Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants


Agricultural, pharmaceutical, and other biologically active substances are emptied or leach into waterways and groundwater, where they can dose-relatedly cause pharmacologic or toxic effects on the resident or dependent animal species. Standard methods can be used to evaluate the effects of individual substances, but evaluation of combinations of substances is more difficult. The mathematically rigorous method of isobolographic analysis was coupled with a simple in vivo invertebrate model. Planarians were selected because they are the lowest extant species with a centralized nervous system. Neostigmine bromide and monopotassium phosphate (KH2PO4) were selected as representative of two types of potential pollutants. Neostigmine bromide and KH2PO4 individually produced dose-related lethality over a 60-minute observation period with LD50 values of 122 and 70 mM, respectively. The LD50 value of a 1:1 combination of the two was significantly different (p < 0.05) from the isobolographic line of additivity. We used planarians as a representative fresh-water species and joint-action (‘isobolographic’) analysis to examine possible interaction between pollutants. In the demonstrative example reported here, there was a subadditive interaction between a 1:1 fixed-ratio combination of neostigmine bromide (as a representative acetylcholinesterase inhibitor used in pesticides) and potassium phosphate (used in fertilizers and detergents).

Share and Cite:

R. Raffa, D. Gallo, C. Tallarida, S. Rawls and R. Tallarida, "Isobolographic Method and Invertebrate (Planarian) Model for Evaluating Combinations of Waterways Pollutants," Pharmacology & Pharmacy, Vol. 3 No. 4, 2012, pp. 381-387. doi: 10.4236/pp.2012.34051.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] P. D. Anderson, V. J. D'Aco, P. Shanahan, S. C. Chapra M. E. Buzby, V. L. Cunningham, B. M. Duplessie, E. P. Hayes, F. J. Mastrocco, N. J. Parke, J. C. Rader, J. H. Samuelian and B. W. Schwab, "Screening analysis of human pharmaceutical compounds in U.S. surface waters," Environ Sci Technol, Vol. 38, 2004, pp. 838- 849.
[2] S. Mompelat, O. Thomas and B. Le Bot, "Contamination levels of human pharmaceutical compounds in French surface and drinking water," J Environ Monit, Vol. 13, 2011, pp. 2929-2939.
[3] J. L. Martinez, "Environmental pollution by antibiotics and by antibiotic resistance determinants," Environ Pollut, Vol. 157, 2009, pp. 2893-2902.
[4] S. Zheng, X. Qiu, B. Chen, X. Yu, Z. Liu, G. Zhong, H. Li, M. Chen, G. Sun, H. Huang, W. Yu and D. Free-stone, "Antibiotics pollution in Jiulong River estuary: source, distribution and bacterial resistance," Chemosphere, Vol. 84, 2011, pp. 1677-1685.
[5] B. Meyer, J. Y. Pailler, C. Guignard, L. Hoffmann and A. Krein, "Concentrations of dissolved herbicides and pharmaceuticals in a small river in Luxembourg," Environ Monit Assess, Vol. 180, 2011, pp. 127-146.
[6] J. Martin, D. Camacho-Munoz, J. L. Santos, I. Aparicio and E. Alonso, "Monitoring of pharmaceutically active compounds on the Guadalquivir River basin (Spain): occurrence and risk assessment," J Environ Monit, Vol. 13, 2011, pp. 2042-2049.
[7] Q. Sui, J. Huang, S. Deng, W. Chen and G. Yu, "Seasonal variation in the occurrence and removal of pharmaceuticals and personal care products in different biological wastewater treatment processes," Environ Sci Technol, Vol. 45, 2011, pp. 3341-3348.
[8] S. Kleywegt, V. Pileggi, P. Yang, C. Hao, X. Zhao, C. Rocks, S. Thach, P. Cheung and B. Whitehead, "Pharm-aceuticals, hormones and bisphenol A in untreated source and finished drinking water in Ontario, Canada-occurrence and treatment efficiency," Sci Total Environ, Vol. 409, 2011, pp. 1481-1488.
[9] D. Fatta-Kassinos, S. Meric and A. Nikolaou, "Pharma-ceutical residues in environmental waters and waste-water: current state of knowledge and future research," Anal Bioanal Chem, Vol. 399, 2011, pp. 251-275.
[10] W. J. Sim, J. W. Lee, E. S. Lee, S K. Shin, S. R. Hwang and J. E. Oh, "Occurrence and distribution of pharma-ceuticals in wastewater from households, livestock farms, hospitals and pharmaceutical manufactures," Chemosphere, Vol. 82, 2011, pp. 179-186.
[11] A. Y. Lin, X. H. Wang and C. F. Lin, "Impact of waste-waters and hospital effluents on the occurrence of controlled substances in surface waters," Chemosphere, Vol. 81, 2010, pp. 562-570.
[12] D. G. Larsson, "Release of active pharmaceutical ingredients from manufacturing sites-need for new management strategies," Integr Environ Assess Manag, Vol. 6, 2010, pp. 184-186.
[13] Y. Valcarcel, S. Gonzalez Alonso, J. L. Rodriguez-Gil, A. Gil and M. Catala, "Detection of pharmaceutically active compounds in the rivers and tap water of the Madrid Region (Spain) and potential ecotoxicological risk," Chemosphere, Vol. 84, 2011, pp. 1336-1348.
[14] M. S. Kostich, A. L. Batt, S. T. Glassmeyer and J. M. Lazorchak, "Predicting variability of aquatic concentrations of human pharmaceuticals," Sci Total Environ, Vol. 408, 2010, pp. 4504-4510.
[15] S. Terzic, I. Senta and M. Ahel, "Illicit drugs in waste- water of the city of Zagreb (Croatia)-estimation of drug abuse in a transition country," Environ Pollut, Vol. 158, 2010, pp. 2686-2693.
[16] T. Heberer, "Occurrence, fate, and removal of pharma-ceutical residues in the aquatic environment: a review of recent research data," Toxicol Lett, Vol. 131, 2002, pp. 5-17.
[17] A. Baba and G. Tayfur, "Groundwater contamination and its effect on health in Turkey," Environ Monit Assess, Vol. 183, 2011, pp. 77-94.
[18] K. Mishra, R. C. Sharma and S. Kumar, "Contamination levels and spatial distribution of organochlorine pesticides in soils from India," Ecotoxicol Environ Saf, Vol. 76, 2012, pp. 215-225.
[19] T. A. Edge, A. El-Shaarawi, V. Gannon, C. Jokinen, R. Kent, I. U. Khan, W. Koning, D. Lapen, J. Miller, N. Neumann, R. Phillips, W. Robertson, H. Schreier, A. Scott, I. Shtepani, E. Topp, G. Wilkes and E. van Bochove, "Investigation of an Escherichia coli environmental benchmark for waterborne pathogens in agricultural watersheds in Canada," J Environ Qual, Vol. 41, 2012, pp. 21-30.
[20] F. Zan, S. Huo, B. Xi, J. Su, X. Li, J. Zhang and K. M. Yeager, "A 100 year sedimentary record of heavy metal pollution in a shallow eutrophic lake, Lake Chaohu, China," J Environ Monit, Vol. 13, 2011, pp. 2788-2797.
[21] M. H. Pham, Z. Sebesvari, B. M. Tu, H. V. Pham and F. G. Renaud, "Pesticide pollution in agricultural areas of Northern Vietnam: case study in Hoang Liet and Minh Dai communes," Environ Pollut, Vol. 159, 2011, pp. 3344-3350.
[22] S. S. Kaushal, P. M. Groffman, L. E. Band, E. M Elliott C. A. Shields and C. Kendall "Tracking nonpoint source nitrogen pollution in human-impacted watersheds," Environ Sci Technol, Vol. 45, 2011, pp. 8225-8232.
[23] A. E. Khater, "Uranium and trace elements in phosphate fertilizers-Saudi Arabia," Health Phys, Vol. 102, 2012, pp. 63-70.
[24] E. U. Cantu-Soto, M. M. Meza-Montenegro, A. I. Valenzuela-Quintanar, A. Felix-Fuentes, P. Grajeda-Cota, J. J. Balderas-Cortes, C. L. Osorio-Rosas, G. Acuna-Garcia and M. G. Aguilar-Apodaca, "Residues of organochlorine pesticides in soils from the southern Sonora, Mexico," Bull Environ Contam Toxicol, Vol. 87, 2011, pp. 556-560.
[25] P. Nila Rekha, R. S. Kanwar, A. K. Nayak, C. K. Hoang and C. H Pederson "Nitrate leaching to shallow ground-water systems from agricultural fields with different management practices," J Environ Monit, Vol. 13, 2011, pp. 2550-2558.
[26] S. E. Lewis, J. E. Brodie, Z. T. Bainbridge, K. W. Rohde, A. M. Davis, B. L. Masters, M. Maughan, M. J. Devlin, J. F. Mueller and B. Schaffelke, "Herbicides: a new threat to the Great Barrier Reef," Environ Pollut, Vol. 157, 2009, pp. 2470-2484.
[27] F. Licciardello, M. L. Antoci, L. Brugaletta and G. L. Cirelli, "Evaluation of groundwater contamination in a coastal area of southeastern Sicily," J Environ Sci Health B, Vol. 46, 2011, pp. 498-508.
[28] V. Babu, P. Unnikrishnan, G. Anu and S. M. Nair, "Distribution of organophosphorus pesticides in the bed sediments of a backwater system located in an agricultural watershed: influence of seasonal intrusion of seawater," Arch Environ Contam Toxicol, Vol. 60, 2011, pp. 597-609.
[29] S. Burgert, R. B. Schafer, K. Foit, M. Kattwinkel, L. Metzeling, R. MacEwan, B. J. Kefford and M. Liess, "Modelling aquatic exposure and effects of insecticides-application to southeastern Australia," Sci Total Environ, Vol. 409, 2011, pp. 2807-2814.
[30] S. Loewe, "The problem of synergism and antagonism of combined drugs," Arzneimittelforschung, Vol. 3, 1953, pp. 285-290.
[31] S. Loewe, "Antagonisms and antagonists," Pharmacol Rev, Vol. 9, 1957, pp. 237-242.
[32] R. J. Tallarida, "Statistical analysis of drug combinations for synergism," Pain, Vol. 49, 1992, pp. 93-97.
[33] R. J. Tallarida and R. B. Raffa, "Testing for synergism over a range of fixed ratio drug combinations: replacing the isobologram," Life Sci, Vol. 58, 1996, pp. PL 23-28.
[34] R. J. Tallarida, H. L. Kimmel and S. G. Holtzman, "Theory and statistics of detecting synergism between two active drugs: cocaine and buprenorphine," Psychopharmacology (Berl), Vol. 133, 1997, pp. 378-382.
[35] R. J. Tallarida, F. Porreca and A. Cowan, "Statistical analysis of drug-drug and site-site interactions with isobolograms," Life Sci, Vol. 45, 1989, pp. 947-961.
[36] R. J. Tallarida, D. J. Stone, Jr. and R. B. Raffa, "Efficient designs for studying synergistic drug combinations," Life Sci, Vol. 61, 1997, pp. PL 417-425.
[37] R. J. Tallarida, D. J. Stone, Jr., J. D. McCary and R. B. Raffa "Response surface analysis of synergism between morphine and clonidine," J Pharmacol Exp Ther, Vol. 289, 1999, pp. 8-13.
[38] R. J. Tallarida and R. B. Murray. Manual of Pharmacologic Calculation with Computer Programs. 2nd edn, (Springer Verlag, 1987).
[39] R. J. Tallarida. Drug Synergism and Dose-Effect Data Analysis. (Chapman & Hall/CRC, 2000).
[40] R. J. Tallarida, U. Midic, N. Lamarre and Z. Obradovic, "Searching for synergism among combinations of drugs of abuse and the use of isobolographic analysis," arXiv, Vol. 1202.4742v1[q-bio.QM], 2012.
[41] S. Algeri, A. Carolei, P. Ferretti, C. Gallone, G. Palladini and G. Venturini, "Effects of dopaminergic agents on monoamine levels and motor behaviour in planaria," Comp Biochem Physiol C, Vol. 74, 1983, pp. 27-29.
[42] F. R. Buttarelli, F. E. Pontieri, V. Margotta and G. Palladini, "Acetylcholine/dopamine interaction in planaria," Comp Biochem Physiol C Toxicol Pharmacol, Vol. 125, 2000, pp. 225-231.
[43] A. Carolei, V. Margotta and G. Palladini, "Proposal of a new model with dopaminergic-cholinergic interactions for neuropharmacological investigations," Neuropsychobiology, Vol. 1, 1975, pp. 355-364.
[44] G. Palladini, V. Margotta, A. Carolei, F. Chiarini, M. Del Piano, G. M. Lauro, L. Medolago-Albani and G. Venturini, "The cerebrum of Dugesia gonocephala s.1. Platyhelminthes, Turbellaria, Tricladida. Morphological and functional observations," J Hirnforsch, Vol. 24, 1983, pp. 165-172.
[45] G. Palladini, S. Ruggeri, F. Stocchi, M. F. De Pandis, G. Venturini and V. Margotta, "A pharmacological study of cocaine activity in planaria," Comp Bio-chem Physiol C Pharmacol Toxicol Endocrinol, Vol. 115, 1996, pp. 41-45.
[46] F. Passarelli, A. Merante, F. E. Pontieri, V. Margotta, G. Venturini and G. Palladini, "Opioid-dopamine interaction in planaria: a behavioral study," Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, Vol. 124, 1999, pp. 51-55.
[47] P. Ribeiro, F. El-Shehabi and N. Patocka, "Classical transmitters and their receptors in flatworms," Parasitology, Vol. 131 Suppl, 2005, pp. S19-40.
[48] G. Venturini, A. Carolei, G. Palladini, V. Margotta and M. G. Lauro, "Radioimmunological and immunocyto-chemical demonstration of Metenkephalin in planaria," Comp Biochem Physiol C, Vol. 74, 1983, pp. 23-25.
[49] G. Venturini, F. Stocchi, V. Margotta, S. Ruggieri, D. Bravi, P. Bellantuono and G. Palladini, "A pharmacological study of dopaminergic receptors in planaria," Neuropharmacology, Vol. 28, 1989, pp. 1377-1382.
[50] J. H. Welsh and L. D. Williams, "Monoamine-contain- ing neurons in planaria," J Comp Neurol, Vol. 138, 1970, pp. 103-115.
[51] R. B. Raffa and P. Desai, "Description and quantification of cocaine withdrawal signs in Planaria," Brain Res, Vol. 1032, 2005, pp. 200-202.
[52] R. B. Raffa, K. E. Finno, C. S. Tallarida and S. M. Rawls, "Topiramate-antagonism of L-glutamate- induced paroxysms in planarians," Eur J Pharmacol, Vol. 649, 2010, pp. 150-153.
[53] R. B. Raffa, L. J. Holland and R. J. Schulingkamp, "Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint," J Pharmacol Toxicol Methods, Vol. 45, 2001, pp. 223-226.
[54] R. B. Raffa and A. F. Martley, "Amphetamine-induced increase in planarian locomotor activity and block by UV light," Brain Res, Vol. 1031, 2005, pp. 138-140.
[55] R. B. Raffa and S. M. Rawls. Planaria: A Model for Drug Action and Abuse. (Landes Bioscience, 2008).
[56] R. B. Raffa, M. J. Robinson and R. J. Tallarida, "Ultra-violet light-induced photorelaxation of agonist-contracted rabbit aorta: further characterization and the estimation of drug-receptor rate constants," Drug Development Research, Vol. 5, 1985, pp. 359-369.
[57] R. B. Raffa, G. W. Stagliano and S. Umeda, "kappa-Opioid withdrawal in Planaria," Neurosci Lett, Vol. 349, 2003, pp. 139-142.
[58] R. B. Raffa and J. M. Valdez, "Cocaine withdrawal in Planaria," Eur J Pharmacol, Vol. 430, 2001, pp. 143- 145.
[59] R. B. Raffa, J. M. Valdez, L. J. Holland and R. J. Schulingkamp, "Energy-dependent UV light-induced disruption of (-)sulpiride antagonism of dopamine," Eur J Pharmacol, Vol. 406, 2000, pp. R11-12.
[60] S. M. Rawls, T. Thomas, M. Adeola, T. Patil, N. Raymondi, A. Poles, M. Loo and R. B. Raffa, "Topiramate antagonizes NMDA- and AMPA-induced seizure-like activity in planarians," Pharmacol Biochem Behav, Vol. 93, 2009, pp. 363-367.
[61] S. Umeda, G. W. Stagliano and R. B. Raffa, "Cocaine and kappa-opioid withdrawal in Planaria blocked by D-, but not L-, glucose," Brain Res, Vol. 1018, 2004, pp. 181-185.
[62] D. Bian, M. H. Ossipov, M. Ibrahim, R. B. Raffa, R. J. Tallarida, T. P. Malan, Jr., J. Lai and F. Porreca, "Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antiserum," Brain Res, Vol. 831, 1999, pp. 55-63.
[63] E. A. Bolan, R. J. Tallarida and G. W. Pasternak, "Synergy between mu opioid ligands: evidence for functional interactions among mu opioid receptor subtypes," J Pharmacol Exp Ther, Vol. 303, 2002, pp. 557-562.
[64] E. E. Codd, R. P. Martinez, L. Molino, K. E. Rogers, D. J. Stone and R. J. Tallarida, "Tramadol and several anticonvulsants synergize in attenuating nerve injury-induced allodynia," Pain, Vol. 134, 2008, pp. 254-262.
[65] C. A. Fairbanks, K. F. Kitto, H. O. Nguyen, L. S. Stone and G. L. Wilcox, "Clonidine and dexmedetomidine produce antinociceptive synergy in mouse spinal cord," Anesthesiology, Vol. 110, 2009, pp. 638-647.
[66] M. J. Field, M. I. Gonzalez, R. J Tallarida and L. Singh, "Gabapentin and the neurokinin(1) receptor antagonist CI-1021 act synergistically in two rat models of neuropathic pain," J Pharmacol Exp Ther, Vol. 303, 2002, pp. 730-735.
[67] D. Mukherjee, S. E. Nissen and E. J. Topol, "Risk of cardiovascular events associated with selective COX-2 inhibitors," JAMA, Vol. 286, 2001, pp. 954-959.
[68] R. B. Raffa, D. J. Stone, Jr. and R. J. Tallarida, "Discovery of "self-synergistic" spinal/supraspinal anti-nociception produced by acetaminophen (paracetamol)" J Pharmacol Exp Ther, Vol. 295, 2000, pp. 291-294.
[69] R. B. Raffa, R. Clark-Vetri, R. J. Tallarida and A. I. Wertheimer, "Combination strategies for pain management," Expert Opin Pharmacother, Vol. 4, 2003, pp. 1697-1708.
[70] D. W. Kolpin, E. T. Furlong, M. T. Meyer, E. M. Thurman, S. D. Zaugg, L. B Barber and H. T Buxton, "Pharmaceuticals, hormones, and other organic waste-water contaminants in U.S. streams, 1999-2000: a national reconnaissance," Environ Sci Technol, Vol. 36, 2002, pp. 1202-1211.
[71] P. E. Stackelberg, E. T. Furlong, M. T. Meyer, S. D. Zaugg, A. K. Henderson and D. B. Reissman, "Persistence of pharmaceutical compounds and other organic wastewater contaminants in a conventional drinking-water-treatment plant," Sci Total Environ, Vol. 329, 2004, pp. 99-113.
[72] K. A. Kidd, P. J. Blanchfield, K. H. Mills, V. P. Palace, R. E. Evans, J. M. Lazorchak and R. W. Flick "Collapse of a fish population after exposure to a synthetic estrogen," Proc Natl Acad Sci U S A, Vol. 104, 2007, pp. 8897-8901.
[73] M. E. Ortiz-Santaliestra, M. J. Fernan-dez-Beneitez, M. Lizana and A. Marco, "Influence of a combination of agricultural chemicals on embryos of the endangered gold-striped salamander (Chioglossa lusitanica)," Arch Environ Contam Toxicol, Vol. 60, 2011, pp. 672-680.
[74] R. A. Relyea and N. Mills, "Predator-induced stress makes the pesticide carbaryl more deadly to gray tree-frog tadpoles (Hyla versicolor)," Proc Natl Acad Sci U S A, Vol. 98, 2001, pp. 2491-2496.
[75] M. D. Boone, C. M. Bridges, J. F. Fairchild and E. E. Little, "Multiple sublethal chemicals negatively affect tadpoles of the green frog, Rana clamitans," Environ Toxicol Chem, Vol. 24, 2005, pp. 1267-1272.
[76] M. D. Boone, R. D. Semlitsch, E. E. Little and M. C. Doyle, "Multiple stressors in amphibian communities: effects of chemical contamination, bullfrogs, and fish," Ecol Appl, Vol. 17, 2007, pp. 291-301.
[77] P. T. Johnson, E. R. Preu, D. R. Sutherland, J. M. Romansic, B. Han and A. R. Blaustein, "Adding infection to injury: synergistic effects of predation and parasitism on amphibian malformations," Ecology, Vol. 87, 2006, pp. 2227-2235.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.