Accumulation of endocrine-disrupting compounds (EDCs) in Procambarus Virginalis tissue in Dnipro river: ecological and hygienic aspects

At present among environmental toxicants, endocrine-disrupting compounds (EDCs) gained the widest distribution due to their presence in chemicals in consumer products and food contact materials, pharmaceuticals, personal hygiene products, and as mycotoxins and phytoestrogens. They are used in animal husbandry as growth promoters and in crop production as pesticides and herbicides. EDCs pollution of water sources is becoming alarming due to the imperfection and difficulty of removing pollutants using traditional water and wastewater treatment processes. Given the EDS activity and genotoxicity, the discharge of these effluents may realize a high risk to aquatic organisms in the receiving environment. Natural and synthetic estrogens are among the most potent endocrine-disrupting compounds found in urban wastewater. The purpose of this work was to determine the concentration of hormones, namely estradiol and cortisol, in Procambarus virginalis hydrobionts of different ages from the Dnipro River in the city of Dnipro. In the presented work, based on a review of modern scientific sources and the results of our own research, emphasis is placed on the need to monitor the accumulation of hormone-like compounds in the tissues of aquatic organisms, namely crustaceans as markers of pollution of aquatic ecosystems by EDCs. The influence of substances destroying the endocrine system is especially dangerous during critical periods of the body’s development. In ecological conclusion, EDC accumulation in hydrobiont’s species may have a negative impact on species conservation. In hygienic aspect, these substances can affect humans through the food chain as consumers of fish products and seafood contaminated with such toxicants.


Introduction
Ecosystem pollution is a serious problem worldwide.Aquatic ecosystems are extremely important for biota, fauna, flora, and humans.In recent decades, there has been a worldwide concern for the environment due to the difficulty of removing pollutants through traditional water and wastewater treatment processes.Among environmental toxicants, endocrinedisrupting compounds (EDCs) encompass nearly 800 different chemicals, including both natural and synthetic compounds, including pesticides, chemicals in consumer products and food contact materials, pharmaceuticals, and personal hygiene products.They alter the activation, synthesis, secretion, and binding of endogenous physiological hormones, thereby influencing 1254 (2023) 012014 IOP Publishing doi:10.1088/1755-1315/1254/1/012014 2 several hormonal and metabolic processes.More specifically, they work by mimicking hormones, binding to their receptors and promoting inappropriate responses at inappropriate times or directly blocking their effects [1,2].Their progressive exponential occupation and, as a result, waste determine their diffusion and accumulation in the environment.Some EDCs persist in the environment for long periods of time, even after their ban (as an example, the pesticide DDT), due to their insolubility, lipophilicity, and high resistance to degradation [3].A large number of pollutants that can have an endocrine effect are present in the water environment both individually (in different concentrations) and in complex mixtures [4,5].
The main source of water pollution is wastewater from various industries.Hormonal activity and genotoxicity of various effluents from textile, paint, electronic and electroplating plants, pulp and paper mills, chemical plants, and municipal sewage treatment plants were evaluated.The results showed the presence of EDS in most industrial effluents.The importance of the city as a huge source of pollution was also confirmed [6].Municipal sewage, the pharmaceutical industry and hospitals are the biggest sources of steroid estrogens too.Municipal landfills are also sources of organic pollution and may contain leachates with significant amounts of dissolved organic matter, which is partly composed of steroid hormones and other pollutants.In addition to urban pollution, some treated industrial wastewater from processing plants contained phytoestrogens.The filtrate is able to penetrate into groundwater.
Existing wastewater treatment technologies do not fully address the growing water pollution situation from wastewater discharges, especially with the increased use of personal care products and pharmaceuticals in private households and the healthcare sector.The urgency of solving the problem of organic pollutants is taken into account in Directive 2013/39 / EU.In addition, Decision 2015/495 of March 20, 2015 defined a list of 10 pollutants.This list includes several chemicals, including the pain reliever diclofenac and the hormones 17beta-estradiol and 17alf a-ethinylestradiol.In addition, it is necessary to take into account the formation and transformation of by-products.Unfortunately, most methods currently in use are unable to completely remove such critical compounds [7].
Endocrine-disrupting chemicals have been found in aquatic environments around the world.Given the EDS activity and genotoxicity, the discharge of these effluents may realize a high risk to aquatic organisms in the receiving environment.Aquatic organisms are particularly affected by anthropogenic pollutants, the effects of which can last a lifetime and are mediated through several pathways, including through the skin and gills, or through feeding on contaminated sediments and bioaccumulation.Aquatic pollutants can compromise reproduction, development, immune response, and other physiological processes that can ultimately affect aquaculture survival.In addition to the direct impact of water pollutants on the population of aquatic organisms that are included in the food chain when used by humans and wildlife, they realize a risk to human health and negatively affect the economics of fisheries and aquaculture [8].
Crustaceans are among the most ecologically significant species in aquatic ecosystems and food webs, playing a crucial role in aquatic ecosystems in transporting food web energy.In addition, these species play an important role as bioindicators in toxicological studies of water pollution.Metabolism of many xenobiotics occurs more slowly in invertebrates than in fish, and therefore invertebrates retain higher levels of contaminants.Therefore, EDS, accumulating in organisms, are a danger to the entire ecosystem through the food web [9].
Bisphenol A (BPA), phthalates (DEHP), polyvinyl chloride (PVC), pesticides, herbicides, and organotin compounds show estrogenic activity.Estrogenic disruption of tissue mineralization in aquatic organisms can have a wide range of consequences, such as increased skeletal abnormalities, altered bone density and mineral homeostasis, which can affect swimming and prey capture and predator avoidance.In addition, a violation of homeostasis can affect the reproduction of wild and cultivated species in aquaculture [10].
Along with estrogens, such a hormone as cortisol, which is a key mediator of the hypothalamic-IOP Publishing doi:10.1088/1755-1315/1254/1/0120143 pituitary-adrenal response to stress, has a significant impact on living organisms.While acute changes in its circulating levels are necessary to maintain homeostasis under dynamic environments, its chronic elevation can affect immunity, behavior, and reproduction [10].
The marbled crayfish Procambarus virginalis belongs to freshwater crayfish species.P. virginalis is a popular pet species in Europe and North America.P. virginalis is an invasive species in the Dnipro River in Ukraine.Now the species has been introduced into the natural ecosystems of the Dnipro near the city of Dnipro.Through the parthenogenetic method of reproduction and polytrophic omnivorousness, P. virginalis can withstand low winter temperatures in the European temperate zone.
Studies indicate the need for monitoring to assess certain classes of pollutants, such as EDS, which are known to realize serious toxicological hazards to aquatic biota at low concentrations [4].
The purpose of this work was to determine the concentration of hormones, namely estradiol and cortisol, in Procambarus virginalis of different ages as hydrobiont from the Dnipro River in the city of Dnipro.

Methodology
The concentration of estradiol and cortisol was determined by the ELISA method on the Stat Fax 303 plus enzyme immunoassay (Awareness Technology, USA) using standard reagents "EstradiolIFA", "CortisolIFA" (HEMA LLC, Ukraine) according to the instructions for the reagent set.Estradiol level was determined in caviar, embryos, young crustacean carcasses samples of Procambarus virginalis, and the muscles of one-month-old (young) crustaceans, fivemonth-old (mature) and year-old (older) ones.Cortisol level was fixed in gills and muscles of different age crustaceans, roe, embryos and juveniles of marbled crayfish.
The statistical analysis was performed using the program Statistic 6.0 for Windows.The statistical significance was considered at p<0.05.[11,12].

Results and discussion
Endocrine control of homeostasis results from the ability of neurons to integrate diverse sensory and hormonal inputs to coordinate autonomic, behavioral, and endocrine responses in their key regulatory centers responsible for the cascade of hormonal events required to modulate critical body functions such as metabolism, reproduction, growth, water balance etc. [13].Originally, EDCs were thought to exert their effects primarily through nuclear hormone receptors, i.e. estrogen, androgen, progesterone, thyroid, and retinoid receptors.Further studies have highlighted that they also act through non-nuclear receptors for steroid hormones (e.g., membrane estrogen receptors); non-steroidal receptors (for example, serotonin, norepinephrine, or dopamine receptors); orphan receptors (for example, aryl hydrocarbon receptors); enzymatic pathways involved in steroid biosynthesis and/or metabolism, as well as numerous other mechanisms that overlap with the endocrine and reproductive systems.Some EDCs have a genotoxic effect, causing deoxyguanosine hydroxylation and/or DNA strand breaks (both single and double), thereby promoting the malignant transformation of affected cells.Numerous endogenous and exogenous factors, such as environmental pollution and diet lead to the deterioration of the reproductive health of individuals of all links of the food chain [3,14,15].
It is known that reproductive processes in crustaceans are controlled by the endocrine system.These hormones play a significant role in normal growth and development.Water pollutants can impair reproduction, development, immune response, and other physiological processes, which can ultimately affect the survival of aquatic organisms.17beta-estradiol (E2) is important for the development of crustacean ovaries.Ovaries, hepatopancreas, hemolymph, and nervous tissues have been shown to be target organs for compounds with estrogenic activity in crustaceans, and concentrations of 17beta-estradiol in various tissues are closely related to vitellogenesis in the ovary and hepatopancreas during ovarian development [16,17].
Epigenetic programming established during development can be altered by exposure to EDCs during sensitive periods such as the preovulatory and ovulatory phases and cause immediate adverse outcomes.In addition, changes in methylation profiles and acetylation landscapes that occur during these periods may predispose to the development of pathologies that occur later in childhood or adulthood and, more dramatically, may even be transmitted from generation to generation [3].
According to the results of our research, the average level of estradiol in caviar samples of Procambarus virginalis was 2107 ng/g fresh weight, embryos -2876 ng/g, and young crustacean carcasses -4924 ng/g.
In the muscles of one-month-old (young) crustaceans, the average level of estradiol was 4634 ng/g of fresh weight, five-month-old (mature) -4854 ng/g, year-old (older) -4877 ng/g of fresh weight.These results highlight the ubiquitous bioaccumulation of estrogens in aquatic invertebrates depending on the duration of exposure.
The distribution of estradiol in the muscles of Procambarus virginalis can directly and effectively reflect the effects of EDC.Xenoestrogens can act on several mechanisms simultaneously.Estrogenic xenobiotics also disrupt the physiology of aquatic organisms, biochemical processes as triggers of oxidative stress, can affect their reproductive development, cause transcriptional effects, and they are also quite powerful initiators of signal cascades from membranes.Transfer of estradiol from the digestive system of Procambarus virginalis to muscle may pose a potential risk to aquaculture, wildlife and human health associated with the consumption of aquatic meat.Finally, they contribute to the general accumulation of estrogen in the environment.
It has been shown that herbicides, namely atrazine, which is commonly found in water bodies, affect the reproductive process of the corresponding fauna, acting as an endocrine disruptor.In this sense, it is suspected that this herbicide affects the secretion of some neurohormones involved in gonadal growth and also changes the circulating levels of steroid hormones that promote the synthesis of vitellogenin for ovarian growth.In addition, atrazine-induced sexual differentiation of young crayfish towards a greater proportion of females reduced offspring production, as well as several embryonic abnormalities, and had a genotoxic effect on crayfish.Finally, some metabolic imbalances, such as reduced energy stores, are observed in some species along with oxidative stress and histopathological effects.as well as suppression of the cortisol response [18].
Cortisol functions as both a glucocorticoid and a mineralocorticoid, which is explained by the unusual structure of its receptors, particularly in fish.This hormone affects the metabolism of carbohydrates, proteins and fats, and also performs important functions related to osmoregulation, growth and reproduction.It is commonly called the hyperglycemic hormone, which ensures the presence of a certain level of glucose in the blood.In aquatic animals, it is an important component in the stress response.Thanks to the timely correction of metabolic processes by this hormone, there is an even distribution of energy resources among target tissues under critical life conditions.Cortisol regulates the processes of osmoregulation through a mediated increase in the activity of enzymes involved in ion metabolism, in particular, Na-K-ATPase [19].This may indicate its stimulating role as an activator of ion-exchange enzymes, particularly in gill tissues, where the highest activity of these enzymes is observed.It should also be noted that cortisol affects the final maturation of oocytes in the preovulatory period [20].It is believed that the deposition of cortisol by the maternal body together with its local production by the ovaries may participate in the regulation of oogenesis.There are two critical stages when cortisone can have a modulating effect on oogenesis.The first stage occurs during vitogenesis when maternal cortisol is incorporated into the yolk, and the second stage occurs during maturation and ovulation, when cortisol can play a regulatory role, causing maturation [21].It has been shown that the stress-induced increase in cortisol, associated with a decrease in estradiol, affects the functions of granulosa cells in the follicle, worsening the quality of oocytes [15].Recent studies suggest that this hormone may act as a key factor linking the stimuli of the social environment and the onset of sex change, initiating the transition in steroidogenesis from estrogen to androgen.In addition, there are significant differences in glucocorticoid content between marine and freshwater crustaceans.Organotin compounds at different doses can inhibit the activity of 11beta-hydroxysteroid dehydrogenase isoenzyme 2, which is responsible for the inactivation of cortisol, which can increase glucocorticoid levels [13,22].
Crayfish gills play an important role in the transport of respiratory gases, excretion of nitrogen compounds and osmoregulation.The gills are the first barrier to waterborne pollutants.Gill tissue condition is generally considered a good indicator of water quality and suitable for environmental impact assessment.Its changes can lead to a decrease in oxygen consumption and a violation of the function of osmoregulation.A marked decrease in oxygen consumption can lead to internal hypoxia, which affects metabolism and movement [23].
Based on the results of the study, it was established that the level of cortisol in the gills of Procambarus virginalis increases (p < 0.05) (by 5.7 and 10 times) depending on age (figure 1A).In contrast, there was a probable decrease (p < 0.05) in the muscle of 2.2-and 3.2-fold with age, respectively (figure 1B).This is related to the distribution of energy between tissues.Cortisol is known to be involved in the development of stress reactions, regulation of water-salt balance, and carbohydrate metabolism in vertebrates, particularly fish.Significant difference between the cortisol content in roe, tissues of embryos and juveniles of the investigated crustaceans wasn't found (figure 1C).It can be assumed the hormone level changes of in this samples are related to the direct influence of the surrounding water environment.

Conclusions
The obtained results emphasize the widespread bioaccumulation of EDS, namely compounds with estrogenic activity, in aquatic invertebrates depending on the duration of exposure, which may indicate the role of the aquatic environment in the bioaccumulation of xenoestrogens.The study demonstrates the need to monitor exposure to EDC contaminants due to their effects on water quality degradation and serious toxicological effects on aquatic biota and human health to inhibit such potential.Estrogens are clearly necessary for normal fauna and human physiology but can have serious negative consequences if allowed to accumulate in the environment.
A link between chronic exposure to low concentrations of chemicals via the environment or food chain and reproductive health in animals has been demonstrated.Xenobiotics, including parent compounds and their metabolites in aquatic ecosystems, can cause developmental delay, malformations, behavioral changes, and mortality in non-target organisms.The main source of exposure to EDCs for humans is food, particularly of animal origin.Concerns have been raised about estrogens entering the human food chain, which in turn is related to how aquatic organisms consumed as food absorb and metabolize estrogens.In epidemiological studies, EDCs have been associated with immunotoxicity and neurobehavioral deficits both in consumers of contaminated products, including aquaculture, which accumulated these substances directly from the environment and in children exposed to EDCs in utero and through breast milk.The possibility that the bioaccumulate properties of persistent organic chemicals with hormone-like activity and chronic low-level exposure may contribute to the overall risk of breast cancer in women, as well as reproductive and developmental effects in humans, has serious implications for the prevention of these diseases.If consumed at levels above safe thresholds, they can cause metabolic diseases in humans.But, taking into account the fact that hormones act at very low doses, especially during sensitive stages of life, this concept can be extrapolated to EDCs, for which the biological significance of doses of certain EDCs below regulatory "safe" levels has been shown.These findings may have serious public health implications, as exposure to EDCs is still occurring worldwide, possibly affecting future generations and leading to the development of previously unreported diseases.
The tissues of aquatic organisms may contain a mixture of several ecologically toxic compounds, in addition to endocrine-disrupting contaminants, due to the widespread contamination of surface waters with poorly treated municipal and industrial wastes.This phenomenon can have additive, synergistic or antagonistic effects, which can explain their ultimate biological activity.
Thus, it is possible to note two important aspects related to the issue of contamination of aquatic biota with compounds that cause the destruction of the endocrine system.The first aspect concerns the impact of EDCs on the reproduction of wildlife populations, including aquatic organisms, which may have a negative impact on species conservation.The second aspect is that, since the effects of EDCs are realized on humans in different ways, primarily through trophic link food chains, further research is needed for public health policy planning.