**3. Molluscs as "possible and valuable" model animals in environmental tests**

#### **3.1. Sex steroid-like receptors in molluscs**

PRG receptor immunoreactive elements were identified in the reproductive system of the female *Octopus vulgaris* [90]. According to Tosti, the PRG receptors are physiologically active because the external application of PRG stimulates the activation of spermatozoa in *Octopus* [91]. In contrast to cephalopods, no progestogen-like receptors have been identified in snails so far. The androgen-like receptor immunopositive cells has already been described in ovotestis of *Biomphalaria alexandrina*, and there is some (inferred) evidence of a role for androgenlike molecules in the reproductive cycle of molluscs [92–94]. But the fact is that homolog or orthologue sequences were not identified in molluscs despite investigations searching for the androgen receptor gene [95]. In contrast, estrogen receptor orthologues have previously been reported in number of freshwater and marine molluscs, such as *Aplysia californica, Biomphalaria glabrata*, *Bithynia tentaculata, Marisa cornuarietis*, *Potamopyrgus antipodarum*, *Nucella lapillus*, *Chlamys farreri, Crassostrea gigas*, *Lottia gigantean, Mytilus edulis*, *Octopus vulgaris* and *Sepiella japonica* [93, 96, 97]. The existence of an estrogen or estrogen-related receptor has been confirmed in *Lymnaeidae* sp. (e.g. *L. ollula*) [98], however in *L. stagnalis* is not investigated so far. The amino acid sequence of endocrine receptor of oyster (*Saccostrea glomerata*) contains a DNA-binding domain and a ligand-binding domain which are conserved among vertebrate endocrine receptors [99]. However, it is worth to mention that real function of identified estrogen receptors are questionable at present, because ligand studies show that the receptor homolog is non-sensitive to estrogen in the oyster, for example [100]. Even so, many researchers speculate that the most steroid pollutants act through the estrogen or androgen-like receptors in molluscs [93]. It is also known that the endocrine effect of TBT (steroid biocide) appear through binding to a nuclear receptor (the retinoid × receptor) in *Nucella lapillus*. The natural ligand (9-cis-retinoic acid) of the retinoid × receptor induces similar imposex in females of *N. lapillus* than TBT at similar concentration [101, 102]. Despite the contradictory observations and opinions about the presence of steroid-like receptors in molluscs, as well as the limited genetic evidence for steroid receptors, binding proteins for classical vertebrate-type steroids have been described. However, it has not yet been demonstrated that this binding is coupled to an endocrine biological response. Some researchers speculate that vertebrate-like steroids, such as estorgen, can also/just act through non-genomic mechanisms in mollusc. Non-genomic action of steroids are expressed through cell surface membrane receptors (not nuclear receptors) and in this case they also can results direct local "ionotropic" effects (e.g. modification ion fluxes) and/or they can activate second messenger kinase cascade system during "metabotropic" pathway (e.g. cAMP-MAPK-PKC) [103, 104].

#### **3.2. Endocrine steroid system of molluscs: evidences and questions**

For example, in fish, the main natural progestin is 17α,20β-dihydroxy-4-pregnen-3-one (DHP). In females, DHP is responsible for maturation of oocytes [78] and ovulation [79], while in males it is involved in spermiation and sperm motility [80]. Synthetic progestogen contaminations altered hormone levels [81], induced transcriptional effects in adults [82] and embryos [83], altered sex development and induced development of male secondary sexual characteristics in female fish [81, 84]. Therefore, there are evidences that progestogen contamination interferes with endogen steroids and adversely affect fish reproduction. According to literature data, LNG and GES significantly reduce egg production in fathead minnow (*Pimephales promelas*) [81, 84]. At environmental ng/L concentrations, progestogens could interfere with natural pheromones, therefore also impair the physiological responses and spawning behavior in fish [85, 86]. In addition, based on earlier work it has been shown that chronic exposure to a mixture of PRG, LNG, DRO induce complex molecular changes both in brain, liver and serum of roach (*Rutilus rutilus*) [87]. Collectively in vertebrates, progestogens activate nuclear PRG receptors [88], but also may activate other steroid receptors, such as the androgen, estrogen, glucocorticoid and mineralocorticoid receptors, exerting combinations of progestogenic, (anti)androgenic, (anti)

estrogenic, glucocorticoidogenic and anti-mineralocorticoidogenic effects [89].

PRG receptor immunoreactive elements were identified in the reproductive system of the female *Octopus vulgaris* [90]. According to Tosti, the PRG receptors are physiologically active because the external application of PRG stimulates the activation of spermatozoa in *Octopus* [91]. In contrast to cephalopods, no progestogen-like receptors have been identified in snails so far. The androgen-like receptor immunopositive cells has already been described in ovotestis of *Biomphalaria alexandrina*, and there is some (inferred) evidence of a role for androgenlike molecules in the reproductive cycle of molluscs [92–94]. But the fact is that homolog or orthologue sequences were not identified in molluscs despite investigations searching for the androgen receptor gene [95]. In contrast, estrogen receptor orthologues have previously been reported in number of freshwater and marine molluscs, such as *Aplysia californica, Biomphalaria glabrata*, *Bithynia tentaculata, Marisa cornuarietis*, *Potamopyrgus antipodarum*, *Nucella lapillus*, *Chlamys farreri, Crassostrea gigas*, *Lottia gigantean, Mytilus edulis*, *Octopus vulgaris* and *Sepiella japonica* [93, 96, 97]. The existence of an estrogen or estrogen-related receptor has been confirmed in *Lymnaeidae* sp. (e.g. *L. ollula*) [98], however in *L. stagnalis* is not investigated so far. The amino acid sequence of endocrine receptor of oyster (*Saccostrea glomerata*) contains a DNA-binding domain and a ligand-binding domain which are conserved among vertebrate endocrine receptors [99]. However, it is worth to mention that real function of identified estrogen receptors are questionable at present, because ligand studies show that the receptor homolog is non-sensitive to estrogen in the oyster, for example [100]. Even so, many researchers speculate that the most steroid pollutants act through the estrogen or androgen-like receptors in molluscs [93]. It is also known that the endocrine effect of TBT (steroid biocide) appear

**3. Molluscs as "possible and valuable" model animals in** 

**environmental tests**

38 Biological Resources of Water

**3.1. Sex steroid-like receptors in molluscs**

Despite many published studies reporting presence of vertebrate-like sex steroids, steroidogenic enzymes and steroid receptors in molluscs, the endocrine system is the most uncleared and contradictory topic of molluscan research. It is generally accepted that vertebrate-type steroids, as PRG, estradiol or testosterone, are presented in various molluscan tissues (e.g. gonads, haemolymph) and they are physiologically potent molecules performing hormonal functions. Regarding their endogenous biosynthesis, evidences are contradictory. At present is unknown whether vertebrate-type sex steroids are formed endogenously during steroidogenesis or they are taken up from their environment through the feeding because it is known that many plant species contain vertebrate-like sex steroids [105]. Since PRG, estradiol and testosterone as functional hormones in mollusc are the same as those of vertebrates, and vertebrates continuously excrete them not just via urine and faces, but via their body surface or gills (in fish), the other possibility is that observed "molluscan" steroids just come from contamination [95, 106]. At the same time, several papers have been published presenting evidence of steroidogenic activity and steroid metabolism in molluscs [107, 108]. For example, beside other metabolic enzymes (e.g. 5α-reductase, sulfotransferase, and acyl-CoA acyltransferases) the occurrence and activity of two key steroidogenic enzymes 3α/β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD are presented in several molluscan species. The 3α/β-HSD is the key enzyme in conversion of prognenolone (P5) to PRG. This enzyme has been described in *Ariolimax californicus*, *Aplysia depilans*, *Helix pomatia*, *Mytilus edulis* and *Octopus vulgaris*. The 17β-HSD is crucial molecule in the last step of steroid syntheses and the primary metabolism. The 17β-HSD catalyzes the interconversion of androstenedione to testosterone, estrone to 17ß-estradiol and androstenedione to dihydrotestosterone. The 17β-HSD enzyme has been detected in many snails (e.g. *Marisa cornuarietis, Ilyanassa obsolete, Hexaplex trunculus, Bolinus brandaris* and *Helix aspersa*), bivalves (e.g. *Crassostreas gigas, Crassostreas virginica, M. edulis, M. galloprovincialis, Ruditapes decussate* and *Patinopecten yessoensis*) and cephalopods (e.g. *Sepia officinalis and O. vulgaris*) so far. These observations comprise a series of indications about the existence of steroidogenesis in different molluscs [107, 108]. At present, no data are available about the expression of key enzymes in *L. stagnalis*, however the cholesterol which is the direct precursor of P5 has been described in its neurons [109]. According to literature data, *L. stagnalis* was able to transform PRG from injected labeled P5 [110]. The P5 is a key molecule in the biosynthetic pathway of main vertebrate steroids, such as PRG, 17ß-estradiol and testosterone which have also been proposed as functional hormones in molluscs [95].

called inspection when the snail moves along the length of the egg mass brushing it with lips and tentacles. Resting and turning last for about an hour each, oviposition 10 minutes and inspection about 2 minutes. Egg-laying in *L. stagnalis* is an example of a complex behavior that is triggered by the release of multiple neuropeptide transmitters from neuroendocrine centers within the central nervous system that act on other neural circuits controlling egg-laying behavior [12]. Weather the action of described sex steroids is receptor-mediated or not is uncleared in *L. stagnalis* at present. But it is a fact that vertebrate-like sex steroids might have a key role in reproduction in snails. Temporal variation in some steroid titers that coincide with

Pond Snail Reproduction as Model in the Environmental Risk Assessment: Reality and Doubts

http://dx.doi.org/10.5772/intechopen.72216

41

Cyproterone acetate (CPA) is a commonly used synthetic progestogen compound in oral contraceptives, which also has anti-androgen effects in vertebrates. The vinclozolin (VZ) is mainly known as anti-androgen, which can also bind to estrogen and progestogen receptors in vertebrates. Using these progestogens, Ducrot and Giusti published no significant difference in shell length of adult *L. stagnalis* between control and treated groups after 21 days of exposure to any of the tested chemicals [25, 27]. However, Ducrot observed slower growing among juveniles, sub-adults and young adults exposed to the highest concentration of VZ (2500 μg/L) during the first week, but partly recovered during the second and third weeks, so that growth pattern did not lead to a significant decrease in the mean shell size compared to control group. In Giusti's experiments, neither CPA (2–50 μg/L), nor VZ (10–240 ng/L) induced more than 10% mortality. According to Ducrot, significant mortality occurred in treated adult animals exposed to the highest concentration of VZ, whereas the feeding activity was stopped in this group. CPA and VZ had no significant effect on cumulated oviposition and fertility in adult snails in single chemical treatment approach, however 2 and 10 μg/L CPA as well as 10–240 ng/L VZ treatments induced a significant decrease in egg number per egg mass compared to control. However, in this experiment no clear concentration-response relationship was described [27, 106]. CPA, VZ and tributyltin (TBT) were also tested on other three gastropod species, *Marisa cornuarietis*, *Nucella lapillus* and *Nassarius reticulatus* in a chronic experiment. In this investigation, the snails were treated by nominal CPA concentration of 1.25 mg/L for 12 month or by nominal VZ concentration of 0.03–1.0 μg/L for up to 5 month. It was reported that no mortality at used concentrations, but a significant decrease in the length of the penis and accessory male sex organs was observed in both species [106, 115]. Since progestogen contaminations are expected be found in the environment as mixtures, Zrinyi and her co-workers applied them together and environmentally relevant concentration range in *L. stagnalis* reproduction tests [116]. In a 10 ng/L eqi-concentration mixture of PRG, LNG, GES and DRO treatment approach resulted that the oocyte production of individual animals significantly changed in the treated group at the end of the 21-day long experiment. The number of laid oocyte per egg mass of individual animals shows continuous growth in the treated group week by week. Their number at the end of the third week was in average of twofold higher compared to the control. The

control animals produced the same amount of oocytes weekly during experiment.

Beside egg number assessment, egg abnormalities can also be observe (e.g. polyembryonic egg, atrophied albumen, unfertilized oocyte or dead zygote in eggs), which refer to reproductive

reproductive stages have been observed.

**3.4. Progestogen effects in** *L. stagnalis*

Steroidogenesis and steroid metabolism play and important role in the regulation of endogenous steroid level in molluscs. As a result of their endogenous biosynthesis, active P5 (e.g. in *M. edulis, Astacus leptodactylus* and *Nephrops norvegicus*), PRG (e.g. in *M. edulis, Mya arenaria* and *O. vulgaris*), androstenedione (e.g. in *M. edulis, H. aspersa, A. leptodactylus* and *Neomysis integer*), estron (e.g. in *M. edulis, Arion ater rufus, H. aspersa* and *Asterias rubens*) and testosterone (e.g. in *M. edulis, Arion ater rufus, A. leptodactylus* and *N. integer*) have been described in several molluscan species [108]. In addition, it has been published that the physiological concentration of these sex steroids are related to changes in the reproductive cycle and their level are higher in one sex than other, or their level are changed during EDC contamination. Furthermore, another vertebrate-type hormone, the gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of "molluscan"sex steroids from the gonads, and elicited contractions of the oviduct, for example, in cephalopods. This result suggests that octopus-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis [111]. GnRH-like hormone has also been identified in two freshwater snails, *Helisoma trivolvis* and *L. stagnalis*, presumably with a similar control function than in cephalopods [112]. From a phylogenetic point of view, observations of steroidogenesis and vertebrate-type steroids are very interesting because they indicate a common origin of a sex hormonal system between molluscs and vertebrates. However, much more information is needed to fully understand the physiological function of sex steroid hormones in molluscs. But also noticeable that according to valuable Scott's reviews, despite many studies starting over 55 years ago, these data are questionable from several reasons. For example, the mollusc genome (so far known) does not contain genes for key enzymes that are necessary to transform cholesterol (precursor molecule in steroid biosynthesis) [95, 106].

#### **3.3. Reproductive system and behavior of** *L. stagnalis*

The reproductive biology of *L. stagnalis* has been well-studied [14, 15, 113, 114]. It is a hermaphrodite species, but during mating behavior one individual acts as male and the other as female. The snail playing the male role climbs on the shell of the prospective female, moves over the shell in a counter-clockwise direction until he reaches the area of the female gonophore. The preputium (muscular structure that surrounds the penis) is then partially everted through the male pore. This is followed by probing for the female pore by the preputium, insertion of this organ into this pore followed by penis eversion and intromission. Each of the four stages prior to intromission is variable in duration but the intromission is more constant. The whole mating behavior may last for several hours. During oviposition (egg-laying), masses containing 50–100 eggs embedded in a gelatinous mass are deposited on the substrate, from which juvenile snails of adult form emerge following about 10 days of intracapsular embryogenic development, without any free-living larval stages [113, 114]. Egg-laying consists of a sequence of behavioral events beginning with a rest period when the animal ceases to locomote, then a turning phase characterized by counter-clockwise shell movements and high frequency rasping to clean the substrate, followed by oviposition and a final phase called inspection when the snail moves along the length of the egg mass brushing it with lips and tentacles. Resting and turning last for about an hour each, oviposition 10 minutes and inspection about 2 minutes. Egg-laying in *L. stagnalis* is an example of a complex behavior that is triggered by the release of multiple neuropeptide transmitters from neuroendocrine centers within the central nervous system that act on other neural circuits controlling egg-laying behavior [12]. Weather the action of described sex steroids is receptor-mediated or not is uncleared in *L. stagnalis* at present. But it is a fact that vertebrate-like sex steroids might have a key role in reproduction in snails. Temporal variation in some steroid titers that coincide with reproductive stages have been observed.

#### **3.4. Progestogen effects in** *L. stagnalis*

data, *L. stagnalis* was able to transform PRG from injected labeled P5 [110]. The P5 is a key molecule in the biosynthetic pathway of main vertebrate steroids, such as PRG, 17ß-estradiol and testosterone which have also been proposed as functional hormones in molluscs [95].

Steroidogenesis and steroid metabolism play and important role in the regulation of endogenous steroid level in molluscs. As a result of their endogenous biosynthesis, active P5 (e.g. in *M. edulis, Astacus leptodactylus* and *Nephrops norvegicus*), PRG (e.g. in *M. edulis, Mya arenaria* and *O. vulgaris*), androstenedione (e.g. in *M. edulis, H. aspersa, A. leptodactylus* and *Neomysis integer*), estron (e.g. in *M. edulis, Arion ater rufus, H. aspersa* and *Asterias rubens*) and testosterone (e.g. in *M. edulis, Arion ater rufus, A. leptodactylus* and *N. integer*) have been described in several molluscan species [108]. In addition, it has been published that the physiological concentration of these sex steroids are related to changes in the reproductive cycle and their level are higher in one sex than other, or their level are changed during EDC contamination. Furthermore, another vertebrate-type hormone, the gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of "molluscan"sex steroids from the gonads, and elicited contractions of the oviduct, for example, in cephalopods. This result suggests that octopus-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis [111]. GnRH-like hormone has also been identified in two freshwater snails, *Helisoma trivolvis* and *L. stagnalis*, presumably with a similar control function than in cephalopods [112]. From a phylogenetic point of view, observations of steroidogenesis and vertebrate-type steroids are very interesting because they indicate a common origin of a sex hormonal system between molluscs and vertebrates. However, much more information is needed to fully understand the physiological function of sex steroid hormones in molluscs. But also noticeable that according to valuable Scott's reviews, despite many studies starting over 55 years ago, these data are questionable from several reasons. For example, the mollusc genome (so far known) does not contain genes for key enzymes that are necessary to transform cholesterol (precursor molecule

The reproductive biology of *L. stagnalis* has been well-studied [14, 15, 113, 114]. It is a hermaphrodite species, but during mating behavior one individual acts as male and the other as female. The snail playing the male role climbs on the shell of the prospective female, moves over the shell in a counter-clockwise direction until he reaches the area of the female gonophore. The preputium (muscular structure that surrounds the penis) is then partially everted through the male pore. This is followed by probing for the female pore by the preputium, insertion of this organ into this pore followed by penis eversion and intromission. Each of the four stages prior to intromission is variable in duration but the intromission is more constant. The whole mating behavior may last for several hours. During oviposition (egg-laying), masses containing 50–100 eggs embedded in a gelatinous mass are deposited on the substrate, from which juvenile snails of adult form emerge following about 10 days of intracapsular embryogenic development, without any free-living larval stages [113, 114]. Egg-laying consists of a sequence of behavioral events beginning with a rest period when the animal ceases to locomote, then a turning phase characterized by counter-clockwise shell movements and high frequency rasping to clean the substrate, followed by oviposition and a final phase

in steroid biosynthesis) [95, 106].

40 Biological Resources of Water

**3.3. Reproductive system and behavior of** *L. stagnalis*

Cyproterone acetate (CPA) is a commonly used synthetic progestogen compound in oral contraceptives, which also has anti-androgen effects in vertebrates. The vinclozolin (VZ) is mainly known as anti-androgen, which can also bind to estrogen and progestogen receptors in vertebrates. Using these progestogens, Ducrot and Giusti published no significant difference in shell length of adult *L. stagnalis* between control and treated groups after 21 days of exposure to any of the tested chemicals [25, 27]. However, Ducrot observed slower growing among juveniles, sub-adults and young adults exposed to the highest concentration of VZ (2500 μg/L) during the first week, but partly recovered during the second and third weeks, so that growth pattern did not lead to a significant decrease in the mean shell size compared to control group. In Giusti's experiments, neither CPA (2–50 μg/L), nor VZ (10–240 ng/L) induced more than 10% mortality. According to Ducrot, significant mortality occurred in treated adult animals exposed to the highest concentration of VZ, whereas the feeding activity was stopped in this group. CPA and VZ had no significant effect on cumulated oviposition and fertility in adult snails in single chemical treatment approach, however 2 and 10 μg/L CPA as well as 10–240 ng/L VZ treatments induced a significant decrease in egg number per egg mass compared to control. However, in this experiment no clear concentration-response relationship was described [27, 106]. CPA, VZ and tributyltin (TBT) were also tested on other three gastropod species, *Marisa cornuarietis*, *Nucella lapillus* and *Nassarius reticulatus* in a chronic experiment. In this investigation, the snails were treated by nominal CPA concentration of 1.25 mg/L for 12 month or by nominal VZ concentration of 0.03–1.0 μg/L for up to 5 month. It was reported that no mortality at used concentrations, but a significant decrease in the length of the penis and accessory male sex organs was observed in both species [106, 115]. Since progestogen contaminations are expected be found in the environment as mixtures, Zrinyi and her co-workers applied them together and environmentally relevant concentration range in *L. stagnalis* reproduction tests [116]. In a 10 ng/L eqi-concentration mixture of PRG, LNG, GES and DRO treatment approach resulted that the oocyte production of individual animals significantly changed in the treated group at the end of the 21-day long experiment. The number of laid oocyte per egg mass of individual animals shows continuous growth in the treated group week by week. Their number at the end of the third week was in average of twofold higher compared to the control. The control animals produced the same amount of oocytes weekly during experiment.

Beside egg number assessment, egg abnormalities can also be observe (e.g. polyembryonic egg, atrophied albumen, unfertilized oocyte or dead zygote in eggs), which refer to reproductive status and can determine the egg quality. Egg quality showed no significant difference in CPA and VZ treatments, however polyembryony was the most frequent phenomena. A 3-week long CPA treatment resulted in significant increase of the frequency of polyembryony in concentration dependent manner from 2 μg/L concentration [27]. In addition, the whole egg mass quality was also assessed in progestogen mixture treatment of adult snails with a three-graded scheme, which integrates the number of polyembryonic eggs with eggs containing dead zygotes. This endpoint resulted in a significant difference by the first week (**Figure 1**), but did not show difference at the end of a 3-week long experiment [116]. Based on published data, following the time window of early embryonic development in *L. stagnalis* could be able to another well endpoint for investigation of external progestogen contaminations. Progestogen mixture applied in environmental relevant concentration (10 ng/L) had significant effect on cell proliferation during early embryonic development. At the end of a 3-week long treatment period of the adult snails, the freshly laid zygotes were observed from the single-cell to the eight-cell stage. In zygotes obtained from hormone-treated adults, a significantly different accelerated cell proliferation could be noticed compared to controls, however the hatching time was unchanged [116]. In single-cell zygotes as well as egg albumen, a partial metabolomic analysis was also carried out using capillary microsampling combined ion mobility separation mass spectrometric technique. It was observed that the molecular composition of zygotes or egg albumen does not differ after steroid treatment of adults, but some semi-quantitative metabolic ratio (e.g. adenylate energy charge (AEC), redox ratio or hexose utilization) can express difference between the groups. These ratios could be used as marked endpoint in assessment of progestogen exposition in snails. The hexose utilization defined by UDP-hexNAc/UDP-hex ratio significantly decreased in single-cell zygote cytoplasm after a 3-week long progestogen mixture treatment of adults. This result partly could explain the observed accelerated cell division in zygotes obtained from treated parents. At the same time, AEC indicating the energy state of the cell was unchanged. This endpoint was significantly increased only in the albumen (obtained from treated adults) during the metamorphosis, which is the half-time of the average hatching time [116].

Based on several published data, we conclude that progestogen contaminations in water ecosystem are harmful for reproduction and early stage development of *L. stagnalis*. But, taken separately, progestogens might not present a risk for the snails, however since they are expected be found in the environment as mixtures (similar then in earlier experiment), there is a risk of additive or even synergistic effects [41]. Together with synthetic estrogenic steroids, progestogens are among the most important group of environmental pharmaceuticals of concern. However, in contrast to estrogens, progestogens have received only little attention so far and their environmental risks are not sufficiently known. Further investigations are needed to fully understand the synergistic effects of mixed progestogens or combined effects of progestogens

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The OECD Conceptual Framework for Testing and Assessment of Endocrine Disruptors is available from 2004 (revised and completed with mollusc's tests in 2012) [117]. This recommendation provides a guide with a five-level assessment but not indented to be a testing strategy of various EDCs. Another OECD reproductive toxicity test guideline with the pond snail *L. stagnalis* is also available from 2010 [25] and this optimized in 2016 using the steroidal TBT [30]. Several studies published data with different endpoints (number of egg mass, eggs, survival and shell size) recommended in OECD guidelines in progestogen exposure of *L. stagnalis* [23–28, 116]. Based on endpoint results coming from steroid, progestogen effects in snail reproduction, the pond snail, *L. stagnalis*, beside a mudsnail, *Potamopyrgus antipodarum*, has been the first aquatic non-arthropod-tests, which were successfully validated within the Conceptual Framework for

and estrogens in freshwater organisms, such as molluscs.

Endocrine Disrupters [3].

**3.5.** *L. stagnalis* **became a "real" test animal in EDC experiment**

**4. General considerations: ecotoxicologist versus physiologist**

Nowadays, we realized that a wide variety of environmental contaminants have specific effects on neuroendocrine system of aquatic species, including snails. For among them, *L. stagnalis* has been used as non-target model organisms in studying environmental contamination long time because they are sensitive to anthropogenic steroids, such as progestogens. Investigating a variety of reproductive endpoints, such as fecundity, oocyte production, egg mass production, the quality of egg masses, the shell size in development and after egglaying, the time window of cell division in the offspring, the metabolite content of single-cell zygotes and egg albumen, it is concluded that progestogen contaminations in water are detrimental for reproduction and early stage development of *L. stagnalis*. Based on its endpoint results, the *L. stagnalis* has become the first aquatic non-arthropod-tests, validated successfully within the Conceptual Framework for Endocrine Disrupters. In this context, the proposed model is ecotoxicologically correct because it has well detectable effects. But if we are interested in physiological mechanisms of steroids (progestogen), many uncleared questions and contradictory observations are detected. For example, how progestogen contamination influences the *Lymnaea* reproduction is difficult to explain because progestogen and androgen

**Figure 1.** Evaluation of the egg mass quality in *Lymnaea*. Figure demonstrates the categories of the egg mass quality in the control and with the 10 ng/L progestogen-treated groups. The egg mass was presented in good (white), fair (gray) and poor (black) quality in the 1st, 2nd and 3rd weeks in both groups. On the 1st week, the egg mass quality was significantly different between the groups (Kruskal-Wallis Chi<sup>2</sup> = 6.31; P < 0.05; n = 41), while this was statistically not different in next 2 weeks (Kruskal-Wallis Chi2 = 3.65; P = 0.56; n = 50 and Chi2 = 0.15; P = 0.70; n = 43). P < 0.05 is signed by asterisk (\*).

Based on several published data, we conclude that progestogen contaminations in water ecosystem are harmful for reproduction and early stage development of *L. stagnalis*. But, taken separately, progestogens might not present a risk for the snails, however since they are expected be found in the environment as mixtures (similar then in earlier experiment), there is a risk of additive or even synergistic effects [41]. Together with synthetic estrogenic steroids, progestogens are among the most important group of environmental pharmaceuticals of concern. However, in contrast to estrogens, progestogens have received only little attention so far and their environmental risks are not sufficiently known. Further investigations are needed to fully understand the synergistic effects of mixed progestogens or combined effects of progestogens and estrogens in freshwater organisms, such as molluscs.

#### **3.5.** *L. stagnalis* **became a "real" test animal in EDC experiment**

status and can determine the egg quality. Egg quality showed no significant difference in CPA and VZ treatments, however polyembryony was the most frequent phenomena. A 3-week long CPA treatment resulted in significant increase of the frequency of polyembryony in concentration dependent manner from 2 μg/L concentration [27]. In addition, the whole egg mass quality was also assessed in progestogen mixture treatment of adult snails with a three-graded scheme, which integrates the number of polyembryonic eggs with eggs containing dead zygotes. This endpoint resulted in a significant difference by the first week (**Figure 1**), but did not show difference at the end of a 3-week long experiment [116]. Based on published data, following the time window of early embryonic development in *L. stagnalis* could be able to another well endpoint for investigation of external progestogen contaminations. Progestogen mixture applied in environmental relevant concentration (10 ng/L) had significant effect on cell proliferation during early embryonic development. At the end of a 3-week long treatment period of the adult snails, the freshly laid zygotes were observed from the single-cell to the eight-cell stage. In zygotes obtained from hormone-treated adults, a significantly different accelerated cell proliferation could be noticed compared to controls, however the hatching time was unchanged [116]. In single-cell zygotes as well as egg albumen, a partial metabolomic analysis was also carried out using capillary microsampling combined ion mobility separation mass spectrometric technique. It was observed that the molecular composition of zygotes or egg albumen does not differ after steroid treatment of adults, but some semi-quantitative metabolic ratio (e.g. adenylate energy charge (AEC), redox ratio or hexose utilization) can express difference between the groups. These ratios could be used as marked endpoint in assessment of progestogen exposition in snails. The hexose utilization defined by UDP-hexNAc/UDP-hex ratio significantly decreased in single-cell zygote cytoplasm after a 3-week long progestogen mixture treatment of adults. This result partly could explain the observed accelerated cell division in zygotes obtained from treated parents. At the same time, AEC indicating the energy state of the cell was unchanged. This endpoint was significantly increased only in the albumen (obtained from treated adults)

during the metamorphosis, which is the half-time of the average hatching time [116].

**Figure 1.** Evaluation of the egg mass quality in *Lymnaea*. Figure demonstrates the categories of the egg mass quality in the control and with the 10 ng/L progestogen-treated groups. The egg mass was presented in good (white), fair (gray) and poor (black) quality in the 1st, 2nd and 3rd weeks in both groups. On the 1st week, the egg mass quality was significantly

= 3.65; P = 0.56; n = 50 and Chi2

= 6.31; P < 0.05; n = 41), while this was statistically not different in next

= 0.15; P = 0.70; n = 43). P < 0.05 is signed by asterisk (\*).

different between the groups (Kruskal-Wallis Chi<sup>2</sup>

2 weeks (Kruskal-Wallis Chi2

42 Biological Resources of Water

The OECD Conceptual Framework for Testing and Assessment of Endocrine Disruptors is available from 2004 (revised and completed with mollusc's tests in 2012) [117]. This recommendation provides a guide with a five-level assessment but not indented to be a testing strategy of various EDCs. Another OECD reproductive toxicity test guideline with the pond snail *L. stagnalis* is also available from 2010 [25] and this optimized in 2016 using the steroidal TBT [30]. Several studies published data with different endpoints (number of egg mass, eggs, survival and shell size) recommended in OECD guidelines in progestogen exposure of *L. stagnalis* [23–28, 116]. Based on endpoint results coming from steroid, progestogen effects in snail reproduction, the pond snail, *L. stagnalis*, beside a mudsnail, *Potamopyrgus antipodarum*, has been the first aquatic non-arthropod-tests, which were successfully validated within the Conceptual Framework for Endocrine Disrupters [3].
