Journal of Korean Society on Water Environment (한국물환경학회지)

Korean Society on Water Environment (한국물환경학회)
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Impact Assessment of Sewage Effluent on Freshwater Crucian Carp Carassius auratus using Biochemical and Histopathological Biomarkers

생화학적 및 조직병리학적 생체지표를 이용한 하수처리장 방류수의 담수 붕어(Carassius auratus) 영향 평가

  • Samanta, Palas (Division of Environmental Science & Ecological Engineering, Korea University) ;
  • Im, Hyungjoon (Division of Environmental Science & Ecological Engineering, Korea University) ;
  • Lee, Hwanggoo (Department of Biological Science, Sangji University) ;
  • Hwang, Soon-Jin (Department of Environmental Health Science, Konkuk University) ;
  • Kim, Wonky (Ensol Partners Co. Ltd.) ;
  • Ghosh, Apurba Ratan (Department of Environmental Science, The University of Burdwan) ;
  • Jung, Jinho (Division of Environmental Science & Ecological Engineering, Korea University)
  • ;
  • 임형준 (고려대학교 환경생태공학부) ;
  • 이황구 (상지대학교 생명과학과) ;
  • 황순진 (건국대학교 보건환경과학과) ;
  • 김원기 ((주)엔솔파트너스) ;
  • ;
  • 정진호 (고려대학교 환경생태공학부)
  • Received : 2016.05.17
  • Accepted : 2016.09.02
  • Published : 2016.09.30
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Abstract

The aim of this study is to assess the influence of effluent discharge from a sewage treatment plant by evaluating oxidative stress and histopathological alterations in freshwater crucian carp Carassius auratus collected from the Eungcheon stream, located in Korea. Catalase activity in the gills, liver, and kidneys of C. auratus was collected from mixing zones; the downstream site was notably higher of fish than that of the upstream site. In addition, the activity of glutathione-S-transferase in the gills and liver was significantly higher in samples from the mixing zone than in those from the upstream site (p < 0.05). In addition, significantly elevated lipid peroxidation levels were observed in fish livers sampled from the mixing zone than in those from the upstream site (p < 0.05). Significant histopathological alternations were also observed in C. auratus, with the order of magnitude changes being liver > kidney > gills. These findings suggest that the liver is most affected by effluent discharge. The degree of tissue changes (DTC) indicate that the highest level occurred in samples from the mixing zone (30.98 ± 5.40) followed by those from the downstream site (19.28 ± 4.31) and was the lowest in samples from the upstream site (4.83 ± 2.67). These findings indicate that fish collected from the mixing zone are most affected by effluent discharge and both oxidative stress and histopathological indices are useful tools for monitoring contaminated rivers and streams.

이 연구의 목적은 응천에서 채집한 담수 붕어(Carassius auratus)의 산화적 스트레스와 조직병리학적 손상을 분석하여 하수처리장 방류수의 영향을 평가하는 것이다. 방류수 혼합 지점과 하류 지점에서 채집한 붕어의 아가미, 간, 콩팥의 과산화수소 분해효소(catalase)의 활동도는 상류 지점과 비교하여 더 높은 것으로 나타났다. 또한, 혼합 지점의 아가미와 간의 글루타치온 S-전이효소(glutathione-S-transferase)의 활동도는 상류 지점과 비교하여 유의하게 높았으며(p < 0.05), 상류 지점보다 유의하게 더 증가된 간의 지질 과산화(lipid peroxidation)가 혼합 지점에서 관찰되었다. 붕어의 조직병리학적 손상의 정도는 간 > 콩팥 > 아가미순으로 나타났다. 이 결과는 간이 방류수의 영향을 가장 크게 받는다는 것을 의미한다. 붕어의 조직 손상도(degree of tissue changes)는 혼합 지점에서 채집한 시료(30.98 ± 5.40)에서 가장 컸으며, 그 다음은 하류 지점 시료(19.28 ± 4.31)이고 가장 낮은 것은 상류 지점 시료(4.83 ± 2.67)로 나타났다. 이러한 결과는 혼합 지점에서 채집한 붕어가 방류수에 가장 큰 영향을 받았다는 것을 의미하며, 산화적 스트레스와 조직병리학적 지표들이 방류수로 오염된 하천을 모니터링하는데 유용하다는 것을 나타낸다.

Keywords

1. Introduction

Effluents from sewage treatment plants (STPs) are highly complex and release high levels of pollutants into the aquatic environment (Woodworth et al., 1999), including polycyclic aromatic hydrocarbons, solvents, heavy metals, pharmaceuticals, and flame retardants (Halling-Sorensen et al., 1998; Paxeus, 1996). STPs usually process a vast amount of chemicals from households, industries, and hospitals, and, due to insufficient biodegradation, the effluents consist of a complex mixture of compounds (Paxeus 1996; Paxeus and Schroder 1996). Several studies conducted on the effects of STP effluents have revealed that they exert physiological effects (Hoeger et al., 2004; Sepulveda et al., 2004; Svenson et al., 2002), generate immune responses (Larsson et al., 1999; Oakes et al., 2004), cause oxidative stress, and exert estrogenic effects (Burkhardt-Holm et al., 1999; Ma et al., 2005; Oakes et al., 2004; Sole et al., 2002) in fish caged/caught from contaminated water bodies. In this context, the use of sentinel organisms for environmental quality monitoring provides a sensitive and reliable approach to estimate the potential effects of pollutants (Farrington and Tripp, 1995). Among them, fish are recognized as an excellent experimental model for use in toxicological studies as they are at the top of the aquatic food chain and respond strongly to stress conditions (van der Oost et al., 2003).

Biochemical variables, such as antioxidant defense enzymes, have recently been used as indicators of water quality to detect the sublethal effects of pollutants. In particular, several studies have demonstrated that biomarkers of oxidative stress can provide satisfactory information on the response of fish to environmental stressors (Farombi et al., 2007; Miller et al., 2007; Monterio et al., 2007; Pavlović et al., 2010). Oxidative stress results from an imbalance between the formation of reactive oxygen species (ROS) and the activity of the cellular antioxidant defense system, leading to oxidative damage of cellular molecules (Kohen and Nyska, 2002). ROS are generally produced during metabolism, and are advantageous under natural conditions (Dröge, 2002), although they can also cause molecular damage. Exposure to anthropogenic compounds can also increase the production of ROS within the cell. This occurs via the malfunction of a series of enzymes in the cytochrome P450 system, and through the redox cycling of various xenobiotics (Di Giulio et al., 1995). Therefore, sensitive and reliable biomarkers should be used to determine significant contaminant exposure that has exceeded detoxification or compensatory mechanisms and/or has resulted in adverse effects on physiological and biochemical functions (Vijayavel et al., 2004). Biochemical biomarkers represent early diagnostic tools, since they can identify changes at a sub-organismal level (i.e., cellular and molecular, etc.) before becoming evident at higher levels of biological organization (Doherty et al., 2010). Similarly, histopathological changes have been widely used as biomarkers to evaluate the health of fish exposed to contaminants, both in the laboratory (Thophon et al., 2003; Wester and Canton, 1991) and in field studies (Hinton et al., 1992; Schwaiger et al., 1997; Teh et al., 1997) due to target organ toxicity. Furthermore, alterations in organs are normally easier to identify than alterations in function (Fanta et al., 2003), and serve as warning signs of potential damage to animal health (Hinton and Lauren, 1990). Therefore, antioxidant defense and histopathology have been accessed in fish in the field and in laboratory studies (Hinton et al., 1992; Regoli et al., 2002; Schwaiger et al., 1997; Teh et al., 1997; Valavanidis et al., 2006; van der Oost et al., 2003).

Although the oxidative stress parameters are used in field toxicology studies with respect to antioxidant defenses (Regoli et al., 2002; Valavanidis et al., 2006; van der Oost et al., 2003), there are large gaps in knowledge on how these complex mixtures of chemicals affect oxidative stress in aquatic organisms. In addition, limited knowledge is available on histopathological responses in fish to effluents from STPs. Therefore, the aim of the present study was to gain a better understanding of the impacts of STP effluents on freshwater crucian carp Carassius auratus from a contaminated stream by integrating measurements of oxidative damage with histopathology. The crucian carp was selected for use in this study due to a scarcity of information and its position as a dominant species in the stream.

 

2. Materials and Methods

2.1. Fish Sampling

Fish sampling was conducted in June-July 2015 at three selected sites in the Eungcheon stream located in Geumwangeup, Chungcheongbuk-do, Korea. The Geumwang STP treats about 6000 m3/day of sewage and directly discharges effluent into this stream. Fish, (crucian carp Carassius auratus) were collected from the mixing zone (N 37' 00' 16.58" E 127' 35' 40.82") where sewage effluent enters and mixes with the stream, and from two other locations along the stream approximately 150 m upstream (N 37' 00' 12.06" E 127' 35' 40.77") and 800 m downstream (N 37' 00' 44.27" E 127' 35' 46.43") of the effluent discharge point.

Fish samples were collected from various habitats, including riffle, run, and pool at sampling sites using a casting net (5 × 5-mm mesh, 15 times per site) and a skimming net (4 × 4-mm mesh, 40 min per site). Following collection, 10 individual fish (> 4 cm in total length) were randomly selected irrespective of sex from each sampling site, which were then transferred to the laboratory within 2 h. Fish care and handling were performed following the guideline of the Institutional Animal Care and Use Committee of Korea University. For histopathological examination, the gills, liver, and kidney were removed, taking care to keep the tissue as intact as possible, and then fixed with 10% neutral formalin solution in the field. For biochemical analyses, fish were brought to the laboratory alive, and the organs were dissected out and immediately frozen and stored at -80℃.

2.2. Water Sampling and Chemical Analyses

Water samples were collected at the same sites where fish sampling was conducted, transported in polyethylene containers on ice to the laboratory, and then stored at 4℃ throughout the entire study period. Dissolved oxygen (DO) concentration, water temperature, pH, and electrical conductivity (EC) of the samples were measured using a multi-parameter water quality meter (YSI- 556, Yellow Springs Instruments, OH, USA) in the field.

Water quality parameters and hazardous water pollutants under regulation for effluent limits in Korea were analyzed according to the Standard Methods for Examination of Water Quality, Ministry of Environment, Korea (MOE, 2014). The water quality parameters measured were suspended solid (SS), biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP). The water pollutants determined were: Cu, Pb, As, Hg, Cr(VI), Cd, Ni, Zn, Se, cyanide, trichloroethylene (TCE), perchloroethylene (PCE), phenols, benzene, 1,2-dichloroethane, chloroform, organophosphorus compound, carbon tetrachloride, di(2-ethylhexyl) phthalate (DEHP), 1,1-dichloroethylene, 1,4-dioxane, vinyl chloride, and acrylonitrile.

2.3. Oxidative Stress Analyses

Catalase (CAT) activity was measured based on the methods described by Aebi (1974). Briefly, tissue samples were homogenized in a mortar-driven Teflon homogenizer with 50 mM phosphate buffer (pH 7.8). The homogenates were then centrifuged at 4500 rpm for 10 min at 4℃ and the supernatants were taken for further analysis. CAT activity was measured based on the decrease in absorbance at 240 nm due to H2O2 consumption (εmM = 0.0436). The reaction volume contained 20 μL of the supernatant, 1.98 mL of 50 mM phosphate buffer (pH 7.0), and 1 mL of 30 mM H2O2.

Glutathione S-transferases (GST) activity was measured as described by Habig et al. (1974). Briefly, tissue samples were homogenized with 20 mM phosphate buffer (pH 6.5) using a mortar-driven Teflon homogenizer. The homogenates were then centrifuged at 10,000 rpm for 10 min at 4℃ and the supernatants were used for further analysis. GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was determined spectrophotometrically at 340 nm (εmM = 9.6). The reaction volume consisted of 50 μL of the supernatant, 2.5 mL 20 mM phosphate buffer (pH 6.5), 0.3 mL reduced glutathione (10 mM), and 0.15 mL CDNB solution (1.5 mM).

Lipid peroxidation (LPO) was analyzed according to the method described by Barata et al. (2005) using a malondialdehyde (MDA) kit (NWK-MDA01, Northwest Life Science Specialties, Vancouver, WA, USA). Briefly, tissue samples were homogenized in a mortar-driven Teflon homogenizer with 1.15% KCl at pH 7.4 (100 mM phosphate buffer). The homogenates were then centrifuged at 4500 rpm for 10 min at 4℃. The LPO level was evaluated by measuring the production of MDA reacting with thiobarbituric acid at 60℃ for 1 h using a microplate spectrophotometer at 532 nm (BioTek Inc., Winooski, VT, USA) according to manufacturer’s instruction. The protein content of all tissue samples was determined spectrophotometrically based on Lowry et al. (1951) using bovine serum albumin (Bio-Rad Laboratories., Hercules, CA, USA) as a standard, and enzyme activity was recorded in units per mg protein (U mg-1).

2.4. Histological Analyses

Histological examinations were performed as described by Ghosh, (1991). Formalin-fixed organs were dehydrated in a graded series of ethanol solutions (70, 90, and 95%). After being embedded in paraffin at 58-60℃, the tissue specimens were sliced using a microtome (SLEE MAINZ, CUT 5062, Germany), subsequently stained with hematoxylin and eosin (H&E), and examined using an optical light microscope (Fluorescence Upright Microscope, Axio Imager M1, Carl-Zeiss, Germany).

Histological alterations in each organ were evaluated semi-quantitatively using two criteria, mean assessment value (MAV) and degree of tissue changes (DTC), in six randomly selected sections per tissue. MAV was estimated based on the methods described by Schwaiger et al. (1997). Alterations were evaluated based on symptoms in the respective tissues as presented in Table 1. To calculate MAV, a numerical value was given for each pathological alterations: 1 = no pathological alteration; 2 = focal changes (i.e., very low to moderate alterations) and 3 = extended pathological alterations (i.e., highest alterations). Based on these classifications, MAV was calculated for each tissue for each fish and an overall MAV was obtained for each sampling site.

Table 1.Histopathological alterations in the gills, liver, and kidneys of Carassius auratus collected at upstream, mixing, and downstream sites of the Eungcheon stream. Tissue damage was classified as stage I (less damage to the tissues, recovery is still possible), stage II (severe damage to the tissues, normal functions are affected), and stage III (extremely severe, irreparable damage)

DTC was estimated based on the severity of lesions and their possibility of recovery as reported by Poleksić and Mitrović-Tutundžić (1994). Tissue lesions were classified into three progressive stages as shown in Table 1: stage I = less damage to the tissues such that recovery is still possible; stage II = severe damage to the tissues such that normal functions are affected, and stage III = extremely severe damage, causing irreparable effects. DTC was calculated by summing up the number of lesions within each of three stages multiplied by stage index, using the following mathematical equation proposed by Poleksić and Mitrović-Tutundžić (1994):

where I, II, and III are the number of stage I, II, and III lesions, respectively. The DTC value obtained for each sampling site was used to categorize overall tissue damage based on the following five classifications: A (0-10) = normal tissue function; B (11-20) = slight damage; C (21-50) = moderate damage; D (51-100) = severe damage and E (>100) = extremely severe and irreversible.

2.5. Statistical Analysis

One-way analysis of variance (ANOVA) was conducted in SPSS program (version 18.0) to analyze biochemical and histopathological changes among different sampling sites with the least significant difference (LSD) test at the p < 0.05 level.

 

3. Results and Discussion

3.1. Physicochemical Quality of Stream Water

Stream water quality is essential as it plays an important role in regulating different metabolic and physiological processes. The physicochemical properties of stream water were determined at each sampling site, namely the mixing zone, and upstream and downstream sites in the Eungcheon stream, and are presented in Table 2. In the present study, none of the water samples contained DO below 4 mg/L, which is considered critical for fish (Esteves, 1988). Each site had an EC value above 100 μS/cm, which was higher in the mixing zone and downstream site than in the upstream site, indicating impaired water quality (Olsen et al., 2001). In addition, increased loading of particles (SS), organic contaminants (BOD and COD), and nutrients (TN and TP) was detected at the mixing zone and downstream site, indicating the influence of effluent discharge from the Geumwang STP. In general, levels of both organic and inorganic contaminants are increased in the mixing zone due to effluent discharge (Jingsheng et al., 2006; Kim et al., 2014), and gradually decrease downstream through self-purification processes without other disturbances (Giller and Malmqvist, 2006; Hyens, 1960). However, the downstream site in this study showed higher values of EC, SS, BOD, and TP than the mixing zone in the most measured variables, probably due to the influence of sewage discharge from houses located near the stream channel. Overall, the water quality of the study sites was impaired by the influence of effluent discharge.

Table 2.* Detection limit; ** Not detected

Chemical analysis of 23 hazardous water pollutants indicated that only Cu and Zn are present in the Eungcheon stream at levels above the limits of detection (Table 2). Considering that concentrations of Cu and Zn in rivers and streams of Korea (total 113 locations) from 2004 to 2005 were 1.24-46.84 μg/L (mean 12.14 μg/L) and 2.08-293.78 μg/L (mean 52.88 μg/L), respectively, the upstream site was not likely to be contaminated by Cu and Zn (MOE, 2006). However, the concentrations of Cu and Zn (2.5 and 20.0 μg/L, respectively) were substantially higher in the mixing zone than in the upstream site. Yoo et al. (2013) demonstrated that concentrations of Cu and Zn (1.2-21 and 1.53-1668 μg/L, respectively) in Hantan River, Korea were elevated due to it receiving wastewater effluents. Song et al. (2010) also reported that heavy metals were enriched in the Chang Jiang River of China following an increase in the concentration of SS, possibly due to wastewater or soil erosion. Higher concentration of Zn found at the mixing zone may interfere with the structural integrity of fish as well as membrane bound enzyme activity when Zn accumulates in fish beyond threshold optimum concentrations (Tuurala and Soivio, 1982). Likewise, Cu only found at the mixing zone may cause structural damage to fish (Patel and Bahadur, 2010).

3.2. Oxidative Stress in C. auratus

Activities of antioxidant enzymes (CAT and GST) and lipid peroxidation (LPO) in the gills, liver, and kidney of the freshwater crucian carp, C. auratus were analyzed to identify changes in oxidative stress. CAT activity in C. auratus collected from the mixing zone and downstream site of the Eungcheon stream was notably higher than that in samples from the upstream site (Fig. 1(a)). CAT plays a critical role in scavenging H2O2, which is a by-product produced by the dismutation of superoxide anion radicals (O2-.) by oxidative stress (Martinez-Alvarez et al., 2005). Enhanced CAT activity is usually observed in the presence of environmental pollutants (Dautremepuits et al., 2004) since CAT in combination with superoxide dismutase represents the first line of defense against oxidative stress (Bebianno et al., 2004). Therefore, elevated levels of CAT, as observed in the present study, reflect a reinforced antioxidant response generated by sewage effluent. Yildirin et al. (2011) reported that stressful conditions might lead to the formation of excessive free radicals, which are considered a major internal threat to cellular homeostasis in aerobic organisms.

Fig. 1.Levels of (a) catalase (CAT), (b) glutathione S-transferases (GST), and (c) lipid peroxidation (LPO) in the gills, liver, and kidneys of Carassius auratus collected at upstream, mixing, and downstream sites of the Eungcheon stream. Data represent mean ± standard deviation (n = 6). Different letters above the columns indicate significant differences (p < 0.05) between sites.

GST activity in the gills of C. auratus collected from the mixing zone and downstream site of the Eungcheon stream was significantly increased (p < 0.05) compared to that in gills of fish collected from the upstream site (Fig. 1(b)). In the liver, GST activity was also significantly higher in fish from the mixing zone than in those from the upstream site, but significantly decreased activity was observed in fish from the downstream site (p < 0.05). However, significantly decreased GST activity was found in the kidney of fish from both the mixing zone and the downstream site (p < 0.05). GST plays a key role in the biotransformation of xenobiotics and can stimulate electrophile metabolites and glutathione (GSH) to improve their hydrophobicity and subsequent excretion. Additionally, GST can inhibit lipid peroxidation, directly inactivates ROS via SH groups, and indirectly induces DNA repair (Choi et al., 2008). Enhanced levels of GST in the gills and liver suggest an adaptive and protective role of this biomolecule against oxidative stress induced by sewage effluents. These results are consistent with the findings of Di Giulio et al. (1993), who reported higher levels of GST in catfish exposed to polluted waters than in the control, and with those of Pandey et al. (2003), who observed increased GST activity in Wallago attu fish collected from the Panipal River in India. A decline in GST level in the kidney of C. auratus may occur because GSH is a substrate, and was largely consumed. In addition, many intermediate metabolites produced during detoxification could reduce GST activity or competitively inhibit GST substrates (Egaas et al., 1999). Similar results were also reported by Mather-Mihaich and Di Giulio (1986), who observed a decrease in GSH levels in channel catfish exposed to bleached kraft mill effluent. Therefore, decreased GST levels in fish from the mixing zone and the downstream site suggest that the ability to protect against toxicants was reduced due to increased utilization of GSH, which can be converted into oxidized glutathione, and inefficient GSH regeneration.

As shown in Fig. 1(c), the LPO level in the gills and kidney was significantly lower in fish from the mixing zone and the downstream site than in those from the upstream site (p < 0.05). However, the LPO level in the liver was significantly higher in fish from the mixing zone than in those from the upstream site, but was lower in fish from the downstream site (p < 0.05). Oxidative stress may affect biomolecules such as proteins, lipids, and DNA when antioxidant defenses are impaired or overcome (Doherty et al., 2010; Farombi et al., 2007; Pavlović et al. 2010). Increased levels of LPO may be related to poor water quality, and the level directly reflects the degree of oxidative damage (Charissou et al., 2004). Significantly reduced levels of LPO in the gills and kidney samples from the mixing zone and downstream site compared to those from the upstream site indicate lower susceptibility of lipid molecules to ROS and the extent of oxidative damage. Conversely, higher levels of LPO in the livers of fish from the mixing zone than in those from the upstream site could be attributed to the high antioxidant (CAT and GST) activity recorded in this study (Fig. 1(a) and 1(b)). Atli et al. (2006) reported that livers have a higher metabolic activity than other organs, suggesting that the liver is more sensitive to toxic pollutants. In addition, the antioxidant defense system preferentially develops in the liver due to its central role in detoxifying environmental pollutants and metabolic products (Li et al., 2010).

3.3. Histopathological Alterations in C. auratus

Details of histopathological alterations in the gills, liver and kidney of C. auratus from the upstream site, mixing zone, and downstream site are shown in Figs. 2, 3, and 4, respectively. Fish gills are the main respiratory organ for gaseous exchange, osmoregulation, excretion of nitrogenous waste products, and acid-base regulation. Due to their direct contact with the external environment, particularly with water, gills are considered to be the primary target of contaminants (Fernandes and Mazon, 2003; Poleksić and Mitrović-Tutundžić, 1994). Hyperplasia and hypertrophy of the gill epithelium, epithelial lifting of lamellae, and lamellar disorganization were common characteristics of fish gills collected from the upstream site (Fig. 2 and Table 1). However, blood congestion, dilation of the marginal channel, lamellar fusion, rupture of chloride cells, and rupture of the lamellar epithelium, along with aforementioned lesions, were prominent pathological characteristics in gills collected from the mixing zone and downstream site. As indicated in Fig. 5, values of MAV and DTC were significantly higher in gills from fish collected in the mixing zone and downstream site than those collected from the upstream site (p < 0.05). Estimated MAV for samples from the upstream site was 1.17 ± 0.12 indicating very few alterations, while it was 1.82 ± 0.20 and 1.33 ± 0.08 for samples from the mixing zone and downstream site, respectively, demonstrating low to moderate alterations with some high alterations in tissues. Conversely, the DTC for samples from the upstream site was 1.67 ± 1.21, while for samples from the mixing zone and downstream site, the DTC was 9.67 ± 6.65 and 4.00 ± 3.95, respectively. The DTC value in samples from all sites was below 10, indicating normal tissue function (class A) predominantly with stage I changes.

Fig. 2.Histological micrograph of the gill of Carassius auratus collected from the Eungcheon stream at (a) an upstream site, showing fusion of secondary gill lamellae (SGL) (black arrow) and hypertrophy (broken arrow); (b) the mixing zone, showing curling of SGL (square), fusion (black arrow) and damage of chloride cells (white arrow); (c) the mixing zone, showing fusion of secondary gill lamellae (SGL) (black arrow), curling (square) and hypertrophy (broken arrow); and (d) a downstream site, showing fusion of secondary gill lamellae (SGL) (black arrow), curling (square) and damage of chloride cells (white arrow).

Fig. 3.Histological micrograph of the liver of Carassius auratus collected from the Eungcheon stream at (a) an upstream site, showing hepatocytes (black arrow) containing nuclei (white arrow) and reduced vacuolation (broken arrow); (b) the mixing zone, showing degenerating hepatic cells (black arrow) and vacuolation (broken arrow); (c) the mixing zone, showing degenerating hepatocytes (black arrow) with fribrillar inclusion in their cytoplasm (white arrow), pyknotic nuclei (arrow head) and cytoplasmic vacuolation (broken arrow); (d) the mixing zone, showing hepatocytes detaching from the pancreas (P) (arrow head) and damage of pancreas (black arrow); and (e) a downstream site, showing hypertrophic hepatocytes (white arrow), clumping of hepatocytes (black arrow) and less vacuolation (broken arrow).

Fig. 4.Histological micrograph of kidneys of Carassius auratus collected from the Eungcheon stream at (a) an upstream site, showing proximal convoluted tubules (PCT) and distal convoluted tubules (DCT), glomerulus (G), and some tubular damage (black arrow); (b) the mixing zone, showing a degenerative kidney tubule (broken arrow) with fragmented glomerulus (white arrow), narrowing of tubules (arrow head) and damage of tubules (black arrow); (c) the mixing zone, showing tubular damage (black arrow) and shrinkage of glomerulus (white arrow); (d) a downstream site, showing tubular damage (black arrow) and proliferated glomerulus (broken arrow); and (e) a downstream site, showing shrinkage of glomerulus (white arrow) and damage of tubules (black arrow).

Fig. 5.(a) Mean assessment value (MAV) and (b) degree of tissue changes (DTC) in the gills, liver, and kidneys of C. auratus collected at upstream, mixing, and downstream sites of the Eungcheon stream. Data represent mean ± standard deviation (n = 6). Different letters above the columns indicate significant differences (p < 0.05) between sites.

Coutinho and Gokhale (2000) observed similar findings, namely hyperplasia and hypertrophy of the gill epithelium, epithelial lifting of lamellae, and lamellar disorganization in the gills of carps (Cyprinus carpio) and tilapias (Oreochromis mossambicus) exposed to wastewater treatment plant effluents. In addition, Winkaler et al. (2001) reported hyperplasia, hypertrophy, dilation of the marginal channel, and aneurysms in Neotropical fish, Astyanax altiparanae collected from the Cambe stream, and demonstrated that contamination of the stream was responsible for structural damage to the fish gill. Epithelial lifting, lamellar disorganization, and lamellar fusion, as observed in the present investigation, are examples of defense mechanisms, which permit the entry of pollutants by increasing the distance between the external environment and the blood. As a consequence of the increased distance between the water and blood, oxygen uptake can be impaired. However, fish have the capacity to increase their ventilation rate to compensate for low oxygen uptake (Fernandes and Mazon, 2003). A high incidence of dilation of the marginal channel is usually caused by the rupture of pillar cells (Rosety-Rodriguez et al., 2002). This simultaneously leads to blood congestion and sometimes the formation of an aneurysm due to increased blood flow because of the direct effects of effluents (Poleksić and Mitrović-Tutundžić, 1994).

The liver is a primary metabolic organ, which plays an important role in detoxification and subsequent elimination of harmful substances (van der Oost et al., 2003). The normal liver is characterized by a prevalence of regular-shaped hepatocytes surrounding bile ducts and pancreatic cells. Livers collected from fish from the upstream site exhibited very few morphological changes, including nuclear hypertrophy, cellular hypertrophy, and cytoplasmic vacuolation (Fig. 3 and Table 1). Conversely, fish collected from the mixing zone and downstream site showed various pathological alterations, such as irregular shaped cells, irregular shaped nuclei, nuclear hypertrophy, cellular hypertrophy, cytoplasmic degeneration, nuclear degeneration, cellular rupture, pyknotic nuclei, and bile stagnation, and the extent of the alterations were higher in samples from the mixing zone compared to those from the downstream site. As shown in Fig. 5, the observed MAV in liver was significantly higher in fish collected from the mixing zone (1.95 ± 0.17) and downstream site (1.56 ± 0.07) compared to those collected from the upstream site (1.18 ± 0.08) (p < 0.05). Higher MAV values in samples from the mixing zone and downstream site indicate predominately low to moderate alterations, with some high alterations in the investigated tissue. DTC was significantly higher in samples from the mixing zone and downstream site than in those from the upstream site (p < 0.05). For samples from the upstream site, DTC was 2.83 ± 1.33, indicating normal tissue function (class A) with mainly stage I changes. The results for DTC in samples from the mixing zone and downstream site were 42.17 ± 5.60 and 28.33 ± 5.24, respectively, indicating moderate to severe tissue damage (class C) with predominantly stage II lesions as well as stage I changes.

Common pathological lesions including nuclear hypertrophy, cellular hypertrophy, and cytoplasmic vacuolation, as observed in the present study, were also reported by Figueiredo-Fernandes et al. (2007) in Oreochromis niloticus and by Mario et al. (2011) in the liver of both Carassius auratus and Dicentrarchus labrax. Vacuolization, as observed in hepatocytes, indicates an imbalance between the rate of substrate synthesis in the parenchymal cells and the rate of their release into the systemic circulation (Gingerich, 1982). In addition, cellular vacuolization may be attributed to the accumulation of lipids and glycogen due to liver dysfunction. Pacheco and Santos (2002) reported that increased vacuolization of the hepatocytes is a signal of degeneration, which suggests metabolic damage, possibly related to exposure to contaminated water. A prevalence of irregular shaped cells, irregular shaped nuclei, nuclear degeneration, cellular rupture, pyknotic nuclei, and bile stagnation were also found in samples from the mixing zone. Navaraj and Yasmin (2012) reported similar findings in the liver of Oreochromis mossambicus exposed to tannery industry effluents. This observation is further corroborated by the findings of Olojo et al. (2005) in Clarias gariepinus collected from a metal contaminated ecosystem. The histopathological changes in the liver confirmed that these lesions also cause metabolic problems. The presence of bile stagnation in the liver of C. auratus indicates that the organ suffered structural and metabolic damage following exposure to sewage effluent, reinforcing the idea that this environment is substantially impaired (Pacheco and Santos, 2002).

Common alterations observed in the kidneys of fish collected from contaminated streams include nuclear hypertrophy, cellular hypertrophy, dilation of glomerulus capillaries, and cytoplasmic vacuolation (Takashima and Hibiya, 1995). In the mixing zone and downstream site, hyaline droplet degeneration, nuclear degeneration, tubular degeneration, cellular rupture, occlusion of tubule lumen, enlargement of glomerulus, dilatation of glomerulus capillaries, reduction of Bowman’s space, and glomerular degeneration were the most common lesions, and the changes were more severe in samples from the mixing zone than in those from the downstream site (Fig. 4 and Table 1). As indicated in Fig. 5, the MAV and DTC in kidneys were significantly higher in fish collected from the mixing zone and downstream site compared with those collected from the upstream site (p < 0.05). The MAV for fish from the upstream site was 1.33 ± 0.07, while it was 1.65 ± 0.6 and 1.40 ± 0.08 for fish from the mixing zone and downstream site, respectively. Higher MAV values were observed for liver tissue sampled from fish in the mixing zone and downstream site, which indicates predominantly low to moderate alterations with some high level alterations in kidney. The DTC for samples from the upstream site was 10.00 ± 5.48, which indicates normal tissue function (class A), with mainly stage I changes. Conversely, DTC values for samples from the mixing zone and downstream site were 41.12 ± 3.95 and 25.50 ± 3.73, respectively, indicating moderate to severe tissue damage (class C), with mainly stage I and II changes.

Similar findings, including dilation of the kidney tubules, shrinkage of the glomerular tuft, and vacuolation of blood cells have been also reported in Rasbora daniconius exposed to industrial wastewater (Pathan et al., 2009). During hyaline droplet degeneration, irregular-sized eosinophilic granules of may appear in the cytoplasm, and the accumulation of these granules can lead to necrosis. Granules may also be produced within the cell itself or by reabsorption of excess amounts of proteineous substances following filtration through the glomerulus (Takashima and Hibiya, 1995). These types of cellular injuries may result in reduced levels of intracellular ATP, which in turn would impair the action of the cation pump, permitting the influx of sodium, chloride, calcium, and water, increasing the cell volume and damaging the cell membrane, with the efflux of some ions (K+), enzymes and other proteins (Rand, 1995; Takashima and Hibiya, 1995). Occlusion of the proximal or distal segments of the renal tubule was observed frequently in fish from the mixing zone. This can occur in response to the accumulation of certain materials in the lumen (Takashima and Hibiya, 1995) and impairs the flow of the filtrate and delays the processes of reabsorption and secretion in the tubule (Hinton and Lauren, 1990). Since fish osmoregulate a large volume of blood through the kidneys, xenobiotics present in the blood can cause pathological changes to the Bowman’s capsule, including a reduction in Bowman’s space and sometimes rupture (Takashima and Hibiya, 1995). Similar alterations were also observed in the posterior kidney of Barbatula barbatula collected from two streams contaminated with pesticides and heavy metals (Schwaiger et al., 1997), in Prochilodus lineatus after trichlorfon exposure (Veiga et al., 2002), in Salmo trutta and B. barbatula both caged in streams contaminated with pesticides, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and metals (Gernhofer et al., 2001), in Trichomycterus brasiliensis exposed to organic mercury (Oliveira Ribeiro et al., 1996), and in Anguilla anguilla exposed to various concentrations of resin acids and pulp mill effluent (Pacheco and Santos, 2002).

Histopathological alterations observed in the gills, liver, and kidney of the C. auratus in the present study indicate that fish were responding to the direct effects of the contaminants present in the effluent as much as they were responding to the secondary effects caused by stress. It must be emphasized that histopathology is able to evaluate the early effects and responses to acute exposure to chemical stressors. The degree of alteration in fish tissue was in the order liver > kidney > gills. The liver was most affected because it is the organ responsible for the primary metabolism of the xenobiotics and plays a vital role in the biotransformation of these xenobiotics. Of the three sites studied, the mixing zone, which receives sewage effluent, was considered to be the most impaired because of high DTC values found for all three organs examined in fish sampled from this site. The average values determined for all tissues indicate that the highest DTC was observed in fish from the mixing zone (30.98 ± 5.40) followed by the downstream site (19.28 ± 4.31), with the lowest values in fish sampled from the upstream, or reference, site (4.83 ± 2.67). Reduced effects in fish sampled from the downstream site might be due to the distance from the point of effluent discharge, which reduces the effect of hazardous water pollutants by dilution and sedimentation processes.

 

4. Conclusions

The results of this study showed that effluents from a SWP induced oxidative stress in C. auratus living in the contaminated stream, as evidenced by the accumulation of lipid peroxidation as well as the induction of antioxidant enzymes, CAT and GST. In addition, significant histopathological changes in the gills, liver, and kidney of the crucian carp further confirm the adverse effects of the sewage effluent. These findings suggest that the integrated use of both semi-quantitative histopathology and biochemical indices is a useful tool to monitor contaminated rivers and streams. In order to identify the exact causes of damage in C. auratus, further investigations should be performed by measuring the levels of environmental contaminants in fish tissues.

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