Multiple effects of a commercial Roundup® formulation on the soil filamentous fungus Aspergillus nidulans at low doses: evidence of an unexpected impact on energetic metabolism

Abstract

Soil microorganisms are highly exposed to glyphosate-based herbicides (GBH), especially to Roundup® which is widely used worldwide. However, studies on the effects of GBH formulations on specific non-rhizosphere soil microbial species are scarce. We evaluated the toxicity of a commercial formulation of Roundup® (R450), containing 450 g/L of glyphosate (GLY), on the soil filamentous fungus Aspergillus nidulans, an experimental model microorganism. The median lethal dose (LD50) on solid media was between 90 and 112 mg/L GLY (among adjuvants, which are also included in the Roundup® formulation), which corresponds to a dilution percentage about 100 times lower than that used in agriculture. The LOAEL and NOAEL (lowest- and no-observed-adverse-effect levels) associated to morphology and growth were 33.75 and 31.5 mg/L GLY among adjuvants, respectively. The formulation R450 proved to be much more active than technical GLY. At the LD50 and lower concentrations, R450 impaired growth, cellular polarity, endocytosis, and mitochondria (average number, total volume and metabolism). In contrast with the depletion of mitochondrial activities reported in animal studies, R450 caused a stimulation of mitochondrial enzyme activities, thus revealing a different mode of action of Roundup® on energetic metabolism. These mitochondrial disruptions were also evident at a low dose corresponding to the NOAEL for macroscopic parameters, indicating that these mitochondrial biomarkers are more sensitive than those for growth and morphological ones. Altogether, our data indicate that GBH toxic effects on soil filamentous fungi, and thus potential impairment of soil ecosystems, may occur at doses far below recommended agricultural application rate.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Aparicio VC, De Gerónimo E, Marino D, Primost J, Carriquiriborde P, Costa JL (2013) Environmental fate of glyphosate and aminomethylphosphonic acid in surface waters and soil of agricultural basins. Chemosphere 93:1866–1873

    CAS  Article  Google Scholar 

  2. Astiz M, de Alaniz MJ, Marra CA (2009) Effect of pesticides on cell survival in liver and brain rat tissues. Ecotoxicol Environ Saf 72:2025–2032

    CAS  Article  Google Scholar 

  3. Aumaitre LA (2002) New feeds from genetically modified plants: substantial equivalence, nutritional equivalence and safety for animals and animal products. Productions Animales 15:97–108

    Google Scholar 

  4. Bentley R (1990) The shikimate pathway—a metabolic tree with many branches. Crit. Rev. Biochem Mol Biol 25:307–384

    CAS  Article  Google Scholar 

  5. Bøhn T, Cuhra M, Traavik T, Sanden M, Fagan J, Primicerio R (2014) Compositional differences in soybeans on the market: glyphosate accumulates in Roundup Ready GM soybeans. Food Chem 153:207–215

    Article  Google Scholar 

  6. Braconi D, Sotgiu M, Millucci L, Paffetti A, Tasso F, Alisi C, Martini S, Rappuoli R, Lusini P, Sprocati AR, Rossi C, Santucci A (2006) Comparative analysis of the effects of locally used herbicides and their active ingredients on a wild-type wine Saccharomyces cerevisiae strain. J Agric Food Chem 54:3163–3172

    CAS  Article  Google Scholar 

  7. Boocock MR, Coggins JR (1983) Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate. FEBS Lett 154:127–133

    CAS  Article  Google Scholar 

  8. Bringolf RB, Cope WG, Mosher S, Barnhart MC, Shea D (2007) Acute and chronic toxicity of glyphosate compounds to glochidia and juveniles of Lampsilis siliquoidea (Unionidae). Environ Toxicol Chem 26:2094–2100

    CAS  Article  Google Scholar 

  9. Carranza CS, Barberis CL, Chiacchiera SM, Magnoli CE (2014) Influence of the pesticides glyphosate, chlorpyrifos and atrazine on growth parameters of nonochratoxigenic Aspergillus section Nigri strains isolated from agricultural soils. J Environ Sci Health 49:747–755

    CAS  Article  Google Scholar 

  10. Catcheside DE, Storer PJ, Klein B (1985) Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli. Mol Gen Genet 199:446–451

    CAS  Article  Google Scholar 

  11. Charles IG, Keyte JW, Brammar WJ, Smith M, Hawkins AR (1986) The isolation and nucleotide sequence of the complex AROM locus of Aspergillus nidulans. Nucleic Acids Res 14:2201–2213

    CAS  Article  Google Scholar 

  12. Clair E, Linn L, Travert C, Amiel C, Séralini G-E, Panoff J-M (2012) Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus. Curr Microbiol 64:486–491

    CAS  Article  Google Scholar 

  13. Costa R, Ayscough KR (2005) Interactions between Sla1p, Lsb5p and Arf3p in yeast endocytosis. Biochem Soc Trans 33:1273–1275

    CAS  Article  Google Scholar 

  14. Cove DJ (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113:51–56

    CAS  Article  Google Scholar 

  15. Cox C (2004) Herbicide factsheet—glyphosate. J Pesticide Reform 24:10–15

    Google Scholar 

  16. Cuhra M (2015) Review of GMO safety assessment studies: glyphosate residues in Roundup Ready crops is an ignored issue. Env Sci Eur 27:20. doi:10.1186/s12302-015-0052-7

    Article  Google Scholar 

  17. Cuhra M, Traavik T, Bøhn T (2013) Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna. Ecotoxicology 22:251–262

    CAS  Article  Google Scholar 

  18. Duke SO, Baerson SR, Rimando AM (2003) Herbicides: glyphosate. In: Plimmer JR, Gammon DW, Ragsdale NN (eds) Encyclopedia of agrochemicals. John Wiley & Sons, New York, USA, pp 708–869

    Google Scholar 

  19. Duke SO, Powles SB (2008) Glyphosate: a once-in-a-century herbicide. Pest manage Sci 64:319–325

    CAS  Article  Google Scholar 

  20. Englard S, Siegel L (1969) Mitochondrial L-malate dehydrogenase of beef heart:[EC 1.1.1.37 l-Malate: NAD oxidoreductase]. Methods Enzymol 13:99–106

    CAS  Article  Google Scholar 

  21. Fansler B, Lowenstein JM (1969) Aconitase from pig heart. Methods Enzymol 13:26–30

    CAS  Article  Google Scholar 

  22. Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA, Read ND (2000) Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J Microsc 198:246–259

    CAS  Article  Google Scholar 

  23. Flipphi M, Oestreicher N, Nicolas V, Guitton A, Vélot C (2014) The Aspergillus nidulans acuL gene encodes a mitochondrial carrier required for the utilization of carbon sources that are metabolized via the TCA cycle. Fungal Genet Biol 68:9–22

    CAS  Article  Google Scholar 

  24. Funke T, Yang Y, Han H, Healy-Fried M, Olesen S, Becker A, Schönbrunn E (2009) Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli. J Biol Chem 284:9854–9860

    CAS  Article  Google Scholar 

  25. Gunatilleke IAUN, Arst HN Jr, Scazzocchio C (1976) Three genes determine the carboxin sensitivity of mitochondrial succinate oxidation in Aspergillus nidulans. Genet Res 26:297–305

    Article  Google Scholar 

  26. Haney RL, Senseman SA, Hons FM, Zuberer DA (2000) Effect of glyphosate on soil microbial activity and biomass. Weed Sci 48:89–93

    CAS  Article  Google Scholar 

  27. Hedberg D, Wallin M (2010) Effects of Roundup and glyphosate formulations on intracellular transport, microtubules and actin filaments in Xenopus laevis melanophores. Toxicol in Vitro 24:795–802

    CAS  Article  Google Scholar 

  28. Hickey PC, Swift SR, Roca MG, Read ND (2005) Live-cell imaging of filamentous fungi using vital fluorescent dyes and confocal microscopy. In: Savidge T, Pothoulakis C (eds) Methods in microbiology, vol 35, Microbial Imaging. Elsevier, London, pp 63–87

    Google Scholar 

  29. James C (2011) Global status of commercialized biotech/GM crops. ISAAA Brief No 43. ISAAA, Ithaca, NY

  30. Jiraungkoorskul W, Upatham ES, Kruatrachue M, Sahaphong S, Vichasri-Grams S, Pokethitiyook P (2003) Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus). Environ Toxicol 18:260–267

    CAS  Article  Google Scholar 

  31. Kappas A (1988) On the mutagenic and recombinogenic activity of certain herbicides in Salmonella typhimurium and in Aspergillus nidulans. Mutation Res 204:615–621

    CAS  Article  Google Scholar 

  32. Kappas A, Georgopoulos SG, Hastie AC (1974) On the genetic activity of benzimidazole and thiophanate fungicides on diploid Aspergillus nidulans. Mutation Res 26:17–27

    CAS  Article  Google Scholar 

  33. Kregiel D (2012) Succinate dehydrogenase of Saccharomyces cerevisiae—the unique enzyme of TCA cycle—current knowledge and new perspectives. In: Canuto RA (ed) Biochemistry, genetics and molecular biology “dehydrogenases”. InTech, DOI. doi:10.5772/48413

    Google Scholar 

  34. Krzysko-Lupicka T, Sudol T (2008) Interactions between glyphosate and autochthonous soil fungi surviving in aqueous solution of glyphosate. Chemosphere 71:1386–1391

  35. Langiano Vdo C, Martinez CB (2008) Toxicity and effects of a glyphosate-based herbicide on the Neotropical fish Prochilodus lineatus. Comp Biochem Physiol C Toxicol Pharmacol 147:222–231

    Article  Google Scholar 

  36. Lee H-L, Kan C-D, Tsai C-L, Liou M-J, Guo H-R (2009) Comparative effects of the formulation of glyphosate-surfactant herbicides on hemodynamics in swine. Clinical Toxicology 47:651–658

    Article  Google Scholar 

  37. Lee SC, Schmidtke SN, Dangott LJ, Shaw B (2008) Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth. Euk Cell 7:1278–1288

    CAS  Article  Google Scholar 

  38. Li N, Oquendo E, Capaldi RA, Robinson JP, He YD, Hamadeh HK, Afshari CA, Lightfoot-Dunn R, Narayanan PK (2014) A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells. Toxicol Sci 142:261–273

    CAS  Article  Google Scholar 

  39. Lipok J, Studnik H, Gruyaert S (2010) The toxicity of Roundup® 360 SL formulation and its main constituents: glyphosate and isopropylamine towards non-target water photoautotrophs. Ecotoxicol Environ Saf 73:1861–1868

    Article  Google Scholar 

  40. Lupi L, Miglioranza KS, Aparicio VC, Marino D, Bedmar F, Wunderlin DA (2015) Occurrence of glyphosate and AMPA in an agricultural watershed from the southeastern region of Argentina. Sci Total Environ 536:687–694

    CAS  Article  Google Scholar 

  41. Maggio-Hall LA, Keller NP (2004) Mitochondrial beta-oxidation in Aspergillus nidulans. Mol Microbiol 54:1173–1185

    CAS  Article  Google Scholar 

  42. Marc J, Mulner-Lorillon O, Bellé R (2004) Glyphosate-based pesticides affect cell-cycle regulation. Biol Cell 96:245–249

    CAS  Article  Google Scholar 

  43. Martinelli SD, Kinghorn JR (1994) Aspergillus: 50 years on—progress in industrial microbiology, Volume 29. Elsevier, Amsterdam - London - New York - Tokyo

    Google Scholar 

  44. Mesnage R, Defarge N, Spiroux de Vendomois J, Séralini G-E (2015) Potential toxic effects of glyphosate and its commercial formulations below regulatory limits. Food Chem Toxicol. doi:10.1016/j.fct.2015.08.012

    Google Scholar 

  45. Millstone E, Brunner E, Mayer S (1999) Beyond ‘substantial equivalence’. Nature 401:525–526

    CAS  Article  Google Scholar 

  46. Mottier A, Kientz-Bouchart V, Serpentini A, Lebel JM, Jha AN, Costil K (2013) Effects of glyphosate-based herbicides on embryo-larval development and metamorphosis in the Pacific oyster, Crassostrea gigas. Aquat Toxicol 128–129:67–78

    Article  Google Scholar 

  47. Nobels I, Spanoghe P, Haesaert G, Robbens J, Blust R (2011) Toxicity ranking and toxic mode of action evaluation of commonly used agricultural adjuvants on the basis of bacterial gene expression profiles. PLoS One 6, e24139. doi:10.1371/journal.pone.0024139

    CAS  Article  Google Scholar 

  48. Paganelli A, Gnazzo V, Acosta H, López SL, Carrasco AE (2010) Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling. Chem Res Toxicol 23:1586–1595

    CAS  Article  Google Scholar 

  49. Peixoto F (2005) Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation. Chemosphere 61:1115–1122

    CAS  Article  Google Scholar 

  50. Penalva MA (2005) Tracing the endocytic pathway of Aspergillus nidulans with FM4-64. Fungal Genet Biol 42:963–975

    CAS  Article  Google Scholar 

  51. Pereira CV, Moreira AC, Pereira SP, Machado NG, Carvalho FS, Sardao VA, Oliveira PJ (2009) Investigating drug-induced mitochondrial toxicity: a biosensor to increase drug safety? Curr Drug Saf 4:34–54

    CAS  Article  Google Scholar 

  52. Peruzzo PJ, Porta AA, Ronco AE (2008) Levels of glyphosate in surface waters, sediments and soils associated with direct sowing soybean cultivation in north pampasic region of Argentina. Environ Pollut 156:61–66

    CAS  Article  Google Scholar 

  53. Piola L, Fuchs J, Oneto ML, Basack S, Kesten E, Casabé N (2013) Comparative toxicity of two glyphosate-based formulations to Eisenia andrei under laboratory conditions. Chemosphere 91:545–551

    CAS  Article  Google Scholar 

  54. Qiu H, Gen J, Ren H, Xia X, Wang X, Yu Y (2013) Physiological and biochemical responses of Microcystis aeruginosa to glyphosate and its Roundup® formulation. J Hazard Mater 248–249:172–176

    Article  Google Scholar 

  55. Relyea RA (2005) The lethal impacts of Roundup and predatory stress on six species of North American tadpoles. Arch Environ Contam Toxicol 48:351–357

    CAS  Article  Google Scholar 

  56. Riechers DE, Wax LM, Liebl RA, Bush DR (1994) Surfactant-increased glyphosate uptake into plasma membrane vesicles isolated from common lambsquarters leaves. Plant Physiol 105:1419–1425

    Google Scholar 

  57. Roberts CF (1969) Isolation of multiple aromatic amino-acid mutants in A. nidulans. In: Roper JA, d’Azevedo JL (eds) Aspergillus news letter, Vol. 10, Sheffield, pp 19-21

  58. Sailaja KK, Satyaprasad K (2006) Degradation of glyphosate in soil and its effect on fungal population. J Environ Sci Eng 48:189–190

    CAS  Google Scholar 

  59. Simonsen L, Fomsgaard IS, Svensmark B, Spliid NH (2008) Fate and availability of glyphosate and AMPA in agricultural soil. J Environ Sci Health B 43:365–375

    CAS  Article  Google Scholar 

  60. Singer TP, Rocca E, Kearney EB (1966) Flavins and flavoproteins. In: Slater EC (ed), vol 1. Elsevier, Amsterdam, pp 391–426

    Google Scholar 

  61. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    CAS  Article  Google Scholar 

  62. Srere PA, Brazil H, Gonen (1963) Citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta Chem Scand 17:S129–S134

    CAS  Article  Google Scholar 

  63. Tsui MT, Chu LM (2003) Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52:1189–1197

    CAS  Article  Google Scholar 

  64. Wardle DA, Parkinson D (1990) Effects of three herbicides on soil microbial biomass and activity. Plant Soil 122:21–28

    CAS  Article  Google Scholar 

  65. Woodward J, Merrett MJ (1975) Induction potential for glyoxylate cycle enzymes during the cell cycle of Euglena gracilis. Eur J Biochem 55:555–559

    CAS  Article  Google Scholar 

  66. Zaller JG, Heigl F, Ruess L, Grabmaier A (2014) Glyphosate herbicide affects belowground interactions between earthworms and symbiotic mycorrhizal fungi in a model ecosystem. Sci Rep 4:5634

    CAS  Article  Google Scholar 

  67. Zobiole LH, Oliveira RS, Visentainer JV, Kremer RJ, Bellaloui N, Yamada T (2010) Glyphosate affects seed composition in glyphosate-resistant soybean. J Agric Food Chem 58:4517–4522

    CAS  Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the University Paris-Sud (UPSud), the Risk Pole from the University of Caen, the non-governmental organization “Générations Futures” and the Committee for Independent Research and Information on Genetic Engineering (CRIIGEN). It received funding from the Regional Council Ile-de-France and the UPSud. We thank Soraya Hadi for technical assistance. We are grateful to Professor J.-P. Bourdineaud and Dr. R. Mesnage for proofreading the article. We thank Claire Robinson for the English revision of the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Christian Vélot.

Additional information

Responsible editor: Philippe Garrigues

Electronic supplementary material

ESM 1

(PDF 4711 kb)

ESM 2

(MP4 13967 kb)

ESM 3

(MP4 12531 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nicolas, V., Oestreicher, N. & Vélot, C. Multiple effects of a commercial Roundup® formulation on the soil filamentous fungus Aspergillus nidulans at low doses: evidence of an unexpected impact on energetic metabolism. Environ Sci Pollut Res 23, 14393–14404 (2016). https://doi.org/10.1007/s11356-016-6596-2

Download citation

Keywords

  • Glyphosate
  • Roundup®
  • Soil
  • Aspergillus nidulans
  • Toxicity
  • Mitochondrial metabolism
  • Low doses