Histopathological effects of the antibiotic erythromycin on the freshwater fish species Oncorhynchus mykiss

Ecotoxicology and Environmental Safety 181 (2019) 1–10

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Histopathological effects of the antibiotic erythromycin on the freshwater fish species Oncorhynchus mykiss

T

Sara Rodriguesa,b, Sara Cristina Antunesa,b, Bruno Nunesc,d, Alberto Teodorico Correiab,e,∗ a

Departamento de Biologia da Faculdade de Ciências da Universidade do Porto (FCUP), Rua do Campo Alegre s/n, 4169-007 Porto, Portugal Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4450-208 Matosinhos, Portugal c Centro de Estudos do Ambiente e do Mar (CESAM), Campus de Santiago, Universidade de Aveiro, 3810-193, Aveiro, Portugal d Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal e Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Rua Carlos da Maia, 296, 4200-150, Porto, Portugal b

A R T I C LE I N FO

A B S T R A C T

Keywords: Fish Acute and chronic exposures Antibiotics Tissue damages

Pharmaceuticals are found in the aquatic compartment due to their continuous release in wastewater effluents or direct dispersal in aquaculture practices raising serious threats to human and environmental health. Erythromycin (ERY) is a macrolide antibiotic widely prescribed in human and veterinary medicine to threat a number of bacterial infections, being consequently found in the aquatic environment. The present work intends to evaluate the sub-lethal effects of ERY on juveniles of rainbow trout (Oncorhynchus mykiss) in terms of tissue damage using histochemical staining procedures. Individuals were exposed for 96 h (acute exposure: 0.001–10 mg/L) and 28 days (chronic exposure: 0.05–0.8 μg/L) to environmentally realistic concentrations of ERY. Qualitative and quantitative approaches were used to assess O. mykiss gills and liver tissue alterations after exposure to ERY. For both exposures the most common gill changes recorded were progressive (e.g. hypertrophy of mucous cells and hyperplasia of the epithelial cells). However, circulatory (e.g. aneurysms and oedemas) and regressive (e.g. epithelial lifting of lamellae and lamellar fusion) changes were also observed in the acute assay. Gill morphometric analysis revealed to be a good indicator of subtle alterations in gill architecture in agreement with the qualitative scoring system. In liver, regressive (e.g. cytoplasmic vacuolization, pyknotic nucleus and hepatocellular degeneration) and circulatory disturbances (e.g. hemorrhage and increase of sinusoidal space) were the most frequently observed alterations, but only for the acute assay. Furthermore, all histological changes observed contributed to a significant increase in the pathological index for both organs. The current data demonstrate the existence of a direct dose-effect relationship between the exposure to this specific macrolide antibiotic and the histological disorders recorded in different tissues of the exposed fish. The histopathological findings observed in this study may have been the result of several physio-metabolic dysfunctions. However, the observed tissue lesions were of minimal or moderate pathological importance, non-specific and reversible. Further investigation into the cellular mechanism of action of ERY is needed.

1. Introduction Erythromycin (ERY) is a macrolide antibiotic used in human medicine to treat acute pelvic inflammatory diseases, skin and respiratory tract infections (Liu et al., 2014). ERY is also used in veterinary medicine, as well as in aquaculture (Serdoz et al., 2011; Rodrigues et al., 2016). In fish farms, ERY is usually used against gram-positive cocci, the main concern for trout farming (Serdoz et al., 2011). ERY appears to be excreted unchanged in substantial amounts, a factor that stresses the

environmental importance of this specific compound (Zuccato et al., 2000). ERY is detected in surface water in levels between several ng/L and μg/L, as reported in a previous literature review, carried out by Rodrigues et al. (2016). ERY has been identified as an antibiotic of particular concern for the aquatic compartment due to its pattern of consumption, continuous discharge, environmental persistence and toxic properties (Johnson et al., 2015). Furthermore ERY is included in the list of class I – high priority pharmaceuticals, requiring future monitoring and the development of specific ecotoxicological studies to

∗ Corresponding author. Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4450-208, Matosinhos, Portugal. E-mail address: [email protected] (A.T. Correia).

https://doi.org/10.1016/j.ecoenv.2019.05.067 Received 10 May 2018; Received in revised form 20 May 2019; Accepted 23 May 2019 Available online 30 May 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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to the laboratory in refrigerated and aerated plastic boxes. Individuals were maintained under laboratory controlled conditions for three weeks. The quarantine period took place in 500 L tanks with recirculation of aerated and dechlorinated tap water, with controlled photoperiod (12L:12D), and constant temperature (15.0 ± 0.3 °C). Fish were fed ad libitum with commercial pellets every two days. Fish were checked daily and sick or dead individuals were discarded immediately. Feeding was halted 24 h before the experiments. Experiments were prior authorized by the Ethical Committee of the host institution (ORBEA/CIIMAR). Furthermore, this work took into consideration the Portuguese animal welfare testing regulations (Decree-Law 113/2013).

address their toxic effects (Voogt et al., 2009). Several works have showed that ERY residues could exert deleterious effects on different non-target aquatic organisms, such as a decrease in growth rate, reproduction and survival, namely in cyanobacteria, algae, rotifers, crustaceans and fish (Isidori et al., 2005; Kim et al., 2009). Some studies evaluated the effects of ERY in fish demonstrating that it may affect wildlife by inducing harmful effects even at residual concentrations (Liu et al., 2014; Rodrigues et al., 2016 and 2019a). ERY was also capable to cause hepatotoxicity in experimental animals such as rats (Venkateswaran et al., 1997; Pari and Murugan, 2004; Abdel-Hameid, 2007). Moreover, recently, it has been shown that ERY can induce genotoxic damage and oxidative stress in O. mykiss individuals exposed to environmentally relevant concentrations (0.05–1 μg/L) (Rodrigues et al., 2016). The exposure of fish to environmental stressors, such as antibiotics, can elicit a complex network of biological responses. Molecular interactions between pollutants and cellular components can result in tissue damage which can be easily assessed through histological analysis (Schwaiger et al., 1997; Rodrigues et al., 2017). Tissue lesions, due to sublethal effects of xenobiotic compounds, on different organs of exposed fish, are useful tools for ecotoxicological studies and for monitoring water pollution (Schwaiger et al., 1996). Histological examination of suitable organs in fish is considered a rapid and cost effective method to assess aquatic pollution (Bernet et al., 1999). Several studies have reported histopathological effects in gills, spleen, liver and kidney of fish after exposure to several xenobiotics as nitrogen compounds (Capkin et al., 2009), pesticides (Altinok and Capkin, 2007; Boran et al., 2010), polychlorinated n-alkanes (Uguz et al., 2003; Monfared and Salati, 2013), metals (Al-Bairuty et al., 2013; Topal et al., 2013) and pharmaceuticals (Nunes et al., 2015; Rodrigues et al., 2017 and 2019b). Although the qualitative data are mainly used to study these tissue damages, quantitative data allows a better understanding of the process behind the interaction between organism and pollutants at the molecular level (Pal et al., 2012). A histological disorder is a higherlevel of biological organization response resulting from a previous physiological and/or biochemical malfunction (Nero et al., 2006). Furthermore, morphological changes recorded in some fish tissues and organs could represent adaptive strategies to maintain body homeostasis (Puvaneswari and Jiyavudeen, 2015). The rainbow-trout, Oncorhynchus mykiss, is a freshwater fish species with worldwide economic and ecological importance (Ortega and Valladares, 2013). Furthermore, because of its sensitivity towards numerous compounds, it is one of the most used test organisms in ecotoxicological studies, being recommended by standardized testing guidelines (OECD, 1992; OECD, 2000). This study aimed to determine the histopathological effects of acute and chronic exposure to ERY in gills and liver of rainbow trout (Oncorhynchus mykiss), in order to understand the nature, consequences and biological significance of the observed tissue alterations.

2.3. Exposure conditions 2.3.1. Acute exposure Exposures were performed under the same laboratory-controlled conditions than those observed for the quarantine period. Fish [weight of 10.9 ± 0.3 g and length of 9.03 ± 0.08 cm; Fulton's condition factor (K) – good (Fulton, 1902): K = 1.46 ± 0.02] were exposed to five distinct concentrations of erythromycin (0.001, 0.01, 0.1, 1 and 10 mg/L); an additional control group (without chemical) was included in the experimental design. Exposure occurred according to OECD test guideline 203 for a period of 96 h (OECD, 1992). The concentrations were sublethal, selected based on previously published studies showing that ERY concentrations in the environment were around 1 μg/L (Hirsch et al., 1998, 1999), as well as studies that evaluated distinct effects on different levels of biological organization (Isidori et al., 2005; Kim et al., 2009), which referred LC50 values above 100 mg/L for fish species. The experimental design was similar to that described by Rodrigues et al. (2016). Briefly, a total of 90 individuals were divided and placed into eighteen 50 L aquaria, with three replicates per treatment each one with five fish (3 aquaria X 6 treatments X 5 fish). Aquaria were randomly distributed in the exposure room and 80% of the medium was renewed each 48 h. Fish were not fed during the acute exposure. 2.3.2. Chronic exposure Long-term exposure was carried out in agreement with OECD guideline 215 (OECD, 2000). Fish [13.1 ± 0.3 g, 9.17 ± 0.08 cm, and Fulton's condition factor (K) – excellent (Fulton, 1902): K = 1.69 ± 0.03] were exposed similarly to what was previously described for the acute exposure (five fish per aquaria with three replicates per treatment), for 28 days, to a range of sub-lethal erythromycin concentrations: 0.05, 0.1, 0.2, 0.4 and 0.8 μg/L. The selected concentrations for the chronic assay were environmentally realistic and similar to ERY levels already reported in the aquatic environment and near 1 μg/L (Hirsch et al., 1998, 1999; Ginebreda et al., 2010). Fish were fed until satiety each two days. Medium renovation (80%) took place every 48 h.

2. Material and methods

2.3.3. Water quality control Water quality was monitored every 48 h during the exposures assays, as recommended by OECD guidelines (nº 203 and 215). Water temperature (15.33 ± 0.05 °C), conductivity (233.46 ± 3.04 μS/cm), dissolved oxygen (7.82 ± 0.03 mg/L) and pH (6.83 ± 0.01) were measured using a multiparameter probe (YSI, 556 MPS). Ammonium (NH3) (1.11 ± 0.08 mg/L) and nitrites (NO2) (0.008 ± 0.01 mg/L) were quantified used a photometer (YSI, 9300 Photometer) with water test tablets (Palintest, NH3 and NO2).

2.1. Chemicals The preparation of the stock and test solutions was made according to Rodrigues et al. (2016). For each bioassay a stock solution (2 g/L and 16 mg/L for acute and chronic exposures, respectively) of ERY (Sigma Aldrich, CAS: 114-07-8) was made by dilution in dechlorinated tap water, using the magnetic stirrer. All test solutions were thereafter prepared at the onset of each assay and medium renewal by successive and appropriate dilutions of the stock solution.

2.4. Sampling and tissue/organ preparation

2.2. Test organisms

After exposures, individuals were anesthetized through immersion in a de-chlorinated ice-water bath until fish lost the ability to swim and lost the righting reflex and operculum movements. It is well known that the most common chemical anesthetics (e.g. clove oil, MS222 and 2-

Juvenile rainbow trouts were acquired at an aquaculture facility in northern Portugal (Posto Aquícola do Torno - Marão) and transported 2

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basal epithelial thickness (BET) (e.g. Fig. 1A) (measurements not near the tips of the filament but representative of the filament) using a free software program (Measure IT, Olympus). For SLW and ID three measurements were made for each secondary lamella: on the proximal (near the basal lamina), medial (middle) and distal (trailing edge) regions. For BET three measurements were made for each filament: measurements at both ends and in the middle of the primary lamellae. For SLL measurements were made in the middle zone of the gill filaments and by choosing secondary lamellae with no apparent changes in its architecture (e.g. curling). Furthermore, attention has been taken to choose the angle of the section in relation to the filament (ideally 90°), by avoiding oblique sections that could bias the measurements. These variables represent the major dimensions of gill's architecture that influence the diffusion distance (gas exchange) in fish (Hughes and Perry, 1976). Subsequently, the proportion of the secondary lamellae available for gas exchange (PAGE) was averaged for each filament of an individual and calculated as: PAGE (%) = 100 x [SLL/(BET + SLL)] (Nero et al., 2006). It was assumed that these morphometric measures were related with the total gas exchange area of fish gills (Stevens, 1992).

phenoxyethanol) can cause severe alterations in metabolic, enzymatic and oxidative stress biomarkers, as reported for rainbow trout (Velisek et al., 2011). Under these circumstances, the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals allow the use of physical methods, such as decapitation. Euthanasia was performed by skilled personnel who have been trained and certified. After the sedation period, fish were decapitated and tissues were immediately collected for further histopathological preparation and analysis. This experiment was also used to evaluate several biochemical biomarkers (Rodrigues et al., 2016, 2019a). 2.5. Histopathological procedure A portion of liver and gills (second gill arch) were chemically fixed in Bouin during 24 h, followed by decalcification (Histofix Decalcifier 1, PanReacAppliChem) (gills only) for 24 h. Then, samples were dehydrated using ethanol solutions of increasing degree (70%, 80%, 90% and 100%). This was followed by diafanization of the samples with a xylene solution for 2 h, thereafter sample were embedded in paraffin wax (56–58 °C), and then manually sectioned (5–8 μm) in the parasagittal plane using a microtome (Leica: Reichert-Jung 2030). Finally, sections were stained with standard (hematoxylin-eosin, HE) and histochemical (Alcian Blue pH 2.5 - Periodic Acid Schiff, AB-PAS) procedures and mounted in DPX. This differential coloration allows the identification of mucous cells in the gill tissue (AB-PAS +: magenta color of neutral mucopolysaccharides) and the presence of glycogen deposits (PAS +: magenta color of cytoplasmic granules) and lipid droplets (translucent color) in hepatocytes. Randomly selected slides from each fish/tissue were blind examined by two experienced readers under a light microscope (Olympus CX41). Photomicrographs were taken using a USB digital camera (Olympus, SC30) at a medium/higher power magnification (100x to 1000x).

2.6. Statistical analyses Data were checked for normality (Shapiro-Wilk test) and homogeneity of variances (Levene test) prior to statistical analysis. Nested ANOVA was run for each variable to test the differences among erythromycin concentrations across experimental groups (random nested factor). If needed (p < 0.05), Dunnett test was performed to compare each treatment with the control group. Statistics were performed using SPSS (v23) and MS Excel using a significance level of 0.05. 3. Results During exposures, no mortality was recorded in the control group [complying with the OECD (1992 and 2000) requirements: mortality < 10%].

2.5.1. Qualitative and semi-quantitative assessment of organ/tissue The accurate identification of the tissue changes was based on previous published works, such as Takashima and Hibiya (1995) and Mumford et al. (2007). Histopathological condition indices (I) for both organs (gills and liver) were adapted from Bernet et al. (1999). Histological changes in organs (org) were classified into 5 different classes: circulatory, regressive (also named degenerative), progressive (also named proliferative), inflammatory and neoplastic reaction patterns (rp). The pathological importance of the observed alteration (alt) was defined as an ‘‘importance factor’’ (w), classified as 1, 2 or 3, corresponding to minimal (reversible pathological lesions), moderate (lesions that in most cases revert after neutralization of the stressor agent) and severe (often irreversible lesions that cause partial or total loss of function of the affected organ) pathological importance, respectively (Table 1). Each alteration was also assessed using a “score value” (a) ranging from 1 to 6 (mild to severe occurrence) depending on the degree and extent of the alteration (i.e. percentage of areas, in gills or liver, exhibiting this specific alteration). Using “importance factors” and “score values” two indices were calculated. The reaction pattern index (I org cat) and the organ index (I org) were calculated following the equations: I org cat = Σ alt (a org rp al × w org rp alt) and I org = Σ rp Σ alt (a org rp al × w org rp alt). The histopathological condition indices, which represent the significance of the lesions and the degree of damage, allowed us to make a statistical analysis. Reaction pattern and organ pathological indices data of liver and gills for each treatment are presented as the mean ± standard error.

3.1. Gills histopathology O. mykiss from the control groups presented gill filaments with a normal morphology (Fig. 1 A). In the regressive lesions category, several modifications were observed in both exposure periods, such as lamellar fusion (Fig. 1 B and C), epithelial lamellar lifting (Fig. 1 D and H) and desquamation (Fig. 1 D). In the progressive lesions category the proliferation and hypertrophy of mucous cells (Fig. 1 C) and hyperplasia of the epithelial cells (Fig. 1. B, E and H) leading to a partial or complete fusion of secondary lamellae were observed. In some cases, the high severity of the gill progressive alterations (severe hyperplasia) resulted in total fusion of secondary lamellae and enclosing the filament (Fig. 1 G). Mucous cells showed a strong positive reaction with AB-PAS and occurred mainly at the tips of the filaments, but also at the midfilamental (inter-lamellar) position (Fig. 2A). Circulatory changes were also evidenced by the presence of aneurysms (Fig. 1 F) and oedemas (Fig. 1 H) in the exposed individuals. The prevalence of each specific lesion for both exposures is shown in Table 1 and corresponds only to ERY-exposed organisms. The progressive alterations were the most predominant lesions in the gills, in both exposures, as shown in Fig. 3. In acute exposure, it was also possible to observe circulatory and regressive changes. Individuals exposed to 1.00 and 10.00 mg/L presented a significant increase of regressive lesions (F[5, 12] = 10.899; p < 0.001) relatively to control (Fig. 3). There was also a significant increase in the progressive lesions (F[5, 12] = 4.292; p = 0.018) of individuals that were exposed to 0.100 and 1.000 mg/L of ERY (Fig. 3). Individuals exposed to 1.000 mg/L showed also a significant increase in circulatory alterations (F[5, 12] = 3.118; p = 0.049) (Fig. 3). No significant alterations were

2.5.2. Gill morphometric analysis Gills morphometric analysis were made using a modified version of the protocol of Nero et al. (2006). Photomicrographs of gills were randomly taken for all fish under high magnification (200×). Three gill filaments per photomicrograph per fish were measured for secondary lamellar length (SLL) and width (SLW), interlamellar distance (ID) and 3

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Table 1 Descriptions of the category of alterations and examples of specific lesions recorded in this study and assigned to each category. The pathological importance of the observed alteration (importance factor = w) was classified as 1, 2 or 3. For each specific change, results of percent alteration prevalence are presented, in a total of 75 exposed organisms (except control), five fish per tank, with three replicates (n = 3) per concentration. For more details see M&M. Category of alteration

Description of general response

Examples of specific tissue alterations Gills

Liver

w - Specific alteration

CIRCULATORY

REGRESSIVE

PROGRESSIVE

INFLAMMATORY

NEOPLASTIC

Disturbances result from a pathological condition of blood and tissue fluid flow

Breakdown of tissue and/or cells which terminate in a functional reduction or loss of an organ. Changes in tissue architecture

Increase in the number of specific cell types or structures

Increased presence of cells used in tissue repair; response to damaged tissue Uncontrolled cell and tissue proliferation

Prevalence (%) Acute

Chronic

1 -Vasodilation

27

1

1 - Hemorrhage 1 - Aneurysm 1 – Oedema 1 - Ephitelial lifting/Lamellar epithelial desquamation

5 0 28 87

0 36 47 55

1 - Lamellar fusion 3 - Necrosis

43 51 Non observed

1 - Hypertrophy of the gill epithelium or mucous cells 2 - Hyperplasia of the gill epithelium 2 - Leucocytes infiltration

7

9

89 7

Non Observed

w - Specific alteration

Prevalence (%) Acute

Chronic

1 - Increase of sinusoidal space 1 - Oedema 1 - Hemorrhage

65

68

63

29

37 23 9 0 33

21 19 32 1 58

89

1 - Irregular nucleus/ hepatocyte 1 - Cytoplasmic vacuolization 2 - Nuclear degeneration 2 - Pyknotic nucleus 3 - Hepatocellular necrosis 1 – Nuclear/cellular hypertrophy of hepatocyte 2 - Cellular hyperplasia

1

2 - Leucocytes infiltration

5

Non observed 3 16

Non observed 11

Non Observed

(Fig. 4 A). Fish exposed to ERY revealed several tissue alterations after short and long exposure periods, namely regressive, including cytoplasmatic vacuolization (Fig. 4 B), namely of glycogen and lipid deposits (vacuolization) (Fig. 2 B), pyknotic nucleus (Fig. 4 C and E) and hepatocellular degeneration (Fig. 4 D); some alterations were progressive, as nuclear hypertrophy (Fig. 4 D) and hypertrophy of hepatocytes (Fig. 4 E); circulatory as hemorrhage (Fig. 4 H) and increase of sinusoidal space (Fig. 4 C e G); and inflammatory as leucocytes infiltration (Fig. 4 F). O. mykiss acutely exposed to erythromycin presented higher levels of regressive and circulatory alterations and lower levels of progressive alterations compared to the controls (Fig. 5). Regressive lesions were significantly higher (F[5,12] = 5.205; p = 0.009) in individuals acutely exposed to the high concentration tested (Fig. 5). In the acute assay, individuals exposed to ERY had no significant changes in the categories of progressive (F[5, 12] = 1.714; p = 0.206), circulatory (F[5, 12] = 2.410; p = 0.098) and inflammatory (F[5, 12] = 1.200; p = 0.366) damages. Although there were no significant differences in categorical liver pathological indices (except for regressive alterations) between control and acute treatments, it was possible to observe a higher total liver pathological index for fish exposed to ERY (F[5, 12] = 5.248; p = 0.009), with significant differences for the three highest concentrations tested (0.1, 1 and 10 mg/L) (Fig. 5). The same pattern of response was also observed after chronic exposure, with regressive changes and circulatory alterations predominating over the other types. The here obtained data showed that regressive (F[5, 12] = 4.746; p = 0.013) and circulatory (F[5, 12] = 9.579; p = 0.001) alterations were significantly increased in fish chronically exposed to 0.4 μg/L and 0.8 μg/L, respectively. No significant differences were however observed for progressive (F[5, 12] = 1.139; p = 0.392) and inflammatory (F[5, 12] = 2.667; p = 0.076) changes. The liver pathological index was significantly higher in organisms exposed to levels of 0.4 and 0.8 μg/L of ERY.

however observed for inflammatory alterations (F[5, 12] = 1.325; p = 0.318). In both assays, neoplastic changes were not found. Overall, fish exposed to the highest concentrations of ERY (0.100, 1.000 and 10.00 mg/L) presented higher total gill pathological indices than control (F[5, 12] = 9.884; p = 0.001), after an acute exposure period (Fig. 3). Regarding the chronic exposure, fish exposed to 0.10–0.80 μg/ L of erythromycin presented significant differences only for progressive disorders (F[5, 12] = 18.680; p < 0.001; Fig. 3). No significant differences were observed in circulatory (F[5, 12] = 1.697; p = 0.210), regressive (F[5, 12] = 2.315; p = 0.108) or inflammatory alterations (F[5, 12] = 1.000; p = 0.458). Fish exposed to 0.10–0.80 μg/L of erythromycin revealed significantly higher gill pathological indices (F[5, 12] = 36.926; p < 0.001; Fig. 3). In both exposures, there was a significant increase of the total changes (gills pathological index). Individuals exposed to erythromycin had significant alterations in gill's morphometric measurements after both exposures (Table 2). A significant reduction in interlamellar distance (ID - F[5,12] = 18.836; p < 0.001), in all concentrations and a significant increase of secondary lamellar width (SLW - F[5, 12] = 8.824; p = 0.001) in fish exposed to the highest concentrations were observed after acute exposure (Table 2). No significant changes were found in BET (F[5, 12] = 0.954; p = 0.482) and SLL (F[5, 12] = 1.629; p = 0.226). Fish exposed during 28 days to 0.40 and 0.80 μg/L of ERY showed significantly gills morphometric alterations with shorter (SLL - F[5, 12] = 7.427; p = 0.002) and wider (SLW - F[5, 12] = 15.004; p < 0.001) secondary lamellae, and reduced interlamellar distance (ID - F[5, 12] = 12.391; p < 0.001) (Table 2). The basal epithelium thickness was also significantly increased (BET - F[5, 12] = 4.825; p = 0.012) in fish exposed to the highest concentrations tested (0.2–0.8 μg/L) (Table 2). Regarding PAGE index and following an acute exposure no statistical differences were observed between the control and exposed groups (F[5, 12] = 2.530; p = 0.087, Table 2). However PAGE was significantly decreased in chronic exposure, for ERY levels of 0.4 μg/L and 0.8 μg/L (F[5, 12] = 10.831; p < 0.001, Table 2).

3.2. Liver histopathology Fish from the control groups showed a normal liver architecture 4

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Fig. 1. Gill histological sections of rainbow trout (O. mykiss) after acute (A–D) and chronic (E–H) exposures to erythromycin. Photographs of organisms from control (A) and exposed to 0.100 mg/L (B and C), 10.00 mg/L (D) – acute exposure; 0.10 μg/L (E), 0.20 μg/L (F), 0.40 μg/L (G), 0.80 μg/L (H) – chronic exposure. (A) Normal gill histology identifying the gill filament (f) and secondary lamellae (sl) on filaments 1 and 2. The four gill measurements that includes secondary lamellar length (SLL), secondary lamellar width (SLW), interlamellar distance (ID), basal epithelial thickness (BET) were examined for morphometric analysis. (B, C, E, H) Hyperplasia of epithelial cells (one asterisk). (B and C) Lamellar fusion (solid circles). (C) Hypertrophy of mucous cells both at the base and along the edges of the secondary lamellae (black arrows). (D) Epithelial lifting of lamellae (black arrow head). (B and H) Oedema (black square). (D) Desquamation (black star). (E) Changes in tissue architecture - curling of secondary lamellae (doted circle). (F) Aneurysm (white arrows). (G) Severe hyperplasia of the epithelium and fusion of secondary lamellae (two asterisks). [H&E Stain; 200 × (A) and 400 × (B–H)].

4. Discussion

low toxic substances, moderate contamination level or shorter exposure periods (Schwaiger et al., 1997). The tissue damages observed in fish gills exposed to ERY in the present study are similar to those already reported in O. mykiss after exposure to a variety of pollutants, including metals (Al-Bairuty et al., 2013), pesticides (Altinok and Capkin, 2007; Boran et al., 2010) and pharmaceuticals (Rodrigues et al., 2017, 2019b). In general, the recorded lesions, more severe in individuals exposed to higher doses, may be categorized in two types (Mallat, 1985). The first type is caused by the direct and earlier effect of a toxic stimulus, such as aneurisms, oedema and the thinning of filament epithelium. The second type occurs due to an adaptive mechanism to reduce the contaminant uptake, such

4.1. Gills histopathology Gills are crucial organs for the direct action of waterborne xenobiotics since they are involved in several vital physiological functions in fish, such as gases exchange, ionic regulation, acid-base balance and excretion of nitrogenous compounds (Evans, 1987; Bernet et al., 1999; Nero et al., 2006). Additionally, gills provide a significant area in contact with the surrounding water, facilitating greater toxicant interaction and absorption (Cinar et al., 2009). Unlike other fish internal organs, tissue damage on gills could be an outcome from exposure to 5

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Fig. 2. Histological sections of gill and liver of rainbow trout (O. mykiss) chronically exposed (0.2 and 0.4 μg/L for gills and liver, respectively). (A) Gill section: the black arrows indicate some of mucous cells (magenta color due to neutral mucins) and strongly PAS stained. (B) Liver section: the white arrows indicate some positive PAS reaction for glycogen granules and the black arrows indicate the lipid droplets (translucent). [AB-PAS Stain; gills200x; liver-1000x]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Although circulatory and regressive changes were also observed after chronic test, these appeared with the highest prevalence after acute exposure. With regard to circulatory changes, gills of O. mykiss exhibited significant disturbances of blood flow, including vascular congestion, aneurisms (also known as telangiectasis) and oedemas. Vascular congestion was punctual, in this study. Aneurysms results from the collapse of the pillar cells and weakness of vascular integrity with the appearance of an abnormal blood-filled vessel that push the lamellar epithelium outward (Pal et al., 2012; Ahmed et al., 2013). Oedema can be interpreted as a direct response to toxicants action (Nero et al., 2006), and it is usually referred to as a first sign of pathology (Omar et al., 2014). Again, similar circulatory disorders, including aneurysms and oedema, were already observed in fish after exposure to several pharmaceuticals (Schwaiger et al., 2004; Nunes et al., 2015; Rodrigues et al., 2017 and 2019b). The circulatory disorders found in the current study are considered changes of low pathological importance, being generally reversible when exposure to the pollutant ends (Bernet et al., 1999; Altinok and Capkin, 2007). Therefore they indicate weak toxicity of the ERY. Common regressive lesions on the gills of O. mykiss included changes in tissue architecture as well as thinning (desquamation) and lifting of epithelial cells. The observed epithelial desquamation of the gills is suggested to be a direct response (Ahmed et al., 2013), and usually occurs associated to circulatory disturbances (Barišić et al., 2015). However the lifting of lamellar epithelium does not necessarily represent a “true” oedema because the sub-epithelial fluid might come primarily from the ambient water (e.g. freshwater) rather than originating as a blood exudate (Mallat, 1985). Furthermore the current observed regressive changes in the individuals exposed to ERY are considered as lesions of moderate severity and reversible, if the pollutant is removed (Bernet et al., 1999). Progressive changes were significantly higher and showed greater occurrence after the chronic exposure, although they have also been observed following an acute exposure. These changes were those that most contributed to the total pathological index observed. The progressive changes, such as hyperplasia and hypertrophy of gill cells, can be considered as common protective processes against pollutants (Barišić et al., 2015). Epithelial thickening, observed here (increase of basal epithelial thickness - BET), could be consequence of the appearance of some defense cells (e.g. macrophages and leucocytes) as part of a compensatory mechanism of tissue repair (Monteiro et al., 2008). An additional protective mechanism is the proliferation and hypertrophy of mucous cells (observed here) since mucus (a layer of glycolipids and glycoproteins) could acts as a protective barrier over gill epithelium (Pereira et al., 2012; Senol, 2014). This seems to be a long-term adaptation mechanism (Nero et al., 2006), since the excessive mucus secretion on the gill filaments stimulates the operculum movement leading to an increase of the breathing rate (Ramzy, 2014). After both exposures, the scattered gill lesions recorded and the observed increase of dose-dependent prevalence of lesions suggest that gill's function is being affected by ERY. Furthermore, from the obtained results, a higher total gill pathological index at higher concentrations of

Fig. 3. Rainbow trout total and categorical (circulatory, regressive, progressive and inflammatory) pathological indices for gills, after acute and chronic exposures to erythromycin. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates (n = 3) per concentration. * represents categorical indices that are significantly different from the control fish group (p < 0.05).

as mucous-cells proliferation, lamellar lifting, epithelial hyperplasia and lamellar fusion, since they minimize the gill's surface area, increase the diffusion distance and decreases the interlamellar distance (Monteiro et al., 2008; Pal et al., 2012). After acute exposure significant changes in all classes of lesions were observed, with the exception of inflammatory alterations.

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Table 2 Basal epithelium thickness (BET; μm), interlamellar distance (ID; μm), secondary lamellar length (SLL; μm) and width (SLW; μm), and proportion of the secondary lamellae available for gas exchange (PAGE %) of rainbow trout gills following acute and chronic exposure of erythromycin. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates (n = 3) per concentration. * represents significant differences compared to the respective control (p < 0.05). Erythromycin concentrations Acute exposure (mg/L)

Chronic exposure (μg/L)

CTRL 0.001 0.010 0.100 1.000 10.00 CTRL 0.05 0.10 0.20 0.40 0.80

BET

ID

51.2 ± 1.3 55.6 ± 2.3 55.5 ± 2.6 56.69 ± 3.09 57.71 ± 3.03 56.4 ± 0.5 49.9 ± 0.9 51.1 ± 1.1 51.7 ± 0.5 54.8 ± 1.8* 54.5 ± 0.5* 55.6 ± 0.8*

23.8 21.3 19.8 17.9 17.2 17.5 24.7 23.8 23.3 22.2 20.0 18.4

± 0.9 ± 0.5* ± 0.2* ± 0.2* ± 0.8* ± 0.4* ± 0.9 ± 0.6 ± 0.6 ± 0.9 ± 0.6* ± 0.3*

SLL

SLW

96.3 ± 6.5 95.6 ± 0.4 97.8 ± 2.5 91.6 ± 2.9 87.9 ± 1.9 86.8 ± 4.3 97.4 ± 2.9 94.8 ± 1.4 96.23 ± 1.09 97.1 ± 0.4 90.6 ± 2.2* 88.1 ± 0.4*

11.2 11.2 13.3 12.5 15.6 14.2 11.8 11.3 11.6 12.0 13.2 14.5

PAGE ± 0.7 ± 0.4 ± 0.3 ± 0.5 ± 0.8* ± 0.5* ± 0.5 ± 0.2 ± 0.3 ± 0.3 ± 0.2* ± 0.3*

65.2 63.2 63.8 61.8 60.4 60.5 66.1 64.9 65.5 63.9 62.4 61.3

± 1.5 ± 0.9 ± 0.6 ± 1.7 ± 1.2 ± 1.1 ± 0.6 ± 0.2 ± 0.2 ± 0.8 ± 0.8* ± 0.4*

drug- or chemical-induced hepatic hypertrophy can be caused by the proliferation of endoplasmic membranes (Nero et al., 2006), increase of biotransformation processes (Schramm et al., 1998), and/or drug metabolizing intracellular enzymes (Hall et al., 2012). However, this effect can be simply the result from the accumulation of glycogen and lipids in cytoplasm of hepatocytes (Wolf and Wolfe, 2005), which is corroborated hereby by the AB-PAS coloration. Among the inflammatory changes, leukocyte infiltration is a morphological disorder of moderate severity found in liver of individuals exposed to ERY, often associated with other alterations, which appears to be an earlier immunological defense due to cell death (Bernet et al., 1999). Furthermore, inflammatory infiltrates can be associated to the hepatocellular degeneration, since necrosis seems to trigger formation of inflammatory foci, with macrophages replacing hepatocytes (Ding et al., 2010). Moreover, rats treated with erythromycin showed a liver parenchyma with inflammatory cell infiltration, as macrophages, and oedema (Sambo et al., 2009). The current study evidenced intense regressive changes in liver of O. mykiss, particularly at higher concentrations, for both exposures, although with higher prevalence after the acute exposure. The here-observed vacuolization of hepatocytes, as a result of glycogen and lipids deposits, can be associated with the inhibition of protein synthesis, energy depletion, disaggregation of microtubules, or shifts in substrate utilization (Ahmed et al., 2013; Younis et al., 2013; Rodrigues et al., 2019b). ERY causes phospholipidosis (excessive accumulation of phospholipids and drug in lysosomes), which may cause modifications in the cell membrane integrity and intracellular accumulation of ERY or its metabolite in rat tissues (Singh et al., 2014a). Fish use glycogen less effectively than mammals; as a result, their hepatocytes tend to be more vacuolated, corresponding to a relatively higher glycogen and/or lipid content (Gingerich, 1982; Ferguson, 1989). Furthermore, O. mykiss liver stores primarily glycogen (Hinton et al., 2001), and therefore an increase in glycogen levels points toward a compromised glycogen breakdown rather than to a stimulation of glycogen production (Schwaiger et al., 1996). Vacuolization of hepatocytes can indicate a discrepancy between the rate of synthesis of substances in the parenchymal cells and the rate of their release into the circulatory system (Younis et al., 2013). Some authors have also discussed that vacuoles in hepatocytes may accumulate insoluble contaminants or their by-products (Köhler, 1990), and this type of finding has been considered as a general failure in lipid metabolism (Van Dyk et al., 2007). Metabolic disturbances can be associated to ERY toxicological effects on the liver through oxidative stress (Rodrigues et al., 2016). During metabolism of ERY it is well known that significant quantities of reactive oxygen species are generated (Venkateswaran et al., 1997; Pari and Murugan, 2004). Hepatocellular degeneration is strongly associated with oxidative stress where lipid peroxidation is a clear source of membrane bilayer susceptibility (Ribeiro et al., 2005).

ERY for both exposure times was made clear. A similar pattern for both exposure periods was the fact that progressive alterations were the more popular and showed the highest indices, followed by the circulatory disorders and regressive changes. The former changes only showed significantly higher indices, after acute exposure. The occurrence of these changes may represent a considerable increase in the distance of water diffusion to the blood and consequently a reduction in oxygen uptake. Tissue damages in fish gills, as reported here due to ERY exposure, may reduce the oxygen up-take and disturb the osmoregulatory function of the fish (Varadarajan et al., 2014). Moreover, gills histological alterations may merely be reflections of generalized stress responses, often secondary to a failure of gill cellular osmoregulation in freshwater species (Evans, 1987). Lastly, the toxic effect of ERY on fish gills can be attributed to the disturbance of the oxidative stress equilibrium and consequent disruption of the cellular and tissue organization, as recently suggested by Rodrigues et al. (2019b). 4.2. Liver histopathology Fish liver is considered the main organ of storage, biotransformation and excretion of xenobiotics and its evaluation could reveal the general health condition of fish (Wolf and Wolfe, 2005; Rodrigues et al., 2019b). Liver is also especially vulnerable and sensitive to environmental stressors, which inevitably reflects on its parenchyma integrity (Roberts, 1978). Furthermore, the toxicological effects of ERY in mammals have been already studied, and include the induction of hepatoxicity (Abdel-Hameid, 2007; Singh et al., 2014a). It means that the occurrence of similar metabolic pathways in fish can lead to the establishment of a comparable scenario that is important to evaluate. The current observed alterations in the livers of exposed fish indicate some physiological disturbances with minimal severity as a result of hepatocellular alterations. Individuals not exposed to ERY mostly exhibited a normal liver architecture. However, some lesions were observed for all experimental groups, including a few individuals from the control group, but in this case with very low prevalence. Fish exposed to ERY revealed some regressive changes in livers such as hepatocellular degeneration, cytoplasmic vacuolization and pyknotic nuclei. Additionally, some liver lesions, namely circulatory disturbances (enlargement of sinusoidal capillaries and hemorrhage) and progressive changes (nuclear and hepatocellular hypertrophy) were observed. Although these last changes were observed in both exposures, its occurrence has not been shown significantly different from respective control animals. It has been recently suggested that nuclear and hepatocyte hypertrophy may occur either directly by denaturation of volume-regulating ATPases, or indirectly through disruption of the cellular energy transfer processes required for ionic regulation, as well as increased cellular activity (Ahmed et al., 2013). Moreover, several studies showed that 7

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Fig. 4. Liver histological sections of rainbow trout (O. mykiss) after acute (A–D) and chronic (E–H) exposures to erythromycin. Photographs of organisms from control (A) and exposed to 0.01 mg/L (B), 0.1 mg/L (C), 1 mg/L (D) – acute exposure; 0.10 μg/L (E), 0.20 μg/L (F), 0.40 μg/L (G), 0.80 μg/L (H) – chronic exposure. (A) Normal liver architecture identifying the hepatocytes (h) and hepatocyte nucleus (nu). Vacuolization (one asterisk). Increase of sinusoidal space (three asterisks). Pyknotic nucleus (solid circles). Nuclear hypertrophy of hepatocytes (Black arrows). Hepatocellular degeneration (black square). Hypertrophy of hepatocytes (Black arrow head). Leucocytes infiltration (white arrow). Hemorrhage (doted circle). [H&E Stain; 400x (B and F) and 1000 × (A, C-E, G and H)].

reversible disturbances, which may not alter the normal functioning of the tissue (Bernet et al., 1999). Similar histological lesions have been reported in O. mykiss exposed to other antibiotic and were considered indicative of the impact of the contaminants on the health of these fish (Rodrigues et al., 2017 and 2019b). This could be possibly related to the fact that the liver is highly supplied with blood (Abdel-Hameid, 2007). The increased blood flow may have led to rupture of sinusoid causing occasional hemorrhages. The cellular degeneration in the liver might be due to oxygen deficiency as a result of gill degeneration (Younis et al., 2013). Several general theories have been proposed to explain causes of histological damages on fish resulting from pollutants, including cumulative effects of oxidative stress (e.g. Rodrigues et al., 2019b). One

Another change moderately observed in the exposed individuals was nuclear pyknosis. Pyknotic nucleus, i.e. irreversible condensed form of chromatin material in the nucleus, were found in both exposures, suggesting a severe and irreversible injury of fish liver (Pal et al., 2012). This change may be associated with the observed hepatocellular degeneration process, and may be possibly indicative of future necrosis. Environmental stressors are able to induce disarrangement of the molecular organization of the cell membranes leading to their degradation (Hadi and Alwan, 2012). Circulatory changes in liver occurred after both exposures. However, only the chronic exposure yielded significant differences in relation to control animals. Sinusoid dilation and hemorrhage are easily 8

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detoxification capacity, thus compromising other organs and/or systems. 5. Conclusions The observed histological features of gills and liver demonstrated some adverse effects of ERY in O. mykiss. ERY toxicity increased with higher chemical concentrations, as well as with larger exposure periods. The majority of alterations were mild or moderate, reversible and nonspecific. However, the total pathological indexes were comparatively more pronounced in gills than liver, comparatively to the control group. Moreover, the current data shows that histological assessment could easily reveal the deleterious effects on fish as result of acute and chronic exposures to environmental stressors, including drugs. However, the underlying mechanisms behind ERY toxicity in fish require further studies. Conflicts of interest The authors declare that they have no competing interests. Acknowledgments Sara Rodrigues and Sara C. Antunes received a Ph.D. fellowship (SFRH/BD/84061/2012) and a post doc grant (SFRH/BPD/109951/ 2015), respectively, by the Portuguese Foundation for Science and Technology (FCT). Bruno Nunes was hired through the Investigator FCT program (IF/01744/2013). This research was partially supported by the Strategic Funding UID/Multi/04423/2019 and UID/AMB/50017 POCI-01-0145-FEDER-007638, through national funds provided by FCT, in the framework of the programme PT 2020. References Abdel-Hameid, N.-A.H., 2007. Protective role of dimethyl diphenyl bicarboxylate (DDB) against erythromycin induced hepatotoxicity in male rats. Toxicol. Vitro 21 (4), 618–625. Ahmed, M.K., Al-Mamun, M.H., Parvin, E., Akter, M.S., Khan, M.S., 2013. Arsenic induced toxicity and histopathological changes in gill and liver tissue of freshwater fish, Tilapia (Oreochromis mossambicus). Exp. Toxicol. Pathol. 65 (6), 903–909. Al-Bairuty, G.A., Shaw, B.J., Handy, R.D., Henry, T.B., 2013. Histopathological effects of waterborne copper nanoparticles and copper sulphate on the organs of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 126, 104–115. Altinok, I., Capkin, E., 2007. Histopathology of rainbow trout exposed to sublethal concentrations of methiocarb or endosulfan. Toxicol. Pathol. 35 (3), 405–410. Barišić, J., Dragun, Z., Ramani, S., Filipović-Marijić, V., Krasnići, N., Čož-Rakovac, R., Kostov, V., Rebok, K., Jordanova, M., 2015. Evaluation of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) as an indicator of river pollution. Ecotoxicol. Environ. Saf. 118, 158–166. Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T., 1999. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis. 22 (1), 25–34. Boran, H., Altinok, I., Capkin, E., 2010. Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss. Tissue Cell 42 (3), 158–164. Capkin, E., Birincioglu, S., Altinok, I., 2009. Histopathological changes in rainbow trout (Oncorhynchus mykiss) after exposure to sublethal composite nitrogen fertilizers. Ecotoxicol. Environ. Saf. 72 (7), 1999–2004. Cinar, K., Aksoy, A., Emre, Y., Aşti, R.N., 2009. The histology and histochemical aspects of gills of the flower fish, Pseudophoxinus antalyae. Vet. Res. Commun. 33 (5), 453–460. Decree-Law 113, 2013. de 7 de agosto. D.R. 151, Série I. Relativo à proteção dos animais utilizados para fins científicos. Ministério da Agricultura, do Mar, do Ambiente e do Ordenamento do Território. Ding, L., Kuhne, W.W., Hinton, D.E., Song, J., Dynan, W.S., 2010. Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes). PLoS One 5 (10), e13287. Evans, D.H., 1987. The fish gill: site of action and model for toxic effects of environmental pollutants. Environ. Health Perspect. 71 (8), 47–58. Ferguson, H.W., 1989. Systemic Pathology of Fish: A Text and Atlas of Comparative Tissue Responses in Diseases of Teleosts. Iowa State University Press, Ames, IA, pp. 263. Fulton, T.W., 1902. The rate of growth of fishes. Sci. Invest. Fish. Div. Scot. Rept. 3, 326–446. Ginebreda, A., Munoz, I., Lopez de Alda, M., Brix, R., Lopez-Doval, J., Barceló, D., 2010. Environmental risk assessment of pharmaceuticals in rivers: relationships between hazard indexes and aquatic macroinvertebrate diversity indexes in the Llobregat

Fig. 5. Rainbow trout total and categorical (circulatory, regressive, progressive and inflammatory) pathological indices for liver, after acute and chronic exposures to erythromycin. Data are expressed as mean ± standard error (SE), five fish per tank, with three replicates (n = 3) per concentration. * represents categorical indices that are significantly different from the control fish group (p < 0.05).

consequence is organelle/cellular damage, which can directly affect key cellular responses. ERY was shown to be hepatotoxic to rats (AbdelHameid, 2007), larval zebrafish (He et al., 2013) and gilthead seabream (Rodrigues et al., 2019b), possibly due to the metabolism of ERY, which generates toxic free radicals (Singh et al., 2014a and b). However a straightforward pattern evidencing oxidative stress in O. mykiss liver exposed to ERY was not yet proved (Rodrigues et al., 2016). As described for gills, ERY treatment induced significant increase of the liver pathological indices, compared with those of control fish. This may be mainly attributed to the regressive and circulatory effects (only in chronic exposure) induced in this organ. The results of the semiquantitative histological assessment showed that the organ index value for the liver of the control group, was considerably lower than for exposed organisms, increasing to more than double after both exposures, especially to higher concentrations. For the case of chronic exposure, it was possible to devise a consistent tendency to increase with increasing concentration of ERY. The presence of a histopathological alterations in the liver after ERY exposure can compromise its regular functionality. One must bear in mind that fish liver plays an important role in irreplaceable and vital functions, and any change or damage to its physiology and morphology could result in a decrease in its metabolic and 9

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