Effects of Endocrine Disrupting Compounds and Temperature on the Moulting Frequency of the Freshwater Isopod Asellus Aquaticus L

Effects on the moulting frequency of Asellus aquaticus 105

ACTA BIOLOGICA BENRODIS 13 (2006): 105-115

Effects of endocrine disrupting compounds and temperature on the moulting frequency of the freshwater isopod Asellus aquaticus L. (Isopoda: Asellota)*

Effekte von hormonähnlich wirksamen Substanzen und Temperatur auf die Häutungsfrequenz der Süßwasserassel Asellus aquaticus L. (Isopoda: Asellota)

LENNART WELTJE 1, 2 & JÖRG OEHLMANN 2 1 International Graduate School (IHI), Ecotoxicology Group, Markt 23, D-02763 Zittau, Germany, [email protected], corresponding author 2 J.W. Goethe University, Department of Ecology and Evolution – Ecotoxicology, Siesmayerstraße 70, D-60323 Frankfurt am Main, Germany

Summary: The effects of the vertebrate endocrine disrupting compounds diuron, linuron, vinclozolin (pesticides), 17α-ethinylestradiol and tamoxifen (pharmaceuticals) on the moulting frequency of the freshwater isopod Asellus aquaticus (L.) are described. In addition, the influence of temperature on moulting frequency was studied. Since moulting is under the control of the steroid hormone 20-hydroxyecdysone, effects may be expected of substances known to influence the steroid sex hormones (i.e. estradiol and testosterone) of vertebrates. Although the chemicals induced changes in moulting frequency (both stimulating and inhibiting effects were found), significant differences were not established. At 20 ºC, the moulting frequency was higher than at 15 °C, but again the difference was not significant. Consequently, improvements in the experimental set-up are discussed. It is concluded that experiments are best performed at a constant temperature, with young, isolated individuals of similar size and sex.

Pesticides, moulting frequency, Asellus aquaticus

Zusammenfassung: Effekte der wirbeltierhormonähnlich wirksamen Substanzen Diuron, Linu- ron, Vinclozolin (Pflanzenschutzmittel), 17α-Ethinylöstradiol und Tamoxifen (Arzneimittel) auf die Häutungsfrequenz der Süßwasserassel Asellus aquaticus (L.) werden beschrieben. Zusätzlich wur- de der Einfluss der Temperatur auf die Häutungsfrequenz untersucht. Da die Häutung über das Steroidhormon 20-Hydroxyecdyson kontrolliert wird, können Effekte von Substanzen erwartet werden, von denen bekannt ist, dass sie die steroiden Sexualhormone von Vertebraten (d.h. Östradiol und Testosteron) beeinflussen. Obwohl die getesteten Chemikalien einen Einfluss auf die Häutungs- frequenz hatten (sowohl fördernde als auch hemmende Effekte wurden beobachtet), wurden keine signifikanten Unterschiede gefunden. Bei 20 °C war die Häutungsfrequenz höher als bei 15 °C, aber der Unterschied war ebenfalls nicht signifikant. Deshalb werden Verbesserungen des experimentellen Aufbaus diskutiert. Daraus wird geschlossen, dass die Versuche am besten bei konstanter Temperatur mit jungen, isolierten Individuen von gleicher Größe und Geschlecht durchgeführt werden.

Pflanzenschutzmittel, Häutungsfrequenz, Asellus aquaticus * Contribution to the First European Workshop on Isopods, Düsseldorf 14.-16. November 2003, Germany

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1. Introduction ca), but it has not been established that they fulfil a functional role (VANDENBERGH et 1.1. Endocrine disruptions in invertebrates al.2003; DEFUR et al. 1999). It may therefore be unrealistic to expect vertebrate xeno-estro- At present, research on the disruption of gens to have estrogenic effects in crustaceans, hormonal processes in animals is mainly fo- but they could affect other endocrine pro- cussed on vertebrates and on in vitro systems cesses, such as moulting (ZOU & FINGERMAN derived from them. The vertebrate endocrine 1997). A possible working mechanism for system is quite well understood and the char- such EDCs is an interaction with the ecdyste- acterisation of its steroid hormones and as- roid receptor, and for bisphenol A and 17α- sociated receptors is on a relatively high level. ethinylestradiol, ecdysteroid antagonistic ac- A fair number of recent in vivo studies with tivity has been shown in a Drosophila melano- chemicals known to interact with the verte- gaster cell line (DINAN et al. 2001). Further, MU brate endocrine system (particularly the xeno- & LEBLANC (2002) have shown that the devel- estrogens bisphenol A, 4-n-nonylphenol and opmental toxicity of testosterone to the 17α-ethinylestradiol), have also demonstrated crustacean Daphnia magna involves anti-ecdy- effects in invertebrates, including molluscs, steroidal activity. DE LOOF & HUYBRECHTS insects and crustaceans (BALDWIN et al. 1997; (1998) suggested in their review that ecdyste- BROWN et al. 1999; OEHLMANN et al. 2000; roids, which are also involved in vitellogen- HAHN et al. 2002; WATTS et al. 2002; VANDEN- esis, may function as the counterparts of ver- BERGH et al. 2003; this list is not exhaustive). tebrate sex steroids in insects. This implies The difficulty with most of these invertebrate that interactions of vertebrate EDCs with the studies is to unequivocally prove that the ef- ecdysteroid receptor may elicit (anti-)estrogenic fect shown is due to the disturbance of an type of effects, not per se related to the pres- endocrine mechanism and not, for instance, ence of an estrogen receptor. On the contrary, by general toxicity (SEGNER et al. 2003). How- other invertebrate groups, notably molluscs ever, such mechanistic proof is required, and echinoderms, do seem to have a func- since it forms the very basis by which an en- tional role for some vertebrate-type steroids, docrine disrupting compound (EDC) is de- hence working mechanisms similar to those fined. To gather this proof, a reasonable in vertebrates may apply. Finally, the experi- amount of knowledge should be available mental design often does not allow for a de- on the endocrinology of the organism under finitive conclusion on the (endocrine) wor- study. In the case of invertebrates, this know- king mechanism of a compound. Hence, ledge is often insufficient. many cases point towards a possibly endo- There are three main issues that hamper crine-mediated effect (e.g. increased reproduc- the interpretation of invertebrate EDC data: tive output, altered moulting behaviour, The first problem is the large diversity in endo- shifted sex-ratios), but evidence remains cir- crine mechanisms, hormones and processes, cumstantial. existing within the invertebrate world, which Although it is evident, from the meanwhile comprises about thirty phyla, against one ‘classic’ example of imposex in gastropods, single phylum of vertebrates (DEFUR et al. caused by organotin compounds, such as tri- 1999). Many mechanisms of endocrine con- butyltin (TBT) and triphenyltin (TPhT) (for trol are simply not known or not fully inves- an overview see CSTEE, 1999), that endocri- tigated yet. Secondly, vertebrate-type steroids ne disruption does occur in natural popula- (estradiol, testosterone, progesterone) have tions of invertebrates, they have not received been detected in a number of invertebrate the amount of attention they deserve within species, e.g. in higher crustaceans (Malacostra- the framework of EDC research. Especially Effects on the moulting frequency of Asellus aquaticus 107 if one considers that at least 95% of the 4) Crustaceans have readily measurable end- known animal species consists of inverte- points, such as moulting and regenerati- brates, which comprise keystone species for on, which are coupled processes under the functioning of ecosystems and food- neuroendocrine control (WEIS et al. 1992; webs, a greater effort should be undertaken FINGERMAN et al. 1998). to investigate to what extent endocrine dis- 5) A useful endpoint could be the forma- ruption really affects invertebrates. tion of oostegites in female isopods. Oo- stegites are ventral overlapping plates, 1.2. Isopods as model organisms to forming the marsupium or brood pouch, assess endocrine disruption which develop after a special moult, the so-called parturial moult, which preceeds Isopods belong to the higher crustaceans fertilisation. The latter can only take place (Malacostraca), the largest class of the sub- when the exoskeleton is still flexible in phylum Crustacea, which are part of the phy- the hours directly after moulting, and is lum Arthropoda. Insects are the other main the main reason why male aquatic isopods arthropod class, and in terms of species monopolize females by carrying them on numbers, arthropods are the most success- their ventral surface (a precopula behaviour ful group of invertebrates, hence important called mate guarding) (ZIMMER 2001). for global biodiversity research. Arthropods 6) Field evidence exists for abnormal sexual are a good starting point to investigate endo- development in amphipod (Malacostra- crine disruption for a number of reasons: ca) populations (GROSS et al. 2001; NA- 1) The endocrine system of insects is described GEL et al. 2002). Various degrees of inter- to a much greater extent than that of any sex have also been found in natural iso- other invertebrate group, mainly because of pod populations (see CHARNIAUX-COTTON the silkworm industry and pest control of 1960), but a relation with environmental insects (DEFUR et al. 1999). A substantial chemicals has not been established. part of the knowledge gathered on insects Isopods occupy key positions as decom- is also applicable to crustaceans. posers in freshwater, terrestrial and marine 2) Insect pest control initiated the develop- ecosystems and may thus provide sentinel ment of third generation pesticides, which species in endocrine disruptor research across have been designed to disrupt the action all environments. This is further underlined of specific arthropod hormones, namely by the global occurrence of the species in the ecdysteroids and juvenile hormones. This present paper, Asellus aquaticus, making it ideal implies that there are specific positive con- for monitoring purposes (and such species trol substances available (agonists and are also available for terrestrial environments, antagonists), which are a necessary tool in for instance Armadillidium vulgare or Porcellio experiments, designed to evaluate the scaber). Finally, it should be added that litera- working mechanism of suspected EDCs. ture on isopods and endocrine disruptors is 3) A unique androgenic gland and associated virtually non-existent and presents therefore androgenic hormone (MARTIN et al. 1999) a completely open and challenging field. have been shown in isopods and other Malacostraca. Androgenic hormone con- 1.3. Aim of the present work trols the development of primary and secondary male characteristics and has a The experiments described in this paper negative regulatory control on vitellogen- are the first in a series on the effects of endo- esis (DE LOOF & HUYBRECHTS 1998; DE- crine disruptors on freshwater isopods. In FUR et al. 1999). these experiments, the effects of two phar-

ACTA BIOLOGICA BENRODIS 13 (2006) 108 LENNART WELTJE & JÖRG OEHLMANN maceuticals (tamoxifen and 17α-ethinylestradiol) and three pesticides (the herbicides linuron and diuron and the fungicide vinclozolin) on the moulting frequency of Asellus b aquaticus are investigated. The selected chemicals are known to interact with vertebrate receptors for the steroids testosterone and estra- a diol. The working mechanisms of these chemicals are: estrogenic (17α-ethinylestradiol), anti-estrogenic (tamoxifen) and anti-an- c drogenic (linuron, vinclozolin and diuron). Their endocrine effects come about by direct i.e. receptor-mediated action, implying that these chemicals bind to the estrogen or to the androgen receptor. In the case of vinclozolin, the effects are mainly due to two metabo- lites of vinclozolin which have greater affin- d ity for the androgen receptor than vinclozolin itself. Since moulting in isopods is under the control of the steroid hormone 20-hydroxyecdysone (also known as β-ecdysone or ecdysterone), effects may be expected from exposure to these compounds. Since it is known that temperature has an effect on moulting, the moulting interval e generally decreases with temperature (BOU- Fig. 1: Chemical structures of the tested com- CHON et al. 1992; MOMMERTZ 1993), we per- pounds. a) vinclozolin, b) diuron, c) linuron, formed an additional experiment, wherein d) tamoxifen, e) 17α-ethinylestradiol. isopod moulting was studied at two diffe- Abb. 1: Strukturformeln der getesteten Substan- rent temperatures, 15 ºC and 20 ºC. This ex- zen. a) Vinclozolin, b) Diuron, c) Linuron, periment serves as a positive control, which d) Tamoxifen, e) 17α-Ethinylöstradiol. should demonstrate that our lab-cultured isopods are sensitive to manipulations in en- standards (purity > 99%). Tamoxifen (pu- vironmental parameters influencing the rity ≥ 99%) and 17α-ethinylestradiol (purity moulting frequency. ≥ 98%) were purchased from Sigma. The Because the aim of this work is to explore structures of these five substances are given the possibility of using freshwater isopods in figure 1. Ethanol, denatured with methyl- for the assessment of EDC effects, they can ethylketone (purity ≥ 99.5%, Merck, Darm- be used to guide and improve future work stadt) was used as a solvent. Chemicals that on this topic. were used to prepare the artificial water (according to DUFT et al. 2003) were all of re- 2. Materials and methods agent grade.

2.1. Chemicals 2.2. Organisms

Vinclozolin, diuron and linuron were Freshwater isopods (Asellus aquaticus) were purchased from Riedel-de Haën as Pestanal collected in the municipality of Zittau (Saxo- Effects on the moulting frequency of Asellus aquaticus 109 ny, Germany) from a small drainage ditch water in volumetric PMP flasks (VIT-LAB, flowing through agricultural land. Leaves of Seeheim-Jugenheim, Germany). This was local alder trees (Alnus glutinosa) on which Asel- diluted two- and tenfold with Milli-Q water lus aquaticus feeds, were sampled to serve as or 3% HNO3 (Suprapur, Merck, Darmstadt, food and substrate in the laboratory mass Germany) respectively, to achieve the required culture and in the experiments. The leaves elemental intensities for the analyses. Micro- were analysed for heavy metals and polycyclic element concentrations in leave digests were aromatic hydrocarbons (PAHs) (see below) measured with a quadrupole Perkin-Elmer to check the pollution state of the sampling Elan 6000 ICP-MS equipped with a cross- site and also the exposure history of the iso- flow nebulizer. Prior to the measurements, pod population. The mass culture was estab- the ICP-MS was optimized for oxide forma- lished in glass aquaria (30 × 30 × 50 cm) con- tion, by means of the ratio CeO+/Ce+, and taining gently aerated artificial water of pH for formation of double-charged ions, by 8.2 ± 0.2 with a depth of 15 - 20 cm and was means of the Ba2+/Ba+ couple (for more de- maintained at 15 °C in a climate room with a tails on instrumental parameters see IVANOVA day-night rhythm of 16:8 h. The culture was et al. 2001). External calibration was per- mainly fed with alder leaves, which also formed with a multi-element standard solu- served as a substrate. Additional food con- tion (Merck, Darmstadt, Germany). Each sisted of TetraPhyll (Melle, Germany), and sample was measured with three repetitions occasionally slices of cucumber and carrot. to generate a measurement average with standard deviation. Mathematical corrections for 2.3. Chemical analyses of the leaves isotopic overlap were applied, using the built-in Perkin-Elmer sofware routines. The field-collected alder (Alnus glutinosa) Macro-elements were measured with a Per- leaves with which the animals were fed in both kin-Elmer Optima 3000 ICP-OES. The refe- the cultures and the experiments, were ana- rence material SRM 1515 Apple Leaves (Nati- lysed to determine the concentration of onal Institute of Standards and Technology, seventeen elements and also of the sixteen Gaithersburg, MD, USA) was used for evalu- PAHs that are on the U.S. EPA’s list of prior- ation of the applied method. ity pollutants. The analyses were done to as- Analyses of PAHs was done with HPLC, sure that the leaves had a normal (background) according to the procedure described for se- contamination and thus did not pose an diment by DUFT et al. (2002) and following extra chemical stress for the animals. DIN 38414 S 21 (DIN 1996). Prior to analy- Analyses of the elements were as follows. sis, the leaves were freeze-dried (ALPHA 1-4, The leaves were dried at 45°C overnight and Christ, Osterode, Germany), milled (Vibra- milled (Vibratory Disc Mill RS 1 of Specialty tory Disc Mill RS 1 of Specialty steel, Retsch, steel, Retsch, Haan, Germany). About 0.3 g Haan, Germany) and subjected to accelerated dry plant material was placed in a PTFE pres- solvent extraction (ASE) using cyclohexane. sure vessel and 4 mL 65% HNO3 and 2 ml

30% H2O2 (both Merck, Suprapur) were 2.4. Experiments added. Digestion of the sample took place in a microwave (Ethos plus, MLS GmbH, Leut- The experiments with 17α-ethinylestradiol, kirch, Germany) which was heated in 3 min vinclozolin, tamoxifen, diuron and linuron to 85°C, then in 5 min to 125°C, in 5 min to were conducted simultaneously in a climate 200°C and this temperature was held for 15 room at 15 °C and shared three solvent (etha- min. After cooling down, the samples were nol) control treatments. Experiments were brought to a volume of 50 ml with Milli-Q conducted in glass beakers of 500 ml, contain-

ACTA BIOLOGICA BENRODIS 13 (2006) 110 LENNART WELTJE & JÖRG OEHLMANN ing 300 ml of artificial water, 10 adult iso- An ANOVA with DUNNETT’s post test pods (body length between 4 and 8 mm) and was performed to test for substance effects a 5 × 5 cm piece of alder leaf. For vinclozolin, in comparison to the solvent control. For tamoxifen, diuron and linuron, concentrations the temperature experiment a t-test was of 10 ng/l and 10 µg/l were tested in dupli- used to test for significant differences cate (n = 2). For 17α-ethinylestradiol, concen- between the two temperatures. In this ex- trations of 10 ng/l and 100 ng/l were tested periment, the pleotelson length and body (n = 1). Solutions were replaced at least once length of all individuals was measured and every week. Leaf pieces were replaced when in the correlation of these measures was one of the treatments the leaf piece was studied. Since the data were normally dis- eaten for about 75%. Over a period of 63 tributed (D’AGOSTINO and PEARSON omni- days, daily observations were made on the bus normality test), correlation was calcu- moulting state of the animals. Exuviae and lated according to PEARSON. For all statisti- dead individuals were removed. cal tests, P = 0.05. All calculations were per- The temperature experiment was con- formed with the software program Prism ducted at 15 °C and 20 °C. At each tempera- 4.02 (GraphPad, San Diego, CA, USA). ture, one 12 wells plate (Nunc, Wiesbaden, Germany) was tested, with in each well 2 ml Table 1: Concentrations (mg/kg dry wt) of 17 of artificial water, one juvenile isopod (body elements with relative standard deviations in lea- length < 3 mm) and a 1.5 × 1.5 cm piece of ves of Alnus glutinosa, collected in 2002. Ele- alder leaf (n = 12). Solutions were replaced at ments in italics were measured with ICP-OES, least once every week. Leaf pieces were replaced others with ICP-MS. when in one of the treatments the leaf piece Tab. 1: Konzentrationen (mg/kg Trockengewicht) von 17 Elementen mit relativer Standardabwei- was eaten for about 75%. Over a period of chung in Blättern von Alnus glutinosus (gesammelt 73 days, daily observations were made on the 2002). Elemente in Kursivschrift wurden mit moulting state of each individual. Exuviae ICP-OES, die anderen mit ICP-MS bestimmt. and dead individuals were removed. In addition, individuals were sexed and measured (pleotelson length and body length) at the end of this experiment or alternately on the day after they had died during the experiment.

2.5. Data handling and statistics

The moulting frequency was calculated as the number of moults per isopod per day. Data were corrected for mortality in the vinclozolin, tamoxifen, diuron, linuron and 17α-ethinylestradiol experiments, because ten individuals were exposed simultaneously and the exuviae could not be assigned to certain individuals. In case of the temperature experiment, isopods could be studied indi- vidually. Individuals in this experiment that died within 14 days after the start of the experiment were omitted from the calculations. Effects on the moulting frequency of Asellus aquaticus 111

Table 2: Concentrations (mg/kg dry wt) of 16 PAHs (listed in elution order) in leaves of Alnus glutinosa, collected in 2002; nd = not detected. Tab. 2: Konzentrationen (mg/kg Trockenge- wicht) von 16 PAHs (in der Reihenfolge ihrer Elution aufgelistet) in Blättern von Alnus glutinosus (gesammelt 2002); nd = nicht nachgewiesen.

3. Results

The chemical analysis of the alder leaves (see table 1 and 2) demonstrated that the site where the isopods and the alder leaves were collected has only a background pollution level. Therefore, and as far as these analyses per- mit, we may conclude that the isopods have no previous history of exposure to pollution beyond a background level. Fig. 2: Effects of diuron, linuron and vinclozo- Exposure to the selected vertebrate endo- lin (10 ng/l and 10 µg/l) on the moulting fre- crine disruptors demonstrated effects on quency of freshwater isopods. Abb. 2: Effekte von Diuron, Linuron und Vin- moulting, albeit non significantly (P > 0.05, clozolin (10 ng/l und 10 µg/l) auf die Häutungs- ANOVA, followed by Dunnett’s test). The frequenz von Süsswasserasseln. results are presented in figs. 2 and 3. Expo- sure to the anti-androgenic pesticides diuron, 10 ng/l than at 10 µg/l. Exposure of the iso- linuron and vinclozolin induced higher pods to the estrogenic drug 17α-ethinylestra- moulting frequencies (linuron in a concentra- diol at 10 ng/l lowered the moulting fre- tion-dependent manner, see fig. 2), whereas quency, while at 100 ng/l moulting was the anti-estrogenic drug tamoxifen lowered stimulated (fig. 3). Unfortunately, none of the moulting frequency in a concentration- these results proved to be significant, due to dependent manner (fig. 3). Effects of diuron several causes which will be dealt with in the and vinclozolin were slightly higher at next section.

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As expected, the moulting frequency was higher at 20 ºC than at 15 ºC (fig. 4). However, also these results were not significant (P > 0.05, t-test). The pleotelson length and the body length of the animals in the temperature experiment were highly correlated (for pooled data, Pearson r = 0.85, P < 0.0001), which implies that one of these measurements is sufficient to characterise the size of the animals. The relation between body length and pleotelson length is depicted in figure 5.

Fig. 3: Effects of tamoxifen (10 ng/l and 10 µg/l) and of 17α-ethinylestradiol (10 ng/l and Fig. 5: Relation between body length and pleo- 100 ng/L) on the moulting frequency of fresh- telson length of juvenile freshwater isopods from water isopods. the temperature experiment. Abb. 3: Effekte von Tamoxifen (10 ng/l und Abb. 5: Zusammenhang zwischen Körperlänge 10 µg/l) und von 17α-Ethinylöstradiol (10 ng/l und Länge des Pleotelsons von juvenilen Süß- und 100 ng/l) auf die Häutungsfrequenz von wasserasseln aus dem Temperaturexperiment. Süßwasserasseln. 4. Discussion and conclusions

Because no significant differences for any of the treatments could be established, it is ob- vious that either the experimental set-up needs to be improved, or alternately, that moulting frequency is not a sensitive endpoint regard- ing exposure to EDCs or to temperature differences. The experimental set-up could certainly be improved. The number of replicates, as well as the homogeneity of the exposed isopod groups, can be optimised. This implies to test a higher number of replicates; preferably Fig. 4: Effects of temperature (15 and 20 ºC) on isolated individuals, of similar size and sex. the moulting frequency of freshwater isopods. Increasing the number of exposed individuals Abb. 4: Effekte der Temperatur (15 und 20 ºC) will lead to a higher statistical power of the auf die Häutungsfrequenz von Süsswasserasseln. test, which is obviously needed for a variable Effects on the moulting frequency of Asellus aquaticus 113 endpoint such as moulting. Isolating the iso- e.g. 20-hydroxyecdysone or tebufenozide, it pods assures an exact recording of the inter- may be elucidated if there is an endocrine moult period of each individual and yields working mechanism involved (see MU & LE- statistically independent observations. Since BLANC 2002). the intermoult period increases with the age Our observations agree with the anti-ec- of the individuals, the sensitivity of the test dysteroid activity of testosterone in the may be increased by testing young, i.e. small, crustacean Daphnia magna (MU & LEBLANC isopods. However, there was hardly any dif- 2002). The only exception is formed by the ference between the moulting frequency of observed lower moulting frequency in the the control groups in the substance and tem- lowest 17α-ethinylestradiol treatment, perature experiments (compare figs. 3 and 4; which does not fit into this series. How- moulting frequency ~0.022 d-1), although the ever, they may be explained by the slight isopods in the temperature experiment were ecdysteroid antagonistic activity that smaller than the ones in the substance ex- 17α-ethinylestradiol showed in an in vitro periments. Because the sex of young isopods cell line (DINAN et al., 2001). is not easily determined, it is advisable to in- Overall, the work presented here did not crease the number of exposed individuals, produce any conclusive evidence on the in- and evaluate the data for each sex separately fluence of vertebrate EDCs on the moult- after the experiment has finished and the ani- ing frequency of the freshwater isopod Asel- mals have been sexed. The importance in dis- lus aquaticus. The results of the experiments tinguishing the sexes is related to the diffe- with 17α-ethinylestradiol and tamoxifen are rent moulting patterns of males and females hard to interpret, because of irregularities (MOMMERTZ 1993). Whereas males have a in the concentration-response relation and regular moulting pattern, females have two the possible involvement of general toxi- types of moults which alternate; “normal” city, respectively. On the other hand, the moults and so-called parturial moults in which experiments with the pesticides vinclozo- the oostegites are formed. In addition, the lin, diuron and linuron hint at ecdysteroid temperature should be controlled with some agonistic activity of these vertebrate anti- precision, because this parameter will influ- androgens. This should be investigated in ence the moulting behaviour of the exposed more detail in experiments with an im- individuals. proved lay-out. The effects of the chosen substances were stimulating in case of the anti-androgenic Acknowledgements pesticides vinclozolin, diuron and linuron but also in the highest concentration of the estro- The authors thank Constanze Stark, Simo- genic drug 17α-ethinylestradiol. If interaction ne Ziebart, Gertraud Wirzinger, Conny with the ecdysteroid receptor is explaining Scholz and Christian Vogt for assistance in these observations, then the previously men- the laboratory and in the field. Heike Hei- tioned compounds are acting as ecdysteroid denreich and Gerlinde Liepelt are gratefully agonists. Opposite effects were observed for acknowledged for chemical analyses of the the anti-estrogenic drug tamoxifen, which alder leaves. This research has been sup- may indicate ecdysteroid antagonism, but ported by a Marie-Curie Fellowship of the could also be indicative for general toxicity, European Community programme Human which has been reported for zinc effects on Potential, under contract number HPMD- the moulting frequency of the terrestrial iso- CT-2000-00039, fellow reference number pod Porcellio scaber (DROBNE & ŠTRUS 1996). HPMD-GH-00-00039-02 to the first By co-exposure with an ecdysteroid agonist, author.

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(Accepted: August 5th, 2005)

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