assessment-fidelino.pmd Acute toxicity of thiamethoxam to Achatina fulica 21Science Diliman (July-December 2012) 24:2, 21-27 ABSTRACT Assessment of acute toxicity of thiamethoxam (Actara® 25WG) to Achatina fulica and its potential ecological applications Maria Kim Feliz N. Abog, Jose Jaime Lorenzo C. de Rivera, Sarah Christina W. Estacio, Jay S. Fidelino*, Orlie John Y. Lavilla, John Christopher A. Pilapil, and Ma. Dolores C. Tongco Institute of Biology, College of Science, University of the Philippines Diliman *Corresponding author: Institute of Biology, University of the Philippines Tel.: (02) 981 8500 loc. 5713; E-mail: jsfidelino@gmail.com Achatina fulica is considered as one of the world’s worst invasive species and is known to cause ecological disruption as well as agricultural and health problems in the Philippines. The study evaluated the use of the insecticide thiamethoxam (Actara® 25WG) in the control of A. fulica populations. This was done by performing an acute toxicity test of thiamethoxam to A. fulica. Six individuals each were exposed to thiamethoxam concentrations of 0, 50, 100, 200, and 400 µg/L for 72 hours. The percent mortality was then determined after the exposure period. Probit analysis was used to determine the LD 50 of thiamethoxam to A. fulica. The LD 50 was found to be 662.95 ± 172.98 µg/L. Using the mean body weight of the snails, the LD 50 per body weight of A. fulica was determined to be 90.09 ± 23.60 µg/kg. This is 70 times lower than the recommended application of thiamethoxam on field. Thus, normal application would eliminate A. fulica. However, because the LD 50 to A. fulica is higher than that for other beneficial non-target species such as honey bees (0.03 µg/bee), the use of thiamethoxam in the control of A. fulica populations is only recommended when in conjunction with the control of target pest insects. Keywords: Ecotoxicology, acute toxicity Fidelino, J.S. and others 22 Science Diliman (July-December 2012) 24:2, 21-27 INTRODUCTION The prevalence of invasive alien species is a global trend and is considered to be one of the major causes of biodiversity loss, economic damage (Lowe and others 2000), and potentially detrimental environmental changes (Leung and others 2002). One such species is Achatina fulica (Achatinidae), the giant African snail, a terrestrial gastropod with a widespread distribution in the humid tropics (Fontanilla 2010). The International Union for Conservation of Nature lists A. fulica as one of the 100 world’s worst invasive species (Lowe and others 2000). A. fulica originated in East Africa but now occupies the Indian subcontinent, Southeast Asia, the Pacific, and the Caribbean (Fontanilla 2010). Its introduction into non-native habitats has been documented to be both intentional (i.e. as a food source) and accidental (Venette and Larson 2004). The reproductive biology of A. fulica has largely aided its success as an invasive species, with the snail reaching maturity at five to eight months and producing 10 to 400 eggs per clutch and up to 1800 eggs per year (Fontanilla 2010). It is also a voracious herbivore and its wide host range makes it a threat to native flora as well as agricultural crops (Venette and Larson 2004). A. fulica may also spread diseases, as it is known to be an intermediate host of the rat lungworm Angiostrongylus cantonensis, which can infect humans in its third juvenile stage. A. cantonensis can cause eosinophilic meningo- encephalitis (EME) or angiostrongyliasis (Fontanilla 2010). Black pod disease, a plant disease caused by Phytophthora palmivora, may also be spread through the snail’s feces (Venette and Larson 2004). In the Philippines, A. fulica populations are known to attack and feed on grown crops, gardens and agricultural farms of ampalaya (Momordica charantia), arrowroot (Canna edulis), banana (Musa paradisiaca), cabbage (Brassica spp.), Canna sp., cassava (Manihot esculenta), Citrus sp., coffee (Coffea spp.), sunflower (Cosmos spp.), cucumber (Cucumis sativus), eggplant (Solanum melongena), upo (gourd: Lagenaria leucantha), gumamela (Hibiscus spp.), malunggay (Moringa olifeira), papaya (Carica papaya), ramie (Boehmeria nivea), squash (Cucurbita sp.), sweet potato (Ipomoea batatas), patola (Luffa sp.), and yam (Dioscorea alata) among others (Sherley 2000, Raut and Barker 2002, Fontanilla 2010). Various strategies for controlling A. fulica populations have been proposed and attempted, but so far, no single control measure has been successful in the eradication of the pest. One strategy that has been attempted in the Philippines involved the purposeful introduction o f the Platydemus manokwari (Turbellaria: Rhynchodemidae), a non-native invertebrate enemy of A. fulica, to Bugsuk Island. Although the giant African snails were eradicated in some areas, the introduction of P. manokwari was said to have adverse effect on indigenous gastropod fauna (Sherley 2000). A more common strategy involves chemical control with the use of toxicants, repellents, or pesticides. Despite the development of new molluscicidal agents, very few are targeted against A. fulica (Raut and Barker 2002). However, even non-target pesticides such as brodifacoum, which is aimed at rodents and vertebrate pests, have been found to be toxic to A. fulica (Booth and others 2001, Hoare and Hare 2006). Thus, using non-target pesticides as chemical control agents for A. fulica may have great potential. The median lethal dose (LD 50 ) is the most frequently used measure of the acute toxicity of a substance. Expressing toxicity as LD 50 provides a relative measure that can be used to compare substances with different mechanisms based solely on their lethal effect. The measurement of LD 50 also assumes that it estimates the toxicity of the most hazardous constituent in the mixture (Spencer and Colonna 2003). A smaller LD 50 value means relatively greater toxicity, indicating that a smaller amount of the substance is required for the death of the test organism (Girard 2010). This study aimed to determine the acute toxicity, expressed as median lethal dose (LD 50 ), of thiamethoxam (Actara® 25WG) to Achatina fulica, which is a non-target species of the insecticide. The study also assessed whether thiamethoxam would be effective in the pest control of the snail. Acute toxicity of thiamethoxam to Achatina fulica 23Science Diliman (July-December 2012) 24:2, 21-27 MATERIALS AND METHODS Achatina fulica Ninety Achatina fulica individuals were obtained from around the University of the Philippines Diliman campus, weighed, and then placed in 15 identical glass containers (30 cm x 30 cm x 45 cm), with six individuals in each container. Nylon tulle netting was used as lids to contain the samples. Three centimeters of loamy soil were added to the container and kept moist throughout the study by spraying with distilled water. Ten grams of tomato leaves were also placed per container to serve as food for the snails for the entire duration of the study. The snails were given a 24-hour acclimatization period before the experiment proper. Thiamethoxam Thiamethoxam, 3-(2chloro-thiazol-5-ylmethyl)-5- methyl-{1,3,5}oxadiazinan-4-yldene-N-nitroamine, was obtained from Syngenta Philippines, Inc. (Makati City, Philippines). Formulated granules of thiamethoxam (Actara® 25WG) were dissolved in distilled water and thiamethoxam solutions were prepared in four concentrations of 50, 100, 200, and 400 µg/L each. The concentrations were calculated based on the formulation of the granules used (250 g thiamethoxam per kg). The pH and temperature of the solutions were then measured. Toxicity test Toxicity tests were performed to determine the LD 50 of thiamethoxam to A. fulica . Ten mL of the thiamethoxam solutions was applied by spraying on the soil surface and the sides of the containers. The snails were left to feed on tomato leaves during the 72-hour toxicity test period. Concentrations tested were 50, 100, 200, and 400 µg/L of thiamethoxam in the insecticide solutions applied per container. A control treatment was also made, with distilled water used as control. After 72 hours, percent mortality was determined by examining the containers for dead snails. Three replicates of the toxicity test were performed. Using the mean weight of A. fulica used per treatment, the thiamethoxam concentrations were also expressed in Treatment Mean Weight (kg) Thiamethoxam Concentration (µg/kg) Control 0.0130 ± 0.0545 0 50 µg/L 0.0109 ± 0.0519 7.678532 100 µg/L 0.0137 ± 0.4650 13.79039 200 µg/L 0.0149 ± 0.0264 22.33958 400 µg/L 0.0121 ± 0.3497 59.18589 Table 1. Concentration of thiamethoxam applied per body weight of Achatina fulica µg/kg (Table 1). Using IBM SPSS Statistic v.19, the LD 50 (in µg/L and µg/kg) was determined by probit analysis. Chi-square goodness-of-fit test was also performed in SPSS to check if the probit model fits the data adequately. RESULTS The temperature of the insecticide solutions ranged from 23.6-23.7°C, while a pH of 6.87 was measured for all the solutions. The percent mortality of A. fulica in the different treatments is shown in Table 2. The probit transformation output from SPSS Statistic v.19 is shown in Figure 1 (thiamethoxam concentration expressed in µg/L) and Figure 2 (thiamethoxam concentration expressed in µg/kg). From the probit analysis graph, the 72-hr median lethal dose (LD 50 ) of thiamethoxam to A. fulica was determined to be 662.95 ± 172.98 µg/ L (90.09 ± 23.60 µg/kg). A Chi-square goodness-of-fit test was also performed with a null hypothesis that the probit analysis model fits the dose response data (Table 3). DISCUSSION Thiamethoxam is a neonicotinoid that is capable of mimicking acetylcholine. It acts selectively on the nicotinic acetylcholine receptors of insects, ultimately damaging the nervous system and leading to the death of the organism (Nauen and others 2003). Neonicotinoids are popular insecticides because the neural pathway affected is more common in invertebrates than in other animal groups (Kindemba 2009). Fidelino, J.S. and others 24 Science Diliman (July-December 2012) 24:2, 21-27 The toxicity (LD 50 ) of thiamethoxam has been previously determined in other species. The 24-hr LD 50 was found to be 0.46 µg/g (460 µg/kbw) in houseflies (Musca domestica), 10.84 µg/g (10,840 µg/kbw) in German cockroaches (Blatella germanica) (Eremina and Lopatina 2005), 0.03 µg/bee in honey bees (Apis mellifera) (Iwasa and others 2004), and 0.024 µg/g (24 µg/kbw) in bumblebees (Bombus terrestris) (NRA 2001). For mice and rats, the LD 50 values were 1,563 mg/kbw (1,563,000 µg/kbw for 59-day exposure) in rats, 783 mg/kbw (783,000 µg/kbw for 14-day exposure) in male mice, and 964 mg/kbw (964,000 µg/kbw for 14- day exposure) in female mice (NRA 2001). Thiamethoxam applied by contact or ingestion is particularly highly toxic to honey bees and bumblebees, and has been implicated along with other pesticides as possible cause of colony collapse disorder (Kindemba 2009, NRA 2001). LD 50 Chi-Square Significance 662.95 ± 172.98 µg/L 0.980 90.09 ± 23.60 µg/kg 0.983 Table 3. The 72-hr median lethal dose (LD 50 ) of thiamethoxam to Achatina fulica Percent Treatment Mortality Control 0 50 µg/L 5.555556 100 µg/L 16.66667 200 µg/L 22.22222 400 µg/L 38.88889 Table 2. Percent mortality of Achatina fulica in the five treatments of thiamethoxam Figure 1. Probit transformation output for the determination of the 72-hour median lethal dose (LD 50 ) (µg/L) of thiamethoxam to A. fulica Acute toxicity of thiamethoxam to Achatina fulica 25Science Diliman (July-December 2012) 24:2, 21-27 The recommended application of Actara® 25WG for tomatoes, potatoes, and sugar beets is 20 g/hl (Syngenta 2011), which is effectively 50,000 µg/L thiamethoxam. Degradation of thiamethoxam is primarily caused by aqueous photolysis, while photolytic degradation of thiamethoxam absorbed into the soil is not significant. In soil/water systems, thiamethoxam accumulates in the sediment phase while degradation is continuous (NRA 2001). The half-life of thiamethoxam in soil ranges from 7 to 109 days, with longer persistence under dry conditions. Thiamethoxam also has the potential to leach down under heavy rainfall conditions (NRA 2001, Gupta and others 2008 ). The LD 50 of thiamethoxam to A. fulica obtained was not within the range of concentrations used in the study (50 to 400 µg/L). Typically, a range-finding experiment is performed to find a more specific range of concentrations prior to the toxicity test proper. The range-finding experiment was not performed because of scarcity of test animals due to the dry weather during the entire study period. This was also the reason why only six individuals were used per dose of thiamethoxam. Instead of a range-finding experiment, previous literature on the toxicity of thiamethoxam and other similar insecticides on other land snails and mollusks were used as the basis for the range of concentrations prepared (Booth and others 2001, Salama and others 2005, Minakshi and Mahajan 2012). The recommended application is about 70 times the LD 50 determined in this study. This prescribed application would be able to eliminate A. fulica pests, if present. However, the use of thiamethoxam solely Figure 2. Probit transformation output for the determination of the 72-hour median lethal dose (LD 50 ) (µg/kg) of thiamethoxam to A. fulica per body weight (µg/kg) Fidelino, J.S. and others 26 Science Diliman (July-December 2012) 24:2, 21-27 as a molluscicidal to A. fulica is not recommended due to the lower LD 50 of the insecticide to other non-target organisms and to the persistence of thiamethoxam, especially in dry soil. Furthermore, since thiamethoxam may potentially leach under heavy rainfall, contamination of nearby water bodies and other ecosystems becomes a concern. The potential effect on other non-target organisms, especially native and beneficial species, should also be considered. Thus, the use of thiamethoxam in the control of A. fulica populations is only recommended in conjunction with the control of target pest insects. ACKNOWLEDGMENTS The researchers would like to thank the late Mang Oca, for whom this paper is written, for the snail supply, and the Institute of Biology, for use of equipment and of the Ecology and Taxonomy Laboratory. REFERENCES Booth LH, Eason CT, Spurr EB. 2001. Literature review of the acute toxicity and persistence of brodifacoum to invertebrates. In: Department of Conservation, editor. Literature review of the acute toxicity and persistence of brodifacoum to invertebrates and studies of residue risks to wildlife and people. Science for Conservation 177. p 1-9. Eremina O, Lopatina Y. 2005. Investigation of neonicotinoid insecticides against house fly Musca domestica (Diptrea: Muscidae) and German cockroach Blatella germanica (Blattodea: Blattelidae). In: Proceedings of the Fifth International Conference on Urban Pests [cited 2012 March 27]. Available from http://www.icup.org.uk/reports/ ICUP090.pdf Fontanilla IKC. 2010. Achatina (Lissachatina) fulica Bowdich: its molecular phylogeny, genetic variation in global populations, and its possible role in the spread of the rat lungworm Angiostrongylus cantonensis (Chen) [PhD thesis]. UK: The University of Nottingham. 633 leaves. Girard JE. 2010. Principles of environmental chemistry. 2nd ed. Sudbury, MA: Jones and Barlett Publishers. 687 p. Gupta S, Gajbhiye VT, Gupta RK. 2008. Soil dissipation and leaching behavior of a neonicotinoid insecticide thiamethoxam. Bull Environ Contam Toxicol 80: 431-37. Hoare JM, Hare KM. 2006. The impact of brodifacoum on non-target wildlife: gaps in knowledge. New Zealand Journal of Ecology 30(2): 157-67. Iwasa T, Motoyama N, Ambrose JT, Roe MR. 2004. Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Prot. 23: 371-78. Kindemba V. 2009. The impact of neonicotinoid insecticdes on bumblebees, honey bees and other non-target invertebrates. UK: Buglife – The Invertebrate Conservation Trust. 52 p. Leung B, Lodge DM, Finoff D, Shogren JF, Lewis MA, Lamberti G. 2002. An ounce of prevention or a pound of cure: bioeconomic risk analysis of invasive species. Proc. R. Soc. Lond. B. 269: 2407-13. Lowe S, Browne M, Boudjelas S, De Poorter M. 2000. 100 of the world’s worst invasive alien species – a selection from the Global Invasive Species Database. Switzerland: Invasive Species Specialist Group, Species Survival Commission, World Conservation Union. 12 p. Mikashi R, Mahajan AY. 2012. Toxicity evaluation of thiamethoxam and triazophos to the freshwater bivalve Lamellidens marginalis (Lamark). Trends in life Sciences 1(3): 29-33. National Registration Authority (NRA). 2001. Evaluation of the new active thiamethoxam in the product Cruiser 350 FS insecticide seed treatment. Canberra, Australia: National Registration Authority for Agricultural and Veterinary Chemicals. 51 p. Nauen R, Ebbinghaus-Kintscher U, Salgado VL, Kaussmann M. 2003. Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants. Pesticide Biochemistry and Physiology 76: 55-69. Acute toxicity of thiamethoxam to Achatina fulica 27Science Diliman (July-December 2012) 24:2, 21-27 Raut SK, Barker GM. 2002. Achatina fulica Bowdich and other Achatinidae as pests in tropical agriculture. In: Barker GM, editor. Molluscs as crop pests. Wallingford; CABI Publishing. p. 84-87. Salama AK, Osman KA, Saber NA, Soliman SA. 2005. Oxidative stress induced by different pesticides in the land snails Helix aspersa. Pakistan Journal of Biological Sciences 8(1): 92-96. Sherley G. 2000. Invasive species in the Pacific: a technical review and draft regional strategy. Samoa: South Pacific Regional Environment Programme. 149 p. Spencer AB, Colonna GR. 2003. NFPA pocket guide to hazardous materials. Massachusetts: National Fire Protection Association, Inc. 220 p. Syngenta. 2011. Actara  25WG fact sheet [cited 2012 March 27]. Available from http://www.syngetna.com/country/eg/ e n / c r o p p r p t e c t i o n / o u r p r o d u c t s / i n s e c t i c i d e s / P a g e s / Actara25 WG.aspx Venette RC, Larson M. 2004. Mini risk assessment: giant African snail, Achatina fulica Bowdich [Gastropoda: Achatinidae] [cited 2012 March 2]/ Available from http:// www.aphis.usda.gov/plant_health/plant_pest_info/ pest_detection/downloads/pra/afulicapra.pdf