**2. Methodological approach**

mechanisms through regulatory actions positive and negative that allows keeping hormone

Several natural and synthetic chemical compounds released into the environment by human activities have the ability to interfere with the normal functioning of the endocrine system [2–4, 7, 8, 12–18]; these compounds are called endocrine disruptors (EDCs). The term endocrine disruptor defines, by both, to a compound or set of exogenous chemical compounds (natural or synthetics) that alter the normal functioning of the endocrine system through alterations in hormonal balance, causing effects on exposed organisms and including about

A wide variety of EDC sources have been documented, including municipal sewage discharges and industrial effluents (e.g., pulp mills), as well as some pesticides and their metabolites [1, 7, 8]. Some of these compounds are persistent and lipophilic, and their concentrations are higher in sediments than in river water [9]. Growing scientific evidence shows that the EDCs can exert estrogenic, androgenic, antiandrogenic, and antithyroid actions on aquatic organisms and can induce alterations in the functional development and reproduction of fish [10–13]. Several studies have detected high concentrations of EDCs in sediments, suggesting that the sediments could be responsible for the observed alterations; however, bioavailability of EDCs is complex. Sediments could be acting as a sink and reducing EDC bioavailability or rereleasing the chemical compounds into the water and acting as a source. The possible exposure routes to aquatic organisms include direct uptake of free compounds across the gills or skin and ingestion of sediment particles [9]. Several laboratories and field studies have reported that fish exposed to sediments experience significant alterations in endocrine functions [14–19].

Several approaches have been employed to show effects derived from the exposure to chemical compounds; however, those based on the use of biomarkers are the most widely used. These have been defined as "xenobiotically induced variations in cellular or biochemical components, processes, structures or functions that are quantifiable in a biological system" [20]. The first level of action of a compound or mixture of chemical compounds occurs in the biochemical-molecular component, triggering responses that tend to maintain the functioning of the organism within the homeostatic levels. If the exposure concentrations are high or are maintained during periods of prolonged time, the answers may not be enough to counteract the effect. In such a case, the agency triggers in the first instance mechanisms of compensation and then repair [21].

The Uruguay river is the natural border between Uruguay from Argentina and supports intensive agricultural and forest production and receives a variety of municipal sewage discharges and industrial effluents [22–24]. Since 1992, studies carried out by the Administration Commission of the Uruguay River (CARU) show the presence of some EDCs like chlorinated compounds (aldrin, dieldrin, HCH, HCB, DDT and its derivatives) and PCBs in fish [25]. Other works from the lower Uruguay river, detected in water and sediments several chemicals such as resin acids, phytosterols, polychlorinated dibenzo-p-dioxins, and dibenzofurans [26, 27]. At the time of the study was carried out, a large pulp mill was under construction at Fray Bentos along the Uruguay river and was associated with considerable controversy [28]. Pulp mill effluents have been associated with endocrine impacts in Canada as well as in other countries [29]. The main objective of this study was to determine whether exposure of

levels in balance [1].

18 Endocrine Disruptors

his progeny [1, 5, 6].

The study was implemented applying a combination of field activities and laboratory, in order to evaluate the potential of the sediments of the Uruguay river of interfere with the normal functioning of the endocrine system and generate effects to reproductive level in fish.

Sediments have been selected as exposure matrix since that several compounds cataloged as endocrine disruptors have high affinity for them and that they have been identified in previous studies as one of the main sources of persistent estrogenic contaminants [6]. On the other hand, several crop protection compounds recognized as endocrine disruptors have been detected in water and fish in the Uruguay River [25, 30].

A battery of biomarkers was selected for the study, which included early warning signs (plasma vitellogenin levels) and late ones (condition factor, hepato and gonadosomatic indices and histological analysis of gonads). These were evaluated under controlled laboratory conditions by exposing juvenile individuals of *Cyprinus carpio* to sediments from different sectors of the Uruguay river.

The presence of vitellogenin, an estrogen-inducible protein, in plasma indicates a high internal concentration of estrogenic compounds, both of endogenous origin as exogenous (xenoestrogens) [31]. Therefore, the presence of detectable concentrations of vitellogenin in plasma of males or immature individuals has been proposed as a biomarker of exposure to xenoestrogenic compounds [1, 31–34]. Several studies have shown the interference of various compounds or mixture of these on the functioning of the endocrine system. Such is the case, where *Cyprinus carpio* males captured in the effluent channel of a plant of household waste treatment, presented levels of plasma vitellogenin significantly elevated [32]. These effects can lead to alterations in the structure of the trophic webs, causing changes in the transfer flows of matter and energy to and from the aquatic ecosystem. They can also cause a stock reduction or a loss in the quality of commercially exploitable species, by the accumulation of xenobiotics in tissues (bioaccumulation and/or biomagnification) [3, 35].

Gonadosomatic (GSI) and hepatosomatic indices (HSI) reflect the dynamics of the use of energy by organisms. Changes in IGS are directly related to sex, age and reproductive stage [30]. Therefore, an acceleration of the maturation of the gonadal cells by exposure to xenoestrogens will be reflected in an increase in the IGS and vice versa. While the changes in the IHS are linked to alterations in the main functions of the liver as the synthesis and degradation of hormones and detoxification of xenobiotics. In this sense, exposure to xenobiotics can cause an increase in the size of the liver [37].

Although through changes in somatic indices, it is possible to demonstrate the existence of effects, it is not possible to elucidate the mechanism (s) by which changes were generated. In this sense, by analyzing histological sections of the gonads, it is possible to determine if the increase in size is due, for example, to acceleration in the maturation of the reproductive cells induced by exposure to xenoestrogens.

(d) Juan Lacaze–JL (S 34°26' W 57°27′): located in the Río de la Plata river (Colonia Department). It is an urban-industrial area and is directly influenced by discharge form an elemental chlo-

First Approach to Screening Endocrine Disruption Activity in Sediments from the Uruguay River…

http://dx.doi.org/10.5772/intechopen.78736

21

The assay was performed in January 2007. Immature common carp were obtained from the National Direction of Aquatic Resources Fish Hatchery (DINARA – Villa Constitucion station). They were transported in plastic bags, which were injected with oxygen and placed in ice. Once received in the laboratory, total weight (g), total and standard length (cm) of the totality of the individuals were recorded (mean body length, 7.2 ± 0.9 cm; mean weight, 9.0 ± 3.2 g) The fish were acclimatized for 15 days in an aerated pool (800 L of dechlorinated water, renewed every 2 days). During the acclimation period as well as during the assay, they were fed *ad libitum* with commercial feed (Marplatense S.A., Montevideo, Uruguay) and were kept under controlled temperature conditions (22 +/− 1°C), light: dark cycle (12:12 h) and

**Figure 1.** Study area and location of the sediment sampling zones. PY Paysandu, NB Nuevo Berlin, LC Las Cañas, JL

rine bleached Kraft pulp mill (was considered a positive control).

**3.2. Experimental design**

Juan Lacaze.

percentage of dissolved oxygen (89 +/− 1%).

Finally, the condition factor reflects the degree of adaptability of the organism to environment, in terms of an adequate energy balance between physiological needs and the increase in body biomass. Therefore, exposure to natural or artificial stressors will cause changes in the storage and transfer of lipids and proteins tending to counteract the effect of the stressor in detriment of the increase in body weight [38].

*Cyprinus carpio* "common carp" is a teleost fish belonging to the Cyprinidae family. It is originally from Asia with a great ability to adapt to different media, has been widely introduced worldwide. In our country, the introduction was made for commercial purposes in the 1960s from Brazil [39]. It has wide ranges of temperature tolerances (12–32°C) and acidity (pH 5–10), resists low levels of dissolved oxygen (1–2 mg/l) and high turbidity. It is an omnivorous species, mainly benthophages [36]. About its reproductive characteristics, this species reaches its sexual maturity between 18 months and 2 years of life, depending mainly on the temperature of the water. The gonadal differentiation is within the category of differentiated gonochorist, starting at 50 days post-birth [40–42]. The Organisation for Economic Co-Operation and Development (OECD) and the United States Environmental Protection Agency (EPA) consider *Cyprinus carpio* as a good bioindicator specie for evaluation effects of endocrine disruption [43, 44] and has already been used in several studies [32, 40, 41, 45–48]. This allows the comparison of the results obtained with those generated in other studies.
