evaluate biochemical and physiological effects in Danio rerio after a short term (2 days) and prolonged (21 days) exposures to sediment spiked with TCS acting as the source of the pollutant in the assay. The activities of catalase (CAT), glutathione-s transferase (GST) and superoxide dismutase (SOD), lipid peroxidation levels (LPO), total capacity against peroxyl radicals (ACAP), and acetylcholinesterase enzymatic activity (AChE) were measured in liver, gills, and brain. Most of TCS on the spiked sediment was biotransformed to MeTCS and promoted different adverse effects on D. rerio. Gills were the most sensitive organ after 2 day-exposure, showing lipid damage and increased SOD activity. After 21 days of exposure, liver was the most sensitive organ, showing lower ACAP, increased LPO levels, and SOD and CAT activities. This is the first study reporting the effects on biochemical markers in D. rerio from a MeTCS sink resulting from sediment spiked with TCS.

Key words
Methyl-triclosan; multivariate analysis; oxidative stress; whole sediment test; zebrafish

INTRODUCTION

Personal care products (PCPs) are one of the main emerging urban pollutants that were proved to have a negative impact on the environment. Their increasing production, because of their wide spectra of applications, inevitably leads to their uncontrolled release into aquatic ecosystems. Moreover, the lack of efficient urban wastewater treatment leads to the presence of these compounds and their metabolites in these ecosystems, all the while their toxic effects on biota remain unclear (Montes-Grajales et al. 2017MONTES-GRAJALES D, FENNIX-AGUDELO M & MIRANDA-CASTRO W. 2017. Occurrence of personal care products as emerging chemicals of concern in water resources: A review. Sci Total Environ 595: 601-614. https://doi.org/10.1016/j.scitotenv.2017.03.286.). Among PCPs, triclosan (TCS) is widely used as antimicrobial ingredient in toothpastes, soaps, and medical disinfectants (Chen et al. 2018CHEN ZF, WEN HB, DAI X, YAN SC, ZHANG H, CHEN YY, DU Z, LIU G & CAI Z. 2018. Contamination and risk profiles of triclosan and triclocarban in sediments from a less urbanized region in China. J Hazard Mat 357: 376-383. https://doi.org/10.1016/j.jhazmat.2018.06.020.). After its use, TCS is washed or rinsed off and may enter the aquatic environment via local wastewater treatment plants where it is partially removed as result of biodegradation, photolysis, and sorption (Capdevielle et al. 2008CAPDEVIELLE M, VAN EGMOND R, WHELAN M, VERSTEEG D, HOFMANN-KAMENSKY M, INAUEN J, CUNNINGHAM V & WOLTERING D. 2008. Consideration of exposure and species sensitivity of triclosan in the freshwater environment. Integrate Environ Assessment Manag 4(1): 15-23. https://doi.org/10.1897/IEAM_2007-022.1.). Methyl-triclosan (MeTCS), one degradation product of TCS, is a more stable and persistent compound, with a much slower kinetic degradation rate (Lindström et al. 2002LINDSTRÖM A, BUERGE IJ, POIGER T, BERGQVIST PA, MÜLLER MD & BUSER HR. 2002. Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater. Environ Sci Tech 36(11): 2322-2329. https://doi.org/10.1021/es0114254., Bedoux et al. 2012BEDOUX G, ROIG B, THOMAS O, DUPONT V & BOT LE. 2012. Occurrence and toxicity of antimicrobial triclosan and by-products in the environment. Environ Sci Pollut Res 19: 1044-1065. https://doi.org/10.1007/s11356-011-0632-z.). The fate of this kind of compounds in environment compartments depends on their physicochemical properties, which in turn impact on their degradation and transformation (Singh et al. 2013SINGH K, SINGH B & SINGH RR. 2013. Effect of land rehabilitation on physicochemical and microbial properties of a sodic soil. Catena 109: 49-57. https://doi.org/10.1016/j.catena.2013.05.006.). Because of their high hydrophobicity, TCS and MeTCS would be expected to be adsorbed to organic matter and sediment. Currently, they have been detected not only in surface or ground water, but also in sludges, biosolids, and aquatic sediments (Wang et al. 2019WANG F, WANG R, LIU F & CHEN W. 2019. Gene expression profiles in brain of male juvenile zebrafish (Danio rerio) treated with triclosan. Toxicol Apli Pharm 362: 35-42. https://doi.org/10.1016/j.taap.2018.10.014.).

Several studies have shown that TCS is toxic to many aquatic organisms, such as algae, insect larvae, benthic organisms, and fish (Orvos et al. 2002ORVOS DR, VERSTEEG DJ, INAUEN J, CAPDEVIELLE M, ROTHENSTEIN A & CUNNINGHAM V. 2002. Aquatic toxicity of triclosan. Environ Tox Chem 21(7): 1338-1349. https://doi.org/10.1002/etc.5620210703., Dussault et al. 2008DUSSAULT ÈB, BALAKRISHNAN VK, SVERKO ED, SOLOMON KR & SIBLEY PK. 2008. Toxicity of human pharmaceuticals and personal care products to benthic invertebrates. Environ Tox Chem 27(2): 425-432. https://doi.org/10.1897/07-354R.1., Oliveira et al. 2009OLIVEIRA R, DOMINGUES I & KOPPE GRISOLIA C. 2009. Effects of triclosan on zebrafish early-life stages and adults. Environ Sci Pollut Res 16: 679-688. https://doi.org/10.1007/s11356-009-0119-3., Perron et al. 2012PERRON MM, HO KT, CANTWELL MG, BURGESS RM & PELLETIER MC. 2012. Effects of triclosan on marine benthic and epibenthic organisms. Environ Tox Chem 31(8): 1861-1866. https://doi.org/10.1002/etc.1884.). Deleterious effects of TCS have been reported in Danio rerio in terms of gene expression (Wang et al. 2019WANG F, WANG R, LIU F & CHEN W. 2019. Gene expression profiles in brain of male juvenile zebrafish (Danio rerio) treated with triclosan. Toxicol Apli Pharm 362: 35-42. https://doi.org/10.1016/j.taap.2018.10.014.), metabolism (Falisse et al. 2017FALISSE E, VOISIN AS & SILVESTRE F. 2017. Impacts of triclosan exposure on zebrafish early-life stage: toxicity and acclimation mechanisms. Aquat Toxicol 189: 97-107. https://doi.org/10.1016/j.aquatox.2017.06.003., Macedo et al. 2017MACEDO S, TORRES T & SANTOS MM. 2017. Methyl-triclosan and triclosan impact embryonic development of Danio rerio and Paracentrotus lividus. Ecotoxicology 26: 482-489. https://doi.org/10.1007/s10646-017-1778-3., Fu et al. 2019FU J, ZHIYUAN G & BARRY CK. 2019. Metabolomic profiling of zebrafish (Danio rerio) embryos exposed to the antibacterial agent Triclosan. Environ Toxicol Chem 38(1): 240-249. https://doi.org/10.1002/etc.4292.), histological alterations (Gyimah et al. 2020GYIMAH E, DONG X, QIU W, ZHANG Z & XU H. 2020. Sublethal concentrations of triclosan elicited oxidative stress, DNA damage, and histological alterations in the liver and brain of adult zebrafish. Environ Sci Pol Res: 1-10. https://doi.org/10.1007/s11356-020-08232-2.), metamorphosis, and reproductive fitness (Stenzel et al. 2019STENZEL A, WIRT H, PATTEN A, THEODORE B & KING-HEIDEN T. 2019. Larval exposure to environmentally relevant concentrations of triclosan impairs metamorphosis and reproductive fitness in zebrafish. Repro Tox 87: 79-86. https://doi.org/10.1016/j.reprotox.2019.05.055.). However, although MeTCS is more persistent in aquatic environments, little information is available about its adverse effects on aquatic biota. MeTCS mechanism of action is similar to TCS and can occur at quantifiable concentrations even when TCS is below the limit of detection (Lindström et al. 2002LINDSTRÖM A, BUERGE IJ, POIGER T, BERGQVIST PA, MÜLLER MD & BUSER HR. 2002. Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater. Environ Sci Tech 36(11): 2322-2329. https://doi.org/10.1021/es0114254.). In this context, MeTCS cytotoxicity was evaluated in invertebrates (DeLorenzo et al. 2007, Gaume et al. 2012GAUME B, BOURGOUGNON N, AUZOUX-BORDENAVE S, ROIG B, LE BOT B & BEDOUX G. 2012. In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata. Comp Bio Physio-C Tox Pharm 156(2): 87-94. https://doi.org/10.1016/j.cbpc.2012.04.006.) and bacteria (Farré et al. 2008FARRÉ M, ASPERGER D & KANTIANI L. 2008. Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri. Analy Bioanaly Chem 390(8): 1999-2007. https://doi.org/10.1007/s00216-007-1779-9.), meanwhile when D. rerio was exposed via waterborne to it, malformations in the cardiovascular system, spinal curvature, disrupted nitrogen metabolism pathways, altered energetic metabolisms, and fatty acid synthesis were registered (Macedo et al. 2017MACEDO S, TORRES T & SANTOS MM. 2017. Methyl-triclosan and triclosan impact embryonic development of Danio rerio and Paracentrotus lividus. Ecotoxicology 26: 482-489. https://doi.org/10.1007/s10646-017-1778-3., Fu et al. 2019FU J, ZHIYUAN G & BARRY CK. 2019. Metabolomic profiling of zebrafish (Danio rerio) embryos exposed to the antibacterial agent Triclosan. Environ Toxicol Chem 38(1): 240-249. https://doi.org/10.1002/etc.4292.). However, it still exists a lack of knowledge about the MeTCS effects on redox balance and neurotoxicity, especially after other kind of exposures rather than the waterborne one.

Assays conducted with whole sediment samples are a more realistic approach and have become a useful tool when assessing toxicity on aquatic organisms. Sediment is an inherently complex, heterogeneous geological matrix, with a number of routes by which biota is exposed to its associated contaminants (Davoren et al. 2005DAVOREN M, SHÚILLEABHÁIN SN, HARTL MG, SHEEHAN D, O’BRIEN NM, O’HALLORAN J & MOTHERSILL C. 2005. Assessing the potential of fish cell lines as tools for the cytotoxicity testing of estuarine sediment aqueous elutriates. Tox in vitro 19(3): 421-431. https://doi.org/10.1016/j.tiv.2004.12.002.). Ecotoxicological sediment assessments have been focused on many invertebrate benthic organisms (Giusto et al. 2012GIUSTO A, SOMMA L & FERRARI L. 2012. Cadmium toxicity assessment in juveniles of the Austral South America amphipod Hyalella curvispina. Ecotox Environ Safety 79: 163-169. https://doi.org/10.1016/j.ecoenv.2011.12.020., Perron et al. 2012PERRON MM, HO KT, CANTWELL MG, BURGESS RM & PELLETIER MC. 2012. Effects of triclosan on marine benthic and epibenthic organisms. Environ Tox Chem 31(8): 1861-1866. https://doi.org/10.1002/etc.1884., Ho et al. 2013HO KT, CHARITON AA, PORTIS LM, PROESTOU D, CANTWELL MG, BAGULEY JG & KAMIKAWA A. 2013. Use of a novel sediment exposure to determine the effects of triclosan on estuarine benthic communities. Environ Tox Chem 32(2): 384-392. https://doi.org/10.1002/etc.2067.). However, apart from representing a major sink of persistent of toxic substances, sediments are considered a source of contaminants that could reach the water column, and then, those substances became available to non-benthonic organisms (Rocha et al. 2011ROCHA PS, BERNECKER C, STRECKER R, MARIANI CF, POMPÊO MLM, STORCH V & BRAUNBECK T. 2011. Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil). Ecotox Environ Safety 74(7): 1951-1959. https://doi.org/10.1016/j.ecoenv.2011.07.009.). Exposure to sediment spiked with TCS generated deleterious effects on embryo-larval development in sea urchin Lytechinus variegatus and in the bivalve Perna perna (Pusceddu et al. 2018PUSCEDDU FH, CHOUERI RB, PEREIRA CDS, CORTEZ FS, SANTOS DRA, MORENO B & CESAR A. 2018. Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints. Environ Pollut 232: 274-283. https://doi.org/10.1016/j.envpol.2017.09.046.), and also changed the composition of meio and macrofauna in marine benthic communities (Ho et al. 2013HO KT, CHARITON AA, PORTIS LM, PROESTOU D, CANTWELL MG, BAGULEY JG & KAMIKAWA A. 2013. Use of a novel sediment exposure to determine the effects of triclosan on estuarine benthic communities. Environ Tox Chem 32(2): 384-392. https://doi.org/10.1002/etc.2067.). To the best of our knowledge, no studies employing fish as test species have been developed.

Fish are organisms widely used for measurement of biomarkers and their size allows obtaining several tissues that provide a more systemic scenario of the toxicity of a compound (Ale et al. 2018aALE A, ROSSI A, BACCHETTA C, GERVASIO S, DE LA TORRE FR & CAZENAVE J. 2018a. Integrative assessment of silver nanoparticles toxicity in Prochilodus lineatus fish. Ecol Indic 93: 1190-1198. https://doi.org/10.1016/j.ecolind.2018.06.023.). Danio rerio is a cyprinid species that inhabits and feeds in whole of the vertical water column (Spence et al. 2007SPENCE R, FATEMA MK, ELLIS S, AHMED ZF & SMITH C. 2007 The diet, growth and recruitment of wild zebrafish (Danio rerio) in Bangladesh. J Fish Biol 71(1): 304-309. doi:10.1111/j.1095-8649.2007.01492.x.). This fish species is broadly used as a model organism to assess ecotoxicological research because its easy accessibility, widely known farming characteristics, and its sensitivity to contaminants. As the impact of TCS on fish is poorly understood, this study aimed to investigate biochemical and physiological effects of sediment spiked with TCS after a short term (2 days) and prolonged (21 days) exposures in the standardized fish species D. rerio. We analyzed multiple biomarker responses including morphological indices, neurotoxicity (acetylcholinesterase enzymatic activity) and oxidative stress (superoxide dismutase, catalase, glutathione-S-transferase antioxidant enzymatic activities, lipid peroxidation levels, and total antioxidant competence against peroxyl radicals) in liver, gills, and brain of fish, and, finally, we performed a multivariate analysis to obtain a holistic view of the effects generated by this emergent pollutant.

MATERIALS AND METHODS

Tests organisms

Wild-type adults of D. rerio (n=128; 0.50 ± 0.05 g body weight and 3.6 ± 0.2 cm total length) were reared in the GECAP (Grupo de Estudios de Contaminación Antrópica en Peces) laboratory. During the acclimation period, fish were daily fed with commercial pellets and maintained in aquaria with continuous flow of unchlorinated freshwater (dissolved oxygen 8.0 ± 0.6 mg L^-1, pH 8.2 ± 0.4, and conductivity 1000 ± 6 μS cm^-1), controlled temperature of 25 ± 2 °C and photoperiod 12:12 light-dark cycles.

Sediment preparation and TCS spiking

Surface sediment (up to 10 cm depth) was taken from an unpolluted site located in Las Flores stream (59° 07´W and 34° 29´S) (Buenos Aires, Argentina). Due to its low anthropic intervention, this site has been assigned as a reference by other authors (Ronco et al. 2008RONCO A, PELUSO L, JURADO M, ROSSINI GB & SALIBIAN A. 2008. Screening of sediment pollution in tributaries from the southwestern coast of the Río de la Plata estuary. Lat Amer J Sed Basin A 15(1): 67-75., Giusto et al. 2012GIUSTO A, SOMMA L & FERRARI L. 2012. Cadmium toxicity assessment in juveniles of the Austral South America amphipod Hyalella curvispina. Ecotox Environ Safety 79: 163-169. https://doi.org/10.1016/j.ecoenv.2011.12.020., Peluso et al. 2013PELUSO L, ROSSINI GB, SALIBIÁN A & RONCO A. 2013. Physicochemical and ecotoxicological based assessment of bottom sediments from the Luján River basin, Buenos Aires, Argentina. Environ Monit Asses 185(7): 5993-6002. https://doi.org/10.1007/s10661-012-3000-7.). Sediment grain size distribution (sand 61%, clay 12% and silt 27%) has been previously characterized (Giusto et al. 2012GIUSTO A, SOMMA L & FERRARI L. 2012. Cadmium toxicity assessment in juveniles of the Austral South America amphipod Hyalella curvispina. Ecotox Environ Safety 79: 163-169. https://doi.org/10.1016/j.ecoenv.2011.12.020., 2014GIUSTO A, SALIBIÁN A & FERRARI L. 2014. Biomonitoring toxicity of natural sediments using juvenile Hyalella curvispina (Amphipoda) as test species: evaluation of early effect endpoints. Ecotoxicology 23(2): https://doi.org/10.1016/j.ecoenv.2011.07.009., Scarcia et al. 2014SCARCIA P, CALAMANTE G & DE LA TORRE F. 2014. Responses of biomarkers of a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) fish species after in situ exposure in a periurban zone of Luján river (Argentina). Environ Tox 29(5): 545-557. https://doi.org/10.1002/tox.21780.). Subsamples from the sediment were taken to determine humidity and total organic matter by combustion at 500 °C for 5 hours (modified from Gordon 2000GORDON E. 2000. Dinámica de la vegetación y del banco de semillas en un humedal herbáceo lacustrino. Rev Biol Trop 48(1): 25-42.).

Triclosan (Irgasan, 5-chloro-2-(2, 4-dichlorophenoxy) phenol, ≥97.0% purity, HPLC) was purchased from Sigma-Aldrich. According to previous works (Chen et al. 2011CHEN X, NIELSEN JL, FURGAL K, LIU Y, LOLAS IB & BESTER K. 2011. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. Chemosphere 84(4): 452-456. https://doi.org/10.1016/j.chemosphere.2011.03.042., Ho et al. 2013HO KT, CHARITON AA, PORTIS LM, PROESTOU D, CANTWELL MG, BAGULEY JG & KAMIKAWA A. 2013. Use of a novel sediment exposure to determine the effects of triclosan on estuarine benthic communities. Environ Tox Chem 32(2): 384-392. https://doi.org/10.1002/etc.2067.) degradation processes of TCS can occur during spiking sediment procedure; then, the nominal concentration was set as 2.9 mg kg^-1 dry weight (dw). This concentration was 3 times greater than the maximum values of TCS reported in the United States: 0.7-0.8 mg kg^-1 (Miller et al. 2008MILLER TR, HEIDLER J, CHILLRUD SN, DELAQUIL A, RITCHIE JC, MIHALIC JN & HALDEN RU. 2008. Fate of triclosan and evidence for reductive dechlorination of triclocarban in estuarine sediments. Environ Sci Tech 42(12): 4570-4576. https://doi.org/10.1021/es702882g.).

Spiked sediment was prepared by plating 2 mL of acetone TCS concentrated solution onto a glass mortar and thoroughly mixed with 1 kg dry sediment and transferred into a dark amber glass bottle which was kept in a continuous rolling system for 4 days (Pusceddu et al. 2018PUSCEDDU FH, CHOUERI RB, PEREIRA CDS, CORTEZ FS, SANTOS DRA, MORENO B & CESAR A. 2018. Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints. Environ Pollut 232: 274-283. https://doi.org/10.1016/j.envpol.2017.09.046.). Finally, the sediment was stored at 4 °C in the dark for 2 days to establish a chemical equilibrium between TCS and the sediment (Löffler et al. 2005LÖFFLER D, RÖMBKE J, MELLER M & TERNES TA. 2005. Environmental fate of phar- maceuticals in water/sediment systems. Environ Sci Technol 39(14): 5209-5218. https://doi.org/10.1021/es0484146.).

Experimental design

Whole sediment static bioassays were conducted in glass aquaria according to the following conditions: 15 L of unchlorinated tap water and 1 kg of unspiked sediment (serving as the control, Ctrl) or TCS spiked sediment. After the addition of the sediment, the system was allowed to stabilize for 24 h before adding the fish. The animals were randomly distributed to each experimental treatment (n=32) and remained exposed for 2 and 21 days under constant aeration and photoperiod (12:12). In addition of aeration, aquaria design included a closed water circuit in order to recirculate and mix the water column of aquaria. Fish were daily fed with commercial pellets, and after the times of exposures the organisms were anesthetized in ice chilled water, weighed, measured, and sacrificed by incision behind the operculum. Gills, liver, and brain were excised and kept in -80 °C until further determinations. This method follows the recommendation of the local and National Institutes of Health Guidelines (Resolution 672-15, National University of Lujan). All experiments were conducted in accordance with national and institutional guidelines (CONICET 2005CONICET - CONSEJO NACIONAL DE INVESTIGACIONES CIENTÍFICAS Y TÉCNICAS. 2005. Marco Ético de Referencia para las Investigaciones Biomédicas en Animales de laboratorio, de granja y obtenidos de la naturaleza. Buenos Aires, Argentina. www.conicet.gov.ar/wp-content/uploads/OCR-RD-20050701-1047.pdf.
www.conicet.gov.ar/wp-content/uploads/OC... ) for the protection of animal welfare.

Physicochemical parameters in water

Dissolved oxygen (OD), pH, conductivity and temperature were registered with a multiparametric Hach (HQ30D) probe with OD laser probe; measurements were made in triplicate and data were taken three times during the 2 days period and four times for the 21 days of exposure (0, 7, 14 and 21 days).

Ammonium (µg N-NH₄ ⁺ L^-1) present in water was determined during the exposure period according to the phenol method based on the formation of indophenol from the reaction of ammonium with hypochlorite and phenol in an alkaline medium. The determinations were made at 635 nm (APHA 2005APHA - AMERICAN PUBLIC HEALTH ASSOCIATION. 2005. Standard Methods for the Examination of Water and Wastewater. Washington, D.C., USA.). For this purpose, water samples were collected three times during the test and filtered with fiberglass filters Munkell® MF/C, which were previously weighed for the determination of suspended particulate matter (SPM).

Quantification of TCS and Me-TCS in water, sediment, and suspended particulate matter.

For the determinations of TCS and MeTCS concentrations in water, sediment and suspended particulate matter, samples were taken in triplicate at each time of exposure during the test. Also, two samples of TCS spiked sediment were collected to determine initial sediment concentrations of TCS and MeTCS.

Extractions

For MeTCS and TCS analysis in water, samples were extracted by passage through a Waters® Oasis C18 HLB 60 mg cartridge (Canosa et al. 2005CANOSA P, RODRIGUEZ I, RUBÍ E & CELA R. 2005. Optimization of solid-phase microextraction conditions for the determination of triclosan and possible related compounds in water samples. J Chrom A 1072(1): 107-115. https://doi:10.1016/j.chroma.2004.11.032., Elorriaga et al. 2013ELORRIAGA Y, MARINO DJ, CARRIQUIRIBORDE P & RONCO A. 2013. Human pharmaceuticals in wastewaters from urbanized areas of Argentina. Bull Environ Cont Tox 90(4): 397-400. https://doi.org/10.1007/s00128-012-0919-x.). The SPE cartridges were first conditioned with 5 mL of methanol, followed by 5 mL of nanopure water. Water samples were extracted at a 5 mL min^-1 flow rate under moderate vacuum in a Visiprep™ SPE Vacuum Manifold (Supelco, Bellefonte, PA). After the extraction, the cartridges were washed with 5 mL of nanopure water, and then air dried for 20 min under vacuum. Retained analytes were eluted with 5 mL of methanol. The elutes were dried under a gentle stream of nitrogen, reconstituted in acetonitrile, passed through 0.45 µm filters, and transferred to amber chromatographic vials.

Suspended particulate matter was extracted by placing the filters in 15 mL polypropylene tubes and adding 5 mL of acetonitrile. Two 10 min sonication cycles were performed, and then centrifuged for 10 min at 3000 g. Lastly, 1 mL of the extract was filtered through 0.45 µm membranes and then placed into amber vials.

Sediments were extracted using a modified QuEChERS procedure (Mac Loughlin et al. 2017MAC LOUGHLIN TM, PELUSO L & MARINO DJ. 2017. Pesticide impact study in the peri-urban horticultural area of Gran La Plata, Argentina. Sci Total Environ 598: 572-580. https://doi.org/10.1016/j.scitotenv.2017.04.116.). Briefly, 7 g of wet sediment were weighed into a 50 mL polypropylene tube and extracted with 15 mL of acetonitrile. After adding solvent, the tubes were vigorously shaken and then sonicated for 10 min, shaken for 1 min and sonicated again. Extraction salts (2 g of NaCl and 6 g of anhydrous MgSO₄) were added and shaken manually for 2 minutes. Finally, the tubes were centrifuged for 10 min at 3000 g. From the supernatant (acetonitrile), 1 mL aliquots were filtered through 0.45 µm filters, and placed in amber chromatographic vials. One was stored at -20 °C until MeTCS analysis by GCμECD, while the other was immediately analyzed for TCS by HPLCMS.

Equipment

TCS was identified and quantified by HPLC-MS through the use of an Agilent model 1100 liquid chromatograph coupled to an Agilent model VL single quadrupole mass spectrometer (Agilent Technologies Inc., Miami, FL, USA). For the ionization, an electrospray source was used in negative mode with selective ions m/z 287.7 and 288.7. The chromatograph was equipped with a C₁₈ XSELECT™ column (75 mm × 4.6 mm, and 3 mm pore size, from Waters Corp., Milford), separation was run in isocratic condition of methanol (HPLC grade, J.T. Baker, USA) and nanopure water (formic acid 0.1%, analytical quality, Merck, Germany), with a flow of 0.5 mL min^-1. Detection instrumental limits were 0.1 μg L^-1 and quantification limit was 0.5 μg L^-1.

MeTCS was identified and quantified by GCμECD through the use of an Agilent 6890N gas chromatograph. The system was equipped with a HP-5 (15 m × 0.53 mm i.d. × 1.5 μm film thicknesses) column. A volume of 3 μL was injected in splitless mode, with the injector temperature at 250 °C. The oven ramp was set to an initial temperature of 80 °C, increased to 180 °C at 20 °C min^-1, and then to 250 °C at 10 °C min^-1, with a total acquisition program of 15 min. Hydrogen was used as carrier gas and nitrogen as makeup gas. The detector was set at 250 °C. Instrumental detection and quantification limits were 0.5 and 1 μg L^-1, respectively.

Biomarker determinations

The condition factor (CF) index was calculated as body weight (g)/total body length (cm)³ (Bagenal & Tesch 1978BAGENAL TB & TESCH FW. 1978: Age and growth. In: Methods for assessment of fish production in freshwaters. Bagenal T (Ed), IBP Handbook, 3rd ed., Blackwell Scientific Publications, Oxford, p. 101-136.) and the hepatosomatic index (HSI) was determined as liver weight (g) × 100 x total fish weight (g)^-1 (Sloof et al. 1983SLOOF W, VAN KREIJL CF & BAARS AJ. 1983. Relative liver weights and xenobiotic metabolizing enzymes of fish from polluted surface in the Netherlands. Aquat Toxicol 4: 1-14. https://doi.org/10.1016/0166-445X(83)90057-7.).

D. rerio liver, gills and brain were pooled processed (n= 4 tissues per pool; n= 8 pool per treatment). Tissues were homogenized on ice until total disintegration with buffer pH 7.4 (0.1 M NaH₂PO₄; 0.15 M KCl; 1 mM EDTA; 1 mM DTT; 10% v/v glycerol) according to Nilsen et al. (1998)NILSEN BM, BERG K & GOKSØYR A. 1998. Induction of cytochrome P450 1A (CYP1A) in fish. A biomarker for environmental pollution. Method Mol Biol 107: 423-438. https://doi.org/10.1385/0-89603-519-0:423.. An aliquot was used for the determination of lipid peroxidation levels and the remaining homogenate was centrifuged at 20000 g for 20 min at 4 °C. The obtained supernatant fraction was reserved for the quantification of enzyme activities and protein content.

Lipid peroxidation (LPO) was determined in liver, gills and brain by measuring the formation of thiobarbituric reactive substances under acidity and heat conditions according to Ohkawa et al. (1979)OHKAWA H, OHISHI N & YAGI K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351-358. and Oakes and van der Kraak (2003)OAKES KD & VAN DER KRAAK GJ. 2003. Utility of the TBARS assay in detecting oxidative stress in white sucker (Catostomus commersoni) populations exposed to pulp mill effluent. Aquat Toxicol 63: 447-463. https://doi.org/10.1016/S0166-445X(02)00204-7..

Catalase (CAT, EC 1.11.1.6) activity was determined in liver, gills and brain following the method described by Beutler (1982)BEUTLER E. 1982. Catalase. In: Beutler E (Ed), Red cell metabolism, a manual of biochemical methods. Grune and Stratton Inc, New York, p. 105-106., and Glutathione S-transferase (GST, EC 2.5.1.18) activity was determined according to Habig and Jakoby (1981)HABIG WH & JAKOBY WB. 1981. Assays for differentiation of glutathione S-Transferases. Methods Enzymol 77: 398-405..

Superoxide dismutase (SOD, EC 1.15.1.1) activity was evaluated in liver and gills following an indirect method involving the inhibition of cytochrome c reduction by the competition with SOD for the superoxide anion radical formed by the xanthine/xanthine oxidase system (McCord & Fridovich 1969MCCORD JM & FRIDOVICH I. 1969. Superoxide dismutase: An enzymatic funtion for erythrocuprein (hemocuprein). J Biol Chem 244: 6049-6055.). The activity was expressed as units of SOD*mg^-1 protein, where 1 SOD unit (U) is defined as the enzyme quantity that causes 50% of inhibition of reduction of cytochrome c per minute.

Total antioxidant competence against peroxyl radicals (ACAP) was determined in liver, gills and brain according to Amado et al. (2009)AMADO LL, GARCIA ML, RAMOS PB, FREITAS RF, ZAFALON B, JOSENCLER LUIS RIBAS FERREIRA JLR, YUNES JS & MONSERRAT JM. 2009. A method to measure total an-tioxidant capacity against peroxyl radicals in aquatic organisms: application to evaluate microcystins toxicity. Sci Total Environ: 2115-2123. https://doi.org/10.1016/j.scitotenv.2008.11.038. and further modifications adopted by Monserrat et al. (2014)MONSERRAT JM, GARCIA ML, VENTURA-LIMA J, GONZÁLEZ M, BALLESTEROS ML, MIGLIORANZA KSB, AMÉD MV & WUNDERLIN DA. 2014. Antioxidant, phase II and III responses induced by lipoic acid in the fish Jenynsia multidentata (Anablapidae) and its influence on endolsulfan accumulation and toxicity. Pestic Biochem Phys 108: 8-15. https://doi.org/10.1016/j.pestbp.2013.10.009.. The measure of antioxidant capacity is given by difference in the fluorescence of the samples after 30 min with and without ABAP and is calculated by the following expression: (FU 30 min_{with ABAP} - FU 30 min_{without ABAP})/FU 30 min_{without ABAP}. As high fluorescence levels are obtained after adding ABAP, a high difference is considered to indicate a low antioxidant capacity suggesting a low ability to neutralize peroxyl radicals.

Acetylcholinesterase activity (AChE) (EC 3.1.1.7) was measured in brain according to Ellman et al. (1961)ELLMAN GL, COURTNEY KD, ANDRES V & FEATHERSTONE RM. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharma 7: 88-95. modified for microplate measurement.

The protein content of the supernatant fractions was measured according to Lowry et al. (1951)LOWRY OH, ROSEBROUGH NJ, FARR AL & RANDALL RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275..

Statistical Analysis

Data distributions were tested for normality and variance homogeneity with Kolmogorov–Smirnov and Levene’s tests, respectively. Significant differences between each Ctrl group and exposure treatments were assessed by t-Student with p<0.05. Also, principal component analysis (PCA) was performed to get an overall view of biological responses and to define the most important parameters involved in TCS-MeTCS toxicity for both time of exposure. After excluding outliers, multivariate analysis was carried out considering seven cases per treatment and twelve variables. All statistical analysis was performed by the InfoStat software (Di Rienzo et al. 2020DI RIENZO JA, CASANOVES F, BALZARINI MG, GONZALEZ L, TABLADA M & ROBLEDO CW. 2020. InfoStat versión 2020. Centro de Transferencia InfoStat. Universidad Nacional de Córdoba, Argentina. https://www.infostat.com.ar/.
https://www.infostat.com.ar/... ).

RESULTS

No mortality was registered along the bioassay, neither in the control group nor in TCS exposed groups.

Water quality parameters in aquaria and effective concentration of TCS and MeTCS

Water physicochemical parameters are detailed in Table I. Dissolved oxygen, pH, and conductivity were the parameters that remained with similar values throughout the experimental periods. However, ammonium concentrations between the Ctrl treatments and TCS-MeTCS were variable at both times of exposure.

Effective concentrations of TCS and MeTCS are presented in Table II. Both compounds were present in sediment, however only MeTCS was detected in water. About 1% of the MeTCS detected in the sediment mobilized to the water column. No differences were observed in the concentration of MeTCS in water during exposure times, with average exposure value 12.3 ± 1.1 µg L^-1 (Fig. 1). Neither TCS nor MeTCS were detected both (sediment and water) on the Ctrl.

Sediment humidity was 43 ± 2.4 % and total organic matter was 3.1 ± 0.4 %.

Biomarkers

No significant changes were observed in the CF and HSI of D. rerio (N= 32) after acute and prolonged exposure to sediment spiked with TCS (Table III).

Liver

Effects of TCS-MeTCS on liver biomarkers of D. rerio are presented in Fig. 2. After 2 days, SOD activity increased 36% and ACAP decreased 48% in respect of Ctrl. On the other hand, after the 21 days exposure with sediment spiked with TCS, all liver biomarkers evaluated were altered in respect of the Ctrl: SOD and CAT activity increased (58% and 35% respectively), GST was inhibited (12%), ACAP decreased in 40% and lipid damage increased in 57%.

Gills

The results of acute and prolonged effects in gills of D. rerio are presented in Fig. 3. After 2 days of exposure, LPO levels increased 75% respect to Ctrl. This change was joined with an increased in SOD activity (12%) and inhibition of GST (36%). In prolonged exposure, lipid damage and SOD activity increased 48% and 50% respectively, and ACAP decreased in 68% respect to Ctrl.

Brain

The effects produced in brain by sediment spiked with TCS are presented in Fig. 4. CAT activity decreased 56% in fish after being exposed during 2 days to TCS-MeTCS, and GST increased after 2 and 21 days of exposure (23 and 26% respectively). AChE activity showed no differences between treated organisms and Ctrl after both exposure times.

Principal component analysis

The first and second component of principal component analysis (PCA) accounted 29.9% and 18.4% of the total variance, respectively (Fig. 5). Control group were grouped and differed mainly from fish treated by the principal component (PC) 1. On the other hand, fish that were in short term exposure were grouped and differentiated from fish exposed for 21 days by the PC2. PC1 focuses on ACAP in liver and gills and LPO in gills, while PC2 focuses on the activation of antioxidant enzymes (GST and CAT) in the 3 evaluated organs. Organisms that were exposed for 21 days to sediment with TCS differed from fish exposed for 2 days primarily due to CAT activity in the brain and liver and GST in gills.

DISCUSSION

Research about the toxicity of contaminated sediments has been limited by the complexity of sediment-water column. Whole sediment bioassays are necessary to assess the toxicity of total sediment, including both soluble and solid phases. In this way, they represent the most realistic exposure scenario for considering bioavailability (Hallare et al. 2011HALLARE AV, SEILER TB & HOLLERT H. 2011. The versatile, changing, and advancing roles of fish in sediment toxicity assessment—a review. J Soils Sed 11(1): 141-173. https://doi.org/10.1007/s11368-010-0302-7.). In addition, it is relevant to employ test species that are not in direct contact with the sediment but may be targeted by contaminants released from it.

Dynamic to sediment spiked with TCS

Total organic matter and sediment humidity found in this work were consistent with values reported by Giusto et al. (2012, 2014) for the sediment in “Las Flores” stream (Buenos Aires, Argentina). The fate and effects of an organic pollutant are related to its bioavailability. In sediment, these compounds become more stable and persistent over time, increasing their potential adverse effects on biota (Ronco et al. 2008RONCO A, PELUSO L, JURADO M, ROSSINI GB & SALIBIAN A. 2008. Screening of sediment pollution in tributaries from the southwestern coast of the Río de la Plata estuary. Lat Amer J Sed Basin A 15(1): 67-75., Boulanger et al. 2019BOULANGER E, BARST BD, ALLOY MM, BLAIS S, HOUDE M & HEAD JA. 2019. Assessment of environmentally contaminated sediment using a contact assay with early life stage zebrafish (Danio rerio). Sci Total Environ 659: 950-962. https://doi.org/10.1016/j.scitotenv.2018.12.265.).

TCS presents a water solubility of 12 mg L^-1 and an octanol-water partitioning coefficient log Kow=3.5-4.8 at neutral pH. The half-life of TCS ranges from 4 to 60 days in sediment and depending on the initial concentration and environmental factors such as the pH, oxygen and light (Bedoux et al. 2012BEDOUX G, ROIG B, THOMAS O, DUPONT V & BOT LE. 2012. Occurrence and toxicity of antimicrobial triclosan and by-products in the environment. Environ Sci Pollut Res 19: 1044-1065. https://doi.org/10.1007/s11356-011-0632-z.). In addition, TCS could be susceptible to degradation via aqueous photolysis, with a half-life of <1 hour (SCCS 2010SCCS - SCIENTIFIC COMMITTEE ON CONSUMER SAFETY. 2010. Opinion on triclosan (antimicrobial resistance), ISSN 1831-4767. https://doi:10.2772/11162.
https://doi:10.2772/11162... ). On the other hand, MeTCS has lower water solubility than TCS (0.4 mg L^-1), but a higher octanol-water partitioning (log Kow=5.34), so it is more stable associated to organic matter and in sediments than TCS (Balmer et al. 2005BALMER ME, BUSER HR, MÜLLER MD & POIGER T. 2005. Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss lakes. Environ Sci Tec 39(4): 953-962. https://doi.org/10.1021/es040055r., Chen et al. 2011CHEN X, NIELSEN JL, FURGAL K, LIU Y, LOLAS IB & BESTER K. 2011. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. Chemosphere 84(4): 452-456. https://doi.org/10.1016/j.chemosphere.2011.03.042.). These characteristics led to the observed less persistent presence of TCS in sediment and the higher persistence and stability of MeTCS.

Sediment preparation with TCS involved an initial interaction of the toxicant in order to favor adsorption and stabilization processes. In this work, 6 days after spiking, more than 90% of TCS had degraded and biotransformation to MeTCS occurred in sediment with non-sterile conditions. In agreement with our results, Chen et al. (2011)CHEN X, NIELSEN JL, FURGAL K, LIU Y, LOLAS IB & BESTER K. 2011. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. Chemosphere 84(4): 452-456. https://doi.org/10.1016/j.chemosphere.2011.03.042. reported that 75% of TCS was removed in sediment within 6 days under aerobic conditions. In addition, Ho et al. (2013)HO KT, CHARITON AA, PORTIS LM, PROESTOU D, CANTWELL MG, BAGULEY JG & KAMIKAWA A. 2013. Use of a novel sediment exposure to determine the effects of triclosan on estuarine benthic communities. Environ Tox Chem 32(2): 384-392. https://doi.org/10.1002/etc.2067. showed that TCS in sediment declined to 46 to 60% of nominal concentration after 3 days. In this context, bacteria from the sediment metabolized TCS to MeTCS, further biomethylating it throughout, maintaining the MeTCS concentrantion constant for 21 days. The sediment acted as a contaminant source, continuously releasing them into the water, and keeping the MeTCS concentration stable throughout the exposure period. Likewise, the effective concentration of TCS in sediment tested in our work is environmentally relevant (Davis et al. 2012DAVIS EF, KLOSTERHAUS SL & STAPLETON HM. 2012. Measurement of flame retardants and triclosan in municipal sewage sludge and biosolids. Environ Inter 40: 1-7. https://doi.org/10.1016/j.envint.2011.11.008., Pusceddu et al. 2018PUSCEDDU FH, CHOUERI RB, PEREIRA CDS, CORTEZ FS, SANTOS DRA, MORENO B & CESAR A. 2018. Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints. Environ Pollut 232: 274-283. https://doi.org/10.1016/j.envpol.2017.09.046.).

Even though TCS has been reported in the particulate material (Kumar et al. 2010KUMAR KS, PRIYA SM, PECK AM & SAJWAN KS. 2010. Mass loadings of triclosan and triclocarban from four wastewater treatment plants to three rivers and landfill in Savannah, Georgia, USA. Arch Environ Contam Toxicol 58: 275-285. https://doi.org/10.1007/s00244-009-9383-y.), the lack of TCS detection in the matrix in this study could be due to the low resuspension of the sediment provoked by D. rerio. The presence of benthic species could generate greater movement of particulate matter in water, and therefore higher amounts of pollutants for fish.

Physicochemical parameters

Although ammonium showed variability even in the control group, these values were lower than the maximum permitted quantity for protection of freshwater life (1130 µg N-NH₄ ⁺ L^-1) according to recommended aquatic life prolonged criteria by USEPA (1999)USEPA - UNITED STATES ENVIRONMENTAL PROTECTION AGENCY. 1999. Update of ambient water quality criteria for ammonia. Washington, DC: Office of Water, Office of Science and Technology.. The experimental conditions (e.g. permanent nutrient enrichment by fish and presence or not of TCS) could explain the variability of ammonium values observed in our work (Stief et al. 2003STIEF P, SCHRAMM A, ALTMANN D & DE BEER D. 2003. Temporal variation of nitrification rates in experimental freshwater sediments enriched with ammonia or nitrite. FEMS Microbio Eco 46(1): 63-71. https://doi.org/10.1016/S0168-6496(03)00193-4.). Also, the pH and conductivity were similar to the values of fish breeding and maintenance, and the conductivity was found in the optimal range (300-1500 µS cm^-1) for D. rerio (Avdesh et al. 2012AVDESH A ET AL. 2012. Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J Vis Exp 69: doi:10.3791/4196.).

Biomarkers

Taking into account that 1% of MeTCS became available in the water column, our results showed that TCS spiked sediment was able to exert adverse effects on D. rerio biochemical parameters.

One of the main functions of liver is to metabolize lipophilic substances, including xenobiotics (Di Giulio & Hinton 2008DI GIULIO RT & HINTON DE. 2008. The toxicology of fishes. Crc Press, p. 1071.), so it is an organ of interest to evaluate toxic effects generated by pollutants. On the other hand, the first enzyme involved in antioxidant defense mechanism is SOD, which catalyzes the dismutation of superoxide anion to hydrogen peroxide and molecular oxygen. The increased activity of SOD observed in liver after the short term exposure to sediment spiked with TCS showed that antioxidant defenses were activated to counteract the superoxide anion and may prevent lipid damage in this tissue. However, the total antioxidant capacity against peroxyl radicals decreased. Besides, the greatest liver damage was observed after 21 days, where activation of both antioxidant enzymes, SOD and CAT, were not sufficient to prevent lipid damage. In addition, the total capacity against peroxyl radicals continued to decrease, evidencing an overall decreased liver antioxidant capacity after a prolonged exposure to sediment spiked with TCS.

Another important detoxification enzyme is CAT, and its activity has been used as a potential biomarker of fishes exposed to toxicants (Zhang et al. 2004ZHANG J, SHEN H, WANG X, WU J & XUE Y. 2004. Effects of chronic exposure of 2, 4-dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus. Chemosphere 55(2): 167-174. https://doi.org.10.1016/j.chemosphere.2003.10.048.). Because CAT degrades H₂O₂ in water and oxygen, the increase in H₂O₂ content generated by SOD activity could explain the increase in CAT activity observed in our study. Similar results were observed in liver of Oreochromis niloticus and Clarias gariepinus fish after exposure to organic pollutants such as butachlor (herbicide) and azinphos-methyl (insecticide) (Oruc et al. 2004ORUC EO, SEVGILER Y & UNER N. 2004. Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl. Comp Biochem Physiol C Toxicol Pharmacol 137: 43-51. https://doi.org/10.1016/j.cca.2003.11.006., Farombi et al. 2008FAROMBI E, AJIMOKO Y & ADELOWO O. 2008. Effect of butachlor on antioxidant enzyme status and lipid peroxidation in fresh water African catfish, (Clarias gariepinus). Inter J Environ Res Pub Health 5(5): 423-427. https://doi.org/10.3390/ijerph5050423.).

Additionally, as GST has a critical role against oxidative damage (Elia et al. 2003ELIA AC, GALARINI R, TATICCHI MI, DÖRR AJ & MANTILACCI L. 2003. Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure. Ecotox Environ Safety 55(2): 162-167. https://doi.org/10.1016/S0147-6513(02)00123-9.), the lower level of this enzyme in fish liver suggests a significant reduction of fish capacity to withstand oxidative stress following 21 days of exposure. In this way, GST mRNA expression decreased over time after TCS exposure in liver of the catfish Pelteobagrus fulvidraco (Ku et al. 2014KU P, WU X, NIE X, OU R, WANG L, SU T & LI Y. 2014. Effects of triclosan on the detoxification system in the yellow catfish (Pelteobagrus fulvidraco): expressions of CYP and GST genes and corresponding enzyme activity in phase I, II and antioxidant system. Comp Bioch Phy Part C: Tox Pharm 166: 105-114. https://doi.org/10.1016/j.cbpc.2014.07.006.). This inhibition could be due to an incomplete degradation of TCS absorbed by the fish. Due to the structural similarity of TCS to polychlorobiphenols, bisphenol A and dioxins, it is thought that TCS could act as a selective inhibitor, as well as a substrate, for phase II enzymes (Wang et al. 2004WANG LQ, FALANY CN & JAMES MO. 2004. Triclosan as a substrate and inhibitor of 3’-phosphoadenosine 5’-phosphosulfate-sulfotransferase and UDP-glucuronosyl transferase in human liver fractions. Drug Metab Dispos 32(10): 1162-1169. https://10.1124/dmd.104.000273.).

ACAP levels allow to emphasize the importance of understanding how antioxidants interact with reactive oxygen species (ROS) through the determination of the total antioxidant capacity, instead of the measurement of limited number of antioxidants (Amado et al. 2009AMADO LL, GARCIA ML, RAMOS PB, FREITAS RF, ZAFALON B, JOSENCLER LUIS RIBAS FERREIRA JLR, YUNES JS & MONSERRAT JM. 2009. A method to measure total an-tioxidant capacity against peroxyl radicals in aquatic organisms: application to evaluate microcystins toxicity. Sci Total Environ: 2115-2123. https://doi.org/10.1016/j.scitotenv.2008.11.038., Ale et al. 2018bALE A, BACCHETTA C, ROSSI A, GALDOPÓRPORA J, DESIMONE M, DE LA TORRE FR, GERVASIO S & CAZENAVE J. 2018b. Nanosilver toxicity in gills of a neotropical fish: metal accumulation, oxidative stress, histopathology and other physiological effects. Ecotox Environ Safety 148: 976-984. https://doi.org/10.1016/j.ecoenv.2017.11.072.). As was mentioned before, ACAP decreased in liver after 2 and 21 days of exposure to sediment spiked with TCS. This could indicate that MeTCS in water impaired the ability of liver to cope with ROS and therefore, leads to a decreased capacity of cells to neutralize peroxyl radicals. This reduction of antioxidant capacity could have led this organ to suffer from oxidative stress. In this sense, lipid damage was found in liver after 21 days, which confirmed that spiked sediment with TCS induced disrupted the redox balance.

Sediment spiked with TCS promoted the same responses in liver of fish as TCS present in water after prolonged exposure. Paul et al. (2020)PAUL T, KUMAR S, SHUKLA SP, PAL P, KUMAR K, POOJARY N & MISHRA A. 2020. A multi-biomarker approach using integrated biomarker response to assess the effect of pH on triclosan toxicity in Pangasianodon hypophthalmus (Sauvage, 1878). Environ Poll 260: 114001. https://doi.org/10.1016/j.envpol.2020.114001. found in P. hypophthalmus a significant increase of SOD, CAT and GST activity in liver, after 30 days exposure to 91 and 182 µg L^-1 of TCS. Likewise, Li et al. (2018)LI C, QU R, CHEN J, ZHANG S, ALLAM AA, AJAREM J & WANG Z. 2018. The pH-dependent toxicity of triclosan to five aquatic organisms (Daphnia magna, Photobacterium phosphoreum, Danio rerio, Limnodrilus hoffmeisteri, and Carassius auratus). Environ Sci Pollut Res 25(10): 9636-9646. https://doi.org/10.1007/s11356-018-1284-z. observed increased liver CAT activity and lipid damage in Carassius auratus after 15 days of exposure to 50 µg L^-1 of TCS. Hemalatha et al. (2019)HEMALATHA D, NATARAJ B, RANGASAMY B, CHELLAPPAN S & MATHAN R. 2019. DNA damage and physiological responses in an Indian major carp Labeo rohita exposed to an antimicrobial agent triclosan. Fish Physiol Biochem 45: 1463-1484. https://doi.org/10.1007/s10695-019-00661-2. reported the same effect in the liver of Labeo rohita after a prolonged exposure to TCS.

Short term toxicity in gills is attributable to TCS entering fish body mainly through passive diffusion across the gill membrane, resulting in an enhanced toxicity in this organ (Khatikarn et al. 2018KHATIKARN J, SATAPORNVANIT K, PRICE OR & DEN BRINK PJV. 2018. Effects of triclosan on aquatic invertebrates in tropics and the influence of pH on its toxicity on micro-algae. Environ Sci Pollut Res 25: 13244-13253. https://doi.org/10.1007/s11356-016-7302-0.). In our study, the gills were the only organ that showed increased lipid peroxidation levels in D. rerio after both times of exposure to sediment spiked with TCS. Gills are the major site of uptake for most waterborne toxicants and the main site of toxic impact for many of them (Ale et al. 2018bALE A, BACCHETTA C, ROSSI A, GALDOPÓRPORA J, DESIMONE M, DE LA TORRE FR, GERVASIO S & CAZENAVE J. 2018b. Nanosilver toxicity in gills of a neotropical fish: metal accumulation, oxidative stress, histopathology and other physiological effects. Ecotox Environ Safety 148: 976-984. https://doi.org/10.1016/j.ecoenv.2017.11.072.). The enzyme GST participates in the defense against oxidative stress detoxifying endogenous harmful compounds like hydroxyalkenals, breakdown products of lipid peroxidation (Cnubeen et al. 2001). In this sense, the increased lipid damage could have generated a great number of metabolites of lipid peroxidation producing the observed GST inhibition after the short term exposure (Farombi et al. 2008FAROMBI E, AJIMOKO Y & ADELOWO O. 2008. Effect of butachlor on antioxidant enzyme status and lipid peroxidation in fresh water African catfish, (Clarias gariepinus). Inter J Environ Res Pub Health 5(5): 423-427. https://doi.org/10.3390/ijerph5050423.). Similar GST responses were reported after 48-hour Channa punctatus exposure to deltamethrin (Sayeed et al. 2003SAYEED I, PARVEZ S, PANDEY S, BIN-HAFEEZ B, HAQUE R & RAISUDDIN S. 2003. Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotox Environ Safety 56(2): 295-301. https://doi.org/10.1016/S0147-6513(03)00009-5.).

The higher SOD activity, observed in gills after both periods of exposure, suggests an increase in superoxide anion and a response to counteract them by the cells. However, it was not enough to prevent lipid damage in this tissue in neither of the two exposure periods tested. Similar results were reported by Paul et al. (2020)PAUL T, KUMAR S, SHUKLA SP, PAL P, KUMAR K, POOJARY N & MISHRA A. 2020. A multi-biomarker approach using integrated biomarker response to assess the effect of pH on triclosan toxicity in Pangasianodon hypophthalmus (Sauvage, 1878). Environ Poll 260: 114001. https://doi.org/10.1016/j.envpol.2020.114001. in gills of P. hypophthalmus after different TCS concentrations and pH values.

Likewise, the decrease in the total capacity against peroxyl radicals after 21 days would allow asserting that after prolonged exposure antioxidant response in gills of D. rerio was decreased. Therefore, the sediment enriched with TCS (and/or MeTCS) produced a decrease ability of the branchial cells to counteract peroxyl radicals. In agreement with our results, Wang et al. (2017)WANG F, XU R, ZHENG F & LIU H. 2017. Effects of triclosan on acute toxicity, genetic toxicity and oxidative stress in goldfish (Carassius auratus). Experi Anim 17-0101: https://doi.org/10.1016/j.chemosphere.2018.01.163. showed in Carassius auratus a decreased in capacity against radicals after 14 days of exposure to TCS. Furthermore, Wang et al. (2019)WANG F, WANG R, LIU F & CHEN W. 2019. Gene expression profiles in brain of male juvenile zebrafish (Danio rerio) treated with triclosan. Toxicol Apli Pharm 362: 35-42. https://doi.org/10.1016/j.taap.2018.10.014. found decreased antioxidant defenses in D. rerio after 42 days of exposure to TCS.

The brain was the less sensitive organ in D. rerio exposed to sediment spiked with TCS. GST activity was increased in both exposure periods and similar results were reported by Araújo et al. (2019)ARAÚJO CVM, QUINTANEIRO AM & MONTEIRO MS. 2019. Chemosphere effects of Triclosan on early development of Solea senegalensis: from Biochemical to individual level. Chemosphere 235: 885-899. https://doi.org/10.1016/j.chemosphere.2019.06.183. in larvae of D. rerio after 22 days of TCS exposure. This mechanism could explain the absence of lipid damage in this tissue. In agreement with our results, Paul et al. (2020)PAUL T, KUMAR S, SHUKLA SP, PAL P, KUMAR K, POOJARY N & MISHRA A. 2020. A multi-biomarker approach using integrated biomarker response to assess the effect of pH on triclosan toxicity in Pangasianodon hypophthalmus (Sauvage, 1878). Environ Poll 260: 114001. https://doi.org/10.1016/j.envpol.2020.114001. showed an increased GST activity in the brain of P. hypophthalmus after 30 days of exposure to TCS, meanwhile Gyimah et al. (2020)GYIMAH E, DONG X, QIU W, ZHANG Z & XU H. 2020. Sublethal concentrations of triclosan elicited oxidative stress, DNA damage, and histological alterations in the liver and brain of adult zebrafish. Environ Sci Pol Res: 1-10. https://doi.org/10.1007/s11356-020-08232-2. found inhibition of CAT activity in brain of D. rerio after exposure to TCS. Additionally, Gyimah et al. (2020)GYIMAH E, DONG X, QIU W, ZHANG Z & XU H. 2020. Sublethal concentrations of triclosan elicited oxidative stress, DNA damage, and histological alterations in the liver and brain of adult zebrafish. Environ Sci Pol Res: 1-10. https://doi.org/10.1007/s11356-020-08232-2. showed absence of brain lipid damage after prolonged exposure to TCS. Overall, contradictory results were shown in brain of fish, therefore more studies considering neurological biomarkers should be carried out.

Furthermore, potential changes occurring in brain could elicit alteration in fish behavior, including their ability to escape from predators, reproduce and compete with other fish (Araújo et al. 2016ARAÚJO CVM, MATILDE MS & RUI R. 2016. Active and Passive Spatial Avoidance by Aquatic Organisms from Environmental Stressors: A Complementary Perspective and a Critical Review. Environ International 92-93: 405-415. https://doi.org/10.1016/j.envint.2016.04.031.). In this sense, AChE is a useful biomarker of neurotoxic effects of chemicals on organisms. In the present study no differences have been observed among treatments. Although several works reported inhibitions of AChE after TCS exposure (Sahu et al. 2018SAHU VK, KARMAKAR S, KUMAR S, SHUKLA SP & KUMAR K. 2018. Triclosan toxicity alters behavioral and hematological parameters and vital antioxidant and neurological enzymes in Pangasianodon hypophthalmus (Sauvage, 1878). Aquat Toxicol 202: 145-152. https://doi.org/10.1016/j.aquatox.2018.07.009., Paul et al. 2019PAUL T, SHUKLA SP, KUMAR K, POOJARY N & KUMAR S. 2019. Effect of temperature on triclosan toxicity in Pangasianodon hypophthalmus (Sauvage, 1878): Hematology, biochemistry and genotoxicity evaluation. Sci Total Environ 668: 104-114. https://doi.org/10.1016/j.scitotenv.2019.02.443., 2020, Li et al. 2018LI C, QU R, CHEN J, ZHANG S, ALLAM AA, AJAREM J & WANG Z. 2018. The pH-dependent toxicity of triclosan to five aquatic organisms (Daphnia magna, Photobacterium phosphoreum, Danio rerio, Limnodrilus hoffmeisteri, and Carassius auratus). Environ Sci Pollut Res 25(10): 9636-9646. https://doi.org/10.1007/s11356-018-1284-z.), these studies were carried out using higher concentrations than the one used in the present work.

According to the principal component analysis, the first principal component (PC1) clearly separated fish exposed to sediment with TCS from the Ctrl, regardless of exposure time, while the effect of exposure time was explained (PC2) by oxidative damage protective enzymes. After the two times of exposure, the organs that were most affected were liver and gills, however the biomarkers that explained the variability were respectively different. After 2 days of exposure, CAT and GST enzymatic activities were the most important biomarkers on the evaluated organs which mean that after the short term exposure D. rerio was able to respond and counteract oxidative damage in liver and brain.

On the other hand, after 21 days of exposure to sediment spiked with TCS, the major changes were observed in ACAP and LPO, so after a prolonged period of exposure organisms lost the ability to counteract ROS, leading to lipid damage.

Carrying out prolonged studies reflecting an environmental scenario such as whole sediment exposures with TCS can be considered as a useful tool in environmental risk assessment. In agreement with our results, Pusceddu et al. (2018)PUSCEDDU FH, CHOUERI RB, PEREIRA CDS, CORTEZ FS, SANTOS DRA, MORENO B & CESAR A. 2018. Environmental risk assessment of triclosan and ibuprofen in marine sediments using individual and sub-individual endpoints. Environ Pollut 232: 274-283. https://doi.org/10.1016/j.envpol.2017.09.046. observed an inhibition of the embryo-larval development in Perna perna after exposure to environmental concentrations (7.5-750 µg kg^-1) of TCS in sediment. Besides, prolonged effects could be related to a multitude of sublethal more sensitive endpoints such as antioxidant responses, ROS generation, behavior and metabolism (Kar et al. 2020KAR S, SANDERSON H, ROY K, BENFENATI E & LESZCZYNSKI J. 2020. Ecotoxicological assessment of pharmaceuticals and personal care products using predictive toxicology approaches. Green Chem 22(5): 1458-1516. https://doi.org/10.1039/C9GC03265G.). Thus, TCS and MeTCS need to be monitored continuously along with their chronic toxicity.

CONCLUSIONS

The battery of biochemical markers assessed in Danio rerio provided a greater understanding of the ecotoxicity of sediments contaminated with TCS. The characterization of compounds such as TCS and MeTCS in the matrices, allowed to make direct inferences about the contaminant and their effects on D. rerio. Liver and gills were the most sensitive organs; while SOD activity, lipid damage, and ACAP levels were the most sensitive biomarkers. Whole sediment exposure assay conducted with a non-benthic model organism also highlighted effects elicited from an important sink of TCS metabolite (MeTCS). To our knowledge, this is the first attempt to evaluate the exposure of sediment spiked with this emergent pollutant and provide a more realistic scenario of exposure, whereas non-target organisms may be threatened and therefore also the whole ecosystem integrity.

ACKNOWLEDGMENTS

This study was funded by the Agencia Nacional de Promoción Científica y Tecnológica, ANPCyT (PICT 2014 Nº 2228, F. de la Torre) and Departamento de Ciencias Básicas e INEDES, Universidad Nacional de Luján. This work is part of the PhD thesis of E. Sager.

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