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

**Pond Snail Reproduction as Model in the** 

Zsolt Pirger, Zita Zrinyi, Gábor Maász, Éva Molnár and

DOI: 10.5772/intechopen.72216

Zsolt Pirger, Zita Zrinyi, Gábor Maász, Éva Molnár and Tibor Kiss Tibor Kiss Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

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

#### **Abstract**

[60] Goodson MS, Kojadinovic M, Troll JV, Scheetz TE, Casavant TL, Soares MB, McFall-Ngai MJ. Identifying components of the NF-kappaB pathway in the beneficial *Euprymna scolopes*-*Vibrio fischeri* light organ symbiosis. Applied and Environmental Microbiology. 2005;**71**(11):6934-

[61] Barbieri E, Barry K, Child A, Wainwright N. Antimicrobial activity in the microbial community of the accessory nidamental gland and egg cases of *Loligo pealei*. The Biological

[62] Gomathi P, Nair JR, Sherief PM. Antibacterial activity in the accessory nidamental gland extracts of the Indian squid, *Loligo duvauceli* orbigny. Indian Journal of Marine Sciences.

[63] Cornet V, Henry J, Goux D, Duval E, Bernay B, Le Corguillé G, Corre E, Zatylny-Gaudin C. How egg case proteins can protect cuttlefish offspring? PLoS One. 2015;**10**(7):e0132836.

[64] Zatylny C, Marvin L, Gagnon J, Henry J. Fertilization in *Sepia officinalis*: The first mollusk sperm-attracting peptide. BBRC. 2002;**296**(5):1186-1193. DOI: 10.1016/S0006-291X(02)

[65] Cornet V, Henry J, Corre E, Le Corguille G, Zanuttini B, Zatylny-Gaudin C. Dual role of the cuttlefish salivary proteome in defense and predation. Journal of Proteomics.

[66] Lemaire J. Table de développement embryonnaire de *Sepia officinalis* L. (mollusque céph-

[67] Cyran N, Staedler Y, Schönenberger J, Klepal W, von Byern J. Hatching glands in cephalopods - A comparative study. Zoologischer Anzeiger. 2013;**253**(1):66-82. DOI: 10.1016/j.

6946. DOI: 10.1128/AEM.71.11.6934-6946

DOI: 10.1371/journal.pone.0132836

2014;**108**:209-222. DOI: 10.1016/j.jprot.2014.05.019

alopode). Bulletin de la Société Zoologique. 1970;**95**:773-782

2010;**39**(1):100-104

32 Biological Resources of Water

02036-3

jcz.2013.04.001

Bulletin. 1997;**193**(2):275-276. DOI: 10.1086/BBLv193n2p275

In European limnetic systems, the most relevant endocrine-disrupting chemicals (EDCs) of steroid type are the natural and synthetic hormones, phytosterols, pesticides, biocides and other chemicals produced by the plastic industry. Their presence in aquatic ecosystems represents a potentially adverse environmental and public health impact. Furthermore, this is a warning signal that the current handling of pharmaceuticals needs to be further improved. Nowadays, it has become clear that EDCs have specific disturbing effects on the neuroendocrine system of invertebrate and vertebrate aquatic animals, particularly gastropods. Among a latter, pond snail (*Lymnaea stagnalis*) has been used as the first aquatic non-arthropod test organism in studying the effect of EDCs because they are sensitive to various anthropogenic steroids, like progestogens. Investigating a variety of reproductive endpoints of *Lymnaea*, such as fecundity, oocyte production, egg mass production, the quality of egg masses, the shell size in development and after egg-laying, the time window of cell division in the offspring, the metabolite content of single-cell zygotes and egg albumen has concluded that progestogen contaminations in water are detrimental for reproduction and early stage development of *Lymnaea*. This chapter is an attempt to show whether *Lymnaea reproduction*, despite many altering reproductive endpoints, is a suitable model for environmental risk assessment or not.

**Keywords:** endocrine-disrupting chemicals, progestogens, molluscs, *Lymnaea stagnalis*, reproduction model

#### **1. Introduction**

In the last few years, it has become clear that a wide variety of environmental contaminants have specific effects on neuroendocrine system of aquatic species. The frequent detection of

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

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 these contaminants.

production, the quality of egg masses, the shell size in successive development and following egg-laying, the time window of cell division in the offspring, the metabolite content of single-

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

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

35

It has been shown that recent research aims to combine molecular level investigation with cellular, organismal, behavior and environmental research. In this chapter, an attempt is made to summarize data particularly obtained on *L. stagnalis* so far, and to discuss the molecular mechanisms, the functional and ecological consequence of EDCs and the advantages of snail preparations as tools for ecotoxicological research. Comparison of the data obtained on molluscs with those obtained on the lower vertebrates, will definitively contribute to the better understanding of the impact caused by EDCs, like steroid hormones, present in our environment.

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,

cell zygotes and egg albumen before and after the treatment of parents.

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

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 production, the quality of egg masses, the shell size in successive development and following egg-laying, the time window of cell division in the offspring, the metabolite content of singlecell zygotes and egg albumen before and after the treatment of parents.

It has been shown that recent research aims to combine molecular level investigation with cellular, organismal, behavior and environmental research. In this chapter, an attempt is made to summarize data particularly obtained on *L. stagnalis* so far, and to discuss the molecular mechanisms, the functional and ecological consequence of EDCs and the advantages of snail preparations as tools for ecotoxicological research. Comparison of the data obtained on molluscs with those obtained on the lower vertebrates, will definitively contribute to the better understanding of the impact caused by EDCs, like steroid hormones, present in our environment.
