**3. Materials and methods**

#### **3.1. Sediment samples**

The sediments samples were collected with an Eckman dredge in December 2006, transported at 4°C to the laboratory, the proportion of organic carbon in each sample was determined by calcination and then were stored at −20°C until bioassay. The zones were selected considering representative sectors of the diverse anthropic activities developed in the basin (**Figure 1**):

(a) Paysandu–PY (S. 32°19' W58°06′): near to the mouth of the Sacra Stream. This stream receives the sewage and industrial effluents of the Paysandu city.

(b) Nuevo Berlin–NB (S 32°59' W 58°04′): located at North of the Fray Bentos city. This site presents an important agricultural use, especially soybean and forestry. On the other hand, according to hydraulic studies carried out under the installation of a pulp mill in the city of Fray Bentos, this one zone would not be influenced by the plume of the effluent. Therefore, it could be considered as a preimpact point in future monitoring.

(c) Las Cañas–LC (S 33°15' W 58°16′): located at South of the Fray Bentos city, is a tourist and agricultural area.

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

#### **3.2. Experimental design**

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

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

*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

The sediments samples were collected with an Eckman dredge in December 2006, transported at 4°C to the laboratory, the proportion of organic carbon in each sample was determined by calcination and then were stored at −20°C until bioassay. The zones were selected considering representative sectors of the diverse anthropic activities developed in the basin (**Figure 1**):

(a) Paysandu–PY (S. 32°19' W58°06′): near to the mouth of the Sacra Stream. This stream

(b) Nuevo Berlin–NB (S 32°59' W 58°04′): located at North of the Fray Bentos city. This site presents an important agricultural use, especially soybean and forestry. On the other hand, according to hydraulic studies carried out under the installation of a pulp mill in the city of Fray Bentos, this one zone would not be influenced by the plume of the effluent. Therefore, it

(c) Las Cañas–LC (S 33°15' W 58°16′): located at South of the Fray Bentos city, is a tourist and

comparison of the results obtained with those generated in other studies.

receives the sewage and industrial effluents of the Paysandu city.

could be considered as a preimpact point in future monitoring.

induced by exposure to xenoestrogens.

20 Endocrine Disruptors

**3. Materials and methods**

**3.1. Sediment samples**

agricultural area.

in detriment of the increase in body weight [38].

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

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

Forty-eight hours prior to the start of the assay, the collected sediments were placed in 30-l tanks in a 1:10 ratio (sediment/water), in order to allow them to decant. Each of the treatments (sectors of the river) was analyzed in triplicate (total 12 fish tanks) and included three tanks with dechlorinated water and no sediment (negative control). Four fishes were placed in each tank randomly and were weighed and measured at the beginning of the assay. The spatial location at de laboratory of the treatments and controls was established randomly by using a table of random numbers. The assay was semi-static, with water replacement every 7 days and an exposure time of 30 days.

*HSI* = (

*GSI* = (

p < 0.05 was considered significant.

**4.1. Plasma VTG levels gonadal histology**

differences were detected among the exposed groups.

**3.6. Statistical analysis**

**4. Results**

\_\_\_\_\_\_\_\_\_ *liver mass*

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

*gonad mass* \_\_\_\_\_\_\_\_\_

Normality and homogeneity of variance were verified, and a single factor analysis of variance or Kruskal-Wallis test was used to determine differences between the physiological indices and plasma VTG levels. Statistical significance was confirmed by Tukey's post hoc test;

Plasma VTG levels are presented in **Figure 2**. The values increased along a latitudinal gradient from Paysandu to Juan Lacaze (means: control = 0.360, Paysandú 6.128 and Juan Lacaze = 9989) accompanied of a reduction in the data dispersion. Only significant differences were observed among the sediment of exposed groups with the control (Tukey's HSD, p < 0.05); however, no

**Figure 2.** Plasma vitellogenin levels in common carp exposed to sediments from the Uruguay River. Vertical bars

indicate the 95% confidence intervals. \*Indicate significant differences (p *<* 0.05).

*body mass*) <sup>×</sup> <sup>100</sup> (2)

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

23

*body mass* ) <sup>×</sup> <sup>100</sup> (3)

#### **3.3. Plasma vitellogenin (VTG) levels**

Once the bioassay was completed, blood samples were extracted from the vena caudalis using heparinized syringes for plasma VTG analysis (heparin solution 20 units/ml). Plasma was separated by centrifugation (Universal 32R, Hettich Zentrifugen) at 1500 rpm for 10 min, and plasma VTG was quantified using antibody pre-coated enzyme-linked immunosorbent assay kits (Biosense Laboratory, Bergen, Norway; product no. V01003402) [49]. The microplates were measured at a wavelength of 492 nm in a Biorad 680 microplate reader spectrophotometer (Hercules, CA, USA). The VTG concentration was calculated based on a standard calibration curve and expressed in μg/ml [50, 51].

#### **3.4. Gonadal histology**

The individuals were sacrificed by cervical dislocation, and the gonad was removed. Gonads were immediately weighed and fixed in 10% phosphate buffered formalin at pH 7.4. Then the tissue was dehydrated gradually through the passage through alcohol 70°, 96°, 100° and chloroform and embedded in paraffin. The cuts were made using a Reichert-Jung microtome at 5 μm. Finally, they were re-hydrated and stained with Harris's hematoxylin and eosin. The fish were sexed, and the reproductive maturity of the gonad cells was determined according to Smith and Walker in 2004 using an optical microscope with 10×, 20× and 40× eyepieces (Olympus Vanox; Tokyo, Japan) and photographed using a digital camera [36, 52–53].

#### **3.5. Physiological indices**

The somatic indices were calculated according to the following morphometric parameters: body weight (g), liver weight (g) and gonad weight (g).

For the condition factor, in first instance, was analyzed the length-weight relationship (log– log curve) from which the slope was obtained (p). This value was the allometric coefficient and was used in the equation Eq. 1.

$$K = \left(\frac{\text{weight}}{\text{st.length}}\right) \times 100\tag{1}$$

The hepatosomatic index (HSI) and gonadosomatic index (GSI) of each fish were determined according to Eqs. (1) and (2)

First Approach to Screening Endocrine Disruption Activity in Sediments from the Uruguay River… http://dx.doi.org/10.5772/intechopen.78736 23

$$HSI = \left(\frac{liver\ mass}{body\ mass}\right) \times 100\tag{2}$$

$$GSI = \left(\frac{gound\ mass}{bady\ mass}\right) \times 100\tag{3}$$

#### **3.6. Statistical analysis**

Normality and homogeneity of variance were verified, and a single factor analysis of variance or Kruskal-Wallis test was used to determine differences between the physiological indices and plasma VTG levels. Statistical significance was confirmed by Tukey's post hoc test; p < 0.05 was considered significant.

#### **4. Results**

Forty-eight hours prior to the start of the assay, the collected sediments were placed in 30-l tanks in a 1:10 ratio (sediment/water), in order to allow them to decant. Each of the treatments (sectors of the river) was analyzed in triplicate (total 12 fish tanks) and included three tanks with dechlorinated water and no sediment (negative control). Four fishes were placed in each tank randomly and were weighed and measured at the beginning of the assay. The spatial location at de laboratory of the treatments and controls was established randomly by using a table of random numbers. The assay was semi-static, with water replacement every 7 days

Once the bioassay was completed, blood samples were extracted from the vena caudalis using heparinized syringes for plasma VTG analysis (heparin solution 20 units/ml). Plasma was separated by centrifugation (Universal 32R, Hettich Zentrifugen) at 1500 rpm for 10 min, and plasma VTG was quantified using antibody pre-coated enzyme-linked immunosorbent assay kits (Biosense Laboratory, Bergen, Norway; product no. V01003402) [49]. The microplates were measured at a wavelength of 492 nm in a Biorad 680 microplate reader spectrophotometer (Hercules, CA, USA). The VTG concentration was calculated based on a standard

The individuals were sacrificed by cervical dislocation, and the gonad was removed. Gonads were immediately weighed and fixed in 10% phosphate buffered formalin at pH 7.4. Then the tissue was dehydrated gradually through the passage through alcohol 70°, 96°, 100° and chloroform and embedded in paraffin. The cuts were made using a Reichert-Jung microtome at 5 μm. Finally, they were re-hydrated and stained with Harris's hematoxylin and eosin. The fish were sexed, and the reproductive maturity of the gonad cells was determined according to Smith and Walker in 2004 using an optical microscope with 10×, 20× and 40× eyepieces

(Olympus Vanox; Tokyo, Japan) and photographed using a digital camera [36, 52–53].

The somatic indices were calculated according to the following morphometric parameters:

For the condition factor, in first instance, was analyzed the length-weight relationship (log– log curve) from which the slope was obtained (p). This value was the allometric coefficient

The hepatosomatic index (HSI) and gonadosomatic index (GSI) of each fish were determined

*st*.*lengthp*) <sup>×</sup> <sup>100</sup> (1)

and an exposure time of 30 days.

22 Endocrine Disruptors

**3.4. Gonadal histology**

**3.5. Physiological indices**

and was used in the equation Eq. 1.

according to Eqs. (1) and (2)

**3.3. Plasma vitellogenin (VTG) levels**

calibration curve and expressed in μg/ml [50, 51].

body weight (g), liver weight (g) and gonad weight (g).

*<sup>K</sup>* <sup>=</sup> ( *weight* \_\_\_\_\_\_\_\_

#### **4.1. Plasma VTG levels gonadal histology**

Plasma VTG levels are presented in **Figure 2**. The values increased along a latitudinal gradient from Paysandu to Juan Lacaze (means: control = 0.360, Paysandú 6.128 and Juan Lacaze = 9989) accompanied of a reduction in the data dispersion. Only significant differences were observed among the sediment of exposed groups with the control (Tukey's HSD, p < 0.05); however, no differences were detected among the exposed groups.

**Figure 2.** Plasma vitellogenin levels in common carp exposed to sediments from the Uruguay River. Vertical bars indicate the 95% confidence intervals. \*Indicate significant differences (p *<* 0.05).

#### **4.2. Gonadal histology**

The gonadal histological analysis of females revealed that oocyte stages was not different between exposed and unexposed groups and that all oocytes were in the previtellogenic and perinucleolar stages according to the classification made by Smith and Walker [36] (**Figure 3C**, **D**). No significant differences were observed in relation to the size of the oocytes (control group range: 59–114 μm, Paysandu range: 79–113 μm, Nuevo Berlin range: 66–109 μm, Las Cañas range: 74–116 μm) nor in the amount of them within the displayed field.

Regarding testicular development, this in general does not have a clear differentiation, and it is only possible to observe a delay in maturity of the exposed individuals respect to

control. This delay is observed by a decrease in the number of accumulations of primary spermatocytes (**Figure 3A**, **B**). Some individuals exposed to sediments from industrial sites (Paysandu and Juan Lacaze) presented oocytes in testicular tissue (testis-ova) (**Table 1**).

Values are given as mean ± standard deviations for each treatment, and respective n in parentheses. \*indicates statistical differences between treatments (p < 0.05). K: condition factor, GSI: gonadosomatic index, HSI: hepatosomatic index.

**Treatment Sex distribution (males/females) Testis-ova (number/males)**

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

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

25

**Table 1.** Sex distribution between treatments and the control and the occurrence of testis-ova.

**Treatment K GSI HSI**

**Table 2.** Physiological indices in the exposed and unexposed groups.

Unexposed 2.6 ± 0.4 (12) 0.5 ± 0.6 (12) 1.3 ± 0.8 (12)\* Paysandu 2.8 ± 0.3 (12) 0.5 ± 0.6 (12) 1.7 ± 0.6 (12) Nuevo Berlin 2.6 ± 0.5 (12) 1.3 ± 1.0 (12) 1.8 ± 0.6 (12) Las Cañas 2.6 ± 0.4 (12) 0.9 ± 0.6 (12) 0.9 ± 0.6 (12)\* Juan Lacaze 2.8 ± 0.4 (12) 0.6 ± 0.8 (12) 2.5 ± 0.4 (12)\*

Unexposed 9/3 0/9 Paysandu 9/3 1/9 Nuevo Berlin 4/8 0/4 Las Cañas 4/8 0/4 Juan Lacaze 10/2 1/10

Values of the physiological indices in the exposed and unexposed groups are given in **Table 2**. No significant differences were observed among the groups for the condition factor (K) or gonadosomatic index (GSI); however, significant differences in HSI were detected. Posthoc comparisons revealed that fish exposed to sediment from Juan Lacaze had significantly increased HSIs compared with those in the control (p = 0.04) and Las Cañas groups (p = 0.02)

The results obtained in plasma vitellogenin levels indicate a marked effect of the treatments. Several known sources of endocrine disruptors are located in the Uruguay river basin, and previous studies have detected some EDCs in sediments [27] that could be responsible for the observed alterations. However, only significant differences were observed in Las Cañas and Juan Lacaze treatments. In the first one, the increase may be due to the contribution of known estrogenic pesticides such as chlorpyrifos, endosulfan, and cypermethrin

**4.3. Physiological indices**

**5. Discussion**

**Figure 3.** Photographs of gonadal histology. (A) male from control (20×); (B) male from Nuevo Berlin (20×); (C) female from the control (20×); (D) female from Nuevo Berlin (10×). PSA primary spermatocyte accumulation, OPP oocytes in previtellogenic and perinucleolar stage, Ct cytoplasm, N nucleus.

First Approach to Screening Endocrine Disruption Activity in Sediments from the Uruguay River… http://dx.doi.org/10.5772/intechopen.78736 25


**Table 1.** Sex distribution between treatments and the control and the occurrence of testis-ova.


Values are given as mean ± standard deviations for each treatment, and respective n in parentheses. \*indicates statistical differences between treatments (p < 0.05). K: condition factor, GSI: gonadosomatic index, HSI: hepatosomatic index.

**Table 2.** Physiological indices in the exposed and unexposed groups.

control. This delay is observed by a decrease in the number of accumulations of primary spermatocytes (**Figure 3A**, **B**). Some individuals exposed to sediments from industrial sites (Paysandu and Juan Lacaze) presented oocytes in testicular tissue (testis-ova) (**Table 1**).

#### **4.3. Physiological indices**

**4.2. Gonadal histology**

24 Endocrine Disruptors

The gonadal histological analysis of females revealed that oocyte stages was not different between exposed and unexposed groups and that all oocytes were in the previtellogenic and perinucleolar stages according to the classification made by Smith and Walker [36] (**Figure 3C**, **D**). No significant differences were observed in relation to the size of the oocytes (control group range: 59–114 μm, Paysandu range: 79–113 μm, Nuevo Berlin range: 66–109 μm, Las Cañas

Regarding testicular development, this in general does not have a clear differentiation, and it is only possible to observe a delay in maturity of the exposed individuals respect to

**Figure 3.** Photographs of gonadal histology. (A) male from control (20×); (B) male from Nuevo Berlin (20×); (C) female from the control (20×); (D) female from Nuevo Berlin (10×). PSA primary spermatocyte accumulation, OPP oocytes in

previtellogenic and perinucleolar stage, Ct cytoplasm, N nucleus.

range: 74–116 μm) nor in the amount of them within the displayed field.

Values of the physiological indices in the exposed and unexposed groups are given in **Table 2**. No significant differences were observed among the groups for the condition factor (K) or gonadosomatic index (GSI); however, significant differences in HSI were detected. Posthoc comparisons revealed that fish exposed to sediment from Juan Lacaze had significantly increased HSIs compared with those in the control (p = 0.04) and Las Cañas groups (p = 0.02)

#### **5. Discussion**

The results obtained in plasma vitellogenin levels indicate a marked effect of the treatments. Several known sources of endocrine disruptors are located in the Uruguay river basin, and previous studies have detected some EDCs in sediments [27] that could be responsible for the observed alterations. However, only significant differences were observed in Las Cañas and Juan Lacaze treatments. In the first one, the increase may be due to the contribution of known estrogenic pesticides such as chlorpyrifos, endosulfan, and cypermethrin from agricultural surrounding areas [54]. Additionally, phytoestrogens released by crops as a defense strategy may be reaching the river in overland runoff. In particular, soybeans contain high levels of genistein, daidzein, and glycitein, which can elicit alterations in endocrine function in wildlife and humans [55]. Juan Lacaze receive untreated municipal sewage effluent containing a complex cocktail of natural (estrone or 17b-estradiol) and synthetic estrogens used in oral contraceptives as well as surfactants used in soaps and detergents (alkylphenols and alkylphenolpolyethoxylates) Furthermore, the plasma VTG concentrations in fish were highest where deposition processes were predominant at Juan Lacaze and where pulp mill effluent was discharged near the sampling site. In that sense, several works worldwide have documented the increase in vitellogenin plasma in juvenile individuals exposed to effluents from cellulose plants [14, 56, 57]. Elevated levels of VTG in males and immature females were clearly an estrogen-mediated response. It is important to note that VTG levels were not affected by the sex ratio, as shown by similar VTG concentrations in the Las Cañas and Juan Lacaze groups with opposite sex ratios, same when comparing the Paysandu and Nuevo Berlin treatments.

EDCs in the sediments. The specific agents responsible for the toxic effects were not identified because it was beyond the scope of this study. Future research is needed to identify the causal agents (natural or synthetic) and to determine exposure routes (e.g., grazing on sediments or bioconcentration from the water column). Finally, in relation to the adequacy of the bioassay developed to be applied as a monitoring tool, since Juan Lacaze sediments generated the greatest changes in the analyzed biomarkers, confirming their inclusion as a positive control. Likewise, the selection of negative controls (without sediment exposure) showed the lowest

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

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

27

We express our thanks to the Departments of Virology and Cellular Biology (Science School) for cooperation with the processing of samples and to Mr. Angel Rosano and the Uruguayan Navy for their assistance in the fieldwork. This study was funded by the National Agricultural Research Institute (INIA) Project SA07 and the Environmental Science Master Program.

Ecology and Environmental Sciences Institute, Sciences College, University of the Republic,

[1] IPCS. Global assessment of the state of the science of endocrine disruptors. World Health Organization. International Programme on Chenical Safety. [Internet]. 2002. Available from: http://www.who.int/ipcs/publications/new\_issues/endocrine\_disruptors/en/ [Accessed:

[2] Ahmed RG. Perinatal TCDD exposure alters developmental neuroendocrine system. Food and Chemical Toxicology Journal. 2011;**49**:1276-1284. DOI: 10.1016/j.fct.2011.03.008

[3] Ahmed RG. Early weaning PCB 95 exposure alters the neonatal endocrine system: Thyroid adipokine dysfunction. Journal of Endocrinology. 2013;**219**(3):205-215. DOI:

levels of changes as well as the lowest dispersion of values between replicates.

**Acknowledgements**

**Conflict of interest**

**Author details**

Montevideo, Uruguay

April 20, 2018]

10.1530/joe.13.0302

**References**

Noelia Rivas-Rivera\* and Gabriela Eguren

\*Address all correspondence to: noeriv@gmail.com

None.

The gonad histology analyses indicated that female fish did not exhibit differences in maturation state. Unlike other works carried out under similar conditions, not differences in the stage of oocyte development were observed [33, 58, 59]. Whereby it could only be a trophoblastic but not protoplasmic growth, this could be checked by increasing the exposure time to see if there are changes in the stages in the individuals with the highest number of cells. However, sediment-exposed males presented delayed testicular maturation than that in the unexposed group. Jobling et al. reported that the induction of VTG in males is negatively correlated with testicular maturation, and Devlin and Nagahama observed retarded gonadal maturation in *C. carpio* males exposed to estrogenic compounds [60, 61]. Changes in sex ratios and intersex individuals have been reported in common carp exposed to EDCs [40, 41, 61]. However, the intersex condition occurs naturally in approximately 5% of the population in this species [7]. Thus, the presence of individuals with testis-ova observed in our study was possibly a natural phenomenon and may not have been caused by exposure to contaminated sediments.

The condition factor showed higher values in all the treatments with respect to the control group; however, the differences are not statistically significant, and this agrees with the results obtained by Orrego et al. [14]. The significant increase in liver mass at Juan Lacaze may have been caused by induction of the hepatic mixed function oxidase system in response to discharge of persistent organic compounds from the pulp mill effluent [62–64]. Increased protein synthesis generates proliferation of endoplasmic reticulum, which can be reflected in increased hepatocyte size [65–69].
