Integrated Use of Oxidative Stress and Histological Biomarkers of T. tinca as Indicators of 17‐Alpha‐Ethynylestradiol Exposure

ABSTRACT

The potential adverse effects of 17‐alpha‐ethynylestradiol (50, 100, and 500 μg EE₂/kg b.w., for 30 days) on tench ( Tinca tinca ) were evaluated by integrating biomarkers including physiological (hepato‐somatic index, spleen‐somatic index, and hematocrit), oxidative stress (catalase, glutathione peroxidase, and glutathione reductase activities; total glutathione level, and lipid peroxidation), metabolic (glutathione S‐transferase activity), as well as histopathological (descriptive and analytical studies) responses. The general health status of the EE₂‐exposed tench was disturbed based on the increase of somatic indices at high tested doses, and the development of anemia in all exposed individuals. Effective control of reactive oxygen species by the antioxidant defense system of the tench exposed to EE₂ should have occurred because the lipid peroxidation process was irrelevant. Histopathological study revealed the presence of regressive changes in the liver (vacuolar degeneration, and deposits of eosinophilic material), regressive (deposits of eosinophilic material), and progressive (hyperplasia of reticuloendothelial cells) changes in the spleens of exposed fish. The severity of some lesions was dose dependent. The identified injuries did not compromise the functions of these organs. Finally, a common pattern of correlation between parameters of oxidative stress and morphological changes was not detected in the current study.

1. Introduction

In recent decades, the occurrence of emerging pollutants in wastewater and environmental waters, as well as their effects on aquatic organisms, has become a matter of considerable concern. This group of substances includes endocrine disrupting compounds (EDCs) that have been extensively studied because of their effects on the endocrine systems of aquatic organisms, especially on their growth and reproductive function.

The European Union established a priority list of substances that needed to be monitored in the aquatic environment (Commission Implementing Decision‐EU‐2015/495), and their effects assessed on aquatic organisms to elucidate their potential environmental risk. One of the compounds included in the cited list is the synthetic estrogen 17‐alpha‐ethynylestradiol (EE₂), which is commonly used in human and livestock medication and consequently released into the aquatic environment through their excretion [1]. In fact, this compound has been found in surface waters at concentrations of ng/L [2, 3, 4, 5], and in sediments, which is a sink for this compound due to its hydrophobic properties and high octanol–water partitioning coefficient (Kow = 4.15), at a rank of ng/g d.w. [6]. EE₂ shows high chemical stability with a long half‐life in the aquatic compartment (t _1/2 = 92 days), which increases its bioavailability and bioaccumulation in aquatic organisms. In addition, this compound undergoes enterohepatic circulation, which amplifies its toxic effects [7]. Many researchers have confirmed that EE₂ can accumulate in different fish organs (liver, gall bladder, intestine, spleen, muscle, gills and gonads) at concentrations of ng/g d.w. [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18], and therefore could exert adverse effects in them. Interestingly, it is noteworthy that the concentration of this substance in the fish body can be 332‐fold higher than that detected in aquatic sites [19], and its potency can be 10–50‐fold higher than that of natural estrogens [20].

Most of the investigations into this compound have focused on the study of its effects on the reproductive function of aquatic organisms. Nevertheless, several studies have reported the effects of this substance on organs not directly related to reproduction, such as the liver, spleen, and kidney of fish, which could compromise the physiological functions regulated by these organs. The liver is a detoxification organ involved in the metabolism and excretion of xenobiotics, and it is involved in the synthesis of different compounds [21]. This organ, together with the spleen, kidney, and lymphoid organs, plays a key role in the immunological function of an organism [22]. The liver and spleen contain estrogen receptors, which are important targets of estrogenic compounds [23, 24].

The metabolism of estrogens generates intermediates which have electrophilic and oxidant characteristics [25]. According to Halliwell and Gutteridge [26], the increased production of reactive oxygen species (ROS) can lead to disturbances in the body's antioxidant defense system, DNA and protein damage, and lipid peroxidation (LPO). In fact, there are several studies that reflect the involvement of EE₂ on oxidative processes generation in fish. Usually, biochemical disturbances often precede the occurrence of pathological alterations in the tissues. Excess in the level of ROS production, apart from altering the antioxidant defense system, could lead to cellular lesions caused by increased vulnerability of the membrane bilayer to LPO and by the tendency of EE₂ to associate with lipids because of its high lipophilicity. Therefore, this substance can cause structural alterations in the tissues. Different authors have observed the development of structural changes in non‐endocrine tissues (liver, spleen and kidney) of fish exposed to this agent [27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39]. Despite this knowledge, there is still a lack of integrative studies specifically evaluating both oxidative and histological biomarkers in tench (Tinca tinca) , a species of ecological and economic importance.

Oxidative stress biomarkers can complement histological biomarkers by indicating ongoing cellular damage, understanding action mechanisms of pollutants, and the organism's physiological response to exposure. Therefore, the integration of oxidative and histological biomarkers offers a powerful and holistic approach to assessing the health of aquatic organisms in the face of environmental pollutants, allowing for more sensitivity and specificity in biomonitoring programs.

Considering these concerns, the present work aimed to investigate the alterations caused by EE₂ in biochemical parameters related to oxidative stress, and in the histological structure of the liver and spleen of tench to evaluate their health status. Moreover, relationships between biochemical parameters of oxidative stress and morphological changes will be established.

2. Materials and Methods

2.1. Animal Husbandry

The experiment was conducted at the Aquaculture Center “Vegas del Guadiana” (38°53′22.1′′ N 6°52′37.3′′ W, Government of Extremadura, Spain) by applying technical procedures according to the legal requirements established in the European Parliament and Council Directive 2010/63/UE for the protection of animals used for scientific purposes.

Tench is a freshwater cyprinid with widespread geographic distribution in Europe and Asia [40]. They feed and live at the bottom of bodies of water in contact with sediments where EE₂ tends to accumulate [6]. It represents a species of considerable value to the aquaculture industry and is used as a sensitive bioindicator for assessing exposure to and effects of environmental pollutants [32, 41, 42, 43, 44, 45, 46]. Therefore, this species has a relevant economic (food species), ecological, and sports value.

Tench were bred in the Aquaculture Center according to Council Directive 98/58/EC of 20 July 1998 concerning the protection of animals kept for farming purposes and following recommendations about the welfare of farmed fish (Aquatic Animal Health Code, World Organization for Animal Health). Individuals were carefully selected by qualified staff from Aquaculture Center, being healthy and free from toxic exposure, and they were checked every day.

For our experiment, tench were housed in outdoor tanks (3 m × 3 m × 1 m), provided with artificial vegetation over a sediment layer to simulate natural conditions, supplied with a flow of water of 40 L/h to assure a 10% water exchange every day. Water parameters are as follows: dissolved oxygen (7.7 ± 0.8 mg/L; 84%); pH (7.7 ± 0.34); temperature (19.2°C ± 1.02°C); total hardness (193 ± 12 mg/L as CaCO₃); conductivity (1050 μS/cm); ammonium (< 0.1 mg/L), nitrates (18 ± 0.3 mg/L); nitrites (< 0.02 mg/L) and phosphates (1.7 ± 0.5 mg/L).

Fish with 488.28 ± 26.35 g in weight and 27.36 ± 0.64 cm in length (mean ± standard error of mean) were daily fed at a rate of 1% of their mean body weight with a free EDCs commercial pellet diet for cyprinids (DIBAQ CYPRINIDS 4.5 MM—analytical constituents and additives: crude protein (42%), total ashes [13.50%], crude fiber [5%], crude fat [6%], phosphorus [1.5%], A vit [10 000 UI/kg], D3 vit [1700 UI/kg], E vit [200 UI/kg], cooper [11 mg/kg], and antioxidants).

2.2. EE₂ Exposure

After an acclimation period (15 days), 70 male fish were subdivided into 10 groups of seven individuals each: control group (composed of unexposed fish), solvent‐control group (composed of corn oil plus ethanol‐exposed fish), and three experimental groups exposed to EE₂. The study was performed in duplicate chambers for the control and EE₂ exposure groups.

EE₂ (≥ 98% purity; Sigma‐Aldrich, Spain) was tested at 50, 100, and 500 μg/kg b.w. for 30 days. Exposure solutions were prepared in the Toxicology laboratory and stored under appropriate conditions until subsequent use at the Aquaculture Center. Fish were injected with a volume of 0.5 mL/g b.w. for each dose. The doses were selected based on previously published data on other cyprinid species, common carp ( Cyprinus carpio ), where chronic exposure to 500 μg/kg EE₂ caused alterations in hematological parameters, histopathological changes in the liver, spleen, and kidney, and changes in enzymes belonging to the antioxidant defense system [33, 47]. On the other hand, Maria et al. [48] investigated the effects of E₂, the naturally occurring estrogen, on the antioxidant system of sea bass ( Lateolabrax japonicus ), employing two distinct routes of exposure (E₂ water diluted: 200 ng/L and 2000 ng/L, and intraperitoneal injection ‐i.p.‐: 0.5 and 5 mg/kg) for comparative analysis. The authors conducted comparisons between effects caused by E₂ concentrations and E₂ i.p. doses of exposure, concluding that i.p. doses caused higher induction of oxidative stress in the liver than E₂ concentrations. According to this comparative study and the maximum concentration of EE₂ reported in the surface water samples by Duong et al. [3], the dose of 50 μg/kg used in our study could be similar to an environmentally realistic concentration (28.6 ng/L) [3]. Solvent‐control fish were injected with corn oil plus ethanol. The solvent‐control and exposed fish were intraperitoneally injected (in the abdomen at angle of 45° between the tail fins and the anal opening using a needle size of 25G) initially and again once per week throughout the duration of the study. The intraperitoneal route of administration for EE₂ was selected due to its potential to facilitate more direct contact of the compound with the gastrointestinal tract, thereby simulating uptake via food ingestion and ensuring substantial systemic absorption. It is pertinent to note that the EE₂ exhibits a tendency for accumulation in sediments, which constitute a primary habitat and foraging ground (including sediment‐dwelling organisms as chironomids) for tench [6]. EE₂ undergoes extensive enterohepatic recirculation [7] and accumulation in different fish organs [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. Its elimination phase half‐life has been reported to be 13–27 h [49]. Considering these factors and with the aim of minimizing manipulation and reducing animal stress, a once‐weekly injection frequency for EE₂ administration was considered appropriate.

2.3. Sampling

Thirty days after the beginning of exposure, the tench were anesthetized by immersion in an aqueous solution of tricaine methanesulfonate (MS222), at a concentration of 100 mg/L. Immediately after anesthesia, blood samples were collected directly from the heart with a heparinized syringe (1 mL) and refrigerated until hematocrit determination. Finally, fish were sacrificed by decapitation, and necropsy was performed in accordance with the procedure described by Ferguson [50] to obtain liver and spleen samples that were frozen and stored at −80°C until further analysis. The body weight (BW) (g), total length (L) (cm), and liver and spleen weights were taken to estimate the condition factor (CF: wet body weight (g)/[length (cm)]³ * 100), the hepato‐somatic index (HSI: liver w.t. (g)/body w.t. (g) * 100), and the spleen‐somatic index (SSI: spleen w.t. (g)/body w.t. (g) * 100). Blood was centrifuged at 12,000 × g for 5 min in microcapillary tubes for hematocrit determination. Sampling was conducted during the morning hours, and all tench within the different experimental groups were sampled for biochemical and histological analyses.

2.4. Biochemistry

The livers were divided into two portions; one portion intended for use in the biochemistry analysis and the other for the histology study. This organ is characterized by its high oxidative potential, resulting in a higher activity of the antioxidant defense system than in other organs [51]. Therefore, it is an appropriate organ for assessing oxidative stress and antioxidant defense.

The liver was homogenized (1:10, w/v) in potassium phosphate buffer (pH 7.4, 0.1 M) on ice using an HG‐15D Witeg—German homogenizer. A volume of this homogenate was reserved to determine LPO, and the remaining homogenate was centrifuged for 20 min at 12,000 × g and 4°C using a 5424/5424R Eppendorf centrifuge to obtain the supernatants that were used for enzymatic activities and protein concentration measurements.

LPO was determined by measuring the thiobarbituric acid reactive substances (TBARS) in homogenates at 535 nm, as described by Ohkawa et al. [52] and Bird and Draper [53]. LPO was expressed as nmol TBARS/mg protein. Total glutathione (TG) content (reduced + oxidized glutathione) was determined in supernatants at 412 nm using a recycling reaction of reduced glutathione with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) in the presence of excess glutathione reductase [54]. The TG level was expressed as mol/mg protein. The activity of antioxidant enzymes was determined in the supernatants. Catalase (CAT) activity was determined by the decomposition of the substrate H₂O₂ at 240 nm [55]. Glutathione peroxidase (GPx) activity was determined by measuring the decrease in NADPH at 340 nm, using H₂O₂ as a substrate [56]. Glutathione reductase (GR) activity was determined according to the method of Smith et al. [57], adapted to microplate by Cribb et al. [58], which is based on the increase in absorbance at 412 nm when 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB) is reduced by reduced glutathione (GSH). Glutathione‐S‐transferase (GST) activity was determined following the conjugation of GSH with 1‐chloro‐2,4‐dinitrobenzene (CDNB) at 340 nm as described by Habig et al. [59]. Enzymatic activity was expressed as μmol/min mg protein. Protein concentration, needed for the normalization of the measured parameters, was determined at 590 nm according to the Bradford method [60] and with bovine γ‐globulin as standard. Measurements for each assay were taken in triplicate, and blank samples were used.

Measurements of GPx, GR, GST activities, TBARS, and protein concentrations were optimized for microtiter plates [61] and performed using a microplate reader BioTek Power‐wave 340 (BioTek Instruments Inc., Vermont, USA). CAT activity was determined using a SHIMADZU UV‐1800 spectrophotometer (SHIMADZU USA Manufacturing Inc.). All measurements were performed at 25°C.

2.5. Histopathological Damage Evaluation

Tissues (liver and spleen) were immediately fixed in Bouin solution (24 h, at room temperature), washed with running tap water, processed, and dehydrated in graded alcohol solutions using an automatic tissue processor (Leica RM 2255, Leica Microsystem, Spain), and finally embedded in paraffin. Each block was sectioned into 5 μm thick serial sections using a microtome (Leica RM 2255, Leica Microsystem, Spain). Sections were stained with hematoxylin–eosin (H‐E) and Prussian blue dye that stains hemosiderin (Table 1).

TABLE 1.

Staining protocols for tench ( Tinca tinca ) tissues.

Hematoxylin‐Eosin stain
Deparaffinize and hydrate	Previous
Hematoxylin	10 min
Destilled water	Washed
Eosine	4 min
Destilled water	Washed
Dehydrate and mount	—

Prussian blue stain
Deparaffinize and hydrate	Previous
Potassium ferrocyanide (2%) and hydrochloric acid (2%) (1:1)	20 min
Alcohol 70%	Until loss of color
Distilled water	5 min
Hematoxylin	10 min
Distilled water	5 min
Dehydrate and mount	—

Qualitative and quantitative analysis of the tissues were performed using a photomicroscope (Eclipse 80, Nikon, Tokyo, Japan), and an image analyzer (Nis‐elements, Nikon, Tokyo, Japan). Observation of pathological changes, measurements, and recounts were performed in all individuals per group using 10 non‐overlapping, randomly chosen images of each organ and animal. To prevent interobserver variability, a blind study was conducted. Prior to the study, two of the authors underwent training. Subsequently, both independently evaluated all images (10 per animal) from all animals and from each organ. After this independent assessment, the data were pooled, revealing a similarity of results exceeding 95%. In cases in which a high concordance was not observed, re‐evaluation would have been performed. Eosinophilic material refers to protein (liver and spleen), and proliferation of reticuloendothelial cells (spleen) were determined by measuring the area occupied in each image at 200× magnification (area/field: 250000 μm²). For liver degeneration, cells showing signs of degeneration were counted in each image at 400× magnification (area/field: 62500 μm²).

The pathological condition of each organ was characterized according to a standardized semi‐quantitative histopathological method proposed by Bernet et al. [62], with slight modifications (adapted for the spleen). Two indices were estimated: the organ index (Iorg), which represents the degree of damage to an organ, and the total index (Tot‐I), that measures the overall health status based on the histological lesions. Iorg was calculated based on the lesion type and was grouped into reaction patterns (circulatory, regressive, progressive, inflammatory, and tumor). Thus, the level of damage to identified lesions was established based on an importance factor (w), corresponding to minimal (1), moderate (2), and severe (3) pathological importance. The extent of the pathological alteration was represented by a score value (a) namely: (0) unchanged, (2) mild, (4) moderate, and (6) severe occurrence. Finally, the Iorg was estimated by the sum of the multiplied importance factors and score values of all alterations found in each analyzed organ, and the Tot‐I was calculated by adding up all organ indices of an individual fish.

2.6. Statistical Analysis

Data for the biochemical and histological assays were checked for normality and variance homogeneity by the Shapiro–Wilk and Levene's tests, respectively. As the ANOVA assumptions were not met, data were analyzed non‐parametrically using the Kruskal–Wallis ANOVA by rank test. When significant differences were found (p ≤ 0.05), a post hoc Dwass‐Steel‐Critchlow‐Fligner pairwise comparison test was applied to compare groups of treated fish with the control group, with a p value of 0.05 as the minimum. As no significant differences were observed between the control and solvent control groups for any of the studied parameters, all statistical comparisons were performed against the total control group formed by the addition of control fish and solvent‐exposed fish. The relationship between qualitative variables (severity of lesions and level of EE₂ exposure) was established using the exact test of Fisher. A correlation study was performed to investigate the relationship between the organic indices, hematocrit, tissue alterations, and biochemical parameters of oxidative stress using Spearman's correlation test. All statistical analyses were performed with the Statistic software Jamovi (Version 2.2.5 [63]) and R Core Team (Version 4.4.2 [64]).

3. Results and Discussion

3.1. Fish Health Indicators

Fish from all groups (control and exposed) showed normal behavior (swimming or respiratory behavior and eating or social habits), and there was no mortality during the test.

Assessment of general health status and potential toxicological effects of EE₂ exposure was conducted through the evaluation of condition factor, organosomatic indices, and hematological parameters.

No significant differences between exposed and control groups were observed when the CF was estimated. This endpoint has been utilized to indicate the overall condition or the growth status of fish [65]. Previous research has produced results comparable to those reported in this study. The CF remained unaffected in flounder ( Solea senegalensis ) following i.p. injection of 1 mg/kg t.w for 48 h (Sole et al. 2014) and sheephead minnows ( Cyprinodon variegatus ) exposed to 0.2–3200 ng EE₂/L for 59 days [39]. Likewise, the CF of juvenile fathead minnows ( Pimephales promelas ) remained unchanged after exposure to 2.5 or 20 ng EE2/L for a duration of 21 days [66]. According to Parrott and Blunt [67], the CF of the same species exposed to EE₂ as high as 32 ng/L was not significantly different from that of the control fish. CF values for all tench groups were close to those reported by Adineh et al. [68] for common carp (range of 1.33–1.86).

Analysis of the organosomatic indices revealed that HSI and SSI of tench exposed to the highest dose (500 μg/kg b.w.) were significantly greater than those of the control group (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.05 and p < 0.01, respectively) (Table 2). However, the estimated HSI values for all tench groups were within the order of magnitude as that reported by Sharma and Ram [69] for common carp that is considered within the physiological range. Similarly, the estimated SSI values of tench are close to the normal range of SSI reported by He et al. [70] for Nile tilapia ( Oreochromis niloticus ).

TABLE 2.

Condition factor (CF), Hepato‐ (HSI) and spleen‐somatic (SSI) indices, hematocrit, and morphometric analysis of the liver and spleen of tench ( Tinca tinca ) after 4 weeks of exposure to EE₂.

Group (μg/kg b.w.)	CF a	HSI b	SSI c	Hematocrit (%)	Eosinophilic material in the liver (μm2/mm2)	Vacuolation in the liver (no. cells/mm2)	Eosinophilic material in the spleen (μm2/mm2)	Hyperplasia of reticuloendothelial cells in the spleen (no. cells/mm2)
Control	2.15 (2.12–2.37)	0.87 (0.81–0.94)	0.10 (0.08–0.12)	38.90 (34.55–42.56)	0.00 (0.00–0.00)	247.00 (184.00–280.00)	1147.44 (838.15–2009.11)	820.72 (646.27–1272.06)
50	2.32 (2.15–2.40)	0.86 (0.73–0.98)	0.11 (0.09–0.15)	29.73 (27.45–33.67)**	0.00 (0.00–0.00)	146.00 (104.75–172.88)*	3579.32 (2361.25–6485.36)*	15616.07 (12986.73–23562.66)***
100	2.21 (2.11–2.25)	0.92 (0.76–0.99)	0.12 (0.11–0.14)	28.65 (25.00–33.73)**	0.00 (0.00–0.00)	173.50 (159.50–224.00)	3564.56 (1530.66–4924.45)	10160.51 (6581.77–14754.50)***
500	2.23 (2.07–2.40)	1.10 (0.96–1.19)*	0.15 (0.13–0.19)**	31.10 (28.95–32.20)**	387.85 (166.14–537.60)***	173.75 (128.88–238.38)	4154.88 (2841.15–9213.51)*	14855.21 (11804.27–18373.42)***

Note: Data are expressed as median and 25th–75th percentile. Asterisk indicates significant differences from the control (Dwass‐Steel‐Critchlow‐Fligner pairwise comparisons: *p < 0.05, **p < 0.01, ***p < 0.001).

^a Condition factor (wet body weight (g)/[length (cm)]³ × 100).

^b Hepato‐somatic index (wet liver weight (g)/wet body weight (g) × 100).

^c Spleen‐somatic index (wet spleen weight (g)/wet body weight (g) × 100).

The HSI is commonly employed as an indicator of hepatic energy reserves. When energy demand increases, the mobilization of its reserves is augmented, and the value of this parameter declines [71]. In contrast, the HSI did not decrease in the tench, suggesting that their energetic status remained suitable. The increase in HSI at the highest tested dose could be explained as a result of hypertrophy and hyperplasia of hepatocytes leading to the metabolism of EE₂ or the production of proteins related to reproduction, such as vitellogenin (VTG). A relationship between an increase in HSI and rough endoplasmic reticulum proliferation was found by Madureira et al. [72].

Biswas et al. [73] induced stress by adding cortisol to the feed of Nile tilapia ( Oreochromis niloticus ). These authors observed a significantly lower SSI in fish exposed to cortisol compared to that estimated in control fish, suggesting lower immunity in them. They concluded that stressors in fish can affect spleen health, given that this parameter indicates the well‐being of this organ. SSI values in EE₂‐exposed groups were not lower than those observed in control groups; therefore, our study suggests that tested doses of EE₂ did not induce stress in tench or alter their immunitary system. The augmentation in the SSI in the tench exposed to 500 μg EE₂/kg b.w. could be related to the proliferation of reticuloendothelial cells in the spleen of the treated fish, as described in Section 3.3.2.

According to the available literature, these morphometric indices (HSI and SSI) are affected by exposure to sex steroid compounds. Considering the same EE₂ route of administration, but a minor duration of exposure (15 vs. 30 days) and higher exposure doses (50 and 100 times higher than 500 μg EE₂/kg b.w.) regarding our study, Verslycke et al. [74] reported an increase of HSI in rainbow trout ( Oncorhynchus mykiss ). A similar result was previously obtained in the same species 9 days after an only injection of 5000 μg EE₂/kg b.w. (10 times higher than the highest tested dose) [75]. In feeding experiments, flounder ( Platichthys flesus ) fed with a food paste containing 500 ng EE₂/kg b.w., for 13 days, exhibited an increase in HSI [14]. Colli‐Dula et al. [76] also observed the same effect in female largemouth bass ( Micropterus salmoides ) fed with 0.2 ng EE₂/g food for 60 days. The findings in this index are in line with those of Rodenas et al. [77], who observed an increase in the HSI and SSI in male gilthead seabream ( Sparus aurata ) exposed to 0.005 ng EE₂/g food for 110 days. Similarly, Palace et al. [78] reported a statistically significant augmentation of HSI in lake sturgeons ( Acipenser fulvescens ) treated with 20, 100, and 200 ng EE₂/L for 25 days. Consistent with these findings, a significant elevation in HSI at the highest concentration was detected in males of rare minnow ( Gobiocypris rarus ) exposed to 1, 5, and 25 ng EE₂/L [38]. A subchronic test with EE₂ in yellow catfish ( Pelteobagrus fulvidraco ) showed that the HSI was higher than that reported in the control group at 0.1 and 1 ng EE₂/L [79], and similarly Zhou et al. [18] reported a significant increase in HSI in crucian carp ( Carassius auratus ) after exposure to 17.1 μg EE₂/L for 27 days.

On the other hand, the hematocrit from the EE₂‐exposed tench was significantly decreased at all tested doses in comparison with the control group (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.01) (Table 2), indicating that the EE₂‐treated fish had anemia. Anemia in fish can impact their performance and survival by compromising their ability to transport oxygen to the tissues, which results in impaired activity, decreased growth and reproduction, respiratory distress, increased vulnerability to predation, diseases, and toxic chemicals. Therefore, the general health status of the EE₂‐exposed tench could be disturbed. This finding is in good agreement with those observed by Schwaiger et al. [33] in carps injected three times over a 70‐day period with 500 μg EE₂/kg b.w. and Rehberger et al. [80] in rainbow trout exposed to 1.5 and 5.5 EE₂ ng/L. Landshman and Bleiberg [81] argued that EE₂ causes inhibition of hematopoietic processes.

3.2. Biochemistry

The liver is a multifunctional organ that plays an important role in the physiology of fish. It is involved in anabolic, catabolic, and storage processes, as well as in the immune function [21].

Organisms have developed defense mechanisms to neutralize the effects of oxyradicals generated under physiological or toxic conditions. The antioxidant defense system includes enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), as well as endogenous substrates such as the reduced glutathione (GSH). When the balance between the antioxidant capability and the pro‐oxidative activity is disturbed in favor of the latter, an oxidative stress process is initiated in the cells; LPO is a consequence of the vulnerability of biological membranes to ROS. In this context, biochemical responses to oxidative stress processes are earlier alarms that warn of potential physiological and tissue alterations in individuals.

According to Tollefsen et al. [24], EE₂ is linked to the hepatic estrogen receptor from Atlantic salmon ( Salmo salar ) and rainbow trout. As a result, the physiology of this organ may be altered. In addition, the liver in conjunction with the kidney and gills is the organ most susceptible to free radicals attack [82]. Thus, the redox status of fish is influenced by exposure to EE₂. This observation is supported by evidence such as the increased mRNA levels of the antioxidant enzymes, including CAT, SOD, and GST [76, 83], or conversely, the decreased mRNA levels of GPx [76].

CAT is associated with the peroxisomes of cells. Its main function is to detoxify the H₂O₂. In this study, CAT activity increased in the groups exposed to 50 and 500 μg EE₂/kg b.w., and decreased in tench exposed to 100 μg EE₂/kg b.w. (Figure 1A). Although not statistically significant, CAT activity tended to increase at the lowest and highest tested doses (50 and 500 μg EE₂/kg b.w., respectively) in comparison to the control group, resulting in the maintenance of a normal H₂O₂ level in hepatocytes, reflecting a compensatory mechanism against the suspected production of superoxide radicals and H₂O₂ by EE₂.

FIGURE 1.

Biochemical biomarkers of oxidative stress in the liver of tench ( Tinca tinca ) after 4 weeks of exposure to EE₂. (Dose I: 50 μg/kg b.w.; Dose II: 100 μg/kg b.w., and Dose III: 500 μg/kg b.w.). Enzymatic activities (μmol/min mg protein): Catalase (CAT) (A), glutathione peroxidase (GPx) (B), glutathione reductase (GR) (C), and glutathione S‐transferase (GST) (E); Levels: Total glutathione (GT; mol/mg protein) (1D), and lipid peroxidation (LPO; nmol TBARS/mg protein) (F). Horizontal line in box plots is the median, the boundaries of the box represent the 25th and 75th percentiles and the minimum and maximum data points are represented by the whiskers. Asterisks indicate significant differences from the control (Dwass‐Steel‐Critchlow‐Fligner pairwise comparisons: *p < 0.05, **p < 0.01, ***p < 0.001).

The effects of EE₂ or analogue compounds on CAT activity of fish have already been shown by previous studies. Ramírez‐Montero et al. [84] assessed the acute effect of EE₂ (36–106 ng/L) on the oxidative state of zebrafish ( Danio rerio ) embryos. They showed that EE₂ significantly increased CAT activity in the liver at all tested concentrations. The same response was observed in the brain of adult zebrafish after exposure to EE₂ (0.05, 0.5, 5, 50, and 75 ng/L) for 15 days [17]. These authors, evaluating the dietary transference of EE₂ to zebrafish from Artemia, also found this finding in the gills, gut, and brain of fish fed 500 ng EE₂/L [16]. Regarding E₂, Japanese sea bass underwent an increase in hepatic CAT activity after 30 days of exposure to 2000 ng E₂/L [85]. Khorshidi et al. [86] documented an increased activity of CAT, but not statistically significantly regarding the control group, in hepatocytes of Siberian sturgeon ( Acipenser baerii ) intraperitoneally injected with 5000 μg E₂/kg b.w. In contrast, EE₂ decreased liver CAT activity, although it did not reach statistical significance, in carp 8 days after intraperitoneal injection with 500 mg EE₂/kg b.w. [47]. The activity of this enzyme was significantly lower than the control in flounder intraperitoneally injected with 1 mg EE₂/kg b.w. [87]. In the same line, a slight reduction in CAT activity was noted in yellow catfish exposed to 0.1 and 1 ng EE₂/L [79]. A similar result, but in this case in serum, was observed by Mo et al. [88], who reported significant inhibition of CAT activity in yellow catfish ( Pelteobagrus fulvidraco ) exposed to 1000 ng EE₂/L for 56 days.

GPx is a cytosolic enzyme that reduces H₂O₂ and lipid peroxides. Its activity was slightly inhibited in EE₂‐treated animals, although this decrease did not reach significance compared to that in the control (Figure 1B). The minimal changes observed in this enzymatic activity could imply that low H₂O₂ or lipid peroxide levels were generated under our experimental conditions of exposure to EE₂. Our result supports previous studies that also found no significant decrease in liver GPx activity in carp exposed to 500 mg EE₂/kg b.w. [47] and Japanese sea bass after exposure to 200 and 2000 ng E₂/L [85]. Gen related to GPx was depressed in largemouth fed with 70 ng EE₂/g food [76]. In contrast, other studies have shown a significant increase in GPx activity in zebrafish embryos treated with EE₂ [84] or in adult zebrafish fed with 1000 μg EE₂/kg for 6 weeks [89]. The same effect was observed by Ait‐aissa et al. [90] in rainbow trout injected with 0.5 mg E₂/kg b.w. after 21 days. GPx expression was induced by E₂ (10⁻⁷ M) for 3 weeks in zebrafish [91]. The activity of this enzyme exhibited a light but not significant increase in Siberian sturgeons treated with E₂ [86].

GR catalyzes the reduction of glutathione disulfide (GSSG) to the sulfhydryl form of glutathione (GSH) at the cost of NADPH, and it is also involved in detoxification processes mediated by GSH. As can be seen from Figure 1C, GR activity decreased in fish exposed to 100 μg EE₂/kg b.w., with a statistically significant value (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.01); instead, slight but no significant increases of this activity were found at 50 and 500 μg EE₂/kg b.w. when compared with the control group. As it will be indicated later, TG levels were similar (at 100 μg EE₂/kg b.w.) or elevated (at 50 and 500 μg EE₂/kg b.w.) in comparison to the control fish (Figure 1D). This condition might suggest that the oxidative use of GSH as a scavenger of ROS did not take place in an important way; consequently, the GSSG levels were low, and it was not necessary to induce a significant induction of GR activity to convert the oxidized to reduced form of this substrate. Studies previously conducted with EE₂ in fish provided evidence that liver GR was significantly inhibited in comparison with the control group [87, 89], suggesting that GSH could not be regenerated to restore its physiological level. However, Ait‐aissa et al. [90] found no impairment in this enzymatic activity in rainbow trout treated with E₂.

TG plays an important role in the regulation of the cell redox state by acting as a scavenger of ROS and as a co‐substrate for GPx, and is also involved in detoxification processes catalyzed by GSTs. In the present study, GT levels markedly increased at lower doses (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.001), while a slight drop and rise were observed at medium and high doses of EE₂, respectively (Figure 1D). As stated by Zhang et al. [92], tissue GSH levels can be elevated during a moderate oxidative stress as an adaptive mechanism through increased biosynthesis. In this respect, Zhou et al. [18] revealed an increase in the glutamate and glycine amino acids, constituents of glutathione, in the kidney of crucian carp after EE₂ exposure. An in vitro assay based on the exposure of rat hepatocytes to 1 mM EE₂ was unable to deplete GSH levels in these cells, but this defense was decreased in an analogous experiment performed with E₂ [93]. In vivo assays using E₂ also showed the same effect. Thus, Thilagam et al. [85] observed a reduction in GSH levels in Japanese sea bass exposed to 200 and 2000 ng E₂/L. This finding is in line with that obtained by Ait‐aissa et al. [90] for rainbow trout.

GST is an enzyme in phase II of detoxification processes that catalyzes the conjugation of exogenous and endogenous compounds, including ROS, with GSH, promoting their excretion. In the current study, exposure to EE₂ led to no significant increase in GST activities in the livers of the exposed organisms (Figure 1E). This observation could indicate that the organism facilitates the excretion of EE₂ or ROS through conjugation with GSH, whose levels were raised at least in tench exposed to 50 and 500 μg EE₂/kg b.w., protecting cells against oxidative damage. Similarly to our results, no statistically significant elevation in liver GST activity was noted in carps injected with 500 mg EE₂/kg [47]. Hepatic GST mRNA expression was significantly increased in largemouth bass fed with 70 ng EE₂/g [76]. Along the same line, gill GST activity was increased in zebrafish fed with 500 ng EE₂/L [16]. In contrast to the induction of GST activity reported in the cited studies, Weiserova et al. [89] described a decrease in this enzymatic activity in the whole body of zebrafish after exposure to 1000 μg EE₂/kg. Concerning E₂, Jin et al. [31] detected that GST proteins were expressed in the liver of zebrafish exposed to E₂ at concentrations of 0.1 and 1 nM for 14 days, considering that this response could be responsible for limiting the damage caused by exposure to this estrogen. Similar results were provided by Ruggeri et al. [91] for this species exposed to 27 μg E₂/L. Teles et al. [94] reported that liver GST activity was significantly increased in gilthead seabream ( Sparus aurata ) exposed to 4000 ng E₂/L for 8 and 12 h, suggesting increased E₂ catabolism. In this context, the activity of this metabolic enzyme was augmented in Japanese sea bass exposed to 200 and 2000 ng E₂/L [85].

LPO, measured as membrane lipid byproducts (TBARS), is one of the principal mechanisms involved in structural and functional cellular damage when an oxidative process is established. A diverse trend was observed in TBARS content in EE₂‐exposed tench: it was significantly increased at a low dose (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.05), whereas medium and high doses caused a decrease (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.01 in 100 μg EE₂/kg b.w.) (Figure 1F). The behavior observed in the TBARS levels at 100 and 500 μg EE₂/kg b.w. could be explained based on an efficient antioxidant response that might have contributed to preventing the attack caused by ROS or by a reduction in the liver lipid content (vacuolization) (Tables 2 and 3), because, according to Miller et al. [95], LPO levels depend on the presence of lipid substrates. Another plausible explanation is related to the ability of estrogens to neutralize radicals mediated by their phenolic ring [96], thereby reducing oxidative aggression. Our results are in harmony with some studies that have shown a decrease in LPO levels in animals treated with EE₂. Thus, Klinger et al. [97] identified a statistically significant reduction in LPO levels in the livers of rats exposed to this hormone. More recently, Tamagno et al. [16] found the same result for gill and brain LPO levels of zebrafish exposed to 50 and 100 ng EE₂/L through feeding. Our findings contrast with other studies where induction of LPO, following an increase in the malondialdehyde (MDA) or TBARS levels, has been described in different tissues of fish exposed to estrogens: in the blood of crucian carp (10 μg EE₂/L—[11]), and yellow catfish (1 μg EE₂/L—[88]); in the gill of sea bass (0.5 mg E₂/kg b.w. i.p. injection—[48]); in the brain of zebrafish (75 ng EE₂/L—[17]); in the liver of sea bass (0.5 mg E₂/kg b.w. i.p. injection—[48]), yellow catfish (0.1 and 1 ng EE₂/L—[79]), and Siberian sturgeon (5000 μg E₂/kg b.w. i.p. injection—[86]); in the kidney of sea bass (0.5 mg E₂/kg b.w. i.p. injection—[48]) and in zebrafish embryos (36–106 ng EE₂/L; [84]). All of these authors agree that these estrogens lead to oxidative damage in tissues.

TABLE 3.

Lesions ranking by stage based on histological appearance of the liver and spleen of tench ( Tinca tinca ) after 4 weeks of exposure to EE₂.

Group (μg/kg b.w.)	Eosinophilic material in the liver		Vacuolation in the liver		Eosinophilic material in the spleen		Hyperplasia of reticuloendothelial cells in the spleen
	Individuals that showed this lesion (%)	Frequency of severity lesion a per no. of fish	Individuals that showed this lesion (%)	Frequency of severity lesion a per no. of fish	Individuals that showed this lesion (%)	Frequency of severity lesion a per no. of fish	Individuals that showed this lesion (%)	Frequency of severity lesion a per no. of fish
Control	7	0: 26 2: 2 4: 0 6: 0	100	0: 0 2: 0 4: 4 6: 24	71	0: 8 2: 16 4: 2 6: 2	43	0: 16 2: 6 4: 6 6: 0
50	14	0: 12 2: 1 4: 1 6: 0	71	0: 0 2: 8 4: 5 6: 1	100	0: 0 2: 8 4: 5 6: 1	93	0: 1 2: 6 4: 4 6: 3
100	7	0: 13 2: 1 4: 0 6: 0	64	0: 5 2: 3 4: 3 6: 3	86	0: 2 2: 7 4: 5 6: 0	100	0: 0 2: 6 4: 7 6: 1
500	43	0: 8 2: 6 4: 0 6: 0	57	0: 6 2: 5 4: 2 6: 1	100	0: 0 2: 5 4: 7 6: 2	100	0: 0 2: 1 4: 8 6: 5

Note: Scoring system of histopathological changes in the liver and spleen: 0 (unchanged), 2 (mild occurrence), 4 (moderate occurrence), and 6 (severe occurrence).

^a Frequency of severity.

Thus, there is not an established pattern of response of the antioxidant defense system to EE₂ exposure in fish. Except for GST, which generally exhibits an increase in its activity (as it was observed in our study), and LPO level that, with some exceptions as in the current work, is increased, the rest of the defenses (CAT, GPx, GR activities and GT level) can experience induction, inhibition, or remain unchanged after exposure to EE₂. The variability of responses probably depends on the doses or concentrations of the compound, route, and duration of exposure. Another factor to consider is the fish species.

3.3. Histology

The histopathological examination is an important biomarker that reflects the stress/health status of fish because of the exposure to sub‐lethal doses or concentrations of environmental contaminants. This tool allows the detection of preliminary sublethal effects that can result in physiological and cellular disturbances [62, 98].

To determine their overall health status, pathological changes in the liver and spleen of EE₂‐exposed tench were analyzed both qualitatively and quantitatively. Additionally, the extent of EE₂‐induced damage and the health status of these organs were evaluated by estimating the Iorg and the Tot‐I. As outlined in Section 2.5, a scoring system was implemented to classify the severity of lesions observed in the liver and spleen (0 [unchanged], 2 [mild occurrence], 4 [moderate occurrence], and 6 [severe occurrence]). In addition, the Iorg was calculated for each organ by summing the products of importance factors and score values of all detected alterations. The Tot‐I represented the sum of these organ indices for each fish.

3.3.1. Liver

The liver is frequently chosen as a histological reference point for evaluating tissue damage due to environmental pollutants. Lesions in this organ can alter its physiological functions.

The liver from control tench, in general, showed the normal architecture of cyprinids, but with hepatocytes less arranged in cords and lobules. The pancreas is located around the portal vein in this family of teleost fish; therefore, it is referred to as hepatopancreatic tissue [99]. Large and spherical hepatocytes contain abundant lipids in their cytoplasm (typical pattern of fatty vacuolization). This finding was noted in all control fish, showing mainly severe occurrence (Tables 2 and 3, Figure 2A).

FIGURE 2.

Histopathological lesions found in the liver and spleen of tench ( Tinca tinca ) after 4 weeks of exposure to EE₂. (A) Control tench. Liver. Vacuolar degeneration. H‐E 400×. (B) Tench exposed to 50 μg/kg p.w. Liver. Vacuolar degeneration. Eosinophilic material in low quantity (stars). H‐E 400×. (C) Tench exposed to 500 μg/kg p.w. Liver. Vacuolar degeneration. Eosinophilic material in moderate quantity (stars). H‐E 200×. (D) Control tench. Spleen. H‐E 200×. (E) Tench exposed to 500 μg/kg p.w. Spleen. Proliferation of reticuloendothelial cells (asterisk). H‐E 200×. (F) Tench exposed to 500 μg/kg p.w. Spleen. Eosinophilic material (<). H‐E 200×.

In general, the principal histopathological changes in the hepatic tissue observed in EE₂‐exposed fish were the decrease in the number of cells with cytoplasmic vacuoles and the presence of eosinophilic material. Thus, in the liver of tench exposed to 50 μg EE₂/kg b.w., a significant decrease in the severity of the hepatocytic vacuolation was observed (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.05), predominating a mild and moderate occurrence over the severe occurrence detected in the control group (Figure 2B). In this treated group, the accumulation of eosinophilic material was irrelevant (Tables 2 and 3, Figure 2B). Fish exposed to 100 and 500 μg EE₂/kg b.w. also showed a decrease, but not statistically significant, in the hepatocytic vacuolation in relation to the control group. The occurrence range was from mild to severe in individuals exposed to 100 μg EE₂/kg b.w., highlighting mild occurrence in tench injected with 500 μg EE₂/kg b.w. The presence of eosinophilic material within melano‐macrophage centres (MMCs) was a common and significant finding in fish exposed to the highest doses (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.001). Nevertheless, this lesion showed mild occurrence in the affected tench (Tables 2 and 3, Figure 2C). Fisher's exact test revealed that the presence of eosinophilic material was associated with the highest dose of exposure (p < 0.05). In addition, the degree of lipid vacuolization was inversely proportional to doses (p < 0.01). The two lesions, present in all groups although with different magnitudes, were of regressive type (vacuolar degeneration, and deposits of eosinophilic material). These findings had a low importance factor (w = 1), which is a low level of damage, due to the easy reversibility. Therefore, it is possible that the normal structure of the organ could be restored if the exposure conditions cease. Liver Iorg exhibited a decrease in tench exposed to EE₂. In fact, statistically significant differences were observed in groups exposed to medium and high doses compared to the control group (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.01) (Table 4). Therefore, the overall degree of liver damage was reduced in the exposed tench. From this index, we can establish that the histopathological alterations found in the liver of the exposed fish did not disturb its functionality.

TABLE 4.

Median (25th–75th percentile) lesion indices of the liver and spleen of tench ( Tinca tinca ) after 4 weeks of exposure to EE₂.

Group (μg/kg b.w.)	Iorg a		Tot − I b
	Liver	Spleen
Control	6.00 (5.20–6.00)	4.08 (2.00–7.04)	10.08 (7.68–13.04)
50	3.40 (1.70–5.50)	9.04 (7.40–13.20)*	12.08 (9.30–18.16)
100	3.20 (0.80–5.20)**	8.64 (6.96–10.08)	11.44 (9.68–13.60)
500	1.80 (1.60–3.00)**	13.20 (11.26–15.18)***	16.08 (13.74–17.12)*

Note: Asterisks indicate significant differences from the control (Dwass‐Steel‐Critchlow‐Fligner pairwise comparisons: *p < 0.05, **p < 0.01, ***p < 0.001).

^a Organ index (Iorg) by organ and group.

^b Total index (Tot − I) by group.

The most important alteration observed in all groups of fish, both control and EE₂‐exposed, was hepatocyte vacuolation. In fact, this finding was more evident in the control than in the treated fish. Although this result is consistent with studies by Zha et al. [38] and Zha et al. [37], who revealed lipid‐like vacuolization in the hepatocytes of rare minnows ( Gobiocypris rarus ), it contrasts with the vast majority of chronic studies where exposure to EE₂ resulted in an increase in the liver lipogenesis of sheepshead minnow [39], rainbow darters ( Etheostoma caeruleum ) [28], Cnesterodon decemmaculatus [36] and least killifish ( Heterandria formosa ) [30]. In the same way, elevated lipid vacuolization of hepatic cells has been described in zebrafish simultaneously exposed to EE₂ and dibutyl phthalate (plasticizer) [27]. A possible reason for the reduction in hepatic lipid stores in the EE₂‐exposed tench was provided by Zha et al. [37], who attributed this fact to energetic changes associated with xenobiotic metabolism or increased energy requirements for VTG synthesis or metabolic degradation. It is important to note that VTG is a precursor protein of egg yolk in females of oviparous species and can be abnormally expressed and exported to other organs in males exposed to EDCs, such as EE₂ [100]. Moreover, Yepuru et al. [101] reported that E₂ regulates gene expression related to lipid synthesis or catabolism in mice, leading to a depletion of lipid reserves.

Another remarkable finding in 500 μg EE₂/kg b.w. exposed tench was the accumulation of eosinophilic material included in macrophages or as inclusions in hepatocytes, which was also observed in rare minnows exposed to EE₂ [37, 38]. Weber et al. [35] previously described an important accumulation of this material in the abdominal cavity (around liver, kidney, or intestine) of zebrafish exposed to 10 ng EE₂/L. Although this lesion may be related to phagocytosis of ROS [102], we consider it to be closely linked to storage/detoxification of endogenous and exogenous substances, as suggested by Agius and Roberts [103] and Sayed and Younes [104]. Thus, this material could come from hemorrhagic processes, where hemosiderin deposits are present, or from VTG synthesis. Hemosiderin is a material derived from the catabolism of hemoglobin, from lysis of erythrocytes. To investigate this origin, the special histologic stain of prussian blue was applied to stain hemosiderin positively. The most part of this material resulted in negative with the mentioned staining. Therefore, it is suggested that this material may be VTG. This hypothesis is supported by Palace et al. [78] and Zha et al. [38], who described liver induction and accumulation of VTG, associated with eosinophilic material, in lake sturgeons and males of rare minnows exposed to concentrations between 20 and 200 ng EE₂/L. The same finding was found in a multigeneration assay performed in male rare minnows exposed to EE₂ concentrations of 0.2, 1, 4, 16, and 64 ng EE₂/L [37]. From the results obtained in these studies, the authors attributed the increase in HSI of exposed fish to the generation of VTG. More recently, Real et al. [105] described, by using an immunohistochemistry method, the presence of VTG in the cytoplasm of hepatocytes from male platyfish ( Xiphophorus maculatus ) intramuscularly exposed to 25 μg EE₂/g for 14 days. Alterations in the expression of some genes involved in reproduction were detected in fish exposed to EE₂. Thus, an increase of VTG expression in the liver of male zebrafish exposed to 50 ng EE₂/L for 7 days was reported by Zhong et al. [106]. Similarly, Rodenas et al. [77] observed increased VTG mRNA levels in the liver of adult gilthead seabream males after prolonged exposure through diet to 5 μg EE₂/g.

Severe histopathological alterations were not observed in the liver of tench under the experimental conditions used in the current study. Therefore, its physiological function of protein synthesis, including enzymes belonging to the antioxidant defense system, was presumably not affected by exposure to EE₂.

3.3.2. Spleen

The spleen in conjunction with the liver and kidney is a major immune organ in fish. The spleen is involved in hematopoiesis, macromolecule clearance, antibody formation, and B‐cell differentiation [22]. A critical determinant of an organism's fitness is its immunocompetence, which facilitates survival, growth, and reproduction, and further serves to mitigate the fitness cost imposed by infections. Several authors have observed that EE₂ is capable of modulating the fish immune system [23, 80, 107, 108, 109, 110, 111].

Examination of splenic tissue sections from control fish showed no evident lesions in this organ, which had the histological structural component characteristics of teleost fishes. Thus, the spleen consisted of splenic pulp (red and white), blood vessels, capillary vessels (named ellipsoid), and MMCs [50]. In teleost fish, the red and white pulp appear in a diffuse way. The red pulp contains reticuloendothelial cells, erythrocytes, and thrombocytes, and the white pulp is constituted mainly by lymphoid cells and macrophages [99] (Figure 2D).

The main finding observed in EE₂ exposed tench was the significant hypertrophy and proliferation of reticuloendothelial cells at all doses tested compared to those found in the control group (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.001). This lesion showed mild (at 50 μg EE₂/kg b.w.) and moderate (at 100, and 500 μg EE₂/kg b.w.) occurrence. According to the results of the Fisher's exact test, the presence of eosinophilic material was related to the highest dose of exposure (p < 0.05), as it was observed in the liver. An important dose‐dependent relationship was found between the hypertrophy of reticuloendothelial cells and the level of exposure to EE₂ (p < 0.001). The presence of eosinophilic material within MMCs was identified in control and EE₂ exposed fish. The degree of severity of this alteration was increased in exposed individuals (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.05 at 50 and 500 μg EE₂/kg b.w.), expressed in mild (at 50 and 100 μg EE₂/k b.w.), and moderate occurrence (at 500 μg EE₂/kg b.w.) (Tables 2 and 3, Figure 2E,F). Exposure to EE₂ induced regressive (deposits of eosinophilic material) and progressive (hyperplasia of reticuloendothelial cells) changes in the spleen of tench. Concerning the first lesion, the level of damage observed was minimal (w = 1) with the possibility of reversibility, whereas the pathological importance of the second lesion, which implies an increase in the activity of reticuloendothelial cells, was moderate (w = 2).

As can be seen in Table 3, the spleen Iorg revealed an increase in tench exposed to EE₂ when compared with control tench (Dwass‐Steel‐Critchlow‐Fligner test, p < 0.05, and p < 0.001 at 50 and 500 μg EE₂/kg b.w., respectively). Thus, the overall degree of the spleen damage was elevated in exposed tench. According to Zachary and McGavin [112], the current study considered this lesion and its expression in Iorg as evidence of a substantial response to EE₂. Based on a comparison between Iorg of the studied organs, EE₂ had a more severe toxic effect on the spleen structure than on the liver of tench, although this was moderated. Despite lesions found in both organs, the overall health status, represented by Tot—I, only appeared to be significantly affected at the highest dose tested.

The hypertrophy and proliferation of reticuloendothelial cells observed in the current study confirm the previously cited histopathological investigation by Schwaiger et al. [33], who highlighted this finding as the main morphological alteration of the spleen of carps exposed to EE₂. On the contrary, Shved et al. [34] reported a decrease in the number and size of the splenic MMCs in tilapia ( Oreochromis niloticus ) after exposure to 125 μg EE₂/g food over 40 days. MMCs in teleost fish are like lymph nodes in mammals [113].

The reticuloendothelial system, also known as the monocyte–macrophage system, is involved in an animal's various defense mechanisms and includes multiple functions such as phagocytosis and myeloproliferative control [114], making its involvement in the immune response notorious. In this context, there are several authors that have reported interactions between the exposure to EE₂ and alterations in the immune system of fish such as tilapia [115], fathead minnows [34, 116], gilthead seabream [107], (Cabas et al. 2018) and stickleback ( Gasterosteus aculeatus ) [117]. This could be explained by the immunomodulatory role of this compound, whose receptors can also be found in immune tissues such as the spleen [34, 118, 119]. Estrogens are known to modulate the immune system in fish through nuclear and membrane estrogen receptors, which are present in lymphoid tissues, as well as in head kidney monocytes/macrophages, neutrophils, and lymphocytes (Cabas et al. 2018); indeed, they modulate leukocyte proliferation [80]. Since immunity parameters were not assessed in this study, the authors cannot definitively confirm any physiological modifications. Therefore, it can be speculated that the observed changes in this system may be due to an adaptive immune response from the individual. However, further studies could be conducted in subsequent research to evidence this detail.

According to Lochmiller and Deerenberg [120], there is a direct connection between immunocompetence and survival, growth, and fecundity. It is in this sense that fish under EE₂ exposure may be more susceptible to infectious diseases, as indicated by Milla et al. [23] in their revision work about the effects of estrogenic endocrine disruptors on the immune function in fish. This system plays an important role in regulating the reproductive cycle in fish. Bearing in mind that, it is interesting to point out a previous work from the same research project from which the current study is derived, where alterations in the sperm quality and histology of testes in the EE₂‐exposed tench were revealed [42, 43].

Abundant dispersed eosinophilic material or within MMCs was found in control and treated groups. In the same way as indicated in the hepatic tissue, this pigment could be hemosiderin or VTG. Hemosiderin is relatively common in MMCs of the spleen in healthy fish [103]. Nevertheless, these pigments can experience an important accumulation within MMCs when individuals are suffering hemolytic anemia, resulting in a pathological process named hemosiderosis [121, 122]. Considering the hematocrit values in EE₂‐exposed tench (Table 2), it appears that all treated fish suffered hemolytic anemia as reported by Landshman and Bleiberg [81]. This finding suggested a priori that some of the pigments could correspond with hemosiderin. Therefore, the prussian blue staining was applied to the spleen, resulting in a lack of positivity for this tinctorial method. Thus, it was concluded that most of the pigments correspond to VTG. In fact, high VTG mRNA levels have been detected in the spleen of zebrafish in response to exposure to EE₂, suggesting that the spleen, as well as the liver, can synthesize VTG [106]. Reticuloendothelial cells not only can synthesize but also capture and store VTG [123]. In addition, the increase in MMCs in the spleen of EE₂‐exposed fish could respond to the induction of this cellular defense system to eliminate discarded material (hemosiderin or VTG) by increased phagocytic activity, as indicated by Agius and Roberts [103].

Inflammation processes can generate ROS, which are responsible for oxidative damage. Recently, Singh et al. [124] documented correlations between inflammation and oxidative stress in Channa punctata exposed to 0.039 and 0.078 mg HgCl₂ /L over 45 days. In our study, inflammatory lesions in the liver and spleen were not described; therefore, this source of ROS can be excluded, which supports the overall irrelevant alterations in the antioxidant defense system and lipid peroxidation of tench treated with EE₂.

In short, the primary histological lesions observed in the liver and spleen of tench exposed to EE₂ exhibited an inverse and direct dose–response relationship, respectively. The spleen of tench showed greater lesion severity compared to the liver. This substance acts as an estrogen receptor agonist, binding to estrogen receptors located in the liver and spleen of fish, thereby leading to VTG induction. This phenomenon is typically dose‐dependent, meaning higher EE₂ doses drive greater VTG production, consistent with the eosinophilic material observed in the current study being indicative of VTG. Histological lesions in the liver and spleen of fish are relevant to ecological risk assessment because these organs are critical for detoxification, immunity, and overall physiological function. Changes in their microscopic structure can serve as biomarkers of exposure and effect, providing crucial insights into the health of individual fish populations, and by extension, the aquatic ecosystem.

3.4. Correlations Between Biochemical Parameters of Oxidative Stress and Morphological Changes

Finally, relationships among biochemical parameters of oxidative stress and morphological changes were evaluated using the Spearman correlation coefficient (see Supporting Information). This analysis aimed to elucidate potential mechanistic links between oxidative stress responses and organ‐specific histopathological changes in tench following EE₂ exposure.

3.4.1. Control Group

In the control group, positive correlations between CAT/GPx (ρ = 0.63, p < 0.01), CAT/GR (ρ = 0.55, p < 0.05), GPx/GT (ρ = 0.54, p < 0.05), GR/GPx (ρ = 0.55, p < 0.05), GR/GST (ρ = 0.73, p < 0.05), GST/LPO (ρ = 0.70, p < 0.05), and a negative correlation between CAT/Tot‐I (ρ = −0.57, p < 0.05) were detected. The observed positive correlations among antioxidant enzymes (CAT, GPx, and GR), GT, and GST suggest a well‐coordinated and effective antioxidant and detoxification network operating under normal physiological conditions. CAT and GPx are enzymes that detoxify H₂O₂. Their positive correlation suggests they operate complementarily to manage this ROS. GR is crucial for regenerating GSH, which serves as a cofactor for both GPx and GST. The correlations observed between GR and GPx/GST reflect the interdependence of these enzymatic systems in maintaining GSH levels and, consequently, the organism's antioxidant and detoxification capacity. The correlation between GPx and GT is biologically logical, as GT (which includes GSH) is an essential substrate for GPx activity. GST is involved in detoxification, whereas LPO is a result of oxidative stress. This positive correlation could imply that, even under controlled conditions, a basal level of ROS or lipid byproducts exists. Consequently, GST activity correlates with the presence of LPO as part of a physiological response to eliminate oxidative damage products. Tot‐I serves as a measure of overall health status based on histological lesions. The observed negative correlation indicates that increased CAT activity is associated with a lower degree of histopathological damage. This finding underscores the protective role of CAT in maintaining tissue integrity and overall health.

3.4.2. 50 μg EE₂ /kg b.w. Group

The previous correlations were not observed in tench exposed to 50 μg EE₂/kg b.w., where only a positive correlation between Iorg‐liver/GT (ρ = 0.58, p < 0.05) was predominant. The effects of EE₂ already begin to manifest at the tissue level with a specific biochemical response that is likely attributable to the association between increased liver damage and elevated GT levels. This might reflect an early adaptive response by the organism to augment glutathione biosynthesis under light oxidative stress, thereby seeking to protect cells and promote detoxification.

3.4.3. 100 μg EE₂ /kg b.w. Group

Tench injected with 100 μg EE₂/kg b.w. showed a positive correlation between CAT/GPx (ρ = 0.69, p < 0.05) as control individuals. Other positive correlations among the biomarkers of oxidative stress in this group were: GPx/GST (ρ = 0.66, p < 0.05) and GR/LPO (ρ = 0.57, p < 0.05). Concerning relationships among morphological changes and parameters of oxidative stress, a negative correlation between Iorg‐liver/GR (ρ = −0.57, p < 0.05) was detected. The positive correlation between CAT and GPx suggests that the coordination between these two antioxidant enzymes is maintained at this dose. The positive correlation between GPx and GST would point to a coordinated effort in detoxification and antioxidation processes involving the glutathione system. An increase in LPO correlates with an increase in GR activity. This could reflect a compensatory response in which GR is activated to regenerate GSH and combat the occurring LPO. The observed increase in hepatic damage is correlated with a reduction in GR activity. This might indicate that the liver's ability to regenerate GSH is impaired with escalating damage, which could in turn compromise the organ's general detoxification and antioxidant defense mechanisms.

3.4.4. 500 μg EE₂ /Kg b.w. Group

New associations, which were not identified in other groups, were established between CAT/GST (ρ = 0.70, p < 0.05) and Iorg‐liver/GST (ρ = 0.79, p < 0.01) in fish exposed to 500 μg EE₂/kg b.w. The positive correlation between CAT and GST at this dose suggests a coordinated response of the antioxidant defense system at this level of exposure. This could indicate that, in the face of a higher oxidative stress load or EE₂ increased metabolites (which the high dose may imply), the tench's organism is simultaneously activating mechanisms to neutralize H₂O₂ (via CAT) and to conjugate and excrete harmful compounds or ROS (via GST). The positive correlation between Iorg‐liver and GST activity at the highest EE₂ dose suggests that glutathione conjugation might be upregulated in response to EE₂ induced hepatocellular damage.

Except in one case, where a positive correlation was observed between CAT/GPx activities in control and exposed fish at the medium dose of EE₂, there was no common pattern of correlations that could be identified in all groups. The lack of consistent correlation patterns across exposure levels may reflect dose‐dependent shifts in antioxidant defense mechanisms and organ‐specific adaptive responses to EE₂. Solé et al. [87] described positive correlations between enzymatic activities related to glutathione, specifically, GPx, GR, and GST in flounder exposed to 1 mg EE₂/kg b.w. In the current study, we also observed positive correlations, in the range reported by the cited authors, between these parameters in control and 100 μg EE₂/kg b.w. exposed fish. Therefore, in terms of the interactions between these enzymes, the results of the current study confirm patterns similar to those described by Solé et al. [87] which reinforce the importance of this system as a key and functional component of the antioxidant response to exposure to EE₂ in fish.

Although these results provide important insights into the oxidative stress response in tench following parenteral administration of EE₂, caution should be exercised when extrapolating them to environmentally relevant exposure scenarios or other exposure routes (e.g., dietary, aquatic) that may involve different metabolic and distribution patterns.

Future studies could employ advanced molecular techniques such as quantitative polymerase chain reaction (qPCR) and transcriptomics to assess the expression of genes involved in both anti‐oxidant defense mechanisms and the immune response in tench exposed to EE₂. This molecular‐level investigation, combined with the findings from the current study, would contribute to identifying specific molecular, biochemical, or histological biomarkers that reliably reflect EE₂ exposure and its adverse effects in tench. Beyond understanding the problem, future research should prioritize developing solutions. These investigations could be complemented by exploring effective mitigation strategies aimed at reducing EE₂ concentrations in aquatic environments, such as advanced ozonation processes or bioremediation approaches.

4. Conclusions

It can be concluded from this study that:

1. The hepato‐somatic and spleen‐somatic indices showed statistically significant increases at the highest tested dose (500 μg EE₂/kg b.w). The increased hepatosomatic index may be attributed to enhanced EE₂ metabolism or vitellogenin production. The increased spleen‐somatic index may be related to the hyperplasia of reticuloendothelial cells. In addition, anemia was observed in all the exposed individuals (50, 100, and 500 μg EE₂/kg b.w.). Therefore, the general health status of the EE₂‐exposed tench was disturbed.

2. Despite some statistically significant modulations in enzymatic antioxidant activity and glutathione levels, the absence of increased lipid peroxidation suggests that oxidative stress remained minimal in tench exposed to EE₂ for 30 days.

3. Intraperitoneal EE₂ exposure induced regressive changes in the liver (vacuolar degeneration, and deposits of eosinophilic material) and regressive (deposits of eosinophilic material) and progressive (hyperplasia of reticuloendothelial cells) changes in the spleen of tench. Histological lesions such as vacuolar degeneration and hyperplasia of reticuloendothelial cells in the liver and spleen, respectively, were dependent on EE₂ dose. Despite the observed cellular damage in both organs, their functions remained uncompromised.

4. A common pattern of correlations between parameters of oxidative stress and morphological changes was not identified in the current study. The current study revealed positive correlations among the glutathione‐related enzymatic activities in both the control group and at a dose of 100 μg EE₂/kg b.w. Biologically, this indicates that the tench's glutathione system maintains a coordinated and integrated antioxidant and detoxification response to EE₂, with enzymes working in concert to neutralize potential pro‐oxidative effects and facilitate its detoxification. The simultaneous detection of an adaptive response by the antioxidant defense system and the presence of some tissue alterations in the liver and spleen suggest the combined use of these biomarkers as early indicators of EE₂ exposure in aquatic environments.

Author Contributions

Ana L. Oropesa: conceptualization, resources, methodology, investigation, supervision, formal analysis, writing, and editing. Alfonso Ramos: methodology, investigation, and formal analysis. Cesar Fallola: resources and methodology. Luis J. Gomez: resources, methodology, investigation, and supervision.

Ethics Statement

All procedures complied with the European Parliament and Council Directive 2010/63/UE for the protection of animals used for scientific purposes. The toxicity test was conducted in the facility of the Aquaculture Center “Vegas del Guadiana” (Government of Extremadura, Spain). The experimental procedures were reviewed and approved by the Ethical and Animal Welfare Committee (Extremadura University, Spain) with a project approved under number 67/2022.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting information.

Oropesa A. L., Ramos A., Fallola C., and Gomez L. J., “Integrated Use of Oxidative Stress and Histological Biomarkers of T. tinca as Indicators of 17‐Alpha‐Ethynylestradiol Exposure,” Environmental Toxicology 41, no. 1 (2026): 49–67, 10.1002/tox.24557.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.