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Soft tunic syndrome (STS) is a protozoal disease caused by Azumiobodo hoyamushi in the edible ascidian Halocynthia roretzi. Previous studies have proven that combined formalin–hydrogen peroxide (H O ) bath is effective in reducing 2 2 STS progress and mortality. To secure target animal safety for field applications, toxicity of the treatment needs to be evaluated. Healthy ascidians were bathed for 1 week, 1 h a day at various bathing concentrations. Bathing with 5- and 10-fold optimum concentration caused 100% mortality of ascidians, whereas mortality by 0.5- to 2.0-fold solutions was not different from that of control. Of the oxidative damage parameters, MDA levels did not change after 0.5- and 1.0-fold bathing. However, free radical scavenging ability and reducing power were significantly decreased even with the lower-than-optimal 0.5-fold concentration. Glycogen content tended to increase with 1-fold bathing without statistical significance. All changes induced by the 2-fold bathing were completely or partially restored to control levels 48 h post-bathing. Free amino acid analysis revealed a concentration-dependent decline in aspartic acid and cysteine levels. In contrast, alanine and valine levels increased after the 2-fold bath treatment. These data indicate that the currently established effective disinfectant regimen against the parasitic pathogen is generally safe, and the biochemical changes observed are transient, lasting approximately 48 h at most. Low levels of formalin and H O were detectable 1 h post-bathing; however, the compounds were completely undetectable 2 2 after 48 h of bathing. Formalin–H O bathing is effective against STS; however, reasonable care is required in the 2 2 treatment to avoid unwanted toxicity. Drug residues do not present a concern for consumer safety. Keywords: Ascidians, Formalin–hydrogen peroxide combination, Toxicity, Biochemical parameters, Drug residues, Soft tunic syndrome Background rigid cellulose-protein tunic structure (Dache et al. 1992) Soft tunic syndrome (STS) in the ascidian Halocynthia without affecting the cellulose fiber structure itself roretzi has markedly reduced production of this edible (Kimura et al. 2015). Highly active protease enzymes are invertebrate. Official figures indicate a gradual decrease produced and excreted from A. hoyamushi cells (Jang et in production to less than a half of the peak yearly pro- al. 2012). Although the disease spreads very rapidly, safe duction of approximately 22,500 t in 1995 (Kumagai et and effective measures have not been established to con- al. 2010). trol the spread of STS in farms. The cause of STS is infection with a protozoal parasite Chemical biocides are the first line of preventive mea- Azumiobodo hoyamushi, which leads to softening of the sures against infective organisms in the absence of a practical method to deal with the infection. Different classes of biocidal agents have been tested, and forma- * Correspondence: firstname.lastname@example.org lin, H O , bronopol, povidone iodine, and NaOCl were Department of Aquatic Life Medicine, College of Ocean Science and 2 2 Technology, Kunsan National University, 558 Daehak-ro, Miryong-Dong, found effective against the causative parasite (Park et Gunsan City, Jeonbuk 54150, Republic of Korea al. 2014; Lee et al. 2016; Kumagai et al. 2016). The Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Fisheries and Aquatic Sciences (2017) 20:12 Page 2 of 7 combination of two anti-infective chemicals, formalin Assessment of oxidative damage and oxido-reductive and H O , was the most promising choice of treatment potential 2 2 owing to their synergistic efficacy (Park et al. 2014). De- To estimate the influence of the treatment on oxido- tailed results from treatment trials were published in a reductive potential in treated animals, three different pa- previous issue of this journal (Lee et al. 2016), and this rameters were assessed: malondialdehyde (MDA) con- paper thus constitutes an important counterpart com- tent, free radical scavenging activity, and reducing power panion to it. of ascidian soft tissues. Bathing ascidians with formalin and H O suggested MDA content was assessed by the thiobarbituric acid- 2 2 a possible use for the combination in treating infected reactive substance (TBARS) method (Ohakawa et al. ascidians; however, the possible side effects, except 1979), using 10 g of tissues after homogenization in 20% mortality, have not been examined. Certain side effects trichloroacetic acid (TCA) solution. For this, the whole are expected, considering the non-selective mechanisms soft tissue of one individual was homogenized and 10 g of action of these agents. The degree and recovery from was taken. 2-Thiobarbituric acid (5 mL, 5 mM; Sigma) toxicity should be considered in deciding the value of a was added to an equal volume of tissue homogenates treatment regimen. To determine the toxicity and safety and stored in a refrigerator before absorbance measure- of the formalin–H O treatment, overall mortality, bio- ments at 530 nm (Optozen POP UV/Vis spectrophotom- 2 2 chemical changes, and drug residue levels were assessed eter, Meacasys, Seoul, Korea). Free radical scavenging after a 1-week bathing treatment schedule in healthy activity was determined according to the DPPH scaven- ascidians. ging method (Blios 1958). For this, 6 g of soft tissues were homogenized in 100 mL methanol followed by addition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) dissolved in Methods methanol. The mixture was reacted for 10 min at room Chemicals temperature, and absorbance was measured at 517 nm Formalin and H O were purchased from Sigma (St. 2 2 with a spectrophotometer. Louis, MO, USA), and actual concentrations were Reducing power was determined (Oyaizu 1986) using assessed before use by HPLC–UV (Soman et al. 2008) the methanol-added homogenates described in the and peroxidase–H O analysis kit (Cell Biolabs, San 2 2 section describing the determination of free radical Diego, CA), respectively. All other reagents were pur- scavenging activity. Phosphate buffer (1 mL, 200 mM, chased from Sigma if not specified otherwise. pH 6.6) was added to 1 mL of tissue homogenate and mixed with 1% potassium ferricyanide solution (1 mL). Test animals: Halocynthia roretzi After incubation at 50 °C for 20 min, 10% TCA was Healthy ascidians (114.7 ± 21.9 g, 90.9 ± 15.5 cm long) added to stop the reaction. Absorbance was measured were obtained from a local dealer and acclimated to la- at 700 nm using a spectrophotometer. Positive controls boratory conditions for 1 week before commencing the contained 10 μM ascorbic acid (vitamin C) instead of experiment. The absence of A. hoyamushi was verified ascidian tissues. by polymerase chain reaction (Shin et al. 2014) with 10 randomly sampled ascidians. Animals were maintained Glycogen content at 15 °C, the temperature at which STS is most likely to Glycogen content was analyzed according to the anthrone occur and treatment administration is expected. The method (Roe and Dailey 1966). Minced soft tissues (2 g) aquaria used were rectangular PVC tanks (L 1.0 m × W were mixed with 30% KOH solution (1 mL) to hydrolyze 0.65 m × H 0.3 m water level, 195 L). During experi- glycogen to glucose. Final colored product obtained in ments, feeding was not performed and water exchange reaction with anthrone was diluted with distilled water to was not needed. appropriate concentrations before spectrophotometric measurements at 620 nm. The standard curve was pre- Treatment procedures pared with D-glucose after identical processing. Drug treatment was performed at 10:00 a.m. for 1 h in separate drug tanks (20 L acryl baths) kept at 15 °C) Free amino acid composition that were artificially aerated. After drug bathing, the as- Free amino acids were analyzed according to the ninhyd- cidians were returned to normal tanks. This treatment rin post-column derivatization method (Friedman 2004) was repeated once daily for a week. Control groups optimized for the Hitachi amino acid analyzer (Hitachi were kept in normal seawater. In toxicity tests, recovery L-8900, Hitachi, Tokyo, Japan). Soft tissues (5 g) were was checked again 48 h after termination of bathing homogenized with distilled water (5 mL) and centrifuged (48 h post-bath group) when tunic signs were detected at 3000 × g for 10 min at 3 °C. Next, to 1 mL of super- in the initial assessment. natant, 5% TCA (0.9 mL) was added to precipitate Lee et al. Fisheries and Aquatic Sciences (2017) 20:12 Page 3 of 7 proteins, followed by centrifugation at 5000 × g for 10 min at 3 °C. After 10-fold dilution of the supernatant with 0.02 N HCl, the samples were filtered through 0.2- μm membrane filters. The amino acids were separated with an ion exchange column (4.6 × 60 mm; Hitachi HPLC Packed Column No. 2622 Li type) installed in an amino acid analyzer and UV detector (Hitachi L-8900). The mobile phase was Wako buffer solution (L-8900 PF- 1,2,3,4, Wako Pure Chemical Industries, Ltd., Osaka, Japan) run at a flow rate of 0.35 mL/min. Amino acid contents were quantified following a post-column nin- hydrin reaction on-line with 0.3 mL/min ninhydrin solu- tion flow. The separation column was kept at 30–70 °C, and the ninhydrin reaction was carried out at 135 °C. In- dividual amino acids were identified against the standard Fig. 1 Mortality of ascidians after combination treatment. Ascidians were exposed to formalin–H O , 4:1 ratio (ppm) 1 h a day for 7 days. 2 2 amino acid mixtures (Wako), with absorbance measured Death was declared in the absence of visually observable siphon at 570 and 440 nm. The volume of the sample injection movement. N = 11 in each group. F formalin, H H O 2 2 was 20 μL. Analyses of formalin and H O in previous studies, see refs. (Park et al. 2014; Lee et al. 2 2 The bathing drug solutions and treated tissues were used 2016)), the treatments corresponded to exposure from for analyses of test drug concentrations. The bathing 0.5- (20:5 ppm) to 10-fold (400:100 ppm) optimal treat- solution was analyzed directly after it was used for ment. The mortality was concentration-dependent. bathing without any further treatment. The ascidian tis- Whereas 0.5- to 2.0-fold treatments caused 10% mor- sues were homogenized in two volumes of distilled tality, not different from that by the non-treated control, water and centrifuged to obtain supernatants. Formalin 5- and 10-fold bathing led to 100% ascidian mortality. content was analyzed by HPLC–UV following complex formation with 2,4-dinitrophenylhydrazine (Soman et Oxidative damage and oxido-reductive potential al. 2008). The limit of detection sensitivity was approxi- Oxidative damage and the effect of treatment on the mately 500 nM. oxido-reductive potential of ascidian soft tissues are H O analysis was performed using the OxiSelect shown in Fig. 2. These parameters were determined 24 h 2 2 hydrogen peroxide colorimetric assay kit (Cell Biolabs, post-bathing after the termination of the 1 week expos- San Diego, CA) in accordance with the manufacturer’s ure schedule. Bathing of ascidians with 2-fold optimum instructions. The detection sensitivity limit was ap- concentration caused a slight but significant elevation of proximately 500 nM. Colored products were detected lipid peroxide levels after 1 week exposure for 1 h a day at 540 nm and quantified by comparison with the (Fig. 2a). In addition, free radical scavenging activity was standard curve. diminished by exposure to treatment: significant reduc- tion was noticed even after 0.5-fold exposure (Fig. 2b). Statistical analysis Along with the reduction in free radical scavenging, a Data are expressed as mean ± standard deviation (SD). significant decrease in reducing power was observed in Statistical analyses performed on biochemical parame- thesametissues(Fig.2c). Addition of vitaminCto ters were conducted by one-way analysis of variance control tissues markedly elevated the reduction poten- followed by Duncan’s multiple comparison tests. Sig- tial, as indicated by elevated free radical scavenging nificance in the difference of means was declared for capacity and reducing power (Figs. 2b, c). These alter- p values <0.05. ations returned to pre-treatment levels after 48-h re- covery in fresh seawater. Results Mortality of ascidians Tissue glycogen content Figure 1 illustrates the mortality of ascidians following A biphasic pattern in glycogen levels was observed bathing treatment with formalin–H O combination. (Fig. 3). The levels increased in a concentration- 2 2 Ascidians were treated for 1 h a day over a week at indi- dependent manner after 0.5- and 1-fold treatments and cated concentrations, and survival was recorded. Since returned to control levels after 2-fold exposure. Glyco- the optimum anti-parasitic treatment under identical gen content stayed unchanged 48 h after exposure conditions was formalin:H O = 40:10 ppm (determined when kept in fresh seawater. 2 2 Lee et al. Fisheries and Aquatic Sciences (2017) 20:12 Page 4 of 7 Fig. 2 Levels of oxidative damage in soft tissues of ascidians exposed to the combination treatment. Each damage parameter was assessed 24 h after the termination of the whole 7-day exposure scheme. Recovery (48 h post-bath column) was assessed in 2-fold exposure (F:H = 80:20 ppm) group after an additional 24 h in fresh seawater. a Malondialdehyde levels. b Free radical scavenging capacity. c Reduction power. F formalin, H H O ., Vit C ascorbic acid (10 μM). 2 2 N =7. Superscripts over bars denote significant statistical difference by Duncan’s multiple comparison tests at p <0.05 Free amino acid composition Free amino acid composition of the edible tissues follow- ing drug bathing is shown in Table 1. Taurine was the most prevalent amino acid-like substance, followed by amino acids proline, glutamic acid, glycine, and histidine. The most evident treatment-induced change was a concentration-dependent, significant decrease in aspartic acid concentration. In addition, significant decreases were noted for cysteine levels after 2-fold treatment and proline levels after 0.5-fold exposure. Notably, a signifi- cant increase in alanine and valine content was observed after 2-fold exposure. Formalin and H O residue concentrations 2 2 Drug residue concentrations in the bathing solution and treated ascidian tissues are shown in Fig. 4. Optimal, 1- fold treatment was used for the residue analysis experi- ment (40 ppm formalin and 10 ppm H O ). Formalin 2 2 concentration in the bathing solution was approximately 30 ppm after use for 1 h and declined slowly over the Fig. 3 Glycogen contents of soft tissues in ascidians exposed to combination treatment. Glycogen content was assessed 24 h after the termination of the whole 7-day exposure scheme. Recovery (48 h post- bath column) was assessed in 2-fold exposure (F:H = 80:20 ppm) group after an additional 24 h in fresh seawater. F formalin, H H O . N =7. 2 2 Superscripts over bars denote significant statistical difference by Duncan’s multiple comparison tests at p <0.05 Lee et al. Fisheries and Aquatic Sciences (2017) 20:12 Page 5 of 7 Table 1 Free amino acid content of edible tissues exposed to the treatment Amino acid content (mg/100 g muscle) Amino acids Control (n =7) 0.5×(n = 10) 1 × (n = 10) 2 × (n =9) Taurine (Tau) 1860.36 ± 357.57 1526.89 ± 186.85 1816.14 ± 242.77 1601.10 ± 247.87 a ab bc c Aspartic acid (Asp) 98.34 ± 47.38 75.68 ± 30.71 57.42 ± 16.54 37.61 ± 28.71 Threonine (Thr) 72.56 ± 29.51 78.14 ± 24.18 70.97 ± 22.51 87.15 ± 31.46 Serine (Ser) 47.49 ± 13.97 47.24 ± 10.11 57.21 ± 14.36 61.54 ± 20.62 Glutamic acid (Glu) 305.64 ± 54.55 248.89 ± 67.42 309.87 ± 67.07 295.48 ± 70.03 Glycine (Gly) 238.45 ± 57.63 183.81 ± 30.83 207.38 ± 33.59 183.10 ± 48.06 a a a b Alanine (Ala) 97.86 ± 22.88 89.12 ± 13.86 98.40 ± 18.56 142.61 ± 45.83 Citrulline (Cit) 0.29 ± 0.76 0.83 ± 2.61 2.26 ± 3.04 0.90 ± 1.45 a ab a b Valine (Val) 23.85 ± 7.56 27.13 ± 10.06 22.67 ± 10.05 36.07 ± 13.19 a ab a b Cysteine (Cys) 5.22 ± 3.00 3.80 ± 3.80 5.38 ± 2.88 1.45 ± 1.66 Methionine (Met) 15.09 ± 6.93 12.47 ± 5.04 11.71 ± 4.42 12.10 ± 3.42 Isoleucine (Ile) 19.08 ± 6.26 20.28 ± 7.14 17.67 ± 6.44 25.44 ± 8.85 Leucine (Leu) 27.92 ± 7.88 34.92 ± 14.07 26.16 ± 9.72 39.49 ± 11.85 Tyrosine (Tyr) 44.50 ± 21.21 27.68 ± 13.18 35.48 ± 28.24 46.99 ± 20.80 Phenylalanine (Phe) 23.20 ± 5.38 32.51 ± 8.87 25.86 ± 6.54 29.99 ± 7.62 Ornithine (Orn) 2.61 ± 0.80 2.49 ± 0.75 2.58 ± 0.74 2.31 ± 0.70 Lysine (Lys) 34.67 ± 8.98 38.28 ± 12.12 36.35 ± 6.27 33.25 ± 5.63 Histidine (His) 144.42 ± 40.33 120.30 ± 37.19 151.46 ± 49.64 145.57 ± 29.00 Arginine (Arg) 15.26 ± 4.53 19.46 ± 7.67 13.99 ± 4.21 12.66 ± 4.80 a b ab ab Proline (Pro) 688.83 ± 286.60 453.67 ± 139.60 607.11 ± 116.11 558.56 ± 96.81 Values with different superscript letters are significantly different (p < 0.05) next 24 h (Fig. 4a). The tissue formalin concentrations in tissues. Significant changes in these parameters were ob- the ascidians were approximately 1/3 of the bath concen- served at optimal treatment concentrations of 40 ppm tration after 1 h and undetectable after 48 h (Fig. 4b). formalin and 10 ppm H O .H O exposure stimulates 2 2 2 2 H O concentrations exhibited a similar pattern to forma- lipid peroxidation, as H O biocidal effects in living or- 2 2 2 2 lin; the agent was stable in the seawater bath and barely ganisms are based on production of free radicals (Siddi- detectable in ascidian tissues after 24 h (Fig. 4c, d). The que et al. 2012; Cavaletto et al. 2002). In addition, lowest concentrations of formalin and H O were about formaldehyde causes lipid peroxidation (Gulec et al. 2 2 0.4 and 0.1 ppm, respectively. These concentrations apply 2006; Saito et al. 2005) directly and via a secondary for both ascidian tissues and culture water. mechanism involving the production of reactive oxygen species (Hancock et al. 2001). Although further studies Discussion are required, it is reasonable to assume that the com- This study was performed to assess the toxicity of com- bined formalin–H O treatment stimulated lipid perox- 2 2 bined formalin–H O treatment in edible ascidians. idation at the 2-fold effective concentration in this 2 2 Formalin–H O combination is very effective against marine invertebrate. It is known that reactive oxygen 2 2 the tunic-infecting parasite A. hoyamushi (Park et al. species deplete endogenous reducing biomaterials in 2014; Lee et al. 2016). The treatment concentrations cells (Lushchak 2014) and glutathione is the representa- tested here were based on concentrations exerting anti- tive reducing agent in marine invertebrates (Conners protozoal effects and used for treating STS. Biochem- 1998). Lipid peroxidation is postponed until reducing ical responses were monitored to evaluate the toxic reserves of the cell are completely exhausted. The ob- effects of the formalin–H O combination. Drug resi- served pronounced decline in free radical scavenging 2 2 due concentrations were analyzed to correlate toxicity ability and reducing power compared to elevation of with drug levels in the tissue. lipid peroxidation could indicate that some biochemical Oxidative damaging effects of the combined agents changes occur than others. were evaluated by examining lipid peroxidation, free rad- Major glycogen deposits in ascidians occur in the pyl- ical scavenging activity, and reduction potential in edible oric gland, which plays a homologous role in the liver Lee et al. Fisheries and Aquatic Sciences (2017) 20:12 Page 6 of 7 Observed amino acid patterns match the typical char- acteristics of edible tissues of this species: high content of taurine, proline, glutamic acid, and glycine, as de- scribed by Watanabe (Watanabe et al. 1983). Aspartic acid concentration was reduced in a concentration- dependent manner. Although aspartic acid levels were relatively high (taurine > proline > glutamic acid, glycine, histidine > aspartic acid, alanine, threonine>serine), the importance of this amino acid in physiology of ascidians is not known. Cysteine content was diminished after 2- fold exposure. Reduced cysteine content may reflect the changes in reducing potential because cysteine is used as a precursor in the synthesis of glutathione, which reactive compounds like formalin and H O may deplete (Poole 2 2 2015). Cysteine protects against free radical damage caused by paraquat (Shoji et al. 1992), although the signifi- cance of cysteine in ascidians is not known. A very interesting phenomenon observed in the free amino acid analysis was the elevation of alanine and valine levels. Alanine is important for intracellular osmolality regulation in Pacific oysters, with salinity changes inducing immediate elevation of alanine levels in mantle tissues (Hosoi et al. 2003). However, the im- portance of alanine and valine in ascidians in relation to stress requires further studies. Biochemical toxic responses were observed 24 h post- bathing; however, associated residue levels of formalin and H O were undetectably low. This finding implies 2 2 that the exposed animals were recovering and further progression of toxicity is not expected. In addition, rapid elimination of treatment agents is ideal from the aspect Fig. 4 Formalin and H O concentration in treatment bath and 2 2 of food safety. In contrast to the rapid decline of forma- treated edible ascidian tissues. Formalin and H O concentrations 2 2 lin and H O residues in edible ascidian tissues, the 2 2 were measured at the termination of 1 h exposure in the treatment compounds are reasonably stable in aquatic media, mak- bath and ascidians. Additional measurements were made at 4, 24, or 48 h. a Formalin concentration; b H O concentration; N =3 2 2 ing daily 1 h bathing treatment possible (Jung et al. 2001; Yamamoto et al. 2011). In addition to their role in in other animals (Ermak 1977). Glycogen storage in the the main purpose of this study, which is examining toxic pyloric gland indicates disturbed metabolic activity responses to formalin–H O bath treatments, biochem- 2 2 (Gaill 1980), and thus, the increasing trend after 1-fold ical parameters assessed can be used to monitor the im- exposure reflects a perturbation in energy balance pact of these chemicals during treatment. caused by the treatment. However, interpretation be- In view of toxicity form this study, formalin–H O 2 2 comes complicated, as the 2-fold exposure did not in- bathing sounds promising to disinfect ascidians against crease glycogen content. It is known that reduction STS-causing parasites. The practice will be specifically status induce changes in glycogen contents in mice useful before landing ascidian seedlings in Korean waters. (Nocito et al. 2015). Free amino acid content is an indicator of toxic re- Conclusions sponse in various aquatic invertebrate animals (Cook et STS is a highly infectious protozoal disease that has se- al. 1972; Hosoi et al. 2003). Changes in free amino acid verely affected ascidian industry in Asian countries. levels in tissues during stress occur because of altered Bathing treatment with formalin–H O combination so- 2 2 amino acid utilization for protein synthesis (Kültz 2005). lution is an effective method for reducing STS mortality. Amino acid changes in ascidians occur seasonally Bathing treatment with optimal drug concentrations in- (Watanabe et al. 1983). 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