Aquatic Toxicology

Aquatic Toxicology 105 (2011) 508–517Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Risks of seawater ozonation in recirculation aquaculture – Effects of oxidative stress on animal welfare of juvenile turbot (Psetta maxima, L.) S. Reiser a,b,∗ , S. Wuertz b,c , J.P. Schroeder a,c , W. Kloas b,d , R. Hanel e,a a Leibniz-Institute of Marine Sciences, IFM-Geomar, Duesternbrooker Weg 20, 24105 Kiel, Germany Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 310, 12587 Berlin, Germany Gesellschaft fuer Marine Aquakultur, Hafentoern 3, 25761 Buesum, Germany d Institute of Biology, Department of Endocrinology, Humboldt University, Invalidenstrasse 43, 10115 Berlin, Germany e Institute of Fisheries Ecology, Johann Heinrich von Thuenen-Institute (vTi), Federal Institute for Rural Areas, Forestry and Fisheries, Palmaille 9, 22767 Hamburg, Germany b c a r t i c l e i n f o a b s t r a c t Ozone is frequently used for water treatment and disinfection in recirculating aquaculture systems. However, due to the fragmentary data on chronic toxicity of ozone produced oxidants (OPO) and its safe concentrations, the daily application of ozone in aquaculture is challenging. To evaluate the chronic effects of sublethal OPO concentrations, juvenile turbot (Psetta maxima, L.) were exposed to OPO concentrations of 0.06, 0.10 and 0.15 mg/l for 21 days. Gills were analysed for histopathological alterations and mRNA expression of heat shock protein 70 (hsp70), hsp90 as well as glutathione-S-transferase (gst) were determined in the gills and the liver after 1 d, 7 d and 21 d. Histopathologic findings confirmed adverse effects at 0.10–0.15 mg/l, but these (necrosis, lamellar clubbing, hypertrophy, hyperplasia) could only be observed after an extended exposure (mostly 21 d), and were considered as irreversible tissue damage. Hsp70 expression in the gills was only significantly increased at the highest OPO concentration (0.15 mg/l) on 1 d and 7 d, and returned to basic levels until day 21. Hsp90 mRNA was already increased at 0.10 mg/l after 1 and 7 days of exposure, and again was comparable to the control group on day 21. In contrast, elevated gst mRNA expression was only observed on day 7 at 0.10 mg and 0.15 mg/l. Although similar trends were observed in the liver for all markers, differences were only significant in exceptional cases due to the high individual variation observed. Thus, mRNA expression in the gills rather than in the liver is recommended as a marker to characterize OPO-induced oxidative stress in turbot. It has to be noted that mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration, suggesting a collapse of adaptive mechanisms as a possible explanation for the observed tissue damage. © 2011 Elsevier B.V. All rights reserved. Article history: Received 30 May 2011 Received in revised form 3 August 2011 Accepted 8 August 2011 Keywords: Turbot RAS GST HSP70 Heat shock protein HSP90 Histology Oxidative stress Ozone 1. Introduction Governed by the perception that many wild stocks are rapidly being depleted and flow-through aquaculture pollutes local water resources, recirculating aquaculture systems (RAS) are increasingly being used to meet the requirements of a sustainable aquaculture production (Waller, 2000; Martins et al., 2010). An effective reduction of pathogens such as bacteria, viruses and fungi is of particular importance in intensive RAS, which is mostly accomplished by the use of oxidizing disinfectants (Liltved et al., 1995). The most frequently applied disinfectant in marine RAS is ozone, comprising additional benefits like the removal of metabolic waste products, toxicants or dissolved organic matter (Rosenthal and ∗ Corresponding author at: Institute for Hydrobiology and Fisheries Science, University of Hamburg, Olbersweg 24, 22767 Hamburg, Germany. Tel.: +49 40 428387874; fax: +49 40 42838 6618. E-mail address: [email protected] (S. Reiser). 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.08.004 Otte, 1979; Summerfelt and Hochheimer, 1997). However, excessive ozonation may severely impact farmed fish by causing adverse effects including histopathologic tissue damage (Richardson et al., 1983; Reiser et al., 2010), alterations in feeding behaviour (Reiser et al., 2010) as well as oxidative stress (Ritola et al., 2000, 2002b; Livingstone, 2003). Although ozone is intensively used in marine aquaculture systems, knowledge on the toxic impact of ozone on marine aquaculture organisms is limited. Findings from freshwater studies cannot be projected to marine conditions, since emerging ozone-induced oxidants differ substantially in marine and freshwater systems (Hoigné et al., 1985; Oemcke and van Leeuwen, 1998). Ozone has a very short half life in seawater of only a few seconds (Haag and Hoigné, 1984). It instantaneously reacts with different chemical compounds resulting in the formation of several reactive species, generally summarized as ‘ozone-produced oxidants’ (OPO). Particularly halogen ions are oxidized to halogenated oxidants by ozone (Haag and Hoigné, 1984; von Gunten, 2003; Perrins et al., 2006). Low concentrations of iodide ions in typical seawater as well as the slow first order rate constant for 1998) with hsp70 and hsp90 being the most frequently studied heat shock proteins. Lewis et al. In unstressed cells. as well as endogenous reactive intermediates produced during cellular oxidative stress like peroxidative products of DNA and lipids (Hayes and Pulford. Reiser et al. 1954. Basu et al. dissolved oxygen concentration: 10. organic peroxides. (van der Oost et al. 1998).. 2000). Gsts conjugate electrophilic xenobiotics and phase-I metabolites. Among several molecules involved. Hsp70 assists the folding of nascent polypeptide chains. Herwig et al. 2007). stocking density (Gornati et al. 2008... there is a high potential for the formation of brominated oxidants in ozonated seawater (Hoigné et al. 2002).16 mg/l.29 ± 0. Ozone-enriched air was injected into the seawater via foam fractionators (Model 1 AH 1100.01. 2. van der Oost et al.. Denmark) and randomly stocked to 12 identical recirculation systems providing triplicates of 23 fish for each OPO treatment and the control group. while phase-II enzymes act on secondary metabolites induced by the primary stressor. Hsp70 and hsp90 have been used intensively as biomarkers for the evaluation of stressors in fish including toxicants (Hassanein et al. Upon exposure of juvenile turbot to three different sublethal OPO concentrations (0. 36. Recirculation systems were set up in a temperature controlled lab in order to ensure constant environmental conditions as recommended by Hurlbert (1984). in particular with regard to chronic effects. juvenile turbot were exposed to OPO concentrations (±SD) of 0. 2004. 2006) and become toxic to the cultivated organisms.93 ± 0..e. Richardson et al. OPO concentrations were expressed as chlorine equivalents. substrate specific. hsp90 and gst mRNA expression in the liver and gills were determined by RT-qPCR. enzymes and steroid hormone receptors (Fink and Goto.. Thus.07.. NHBr2 ) represent the majority of OPO in marine RAS.e. The results were discussed with regard to histopathological alterations and abnormalities of the gills. Perrins et al.. Each recirculation system comprised of a 200 l fiberglass tank (Chemowerk) filled with approximately 150 l of filtered natural seawater and an individual biofilter and foam fractionator (by-pass operated). using the colorimetric N..Ndiethyl-p-phenylenediamine (DPD) method (DPD Total Chlorine powder pillows. it is not stressor specific (Iwama et al.01 and 0. acts as a molecular chaperone and mediates the repair and degradation of altered or denatured proteins. In general.. 1999) which are further specified according to their molecular weight (kDa). Ozone gas was produced by electrical discharge ozone generators (ModelC 200. Materials and methods 2.. thereby affecting respiratory function and osmoregulation (Giese & Christensen..41 ± 0.1. 1989.2 ± 1. as recommended for the quantification of OPO in seawater (American Public Health Association. Hayes and Pulford (1995) suggested that the level of gst expression determines the sensitivity of cells towards a broad spectrum of toxicants. 2003). detoxifying enzymes are specific to a stressor.5 mm. Erwin Sander Elektroapparatebau GmbH) operating in counter-flow mode to ensure maximum diffusion of ozone. OPOs were reported to oxidize membrane lipids of epithelia cells. 1998. . total ammonia-N. Dana Feed) to apparent satiation (accounting for approximately 2% of body weight) in 48 h intervals to minimize fluctuations in OPO concentrations.. Erwin Sander Elektroapparatebau GmbH) using compressed air. Major aim of the present study was to ultimately define a threshold for a safe. etc.. hsps act as an alarm response. 0. 1979). 2007. Bouraoui et al. 1978.53 mg/l and nitrite-N: 0. In stressed cells.57 ± 0. 2005) and poor water quality such as high nitrite loads (Deane and Woo.. 1998. the brominated oxidants free bromine (HOBr/OBr− ) and bromamines (NH2 Br. We used the nomenclature “ozone-produced oxidants” (OPO) to refer to the total residual oxidant concentration.e. salinity. So far.10 ± 0.02 mg/l for 21 days.. Contrary to hsps. Since cellular stress response represents a defence reaction of cells to damage that environmental forces inflict on macromolecules.g.48 ± 0. Boone and Vijayan. Hach) at 530 nm with a DR/2800 Spectrophotometer (Hach Lange). Gst inactivates lipoperoxidation products. hsp90 and gst as biomarkers for oxidative stress. Hsp are involved in numerous aspects of protein metabolism maintaining cellular integrity and homeostasis (Fink and Goto. i. non-hazardous OPO concentration for juvenile turbot. measured spectrophotometrically as “total residual chlorine”. Block et al. we monitored chronic adverse effects on juvenile turbot (Psetta maxima) caused by ozonation and evaluated the suitability of hsp70. Following an acclimation period of 10 days. e. 2003). lipid hydroperoxides and their derivates (Salinas and Wong.2 g wet weight) were purchased from a commercial turbot hatchery (Maximus Fry. pH: 7. 1999. Crecelius. 0. As an enzyme contributing to phase-II metabolism.e.84 mg/l. To safeguard good welfare within aquaculture production systems. ameliorating stressful states by detoxification and re-establishment of homeostasis. The retention time in the foam fractionator was set to approximately 1 min. dissolved oxygen.06 ± 0. with relatively high bromide-ion concentrations of 60–70 mg/l and an ozone reaction rate constant of approximately 160 M/s. with the nucleophilic tripeptide glutathione (GSH) from the intracellular pool (Halliwell and Gutteridge. 1. van der Oost et al.. 1998. maintaining cellular functioning by increased biosynthesis. 1983). i. Pandey et al. OPO concentrations were determined spectrophotometrically in 2 h intervals over the entire experimental period and adjusted if necessary. However. 1976. i.06. 1999). hsp70. total ammonia-N (TAN): 0. Ozonated water was introduced at high flow rates (600 l/h) via vertical spray bar and supply of ozone was controlled and regulated by a redox potential controller (Erwin Sander Elektroapparatebau GmbH) using the redox potential as proxy for the total oxidant concentration (Buchan et al.. salinity: 18. Physical and chemical water parameters (temperature. oxidized lipids. alterations in Gst activity have been observed in fish exposed to different toxicants (Ferrari et al. 1998) and fed with a commercial pellet feed (DAN-EX 1562. 2010). Hence. potential stressors like OPO have to be evaluated.. 1999.. they have a high potential to accumulate within RAS (Oemcke and van Leeuwen. 2002). 2005). In the present study. phase-I enzymes that metabolize the stressor/toxicant in a first step.10 ± 0.4 ± 8. Fish were kept at a 12:12 L:D photoperiod (Pichavant et al.S..15 ± 0. 0. i. Furthermore some OPOs are suggested to enter the cell.. nitrite-N) were measured at 2 day intervals as described (Reiser et al.45 ◦ C. detoxification is divided in two phases. heat shock proteins (hsp) comprise a family of highly conserved cellular proteins upregulated in response to cellular stress.00. damaged macromolecules (Iwama et al. pH.. Buchan et al. Mean water quality (±SD) was: temperature: 14. 1985) make the respective oxidation products less important. 2003).0 × 10−3 M/s) (Hoigné et al. Experimental setup Juvenile turbot (12. 2005).25.15 mg/l) for 21 days. As these OPO are more stable compared to ozone itself (Blogoslawski et al. Hsp90 supports numerous components of the cytoskeleton. 2008) as well as ozone (Ritola et al. 1995.. / Aquatic Toxicology 105 (2011) 508–517 509 ozone’s reaction with chloride ions (k = 3. Glutathione S-transferases (GSTs) represent a multigene family of primarily soluble enzymes that are involved in detoxification of electrophilic and genotoxic xenobiotics. there is a basic production of hsp.10 and 0.0 cm TL. 1985). 2006. 1* Taq buffer. followed by 40 cycles of 95 ◦ C denaturation for 30 s. For histological analysis the second right gill arch was stored in 4% sodiumtetraborate-buffered formalin. / Aquatic Toxicology 105 (2011) 508–517 Table 1 Primers used for real-time RT-qPCR providing NCBI accession code. Data were calculated as mean for each single tank and compared between replicates and treatments (Hurlbert. 7 and 21 days of OPO exposure. RNA (1 ␮g) was transcribed using a poly(dT) primer [CCTGAATTCTAGAGCTCA(T)17] in a two step procedure. 2. A DNAse I treatment was used to assure DNA-free RNA for transcription (Qiagen). position and amplicon size.5 mM dNTP] and denaturation at 94 ◦ C for 3 min.3. Reiser et al.2. 700 ␮l gill) was used for the subsequent extraction. 12000 g. a subsample of the supernatant (300 ␮l liver. Histology Gill arches were dehydrated in a graded series of ethanol. 2. 1 U Taq). Upon centrifugation (15 min. isopropanol was removed. ß-2-mikroglobuline (ß2m).. and only considered if Ct < 0. nucleotide sequence.5 min at 30. (2004). three commonly used housekeeping genes (ß-actin (act). When conformed. A melting curve was included at the end of the PCR to confirm specific amplification. Leica). clubbed lamellar tips. epithelial lifting. 10 U AMV-RT. 2009). Sampling Following 1. After centrifugation (15 min. (1) annealing of poly(dT) primer at 70 ◦ C for 2 min [reaction mix: 0. Act (ß-actin). After dissecting the gill apparatus. If normality or homogeneity . 10 nM of each dNTP. hypertrophy and hyperplasia according to Mallatt (1985).0 l/s using a tissue lyser (Qiagen). the effect of each single factor was investigated by one-way ANOVA. 12000 g. It was consequently used for normalisation by the comparative CT method (Pfaffl. necrotic. tissue samples were homogenized twice in 700 ␮l TRIZOL® for 1. 600 ␮l of TRIZOL® were additionally added.. Real-time quantification Using gene-specific primers (Table 1). Specific amplification of the assays was confirmed by direct sequencing and multiple sequence alignment analysis as previously described (Wuertz et al. ß2m (ß-2-mikroglobuline). 2. samples were vortexed (15 s). RNApolymerases II (pol)) were evaluated comparing CT values of a subset of samples over time. Total RNA content was measured by Ribogreen RNA-quantification kit (Invitrogen) in a Spectrafluor plus microplate reader (Tecan) and RNA integrity was controlled on a RNA 6000 Nano LabChip (Agilent Technologies) as described (Trubiroha et al. RNA extraction Extraction of total RNA from stored tissue samples was performed using TRIZOL® (Invitrogen) according to Kroupova et al. All PCR reactions were run in duplicate. Cross-sections of 2 ␮m were haematoxylin–eosin stained and analysed using a Leica DMRBE microscope equipped with a digital camera (DFC320. Frances et al. For normalisation. 4 ◦ C).5. factorial ANOVAs were conducted and significance was analysed by Tukey HSD test for equal sample sizes or unequal N HSD test. The target transcript expression of genes is presented as fold increase of the control at each day. For each run. fish were randomly sampled by dip-netting and immediately killed in icewater as recommended (FAO. Target act ß2 m pol hsp70 hsp90 gst Primer actF actR ß2mF ß2mR polF polR hsp70F hsp70R hsp90F hsp90R gstF gstR Sequence 5 -AgCACggTATTgTgACCAAC-3 3 -AgCTTggATggCAACgTAC-5 5 -gATCTgCCACgTgAgCg-3 3 -AggAACggCACgCTCTT-5 3 -AgACgCACAgACCTCACg-5 3 -ggTgCCATgTCgACTAgAgAC-5 5 -CTgTCCCTgggTATTgAgAC-3 3 -gAACACCACgAggAgCA-5 5 -CCgCCTACCTCgTTgC-3 3 -gTAgCCgATgAACTgCgAgT-5 5 -gggTTCgCATCgCTTTT-3 3 -ggCCTggTCTCgTCTATgTACT-5 Position 142 327 56 183 96 285 1162 1366 401 610 9 183 Accession nr EU686692 EU878757 EU925140 EF191027 EU099575 DQ848966 Amplification size 185 127 189 204 209 174 2. 1*SYBR-Green I solution. first and second left gill arches were sampled for gene expression analysis and stored in RNAlater. Statistical analysis Data are presented as mean ± standard deviation and were analysed using STATISTICA 6. (1987).510 S. PCR conditions were: 95 ◦ C initial denaturation for 8 min. To assure a high RNA quality. cleared with xylene and infiltrated and embedded in paraffin according to Wuertz (2005). If a significant interaction between the two factors “exposure time” and “OPO concentration” was observed.6. 2009). and incubated for 10 min at room temperature (RT). 2. 56 ◦ C primer annealing for 30 s and 72 ◦ C polymerisation for 40 s. real-time PCR was carried out with a Mx3005p qPCR Cycler (Stratagene) using hot start polymerase (Platinum. 270 ␮l isopropanol (Roth) were added and the sample was incubated at room temperature for 10 min. (2011). respectively. followed by centrifugation for 10 min (12000 g. between and within treatments. 270 ␮l of the aqueous phase were transferred to a new vial.5. Evaluation revealed ß2m as most constantly expressed housekeeping gene. lamellar fusion. hsp70 (heat shock protein 70).3 ml and 250 ␮l of chloroform. Invitrogen) and SYBR Green (25 ␮l reaction volume: 4 ␮l diluted cDNA. hsp90 (heat shock protein 90) and gst (glutathione-S-transferase) genes. The histopathological alterations were recorded as percentage of affected secondary lamellae and assigned to the categories not affected. 0.4. For gene expression analysis. 2007). pol (RNApolymerases II).01%). 2001). (1998) and Gisbert et al. 4 ◦ C). Pellets were redissolved in DEPC-treated water (0. 5 mM MgCl2 . Genomic DNA contamination was excluded by an -RT control where the AMV-RT had been replaced by DEPC treated water. Crespo et al. quantities were calculated from a standard dilution series of a pooled liver and a pooled gill sample compensating for lot-to-lot and run-to-run variations. chilled on ice for 3 min followed by (2) synthesis at 36 ◦ C for 60 min [reaction mix: AMV buffer. 400 nM of each primer. 1984). A liver sample was extracted through an incision in the ventral cavity. Five primary filaments from the second right gill arch per individual were randomly chosen and 20 lamellae per filament were blindly selected and analysed. In brief. approximately 100 mg of liver and gill tissue were immediately incubated in 1 ml RNAlater (100 mg/ml) for 24 h at 4 ◦ C and stored at −20 ◦ C. Adding further TRIZOL® to 1. 4 ◦ C). the pellet was washed with 70% ethanol (300 ␮l) and allowed to dry after the removal of ethanol.1 (StatSoft).625 ␮mol poly(dT)]. Histopathological alterations of the secondary gill filaments of juvenile turbot (Psetta maxima) upon exposure to OPO concentrations of 0. 16) = 0. The total amount of affected lamellae remained almost stable in control animals (Fig. “lamellar clubbing”. In contrast.15 mg/l compared to the control at day 1.98. subsequent analyses within levels of each factor were carried out using Kruskal-Wallis test.05. p > 0. 3.1. p > 0. 1). F(1.57. 1) with the 0. F(1. act and ß2m in the gills were affected neither by OPO concentration (act: two-way ANOVA.S. of variances were not confirmed. pol mRNA levels were significantly higher after 1 d compared to 21 d (two-way ANOVA. ß2m and pol are presented in Figs. lamellar fusion.05. p > 0. p > 0.2.01). 14) = 0. “hypertrophy” and “hyperplasia” as described in the text. data were analysed with the Scheirer-Ray-Hare test (Sokal and Rohlf. F(1. Gill histology Several histopathological deteriorations were detected and categorised as not affected. even after transformation. 16) = 14.001.05).05. 8) = 12. epithelial lifting. p > 0. No significant . F(1.58.10 and 0.22. No significant interaction was observed for both housekeeping genes (p > 0. F(1. F(1. 2 and 3. necrosis. 16) = 0. 16) = 1. F(1. 2). Similarly. In the liver. p < 0.67. p > 0.15 mg/l OPO.02. F(1. 16) = 0.05) (Fig. The major histopathological categories analysed are “not affected”. “necrosis”. 16) = 0. 1). 1.05) nor by the duration of the experiment (act: two-way ANOVA.01) but not by OPO concentration (two-way ANOVA.24. which were not correlated to the OPO concentration and therefore excluded from further analysis. ß2m: two-way ANOVA. p < 0. Necrotic abnormalities were observed in 69. ß2m two-way ANOVA.06. 3. mRNA expression of act and ß2m was not influenced by OPO concentration (act: two-way ANOVA.33. p > 0. 0.15 mg/l exhibited the most pronounced alterations in gill histology.4% of the investigated specimens and were also present in controls. hypertrophy and hyperplasia (Fig. showing a slight increase in hyperplasia at day 21. lamellar clubbing.10 and 0. Here. ß2m: two-way ANOVA. p > 0. 3). Results 3. Fish exposed to OPO concentrations of 0. 14) = 0. / Aquatic Toxicology 105 (2011) 508–517 511 Fig.05).10 and the 0. F(1. Evaluation of housekeeping genes Relative mRNA expression of the commonly used housekeeping genes act.05. 16) = 0.24.06. In OPO-exposed turbot the amount of gill filaments showing no histopathological damage decreased steadily over the experiment (Fig.15 mg/l treatment showing the most pronounced changes in gill histology. Reiser et al. 7 and 21. p > 0. F(1. Lamellar clubbing. 1995). 16) = 3. hypertrophy and hyperplasia occurred in all specimens analysed. Lamellar fusion and epithelial lifting were detected in just few specimens analysed.05) nor by the time of exposure (act: two-way ANOVA. Upon exposure to 0. pol mRNA expression in liver was significantly affected by time of exposure (two-way ANOVA.05) (Fig. F(1. ß2m: two-way ANOVA. ß2 microgobulin (ß2m) and RNA-Polymerases II (pol) in the liver of control specimens and fish exposed to the highest OPO concentration (0. mRNA transcription of the housekeeping genes actin (act).05).05). Expression was normalised to ß2m and set as fold increase of control at each sampling day.61.15 mg/l.01) but not by time of exposure (twoway ANOVA.68 fold increase after 7 d of exposure when compared to control specimens. exposure time: two-way ANOVA. 16) = 13. RNA transcription of the housekeeping genes actin (act).06 mg/l and 0. For hsp70. subsequent Tukey HSD did not Fig. p > 0. In contrast. 24) = 0. In both tissues. Here.15 mg/l after 1 d and a 1.38 fold increase after 7 d. actin and ß2m were thus regarded as suitable housekeeping genes. hsp90 and gst in the liver increased upon OPO exposure compared to control specimens until day 7 and returned to basal levels by day 21 (Fig.07 fold increase at day 7.37. As observed for hsp. Error bars denote standard deviations.37. Reiser et al.05.01) but not by OPO concentration (two-way ANOVA.06 mg/l. ß2 microgobulin (ß2m) and RNA-Polymerases II (pol) in the gills of control specimens and fish exposed to the highest OPO concentration (0. interaction was observed (p > 0. 3.06 fold elevation at day 1 and a 2. 2. 7 and 21. respectively. 24) = 0. 24) = 5. show any differences between the different OPO treatments (p > 0. p > 0. p > 0. F(1. F(1. expression returned to basic levels by day 21 and no significant differences between treatment groups and exposure time were observed (OPO concentration: twoway ANOVA. (b) hsp90 and (c) gst genes in the liver of juvenile turbot P. Again. the mRNA expression of gst returned to basic levels on day 21 and did not show any . gst exhibited a 2. 16) = 2.15 mg/l) after 1 and 21 days of exposure.72 fold increase in hsp90 after 1 d and a 1. F(3. / Aquatic Toxicology 105 (2011) 508–517 Fig. Exposure to an OPO concentration of 0. Fig. However.15 mg/l revealed a 1.15 mg/l compared to the control at day 1. At an OPO concentration of 0. ß2m was chosen for further analyses as actin mRNA expression in the target tissues was slightly more variable than that of ß2m. Different lower case letters indicate significant differences between exposure times.37. gst mRNA increased to 1. a 1. maxima upon exposure to OPO concentrations of 0.05). p < 0. 4).66 at day 1. F(2. Mean (±SD. 4. 3.10 mg/l and 0. F(2.56.93 fold increase was observed at 0.3. p > 0. Different upper case letters indicate significant differences between control and the OPO treatment. 0. Hsp70 was significantly affected by exposure time (two-way ANOVA.84.10 mg/l OPO treatment groups.05). p < 0. mRNA expression in liver All biomarker mRNAs.15 mg/l) after 1 and 21 days of exposure. In the 0.05). 24) = 1. those of hsp70.62 and 1. F(3. n = 3) normalized mRNA expression of (a) hsp70. mRNA expression of pol was significantly affected by OPO concentration (two-way ANOVA.512 S. 8) = 3. 1980.06 mg/l (p < 0.15 mg/l for 7 d was significantly elevated compared to that in control fish (p < 0. hsp90: F(2.28. Error bars denote standard deviations.001). a gradual increase in the amount of gst mRNA with increasing OPO concentration was observed at day 7 (one-way ANOVA.05) but not compared to fish exposed to 0.16 fold more at day 7.05).001.46 fold more hsp90 than control specimens (p < 0. 8) = 16. 8) = 1. due to its high oxidative capacity has been established as an effective water treatment with proven efficacy of disinfection in routine application (Rosenthal and Fig.001. Hsp90 mRNA levels of fish exposed to 0. 8) = 3.10 mg/l treatment (p < 0. as well as to fish of the 0. Turbot exposed to 0. 5b). p > 0.05. p < 0.88 fold more at day 7. 24) = 15.49 fold increase.17.40. 1997). 5c) was not found to be altered by OPO at day 1 (one-way ANOVA. columns with different upper case letters indicate significant differences among concentrations. Fukunaga et al. Expression of hsp70.05) and day 7 (one-way ANOVA. p > 0. 2000. hsp90: F(3. p < 0.S.001). Morita et al. After 21 days of OPO exposure.05). 5. F(3.001.. 1995. Therefore. p < 0.64. 0.33.10 mg/l and 0. mRNA expression in the gills mRNA expression of the investigated biomarker genes in the gills revealed similar patterns as in liver. p < 0. 24) = 6. the gst mRNA level in fish exposed to an OPO concentration of 0. p > 0.06 mg/l. However. p < 0. Fish exposed to an OPO concentration of 0. Hsp90 in fish exposed to an OPO concentration of 0. Discussion In the past 20 years. F(3. 5). Ozonation.15 mg/l at day 7 was significant compared to control specimens (p < 0.. Summerfelt and Hochheimer.27 fold. . gst: F(6. the technical development of RAS for a sustainable marine finfish production has become an emerging technology in aquaculture.05). Paller and Heidinger.10 mg/l were significantly elevated compared to those from control fish (p < 0. Ritola et al.. Mean (±SD.g. Turbot exposed to OPO showed strong alterations in hsp70 compared to control specimens at day 1 (one-way ANOVA.001).05).97. F(3.10 mg/l (p < 0.001.03 fold increase in gst expression compared to control specimens (p < 0. F(3. p > 0.71. 5 a).001.001). hsp90 decreased beyond control levels within all OPO treatments. p < 0.10 mg/l was significantly elevated compared to control specimens (p < 0.05). 24) = 26. Still.02. 24) = 21. (b) hsp90 and (c) gst in the gills of juvenile turbot P.45. However. p < 0. gst: F(2. / Aquatic Toxicology 105 (2011) 508–517 513 statistical differences between the groups (OPO concentration: Scheirer-Ray-Hare two-way ANOVA. where hsp70 was elevated around 1.05. hsp90: F(6.75 fold more hsp70 compared to control specimens (p < 0. 8) = 5..06. F(3.24. even low OPO concentrations can adversely affect the physiology of fishes (Richardson et al. maxima upon exposure to OPO concentrations of 0.37. p > 0. For all three genes.05). H(2) = 2. no significant effect of OPO concentration on hsp90 levels was detected (F(3. a significant effect of OPO concentration on hsp90 mRNA level was detected at day 1 (one-way ANOVA. no significant difference between controls and OPO treatments was observed (one-way ANOVA. 1995.14.15 mg/l compared to the control at day 1. F(3. whereas fish exposed to 0. 1983. to fish exposed to 0. At day 21... F(3. Gst mRNA levels in OPOexposed specimens returned to control levels at day 21. numerous studies focused on acute ozone toxicity addressing freshwater fishes (e.77. 8) = 14. 24) = 6.05).01) and 2. Expression was normalised to ß2m and set as fold increase of control at each sampling day.06 mg/l (p > 0. Reiser et al. Otte. subsequent one-way-ANOVAs were conducted to separately investigate the effects of OPO concentration and time of exposure. Gst expression (Fig.01) showed an ∼1. 8) = 7.05) and day 7 (one-way ANOVA. Letters are not shown if there was no significant difference within one sampling day or between sampling days.05. Following 1 d of exposure.01) as well as by exposure time (hsp70: F(2.15 mg/l exhibited around 1. At the same exposure time. 1979. Reiser et al. 8) = 21. 1979.15 mg/l OPO treatment exhibited around 2. p < 0. p > 0. with regard to the high oxidative potential..05). 24) = 2.06.15 mg/l OPO showed an up to 2. 24) = 8. F(3.001) (Fig. 3. p < 0. As for hsp70. Liltved et al. p < 0. showing no significant differences among treatments (one-way ANOVA.32. n = 3) normalized mRNA expression of (a) hsp70. In addition. hsp90 and gst in the gills were all affected by OPO concentration (hsp70: F(3. 8) = 1. p < 0. a significant interaction occurred (hsp70: F(6.55.05) at day 1 and around 1.58.01). 4.05). exposure time: Scheirer-Ray-Hare two-way ANOVA. 24) = 15.85. different lower case letters indicate significant differences between exposure times. Up to now.06 mg/l (p < 0. gst: F(3. Wedemeyer et al. 24) = 9.01). 2002b). 2010). fish in the 0. p < 0. individual variation as indicated by the standard deviation was lower (Fig. within the same concentration. 7 and 21. H(3) = 2. p < 0.01) (Fig. p < 0. 1992. Here.01) as well as in fish exposed to 0. The increase in hsp70 mRNA in fish exposed to 0.4. means not sharing a common letter are significantly different. The increase of both hsp mRNAs towards significant levels illustrates that OPO concentrations as low as 0. Most prominent. 2004). / Aquatic Toxicology 105 (2011) 508–517 but just a few have considered estuarine and marine finfish species (Toner and Brooks. indicating protein damage and onset of protective mechanisms (Iwama et al. in the present study mRNAs of hsp90 and hsp70 were similarly increased. Iwama et al. revealing a more differentiated expression between OPO concentrations. reaching its climax on day 7..514 S.and concentration-dependent manner.. proliferation of neither mucous cells nor mucus itself has been observed (Reiser et al. Richardson et al. all biomarkers returned to normal on day 21. Richardson et al. ‘safe’ represents a level which does not cause an adverse. For daily aquaculture practice. necroses represent a deterioration of the gill epithelium... usually. Clubbing. Additionally. Cerqueira and Fernandes. thresholds for safe levels have to consider chronic toxicity at sublethal ozone and OPO concentrations. there is a baseline production of reactive oxygen species (ROS) in any cell as ROS arise from numerous cellular processes. Additionally.. (1983) in white perch at comparable OPO levels: massive deteriorations were reported at OPO levels of 0. 1981. This has widely been reported in fish. revealing a concentration-dependent response but not a continuous timedependent increase.. Mallatt. 2003). 2003. 1983). In this context. However.. Insufficiency and collapse of the aforementioned protective mechanisms in the gills causes rupture of the surrounding epithelium and enables infiltration and spread of the irritants through the dehisced cells. van der Oost et al. results obtained from freshwater cannot be projected to fish kept under marine conditions. (2010) reported slightly but not significantly increased haemoglobin and hematocrit levels in turbot upon 1 day of OPO exposure. 2004). the decrease in expression levels at day 7 potentially indicates an exhaustion of the hsp response but this remains speculative. Hsp70 and hsp90 mainly function as molecular chaperones mediating the repair of altered or denatured proteins. which can be explained by the passive and benthic mode of life compared to the highly mobile white perch. 2002)... Jiang et al. 2007) or upon exposure to toxicants (Mallatt. 1971). the turbot used for this study were already adapted to aquaculture conditions potentially making them more robust towards higher OPO concentrations. However. 1983. 2010).10 mg/l can already modulate cellular homeostasis in turbot.10 mg/l and to 0. Respiration is retained at deteriorated gill epithelia as. Still. Reiser et al. these irreversible damages indicate an exhaustion of adaptive countermeasures at OPO concentrations ≥0. oxidative stress due to enhanced formation of ROS involves the production of peroxides and free radicals at the cellular level and results in progressing denaturation of proteins and oxidation of lipids (Livingstone. hyperplasia of epithelia as well as chloride cells have been repeatedly reported indicative for chronic inflammation and distress at the cellular level. Alterations in hsp translation across baseline expression represent a first alarm response towards cellular stress. excessive hyperplasia and hypertrophy could also affect respiration and ultimately gill functionality as diffusion processes like gas-exchange. Hyperplasia..10 mg/l.. hypotrophy and hyperplasia may be regarded as adaptive defence responses towards irritants such as OPO in the process water (Mallatt. The categories of histological abnormalities identified here have been observed in numerous studies on different irritants (reviewed by Mallatt. 1998.e. 1975. e.. 2002) with hsp70 and hsp90 being considered as the most common markers of cellular stress (Lewis et al. transcription of hsp70 and hsp90 genes increased within 1 day. cell proliferation at the tips of the secondary filaments. 1985). Irritants in the water usually affect the gills first as they are just covered with a thin epithelium.. the hsp response is immediately established after proteins are damaged (Iwama et al. Chloride cells may also affect toxicant extrusion or neutralization (Mallatt. Due to the lack of introns in hsp genes. (2004) observed increased hsp70 and hsp90 with a persistent stressor still after 3 months in sea bass reared at high densities contradicting hsp exhaustion after 3 weeks of exposition.. 1985). 1985). 1985). However. 1998.10 mg/l to compensate for reduced oxygen permeability. studies focusing on the acute toxicity are of minor relevance for an ozone application in aquaculture production. increasing in a time. no difference between controls and specimens subjected to 0. hsp90 and gst mRNA congruent to the histological findings were observed. 1998). In the present study. Furthermore. detoxification should be enhanced as the amount of contributing cells is increased. OPO might infiltrate the cells via the irrigated gills. 1985). Upon exposure. lamellar clubbing. providing only a slight barrier between blood and the aquatic environment (Evans. get dispersed via the circulatory system and locally contribute to oxidative processes. Jones et al. play a crucial role in protein folding and support numerous components of the cytoskeleton that might be affected by ROS (Fink and Goto. 1981. suggesting a compensation of reduced oxygen uptake just after immediate contact with OPO. Because of the different ozone chemistry in both media. In general. 2003). data concerning the duration and dynamics of the hsp response towards persistent stressors is limited (Iwama et al. Additionally. Massive proliferation of chloride cells might indicate ion deficiency and therefore represent an attempt to re-establish cellular homeostasis (Laurent & Perry. Hall et al. explaining their high mRNA expression upon short term exposure of just one day in the present study. since ozone concentrations remain far below acutely toxic levels. Heat shock proteins have been identified as components of the secondary stress response cascade (Barton et al. 1985). Following chronic exposure.. Here. The hsp response decreased at day 7 and returned to basal levels by 21 days of exposure. 2004).g. The observed histopathological abnormalities were less severe compared to those found by Richardson et al.. excretion and acid–base regulation are exacerbated through the thickened gill epithelium (Evans et al. having higher oxygen requirements. Among the hsp family. sublethal effect upon long-term exposure (Sprague. In contrast.15 mg/l. Expression profiles of hsp70. which was attributed to the major importance of hsp70 as a molecular chaperone (Iwama et al. irritation of the gill epithelium and cell ruptures entail mucus proliferation and therefore restrict oxygen permeability as shown for white perch exposed to OPO (Richardson et al. . succeeded by gst. 2006). encapsulation of holdfasts of parasites (Lovy et al. Nevertheless. 1987). Richardson and Burton. i. 2005). as well as hypertrophy are considered to be not pathognomonic as they have been described to arise regardless of the irritant (Frances et al. Here excessive mucus production was associated with significantly increased hematocrit at OPO concentrations of ≥0. 1973. 1985).15 mg OPO/l was detected. 1983. Implying disturbances in the normal redox state of tissues. 1998). which disrupt cellular integrity of the tissue and ultimately constrains gill functionality (Mallatt. 1999. Mallatt.. Reiser et al. possibly accounting for increased mucous secretion upon OPO exposure only at the first day. 1991). hsp70 has been reported most sensitive towards stressors compared to hsp90.. In general. This induction of gene expression upon exposure clearly indicates an OPO-induced oxidative stress. Upon chronic exposure of turbot to sublethal OPO concentrations we observed adverse effects in gill histology. Gornati et al. necroses were just observed to a minor extent accumulating in a non-significant manner upon exposure to high concentrations. Although necrotic cells may be replaced (Karan et al. 2001. 2004).. This further confirms the robust nature of turbot (van Ham. they have often been interpreted as a way to block the entry of toxicants by increasing the toxicant–blood diffusion distance. thereby reducing further dispersion via the circulatory system (Morgan and Tovell. Among these abnormalities. Unfortunately. necroses increased at higher concentrations. D. L.R. L.A.g. 1979. Standard Methods for the Examination of Water and Wastewater .). E.. 225–231. Garceau.. evaluation of mRNA levels in liver and gills clearly demonstrate that sublethal OPO concentrations of ≥0. pp. Environ.. Respiratory and osmoregulatory responses of white perch (Morone americana) exposed to chlo and ozone in estuarine waters. Neuparth. Gorchev. Iwama. oxidative stress is locally restricted and the gills might be affected in particular due to the instantaneous contact with the irritant in the medium (Mallatt.H... Measurement of dissolved ozone in seawater: a comparison of methods. Vijayan. cellular adaptation involving an onset of detoxification processes could reduce oxidative stress and decreases the expression of hsp. showing highest transcription levels upon 7 days of OPO exposure. 2002. an upregulation of transcripts with products involved in detoxifying mechanisms has been observed in the present study. Barton.M. 2005). Gene 295. 2007. These enzymes catalyse the scavenging of oxidized radicals and prevent cellular damage by ROS (van der Oost et al. Impact of nitrite exposure in endocrine.. Still.. Washington. Res. . References American Public Health Association. 2002.. Schroeder et al..J. 2000. M. Measurements of oxidants in ozonized seawater and some biological reactions. J.N. Environ. 223–233. 2003. 2005. USA. P. A. Chem. Jamesville. pp. transferases and reductases. Aquat. N. Toxicol. Safe. M. Sala. Hence. 444–453.. 2000. J. 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