527 Environmental Toxicology and Chemistry, Vol. 21, No. 3, pp. 527–531, 2002  2002 SETAC Printed in the USA 0730-7268/02 $9.00 + .00 RESPONSE OF THE AMPHIBIAN TADPOLE (XENOPUS LAEVIS) TO ATRAZINE DURING SEXUAL DIFFERENTIATION OF THE TESTIS LUZ TAVERA-MENDOZA,² S YLVIA RUBY,*² P AULINE BROUSSEAU,‡ MICHEL FOURNIER,‡ DANIEL CYR,‡ and DAVID MARCOGLIESE§ ²Department of Biology, Concordia University, Montreal, Quebec H3G-1M8, Canada ‡INRS-Institut Armand-Frappier, Point-Claire, Quebec H9R-1G6, Canada §Environment Canada, Montreal, Quebec H2Y-2E7 ( Received 11 April 2001; Accepted 10 August 2001) Abstract—Xenopus laevis tadpoles were exposed for 48 h during sexual differentiation to atrazine at 21 g/L under static laboratory conditions at 21  0.5°C. After this exposure period, tadpoles were fixed and the kidney–gonad complex was microdissected. Quantitative histological analysis of the gonad revealed a 57% reduction in testicular volume among atrazine-exposed tadpoles. In addition, primary spermatogonial cell nests that represent germ cells for the life of the organism were reduced by 70%. Nursing cells, which provide nutritive support for the developing germ cells, had declined by 74%. Testicular resorption was observed among 70% and aplasia or failure of full development of the testis was recorded in 10% of the atrazine-exposed tadpoles. Because cell nests represent the pool of primordial germ cells for the reproductive life of the organism, the combined reduction in sper- matogonial cell nests and nursing cells suggest that a pulse exposure to 21 g/L of atrazine during sexual differentiation could significantly reduce reproduction during the reproductive life of these animals. Keywords—Atrazine Amphibian Tadpole Sexual differentiation Testis INTRODUCTION Amphibian population declines exceeding normal popula- tion fluctuations have been perceived globally in recent years and extinction has occurred in some populations [1,2]. Al- though no single cause has been identified, current studies raise concern regarding the potential role that pesticides may play in these declines [3]. Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-tri- azine) is a heavily utilized herbicide in North America [4]. Atrazine primarily is used for weed control in corn and sor- ghum production [5]. Approximately 36,000 tons of atrazine were applied to crops in the United States in 1993. At the same time, more than 584 tons were applied to corn in the province of Ontario in Canada [6]. Atrazine inhibits photo- synthesis by blocking electron transport within the Hill re- action of photosystem II [7]. Reports suggest that atrazine is more toxic to plants than to animals [5]. Atrazine is relatively persistent in naturally occurring freshwater. The half-life of atrazine is variable and ranges from 8 to 350 d, depending upon the ecological factors existing in the ecosystem [5]. The no-observed-ecological-effect concentration for atrazine in surface waters has been estimated at 21gL -1 [6]. Concen- trations of atrazine as high as 21 g/L have been measured in the St. Lawrence River Valley region of Quebec, Canada [8]. Although concentrations in rivers and streams rarely exceed 20 g/L, concentrations at the field edge can reach 250 g/L and concentrations as high as 740 mg/L have been detected in runoff waters from treated cornfields after spring application [9]. Numerous laboratory and field studies have examined the effects of atrazine on amphibian larval development [5,9–13]. * To whom correspondence may be addressed (sruby@alcor.concordia.ca). In a recent study [9], when larvae of leopard frog (Rana pi- piens) were exposed to atrazine from hatching until meta- morphosis at 0, 20, and 200 g/L under laboratory controlled conditions, no significant effect was observed in development rate, percent metamorphosis, time to metamorphosis, percent survival, mass at metamorphosis, or hematocrit. The present investigation is the first attempt in any vertebrate to examine the effects of atrazine on gonadal differentiation and repro- ductive impairment. The gonadal differentiation stage was selected because it marks a critically sensitive period during amphibian larval development. Future gametes originate in the gonad. They arise from relatively few primordial germ cells that form dur- ing this early stage of development [14]. Xenopus laevis was selected as the experimental animal because its comparability to mammalian species in teratogenic response has been doc- umented previously [15]. In addition, this methodology is read- ily transferable to other species of frogs and toads. MATERIALS AND METHODS Xenopus laevis (Niewkoop stage 54) tadpoles, just prior to gonadal differentiation [16], were purchased from Xenopus 1 (Dexter, MI, USA). They were held in 100-L glass aquaria containing 15 L of dechlorinated, aerated, Montreal (Canada) city water held at 21°C under static conditions. A 12:12 h light:dark photoperiod was maintained throughout the accli- mation and exposure period. Tadpoles were acclimated for one week before the experiment. They were exposed to atrazine at 21 g/L for 48 h during gonadal differentiation (stage 56) [16], which occurs during early metamorphosis. This exposure concentration was selected because this concentration of at- razine recently was reported in the St. Lawrence River Valley region of Quebec, Canada [8]. The experimental design included two controls and two