ACCIONES COLECTIVAS

The bony fish represent the largest group of vertebrates both in number of species and in number of individuals. Their evolution and adaptation to different habitats has led to a high diversity of physiological, anatomical, behavioral and ecological strategies in fish (Bernanke and Köhler, 2008). Endocrine disruption has been studied in a variety of freshwater teleost fish species, but cyprinids (e.g. fathead minnow, zebrafish, carp) and salmonids (e.g. rainbow trout, brown trout, Atlantic salmon) are the best represented (Jobling and Tyler, 2003). Chemicals altering the sex hormone levels may induce quantitatively and qualitatively different biological responses and adverse apical effects in various fish species because of the different mechanisms for sexual development and different reproductive strategies (Hutchinson et al., 2006). Moreover, the sex, life stage and the endogenous hormone levels during the reproductive cycle of fish are other important physiological factors that may influence the effects of EDC exposure (Kawai et al., 2003). Therefore, it is important to analyze the differences in the sensitivities of different fish species to effects of EDCs for interspecies extrapolation from laboratory fish tests to wild fish populations (Hutchinson et al., 2006). For the experiments of this thesis, three different fish species with different sexual development and/or different reproductive strategies have been used.

1.6.1. Fathead minnow (Pimephales promelas) and zebrafish (Danio rerio)

Fathead minnow and zebrafish are small fish species currently used for EDC testing. Established methods for maintaining both species in continuous cultures in laboratory exist and the fish are easy to handle (Ankley et al., 2004). Both species have a short life cycle and start to spawn early in their life (Hutchinson et al., 2006). They are fractional spawners and produce a high number of eggs (Mills and Chichester, 2005). Their spawning activity can be precisely controlled by manipulating the temperature, photoperiod, and spawning substrate. Determination of fertility of the eggs can be achieved easily using a microscope. Thus, they are successfully used for partial and full life cycle tests to investigate reproductive effects of xenobiotics (Ankley et al., 2004).

The fathead minnow has a ubiquitous distribution across North America. Adults are approximately 50 to 75 mm long and weigh 2 to 5 g (Jensen et al., 2001). They have a generation time of 4 to 5 months. A reproductively active female fathead minnow typically

Chapter 1 Introduction

22 deposits clutches of 50 to 100 eggs on the bottom of the spawning substrate every 3 to 5 days (Jensen et al. 2001). Embryos hatch in approximately 4 to 5 days at 25°C (Ankley et al, 2004). Fathead minnows exhibit secondary sex characteristics and defined mating behavior, which makes them suitable to investigate phenotypic and behavioral changes associated with EDC exposure (Mills and Chichester, 2005). The gonadal differentiation of fathead minnow starts early in its development, and the gonads can be identified as either male or female from 10 to 25 days post-hatch (van Aerle et al, 2004; Uguz, 2008).

The zebrafish is a small sized fish species native to India and Burma. Adult zebrafish are approximately 40 to 50 mm long, and weigh about 1.5 g. The reproductive cycle takes 3 to 4 months. Reproductively active female zebrafish can spawn almost daily, with relatively large spawns (e.g. >150 eggs) occurring every 5 to 10 days. The eggs are released in the water column and settle to the bottom of the tank. The embryos hatch in about 3 days at 28°C (Ankley et al., 2004). In contrast to the fathead minnow, all juvenile individuals first develop ovary-like gonads. Only after approximately 35 to 45 days post-hatch, the ovarian tissue of male fish regresses and develop into testicular tissue (Maack and Segner, 2003).

1.6.2. Brown trout (Salmo trutta fario)

The brown trout is an important freshwater fish species in European rivers and has a high commercial and social value. Over the last 20 years, however, its catch has dramatically declined in Swiss rivers (Körner et al., 2005). To evaluate the causes for the observed decline, the project ‘Fishnet’ was established in 1998. Within the “fishnet” project, various hypotheses were put forward to explain the reduced catch (Burkhardt-Holm et al., 2002). Among others, a disturbed reproductive health as a consequence of endocrine disruption has been considered.

The brown trout is an annual spawner with a predominant spawning season in November and December. The brown trout is a gonochoristic species, in which gonadal development and differentiation happens during the first two to three years before attaining sexual maturity, with the males usually maturing before females (Billiard, 1987; Elliott, 1994). Signs of sex differentiation appear first in females at approximately 20-30 days post- hatch, and later on in males. According to their reproductive cycle, the morphology of their gonads follows seasonal changes. In males, gonads remain undifferentiated until the initiation of the first spermatogenic cycle (i.e. 1 to 2 years), and the complete

23 spermatogenesis and the first reproductive cycle usually takes place during the second year of life. In contrast, the completion of oogenesis till the ovulation and the first reproductive cycle happens for most females only in the third year of life (Billard, 1987). During the second year of life, some vitellogenic activity can, however, be observed in some females, although final ovulation does not take place. In salmonids, the capacity to synthesize and metabolize sex steroids is active from the time of egg fertilization but changes seasonally throughout the sexual differentiation and development of gonads (Billard et al., 1992; Feist and Schreck, 1996; Yeoh et al., 1996). Sex differentiation is labile during early life stages since sex inversion can easily be obtained by feeding the fry with food containing androgens or estrogens (Hunter and Donaldson, 1983).