1582 Environmental Toxicology and Chemistry, Vol. 22, No. 7, pp. 1582–1588, 2003  2003 SETAC Printed in the USA 0730-7268/03 $12.00 + .00 SUSCEPTIBILITY OF BLACK FLY LARVAE (DIPTERA: SIMULIIDAE) TO LAWN-CARE INSECTICIDES INDIVIDUALLY AND AS MIXTURES JAY P. OVERMYER,*² K EVIN L. ARMBRUST,‡ and RAY NOBLET² ²University of Georgia, Department of Entomology, 413 Biological Sciences Building, Athens, Georgia 30602, USA ‡Mississippi State University, Mississippi State Chemical Laboratory, P.O. Box CR, Mississippi State, Mississippi 39762, USA ( Received 16 July 2002; Accepted 20 December 2002) Abstract—Urban and suburban watersheds have the potential to be highly impacted by chemicals, especially insecticides to control insect pests on lawns, ornamental plants, and home gardens. Three of the most common lawn-care insecticides detected in urban watersheds, carbaryl, chlorpyrifos, and malathion, have been evaluated using an acute orbital shaker toxicity test to determine their respective concentrations that produce 50% mortality (LC50) in Simulium vittatum Zetterstedt cytospecies IS-7 larvae. Results of the 48-h LC50 tests show chlorpyrifos to be the most toxic to black fly larvae (LC50 = 0.28 g/L) followed by carbaryl (LC50 = 23.72 g/L) and malathion (LC50 = 54.20 g/L). These insecticides were also tested as binary and ternary mixtures using the toxic unit (TU) approach. Toxicity was shown to be greater than additive for the ternary mixture of chlorpyrifos–carbaryl–malathion (LC50 = 0.56 TU) and the binary mixtures of chlorpyrifos–malathion (LC50 = 0.72 TU) and carbaryl–malathion (LC50 = 0.78 TU). The binary combination of chlorpyrifos and carbaryl was shown to be additive (LC50 = 0.98 TU). These results indicate that aquatic invertebrate populations in urban and suburban streams may experience a higher-than-expected increase in toxicity- related effects when all three chemicals are present in the waterway. Keywords—Toxicity Insecticides Simulium vittatum Suburban watersheds Insecticide mixtures INTRODUCTION Urban and suburban watersheds have the potential to be highly impacted by chemicals, especially insecticides used to control insect pests on lawns, ornamental plants, and home gardens. An estimated 17 million pounds of insecticidal active ingredients are applied annually in domestic settings for con- trol of insects on lawns and gardens (U.S. Environmental Pro- tection Agency, http://www.epa.gov/oppbead1/pestsales/ 97pestsales/index.htm). Multiple lawn-care insecticides enter urban and suburban waterways primarily through runoff from rain events and are commonly detected in these aquatic sys- tems [1–4]. Consequently, organisms inhabiting streams and rivers receiving insecticide runoff from lawns and gardens are exposed to these chemicals as mixtures, which may lead to possible increases or decreases in the toxic effect of the in- dividual insecticide [5,6]. It has been perceived that chemicals with the same quan- titative structure–activity relationship (QSAR) or mode of ac- tion produce additive toxicity when applied jointly [7–10]. However, studies with organophosphate insecticides applied jointly have shown that these chemicals elicit greater-than- additive effects when organisms are exposed to them as mix- tures [6,11]. Thus, it is erroneous to assume that all structurally and mechanistically similar chemicals behave additively as mixtures. Experimental data are needed on specific chemical mixtures, particularly those commonly occurring in urban and suburban watersheds, to determine their joint activity. The organophosphate insecticides chlorpyrifos (O,O-dieth- yl O-[3,5,6-trichloro-2-pyridyl]-phosphorothioate] and mala- thion (diethyl mercaptosuccinate, O,O-dimethyl-dithiophos- phate) and the carbamate insecticide carbaryl (1-naphthyl-N- * To whom correspondence may be addressed (jayo@arches.uga.edu). methylcarbamate) are registered for use on lawns and gardens for control of insect pests in the United States. Although chlor- pyrifos is no longer registered for domestic use, it is registered for use on golf courses, which are often built in residential communities, to control turf pests. As a result of their wide- spread use, chlorpyrifos, carbaryl, and malathion are three of the most commonly detected insecticides in urban and sub- urban streams, such as the Flint River watershed of Georgia, USA [2]. Thus, it is imperative to determine the cumulative toxicity of these insecticides in order to predict potential im- pacts on organisms inhabiting these streams. The purpose of this study was to evaluate the toxicities of chlorpyrifos, malathion, and carbaryl individually and as mix- tures using black fly larvae, Simulium vittatum Zetterstedt cy- tospecies IS-7, as the test organism in an acute orbital shaker toxicity test. Acute toxicity will be determined for each in- secticide through analyzing the lethal concentration required to kill 50% of the test organisms (LC50), followed by assessing the joint toxicity as mixtures using the modified toxic unit approach of Marking [12] as described by Pape-Lindstom and Lydy [13]. Data from this study should provide a better un- derstanding of the toxicities resulting from the use of multiple lawn-care insecticides. MATERIALS AND METHODS Organisms Black fly larvae (Diptera: Simuliidae) are often dominant species in stream ecosystems. They are holometabolous, filter- feeding insects commonly found on rocks, trailing vegetation, snags, and other substrates in streams and rivers. Their primary role in aquatic ecosystems is converting ultrafine particulate organic matter into fine particulate organic matter that can be utilized by other organisms. Larval simuliids also serve as a food source for fish and other invertebrates [14]. Larvae for