Abstract
Triazoles are among the most widely used fungicides in the world due to their efficacy against fungal crop diseases and their broad spectrum of action. Intensive use of triazoles has resulted in residual contamination in different compartments of agroecosystems and exposes non-target species to potential sublethal effects. Triazoles are known to be immunomodulators in medicine and therapeutic treatments, but very little data is available on their potential effect on immune parameters of non-target vertebrate species living in agroecosystems. In this study, we experimentally examined the impact of tebuconazole on three immune biomarkers (haemagglutination titre (HA), haemolysis titre (HL), and haptoglobin concentration (Hp)), as well as on the body condition of house sparrows (Passer domesticus). Our results suggest that tebuconazole had very little, if any, effect on the studied immune parameters. However, further studies are needed to better assess the effect of tebuconazole on bird immunity because (1) experimental individuals were kept under optimal conditions and the impact of tebuconazole on immunity may occur under suboptimal conditions, (2) only one concentration of tebuconazole was tested and its effect could be dose-dependent and (3) other complementary immunological biomarkers should be studied, given the complexity of the vertebrate immune system. Current knowledge on the potential effects of triazoles on the immunity of wild farmland vertebrates is still largely insufficient. Further physiological and immune studies should be conducted to better understand the effect of triazole fungicides on farmland birds.
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References
Alayash AI (2004) Redox biology of blood. Antioxidants & Redox Signaling 6(6):941–943. https://doi.org/10.1089/ars.2004.6.941
Ali, S, Ullah, MI, Sajjad, A, Shakeel, Q, & Hussain, A (2021). Environmental and Health Effects of Pesticide Residues. In Inamuddin, M. I. Ahamed, & E Lichtfouse (Éds.), Sustainable Agriculture Reviews 48: Pesticide Occurrence, Analysis and Remediation Vol. 2 Analysis (p. 311-336). Springer International Publishing. https://doi.org/10.1007/978-3-030-54719-6_8
Andersen CBF, Stødkilde K, Saederup KL, Kuhlee A, Raunser S, Graversen JH, Moestrup SK (2017) Haptoglobin. Antioxidants & Redox Signaling 26(14):814–831. https://doi.org/10.1089/ars.2016.6793
Angelier F, Tonra CM, Holberton RL, Marra PP (2011) Short‐term changes in body condition in relation to habitat and rainfall abundance in American Redstarts Setophaga ruticilla during the non‐breeding season. Journal of Avian Biology 42(4):335–341
Angelier F, Prouteau L, Brischoux F, Chastel O, Devier M-H, Le Menach K, Martin S, Mohring B, Pardon P, Budzinski H (2023) High contamination of a sentinel vertebrate species by azoles in vineyards: A study of common blackbirds (Turdus merula) in multiple habitats in western France. Environmental Pollution 316:120655. https://doi.org/10.1016/j.envpol.2022.120655
Bartholomew GA, Cade TJ (1963) The water economy of land birds. The Auk 80(4):504–539. https://doi.org/10.2307/4082856
Bellot P, Dupont SM, Brischoux F, Budzinski H, Chastel O, Fritsch C, Lourdais O, Prouteau L, Rocchi S, Angelier F (2022) Experimental exposure to Tebuconazole Affects metabolism and body condition in a Passerine Bird, the House Sparrow (Passer domesticus). Environmental Toxicology and Chemistry 41(10):2500–2511. https://doi.org/10.1002/etc.5446
Berenzen N, Lentzen-Godding A, Probst M, Schulz H, Schulz R, Liess M (2005) A comparison of predicted and measured levels of runoff-related pesticide concentrations in small lowland streams on a landscape level. Chemosphere 58(5):683–691. https://doi.org/10.1016/j.chemosphere.2004.05.009
Boes M (2000) Role of natural and immune IgM antibodies in immune responses. Molecular immunology 37(18):1141–1149. https://doi.org/10.1016/S0161-5890(01)00025-6
Brown, L, Feldstein, H, Haddad, L, Peña, C, & Quisumbing, A (1995). Generating food security in the year 2020: women as producers, gatekeepers, and shock absorbers. https://www.semanticscholar.org/paper/GENERATING-FOOD-SECURITY-IN-THE-YEAR-2020%3A-WOMEN-AS-Brown-Feldstein/01f19be2a78245ec09e5ccde6c7c36d96187a929
Carvalho FP (2006) Agriculture, pesticides, food security and food safety. Environmental Science & Policy 9(7):685–692. https://doi.org/10.1016/j.envsci.2006.08.002
Carvalho FP (2017) Pesticides, environment, and food safety. Food and Energy Security 6(2):48–60. https://doi.org/10.1002/fes3.108
Delves PJ, Roitt IM (2000) The Immune System. New England Journal of Medicine 343(1):37–49. https://doi.org/10.1056/NEJM200007063430107
Désert M, Ravier S, Gille G, Quinapallo A, Armengaud A, Pochet G, Savelli J-L, Wortham H, Quivet E (2018) Spatial and temporal distribution of current-use pesticides in ambient air of Provence-Alpes-Côte-d’Azur Region and Corsica, France. Atmospheric Environment 192:241–256. https://doi.org/10.1016/j.atmosenv.2018.08.054
Dhouib I, Annabi A, Jallouli M, Marzouki S, Gharbi N, Elfazaa S, Lasram MM (2016) Carbamates pesticides induced immunotoxicity and carcinogenicity in human: A review. Journal of Applied Biomedicine 14(2):85–90. https://doi.org/10.1016/j.jab.2016.01.001
Effner R, Hiller J, Eyerich S, Traidl-Hoffmann C, Brockow K, Triggiani M, Behrendt H, Schmidt-Weber CB, Buters JTM (2017) Cytochrome P450s in human immune cells regulate IL-22 and c-Kit via an AHR feedback loop. Scientific Reports 7(1):Art. 1. https://doi.org/10.1038/srep44005
Fernández-Vizcaíno E, Fernández de Mera IG, Mougeot F, Mateo R, Ortiz-Santaliestra ME (2020) Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge. Environmental Research 189:109928. https://doi.org/10.1016/j.envres.2020.109928
Fernández-Vizcaíno E, Ortiz-Santaliestra ME, Fernández-Tizón M, Mateo R, Camarero PR, Mougeot F (2022) Bird exposure to fungicides through the consumption of treated seeds: A study of wild red-legged partridges in central Spain. Environmental Pollution 292:118335. https://doi.org/10.1016/j.envpol.2021.118335
Fernández-Vizcaíno E, Mougeot F, Cabodevilla X, Fernández-Tizón M, Mateo R, Madeira MJ, Ortiz-Santaliestra ME (2023) Diet and Spatial Ecology Influence Red-Legged Partridge Exposure to Pesticides Used as Seed Treatment. Environmental Science & Technology 57(40):14861–14870
Fidan I, Yesilyurt E, Kalkanci A, Sahin N, Aslan SO, Ogan MC, Dizbay M (2014) Immunomodulatory Effects of Voriconazole and Caspofungin on Human Peripheral Blood Mononuclear Cells Stimulated by Candida albicans and Candida krusei. The American Journal of the Medical Sciences 348(3):219–223. https://doi.org/10.1097/MAJ.0000000000000236
Fringuelli R, Schiaffella F, Vecchiarelli A (2001) Antifungal and immunomodulating activities of 1,4-benzothiazine azole derivatives: Review. Journal of Chemotherapy 13(1):9–14. https://doi.org/10.1179/joc.2001.13.1.9
Huang T, Jiang H, Zhao Y, He J, Cheng H, Martyniuk CJ (2022) A comprehensive review of 1, 2, 4-triazole fungicide toxicity in zebrafish (Danio rerio): A mitochondrial and metabolic perspective. Science of The Total Environment 809:151177
Hegemann A, Matson KD, Both C, Tieleman BI (2012) Immune function in a free-living bird varies over the annual cycle, but seasonal patterns differ between years. Oecologia 170(3):605–618. https://doi.org/10.1007/s00442-012-2339-3
Hõrak P, Saks L, Ots I, Kollist H (2002) Repeatability of condition indices in captive greenfinches (Carduelis chloris). Canadian Journal of Zoology 80(4):636–643. https://doi.org/10.1139/z02-038
Hõrak P, Saks L, Ots I, Kullissaar T, Kollist H, Zilmer M (2003) Physiological effects of immune challenge in captive greenfinches (Carduelis chloris). Canadian Journal of Zoology 81(3):371–379. https://doi.org/10.1139/z03-020
Humann-Guilleminot S, Clément S, Desprat J, Binkowski ŁJ, Glauser G, Helfenstein F (2019) A large-scale survey of house sparrows feathers reveals ubiquitous presence of neonicotinoids in farmlands. Science of the Total Environment 660:1091–1097
Janeway Jr, CA, Travers, P, Walport, M, & Shlomchik, MJ (2001). The complement system and innate immunity. In Immunobiology: The Immune System in Health and Disease. 5th edition. Garland Science.
Kahle M, Buerge IJ, Hauser A, Müller MD, Poiger T (2008) Azole fungicides: occurrence and fate in wastewater and surface waters. Environmental Science & Technology 42(19):7193–7200. https://doi.org/10.1021/es8009309
Kalogridi E-C, Christophoridis C, Bizani E, Drimaropoulou G, Fytianos K (2014) Part II: Temporal and spatial distribution of multiclass pesticide residues in lake sediments of northern Greece: application of an optimized MAE-LC-MS/MS pretreatment and analytical method. Environmental Science and Pollution Research 21(12):7252–7262. https://doi.org/10.1007/s11356-014-2794-y
Kharb R, Sharma PC, Yar MS (2011) Pharmacological significance of triazole scaffold. Journal of Enzyme Inhibition and Medicinal Chemistry 26(1):1–21. https://doi.org/10.3109/14756360903524304
Kim K-H, Kabir E, Jahan SA (2017) Exposure to pesticides and the associated human health effects. Science of The Total Environment 575:525–535. https://doi.org/10.1016/j.scitotenv.2016.09.009
Lee G-H, Choi K-C (2020) Adverse effects of pesticides on the functions of immune system. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 235:108789. https://doi.org/10.1016/j.cbpc.2020.108789
Liu F, Li X, Bello BK, Zhang T, Yang H, Wang K, Dong J (2022) Difenoconazole causes spleen tissue damage and immune dysfunction of carp through oxidative stress and apoptosis. Ecotoxicology and Environmental Safety 237:113563. https://doi.org/10.1016/j.ecoenv.2022.113563
Lopez-Antia A, Feliu J, Camarero PR, Ortiz-Santaliestra ME, Mateo R (2016) Risk assessment of pesticide seed treatment for farmland birds using refined field data. Journal of Applied Ecology 53(5):1373–1381. https://doi.org/10.1111/1365-2664.12668
Lopez-Antia A, Ortiz-Santaliestra ME, Mougeot F, Camarero PR, Mateo R (2018) Brood size is reduced by half in birds feeding on flutriafol-treated seeds below the recommended application rate. Environmental Pollution 243:418–426. https://doi.org/10.1016/j.envpol.2018.08.078
Lopez-Antia A, Ortiz-Santaliestra ME, Mougeot F, Camarero PR, Mateo R (2021) Birds feeding on tebuconazole treated seeds have reduced breeding output. Environmental Pollution 271:116292. https://doi.org/10.1016/j.envpol.2020.116292
Lopez-Antia A, Ortiz-Santaliestra ME, Mougeot F, Mateo R (2013) Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole. Ecotoxicology 22(1):125–138. https://doi.org/10.1007/s10646-012-1009-x
Lutnicka H, Bojarski B, Ludwikowska A, Wrońska D, Kamińska T, Szczygieł J, Troszok A, Szambelan K, Formicki G (2016) Hematological Alterations as a Response to Exposure to Selected Fungicides in Common Carp (Cyprinus carpio L.). Folia Biologica 64(4):235–244. https://doi.org/10.3409/fb64_4.235
Lv X, Pan L, Wang J, Lu L, Yan W, Zhu Y, Xu Y, Guo M, Zhuang S (2017) Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity. Environmental Pollution 222:504–512. https://doi.org/10.1016/j.envpol.2016.11.051
Machado-Neves M, Neto MJO, Miranda DC, Souza ACF, Castro MM, Sertorio MN, Carvalho TF, Matta SLP, Freitas MB (2018) Dietary Exposure to Tebuconazole Affects Testicular and Epididymal Histomorphometry in Frugivorous Bats. Bulletin of Environmental Contamination and Toxicology 101(2):197–204. https://doi.org/10.1007/s00128-018-2377-6
Mahmood, I, Imadi, SR, Shazadi, K, Gul, A, & Hakeem, KR (2016). Effects of Pesticides on Environment. In KR Hakeem, MS Akhtar, & SNA Abdullah (Éds.), Plant, Soil and Microbes: Volume 1: Implications in Crop Science (p. 253-269). Springer International Publishing. https://doi.org/10.1007/978-3-319-27455-3_13
Martin LB, Alam JL, Imboma T, Liebl AL (2010) Variation in inflammation as a correlate of range expansion in Kenyan house sparrows. Oecologia 164(2):339–347. https://doi.org/10.1007/s00442-010-1654-9
Matson KD, Horrocks NPC, Versteegh MA, Tieleman BI (2012) Baseline haptoglobin concentrations are repeatable and predictive of certain aspects of a subsequent experimentally-induced inflammatory response. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 162(1):7–15. https://doi.org/10.1016/j.cbpa.2012.01.010
Matson KD, Ricklefs RE, Klasing KC (2005) A hemolysis–hemagglutination assay for characterizing constitutive innate humoral immunity in wild and domestic birds. Developmental & Comparative Immunology 29(3):275–286. https://doi.org/10.1016/j.dci.2004.07.006
Millet S, Bennett J, Lee KA, Hau M, Klasing KC (2007) Quantifying and comparing constitutive immunity across avian species. Developmental & Comparative Immunology 31(2):188–201. https://doi.org/10.1016/j.dci.2006.05.013
Mokarizadeh A, Faryabi MR, Rezvanfar MA, Abdollahi M (2015) A comprehensive review of pesticides and the immune dysregulation: Mechanisms, evidence and consequences. Toxicology Mechanisms and Methods 25(4):258–278. https://doi.org/10.3109/15376516.2015.1020182
Moreno-Rueda G (2011) Trade-off between immune response and body mass in wintering house sparrows (Passer domesticus). Ecological Research 26(5):943–947
Morfin R (2002) Involvement of steroids and cytochromes P450 species in the triggering of immune defenses. The Journal of Steroid Biochemistry and Molecular Biology 80(3):273–290. https://doi.org/10.1016/S0960-0760(02)00025-0
Moser VC, Barone Jr S, Smialowicz RJ, Harris MW, Davis BJ, Overstreet D, Mauney M, Chapin RE (2001) The effects of perinatal tebuconazole exposure on adult neurological, immunological, and reproductive function in rats. Toxicological Sciences 62(2):339–352. https://doi.org/10.1093/toxsci/62.2.339
Mu X, Chai T, Wang K, Zhang J, Zhu L, Li X, Wang C (2015) Occurrence and origin of sensitivity toward difenoconazole in zebrafish (Danio reio) during different life stages. Aquatic Toxicology 160:57–68. https://doi.org/10.1016/j.aquatox.2015.01.001
Naranjo TW, Lopera D, Zuluaga AF, Cano LE (2016) Immunomodulatory activity of itraconazole in lung. Tropical Journal of Pharmaceutical Research 15(12):Art. 12. https://doi.org/10.4314/tjpr.v15i12.10
Ochsenbein AF, Zinkernagel RM (2000) Natural antibodies and complement link innate and acquired immunity. Immunology Today 21(12):624–630. https://doi.org/10.1016/S0167-5699(00)01754-0
Ortiz-Santaliestra ME, Alcaide V, Camarero PR, Mateo R, Mougeot F (2020) Egg overspray with herbicides and fungicides reduces survival of red-legged Partridge chicks. Environmental Science & Technology 54(19):12402–12411. https://doi.org/10.1021/acs.est.0c04203
Pandey SP, Mohanty B (2015) The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupt the pituitary–thyroid axis of a wildlife bird. Chemosphere 122:227–234. https://doi.org/10.1016/j.chemosphere.2014.11.061
Parkin J, Cohen B (2001) An overview of the immune system. The Lancet 357(9270):1777–1789. https://doi.org/10.1016/S0140-6736(00)04904-7
Pelosi C, Bertrand C, Daniele G, Coeurdassier M, Benoit P, Nélieu S, Lafay F, Bretagnolle V, Gaba S, Vulliet E, Fritsch C (2021) Residues of currently used pesticides in soils and earthworms: A silent threat? Agriculture, Ecosystems & Environment 305:107167. https://doi.org/10.1016/j.agee.2020.107167
Pesce, S, Mamy, L, Sanchez, W, Amichot, M, Artigas, J, Aviron, S, … & Leenhardt, S (2023). Main conclusions and perspectives from the collective scientific assessment of the effects of plant protection products on biodiversity and ecosystem services along the land–sea continuum in France and French overseas territories. Environmental Science and Pollution Research, 1-16. https://doi.org/10.1007/s11356-023-26952-z
Peyton L, Gallagher S, Hashemzadeh M (2015) Triazole antifungals: A review. Drugs of today (Barcelona, Spain: 1998) 51:705–718. https://doi.org/10.1358/dot.2015.51.12.2421058
Pimentel D (1996) Green revolution agriculture and chemical hazards. Science of The Total Environment 188:S86–S98. https://doi.org/10.1016/0048-9697(96)05280-1
Pimentel D, Pimentel M (1990) Comment: Adverse environmental consequences of the green revolution. Population and Development Review 16:329–332. https://doi.org/10.2307/2808081
Quaye IK (2008) Haptoglobin, inflammation and disease. Transactions of The Royal Society of Tropical Medicine and Hygiene 102(8):735–742. https://doi.org/10.1016/j.trstmh.2008.04.010
Rokbani O, Fattouch S, Chakir A, Roth E (2019) Heterogeneous oxidation of two triazole pesticides (diniconazole and tebuconazole) by OH-radicals and ozone. Science of The Total Environment 694:133745. https://doi.org/10.1016/j.scitotenv.2019.133745
Sadrzadeh SM, Graf E, Panter SS, Hallaway PE, Eaton JW (1984) Hemoglobin. A biologic fenton reagent. Journal of Biological Chemistry 259(23):14354–14356. https://doi.org/10.1016/S0021-9258(17)42604-4
Saxena AK, Devillers J, Bhunia SS, Bro E (2015) Modelling inhibition of avian aromatase by azole pesticides. SAR and QSAR in Environmental Research 26(7-9):757–782. https://doi.org/10.1080/1062936X.2015.1090749
Shen J, Liu P, Sun Y, Xu X, Guo L, Rao Q, Chen M, Liu X (2021) Embryonic exposure to prothioconazole induces oxidative stress and apoptosis in zebrafish (Danio rerio) early life stage. Science of The Total Environment 756:143859. https://doi.org/10.1016/j.scitotenv.2020.143859
Souders CL, Xavier P, Perez-Rodriguez V, Ector N, Zhang J-L, Martyniuk CJ (2019) Sub-lethal effects of the triazole fungicide propiconazole on zebrafish (Danio rerio) development, oxidative respiration, and larval locomotor activity. Neurotoxicology and Teratology 74:106809. https://doi.org/10.1016/j.ntt.2019.106809
Swanson DL, Olmstead KL (1999) Evidence for a proximate influence of winter temperature on metabolism in passerine birds. Physiological and Biochemical Zoology 72(5):566–575
Taxvig C, Hass U, Axelstad M, Dalgaard M, Boberg J, Andeasen HR, Vinggaard AM (2007) Endocrine-disrupting activities in vivo of the Fungicides Tebuconazole and Epoxiconazole. Toxicological Sciences 100(2):464–473. https://doi.org/10.1093/toxsci/kfm227
Teng M, Zhu W, Wang D, Qi S, Wang Y, Yan J, ... & Wang C (2018) Metabolomics and transcriptomics reveal the toxicity of difenoconazole to the early life stages of zebrafish (Danio rerio). Aquatic Toxicology, 194:112–120
Tian S, Teng M, Meng Z, Yan S, Jia M, Li R, Liu L, Yan J, Zhou Z, Zhu W (2019) Toxicity effects in zebrafish embryos (Danio rerio) induced by prothioconazole. Environmental Pollution 255:113269. https://doi.org/10.1016/j.envpol.2019.113269
Toda M, Beer KD, Kuivila KM, Chiller TM, Jackson BR (2021) Trends in agricultural Triazole fungicide use in the United States, 1992–2016 and possible implications for antifungal-resistant fungi in human disease. Environmental Health Perspectives 129(5):055001. https://doi.org/10.1289/EHP7484
Valadas J, Mocelin R, Sachett A, Marcon M, Zanette RA, Dallegrave E, Herrmann AP, Piato A (2019) Propiconazole induces abnormal behavior and oxidative stress in zebrafish. Environmental Science and Pollution Research 26(27):27808–27815. https://doi.org/10.1007/s11356-019-05977-3
Wakelin, D (1996). Immunity to Parasites: How Parasitic Infections are Controlled. Cambridge University Press.
Yamaguchi H, Abe S, Tokuda Y (1993) Immunomodulating activity of antifungal drugs. Annals of the New York Academy of Sciences 685:447–457. https://doi.org/10.1111/j.1749-6632.1993.tb35905.x
Yu L, Chen M, Liu Y, Gui W, Zhu G (2013) Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole. Aquatic Toxicology 138-139:35–42. https://doi.org/10.1016/j.aquatox.2013.04.001
Zarn JA, Br, üschweiler BJ, Schlatter JR (2003) Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environmental Health Perspectives 111(3):255–261. https://doi.org/10.1289/ehp.5785
Acknowledgements
This research has been funded by the Agence Nationale de la Recherche (ANR project VITIBIRD ANR-20-CE34-0008 to F.A.), by the Région Nouvelle-Aquitaine (MULTISTRESS project), by the Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES project BiodiTox #2019/1/031 to F.B.), by the Centre National de la Recherche Scientifique and by MITI-PEPSAN (mission for transversal and interdisciplinary initiatives to C.F.), by the Contrats de Plan Etat-Région (CPER) – ECONAT and by Fonds Européen de Développement Régional (FEDER) ECOTOXAGRI.
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BP: Conceptualization, Data curation, Formal analysis, Writing – Original Draft, Visualization. CB: Methodology, Data curation, Validation, Writing – Review & Editing. BF: Conceptualization, Validation, Writing – Review & Editing, Funding acquisition. FC: Writing – Review & Editing, Funding acquisition. HS: Data curation, Validation, Writing – Review & Editing. QA: Methodology, Data curation. AF: Conceptualization, Validation, Resources, Writing – Original Draft, Writing – Review & Editing, Supervision, Funding acquisition.
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Bellot, P., Bichet, C., Brischoux, F. et al. Experimental investigation of the effect of tebuconazole on three biomarkers of innate immunity in the house sparrow (Passer domesticus). Ecotoxicology 33, 119–129 (2024). https://doi.org/10.1007/s10646-024-02732-4
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DOI: https://doi.org/10.1007/s10646-024-02732-4