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Effects of antioxidant enzymes and bioaccumulation in eels (Anguilla japonica) by acute exposure of waterborne cadmium

Effects of antioxidant enzymes and bioaccumulation in eels (Anguilla japonica) by acute exposure... This study was conducted to evaluate the acute effects of waterborne cadmium exposure on bioaccumulation and antioxidant enzymes in eels (Anguilla japonica) and to determine the median lethal concentration (LC ). Fish −1 were exposed to different cadmium concentrations (0, 0.15, 0.30, 0.61, 1.83, 3.08, 3.67, 4.29, and 5.51 mg L )for −1 96 h. The LC of A. japonica to cadmium was 3.61 mg L . Cadmium accumulation generally increased in tissues −1 with increasing waterborne cadmium concentrations. At ≥ 1.83 mg L exposure, all tissues accumulated significant cadmium concentrations compared with the control group, in the order of kidney > liver > gill > spleen > muscle. Measurements of variation in actual cadmium concentrations showed that a reduction of the metal in experimental water was related to cadmium accumulation in tissues. As activity alteration of antioxidant enzymes for reactive oxygen −1 species, superoxide dismutase and catalase activities increased at ≥ 0.61 mg L significantly, glutathione peroxidase and glutathione S-transferase activities were not significantly changed. The results of this study suggest that acute exposure to waterborne cadmium is potentially fatal to A. japonica due to the metal’s major accumulation in various tissues and the effect of antioxidant enzyme activity. Keywords: Cadmium, Anguilla japonica, Acute toxicity, LC , Bioaccumulation, Antioxidant enzyme Introduction of gills (Verbost et al. 1987, 1988, 1989; Pinot et al. Metals naturally exist in aquatic ecosystems, but side ef- 2000). Cd redox activity affects antioxidants, thus redu- fects from industrialization have resulted in excessive con- cing protection against oxidative stress, increasing lipid centrations. Exposure to high metal levels may negatively peroxidation, and decreasing DNA synthesis (Okorie affect fish and other aquatic organisms, hampering physio- et al. 2014). In addition, Cd lowers plasma Na, Cl, and logical functions, growth rate, and reproduction, or even K, leading to hyperglycemia and hypermagnesemia increasing mortality (Reddy and Reddy 2013;Öz 2018;Öz (Larsson et al. 1981; Haux and Larsson 1984; Sjöbeck et al. 2018). Cadmium is a particularly widespread and et al. 1984). Even at low concentrations, Cd deforms tis- toxic example that is documented to accumulate in ex- sues and vertebrae, causing respiration abnormalities posed organisms; it is used primarily in alloys, pigments, and death in fish (De Smet and Blust 2001). Cd and electroplating, and batteries (Bryan 1976; Farag et al. 1995; other toxic heavy metals can also accumulate through Adriano 2001;Javed 2003). direct absorption or biomagnification; the resultant in- In fish, Cd disrupts Ca metabolism through competi- hibition of major organ function (i.e., liver, kidney, and tion for transport sites on the basolateral calcium pumps gills) is strongly linked to toxicity. Thus, the degree of accumulation in each organ is used frequently as a bio- monitor for metal contamination (Handy 1992). * Correspondence: jckang@pknu.ac.kr In fish exposed to Cd, reactive oxygen species (ROS), Department of Aquatic Life Medicine, Pukyong National University, Busan such as hydrogen peroxide (H O ), hydroxyl, and oxygen 48513, South Korea 2 2 Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 2 of 10 radical, occur and induce oxidative stress. As a result, kidney, spleen, gills, and muscle) and change of antioxi- the biological system induces antioxidant enzymes, e.g., dant enzymes (SOD, CAT, GPx, and GST) in the liver superoxide dismutase (SOD), catalase (CAT), glutathi- with the determination of LC for Cd in adult A. one peroxidase (GPx), glutathione S-transferase (GST), japonica. to mitigate the attack of ROS. These enzymes are used as stress biomarkers in fish by exposure or contamin- Materials and methods ation of heavy metals and generation of ROS. SOD is Experimental fish and design catalyzing the transformation of superoxide anion radi- Anguilla japonica specimens were collected from the eel cals to H O and oxygen (O ). Catalase (CAT) decom- aquafarm of Paju city, Gyeonggi province, South Korea. 2 2 2 poses toxic H O to O and H O. Glutathione Fish were acclimated to a polyvinyl (PVC) tank for 2 2 2 2 2 peroxidase (GPx) decomposes H O or organic hydro- weeks prior to experiment and food-deprived. Also, we 2 2 peroxide to H O or corresponding alcohols using re- identified no infection of parasites in some fish before duced glutathione (GSH) into oxidized glutathione acclimation and toxicity test to prevent mortality by par- (GSSG). Glutathione S-transferase (GST) detoxifies the asites and used visually healthy fish for the experiment. reactive intermediates and oxygen radicals by catalyzing Acute Cd toxicity test was conducted under laboratory the conjugation of GSH to various electrophilic metabo- conditions. Acclimated fish (n = 90; average weight lites, thereby enhancing water solubility and assisting ex- 186.6 ± 11.9 g) were selected, divided into nine groups cretion (Livingstone 2003). (10 per group), and placed into plastic aquaria (555 × Two standard tests of metal toxicity are acute or 395 × 310 mm) filled with underground water. Table 1 chronic exposure. In many organisms including fish, summarizes the water quality parameters measured for acute toxicity is defined as LC (median lethal concen- the bioassay. Water temperature was maintained with a tration), a concentration that kills approximately 50% of heater at 29 ± 1 °C. To make conditions similar to aqua- a test group after exposure to increasingly higher toxi- farm, the laboratory was kept in 24-h darkness except cant levels for a specified, relatively short time frame when checking fish mortality. During the exposure (Schreck and Moyle 1990; Mason 1991). Acute toxicity period, water was not renewed and fish were not fed. data are supplemented with chronic toxicity tests for the Analytical-grade CdCl (Aldrich, Inc., USA) was dis- same compound, exposing subject organisms to the solved in triple distilled water to prepare stock Cd solu- same low concentration over a longer period. Such tion used for exposure experiments (see the information is useful as a reference when performing en- “Determination of LC and assay of actual Cd levels in vironmental surveys of contaminated areas and deter- experimental water” section). mining the effects of toxicant efflux after industrial accidents. Determination of LC and assay of actual Cd levels in Eels are commonly consumed in Asia and are mostly experimental water produced through aquaculture. Farmed eels are fed paste Experimental fish were exposed to waterborne CdCl −1 that contains a high ratio of fish meal. Thus, Cd accu- treatments of 0.25, 0.5, 1, 3, 5, 6, 7, and 9 mg L , 0.15, −1 mulation can occur if the metal’s concentration in fish 0.30, 0.61, 1.83, 3.08, 3.67, 4.29, and 5.51 mg L as only meal is high. Eels suffer particularly high mortality under Table 1 Quality parameters of water used during acclimation Cd exposure, because their benthic lifestyle increases and experimentation contact with heavy metals that sink to the floor. These Parameters Value factors indicate that we require data on Cd effects in eels pH 7.96 ± 0.10 to ensure food safety and assess environmental contam- Salinity (‰) ≤ 0.1 ination. However, despite the progress made on under- −1 standing the outcome of Cd exposure in several fish Dissolved oxygen (mg L ) 7.75 ± 0.05 −1 species (Handy 1993; Yilmaz et al. 2004; Aldoghachi Chemical oxygen demand (mg L ) < 0.01 et al. 2016), little research has been conducted in eels, −1 NH -N (mg L ) 0.015 ± 0.005 especially Anguilla japonica. Furthermore, many bio- −1 NO -N (mg L ) 0.003 ± 0.001 accumulation studies focus on chronic exposure, despite −1 NO -N (mg L ) 4.3 ± 0.1 the possibility of industrial accidents causing acute Cd 2− −1 SO (mg L ) 8.0 ± 0.5 exposure and accumulation. If Cd in eels is highly accu- 4 3− −1 mulated after acute exposure, it can affect the health of PO (mg L ) 0.25 ± 0.05 −1 humans as food through a catch. Thus, the purpose of SS (mg L)28±2 this study is to assess risk as food, identify the effect on −1 Total hardness (CaCO mg L ) 150 ± 10 fish health, and utilize baseline data for chronic toxicity 2+ −1 Cd (mg L ) ≤ 0.0001 test by investigating accumulation in major tissues (liver, Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 3 of 10 Cd concentrations, for 96 h. A water-only control was in the liver were determined using the method of Brad- also used. Cd exposure concentrations were established ford (1976), with bovine serum albumin as a standard. after pre-experiment by referring to Cd chronic accumu- −1 lation concentration (0.1 mg L ) in eels, Anguilla japon- Statistical analysis ica, through previous study (Yang and Chen 1996). Dead Finney’s probit analysis was used to determine the LC fish were counted every 12 h and removed immediately of Cd in eels, along with confidence limits. Between- from the aquaria. Experimental water was collected to group differences in Cd bioaccumulation and activities measure actual Cd concentrations at 12 and 96 h after of antioxidant enzymes were analyzed using two tests as adding the stock Cd solution. Water samples were di- a one-way ANOVA depending on Levene’s test for equal luted with 2% nitric acid before analysis using ICP-MS variance. Duncan’s multiple range and Games-Howell (inductively coupled plasma mass spectrometry; NexION tests were used at P > 0.05 and P < 0.05 in equality of 300X, Perkin-Elmer Inc., USA). The change rate and variances, respectively. Significance of post hoc test was decrement of actual Cd were calculated as follows: set at P < 0.05. All statistics were performed in SPSS ver- (1) Change rate (%) = 100 × (1 − Cd concentration at sion 20 (IBM co., USA). 96 h ÷ Cd concentration at 12 h) −1 (1) Duncan’s multiple range test: kidney, SOD, CAT, (2) Decrement (mg L ) = Cd concentration at 12 h − GPx, and GST Cd concentration at 96 h (2) Games-Howell test: liver, spleen, gill, muscle Tissue analysis to confirm Cd bioaccumulation After a 96-h Cd exposure, gills, liver, kidney, spleen, and Results muscle samples of live fish were collected and kept at − Median lethal concentrations of Cd in A. japonica 80 °C until analysis. Tissues were freeze-dried and Mortality was first measured at a Cd concentration of ≥ digested with nitric acid (Suprapur grade, Merck, −1 3.08 mg L , and the mortality rate reached 100% at 5.51 Germany) and hydrogen peroxide (bioassay grade, −1 mg L . The number of dead fish increased with increas- Merck, Germany) in a microwave (START D, Milestone, ing Cd concentration. Based on mortality data, LC of Italy). The resultant solutions were diluted with triple 50 Cd in A. japonica after 24, 48, 72, and 96 h was 5.10, distilled water and subjected to ICP-MS (NexION, −1 4.04, 3.67, and 3.61 mg L , respectively (Table 2). Perkin-Elmer Inc., USA). Assay of antioxidant enzymes activity Variation in actual Cd levels of experimental water The Collected liver was homogenized with 0.1 M Variation in the actual Cd concentration of experimental phosphate-buffered saline (PBS) using tissue lyzer (Tis- water was measured to investigate correlations between sueLyser II, QIAGEN, Germany). The homogenate was Cd accumulation in A. japonica and changes to Cd centrifuged at 10,000g for 30 min under 4 °C. The super- levels during the acute toxicity test. We found that ac- natants were obtained and stored at − 80 °C until ana- tual Cd concentrations generally decreased after the 96- lysis. SOD activity was analyzed using the SOD assay kit h exposure (Table 3). Based on measurements from 12-h (Dojindo Co., Japan) measuring 50% inhibition rate for post-exposure, the lowest and highest rate change in the reduction reaction of 2-(4-lodophenyl)-3-(4-nitro- −1 concentration were 5.1% (at exposure to 5.51 mg L Cd) phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monoso- −1 and 16.8% (at 1.83 mg L ), respectively. In contrast, the dium salt (WST-1) and was expressed as U mg −1 lowest and highest absolute decrement of actual Cd con- protein . CAT activity was analyzed using the CAT centration (again measured 12-h post-exposure) were assay kit (Sigma-Aldrich Inc., USA) measuring the ab- −1 −1 −1 0.015 mg L (at 0.15 mg L ) and 0.664 mg L (at 3.67 sorbance of the chromogen versus the amount of re- −1 mg L ), respectively. sidual H O after reaction with samples and was 2 2 −1 expressed as U mg protein . GPx activity was analyzed Table 2 Estimated median lethal concentrations (LC ) and using the GPx cellular activity assay kit (Sigma-Aldrich 50 confidence limits Inc., USA) measuring the change in absorbance at 340 Period Concentration 95% confidence limits nm by the reduction reaction of tert-butyl hydroperoxide −1 −1 (h) (Cd mg L ) and was expressed as U mg protein . GST activity was Lower Upper analyzed using the GST assay kit (Sigma-Aldrich Inc., 24 5.10 4.37 6.84 USA) measuring the change in absorbance at 340 nm by 48 4.04 3.55 4.66 reaction of sample and substrate solution including 1- 72 3.67 3.25 4.10 chloro-2,4-dinitrobenzene (CDNB) and was expressed as 96 3.61 3.19 3.99 −1 −1 μmol min mg protein . Total protein concentrations Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 4 of 10 Table 3 The change rate and decrement of actual Cd japonica mortality increased with increasing Cd concen- concentrations in experimental water at two time points (12- tration and exposure period. Although previous studies and 96-h post-exposure) under different Cd concentrations had attempted to examine acute Cd toxicity in fresh- −1 Exposure Actual Cd conc. (Cd mg L ) Change Decrement water fish (e.g., tilapia, common carp, rasbora) conc. (Cd rate (%) (Cd mg 12 h 96 h (Rehwoldt et al. 1972; Shuhaimi-Othman et al. 2015), −1 −1 mg L ) L ) these were generally not enough for the overall assess- 0.15 0.156 0.141 9.6 0.015 ment of environmental pollution. Nonetheless, we com- 0.30 0.331 0.310 6.3 0.021 pared our results with several previous studies to 0.61 0.662 0.624 5.7 0.039 evaluate Cd acute toxicity (Table 4). In contrast with −1 1.83 2.148 1.786 16.8 0.362 3.61 mg L (after 96-h Cd exposure) in the LC of this study, the LC in tilapia sac fry (Oreochromis niloticus) 3.08 3.064 2.763 9.8 0.302 50 −1 and juvenile (Oreochromis sp.) were 1.6 mg L (after 24- 3.67 4.068 3.404 16.3 0.664 −1 h Cd exposure) and 0.7 mg L (after 96-h Cd exposure), 4.29 4.624 4.177 9.6 0.448 respectively (Andaya and Gotopeng 1982; Aldoghachi 5.51 5.715 5.421 5.1 0.294 et al. 2016). Moreover, the 96-h LC of Cd in adult −1 guppies (Poecilia reticulata) was 30.4 mg L (Yilmaz −1 Bioaccumulation et al. 2004), while it was 7.42 mg L in juvenile piauçu Cd exposure caused a net increase of Cd content in all (Luciobrama microcephalus) (Gomes et al. 2009). These tested A. japonica tissues compared with the control data indicate that between-species differences in life his- (Fig. 1). The order of Cd accumulation in tissues (in- tory, genetic composition, and individual conditions cluding control) was as follows: kidney > liver > gills > have a greater impact on fish tolerance (or sensitivity) to spleen > muscle, with the highest and lowest concentra- Cd toxicity than size and age (Rand et al. 1995). Ideally, −1 tions being 122.190 mg kg in the kidney (at 3.67 mg within-species comparisons would better indicate −1 −1 −1 L ) and 0.049 mg kg in the muscle (at 0.15 mg L )of whether our results are typical of A. japonica. However, exposed groups, respectively. As expected, accumulation although some studies examining acute Cd toxicity do rose with increasing exposure concentration. However, exist for this species, differences in experimental significant differences as compared with control in Cd water conditions (e.g., hardness, pH, temperature) accumulation across all tissues were observable at ≥ complicate the interpretation of any cross-study vari- −1 1.83 mg L Cd exposure. Individually, significant differ- ation (Shuhaimi-Othman et al. 2015). Regardless, this −1 ences were apparent at ≥ 0.15 mg L in the gill and studyprovidesimportant basicdatafor anyfuture −1 −1 muscle, ≥ 0.30 mg L in the liver, ≥ 0.61 mg L in the studyinvestigating chronicCdtoxicityin A. japonica −1 kidney, and ≥ 1.83 mg L in the spleen. and allows for further comparative analyses of Cd tol- erance among fish. Antioxidant enzymes activity Cd accumulation in tissues may differ according to After acute exposure to Cd during 96 h, activities of anti- metal’s form. Inorganic Cd tends to be accumulated in the oxidant enzymes (SOD, CAT, GPx, and GST) in eel’sliver liver, while Cd-thiols are readily accumulated in the kid- were determined (Fig. 2). Activities of SOD and CAT in- ney, considered the organ most sensitive to Cd toxicity creased as compared with control by increasing exposure (Hammond and Foulkes 1986; Woo et al. 1993; Okorie concentration. Significant increase from control (as 1898 et al. 2014). Here, we demonstrated that Cd accumulation −1 Umg protein ) in SOD activity was observable at ≥ 1.83 was higher in the kidney and liver than other tissues, with −1 −1 −1 mg L (as 2811 U mg protein ) with the highest activity significant differences from control at ≥ 0.61 mg L Cd −1 −1 being 3195 U mg protein at 3.08 mg L Cd exposure. exposure. Furthermore, both organs had greater Cd con- −1 Significant increase from control (as 1021 U mg protein ) centrations even in the control condition. This result cor- −1 in CAT activity was observable at ≥ 0.61 mg L with the roborates previous study; in A. japonica exposed to 0.05 −1 −1 −1 highest activity being 1704 U mg protein at 3.67 mg L mg L of Cd, the primary tissues that accumulated Cd Cd exposure. GPx activity from control (as 0.1024 U mg were the kidney and liver (Yang and Chen 1996). The pre- −1 −1 protein ) decreased at 3.08 and 3.67 mg L Cd exposure vious study suggested that the two tissues could function −1 as 0.0644 and 0.0664 U mg protein ,respectively, butitis as indicators of Cd pollution in water, because they appear not significant. GST activity from control (as 0.2551 μmol to be critical sites of Cd accumulation. Field studies in −1 −1 min mg protein ) was not changed at all groups. aquatic ecosystems generally support experimental find- ings. Cd concentrations in the kidney of captured A. ros- Discussion trata and A. anguilla (at two reference and contaminated Like the findings of a general aquatic toxicity study sites) were higher than concentrations in the liver and (Rand et al. 1995), this study demonstrated that A. muscle (Pannetier et al. 2016). Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 5 of 10 ab c d Fig. 1 Cd accumulation in organs of Anguilla japonica exposed to different Cd concentrations. Liver (a), Kidney (b), Spleen (c), Gill (d), and Muscle (e). Superscript asterisks indicate significant differences (P < 0.05) Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 6 of 10 ab cd Fig. 2 Activities of antioxidant enzymes in the liver of Anguilla japonica exposed to different Cd concentrations. SOD (a), CAT (b), GPx (c), and GST (d). Superscript asterisks indicate significant differences (P < 0.05) Table 4 Comparison of LC values of A. japonica with other freshwater fish studied previously −1 Species Live stage Duration (h) LC (mg L ) Reference Aguilla japonica Adult 96 3.61 This study Anguilla rostrata 96 0.82 Rehwoldt et al. (1972) Cyprinus carpio 96 0.24 Rehwoldt et al. (1972) Oreochromis niloticus Sac fry 24 1.6 Andaya and Gotopeng (1982) Oreochromis sp. Juvenile 96 0.7 Aldoghachi et al. (2016) Poecilia reticulata Adult 96 30.4 Yilmaz et al. (2004) Luciobrama macrocephalus Juvenile 96 7.42 Gomes et al. (2009) Channa marulius Fingerling 96 75.70 Batool et al. (2014) Wallago attu Fingerling 96 32.95 Batool et al. (2014) Rasbora sumatrana Adult 96 0.10 Shuhaimi-Othman et al. (2015) Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 7 of 10 We found that Cd accumulation in the gills in- Significance of variance homogeneity was < 0.05 in all creased about fivefold from the control level at 3.67 tissues except for the kidney about bioaccumulation be- −1 mg L Cd exposure, the highest rate of increase ob- tween the test groups. These results were related to −1 served in the experiment. Similarly, in common carp higher dispersion in 3.08 and 3.67 mg L exposure −1 (Cyprinus carpio)exposed to 5mgL of a combined groups than other groups, because low sample number metal solution (Cr, Ni, Cd, and Pb) for 32 days, the by mortality affected the degree of dispersion statisti- gills exhibited a higher rate of increase in Cd accu- cally. The high dispersion means that ability of accumu- mulation compared with the other tested tissues and lation and depuration can differ between individuals, also contained the highest in amount of Cd (followed though the species and environment of the experiment by the liver, kidney, and flesh) (Vinodhini and Nar- are the same. Nevertheless, the kidney may be consid- ayanan 2008). This high Cd level is likely explained ered to be a better selection than other tissues as an in- by the fact that gills are a major point of entry for dicator of bioaccumulation in eels, Anguilla japonica,by Cd, which passively diffuses through gill Ca channels Cd acute exposure, because the significance of variance (Verbost et al. 1989). Also, these results indicate that homogeneity was > 0.05. gills are the most sensitive organ to Cd absorption We also demonstrated that the degree of bioaccumula- and accumulation in freshwater fish. tion reflects variation in waterborne Cd concentrations. Studies are insufficient about accumulation in the For example, all tissues differed significantly in Cd accu- −1 spleen by acute exposure of heavy metals. In our study, mulation (compared with control) at ≥ 1.83 mg L ,a Cd accumulation in the spleen showed a significant in- point that also marked the highest change rate to Cd −1 crease at ≥ 1.83 mg L Cd exposure as a higher concen- concentrations in experimental water. Additionally, Cd tration group than other tissues. It means that Cd was present in all tissues (except for muscle) at the high- −1 depuration in the spleen is higher than other tissues at est concentrations under 3.67 mg L , a point that also the exposure to low Cd concentration. For example, ac- marked the highest Cd decrement in the water. cumulation in the spleen of brook trout exposed to Some studies have suggested that Cd transference −1 waterborne 0.001 mg L Cd as sub-lethal concentration from the digestive canal to the liver (via the portal sys- during 77 days has not increased compared with control tem) does not occur if the fish is exposed to heavy (Sangalang and Freeman 1979). However, when referring metals for only a short term (Handy 1993). In this study, −1 to LC concentrations for Oreochromis species in Table Cd below a certain concentration (≤ 0.30 mg L ) accu- 4, accumulation in the spleen of Oreochromis niloticus mulated primarily in the gills (the main absorption −1 exposed to waterborne 1 mg L Cd as high concentra- route), likely because such levels are quickly removed by tion during 15 days has increased highly from control the liver, the most important organ for detoxification in (Cicik et al. 2004). Depuration ability of heavy metals in acute exposure (Chowdhury et al. 2005). In contrast, Cd the spleen could be related to metallothionein (MT) ex- over a certain concentration (between 0.61 and 3.67 mg −1 pression and positive effect in specific tissues to remove L ) accumulated significantly more in the kidney and non-essential metals in tissues. Fold change of MT liver. Although the water in high-exposure groups (4.29 −1 mRNA levels in the spleen of Korean bitterling, Achei- and 5.51 mg L ) had lower rates of change and decre- lognathus signifier (cyprinidae), exposed to waterborne ment in Cd concentrations than water from low- 0.5 μM copper (Cu) during 48 h was the high increase exposure groups, this was due to high eel mortality from following liver among 6 tissues (Lee et al. 2011). Also, as Cd toxicity before bioaccumulation could occur. There- results which inject MT for detoxification in grass carp, fore, Cd transference in fish exposed to Cd for a short Ctenopharyngodon idellus, on the 4 days after injection term can occur depending on Cd concentrations without of 20 μM/kg CdCl , the increase of Cd accumulation in mortality. the spleen suppressed highly more than head-kidney In this study, activities of SOD and CAT increased (Huang et al. 2019). generally, similar to a significant increase of Cd accumu- Accumulation of heavy metal in the muscle is important, lation in the liver. Significant increase in activities of because it is related to the health of a person by eating SOD and CAT by Cd exposure is related to an increase muscle as food. Cd accumulation in the muscle showed a of ROS in fish. As the highest activities of antioxidant −1 significant increase at ≥ 0.15 mg L Cd exposure. In a previ- enzymes (SOD, CAT), the liver is stronger for oxidative ous study, Cd accumulation in the muscle of Sparus aurata stress than other tissues (Atli et al. 2006). According to −1 was higher than control by acute Cd exposure (0.5 mg L ) a report of Safari (2015), stress from heavy metals in- for short period (2, 4, and 24 h) (Souid et al. 2013). Because duces expression of genes encoding SOD and CAT to of rapid accumulation in the muscle by acute Cd exposure, detoxify ROS (Rastgoo and Alemzadeh 2011). Similar it is necessary to investigate food safety for fishery and a results about SOD and/or CAT reported in various catch of fish surrounding industrial accident of Cd spill. fish species, e.g., sturgeon, murrel, tilapia (Atli and Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 8 of 10 Canli 2007; Dabas et al. 2012;Safari 2015). Results of this and other studies, thus, the activity change of antioxi- −1 study mean that some fish in 3.08 and 3.67 mg L dant enzyme is considered to have limits with experi- Cd exposure maintained life by sufficiently increasing ment species, individuals, kinds of enzymes, etc., even if activities of SOD and CAT to eliminate ROS in the body. it is exposed to heavy metal of high concentration that But some studies reported about the decrease of SOD could cause mortality. and/or CAT activities unlike the results of ours. For ex- Most studies with ours are different in the experimen- ample, CAT activity in the liver of Channa marulius and tal condition, such as fish species, size, and temperature. Wallago attu decreased with increasing exposed Cd con- They are all necessary to establish accurate environmen- centrations, but SOD activity increased (Batool et al. tal pollution indicators. The studies on the effect of anti- 2014). And, in the liver of Cyprinus carpio, both CAT and oxidant enzyme activity and accumulation by heavy SOD activities decreased after Cd exposure during 96 h metal exposure are mostly conducted in chronic toxicity (Karaytug et al. 2011). Roméo et al. (2000)reported that test. So, it is important that our study results have a the decrease of CAT activity is attributed to the direct similar pattern to those of the chronic toxicity test. This binding of Cd in CAT at Cd exposure. These differences is because the effects on chronic toxicity can be inferred can come by fish species, metal species, environmental through acute toxicity test. We expect that our findings factors in the experiment, etc. In the study of Saglam et al. are used as a direct data on food availability of polluted (2014), SOD and CAT activities in the liver after Cd and fish by heavy metal exposure of high level, as our study Cu exposure were different depending on water hardness, was conducted using fish of the size available as food. respectively. Cd induce hepatotoxicity by tightly binding to thiol Conclusions groups of GSH as the first defense line of acute Cd ex- We investigated Cd acute toxicity and bioaccumulation posure. Decrease of GSH induces oxidative stress in the in adult A. japonica after 96 h of exposure. The LC of −1 liver by free radical production and disruption of the cel- Cd after 96 h was 3.61 mg L Cd. Cd accumulation was lular GSH system (Dudley and Klaassen 1984; Liu et al. acute in all measured tissues (following the order kidney 2009). Saglam et al. (2014) reported that GPx activity > liver > gill > spleen > muscle) and corresponded to Cd can be considered complementary to CAT activity, but decrement and change rate in experimental water. Cd −1 its capacity is smaller than CAT activity for decompos- exposure at ≥ 1.83 mg L led to significant increases as ition of the peroxides (Sampaio et al. 2008). In the study compared with control in Cd accumulation for all of Choi et al. (2007), the expression level of GPx mRNA tissues, but the accumulation rate was highest in the in the liver of goldfish decreased after Cd exposure by gills. In activity alteration of antioxidant enzymes as bio- injection and became undetectable after 12 h exposure. markers for oxidative stress, both SOD and CAT activ- −1 GPx activity in the liver of gilthead sea bream decreased ities increased ≥ 1.83 mg L significantly. GPx activity −1 −1 after waterborne Cd exposure at 0.1 mg L concentra- showed a decrease tendency at 3.08 and 3.67 mg L Cd tion for 4 days (Cirillo et al. 2012). Crupkin and Menone exposure, and GST activity was not changed. Our study (2013) reported that GST activity in the liver had no sig- results emphasize the need for additional studies on Cd nificant change in Australoheros facetus exposed to vari- chronic exposure and depuration of A. japonica. The re- ous Cd concentrations except for a group of lower sults obtained could aid in setting standards for the concentration, but GST activity of other tissues (gill, influence investigation of Cd contamination in aquatic brain) altered significantly more than the liver. With environments and processing methods (e.g., instant consideration for these facts and similar studies, we sup- disuse, usage conversion, or long-term acclimation) for pose that our results for decrease tendency of GPx activ- Cd-accumulated eel. ity in high concentrations and changeless of GST Acknowledgements activity were affected by an increase of Cd ion and de- Not applicable crease of GSH in the liver. Authors’ contributions Despite mortality over the majority, the activity change −1 TY Ahn carried out the experiment, analyzed the data, and finalized the of antioxidant enzyme at 3.67 mg L exposure was manuscript. HJ Park and JH Kim analyzed the experimental data and −1 smaller than 3.08 mg L exposure. In a previous study, participated in drafting the manuscript. JC Kang participated in the design of the experiment and drafted the manuscript. All authors read and approved the activity change of antioxidant enzyme was small the final manuscript. against the change of mortality with Cd concentration increase by acute exposure (Batool et al. 2014). Also, the Funding Not applicable graph of antioxidative activity shows bell shape or changeless with Cd concentration increase, even though Availability of data and materials mortality does not occur (Atli et al. 2006; Crupkin and The datasets used and/or analyzed during the current study are available Menone 2013; Souid et al. 2013). On the basis of our from the corresponding author on reasonable request. Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 9 of 10 Ethics approval and consent to participate the Clark Fork River, Montana. Can J Fish Aquat Sci. 1995;52:2038–50. https:// Not applicable doi.org/10.1139/f95-795. Gomes LC, Chippari-Gomes AR, Oss RN, Fernandes LFL, de Almedia Magris R. 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Effects of antioxidant enzymes and bioaccumulation in eels (Anguilla japonica) by acute exposure of waterborne cadmium

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Abstract

This study was conducted to evaluate the acute effects of waterborne cadmium exposure on bioaccumulation and antioxidant enzymes in eels (Anguilla japonica) and to determine the median lethal concentration (LC ). Fish −1 were exposed to different cadmium concentrations (0, 0.15, 0.30, 0.61, 1.83, 3.08, 3.67, 4.29, and 5.51 mg L )for −1 96 h. The LC of A. japonica to cadmium was 3.61 mg L . Cadmium accumulation generally increased in tissues −1 with increasing waterborne cadmium concentrations. At ≥ 1.83 mg L exposure, all tissues accumulated significant cadmium concentrations compared with the control group, in the order of kidney > liver > gill > spleen > muscle. Measurements of variation in actual cadmium concentrations showed that a reduction of the metal in experimental water was related to cadmium accumulation in tissues. As activity alteration of antioxidant enzymes for reactive oxygen −1 species, superoxide dismutase and catalase activities increased at ≥ 0.61 mg L significantly, glutathione peroxidase and glutathione S-transferase activities were not significantly changed. The results of this study suggest that acute exposure to waterborne cadmium is potentially fatal to A. japonica due to the metal’s major accumulation in various tissues and the effect of antioxidant enzyme activity. Keywords: Cadmium, Anguilla japonica, Acute toxicity, LC , Bioaccumulation, Antioxidant enzyme Introduction of gills (Verbost et al. 1987, 1988, 1989; Pinot et al. Metals naturally exist in aquatic ecosystems, but side ef- 2000). Cd redox activity affects antioxidants, thus redu- fects from industrialization have resulted in excessive con- cing protection against oxidative stress, increasing lipid centrations. Exposure to high metal levels may negatively peroxidation, and decreasing DNA synthesis (Okorie affect fish and other aquatic organisms, hampering physio- et al. 2014). In addition, Cd lowers plasma Na, Cl, and logical functions, growth rate, and reproduction, or even K, leading to hyperglycemia and hypermagnesemia increasing mortality (Reddy and Reddy 2013;Öz 2018;Öz (Larsson et al. 1981; Haux and Larsson 1984; Sjöbeck et al. 2018). Cadmium is a particularly widespread and et al. 1984). Even at low concentrations, Cd deforms tis- toxic example that is documented to accumulate in ex- sues and vertebrae, causing respiration abnormalities posed organisms; it is used primarily in alloys, pigments, and death in fish (De Smet and Blust 2001). Cd and electroplating, and batteries (Bryan 1976; Farag et al. 1995; other toxic heavy metals can also accumulate through Adriano 2001;Javed 2003). direct absorption or biomagnification; the resultant in- In fish, Cd disrupts Ca metabolism through competi- hibition of major organ function (i.e., liver, kidney, and tion for transport sites on the basolateral calcium pumps gills) is strongly linked to toxicity. Thus, the degree of accumulation in each organ is used frequently as a bio- monitor for metal contamination (Handy 1992). * Correspondence: jckang@pknu.ac.kr In fish exposed to Cd, reactive oxygen species (ROS), Department of Aquatic Life Medicine, Pukyong National University, Busan such as hydrogen peroxide (H O ), hydroxyl, and oxygen 48513, South Korea 2 2 Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 2 of 10 radical, occur and induce oxidative stress. As a result, kidney, spleen, gills, and muscle) and change of antioxi- the biological system induces antioxidant enzymes, e.g., dant enzymes (SOD, CAT, GPx, and GST) in the liver superoxide dismutase (SOD), catalase (CAT), glutathi- with the determination of LC for Cd in adult A. one peroxidase (GPx), glutathione S-transferase (GST), japonica. to mitigate the attack of ROS. These enzymes are used as stress biomarkers in fish by exposure or contamin- Materials and methods ation of heavy metals and generation of ROS. SOD is Experimental fish and design catalyzing the transformation of superoxide anion radi- Anguilla japonica specimens were collected from the eel cals to H O and oxygen (O ). Catalase (CAT) decom- aquafarm of Paju city, Gyeonggi province, South Korea. 2 2 2 poses toxic H O to O and H O. Glutathione Fish were acclimated to a polyvinyl (PVC) tank for 2 2 2 2 2 peroxidase (GPx) decomposes H O or organic hydro- weeks prior to experiment and food-deprived. Also, we 2 2 peroxide to H O or corresponding alcohols using re- identified no infection of parasites in some fish before duced glutathione (GSH) into oxidized glutathione acclimation and toxicity test to prevent mortality by par- (GSSG). Glutathione S-transferase (GST) detoxifies the asites and used visually healthy fish for the experiment. reactive intermediates and oxygen radicals by catalyzing Acute Cd toxicity test was conducted under laboratory the conjugation of GSH to various electrophilic metabo- conditions. Acclimated fish (n = 90; average weight lites, thereby enhancing water solubility and assisting ex- 186.6 ± 11.9 g) were selected, divided into nine groups cretion (Livingstone 2003). (10 per group), and placed into plastic aquaria (555 × Two standard tests of metal toxicity are acute or 395 × 310 mm) filled with underground water. Table 1 chronic exposure. In many organisms including fish, summarizes the water quality parameters measured for acute toxicity is defined as LC (median lethal concen- the bioassay. Water temperature was maintained with a tration), a concentration that kills approximately 50% of heater at 29 ± 1 °C. To make conditions similar to aqua- a test group after exposure to increasingly higher toxi- farm, the laboratory was kept in 24-h darkness except cant levels for a specified, relatively short time frame when checking fish mortality. During the exposure (Schreck and Moyle 1990; Mason 1991). Acute toxicity period, water was not renewed and fish were not fed. data are supplemented with chronic toxicity tests for the Analytical-grade CdCl (Aldrich, Inc., USA) was dis- same compound, exposing subject organisms to the solved in triple distilled water to prepare stock Cd solu- same low concentration over a longer period. Such tion used for exposure experiments (see the information is useful as a reference when performing en- “Determination of LC and assay of actual Cd levels in vironmental surveys of contaminated areas and deter- experimental water” section). mining the effects of toxicant efflux after industrial accidents. Determination of LC and assay of actual Cd levels in Eels are commonly consumed in Asia and are mostly experimental water produced through aquaculture. Farmed eels are fed paste Experimental fish were exposed to waterborne CdCl −1 that contains a high ratio of fish meal. Thus, Cd accu- treatments of 0.25, 0.5, 1, 3, 5, 6, 7, and 9 mg L , 0.15, −1 mulation can occur if the metal’s concentration in fish 0.30, 0.61, 1.83, 3.08, 3.67, 4.29, and 5.51 mg L as only meal is high. Eels suffer particularly high mortality under Table 1 Quality parameters of water used during acclimation Cd exposure, because their benthic lifestyle increases and experimentation contact with heavy metals that sink to the floor. These Parameters Value factors indicate that we require data on Cd effects in eels pH 7.96 ± 0.10 to ensure food safety and assess environmental contam- Salinity (‰) ≤ 0.1 ination. However, despite the progress made on under- −1 standing the outcome of Cd exposure in several fish Dissolved oxygen (mg L ) 7.75 ± 0.05 −1 species (Handy 1993; Yilmaz et al. 2004; Aldoghachi Chemical oxygen demand (mg L ) < 0.01 et al. 2016), little research has been conducted in eels, −1 NH -N (mg L ) 0.015 ± 0.005 especially Anguilla japonica. Furthermore, many bio- −1 NO -N (mg L ) 0.003 ± 0.001 accumulation studies focus on chronic exposure, despite −1 NO -N (mg L ) 4.3 ± 0.1 the possibility of industrial accidents causing acute Cd 2− −1 SO (mg L ) 8.0 ± 0.5 exposure and accumulation. If Cd in eels is highly accu- 4 3− −1 mulated after acute exposure, it can affect the health of PO (mg L ) 0.25 ± 0.05 −1 humans as food through a catch. Thus, the purpose of SS (mg L)28±2 this study is to assess risk as food, identify the effect on −1 Total hardness (CaCO mg L ) 150 ± 10 fish health, and utilize baseline data for chronic toxicity 2+ −1 Cd (mg L ) ≤ 0.0001 test by investigating accumulation in major tissues (liver, Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 3 of 10 Cd concentrations, for 96 h. A water-only control was in the liver were determined using the method of Brad- also used. Cd exposure concentrations were established ford (1976), with bovine serum albumin as a standard. after pre-experiment by referring to Cd chronic accumu- −1 lation concentration (0.1 mg L ) in eels, Anguilla japon- Statistical analysis ica, through previous study (Yang and Chen 1996). Dead Finney’s probit analysis was used to determine the LC fish were counted every 12 h and removed immediately of Cd in eels, along with confidence limits. Between- from the aquaria. Experimental water was collected to group differences in Cd bioaccumulation and activities measure actual Cd concentrations at 12 and 96 h after of antioxidant enzymes were analyzed using two tests as adding the stock Cd solution. Water samples were di- a one-way ANOVA depending on Levene’s test for equal luted with 2% nitric acid before analysis using ICP-MS variance. Duncan’s multiple range and Games-Howell (inductively coupled plasma mass spectrometry; NexION tests were used at P > 0.05 and P < 0.05 in equality of 300X, Perkin-Elmer Inc., USA). The change rate and variances, respectively. Significance of post hoc test was decrement of actual Cd were calculated as follows: set at P < 0.05. All statistics were performed in SPSS ver- (1) Change rate (%) = 100 × (1 − Cd concentration at sion 20 (IBM co., USA). 96 h ÷ Cd concentration at 12 h) −1 (1) Duncan’s multiple range test: kidney, SOD, CAT, (2) Decrement (mg L ) = Cd concentration at 12 h − GPx, and GST Cd concentration at 96 h (2) Games-Howell test: liver, spleen, gill, muscle Tissue analysis to confirm Cd bioaccumulation After a 96-h Cd exposure, gills, liver, kidney, spleen, and Results muscle samples of live fish were collected and kept at − Median lethal concentrations of Cd in A. japonica 80 °C until analysis. Tissues were freeze-dried and Mortality was first measured at a Cd concentration of ≥ digested with nitric acid (Suprapur grade, Merck, −1 3.08 mg L , and the mortality rate reached 100% at 5.51 Germany) and hydrogen peroxide (bioassay grade, −1 mg L . The number of dead fish increased with increas- Merck, Germany) in a microwave (START D, Milestone, ing Cd concentration. Based on mortality data, LC of Italy). The resultant solutions were diluted with triple 50 Cd in A. japonica after 24, 48, 72, and 96 h was 5.10, distilled water and subjected to ICP-MS (NexION, −1 4.04, 3.67, and 3.61 mg L , respectively (Table 2). Perkin-Elmer Inc., USA). Assay of antioxidant enzymes activity Variation in actual Cd levels of experimental water The Collected liver was homogenized with 0.1 M Variation in the actual Cd concentration of experimental phosphate-buffered saline (PBS) using tissue lyzer (Tis- water was measured to investigate correlations between sueLyser II, QIAGEN, Germany). The homogenate was Cd accumulation in A. japonica and changes to Cd centrifuged at 10,000g for 30 min under 4 °C. The super- levels during the acute toxicity test. We found that ac- natants were obtained and stored at − 80 °C until ana- tual Cd concentrations generally decreased after the 96- lysis. SOD activity was analyzed using the SOD assay kit h exposure (Table 3). Based on measurements from 12-h (Dojindo Co., Japan) measuring 50% inhibition rate for post-exposure, the lowest and highest rate change in the reduction reaction of 2-(4-lodophenyl)-3-(4-nitro- −1 concentration were 5.1% (at exposure to 5.51 mg L Cd) phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monoso- −1 and 16.8% (at 1.83 mg L ), respectively. In contrast, the dium salt (WST-1) and was expressed as U mg −1 lowest and highest absolute decrement of actual Cd con- protein . CAT activity was analyzed using the CAT centration (again measured 12-h post-exposure) were assay kit (Sigma-Aldrich Inc., USA) measuring the ab- −1 −1 −1 0.015 mg L (at 0.15 mg L ) and 0.664 mg L (at 3.67 sorbance of the chromogen versus the amount of re- −1 mg L ), respectively. sidual H O after reaction with samples and was 2 2 −1 expressed as U mg protein . GPx activity was analyzed Table 2 Estimated median lethal concentrations (LC ) and using the GPx cellular activity assay kit (Sigma-Aldrich 50 confidence limits Inc., USA) measuring the change in absorbance at 340 Period Concentration 95% confidence limits nm by the reduction reaction of tert-butyl hydroperoxide −1 −1 (h) (Cd mg L ) and was expressed as U mg protein . GST activity was Lower Upper analyzed using the GST assay kit (Sigma-Aldrich Inc., 24 5.10 4.37 6.84 USA) measuring the change in absorbance at 340 nm by 48 4.04 3.55 4.66 reaction of sample and substrate solution including 1- 72 3.67 3.25 4.10 chloro-2,4-dinitrobenzene (CDNB) and was expressed as 96 3.61 3.19 3.99 −1 −1 μmol min mg protein . Total protein concentrations Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 4 of 10 Table 3 The change rate and decrement of actual Cd japonica mortality increased with increasing Cd concen- concentrations in experimental water at two time points (12- tration and exposure period. Although previous studies and 96-h post-exposure) under different Cd concentrations had attempted to examine acute Cd toxicity in fresh- −1 Exposure Actual Cd conc. (Cd mg L ) Change Decrement water fish (e.g., tilapia, common carp, rasbora) conc. (Cd rate (%) (Cd mg 12 h 96 h (Rehwoldt et al. 1972; Shuhaimi-Othman et al. 2015), −1 −1 mg L ) L ) these were generally not enough for the overall assess- 0.15 0.156 0.141 9.6 0.015 ment of environmental pollution. Nonetheless, we com- 0.30 0.331 0.310 6.3 0.021 pared our results with several previous studies to 0.61 0.662 0.624 5.7 0.039 evaluate Cd acute toxicity (Table 4). In contrast with −1 1.83 2.148 1.786 16.8 0.362 3.61 mg L (after 96-h Cd exposure) in the LC of this study, the LC in tilapia sac fry (Oreochromis niloticus) 3.08 3.064 2.763 9.8 0.302 50 −1 and juvenile (Oreochromis sp.) were 1.6 mg L (after 24- 3.67 4.068 3.404 16.3 0.664 −1 h Cd exposure) and 0.7 mg L (after 96-h Cd exposure), 4.29 4.624 4.177 9.6 0.448 respectively (Andaya and Gotopeng 1982; Aldoghachi 5.51 5.715 5.421 5.1 0.294 et al. 2016). Moreover, the 96-h LC of Cd in adult −1 guppies (Poecilia reticulata) was 30.4 mg L (Yilmaz −1 Bioaccumulation et al. 2004), while it was 7.42 mg L in juvenile piauçu Cd exposure caused a net increase of Cd content in all (Luciobrama microcephalus) (Gomes et al. 2009). These tested A. japonica tissues compared with the control data indicate that between-species differences in life his- (Fig. 1). The order of Cd accumulation in tissues (in- tory, genetic composition, and individual conditions cluding control) was as follows: kidney > liver > gills > have a greater impact on fish tolerance (or sensitivity) to spleen > muscle, with the highest and lowest concentra- Cd toxicity than size and age (Rand et al. 1995). Ideally, −1 tions being 122.190 mg kg in the kidney (at 3.67 mg within-species comparisons would better indicate −1 −1 −1 L ) and 0.049 mg kg in the muscle (at 0.15 mg L )of whether our results are typical of A. japonica. However, exposed groups, respectively. As expected, accumulation although some studies examining acute Cd toxicity do rose with increasing exposure concentration. However, exist for this species, differences in experimental significant differences as compared with control in Cd water conditions (e.g., hardness, pH, temperature) accumulation across all tissues were observable at ≥ complicate the interpretation of any cross-study vari- −1 1.83 mg L Cd exposure. Individually, significant differ- ation (Shuhaimi-Othman et al. 2015). Regardless, this −1 ences were apparent at ≥ 0.15 mg L in the gill and studyprovidesimportant basicdatafor anyfuture −1 −1 muscle, ≥ 0.30 mg L in the liver, ≥ 0.61 mg L in the studyinvestigating chronicCdtoxicityin A. japonica −1 kidney, and ≥ 1.83 mg L in the spleen. and allows for further comparative analyses of Cd tol- erance among fish. Antioxidant enzymes activity Cd accumulation in tissues may differ according to After acute exposure to Cd during 96 h, activities of anti- metal’s form. Inorganic Cd tends to be accumulated in the oxidant enzymes (SOD, CAT, GPx, and GST) in eel’sliver liver, while Cd-thiols are readily accumulated in the kid- were determined (Fig. 2). Activities of SOD and CAT in- ney, considered the organ most sensitive to Cd toxicity creased as compared with control by increasing exposure (Hammond and Foulkes 1986; Woo et al. 1993; Okorie concentration. Significant increase from control (as 1898 et al. 2014). Here, we demonstrated that Cd accumulation −1 Umg protein ) in SOD activity was observable at ≥ 1.83 was higher in the kidney and liver than other tissues, with −1 −1 −1 mg L (as 2811 U mg protein ) with the highest activity significant differences from control at ≥ 0.61 mg L Cd −1 −1 being 3195 U mg protein at 3.08 mg L Cd exposure. exposure. Furthermore, both organs had greater Cd con- −1 Significant increase from control (as 1021 U mg protein ) centrations even in the control condition. This result cor- −1 in CAT activity was observable at ≥ 0.61 mg L with the roborates previous study; in A. japonica exposed to 0.05 −1 −1 −1 highest activity being 1704 U mg protein at 3.67 mg L mg L of Cd, the primary tissues that accumulated Cd Cd exposure. GPx activity from control (as 0.1024 U mg were the kidney and liver (Yang and Chen 1996). The pre- −1 −1 protein ) decreased at 3.08 and 3.67 mg L Cd exposure vious study suggested that the two tissues could function −1 as 0.0644 and 0.0664 U mg protein ,respectively, butitis as indicators of Cd pollution in water, because they appear not significant. GST activity from control (as 0.2551 μmol to be critical sites of Cd accumulation. Field studies in −1 −1 min mg protein ) was not changed at all groups. aquatic ecosystems generally support experimental find- ings. Cd concentrations in the kidney of captured A. ros- Discussion trata and A. anguilla (at two reference and contaminated Like the findings of a general aquatic toxicity study sites) were higher than concentrations in the liver and (Rand et al. 1995), this study demonstrated that A. muscle (Pannetier et al. 2016). Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 5 of 10 ab c d Fig. 1 Cd accumulation in organs of Anguilla japonica exposed to different Cd concentrations. Liver (a), Kidney (b), Spleen (c), Gill (d), and Muscle (e). Superscript asterisks indicate significant differences (P < 0.05) Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 6 of 10 ab cd Fig. 2 Activities of antioxidant enzymes in the liver of Anguilla japonica exposed to different Cd concentrations. SOD (a), CAT (b), GPx (c), and GST (d). Superscript asterisks indicate significant differences (P < 0.05) Table 4 Comparison of LC values of A. japonica with other freshwater fish studied previously −1 Species Live stage Duration (h) LC (mg L ) Reference Aguilla japonica Adult 96 3.61 This study Anguilla rostrata 96 0.82 Rehwoldt et al. (1972) Cyprinus carpio 96 0.24 Rehwoldt et al. (1972) Oreochromis niloticus Sac fry 24 1.6 Andaya and Gotopeng (1982) Oreochromis sp. Juvenile 96 0.7 Aldoghachi et al. (2016) Poecilia reticulata Adult 96 30.4 Yilmaz et al. (2004) Luciobrama macrocephalus Juvenile 96 7.42 Gomes et al. (2009) Channa marulius Fingerling 96 75.70 Batool et al. (2014) Wallago attu Fingerling 96 32.95 Batool et al. (2014) Rasbora sumatrana Adult 96 0.10 Shuhaimi-Othman et al. (2015) Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 7 of 10 We found that Cd accumulation in the gills in- Significance of variance homogeneity was < 0.05 in all creased about fivefold from the control level at 3.67 tissues except for the kidney about bioaccumulation be- −1 mg L Cd exposure, the highest rate of increase ob- tween the test groups. These results were related to −1 served in the experiment. Similarly, in common carp higher dispersion in 3.08 and 3.67 mg L exposure −1 (Cyprinus carpio)exposed to 5mgL of a combined groups than other groups, because low sample number metal solution (Cr, Ni, Cd, and Pb) for 32 days, the by mortality affected the degree of dispersion statisti- gills exhibited a higher rate of increase in Cd accu- cally. The high dispersion means that ability of accumu- mulation compared with the other tested tissues and lation and depuration can differ between individuals, also contained the highest in amount of Cd (followed though the species and environment of the experiment by the liver, kidney, and flesh) (Vinodhini and Nar- are the same. Nevertheless, the kidney may be consid- ayanan 2008). This high Cd level is likely explained ered to be a better selection than other tissues as an in- by the fact that gills are a major point of entry for dicator of bioaccumulation in eels, Anguilla japonica,by Cd, which passively diffuses through gill Ca channels Cd acute exposure, because the significance of variance (Verbost et al. 1989). Also, these results indicate that homogeneity was > 0.05. gills are the most sensitive organ to Cd absorption We also demonstrated that the degree of bioaccumula- and accumulation in freshwater fish. tion reflects variation in waterborne Cd concentrations. Studies are insufficient about accumulation in the For example, all tissues differed significantly in Cd accu- −1 spleen by acute exposure of heavy metals. In our study, mulation (compared with control) at ≥ 1.83 mg L ,a Cd accumulation in the spleen showed a significant in- point that also marked the highest change rate to Cd −1 crease at ≥ 1.83 mg L Cd exposure as a higher concen- concentrations in experimental water. Additionally, Cd tration group than other tissues. It means that Cd was present in all tissues (except for muscle) at the high- −1 depuration in the spleen is higher than other tissues at est concentrations under 3.67 mg L , a point that also the exposure to low Cd concentration. For example, ac- marked the highest Cd decrement in the water. cumulation in the spleen of brook trout exposed to Some studies have suggested that Cd transference −1 waterborne 0.001 mg L Cd as sub-lethal concentration from the digestive canal to the liver (via the portal sys- during 77 days has not increased compared with control tem) does not occur if the fish is exposed to heavy (Sangalang and Freeman 1979). However, when referring metals for only a short term (Handy 1993). In this study, −1 to LC concentrations for Oreochromis species in Table Cd below a certain concentration (≤ 0.30 mg L ) accu- 4, accumulation in the spleen of Oreochromis niloticus mulated primarily in the gills (the main absorption −1 exposed to waterborne 1 mg L Cd as high concentra- route), likely because such levels are quickly removed by tion during 15 days has increased highly from control the liver, the most important organ for detoxification in (Cicik et al. 2004). Depuration ability of heavy metals in acute exposure (Chowdhury et al. 2005). In contrast, Cd the spleen could be related to metallothionein (MT) ex- over a certain concentration (between 0.61 and 3.67 mg −1 pression and positive effect in specific tissues to remove L ) accumulated significantly more in the kidney and non-essential metals in tissues. Fold change of MT liver. Although the water in high-exposure groups (4.29 −1 mRNA levels in the spleen of Korean bitterling, Achei- and 5.51 mg L ) had lower rates of change and decre- lognathus signifier (cyprinidae), exposed to waterborne ment in Cd concentrations than water from low- 0.5 μM copper (Cu) during 48 h was the high increase exposure groups, this was due to high eel mortality from following liver among 6 tissues (Lee et al. 2011). Also, as Cd toxicity before bioaccumulation could occur. There- results which inject MT for detoxification in grass carp, fore, Cd transference in fish exposed to Cd for a short Ctenopharyngodon idellus, on the 4 days after injection term can occur depending on Cd concentrations without of 20 μM/kg CdCl , the increase of Cd accumulation in mortality. the spleen suppressed highly more than head-kidney In this study, activities of SOD and CAT increased (Huang et al. 2019). generally, similar to a significant increase of Cd accumu- Accumulation of heavy metal in the muscle is important, lation in the liver. Significant increase in activities of because it is related to the health of a person by eating SOD and CAT by Cd exposure is related to an increase muscle as food. Cd accumulation in the muscle showed a of ROS in fish. As the highest activities of antioxidant −1 significant increase at ≥ 0.15 mg L Cd exposure. In a previ- enzymes (SOD, CAT), the liver is stronger for oxidative ous study, Cd accumulation in the muscle of Sparus aurata stress than other tissues (Atli et al. 2006). According to −1 was higher than control by acute Cd exposure (0.5 mg L ) a report of Safari (2015), stress from heavy metals in- for short period (2, 4, and 24 h) (Souid et al. 2013). Because duces expression of genes encoding SOD and CAT to of rapid accumulation in the muscle by acute Cd exposure, detoxify ROS (Rastgoo and Alemzadeh 2011). Similar it is necessary to investigate food safety for fishery and a results about SOD and/or CAT reported in various catch of fish surrounding industrial accident of Cd spill. fish species, e.g., sturgeon, murrel, tilapia (Atli and Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 8 of 10 Canli 2007; Dabas et al. 2012;Safari 2015). Results of this and other studies, thus, the activity change of antioxi- −1 study mean that some fish in 3.08 and 3.67 mg L dant enzyme is considered to have limits with experi- Cd exposure maintained life by sufficiently increasing ment species, individuals, kinds of enzymes, etc., even if activities of SOD and CAT to eliminate ROS in the body. it is exposed to heavy metal of high concentration that But some studies reported about the decrease of SOD could cause mortality. and/or CAT activities unlike the results of ours. For ex- Most studies with ours are different in the experimen- ample, CAT activity in the liver of Channa marulius and tal condition, such as fish species, size, and temperature. Wallago attu decreased with increasing exposed Cd con- They are all necessary to establish accurate environmen- centrations, but SOD activity increased (Batool et al. tal pollution indicators. The studies on the effect of anti- 2014). And, in the liver of Cyprinus carpio, both CAT and oxidant enzyme activity and accumulation by heavy SOD activities decreased after Cd exposure during 96 h metal exposure are mostly conducted in chronic toxicity (Karaytug et al. 2011). Roméo et al. (2000)reported that test. So, it is important that our study results have a the decrease of CAT activity is attributed to the direct similar pattern to those of the chronic toxicity test. This binding of Cd in CAT at Cd exposure. These differences is because the effects on chronic toxicity can be inferred can come by fish species, metal species, environmental through acute toxicity test. We expect that our findings factors in the experiment, etc. In the study of Saglam et al. are used as a direct data on food availability of polluted (2014), SOD and CAT activities in the liver after Cd and fish by heavy metal exposure of high level, as our study Cu exposure were different depending on water hardness, was conducted using fish of the size available as food. respectively. Cd induce hepatotoxicity by tightly binding to thiol Conclusions groups of GSH as the first defense line of acute Cd ex- We investigated Cd acute toxicity and bioaccumulation posure. Decrease of GSH induces oxidative stress in the in adult A. japonica after 96 h of exposure. The LC of −1 liver by free radical production and disruption of the cel- Cd after 96 h was 3.61 mg L Cd. Cd accumulation was lular GSH system (Dudley and Klaassen 1984; Liu et al. acute in all measured tissues (following the order kidney 2009). Saglam et al. (2014) reported that GPx activity > liver > gill > spleen > muscle) and corresponded to Cd can be considered complementary to CAT activity, but decrement and change rate in experimental water. Cd −1 its capacity is smaller than CAT activity for decompos- exposure at ≥ 1.83 mg L led to significant increases as ition of the peroxides (Sampaio et al. 2008). In the study compared with control in Cd accumulation for all of Choi et al. (2007), the expression level of GPx mRNA tissues, but the accumulation rate was highest in the in the liver of goldfish decreased after Cd exposure by gills. In activity alteration of antioxidant enzymes as bio- injection and became undetectable after 12 h exposure. markers for oxidative stress, both SOD and CAT activ- −1 GPx activity in the liver of gilthead sea bream decreased ities increased ≥ 1.83 mg L significantly. GPx activity −1 −1 after waterborne Cd exposure at 0.1 mg L concentra- showed a decrease tendency at 3.08 and 3.67 mg L Cd tion for 4 days (Cirillo et al. 2012). Crupkin and Menone exposure, and GST activity was not changed. Our study (2013) reported that GST activity in the liver had no sig- results emphasize the need for additional studies on Cd nificant change in Australoheros facetus exposed to vari- chronic exposure and depuration of A. japonica. The re- ous Cd concentrations except for a group of lower sults obtained could aid in setting standards for the concentration, but GST activity of other tissues (gill, influence investigation of Cd contamination in aquatic brain) altered significantly more than the liver. With environments and processing methods (e.g., instant consideration for these facts and similar studies, we sup- disuse, usage conversion, or long-term acclimation) for pose that our results for decrease tendency of GPx activ- Cd-accumulated eel. ity in high concentrations and changeless of GST Acknowledgements activity were affected by an increase of Cd ion and de- Not applicable crease of GSH in the liver. Authors’ contributions Despite mortality over the majority, the activity change −1 TY Ahn carried out the experiment, analyzed the data, and finalized the of antioxidant enzyme at 3.67 mg L exposure was manuscript. HJ Park and JH Kim analyzed the experimental data and −1 smaller than 3.08 mg L exposure. In a previous study, participated in drafting the manuscript. JC Kang participated in the design of the experiment and drafted the manuscript. All authors read and approved the activity change of antioxidant enzyme was small the final manuscript. against the change of mortality with Cd concentration increase by acute exposure (Batool et al. 2014). Also, the Funding Not applicable graph of antioxidative activity shows bell shape or changeless with Cd concentration increase, even though Availability of data and materials mortality does not occur (Atli et al. 2006; Crupkin and The datasets used and/or analyzed during the current study are available Menone 2013; Souid et al. 2013). On the basis of our from the corresponding author on reasonable request. Ahn et al. Fisheries and Aquatic Sciences (2020) 23:23 Page 9 of 10 Ethics approval and consent to participate the Clark Fork River, Montana. Can J Fish Aquat Sci. 1995;52:2038–50. https:// Not applicable doi.org/10.1139/f95-795. Gomes LC, Chippari-Gomes AR, Oss RN, Fernandes LFL, de Almedia Magris R. 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