THE EFFECT OF POLYCHLORINATED BIPHENYLS MIXTURE ( AROCLOR 1254 ) ON THE EMBRYONIC DEVELOPMENT AND HATCHING OF PRUSSIAN CARP , CARASSIUS GIBELIO , AND COMMON CARP , CYPRINUS CARPIO ( ACTINOPTERYGII : CYPRINIFORMES : CYPRINIDAE )

Aroclor 1254, a polychlorinated biphenyls (PCBs) mixture, belongs to endocrine-disrupting chemicals (EDCs) which are able to affect the neuroendocrine axis (hypothalamo-pituitary-gonadal) involved in the regulation of reproductive function in fish (Khan et al. 2001, Khan and Thomas 2006) and in other vertebrates (Colborn et al. 1993, Gore 2001). The action of polychlorinated biphenyls as endocrine disruptors (usually through the oestrogen or androgen receptors) depends on the degree of their chlorination. Low chlorinated PCBs act in an estrogenic manner, and these with higher (>48%) chlorination act as weak estrogens or as oestrogen receptor antagonist (Kester et al. 2000, Gregoraszczuk et al. 2008). There are data showing that the exposure of adult fish to PCBs, leads to the changes in GnRH neurons, the key cells involved in the control of the secretion of luteinizing hormone (Khan and Thomas 2001). The impairment of LH secretion was found in Atlantic croaker, Micropogonias undulatus (Linnaeus, 1766), and common carp, Cyprinus carpio, after exposure to Aroclor 1254 (Khan and Thomas 2001, Socha et al. 2008a). Khan and Thomas (2001) explain that this neuroendocrine disruption of gonadotropin release is caused, at least in part, by inhibition of hypothalamic monoamine neurotransmitters (5-HT and DA). In vitro culture of pituitary carp cells in the presence of PCBs shows the possibility of direct action of PCB on LH release at the level of pituitary (Socha et al. 2008a). Beside the action on the hypothalamic-pituitary-gonadal axis PCBs are able to change other endocrine glands function linked to the reproduction. In experiments on Atlantic croaker and Nile tilapia, ACTA ICHTHYOLOGICA ET PISCATORIA (2012) 42 (1): 31–35 DOI: 10.3750/AIP2011.42.1.04


INTRODUCTION
Aroclor 1254, a polychlorinated biphenyls (PCBs) mixture, belongs to endocrine-disrupting chemicals (EDCs) which are able to affect the neuroendocrine axis (hypothalamo-pituitary-gonadal) involved in the regulation of reproductive function in fish (Khan et al. 2001, Khan andThomas 2006) and in other vertebrates (Colborn et al. 1993, Gore 2001).The action of polychlorinated biphenyls as endocrine disruptors (usually through the oestrogen or androgen receptors) depends on the degree of their chlorination.Low chlorinated PCBs act in an estrogenic manner, and these with higher (>48%) chlorination act as weak estrogens or as oestrogen receptor antagonist (Kester et al. 2000, Gregoraszczuk et al. 2008).There are data showing that the exposure of adult fish to PCBs, leads to the changes in GnRH neurons, the key cells involved in the control of the secretion of luteinizing hormone (Khan and Thomas 2001).The impairment of LH secretion was found in Atlantic croaker, Micropogonias undulatus (Linnaeus, 1766), and common carp, Cyprinus carpio, after exposure to Aroclor 1254 (Khan andThomas 2001, Socha et al. 2008a).Khan and Thomas (2001) explain that this neuroendocrine disruption of gonadotropin release is caused, at least in part, by inhibition of hypothalamic monoamine neurotransmitters (5-HT and DA).In vitro culture of pituitary carp cells in the presence of PCBs shows the possibility of direct action of PCB on LH release at the level of pituitary (Socha et al. 2008a).Beside the action on the hypothalamic-pituitary-gonadal axis PCBs are able to change other endocrine glands function linked to the reproduction.In experiments on Atlantic croaker and Nile tilapia, Oreochromis niloticus (L.), polychlorinated biphenyls were also found to alter thyroid status by changing circulating levels of T 4 and/or T 3 (LeRoy et al. 2006, Coimbra andReis-Henriques 2007).Thyroid hormones are important in fish early development, growth, and reproduction (Power et al. 2001).PCBs are known to accumulate in the lipid-rich tissues-they were found in liver and gonads of many aquatic organisms.High PCB levels in ovary or eggs have been associated with reproductive failures such as decreased hatching success and high embryonic mortality in Baltic herring, Clupea harengus L., or lake trout, Salvelinus namaycush (Walbaum, 1792) (see: Mac et al. 1993, Hansen et al. 1985).These types of xenobiotics can be transmitted parentally or environmentally, and finally they affect the offspring.Despite a ban on manufacture of PCBs in the late 1970s, they are still present in the aquatic environment and may have an effect on developing embryos.
The aim of this study was to investigate the effect of Aroclor 1254, a polychlorinated biphenyls mixture with a 54% (weight) of chlorine, on the embryonic development of two cyprinid species: Prussian carp, Carassius gibelio (Bloch, 1782), and common carp, Cyprinus carpio L.

MATERIAL AND METHODS
The experiments were conducted at the Department of Ichthyobiology and Fisheries, University of Agriculture in Krakow, Poland in the end of June 2009.
For the first experiment the eggs were obtained from 4 Prussian carp (Carassiuas gibelio) females, stimulated previously with sGnRH analogue (Bachem Feinchemicalien AG, Switzerland) at the dose of 10 mg • kg -1 body weight.The samples of eggs obtained from each female were divided into two Petri dishes (about 150 eggs in each dish) and activated with common carp sperm.After 1 min the water in experimental groups was replaced with 1 or 10 ng • mL -1 of Aroclor1254 (Promochem, Poland), while in control group-with clean water (seasoned tap water supplemented with Fungi Stop Konzentrat (TETRA, Germany).
In the second experiment the eggs were obtained from 4 common carp (Cyprinus carpio) females, which were stimulated with Gonazon TM 32 mg • kg -1 body weight (Intervet International BV, Boxmeer, The Netherlands) to induce ovulation.The eggs were fertilized with 10 µL of common carp sperm.After 1 min the water in experimental groups was replaced with appropriate mixture of Aroclor 1254 (1, 10, 50, or 100 ng • mL -1 water), while in control group-with clean water.Fungi Stop at the concentration of 75 µL -1 of water was used to prevent fungal diseases of incubated eggs.
Water was changed twice a day in each dish.The incubation lasted for about 4 days.The mortality, hatching rate, number of hatched larvae, and number of deformed larvae (with vertebral curvatures, yolk sac malformation) were observed and counted.The results were analysed by means of nonparametric two-tailed Mann-Whitney test.The differences between the means were determined as significant for P < 0.05.

RESULTS
The effect of Aroclor 1254 on the development of activated Prussian carp eggs.After 24 h of incubation there was no significant difference in the percentage of live eggs between the Aroclor 1254 treated groups and the control one (Fig. 1).Statistically significant higher rate of hatching at 75 h of exposure to Aroclor at the concentration of 1 ng • mL -1 was noted (Fig. 2).There were no significant differences in the hatching rate after: 78, 81, 85, 89, and 99 h of exposure (Fig. 2).There was no significant difference in the percentage of deformed larvae between all groups of exposure (Fig. 3).
The efect of Aroclor 1254 on the development of fertilised common carp eggs.The proportion of live eggs ranged from 76.4% in the group incubated with Aroclor 1254 at the concentration of 10 ng • mL -1 to 80.28% in the control group.There was no significant difference in the percentage of living carp eggs between the experimental groups and the control one after the first day of incubation (Fig. 4).
Tested concentration of Aroclor 1254 (1, 10, 50 and 100 ng • mL -1 water) did not change the hatching rate of common carp larvae (Fig. 5).There was no significant time dependent effect on number of hatched larvae.
The highest percentage of deformed larvae was found in the group incubated with Aroclor 1254 at the concentration of 10 ng • mL -1 water (Fig. 6).The percentage of deformed larvae was significantly different in comparison with control group (P < 0.05).

DISCUSSION
Chemical pollutants (heavy metals, PCBs) present in aquatic environment can disrupt vital physiological processes (growth, reproduction, osmoregulation) in fish during their different life stages (Coimbra and Reis-Henriques 2007, Khan and Thomas 2001, Szczerbik et al. 2008).Exposure to PCBs may decline the population in many fish species due to decreased ovarian growth (Khan and Thomas 2001), induction of testicular lesions (Sangalang et al. 1981), decreased motility of sperm (Socha et al. 2008b), and finally due to the reduction in the number of spawned eggs (Örn et al. 1998(Örn et al. , Heiden et al. 2006)).
In this study we have focused on the influence of PCB mixture Aroclor 1254 on embryonic development and hatching of two freshwater species: Prussian and common carp.Aroclor 1254 was added after activation/fertilization of eggs with carp semen, to eliminate the effect of PCBs on sperm motility, because there are data showing that PCBs are able to decrease spermatozoa important parameters (Socha et al. 2008b).In both experiments there was no difference in the percentage of living eggs in the first 24 h of incubation; that period appears to be the most sensitive time for egg viability (Kime 1999).Concerning the hatching time, acceleration of hatching at 75 h at the lowest concentration of Aroclor 1254 (1 ng • mL -1 ), was found only in Carassius gibelio eggs.In the second experiment with fertilised common carp eggs there were no differences of hatching frequency among all groups.Similar results were obtained in zebrafish, Danio rerio (Hamilton, 1822), exposed to the mixture of PCB-no difference of hatching frequency or median hatching time were observed (Örn et al. 1998).In our study, the significantly higher number of deformed common carp larvae was observed after exposure to 10 ng • mL -1 of Aroclor 1254.It seems that common carp eggs are more sensitive to this kind of xenobiotics than eggs of Prussian carp.The lack of larvae deformation in Carassius gibelio, after exposure to the same concentration of Aroclor 1254 (1 and 10 ng • mL -1 ) was noted.It is worth noticing that activated Prussian carp eggs were also resistant to cadmium, another waterborne pollu-tant (Szczerbik et al. 2008).It is known that the outermost layer of the chorion binds the greatest amount of PCBs, antibiotics, or other toxic chemicals and thereby impede entry of these xenobiotics (Kudo andYazawa 1997, Mäenpää et al. 2004).These properties may explain why greater bioaccumulation occurs later in development of fish (Mäenpää et al. 2004) perhaps due to saturation of the chorion binding sites, and why hatched larvae are more sensitive to xenobiotics (Viant et al. 2006).
Summing up, our results showed that Aroclor 1254 (at tested concentrations) is not harmful to the development of activated Prussian carp and common carps eggs, but in the case of common carp embryos, PCB mixture may increase the teratogenic effect.Since fish larvae can be more sensitive to contaminants than developing embryos it is important to evaluate the impact of Aroclor 1254 during early life stages of Prussian carp and common carp.