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A cocktail of contaminants: how mixtures of pesticides at low concentrations affect aquatic communities


The ubiquity of anthropogenic chemicals in nature poses a challenge to understanding how ecological communities are impacted by them. While we are rapidly gaining an understanding of how individual contaminants affect communities, communities are exposed to suites of contaminants yet investigations of the effects of diverse contaminant mixtures in aquatic communities are rare. I examined how a single application of five insecticides (malathion, carbaryl, chlorpyrifos, diazinon, and endosulfan) and five herbicides (glyphosate, atrazine, acetochlor, metolachlor, and 2,4-D) at low concentrations (2–16 p.p.b.) affected aquatic communities composed of zooplankton, phytoplankton, periphyton, and larval amphibians (gray tree frogs, Hyla versicolor, and leopard frogs, Rana pipiens). Using outdoor mesocosms, I examined each pesticide alone, a mix of insecticides, a mix of herbicides, and a mix of all ten pesticides. Individual pesticides had a wide range of direct and indirect effects on all trophic groups. For some taxa (i.e., zooplankton and algae), the impact of pesticide mixtures could largely be predicted from the impacts of individual pesticides; for other taxa (i.e., amphibians) it could not. For amphibians, there was an apparent direct toxic effect of endosulfan that caused 84% mortality of leopard frogs and an indirect effect induced by diazinon that caused 24% mortality of leopard frogs. When pesticides were combined, the mix of herbicides had no negative effects on the survival and metamorphosis of amphibians, but the mix of insecticides and the mix of all ten pesticides eliminated 99% of leopard frogs. Interestingly, these mixtures did not cause mortality in the gray tree frogs and, as a result, the gray tree frogs grew nearly twice as large due to reduced competition with leopard frogs. In short, wetland communities can be dramatically impacted by low concentrations of pesticides (both separate and combined) and these results offer important insights for the conservation of wetland communities.

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  1. Alford RA, Richards SJ (1999) Global amphibian declines: a problem in applied ecology. Annu Rev Ecol Syst 30:133–165

  2. Arar EJ, Collins GB (1997) In vitro determination of chlorophyll a and pheophytin a in marine and freshwater algae by flourescence. Method 445.0. National Exposure Research Laboratory, US EPA, Cincinnati

  3. Aston LS, Seiber JN (1997) Fate of summertime airborne organophosphate pesticide residues in the Sierra Nevada mountains. J Environ Qual 26:1483–1492

  4. Barry MJ, Logan DC (1998) The use of temporary pond microcosms for aquatic toxicity testing: direct and indirect effects on endosulfan on community structure. Aquat Toxicol 41:101–124

  5. Battaglin WA, Thurman EM, Kalkhoff SJ, Porter SD (2003) Herbicides and transformation products in surface waters of the midwestern United States. J Am Water Resourc Assoc 39(August):743–756

  6. Berrill M, Coulson D, McGillivray L, Pauli B (1998) Toxicity of endosulfan to aquatic stages of anuran amphibians. Environ Toxicol Chem 17:1738–1744

  7. Boone MD, Semlitsch RD (2001) Interactions of an insecticide with larval density and predation in experimental amphibian communities. Conserv Biol 15:228–238

  8. Boone MD, Bridges CM, Fairchild JF, Little EE (2005) Multiple sublethal chemicals negatively affect tadpoles of the green frog, Rana clamitans. Environ Toxicol Chem 24:1267–1272

  9. Boone MD, Bridges-Britton CM (2006) Examining multiple sublethal contaminants on the gray treefrog (Hyla versicolor): effects of an insecticide, herbicide, and fertilizer. Environ Toxicol Chem 25:3261–3265

  10. Boone MD, Semlitsch RD, Fairchild JF, Rothermel BB (2004) Effects of an insecticide on amphibians in large-scale experimental ponds. Ecol Appl 14:685–691

  11. Bridges CM, Boone MD (2003) The interactive effects of UV-B and insecticide exposure on tadpole survival, growth and development. Biol Conserv 113:49–54

  12. Britson CA, Threlkeld ST (1998) Abundance, metamorphosis, developmental, and behavioral abnormalities in Hyla chrysoscelis tadpoles following exposure to three agrichemicals and methyl mercury in outdoor mesocosms. Bull Environ Contam Toxicol 61:154–161

  13. Broomhall SD (2002) The effects of endosulfan and variable water temperature on survivorship and subsequent vulnerability to predation in Litoria citropa tadpoles. Aquat Toxicol 61:243–250

  14. California Department of Fish and Game (1982) Monitored aquatic incidents during broadscale aerial application over San Francisco Bay area, 1981. Administrative report 82-2. Sacramento

  15. Christin MS, Gendron AD, Brousseau P, Menard L, Marcogliese DJ, Cyr D, Ruby S, Fournier M (2003) Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection. Environ Toxicol Chem 22:1127–1133

  16. Christin MS, Menard L, Gendron AD, Ruby S, Cyr D, Marcogliese DJ, Rollins-Smith L, Fournier M (2004) Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens. Aquat Toxicol 67:33–43

  17. Dahl T (2000) Status and trends of wetlands in the conterminous united states 1986–1997. US Department of the Interior, Fish and Wildlife Service, Washington, DC

  18. Daly GL, Lei YD, Teixeira A, Muir DCG, Castillo LE, Wania F (2007) Accumulation of current-use pesticides in neotropical montane forests. Environ Sci Technol 41:1118–1123

  19. Davidson C, Knapp RA (2007) Multiple stressors and amphibian declines: dual impacts of pesticides and fish on yellow-legged frogs. Ecol Appl 17(2):587–597

  20. Davidson C, Shafer HB, Jennings MR (2001) Declines of the California red-legged frog: climate, UV-B, habitat, and pesticides hypotheses. Ecol Appl 11:464–479

  21. Davidson C, Shafer HB, Jennings MR (2002) Spatial tests of the pesticide drift, habitat destruction, UV-B, and climate-change hypotheses for California amphibian declines. Conserv Biol 16:1588–1601

  22. de Noyelles F, Dewey SL, Huggins DG, Kettle WD (1994) Aquatic mesocosms in ecological effects testing: detecting direct and indirect effects of pesticides. In: Graney RL, Kennedy JH, Rodgers JH Jr (eds) Aquatic mesocosm studies in ecological risk assessment. Lewis, Boca Raton, pp 577–603

  23. Denver RJ, Mirhadi N, Phillips M (1998) Adaptive plasticity in amphibian metamorphosis: response of Scaphiopus hammondii tadpoles to habitat desiccation. Ecology 19:1859–1872

  24. Edwards WM, Triplett GB Jr, Kramer RM (1980) A watershed study of glyphosate transport in runoff. J Environ Qual 9:661–665

  25. Faust M, Altenburger R, Backhaus T, Blanck H, Boedeker W, Gramatica P, Hamer V, Scholze M, Vighi M, Grime LH (2003) Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action. Aquat Toxicol 63:43–63

  26. Fernandez-Casalderrey A, Ferrando MD, Andreu-Moliner E (1994) Effect of sublethal concentrations of pesticides on the feeding behavior of Daphnia magna. Ecotoxicol Environ Saf 27:82–89

  27. Fleeger JW, Carman KR, Nisbet RM (2003) Indirect effects of contaminants in aquatic ecosystems. Sci Total Environ 317:207–233

  28. Gendron AD, Marcogliese DJ, Barbeau S, Christin MS, Brousseau P, Ruby S, Cyr D, Fournier M (2003) Exposure of leopard frogs to a pesticide mixture affects life history characteristics of the lungworm Rhabdias ranae. Oecologia 135:469–476

  29. Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16:183–190

  30. Gilliom RJ, Barbash JE, Crawford CG, Hamilton PA, Martin JD, Nakagaki N, Nowell LH, Scott JC, Stackelberg PE, Thelin GP, Wolock DM (2007) The quality of our nation’s waters—pesticides in the nation’s streams and ground water, 1992–2001. US Geological Survey circular 1291

  31. Hageman KJ, Simonich SL, Campbell DH, Wilson GR, Landers DH (2006) Atmospheric deposition of current-use and historic-use pesticides in snow at national parks in the western United States. Environ Sci Technol 40:3174–3180

  32. Hanazato T, Yasuno M (1989) Effects of carbaryl on spring zooplankton communities in ponds. Environ Pollut 56:1–10

  33. Hanazato T, Yasuno M (1990) Influence of time of application of an insecticide on recovery patterns of a zooplankton community in experimental ponds. Arch Environ Contam Toxicol 19:77–83

  34. Hanazato T, Yasuno M (1987) Effects of a carbamate insecticide, carbaryl, on summer phyto- and zooplankton communities in ponds. Environ Pollut 48:145–159

  35. Havens KE (1994) An experimental comparison of effects of two chemical stressors on a freshwater zooplankton assemblage. Environ Pollut 84:245–251

  36. Havens KE (1995) Insecticide (carbaryl, 1-napthyl-n-methylcarbamate) effects on a freshwater plankton community: zooplankton size, biomass, and algal abundance. Water Air Soil Pollut 84:1–10

  37. Hayes TB, Case P, Chui S, Chung D, Haeffele C, Haston K, Lee M, Mai VP, Marjuoa Y, Parker J, Tsui M (2006) Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact? Environ Health Perspect 114(1):40–50

  38. Hose GC, van den Brink PJ (2004) Confirming the species-sensitivity distribution for endosulfan using laboratory, mesocosm, and field data. Archiv Environ Contam Toxicol 47:511–5220

  39. Howe GE, Gillis R, Mowbray RC (1998) Effect of chemical synergy and larval stage on the toxicity of atrazine and alachlor to amphibian larvae. Environ Toxicol Chem 17:519–525

  40. Kashian DR, Dodson SI (2002) Effects of common-use pesticides on developmental and reproductive processes in Daphnia. Toxicol Ind Health 18:225–235

  41. Kiely T, Donaldson D, Grube A (2004) Pesticide industry sales and usage: 2000 and 2001 market estimates. US EPA, Washington, DC

  42. LeNoir JS, McConnell LL, Fellers GM, Cahill TM, Seiber JN (1999) Summertime transport of current-use pesticides from California’s Central Valley to the Sierra Nevada mountain range, USA. Environ Toxicol Chem 18:2715–2722

  43. McConnell LL, LeNoir JS, Datta S, Seiber JN (1998) Wet deposition of current-use pesticides in the Sierra Nevada mountain range, California, USA. Environ Toxicol Chem 17:1908–1916

  44. Mills NE, Semlitsch RD (2004) Competition and predation mediate the indirect effects of an insecticide on southern leopard frogs. Ecol Appl 14:1041–1054

  45. Munn MD, Gilliom RJ, Moran PW, Nowell LH (2006) Pesticide toxicity index for freshwater aquatic organisms, 2nd edn. US Geological Survey scientific investigations report 2006-5148

  46. Muschal M (1997) Central and Northwest regions water quality program: 1997/97 report on pesticides monitoring. Department of Land and Water Conservation, Sydney

  47. Norris LA, Lorz HW, Gregory SZ (1983) Influence of forest and range land management on anadromous fish habitat in western North America: forest chemicals. PNW-149. General technical report. USDA. Forest Service, Portland

  48. Relyea RA (2004) The growth and survival of five amphibian species exposed to combinations of pesticides. Environ Toxicol Chem 23:1737–1742

  49. Relyea RA (2005) The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 15:618–627

  50. Relyea RA, Hoverman JT (2006) Assessing the ecology in ecotoxicology: a review and synthesis in freshwater systems. Ecol Lett 9:1157–1171

  51. Relyea RA, Diecks N (2008) An unforeseen chain of events: lethal effects of pesticides at sublethal concentrations. Ecol Appl 18:1728–1742

  52. Ridal JJ, Mazumder A, Lean DRS (2001) Effects of nutrient loading and planktivory on the accumulation of organochlorine pesticides in aquatic food chains. Environ Toxicol Chem 20:1312–1319

  53. Rohr JR, Elskus AA, Shepherd BS, Crowley PH, McCarthy TM, Niedzwiecki JH, Sager T, Sih A, Palmer BD (2003) Lethal and sublethal effects of atrazine, carbaryl, endosulfan, and octylphenol on the streamside salamander (Ambystoma barbouri). Environ Toxicol Chem 22:2385–2392

  54. Rohr JR, Crumrine PW (2005) Effects of a herbicide and an insecticide on pond community structure and processes. Ecol Appl 15:1135–1147

  55. Solomon KR, Baker DB, Richards RP, Dixon KR, Klaine SJ, La Point TW, Kendall RJ, Weisskopf CP, Giddings JM, Giesy JP, Hall LW Jr, Williams WM (1996) Ecological risk assessment of atrazine in North American surface waters. Environ Toxicol Chem 15:31–76

  56. Sparling DW, Fellers GM, McConnell LL (2001) Pesticides and amphibian population declines in California, USA. Environ Toxicol Chem 20:1591–1595

  57. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW (2004) Status and trends of amphibian declines and extinctions worldwide. Science 306:1783–1786

  58. US EPA (2006) 2006 edition of the drinking water standards and health advisories

  59. van Den Brink PJ, Hartgers EM, Glystra R, Bransen F, Brock TCM (2002) Effects of a mixture of two insecticides in freshwater microcosms. II. Responses of plankton and ecological risk assessment. Ecotoxicology 11:181–197

  60. van Wijngaarden RPA, Cuppen JGM, Arts GHP, Crum SJH, van den Hoorn MW, van den Brink PJ, Brock TCM (2004) Aquatic risk assessment of a realistic exposure to pesticides used in bulb crops: a microcosm study. Environ Toxicol Chem 23:1479–1498

  61. van Wijngaarden RPA, Brock TCM, Douglas MT (2005) Effects of chlorpyrifos in freshwater model ecosystems: the influence of experimental conditions on ecotoxicological thresholds. Pest Manage Sci 61:923–935

  62. Wendt-Rasch L, van den Brink PJ, Crum SJH, Woin P (2004) The effects of a pesticide mixture on aquatic ecosystems differing in trophic status: responses of the macrophyte Myriophylum spicatum and the periphytic algal community. Ecotoxicol Environ Saf 57:383–398

  63. Wetzel RG, Likens GE (2000) Limnological analyses. Springer, New York, p 429

  64. Zabik JM, Seiber JN (1993) Atmospheric transport of organophosphate pesticides from California Central Valley to the Sierra Nevada mountains. J Environ Qual 22:80–90

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I thank Josh Auld, Nicole Diecks, Jason Hoverman, Devin Jones, and Aaron Stoler for their assistance in executing the experiments and a special thanks to Devin Jones for collecting so many metamorphs. I also thank Josh Auld, Jason Hoverman, Maya Groner, and the anonymous reviewers for their comments on the manuscript. This work was supported by a grant from the National Science Foundation.

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Correspondence to Rick A. Relyea.

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Communicated by Ross Alford.

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Relyea, R.A. A cocktail of contaminants: how mixtures of pesticides at low concentrations affect aquatic communities. Oecologia 159, 363–376 (2009).

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  • Amphibian decline
  • Pesticide mixture
  • Food web
  • Multiple pesticides
  • Synergistic