2107 Environmental Toxicology and Chemistry, Vol. 19, No. 8, pp. 2107–2113, 2000  2000 SETAC Printed in the USA 0730-7268/00 $9.00 + .00 EFFECTS OF ENDOCRINE-ACTIVE CHEMICALS ON THE DEVELOPMENT OF SEX CHARACTERISTICS OF DAPHNIA MAGNA ALLEN W. OLMSTEAD and GERALD A. LEBLANC* Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633, USA ( Received 23 August 1999; Accepted 11 January 2000) Abstract—Standard reproductive assays with daphnids involve parthenogenetically reproducing females and exclude the assessment of effects on sexual reproduction. The goals of this study were to characterize sexual differentiation of male and female daphnids (Daphnia magna) and to evaluate whether exposure to putative endocrine-disrupting chemicals may perturb the development of sex characteristics. Anatomical sex differences that developed during maturation in males included elongated first antennae and morphologic alterations in the head capsule and carapace edge. Reproductive maturation in females was associated with the development of a brood chamber and abdominal process. Alterations in the growth rates of the first antennae of males and the abdominal process of females were used to evaluate the effects of chemical exposure on the development of these sex characteristics during maturation. Exposure of female daphnids to the nonsteroidal vertebrate estrogen diethylstilbesterol and the insect juvenile hormone analog methoprene at concentrations as low as 3.0 and 0.080 M, respectively, stimulated development of the abdominal process. Exposure of males to the steroidal vertebrate androgen androstenedione (6.0 M) stimulated development of the first antennae. These results demonstrate that the development of secondary sex characteristics in daphnids can be altered by chemical exposure. Keywords—Endocrine disruption Daphnid Sexual differentiation Invertebrate INTRODUCTION In 1996, the U.S. Congress mandated that the U.S. Envi- ronmental Protection Agency develop and implement screen- ing and testing methods for the evaluation of toxicity asso- ciated with endocrine-disrupting chemicals. In response, var- ious testing approaches were considered that would serve to detect endocrine toxicity [1]. A battery of testing methods has been in place for decades, for the evaluation of chemical tox- icity as required under legislation such as the Federal Insec- ticide and Rodenticide Act and the Toxic Substances Control Act [2]. An expeditious means of evaluating the endocrine- disrupting potential of chemicals would be to incorporate en- docrine-relevant endpoints into these existing testing proto- cols. Cladocerans, including Daphnia magna, have been used extensively as representative freshwater invertebrates when evaluating the toxicity of chemicals that pose some risk for contaminating the environment through manufacture, use, or disposal [3]. Daphnia magna populations reproduce by cyclic parthenogenesis involving both sexual and asexual reproduc- tion [4,5]. Laboratory cultures of daphnids are typically main- tained in the parthenogenetic state. Further, life cycle toxicity assessments typically include only evaluations of the effects of a chemical on parthenogenetic reproduction [6]. Evidence of sexual reproduction in cultures for toxicity tests has typi- cally been used as an indicator of poor culturing or testing techniques [3]. Sexual reproduction in daphnids is critical to population viability because fertilized eggs (resting eggs) can sustain freezing or drying and thus provide a means of repopulation in new, more favorable environments or after environmental * To whom correspondence may be addressed (gal@unity.ncsu.edu). adversity [4,7]. Sexual reproduction also provides a means for reshuffling of the gene pool and increasing genetic diversity in a population [4]. Sexual reproduction includes many pu- tative endocrine-regulated processes that are not evaluated when assessing toxicity only to parthenogenetically reproduc- ing organisms. These include sex ratios of offspring, devel- opment of secondary sex characteristics, fertility, and hatching success of resting eggs. The development of sexual characteristics has been shown to be affected by chemical insult in a number of different animals. Alligators from Lake Apopka, Florida, USA, a site contaminated with the organochlorine pesticides dicofol and DDT, were found to have diminutive phalli, poorly organized testes, and significantly lower plasma testosterone concentra- tions when compared to a reference lake, Lake Woodruff, Flor- ida, USA [8]. 4-tert-Octylphenol stimulated vitellogenin pro- duction and caused reproductive impairment in male Japanese medaka [9]. Invertebrates are likewise susceptible to toxicant insult re- sulting in deviations from normal sexual differentiation. The most well-documented account is that of exposure to tributyltin resulting in the formation of a penis in female gastropods, a condition known as imposex [10]. High incidences of inter- sexuality in populations of copepods were reported in areas receiving sewage discharge [11]. Short-term exposure of neo- natal daphnids to t-amyl phenol during a critical window of development has been observed to cause females to develop certain male secondary sex characteristics [12]. Exposure of daphnid populations to atrazine in the laboratory has been shown to alter the sex ratio of the offspring [13]. The specific objectives of this study were to identify ana- tomical traits that are characteristic of normal sexual differ- entiation in daphnids and then evaluate the susceptibility of these parameters to disruption by exposure to a hormonally