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The effects of elevated carbon dioxide and temperature levels on tilapia (Oreochromis mossambicus): Respiratory enzymes, blood pH and hematological parameters
Environmental Toxicology and Pharmacology 44 (2016) 114–119
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The effects of elevated carbon dioxide and temperature levels on tilapia (Oreochromis mossambicus): Respiratory enzymes, blood pH and hematological parameters Hasan Kaya a,∗ , Olcay Hisar a , Sevdan Yılmaz b , Mert Gürkan c , S¸ükriye Aras Hisar d a
Department of Basic Sciences, Marine Sciences and Technology Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey Department of Aquaculture, Marine Sciences and Technology Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey c Department of Biology, Faculty of Arts and Sciences, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey d Department of Food Engineering, Engineering Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey b
a r t i c l e
i n f o
Article history: Received 28 December 2015 Received in revised form 28 April 2016 Accepted 1 May 2016 Available online 3 May 2016 Keywords: Elevated carbon dioxide and temperature Hematology Blood pH Na+ K+ -ATPase Carbonic anhydrase Oreochromis mossambicus
a b s t r a c t Oreochromis mossambicus were exposed to two different temperature and carbon dioxide partial pressure levels for about two weeks, as the ambient (Control; 25 ◦ C, 3.3 mg/L CO2 ), high CO2 (25 ◦ C, 14 mg/L CO2 ), high temperature (30 ◦ C, 3 mg/L CO2 ) and combined (30 ◦ C, 14.1 mg/L CO2 ) groups. No mortality was observed during the experiments. As a result of the study, elevated CO2 concentrations cause negative effects on the hematological parameters. At the end of the study, while the blood Carbonic Anhydrase (CA) activity, in the high CO2 group (25 ◦ C, 14 mg/L CO2 ), statistically increased at the 7th day compared to the control group, it decreased at the 14th day (p < 0.05). In addition, the blood CA activity, in the combined (30 ◦ C, 14.1 mg/L CO2 ) group, showed a decrease at the 14th day compared to the control group (p < 0.05). At the end of study, unlike the blood CA activity, gill, liver and kidney CA activity showed an increase in the tissues compared to the control groups (p < 0.05). Furthermore, the Na+ , K+ -ATPase activities were stimulated significantly in the gills in both high CO2 and temperature groups at day 7, but it showed a significant amount of inhibition at the 14th day compared to the control groups. Overall, increasing carbon dioxide concentration in different temperatures has negative effects on the hematological parameters and respiratory enzyme of the tilapia fish. In addition, it is observed that the fish survive at negative conditions with adaptation mechanisms. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Due to anthropogenic activities, the level of atmospheric CO2 has raised by 280 mg/L from industrial revolution and today reached to 390 mg/L and it’s estimated to reach 421–936 mg/L by the year 2100 (IPCC, 2013). Approximately 1/3 of the excess of CO2 in the atmosphere will be absorbed by surface ocean waters, leading to an estimated drop in pH of 0.3–0.4 units globally by the end of 21st century (IPCC, 2013). Because of global warming, the rise in atmospheric temperatures will range between +1◦ C (0.3 − 1.7 ◦ C) and +3 ◦ C (2.6 − 4.8 ◦ C) (IPCC, 2013) over the next decades, affecting freshwater ecosystems too (Rosset and Oertli, 2011; Foucreau et al., 2014). This increase may induce thermal stress, which would mean alterations in metabolism of organisms especially fish (Pörtner, 2002).
∗ Corresponding author. E-mail address: [email protected] (H. Kaya). http://dx.doi.org/10.1016/j.etap.2016.05.003 1382-6689/© 2016 Elsevier B.V. All rights reserved.
Temperature changes have a strong impact not only on oxygen consumption of poikilothermic organisms and oxygen ligation of hemoglobin but also on the oxygen status of an environment. This essential environmental factor affects metabolism activities of all poikilothermic animals and, thus draws their life boundaries. Studies being conducted today report that global warming in waters has severe effects on the distribution and reproduction of fish species (Perry et al., 2005; Brander, 2007). The tolerance of fish to temperature changes may increase the need for oxygen or affect the oxygen supply capacity to tissues even in low levels apart from the high concentrations (Lannig et al., 2003). The studies reveal that environmental stress sources affect organisms through operating effect mechanisms (synergistic and antagonistic etc.) (Schiedek et al., 2007; Gooding et al., 2009). Possible rises in the levels of temperature and CO2 , as a result of climate change in water, may lead to biochemical changes and, thus may bring irrevocable effects on aquatic organisms. In studies that researched the acidification resulted from CO2 in waters, important calcifying organisms were mostly used. However, the
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Fig. 1. Experimental system.
knowledge about this effect on fish physiology is limited. In most aquatic invertebrates, oxygen consumption falls despite increasing additional energy use due to elevated CO2 levels. Fish, on the other hand, sustain their oxygen consumption on elevated CO2 levels. Nevertheless, the effects on the reproduction, early development, growth, behaviors and physiologies of fish exposed to CO2 for a long term are of importance and take place among the prioritized issues researched. This study aims to investigate the effects of possible elevations in temperature and CO2 levels in waters, being associated with the scenarios on global warming, on respiration and blood physiologies using tilapia(O. mossambicus). In experiments conducted within this scope, changes in hematology, blood pH and respiration enzymes (Na+ , K+ -ATPase and Carbonic Anhydrase) of tilapia fish exposed to elevated CO2 concentrations in two different temperature values were investigated.
of removing chlorine in water and total chlorine level was determined via color test (o-tolidine). The water was taken from the main tank into two different tanks (4–5) via pipes. The temperature was set to 25 ◦ C in the first tank and to 30 ◦ C in the second one through 40 kW heaters. CO2 gas was injected to two different main tanks (6–7) (which were heated again with heaters and aerated with air pump) through ceramic diffuser in order to stabilize CO2 concentration at 14 mg/L. The water taken from these tanks was transferred into 100-L aquaculture tanks (8) [3 tanks for each group; Control (25 ◦ C, 3.3 mg/L CO2 ), high CO2 (25 ◦ C, 14 mg/L CO2 ), high temperature (30 ◦ C, 3 mg/L CO2 ) and combined (30 ◦ C, 14.1 mg/L CO2 )]. During the tests, water quality in tanks (pH, water temperature, dissolved oxygen and carbon dioxide level) was measured three times a day. In addition, behaviors (time allocated for swimming, irregular movements and sudden bounces and so on) and external findings of the fish were observed prior to every feeding session.
2. Material and methods 2.3. Sampling 2.1. Experimental setup In this study, tilapia fish are preferred as test material since they easily adapt into environmental conditions, have more tolerance to changing temperature and oxygen conditions compared to other species. In the present study, a total of 144 tilapia fish (O. mossambicus) weighing at 13–15 g are used. Tilapia has been supplied from the Department of Aquaculture in the Faculty of Marine Sciences and Technology at C¸anakkale Onsekiz Mart University. The fish were adapted into environment conditions for 30 days under 12:12 photoperiod regime at 26 ◦ C within 12 adaptation tanks where the test was conducted. During the test, 12 fish were placed in each tank and the test was conducted for 3 replicates for each test group. Throughout the test lasted for 14 days, the fish were fed twice a day at the rate of 2% of each fish’s body weight. A total of 4 different groups were made in order to detect the effect of carbon dioxide and temperature on fish in the test. 2.2. Experimental system In this study, stabilized tap water which was injected with CO2 under heating and pressure was used (Fig. 1). Initially, tap water has passed through the degassing column (1) and stored in the main tank (2) (Hisar et al., 2007). Afterwards, sodium pyrosulfuryl was added through a peristaltic pump (3) into the tank with the purpose
In the experiment, twelve fish on the first day (fish from the stock), six fish from each aquarium on the 7th and 14th day were used for blood pH and hematology analyses, and tissues were dissected for the determination of Na+ , K+ -ATPase and CA activities analysis. 2.4. Blood analysis For blood sampling, ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma Aldrich Chemical Co., St. Louis, MO, USA) was used to anesthesia the fish (Kaya et al., 2015). They were wiped clean to avoid mucus being mixed into the blood, and then the blood was collected from the caudal vein using a 2.5 mL plastic syringe without harming the fish. An aliquot of blood (200 L) was transferred to EDTA tubes (BD, Oxford, UK) for hematological analysis, and the remainder (300 L) to plastic biochemistry tubes (Vacutest Kima s.r.l., Piove di Sacco, Italy). The tubes were centrifuged at 4000g for 10 min in a centrifuge for serum separation and stored at −80 ◦ C. The serum was used for CA analysis. 2.5. Hematological parameters RBCs (106 mm3 ) were counted with a hemocytometer (Glaswarenfabrik Karl Hecht KG,RhD on, Germany) using Dacie’s
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diluting fluid (Blaxhall and Daisley 1973). The hematocrit ratio (Hct, %) was determined using a capillary hematocrit tube. The hemoglobin (Hb, gdL) concentration was determined by spectrophotometry (Optizen POP UV/VIS Spectrophotometer, Seoul, Republic of Korea) at 540 nm using the cyanomethaemoglobin method (Blaxhall and Daisley 1973). The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the following equations (Lewis et al., 2006): MCV (mm3 ) = Hct (%) × 10/RBC (106 /L); MCH (pg) = (Hb (g/dL) × 10)/RBC (106 /L); MCHC (%) = (Hb (g/dL) × 10)/Hct (%). 2.6. CA activity Liver, gill and kidney were removed and immediately snapfrozen in liquid nitrogen and stored at −80 ◦ C until required. Tissues were homogenized (Stuart SHM1Homogenisator, Staffordshire, UK) in a 25 mmol/ L TRIS-(2-amino-2-hydroxylmethyl-1,3propanediol) HCl buffer, adjusted to pH 8.7. The CO2 hydratase activity of the CA enzyme was assayed colorimetrically by using the method of Wilbur and Anderson (1976). A 0.2 mM phenol red solution was used as an indicator, working at an absorbance of 557 nm, with 20 mM TRIS as a buffer (pH 8.3) and 20 mM NaCl, following the initial rate of the CA catalyzed CO2 hydration for 10–100 s. The CO2 hydratase activity (enzyme units) was calculated as (t0-tc)/tc, where t0 is the time for pH change of the nonenzymatic reaction and tc is the time for pH change of the enzymatic reaction. To calculate specific activities of CA in the serum and tissues (gill, liver and kidney) protein quantities were determined (specific CA activity was expressed as enzyme units per milligram of protein) spectrophotometrically (595 nm) according to Bradford (1976) by using bovine serum albumin as the standard. 2.7. Na+ , K+ -ATPase activities On the first day, 12 fish (fish from the stock tank), and on the 7th and 14th day six fish from each aquarium were used for Na+ , K+ -ATPase and CA activities in the gill, liver and kidney tissues. The fish were anesthetized as given above, liver, gill and
kidney were removed, instantly snap-frozen in liquid nitrogen, and stored at −80 ◦ C until required. Tissues (about 0.5 g) were homogenized (Stuart SHM1Homogenisator, Staffordshire, UK) in five volumes (2.5 mL) of an ice-cold isotonic buffer [300 mmol/L sucrose, 0.1 mmol/L ethylenediamine tetra acetic acid (EDTA), 20 mmol/L (4-(2-hydroxyethyl) piperazine-1- ethane sulfonic acid (HEPES), adjusted to pH 7.8 with a few drops of 2 mol/L TRIS (2-amino-2-hydroxylmethyl-1,3-propanediol)]. Tissues were analyzed for Na+ , K+ -ATPase according to Silva et al. (1977) as follows: each sample (15 L, in triplicate) was put into 400 L of both K+ (containing buffer) and K (free buffer, plus 1.0 mmol/L Quabain), and then incubated at 37 ◦ C for 10 min. The reaction was stopped by adding 1 mL of ice-cold trichloroacetic acid (8.6%, w/v) and 1 mL of a color reagent was added to each tube (9.6%, w/v FeSO4 6H2 O, 1.15%, w/v ammonium heptamolybdate dissolved in 0.66 M H2 SO4 ), and the color allowed to develop for 15 min at room temperature. Absorbance was measured at 660 nm (Optizen POP UV/VIS Spectrophotometer, Seoul, Republic of Korea) against 0–0.5 mmol/L phosphate standards. 2.8. Statistical analysis All data were expressed as means ± standard error of the mean (S.E.M.). In the study, normality of data and homogeneity of variances were checked using Kolmogorove Smirnov and Levene tests respectively. When appropriate, One-Way ANOVA and Tukey sub-test were performed. When normality of variances were not assumed, Kruskal Wallis test was performed. The statistical analysis was made using SPSS 21.0 (SPSS, Chicago, IL, USA), and the significance level was considered to be 0.05. 3. Results In this study, any death case was observed neither in control nor in test group fish. Furthermore, no change was observed in feeding and behaviors of fish in any group. In the study, pH levels fell simultaneously with the inclusion of CO2 to water in groups exposed to CO2 . Throughout the experiment process, pH level of the control group was around 7.6 while it fell down to 6 at the end of the 7th
Table 1 Physico-chemical parameters measured in the experiment.
◦
pH
Dissolved oxygen (mg/L)
Temperature (◦ C)
Carbon dioxide (mg/L)
25 C-Control
Initial 2th day 4th day 7th day 10th day 14th day
7.7 7.6 7.7 7.6 7.7 7.7
7.1 7 7.2 7.1 7.2 7.2
25 25.1 25.4 25 25.2 25.2
3.3 3 3.7 3 3.5 3.2
25 ◦ C-CO2
Initial 2th day 4th day 7th day 10th day 14th day
7.7 7.56 7.18 7 6 5.2
7.1 7 7.2 7.1 7.2 7.2
25 25 25.1 25.2 25 25
3.4 14.1 14.2 13.9 14.1 14
30 ◦ C-Control
Initial 2th day 4th day 7th day 10th day 14th day
7.75 7.64 7.5 7.71 7.8 7.85
7.1 7.2 7 7.2 7.1 7.3
30 30.1 30.2 30.1 30 30
3 3.1 3.2 3 3.4 3.4
30 ◦ C-CO2
Initial 2th day 4th day 7th day 10th day 14th day
7.71 7.61 7.15 7.1 6 5.1
7 7 7.1 7.1 7.2 7.1
30 30.1 30.2 30 30 30.2
3.5 14.1 14 13.9 14.2 14.1
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Table 2 Effect of subchronic exposure to carbon dioxide with different temperature levels on the Red blood cell count (RBC), hematocrit (Hct), hemoglobin (Hb) concentration, corpuscular volume mean (MCV), corpuscular Hb mean (MCH), mean corpuscular Hb concentration (MCHC) and blood pH of O. mossambicus at 0, 7 and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA/Tukey or Kruskal Wallis test were used).
Initial 25 ◦ C-Control 25 ◦ C-CO2 30 ◦ C-Control 30 ◦ C-CO2 25 ◦ C-Control 25 ◦ C-CO2 30 ◦ C-Control 30 ◦ C-CO2
7th day
14th day
RBC(x106 mm3 )
Hb (g/dL)
Hct (%)
MCV (fL)
MCH (pg)
MCHC (%)
Blood pH
3.16 ± 0.11
10.52 ± 0.22
20.60 ± 0.51
65.70 ± 3.86
33.51 ± 1.70
51.19 ± 1.70
7.14 ± 0.03
3.17 ± 0.15a 2.42 ± 0.04b 2.16 ± 0.22b 2.00 ± 0.24b 3.19 ± 0.13a 3.47 ± 0.06a 3.32 ± 0.12a 3.02 ± 0.24a
9.48 ± 0.41b 12.80 ± 0.18a 10.11 ± 0.21b 10.21 ± 0.71b 10.77 ± 0.36a 9.76 ± 0.62ab 7.98 ± 1.02c 10.31 ± 0.86ab
21.75 ± 0.85a 20.00 ± 1.08ab 17.02 ± 1.08b 17.75 ± 0.85b 21.01 ± 0.84a 21.60 ± 0.93a 20.20 ± 0.58ab 17.60 ± 1.54b
69.61 ± 6.38a 82.71 ± 3.67a 79.92 ± 4.73a 95.49 ± 17.50a 66.63 ± 5.02a 62.15 ± 2.30 a 61.10 ± 2.15a 58.15 ± 0.78a
30.27 ± 2.47b 53.05 ± 1.11a 48.04 ± 4.04a 54.37 ± 10.07a 34.16 ± 2.45a 28.08 ± 1.70ab 23.80 ± 2.16b 34.38 ± 2.18a
43.67 ± 1.82b 64.70 ± 4.32a 59.96 ± 2.69a 56.88 ± 2.71a 51.61 ± 2.70 ab 45.30 ± 2.65bc 39.25 ± 4.30c 59.12 ± 3.68a
7.19 ± 0.02b 7.32 ± 0.03a 7.14 ± 0.01b 7.16 ± 0.03b 7.15 ± 0.02a 7.09 ± 0.11a 7.11 ± 0.02a 7.12 ± 0.01a
Fig. 2. Effect of subchronic exposure to carbon dioxide with different temperature levels on the carbonic anhydrase (unit/mg protein) activity in the a) blood b) liver c) gill and d) kidney tissues of O. mossambicus at 0, 7, and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA followed by Tukey post-hoc test).
day; to 5 at the end of the 14th day in groups exposed to CO2 . On the other hand, CO2 level of the control group was 3.27 mg/L at 25 ◦ C and 3.18 mg/L in average at 30 ◦ C. Nevertheless, CO2 level was stable around 14 mg/L following the 2nd day in groups exposed to CO2 . In the study, oxygen values were at the same level in all tanks for the control groups and those exposed to carbon dioxide. Findings related to pH, carbon dioxide, dissolved oxygen and temperature measured in water during the experiment are presented in Table 1. The variations in hematological parameters and blood pH as a result of 14-day carbon dioxide exposure are illustrated in Table 2. On the 7th day of the experiment, a statistically significant decline was observed in the number of erythrocyte in groups exposed to carbon dioxide at 25 ◦ C and 30 ◦ C compared to the control group at the given temperature values (p < 0.05), while an increase was observed in MCH, MCHC, Hb and blood pH in the group exposed to carbon dioxide at 25 ◦ C compared to the control group at the same temperature level (p < 0.05). In the group exposed to carbon dioxide at 25 ◦ C, CA enzyme activity in blood increased statistically compared to the control group on the 7th day of the test while in both temperature values it fell down on the 14th day (p < 0.05). In groups exposed to carbon dioxide at 25 and 30 ◦ C, liver CA activity increased on the 7th and 14th days compared to the control groups. CA enzyme activity in
gills at the group exposed to 25 ◦ C increased statistically at day 7 compared to the control group but in the group exposed to CO2 at 30 ◦ C an increase was observed at day 14. The most interesting findings of CA activity were found in kidneys in groups exposed to carbon dioxide. While, in the group exposed to carbon dioxide at 25 ◦ C, CA activity increased on the 7th day (p < 0.05); it started to fall down on the 14th day (no statistically significant increase). In the group exposed to carbon dioxide at 30 ◦ C, on the contrary, CA activity declined on the 7th day; it increased on the 14th day (Fig. 2). According to this study, Na+ , K+ -ATPase findings on gill, liver and kidney tissue are presented in Fig. 3. While in groups exposed to carbon dioxide at 25 and 30 ◦ C, gill Na+ , K+ -ATPase activity increased on the 7th day compared to the control group; they were inhibited on the 14th day. On the other hand, liver Na+ , K+ -ATPase activity increased in the group exposed to carbon dioxide at 25 ◦ C on the 14th day compared to their control group; it fell down in the group exposed to carbon dioxide at 30 ◦ C at day 7. While, in groups exposed to carbon dioxide at 25 and 30 ◦ C, kidney Na+ , K+ -ATPase activity declined on the 7th day compared to control groups; on the 14th day, this decline sustained in the group exposed to CO2 at 25 ◦ C; it reached the equal level with the control group in the one exposed to CO2 at 30 ◦ C.
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Fig. 3. Effect of subchronic exposure to carbon dioxide with different temperature levels on the Na+ , K+ -ATPase (Pi mol/mg protein/h) activity in the a) gill b) liver and c) kidney tissues of O. mossambicus at 0, 7, and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA followed by Tukey post-hoc test).
4. Discussion Temperature is one of the fundamental factors being effective in performance, metabolism and zoogeographic distribution in ectodermic animals including fish (Pörtner, 2010). The elevated carbon dioxide level in waters may affect organism physiology directly by reducing pH of waters or indirectly by changing the composition of metal ion in water (Lannig et al., 2010). Today, the observable effects in physiology of aquatic organisms are not discovered completely yet, due to climate changes. One of the most important reasons for this is the limited number of measurable argument related to physiological phenomenon on the ecosystem level (Pörtner, 2010). Biologic stress that might occur in organisms manifests itself in different ways (Barton, 1997). There are studies that have been conducted so far on the effects of elevated carbon dioxide levels in cold-water fish such as trout and salmon (Fivelstad et al., 1999, 2007; Kaya et al., 2013). On the other hand, no study was found about the effects of both elevated carbon dioxide and changing temperature levels in tropical fish species. In this study, the number of erythrocyte (RBC), hemoglobin, hematocrit, MCV, MCH, MCHC and blood pH levels are investigated in samples of O. mossambicus. As a result of elevated carbon dioxide concentration in the water, hypercapnia is observed due to abnormally high level carbon dioxide in the blood (Heisler, 1984). The increase in blood partial carbon dioxide pressure can cause a respiratory acidosis in fish (Goss et al., 1994). Because increased carbon dioxide concentrations cause acidification in the blood and tissue respiratory pigment, it causes a reduction in oxygen intake and delivery (Pörtner et al., 2004). In cases where acidification occur and blood pH fall, fish can compensate the reduced red blood cell pH with Na+ /H + ion exchange by catecholamine proteins (Randall and Brauner, 1998). In our study also, in the group exposed to CO2 at 25 ◦ C changes seen in hematology at the end of the first week returned to the normal values at the end of trial, this can be explained by the operation of the adaptation mechanism. A similar situation was also seen in our previous study on CO2 exposed trout (Oncorhynchus mykiss). RBC, hemoglobin and hematocrit values of the cold water fish exposed to CO2 showed a decrease at the end
of the first week like the present study and it came to similar situations (excluding hematocrit) with the control at the end of the experiment. On the other hand, there are some studies investigating high carbon dioxide under aquaculture conditions. In these studies, Fivelstad et al. (2007) exposed Atlantic salmons to high level of CO2 (12 mm Hg) at two different temperature values (5 and 15 ◦ C) and detected that RBC amount and MCH value did not change compared to the control group at the end of 39 days. In addition, hematocrit amount and MCV values fell down at both temperature values (5 and 15 ◦ C) in fish exposed to high level of CO2. In the same study, hemoglobin decreased in fish exposed to CO2 at 5 ◦ C while no change occurred at 15 ◦ C. Similarly in our study, no change occurred in RBC value at the end of 14 days while MCV and MCH value decreased at the end of the test. Fluctuations in hematologic parameters during the study can be associated with the adaptation of fish into environment conditions including high level of CO2. Studies indicate a similarity between the control group and the group of fish exposed to CO2 for a long period of time in terms of hematological parameters. For example, in a study conducted by Fivelstad et al. (2003) on Atlantic salmons, fish were exposed to 16 and 24 mg/L CO2 for 57 days and at the end of the test, no difference was detected in hematologic parameters (Hct, Hb and MCH) of fish compared to the control group. Similarly, Fivelstad et al. (1998) exposed Atlantic salmons to three different concentrations of CO2 (10, 26 and 44 mg/L CO2 ) and found that Hct value never changed at the end of 57 days. However, the lack of any change in hematologic parameters at the end of high levels of CO2 exposure should not be understood as the fish health is not adversely affected. At this point, analysis of various physiologic changes will ensure to assess fish health as a whole in physiologic terms. As osmoregulatory organs in fish; gill, kidney and intestine are specialized in acid-base regulation associated with the active ion transportation (Melzner et al., 2009). Especially gill in fish has a significant role in ensuring osmotic regulation apart from its task in transferring Na+ , K+ -ATPase enzymes taking place in epithelia cells into active electrolyte (Kaya et al., 2016). Na+ , K+ -ATPase enzyme regulates ionic and electric gradient required for transepithelial
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salt movements in osmoregulation. Carbonic anhydrase enzyme, on the other hand, plays a significant role in maintaining acidbase balance and transferring and eliminating blood carbon dioxide (CO2 ) (by means of catalyzing H+ and HCO3 ) in fish (Henry and Cameron, 1982). In reviewing the literature available in terms of mentioned enzyme activities, it has been found that the studies usually performed on marine organisms. However, similar studies using freshwater organisms are limited (Kaya et al., 2013). Of respiratory enzymes analyzed in this study, CA activity significantly decreased in blood samples of groups exposed to carbon dioxide at the end of the test while it increased in osmoregulatory organs (gill and kidney). Similarly, it was detected that Na+ , K+ -ATPase enzyme activity of gill and kidney being among osmoregulatory organs was significantly inhibited in groups exposed to carbon dioxide (on the 14th day, except for 30CO2 ). The data obtained from this study show that carbonic anhydrase and Na+ , K+ -ATPase enzyme activities in osmoregulatory tissues tried to overcome the problems occurred in acid-base balance due to hypercapnia conditions. The long term effects of changes in carbon dioxide and temperature in waters are not completely known, yet. However, acid-base disorders may lead to obvious consequences at biochemical levels. Changes in Na+ , K+ ATPase enzyme activity may lead to adverse results on physiologic level due to the impairment of extracellular fluid (therefore, cell fluid is impaired as well). Impairments in acid-base balance and/or ionic/osmotic balance in organisms living in all aqua-ecosystems (including fresh water, sea water and brackish water) may affect the survival of organisms. To conclude, it was observed that elevated carbon dioxide and temperature levels have acute adverse effect on some hematologic parameters and blood pH in tilapia and at the end of this study, the given parameters were found to be closer to normal values. In addition, fluctuations in CA and Na+ , K+ -ATPase enzyme activity show that organisms give reaction to stress conditions through their adaptation mechanisms. We believe that further chronic studies that will also consider acidification apart from the elevated carbon dioxide and temperature levels in sea and freshwater fish species should be conducted. Acknowledgements This study was supported by The Scientific and Technological Council of Turkey (TUBITAK, Project Number: 113O220, coordinated by H Kaya). References Barton, B.E., 1997. IL-6: insights into novel biological activities. Clin. Immun. Immunopathol. 85 (1), 16–20. Blaxhall, P.C., Daisley, K.W., 1973. Routine haematological methods for use with fish blood. J. Fish Biol. 5 (6), 771–781. Bradford, M.M., 1976. A rapid and sensitive method for the quantitiation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brander, K.M., 2007. Global fish production and climate change. Proc. Natl. Acad. Sci. 104 (50), 19709–19714. Fivelstad, S., Haavik, H., Lovik, G., Olsen, A.B., 1998. Sublethal effects and safe levels of carbon dioxide for atlantic salmon postsmolts (Salmo salar L.). Aquaculture 160, 305–316. Fivelstad, S., Olsen, A.B., Kløften, H., Ski, H., Steffanson, S., 1999. Effects of carbon dioxide on atlantic salmon (Salmo salar L.) smolts at constant pH in bicarbonate rich freshwater. Aquaculture 178, 171–187. Fivelstad, S., Olsen, A.B., Waagbo, R., Zeitz, S., Hosfeld, C.D., Stefansson, S., 2003. A major water quality problem in smolt farms: combined effects of carbon dioxide and reduced pH and aluminium on atlantic salmon (Salmo salar L.) smolts: physiology and growth. Aquaculture 215, 339–357.
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Fivelstad, S., Waagbo, R., Stefansson, S., Olsen, A.B., 2007. Impacts of elevated water carbon dioxide partial pressure at two temperatures on atlantic salmon (Salmo salar L.) parr growth and haematology. Aquaculture 269, 241–249. Foucreau, N., Cottin, D., Piscart, C., Hervant, F., 2014. Physiological and metabolic responses to rising temperature in Gammarus pulex (Crustacea) populations living under continental or Mediterranean climates. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 168, 69–75. Gooding, R.A., Harley, C.D., Tang, E., 2009. Elevated water temperature and carbon dioxide concentration increase the growth of a keystone echinoderm. Proc. Natl. Acad. Sci. 106 (23), 9316–9321. Goss, G.G., Laurent, P., Perry, S.F., 1994. Gill morphology during hypercapnia in brown bullhead (Ictalurus nebulosus): role of chloride cells and pavement cells in acid–base regulation. J. Fish Biol. 45, 705–718. Henry, R.P., Cameron, J.N., 1982. The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustaceans. J. Exp. Zool. 221, 309–321. Heisler, N., 1984. Acid-base regulation in fishes. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, XA. Academic Press, New York, pp. 315–399. Hisar, O., Hisar, S¸.A., Sirkecioglu, A.N., Karatas, M., Yanik, T., 2007. Using an oxygenating and degassing column to improve water quality in salmonid hatcheries. Int. J. Nat. Eng. Sci. 1, 63–64. IPCC, 2013. Intergovernmental Panel on Climate Change (IPCC) summary for policymakers contribution of working group I to the fifth assessment report of the Intergovernmental panel on climate change. In: Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S.K., Boschung, J. (Eds.), Climate Change 2013: The Physical Science Basis. Cambridge University Press, Cambridge New York. Kaya, H., Yılmaz, S., Gürkan, M., Hisar, O., 2013. Effects of environmental hypercapnia on hemato-immunological parameters, carbonic anhydrase, and Na+ , K+ -ATPase enzyme activities in rainbow trout (Oncorhynchus mykiss) tissues. Toxicol. Environ. Chem. 95 (8), 1395–1407. Kaya, H., Aydın, F., Gürkan, M., Yılmaz, S., Ates, M., Demir, V., Arslan, Z., 2015. Effects of zinc oxide nanoparticles on bioaccumulation and oxidative stress in different organs of tilapia (Oreochromis niloticus). Environ. Toxicol. Pharmacol. 40 (3), 936–947. Kaya, H., Aydın, F., Gürkan, M., Yılmaz, S., Ates, M., Demir, V., Arslan, Z., 2016. A comparative toxicity study between small and large size zinc oxide nanoparticles in tilapia (Oreochromis niloticus): organ pathologies, osmoregulatory responses and immunological parameters. Chemosphere 144, 571–582. Lannig, G., Eckerle, L., Serendero, I., Sartoris, F.J., Fischer, T., Knust, R., Pörtner, H.O., 2003. Temperature adaptation in eurythermal cod (Gadus morhua): a comparison of mitochondrial enzyme capacities in boreal and Arctic populations. Mar. Biol. 142 (3), 589–599. Lannig, G., Eilers, S., Pörtner, H., Sokolova, I.M., Bock, C., 2010. Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas—changes in metabolic pathways and thermal response. Mar. Drugs 8 (8), 2318–2339. Lewis, S.M., Bain, B.J., Bates, I., 2006. Dacie and Lewis Practical Haematology, 10th ed. Churchill Livingstone Elsevier, Philadelphia, PA. Melzner, F., Gutowska, M.A., Langenbuch, M., Dupont, S., Lucassen, M., Thorndyke, M.C., Pörtner, H.O., 2009. Physiological basis for high CO2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? Biogeosciences 6 (10), 2313–2331. Pörtner, H.O., 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Biochem. Physiol. A Mol. Integr. Physiol. 132 (4), 739–761. Pörtner, 2010. Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893. Perry, A.L., Low, P.J., Ellis, J.R., Reynolds, J.D., 2005. Climate change and distribution shifts in marine fishes. Science 308 (5730), 1912–1915. Pörtner, H.O., Langenbuch, M., Reipschlager, A., 2004. Biological impact of elevated ocean CO2 concentrations: lessons from animal physiology and earth history. J. Oceanogr. 60, 705–718. Rosset, V., Oertli, B., 2011. Freshwater biodiversity under climate warming pressure: identifying the winners and losers in temperate standing waterbodies. Biol. Conserv. 144 (9), 2311–2319. Schiedek, D., Sundelin, B., Readman, J.W., Macdonald, R.W., 2007. Interactions between climate change and contaminants. Mar. Pollut. Bull. 54 (12), 1845–1856. Silva, P., Solomon, R., Spokes, K., Epstein, F.H., 1977. Ouabain inhibition of gill Na, K-ATPase: relationship to active chloride transport. J. Exp. Zool. 199, 419–426. Wilbur, K.M., Anderson, N.G., 1976. Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176, 147–154.
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The effects of elevated carbon dioxide and temperature levels on tilapia (Oreochromis mossambicus): Respiratory enzymes, blood pH and hematological parameters Hasan Kaya a,∗ , Olcay Hisar a , Sevdan Yılmaz b , Mert Gürkan c , S¸ükriye Aras Hisar d a
Department of Basic Sciences, Marine Sciences and Technology Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey Department of Aquaculture, Marine Sciences and Technology Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey c Department of Biology, Faculty of Arts and Sciences, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey d Department of Food Engineering, Engineering Faculty, C¸anakkale Onsekiz Mart University, C¸anakkale, Turkey b
a r t i c l e
i n f o
Article history: Received 28 December 2015 Received in revised form 28 April 2016 Accepted 1 May 2016 Available online 3 May 2016 Keywords: Elevated carbon dioxide and temperature Hematology Blood pH Na+ K+ -ATPase Carbonic anhydrase Oreochromis mossambicus
a b s t r a c t Oreochromis mossambicus were exposed to two different temperature and carbon dioxide partial pressure levels for about two weeks, as the ambient (Control; 25 ◦ C, 3.3 mg/L CO2 ), high CO2 (25 ◦ C, 14 mg/L CO2 ), high temperature (30 ◦ C, 3 mg/L CO2 ) and combined (30 ◦ C, 14.1 mg/L CO2 ) groups. No mortality was observed during the experiments. As a result of the study, elevated CO2 concentrations cause negative effects on the hematological parameters. At the end of the study, while the blood Carbonic Anhydrase (CA) activity, in the high CO2 group (25 ◦ C, 14 mg/L CO2 ), statistically increased at the 7th day compared to the control group, it decreased at the 14th day (p < 0.05). In addition, the blood CA activity, in the combined (30 ◦ C, 14.1 mg/L CO2 ) group, showed a decrease at the 14th day compared to the control group (p < 0.05). At the end of study, unlike the blood CA activity, gill, liver and kidney CA activity showed an increase in the tissues compared to the control groups (p < 0.05). Furthermore, the Na+ , K+ -ATPase activities were stimulated significantly in the gills in both high CO2 and temperature groups at day 7, but it showed a significant amount of inhibition at the 14th day compared to the control groups. Overall, increasing carbon dioxide concentration in different temperatures has negative effects on the hematological parameters and respiratory enzyme of the tilapia fish. In addition, it is observed that the fish survive at negative conditions with adaptation mechanisms. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Due to anthropogenic activities, the level of atmospheric CO2 has raised by 280 mg/L from industrial revolution and today reached to 390 mg/L and it’s estimated to reach 421–936 mg/L by the year 2100 (IPCC, 2013). Approximately 1/3 of the excess of CO2 in the atmosphere will be absorbed by surface ocean waters, leading to an estimated drop in pH of 0.3–0.4 units globally by the end of 21st century (IPCC, 2013). Because of global warming, the rise in atmospheric temperatures will range between +1◦ C (0.3 − 1.7 ◦ C) and +3 ◦ C (2.6 − 4.8 ◦ C) (IPCC, 2013) over the next decades, affecting freshwater ecosystems too (Rosset and Oertli, 2011; Foucreau et al., 2014). This increase may induce thermal stress, which would mean alterations in metabolism of organisms especially fish (Pörtner, 2002).
∗ Corresponding author. E-mail address: [email protected] (H. Kaya). http://dx.doi.org/10.1016/j.etap.2016.05.003 1382-6689/© 2016 Elsevier B.V. All rights reserved.
Temperature changes have a strong impact not only on oxygen consumption of poikilothermic organisms and oxygen ligation of hemoglobin but also on the oxygen status of an environment. This essential environmental factor affects metabolism activities of all poikilothermic animals and, thus draws their life boundaries. Studies being conducted today report that global warming in waters has severe effects on the distribution and reproduction of fish species (Perry et al., 2005; Brander, 2007). The tolerance of fish to temperature changes may increase the need for oxygen or affect the oxygen supply capacity to tissues even in low levels apart from the high concentrations (Lannig et al., 2003). The studies reveal that environmental stress sources affect organisms through operating effect mechanisms (synergistic and antagonistic etc.) (Schiedek et al., 2007; Gooding et al., 2009). Possible rises in the levels of temperature and CO2 , as a result of climate change in water, may lead to biochemical changes and, thus may bring irrevocable effects on aquatic organisms. In studies that researched the acidification resulted from CO2 in waters, important calcifying organisms were mostly used. However, the
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Fig. 1. Experimental system.
knowledge about this effect on fish physiology is limited. In most aquatic invertebrates, oxygen consumption falls despite increasing additional energy use due to elevated CO2 levels. Fish, on the other hand, sustain their oxygen consumption on elevated CO2 levels. Nevertheless, the effects on the reproduction, early development, growth, behaviors and physiologies of fish exposed to CO2 for a long term are of importance and take place among the prioritized issues researched. This study aims to investigate the effects of possible elevations in temperature and CO2 levels in waters, being associated with the scenarios on global warming, on respiration and blood physiologies using tilapia(O. mossambicus). In experiments conducted within this scope, changes in hematology, blood pH and respiration enzymes (Na+ , K+ -ATPase and Carbonic Anhydrase) of tilapia fish exposed to elevated CO2 concentrations in two different temperature values were investigated.
of removing chlorine in water and total chlorine level was determined via color test (o-tolidine). The water was taken from the main tank into two different tanks (4–5) via pipes. The temperature was set to 25 ◦ C in the first tank and to 30 ◦ C in the second one through 40 kW heaters. CO2 gas was injected to two different main tanks (6–7) (which were heated again with heaters and aerated with air pump) through ceramic diffuser in order to stabilize CO2 concentration at 14 mg/L. The water taken from these tanks was transferred into 100-L aquaculture tanks (8) [3 tanks for each group; Control (25 ◦ C, 3.3 mg/L CO2 ), high CO2 (25 ◦ C, 14 mg/L CO2 ), high temperature (30 ◦ C, 3 mg/L CO2 ) and combined (30 ◦ C, 14.1 mg/L CO2 )]. During the tests, water quality in tanks (pH, water temperature, dissolved oxygen and carbon dioxide level) was measured three times a day. In addition, behaviors (time allocated for swimming, irregular movements and sudden bounces and so on) and external findings of the fish were observed prior to every feeding session.
2. Material and methods 2.3. Sampling 2.1. Experimental setup In this study, tilapia fish are preferred as test material since they easily adapt into environmental conditions, have more tolerance to changing temperature and oxygen conditions compared to other species. In the present study, a total of 144 tilapia fish (O. mossambicus) weighing at 13–15 g are used. Tilapia has been supplied from the Department of Aquaculture in the Faculty of Marine Sciences and Technology at C¸anakkale Onsekiz Mart University. The fish were adapted into environment conditions for 30 days under 12:12 photoperiod regime at 26 ◦ C within 12 adaptation tanks where the test was conducted. During the test, 12 fish were placed in each tank and the test was conducted for 3 replicates for each test group. Throughout the test lasted for 14 days, the fish were fed twice a day at the rate of 2% of each fish’s body weight. A total of 4 different groups were made in order to detect the effect of carbon dioxide and temperature on fish in the test. 2.2. Experimental system In this study, stabilized tap water which was injected with CO2 under heating and pressure was used (Fig. 1). Initially, tap water has passed through the degassing column (1) and stored in the main tank (2) (Hisar et al., 2007). Afterwards, sodium pyrosulfuryl was added through a peristaltic pump (3) into the tank with the purpose
In the experiment, twelve fish on the first day (fish from the stock), six fish from each aquarium on the 7th and 14th day were used for blood pH and hematology analyses, and tissues were dissected for the determination of Na+ , K+ -ATPase and CA activities analysis. 2.4. Blood analysis For blood sampling, ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma Aldrich Chemical Co., St. Louis, MO, USA) was used to anesthesia the fish (Kaya et al., 2015). They were wiped clean to avoid mucus being mixed into the blood, and then the blood was collected from the caudal vein using a 2.5 mL plastic syringe without harming the fish. An aliquot of blood (200 L) was transferred to EDTA tubes (BD, Oxford, UK) for hematological analysis, and the remainder (300 L) to plastic biochemistry tubes (Vacutest Kima s.r.l., Piove di Sacco, Italy). The tubes were centrifuged at 4000g for 10 min in a centrifuge for serum separation and stored at −80 ◦ C. The serum was used for CA analysis. 2.5. Hematological parameters RBCs (106 mm3 ) were counted with a hemocytometer (Glaswarenfabrik Karl Hecht KG,RhD on, Germany) using Dacie’s
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diluting fluid (Blaxhall and Daisley 1973). The hematocrit ratio (Hct, %) was determined using a capillary hematocrit tube. The hemoglobin (Hb, gdL) concentration was determined by spectrophotometry (Optizen POP UV/VIS Spectrophotometer, Seoul, Republic of Korea) at 540 nm using the cyanomethaemoglobin method (Blaxhall and Daisley 1973). The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the following equations (Lewis et al., 2006): MCV (mm3 ) = Hct (%) × 10/RBC (106 /L); MCH (pg) = (Hb (g/dL) × 10)/RBC (106 /L); MCHC (%) = (Hb (g/dL) × 10)/Hct (%). 2.6. CA activity Liver, gill and kidney were removed and immediately snapfrozen in liquid nitrogen and stored at −80 ◦ C until required. Tissues were homogenized (Stuart SHM1Homogenisator, Staffordshire, UK) in a 25 mmol/ L TRIS-(2-amino-2-hydroxylmethyl-1,3propanediol) HCl buffer, adjusted to pH 8.7. The CO2 hydratase activity of the CA enzyme was assayed colorimetrically by using the method of Wilbur and Anderson (1976). A 0.2 mM phenol red solution was used as an indicator, working at an absorbance of 557 nm, with 20 mM TRIS as a buffer (pH 8.3) and 20 mM NaCl, following the initial rate of the CA catalyzed CO2 hydration for 10–100 s. The CO2 hydratase activity (enzyme units) was calculated as (t0-tc)/tc, where t0 is the time for pH change of the nonenzymatic reaction and tc is the time for pH change of the enzymatic reaction. To calculate specific activities of CA in the serum and tissues (gill, liver and kidney) protein quantities were determined (specific CA activity was expressed as enzyme units per milligram of protein) spectrophotometrically (595 nm) according to Bradford (1976) by using bovine serum albumin as the standard. 2.7. Na+ , K+ -ATPase activities On the first day, 12 fish (fish from the stock tank), and on the 7th and 14th day six fish from each aquarium were used for Na+ , K+ -ATPase and CA activities in the gill, liver and kidney tissues. The fish were anesthetized as given above, liver, gill and
kidney were removed, instantly snap-frozen in liquid nitrogen, and stored at −80 ◦ C until required. Tissues (about 0.5 g) were homogenized (Stuart SHM1Homogenisator, Staffordshire, UK) in five volumes (2.5 mL) of an ice-cold isotonic buffer [300 mmol/L sucrose, 0.1 mmol/L ethylenediamine tetra acetic acid (EDTA), 20 mmol/L (4-(2-hydroxyethyl) piperazine-1- ethane sulfonic acid (HEPES), adjusted to pH 7.8 with a few drops of 2 mol/L TRIS (2-amino-2-hydroxylmethyl-1,3-propanediol)]. Tissues were analyzed for Na+ , K+ -ATPase according to Silva et al. (1977) as follows: each sample (15 L, in triplicate) was put into 400 L of both K+ (containing buffer) and K (free buffer, plus 1.0 mmol/L Quabain), and then incubated at 37 ◦ C for 10 min. The reaction was stopped by adding 1 mL of ice-cold trichloroacetic acid (8.6%, w/v) and 1 mL of a color reagent was added to each tube (9.6%, w/v FeSO4 6H2 O, 1.15%, w/v ammonium heptamolybdate dissolved in 0.66 M H2 SO4 ), and the color allowed to develop for 15 min at room temperature. Absorbance was measured at 660 nm (Optizen POP UV/VIS Spectrophotometer, Seoul, Republic of Korea) against 0–0.5 mmol/L phosphate standards. 2.8. Statistical analysis All data were expressed as means ± standard error of the mean (S.E.M.). In the study, normality of data and homogeneity of variances were checked using Kolmogorove Smirnov and Levene tests respectively. When appropriate, One-Way ANOVA and Tukey sub-test were performed. When normality of variances were not assumed, Kruskal Wallis test was performed. The statistical analysis was made using SPSS 21.0 (SPSS, Chicago, IL, USA), and the significance level was considered to be 0.05. 3. Results In this study, any death case was observed neither in control nor in test group fish. Furthermore, no change was observed in feeding and behaviors of fish in any group. In the study, pH levels fell simultaneously with the inclusion of CO2 to water in groups exposed to CO2 . Throughout the experiment process, pH level of the control group was around 7.6 while it fell down to 6 at the end of the 7th
Table 1 Physico-chemical parameters measured in the experiment.
◦
pH
Dissolved oxygen (mg/L)
Temperature (◦ C)
Carbon dioxide (mg/L)
25 C-Control
Initial 2th day 4th day 7th day 10th day 14th day
7.7 7.6 7.7 7.6 7.7 7.7
7.1 7 7.2 7.1 7.2 7.2
25 25.1 25.4 25 25.2 25.2
3.3 3 3.7 3 3.5 3.2
25 ◦ C-CO2
Initial 2th day 4th day 7th day 10th day 14th day
7.7 7.56 7.18 7 6 5.2
7.1 7 7.2 7.1 7.2 7.2
25 25 25.1 25.2 25 25
3.4 14.1 14.2 13.9 14.1 14
30 ◦ C-Control
Initial 2th day 4th day 7th day 10th day 14th day
7.75 7.64 7.5 7.71 7.8 7.85
7.1 7.2 7 7.2 7.1 7.3
30 30.1 30.2 30.1 30 30
3 3.1 3.2 3 3.4 3.4
30 ◦ C-CO2
Initial 2th day 4th day 7th day 10th day 14th day
7.71 7.61 7.15 7.1 6 5.1
7 7 7.1 7.1 7.2 7.1
30 30.1 30.2 30 30 30.2
3.5 14.1 14 13.9 14.2 14.1
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Table 2 Effect of subchronic exposure to carbon dioxide with different temperature levels on the Red blood cell count (RBC), hematocrit (Hct), hemoglobin (Hb) concentration, corpuscular volume mean (MCV), corpuscular Hb mean (MCH), mean corpuscular Hb concentration (MCHC) and blood pH of O. mossambicus at 0, 7 and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA/Tukey or Kruskal Wallis test were used).
Initial 25 ◦ C-Control 25 ◦ C-CO2 30 ◦ C-Control 30 ◦ C-CO2 25 ◦ C-Control 25 ◦ C-CO2 30 ◦ C-Control 30 ◦ C-CO2
7th day
14th day
RBC(x106 mm3 )
Hb (g/dL)
Hct (%)
MCV (fL)
MCH (pg)
MCHC (%)
Blood pH
3.16 ± 0.11
10.52 ± 0.22
20.60 ± 0.51
65.70 ± 3.86
33.51 ± 1.70
51.19 ± 1.70
7.14 ± 0.03
3.17 ± 0.15a 2.42 ± 0.04b 2.16 ± 0.22b 2.00 ± 0.24b 3.19 ± 0.13a 3.47 ± 0.06a 3.32 ± 0.12a 3.02 ± 0.24a
9.48 ± 0.41b 12.80 ± 0.18a 10.11 ± 0.21b 10.21 ± 0.71b 10.77 ± 0.36a 9.76 ± 0.62ab 7.98 ± 1.02c 10.31 ± 0.86ab
21.75 ± 0.85a 20.00 ± 1.08ab 17.02 ± 1.08b 17.75 ± 0.85b 21.01 ± 0.84a 21.60 ± 0.93a 20.20 ± 0.58ab 17.60 ± 1.54b
69.61 ± 6.38a 82.71 ± 3.67a 79.92 ± 4.73a 95.49 ± 17.50a 66.63 ± 5.02a 62.15 ± 2.30 a 61.10 ± 2.15a 58.15 ± 0.78a
30.27 ± 2.47b 53.05 ± 1.11a 48.04 ± 4.04a 54.37 ± 10.07a 34.16 ± 2.45a 28.08 ± 1.70ab 23.80 ± 2.16b 34.38 ± 2.18a
43.67 ± 1.82b 64.70 ± 4.32a 59.96 ± 2.69a 56.88 ± 2.71a 51.61 ± 2.70 ab 45.30 ± 2.65bc 39.25 ± 4.30c 59.12 ± 3.68a
7.19 ± 0.02b 7.32 ± 0.03a 7.14 ± 0.01b 7.16 ± 0.03b 7.15 ± 0.02a 7.09 ± 0.11a 7.11 ± 0.02a 7.12 ± 0.01a
Fig. 2. Effect of subchronic exposure to carbon dioxide with different temperature levels on the carbonic anhydrase (unit/mg protein) activity in the a) blood b) liver c) gill and d) kidney tissues of O. mossambicus at 0, 7, and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA followed by Tukey post-hoc test).
day; to 5 at the end of the 14th day in groups exposed to CO2 . On the other hand, CO2 level of the control group was 3.27 mg/L at 25 ◦ C and 3.18 mg/L in average at 30 ◦ C. Nevertheless, CO2 level was stable around 14 mg/L following the 2nd day in groups exposed to CO2 . In the study, oxygen values were at the same level in all tanks for the control groups and those exposed to carbon dioxide. Findings related to pH, carbon dioxide, dissolved oxygen and temperature measured in water during the experiment are presented in Table 1. The variations in hematological parameters and blood pH as a result of 14-day carbon dioxide exposure are illustrated in Table 2. On the 7th day of the experiment, a statistically significant decline was observed in the number of erythrocyte in groups exposed to carbon dioxide at 25 ◦ C and 30 ◦ C compared to the control group at the given temperature values (p < 0.05), while an increase was observed in MCH, MCHC, Hb and blood pH in the group exposed to carbon dioxide at 25 ◦ C compared to the control group at the same temperature level (p < 0.05). In the group exposed to carbon dioxide at 25 ◦ C, CA enzyme activity in blood increased statistically compared to the control group on the 7th day of the test while in both temperature values it fell down on the 14th day (p < 0.05). In groups exposed to carbon dioxide at 25 and 30 ◦ C, liver CA activity increased on the 7th and 14th days compared to the control groups. CA enzyme activity in
gills at the group exposed to 25 ◦ C increased statistically at day 7 compared to the control group but in the group exposed to CO2 at 30 ◦ C an increase was observed at day 14. The most interesting findings of CA activity were found in kidneys in groups exposed to carbon dioxide. While, in the group exposed to carbon dioxide at 25 ◦ C, CA activity increased on the 7th day (p < 0.05); it started to fall down on the 14th day (no statistically significant increase). In the group exposed to carbon dioxide at 30 ◦ C, on the contrary, CA activity declined on the 7th day; it increased on the 14th day (Fig. 2). According to this study, Na+ , K+ -ATPase findings on gill, liver and kidney tissue are presented in Fig. 3. While in groups exposed to carbon dioxide at 25 and 30 ◦ C, gill Na+ , K+ -ATPase activity increased on the 7th day compared to the control group; they were inhibited on the 14th day. On the other hand, liver Na+ , K+ -ATPase activity increased in the group exposed to carbon dioxide at 25 ◦ C on the 14th day compared to their control group; it fell down in the group exposed to carbon dioxide at 30 ◦ C at day 7. While, in groups exposed to carbon dioxide at 25 and 30 ◦ C, kidney Na+ , K+ -ATPase activity declined on the 7th day compared to control groups; on the 14th day, this decline sustained in the group exposed to CO2 at 25 ◦ C; it reached the equal level with the control group in the one exposed to CO2 at 30 ◦ C.
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Fig. 3. Effect of subchronic exposure to carbon dioxide with different temperature levels on the Na+ , K+ -ATPase (Pi mol/mg protein/h) activity in the a) gill b) liver and c) kidney tissues of O. mossambicus at 0, 7, and 14 d postexposure. For given times, concentrations with different lowercase letters are significantly different (p < 0.05); one-way ANOVA followed by Tukey post-hoc test).
4. Discussion Temperature is one of the fundamental factors being effective in performance, metabolism and zoogeographic distribution in ectodermic animals including fish (Pörtner, 2010). The elevated carbon dioxide level in waters may affect organism physiology directly by reducing pH of waters or indirectly by changing the composition of metal ion in water (Lannig et al., 2010). Today, the observable effects in physiology of aquatic organisms are not discovered completely yet, due to climate changes. One of the most important reasons for this is the limited number of measurable argument related to physiological phenomenon on the ecosystem level (Pörtner, 2010). Biologic stress that might occur in organisms manifests itself in different ways (Barton, 1997). There are studies that have been conducted so far on the effects of elevated carbon dioxide levels in cold-water fish such as trout and salmon (Fivelstad et al., 1999, 2007; Kaya et al., 2013). On the other hand, no study was found about the effects of both elevated carbon dioxide and changing temperature levels in tropical fish species. In this study, the number of erythrocyte (RBC), hemoglobin, hematocrit, MCV, MCH, MCHC and blood pH levels are investigated in samples of O. mossambicus. As a result of elevated carbon dioxide concentration in the water, hypercapnia is observed due to abnormally high level carbon dioxide in the blood (Heisler, 1984). The increase in blood partial carbon dioxide pressure can cause a respiratory acidosis in fish (Goss et al., 1994). Because increased carbon dioxide concentrations cause acidification in the blood and tissue respiratory pigment, it causes a reduction in oxygen intake and delivery (Pörtner et al., 2004). In cases where acidification occur and blood pH fall, fish can compensate the reduced red blood cell pH with Na+ /H + ion exchange by catecholamine proteins (Randall and Brauner, 1998). In our study also, in the group exposed to CO2 at 25 ◦ C changes seen in hematology at the end of the first week returned to the normal values at the end of trial, this can be explained by the operation of the adaptation mechanism. A similar situation was also seen in our previous study on CO2 exposed trout (Oncorhynchus mykiss). RBC, hemoglobin and hematocrit values of the cold water fish exposed to CO2 showed a decrease at the end
of the first week like the present study and it came to similar situations (excluding hematocrit) with the control at the end of the experiment. On the other hand, there are some studies investigating high carbon dioxide under aquaculture conditions. In these studies, Fivelstad et al. (2007) exposed Atlantic salmons to high level of CO2 (12 mm Hg) at two different temperature values (5 and 15 ◦ C) and detected that RBC amount and MCH value did not change compared to the control group at the end of 39 days. In addition, hematocrit amount and MCV values fell down at both temperature values (5 and 15 ◦ C) in fish exposed to high level of CO2. In the same study, hemoglobin decreased in fish exposed to CO2 at 5 ◦ C while no change occurred at 15 ◦ C. Similarly in our study, no change occurred in RBC value at the end of 14 days while MCV and MCH value decreased at the end of the test. Fluctuations in hematologic parameters during the study can be associated with the adaptation of fish into environment conditions including high level of CO2. Studies indicate a similarity between the control group and the group of fish exposed to CO2 for a long period of time in terms of hematological parameters. For example, in a study conducted by Fivelstad et al. (2003) on Atlantic salmons, fish were exposed to 16 and 24 mg/L CO2 for 57 days and at the end of the test, no difference was detected in hematologic parameters (Hct, Hb and MCH) of fish compared to the control group. Similarly, Fivelstad et al. (1998) exposed Atlantic salmons to three different concentrations of CO2 (10, 26 and 44 mg/L CO2 ) and found that Hct value never changed at the end of 57 days. However, the lack of any change in hematologic parameters at the end of high levels of CO2 exposure should not be understood as the fish health is not adversely affected. At this point, analysis of various physiologic changes will ensure to assess fish health as a whole in physiologic terms. As osmoregulatory organs in fish; gill, kidney and intestine are specialized in acid-base regulation associated with the active ion transportation (Melzner et al., 2009). Especially gill in fish has a significant role in ensuring osmotic regulation apart from its task in transferring Na+ , K+ -ATPase enzymes taking place in epithelia cells into active electrolyte (Kaya et al., 2016). Na+ , K+ -ATPase enzyme regulates ionic and electric gradient required for transepithelial
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salt movements in osmoregulation. Carbonic anhydrase enzyme, on the other hand, plays a significant role in maintaining acidbase balance and transferring and eliminating blood carbon dioxide (CO2 ) (by means of catalyzing H+ and HCO3 ) in fish (Henry and Cameron, 1982). In reviewing the literature available in terms of mentioned enzyme activities, it has been found that the studies usually performed on marine organisms. However, similar studies using freshwater organisms are limited (Kaya et al., 2013). Of respiratory enzymes analyzed in this study, CA activity significantly decreased in blood samples of groups exposed to carbon dioxide at the end of the test while it increased in osmoregulatory organs (gill and kidney). Similarly, it was detected that Na+ , K+ -ATPase enzyme activity of gill and kidney being among osmoregulatory organs was significantly inhibited in groups exposed to carbon dioxide (on the 14th day, except for 30CO2 ). The data obtained from this study show that carbonic anhydrase and Na+ , K+ -ATPase enzyme activities in osmoregulatory tissues tried to overcome the problems occurred in acid-base balance due to hypercapnia conditions. The long term effects of changes in carbon dioxide and temperature in waters are not completely known, yet. However, acid-base disorders may lead to obvious consequences at biochemical levels. Changes in Na+ , K+ ATPase enzyme activity may lead to adverse results on physiologic level due to the impairment of extracellular fluid (therefore, cell fluid is impaired as well). Impairments in acid-base balance and/or ionic/osmotic balance in organisms living in all aqua-ecosystems (including fresh water, sea water and brackish water) may affect the survival of organisms. To conclude, it was observed that elevated carbon dioxide and temperature levels have acute adverse effect on some hematologic parameters and blood pH in tilapia and at the end of this study, the given parameters were found to be closer to normal values. In addition, fluctuations in CA and Na+ , K+ -ATPase enzyme activity show that organisms give reaction to stress conditions through their adaptation mechanisms. We believe that further chronic studies that will also consider acidification apart from the elevated carbon dioxide and temperature levels in sea and freshwater fish species should be conducted. Acknowledgements This study was supported by The Scientific and Technological Council of Turkey (TUBITAK, Project Number: 113O220, coordinated by H Kaya). References Barton, B.E., 1997. IL-6: insights into novel biological activities. Clin. Immun. Immunopathol. 85 (1), 16–20. Blaxhall, P.C., Daisley, K.W., 1973. Routine haematological methods for use with fish blood. J. Fish Biol. 5 (6), 771–781. Bradford, M.M., 1976. A rapid and sensitive method for the quantitiation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brander, K.M., 2007. Global fish production and climate change. Proc. Natl. Acad. Sci. 104 (50), 19709–19714. Fivelstad, S., Haavik, H., Lovik, G., Olsen, A.B., 1998. Sublethal effects and safe levels of carbon dioxide for atlantic salmon postsmolts (Salmo salar L.). Aquaculture 160, 305–316. Fivelstad, S., Olsen, A.B., Kløften, H., Ski, H., Steffanson, S., 1999. Effects of carbon dioxide on atlantic salmon (Salmo salar L.) smolts at constant pH in bicarbonate rich freshwater. Aquaculture 178, 171–187. Fivelstad, S., Olsen, A.B., Waagbo, R., Zeitz, S., Hosfeld, C.D., Stefansson, S., 2003. A major water quality problem in smolt farms: combined effects of carbon dioxide and reduced pH and aluminium on atlantic salmon (Salmo salar L.) smolts: physiology and growth. Aquaculture 215, 339–357.
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Fivelstad, S., Waagbo, R., Stefansson, S., Olsen, A.B., 2007. Impacts of elevated water carbon dioxide partial pressure at two temperatures on atlantic salmon (Salmo salar L.) parr growth and haematology. Aquaculture 269, 241–249. Foucreau, N., Cottin, D., Piscart, C., Hervant, F., 2014. Physiological and metabolic responses to rising temperature in Gammarus pulex (Crustacea) populations living under continental or Mediterranean climates. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 168, 69–75. Gooding, R.A., Harley, C.D., Tang, E., 2009. Elevated water temperature and carbon dioxide concentration increase the growth of a keystone echinoderm. Proc. Natl. Acad. Sci. 106 (23), 9316–9321. Goss, G.G., Laurent, P., Perry, S.F., 1994. Gill morphology during hypercapnia in brown bullhead (Ictalurus nebulosus): role of chloride cells and pavement cells in acid–base regulation. J. Fish Biol. 45, 705–718. Henry, R.P., Cameron, J.N., 1982. The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustaceans. J. Exp. Zool. 221, 309–321. Heisler, N., 1984. Acid-base regulation in fishes. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, XA. Academic Press, New York, pp. 315–399. Hisar, O., Hisar, S¸.A., Sirkecioglu, A.N., Karatas, M., Yanik, T., 2007. Using an oxygenating and degassing column to improve water quality in salmonid hatcheries. Int. J. Nat. Eng. Sci. 1, 63–64. IPCC, 2013. Intergovernmental Panel on Climate Change (IPCC) summary for policymakers contribution of working group I to the fifth assessment report of the Intergovernmental panel on climate change. In: Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S.K., Boschung, J. (Eds.), Climate Change 2013: The Physical Science Basis. Cambridge University Press, Cambridge New York. Kaya, H., Yılmaz, S., Gürkan, M., Hisar, O., 2013. Effects of environmental hypercapnia on hemato-immunological parameters, carbonic anhydrase, and Na+ , K+ -ATPase enzyme activities in rainbow trout (Oncorhynchus mykiss) tissues. Toxicol. Environ. Chem. 95 (8), 1395–1407. Kaya, H., Aydın, F., Gürkan, M., Yılmaz, S., Ates, M., Demir, V., Arslan, Z., 2015. Effects of zinc oxide nanoparticles on bioaccumulation and oxidative stress in different organs of tilapia (Oreochromis niloticus). Environ. Toxicol. Pharmacol. 40 (3), 936–947. Kaya, H., Aydın, F., Gürkan, M., Yılmaz, S., Ates, M., Demir, V., Arslan, Z., 2016. A comparative toxicity study between small and large size zinc oxide nanoparticles in tilapia (Oreochromis niloticus): organ pathologies, osmoregulatory responses and immunological parameters. Chemosphere 144, 571–582. Lannig, G., Eckerle, L., Serendero, I., Sartoris, F.J., Fischer, T., Knust, R., Pörtner, H.O., 2003. Temperature adaptation in eurythermal cod (Gadus morhua): a comparison of mitochondrial enzyme capacities in boreal and Arctic populations. Mar. Biol. 142 (3), 589–599. Lannig, G., Eilers, S., Pörtner, H., Sokolova, I.M., Bock, C., 2010. Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas—changes in metabolic pathways and thermal response. Mar. Drugs 8 (8), 2318–2339. Lewis, S.M., Bain, B.J., Bates, I., 2006. Dacie and Lewis Practical Haematology, 10th ed. Churchill Livingstone Elsevier, Philadelphia, PA. Melzner, F., Gutowska, M.A., Langenbuch, M., Dupont, S., Lucassen, M., Thorndyke, M.C., Pörtner, H.O., 2009. Physiological basis for high CO2 tolerance in marine ectothermic animals: pre-adaptation through lifestyle and ontogeny? Biogeosciences 6 (10), 2313–2331. Pörtner, H.O., 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals. Biochem. Physiol. A Mol. Integr. Physiol. 132 (4), 739–761. Pörtner, 2010. Oxygen- and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. J. Exp. Biol. 213, 881–893. Perry, A.L., Low, P.J., Ellis, J.R., Reynolds, J.D., 2005. Climate change and distribution shifts in marine fishes. Science 308 (5730), 1912–1915. Pörtner, H.O., Langenbuch, M., Reipschlager, A., 2004. Biological impact of elevated ocean CO2 concentrations: lessons from animal physiology and earth history. J. Oceanogr. 60, 705–718. Rosset, V., Oertli, B., 2011. Freshwater biodiversity under climate warming pressure: identifying the winners and losers in temperate standing waterbodies. Biol. Conserv. 144 (9), 2311–2319. Schiedek, D., Sundelin, B., Readman, J.W., Macdonald, R.W., 2007. Interactions between climate change and contaminants. Mar. Pollut. Bull. 54 (12), 1845–1856. Silva, P., Solomon, R., Spokes, K., Epstein, F.H., 1977. Ouabain inhibition of gill Na, K-ATPase: relationship to active chloride transport. J. Exp. Zool. 199, 419–426. Wilbur, K.M., Anderson, N.G., 1976. Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176, 147–154.