**2. Steroid type EDCs in the aquatic environment**

endocrine-disrupting chemicals (EDCs) in the aquatic environment and a high consumption of contraceptives all over the world reflect a rapidly growing concern on their environmental impact. EDCs interfere with the body's endocrine system mimicking or partly mimicking naturally occurring hormones in the body and induce adverse developmental, reproductive, neurological (cognitive and behavior) and immune effects in both humans and wildlife [1]. In addition, the high frequencies of detection of these contaminants in aquatic environments and the incomplete removal of them during passage through sewage treatment plants may pose the greatest risk during prenatal and early postnatal development when organ and neural systems are developing. The increasing and continuous occurrence of steroidal estrogen and progestogen compounds in the environment can lead to toxicological effects on non-target organisms, therefore, it is important on the whole to assess the environmental risk posed by

Molluscs like gastropods and bivalves have been used as non-target model organisms in studying environmental contamination for a long. They proved to be effective model animals because they are ubiquitous, have highly conserved control and regulatory biochemical pathways that are often homologous to vertebrate systems and they are extremely sensitive to anthropogenic inputs [2–4]. For example, the bivalves, by virtue their ability to accumulate toxic substances (due to their sessile and filtering life style) in their body are considered as excellent indicators of ecosystem health [5]. Furthermore, molluscs are ecologically crucial organisms, which are essential to the biosphere and to the human economy. They are the second most diverse animal group (10 taxonomic classes) encompassing more than 400,000 species, they are ecologically and commercially important as food and non-food resources. Among them terrestrial gastropods are destructive agricultural pests causing economic damage to a wide variety of plants including horticulture, field crops and forestry. In addition they are of importance in medical and veterinary practice, since they serve as intermediate hosts for several human and animal diseases, such as schistosomiasis and helminth diseases [6]. Both terrestrial (e.g. *Helix pomatia*), marine (e.g. *Aplysia californica*) and freshwater (e.g. *Lymnaea stagnalis*) snails have proved to be excellent models, due to their "simple" nervous system, in neurophysiology and behavioral ecology [7–12]. Gastropod model organisms play an important role for immunology [13], reproductive and developmental biology (which is facilitated by several genome and transcriptome projects that are currently underway) [14–16], neurobiology, especially on learning and memory formation [17–22]. Some species, in particular simple pond snail (*Lymnaea stagnalis*) have been widely applied in pollution biomonitoring programs, and widely used in a variety of ecotoxicological studies [23–28]. Based on earlier investigations the reproduction test of *L. stagnalis* was officially approved by the national coordinators of the Organization for Economic Cooperation and Development (OECD) member countries as test guidelines. *L. stagnalis* and the New Zealand mudsnail (*Potamopyrgus antipodarum*) have been the first aquatic non-arthropod-tests, which were successfully validated within the Conceptual Framework for Endocrine Disrupters [3, 29, 30]. Therefore, in this chapter one of the most relevant mollusc of European limnetic systems, the hermaphroditic *L. stagnalis* is particularly presented to model the various physiological effects on its reproductive and developmental parameters eliciting by acute or chronic exposures of endocrine-disrupting substances. A variety of endpoints are assessed and collected, including fecundity, oocyte production, egg mass

these contaminants.

34 Biological Resources of Water

The release of human pharmaceuticals (as xenobiotics) into aquatic ecosystems is a serious environmental risk which results in an acute and chronic contamination of non-target invertebrate (e.g. molluscs) and vertebrate (e.g. fish) freshwater organisms [31]. Among the most critical environment contaminants are EDCs, which are defined as an exogenous substance that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism. It is concluded that endocrine disruption is not considered a toxicological end point per se but a functional change that may lead to adverse effects in both non-target and target organisms, as well. EDCs act as agonist or antagonists at multiple sites via complex mechanisms of action including: receptor-mediated mechanisms, synthesis and/or metabolism of hormones, neuropeptides and neurotransmitters, as well as transport pathways [32].

In European limnetic system, the most relevant steroid type EDCs are follows: natural (e.g. progesterone, estradiol, testosterone [33–35] and synthetic (e.g. drospirenone, levonorgestrel, ethinylestradiol, cyproterone acetate (CPA), t-methyltestosterone [23, 33–35]) hormones, phytosterols (e.g. β-sitosterol [23]), pesticides (e.g. octylphenol, chlordecone [35, 36]), fungicides (e.g. vinclozolin (VZ), pyraclostrobin [25, 28]), biocides (e.g. tributyltin [23, 36]) and other chemicals produced in the plastic industry (e.g. bisphenol A [36]). One of the most cited examples to steroidal EDCs is the tributyltin (TBT) in molluscs. It caused imposex and intersex development as two masculinization phenomena in more than 260 species of gastropod worldwide, and severe losses of invertebrate biodiversity in waters [5, 37]. Several studies on perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of steroid type EDCs have already been reported. These more recent studies collectively provide evidence for possible detrimental effects of steroidal EDCs on *L. stagnalis* reproduction and embryonic development. However, the underlying mechanisms between exposure to EDCs and a variety of biologic outcomes, their potential long-term side effects of these molecules on molluscs remain largely unknown. This book chapter is mainly focused on synthetic steroids because they have become one of the most harmful pharmaceutical pollutants in molluscs.

Synthetic steroids, like estrogens and progestogens, are potent endocrine disrupters, which can modify diverse physiological, hormonal and behavioral processes in freshwater species, and subsequently affect their capacity to reproduce, develop and grow [38, 39]. Estrogens and progestogens in combination are widely used as synthetic oral contraceptives (SOCs) [40]. SOC residues or their metabolites are eliminated from the human body unchanged or in the form of active metabolites in a remarkable amount [41, 42]. These biologically active agents enter into the waste water treatment plants (WWTP) where the generally applied treatment process is not suitable to eliminate them perfectly [42–45]. Consequently, synthetic steroid hormone residues enter the aquatic environment (e.g. surface waters) manly through cleaned effluents. The first review, which describes the presence of estrogen and progestogen hormones in natural surface waters was published by Richardson and Bowron [46]. In fact, very few pharmaceutical chemicals were identified due to the limitations of the early gas chromatography and HPLC techniques. The development of analytical techniques (e.g. liquid chromatographic-mass spectrometric method with solid-phase extraction, see later) decreased the limit of detection, resulting in an increasing number of detectable SOCs in surface and ground water, as well [47, 48]. Nowadays, their reported presence are in a concentration range from a few ng/L to often tens or hundreds of ng/L (estrogens: 0.20–480.00 ng/L, progestogens: 0.07–22.20 ng/L) in surface waters [47, 49–51]. The catchment area of the largest shallow lake of Central Europe is a habitat of several molluscs (e.g. *L. stagnalis, Anodonta cygnaea, Dreissena polymorpha*) and fish species (e.g. *Rutilus rutilus, Alburnus alburnus, Abramis brama, Carassius carassius, Cyprinus carpio, Perca fluviatilis*), where the estrogen and progestogen concentrations were found between 0.07–0.68 ng/L and 0.23–13.67 ng/L, respectively [33, 34]. The presence of steroid hormones has also been found in the drinking water, which is a warning sign that the current handling of pharmaceuticals may lead to future global human health problems [52–56]. It has already been described that exogenous steroid contaminations have wide range genotoxicity, neurotoxicity and germ cell-damaging effects in humans. For example, ethinylestradiol may modify brain structure, function, and consequently, behaviors pattern during the female brain development [57]. Furthermore, accumulating evidence suggest that human exposure to steroids is related to the impairment of male reproductive function (e.g. decreased sperm number) and can interrupt other hormonally regulated metabolic processes, particularly if exposure occurs during early development [58].

Multi-residue analysis, as a field of study encompassing steroid EDCs residue analysis, has made considerable advances regarding selectivity and detection limits. Before analytical procedures, in order to keep track levels of EDCs, it is recommended that (e.g. deuterated) internal standards are added to the water or solid samples [64]. In general, there are several extraction methods, such as liquid-liquid extraction (LLE), solid-phase extraction (SPE), solidphase micro-extraction (SPME), stir-bar sorptive extraction (SBSE), selective pressurized liquid extraction (SPLE), Soxhlet extraction (SE), ultrasonic extraction (USE), microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) [65–68]. The majority of current analytical methods for separation and detection of various steroidal EDCs, for example, use liquid chromatography-tandem mass spectrometry (LC-MS/MS) because its versatility, specificity and selectivity are very well [69]. Other possibility to detection and quantitative measurement of steroidal EDCs is also offered by gas chromatography (GC) with electron capture detection

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37

In case of water samples, the main steps of analytical methods are the filtration (e.g. glass microfiber filters), extraction and purification (e.g. SPE), finally quantitative measured by using LC-MS/MS. Generally, around 0.1 ng/L limit of quantification (LOQ) value are achieved [33, 34, 71, 72]. The detection of steroid EDCs from various solid samples are complicated because more sample preparation steps are required (drying, homogenization, destruction, extraction and purification). The most commonly applied extraction methods are USE, MAE and SPLE for solid environmental matrices, such as sediment or biological tissues [64–68]. After extrac-

tion procedure, off-line SPE and LC-MS/MS are utilized for EDCs analysis [64, 73, 74].

Together with synthetic estrogenic steroids, progestogens are among the most important group of environmental pharmaceuticals of concern. A large number of studies investigating the occurrence and effects of natural and synthetic estrogen hormones (e.g. ethinylestradiol, estradiol, estrone and estriol), and the risk is now well documented [47, 49, 75, 76]. Several studies have also been conducted on the risk related to anti-androgens [77], but contrast to these, surprisingly, relatively few data are published about the occurrence of progestogens in different waters [34, 41, 49] and mainly their neuroendocrine effects on non-target freshwater organisms,

Progesterone (PRG) is an endogenous steroid hormone involved in the female menstrual cycle, pregnancy and the embryogenesis of humans and other vertebrate species. In turn progestins are a group of natural and synthetic molecules that have effects similar to those exerted by PRG. The endogenous PRG and its analogue progestins together are generally referred to as progestogens (or gestagens). The most important and frequent synthetic progestogens are the follows drospirenone (DRO), levonorgestrel (LNG), gestodene (GES), norethindrone (NET) and ciproterone acetate (CPA). The progestogens that are used in hormonal contraceptives are LNG (e.g. Alesse, Trivora-28, Plan B, Mirena), DRO (e.g. Yasmin, Yasminelle), GES (e.g. Femodene) and CPA (e.g. Diane-35, Dianette). There are approximately 20 different progestogens used in human and veterinary medicine. Despite significant use, their ecotoxicological implications are poorly understood in environment. According to Fent, only about 50% of the progestogens in use have been analyzed for their environmental occurrence and effects in aquatic organisms [49].

**2.2. Progestogens as neuroendocrine disruptors: an outlook on the world of fish**

and confirmation by MS [70].

including particularly invertebrates [49].

#### **2.1. Methods in detection of steroidal EDCs**

Measurements of multi-residue analysis require a rapid, sensitive, robust and reliable method with fast response time (high-throughput). These analytical measurements are essentially determined by two crucial things, one is the limit of detection, and the other is the sample (matrix) complexity. The subject of detection limits in analytical chemistry has improved since the 1970s and these resulted that the amount of detectable analytes, such as EDCs, are decreased [59–62]. Nowadays, the mass spectrometry based methods are extended and their detection limits are almost low ppm or ppb, which are below the environmentally relevant concentrations at the time. Other problem with the detection and quantification of an analyte can result from different matrix effects, sample concentration or other conditions, such as instrument sensitivity and reagent purity. In general, these matrices have different type of waters (e.g. wastewater influents or effluents, ground-, surface- and tap waters) and various solid samples (sediment, sludge, biological matrices). Sample preparation techniques can enhance the performance results for better recovery, increased sensitivity and lower detection limits [63].

Multi-residue analysis, as a field of study encompassing steroid EDCs residue analysis, has made considerable advances regarding selectivity and detection limits. Before analytical procedures, in order to keep track levels of EDCs, it is recommended that (e.g. deuterated) internal standards are added to the water or solid samples [64]. In general, there are several extraction methods, such as liquid-liquid extraction (LLE), solid-phase extraction (SPE), solidphase micro-extraction (SPME), stir-bar sorptive extraction (SBSE), selective pressurized liquid extraction (SPLE), Soxhlet extraction (SE), ultrasonic extraction (USE), microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) [65–68]. The majority of current analytical methods for separation and detection of various steroidal EDCs, for example, use liquid chromatography-tandem mass spectrometry (LC-MS/MS) because its versatility, specificity and selectivity are very well [69]. Other possibility to detection and quantitative measurement of steroidal EDCs is also offered by gas chromatography (GC) with electron capture detection and confirmation by MS [70].

and subsequently affect their capacity to reproduce, develop and grow [38, 39]. Estrogens and progestogens in combination are widely used as synthetic oral contraceptives (SOCs) [40]. SOC residues or their metabolites are eliminated from the human body unchanged or in the form of active metabolites in a remarkable amount [41, 42]. These biologically active agents enter into the waste water treatment plants (WWTP) where the generally applied treatment process is not suitable to eliminate them perfectly [42–45]. Consequently, synthetic steroid hormone residues enter the aquatic environment (e.g. surface waters) manly through cleaned effluents. The first review, which describes the presence of estrogen and progestogen hormones in natural surface waters was published by Richardson and Bowron [46]. In fact, very few pharmaceutical chemicals were identified due to the limitations of the early gas chromatography and HPLC techniques. The development of analytical techniques (e.g. liquid chromatographic-mass spectrometric method with solid-phase extraction, see later) decreased the limit of detection, resulting in an increasing number of detectable SOCs in surface and ground water, as well [47, 48]. Nowadays, their reported presence are in a concentration range from a few ng/L to often tens or hundreds of ng/L (estrogens: 0.20–480.00 ng/L, progestogens: 0.07–22.20 ng/L) in surface waters [47, 49–51]. The catchment area of the largest shallow lake of Central Europe is a habitat of several molluscs (e.g. *L. stagnalis, Anodonta cygnaea, Dreissena polymorpha*) and fish species (e.g. *Rutilus rutilus, Alburnus alburnus, Abramis brama, Carassius carassius, Cyprinus carpio, Perca fluviatilis*), where the estrogen and progestogen concentrations were found between 0.07–0.68 ng/L and 0.23–13.67 ng/L, respectively [33, 34]. The presence of steroid hormones has also been found in the drinking water, which is a warning sign that the current handling of pharmaceuticals may lead to future global human health problems [52–56]. It has already been described that exogenous steroid contaminations have wide range genotoxicity, neurotoxicity and germ cell-damaging effects in humans. For example, ethinylestradiol may modify brain structure, function, and consequently, behaviors pattern during the female brain development [57]. Furthermore, accumulating evidence suggest that human exposure to steroids is related to the impairment of male reproductive function (e.g. decreased sperm number) and can interrupt other hormonally regulated metabolic processes,

particularly if exposure occurs during early development [58].

recovery, increased sensitivity and lower detection limits [63].

Measurements of multi-residue analysis require a rapid, sensitive, robust and reliable method with fast response time (high-throughput). These analytical measurements are essentially determined by two crucial things, one is the limit of detection, and the other is the sample (matrix) complexity. The subject of detection limits in analytical chemistry has improved since the 1970s and these resulted that the amount of detectable analytes, such as EDCs, are decreased [59–62]. Nowadays, the mass spectrometry based methods are extended and their detection limits are almost low ppm or ppb, which are below the environmentally relevant concentrations at the time. Other problem with the detection and quantification of an analyte can result from different matrix effects, sample concentration or other conditions, such as instrument sensitivity and reagent purity. In general, these matrices have different type of waters (e.g. wastewater influents or effluents, ground-, surface- and tap waters) and various solid samples (sediment, sludge, biological matrices). Sample preparation techniques can enhance the performance results for better

**2.1. Methods in detection of steroidal EDCs**

36 Biological Resources of Water

In case of water samples, the main steps of analytical methods are the filtration (e.g. glass microfiber filters), extraction and purification (e.g. SPE), finally quantitative measured by using LC-MS/MS. Generally, around 0.1 ng/L limit of quantification (LOQ) value are achieved [33, 34, 71, 72]. The detection of steroid EDCs from various solid samples are complicated because more sample preparation steps are required (drying, homogenization, destruction, extraction and purification). The most commonly applied extraction methods are USE, MAE and SPLE for solid environmental matrices, such as sediment or biological tissues [64–68]. After extraction procedure, off-line SPE and LC-MS/MS are utilized for EDCs analysis [64, 73, 74].

#### **2.2. Progestogens as neuroendocrine disruptors: an outlook on the world of fish**

Together with synthetic estrogenic steroids, progestogens are among the most important group of environmental pharmaceuticals of concern. A large number of studies investigating the occurrence and effects of natural and synthetic estrogen hormones (e.g. ethinylestradiol, estradiol, estrone and estriol), and the risk is now well documented [47, 49, 75, 76]. Several studies have also been conducted on the risk related to anti-androgens [77], but contrast to these, surprisingly, relatively few data are published about the occurrence of progestogens in different waters [34, 41, 49] and mainly their neuroendocrine effects on non-target freshwater organisms, including particularly invertebrates [49].

Progesterone (PRG) is an endogenous steroid hormone involved in the female menstrual cycle, pregnancy and the embryogenesis of humans and other vertebrate species. In turn progestins are a group of natural and synthetic molecules that have effects similar to those exerted by PRG. The endogenous PRG and its analogue progestins together are generally referred to as progestogens (or gestagens). The most important and frequent synthetic progestogens are the follows drospirenone (DRO), levonorgestrel (LNG), gestodene (GES), norethindrone (NET) and ciproterone acetate (CPA). The progestogens that are used in hormonal contraceptives are LNG (e.g. Alesse, Trivora-28, Plan B, Mirena), DRO (e.g. Yasmin, Yasminelle), GES (e.g. Femodene) and CPA (e.g. Diane-35, Dianette). There are approximately 20 different progestogens used in human and veterinary medicine. Despite significant use, their ecotoxicological implications are poorly understood in environment. According to Fent, only about 50% of the progestogens in use have been analyzed for their environmental occurrence and effects in aquatic organisms [49].

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].

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

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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

during "metabotropic" pathway (e.g. cAMP-MAPK-PKC) [103, 104].

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