Experimental Biology Online (EBO) 1999 4 : 5
© Springer-Verlag 1999
Cardio-respiratory effects of chloramine-T exposure in rainbow trout Mark D. Powell 1,2 · Steve F. Perry2 1 2
School of Aquaculture, University of Tasmania, Launceston, Tasmania 7250 Australia Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, K1N 6N5 Canada
Received: October 11 1999 / Accepted: November 24 1999 / Published: December 13 1999
Abstract. In order to establish whether the blood gas respiratory disturbances noted with exposure to chloramine-T are due to differences in the rates of uptake of O2 and excretion of CO2 or gill blood flow, adult rainbow trout (Oncorhynchus mykiss) were fitted with dorsal aorta and bulbus arteriosus catheters to facilitate blood pressure recordings, an ultrasonic blood flow probe and opercular impedance electrodes. Fish received either a 45-min static exposure to 9 mg l-1 chloramine-T or tap water (control) and continuous recordings of blood pressure, and ventilation frequency and amplitude were made. Pre- and post-exposure arterial and venous blood samples were taken and analyzed for O2 and CO2 content, hemoglobin concentration and hematocrit. Chloramine-T exposure had no effect on any of the continuously recorded parameters. However, individual measurements (made immediately prior to and following exposure) of cardiac output and O2 uptake rates increased significantly following exposure to chloramine-T compared to before exposure. CO2 excretion rates were unaffected by chloramine-T exposure. Calculation of the perfusion convection requirement showed a significant increase for CO2 but not for O2. It was concluded that increases in O2 uptake resulted from increased cardiac output but that CO2 excretion, a diffusion-limited process, was not increased due to additional diffusive limitations caused by the irritant effect of chloramine-T. Key words
Cardiovascular physiology · Chloramine-T · Gas exchange Rainbow trout · Respiration
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Experimental Biology Online (EBO) 1999 Vol. 4:5 • ISSN: 1430-3418
Introduction Chloramine-T is a widely used chemotherapeutic agent for the treatment of bacterial gill disease in the freshwater aquaculture industry (Thorburn and Moccia, 1993). It is believed that chloramine-T degrades in solution to paratoluenesulfonamide and sodium hypochlorite. In doing so, this results in the liberation of hypochlorite and free chlorine which acts as an irritant. Studies have demonstrated that chloramine-T exposure induces acute respiratory and acid-base disturbances in rainbow trout blood (Powell and Perry, 1996; 1997) that are believed to be a consequence of increased mucus secretion owing to the irritant effects of hypochlorite on the gill. When exposed acutely, trout showed an increase in arterial carbon dioxide tension (PCO2) but arterial oxygen tension (PO2) was unaffected (Powell and Perry, 1996). This rise in PCO2 occurred even though the fish were hyperventilating in response to the chemical exposure (Powell and Perry, 1996). However, forced ventilation through exposure to moderate hypoxia eliminated any rise in arterial PCO2 and any additional hyperventilation that also occurred when fish were exposed to hyperoxic conditions (Powell and Perry, 1997). This has led to the inference that the secretion of branchial mucus impairs CO2 excretion without hindering O2 uptake because CO2 excretion is more sensitive to changes in the diffusional properties of the gill as compared with O2 uptake. However, this is speculative and to date the evidence is circumstantial. The exchange of respiratory gases is directly affected by changes in branchial perfusion. The main parameters affecting gill perfusion are cardiac output and the pressure drop across the gills (ventral-dorsal aortic blood pressures) which determines branchial vascular resistance. This assumes, however, that all the blood goes through the arterio-arterial pathway. An overall increase in ventral aortic blood pressure is believed to elicit lamellar recruitment and redistribution of intralamellar blood flow to increase the functional surface area over which gas exchange may occur (as reviewed by Farrell, 1993). This increase in ventral aortic pressure may arise through increases in systemic and/or branchial vascular resistance or through increases in cardiac output or any combination thereof. Previous studies, while attributing the effects of chloramine-T on CO2 excretion to increases in the diffusive limitations, could not exclude the contribution of changes in the pattern of branchial perfusion. Thus, the aim of this study was to examine the cardiovascular responses of rainbow trout during an acute exposure to a gill irritant — chloramine-T. Chloramine-T was used as a gill irritant because of its potential for increasing branchial mucus secretion and its previously described effects on respiratory gas tensions in the blood (Powell and Perry, 1996; 1997) and its commercial relevance as a chemotherapeutic treatment for fish gill diseases. We hypothesized that chloramine-T exposure would not increase branchial vascular resistance, therefore supporting the notion that the primary effects on CO2 transfer reflect changes in gill diffusive conductance.
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Materials and methods Fish Adult rainbow trout [748.8±30.0 g (mean ±SE)] were purchased from Linwood Acres Trout Farm, Cambellcroft, Ontario and acclimated to laboratory conditions for at least 14 days prior to use. During the acclimation period fish were maintained in dechlorinated city of Ottawa tap water under a natural photoperiod at 15°C and fed ad libitum on a commercial pelleted diet. Surgical procedures Fish were anesthetized with a chilled oxygenated (dechlorinated) solution of 100 mg l-1 tricaine methanesulfonate (TMS Sindel, Vancouver, Canada) adjusted to ≅ pH 7 with 20 mg l-1 sodium bicarbonate. The gills were constantly irrigated with the same solution throughout the following surgical procedures. A PE 160 (Clay Adams) catheter was implanted into the buccal cavity to facilitate the measurement of inspired water PO2.A catheter (PE 50) was implanted in the dorsal aorta according to Soivio et al. (1975). A small incision was made in the ventral surface anterior to the branchiostegal region, the pericardium was dissected and the ventricle and bulbus arteriosus were exposed. A silicone catheter (I.D. 0.51 mm; O.D. 0.94 mm) was inserted into the bulbous using an internal trochar and attached to the bulbus arteriosus using a small drop of cyanoacrylate adhesive (Olsen et al., 1997). The catheter was then connected to a length of polyethylene tubing (PE 60; Clay Adams). All catheters were filled with non-heparinized Cortland's saline (Wolf, 1963) prior to insertion. A 3-s or 4-s ultrasonic flow probe (Transonics Systems, Ithaca, N.Y., USA) was then attached non-occlusively around the bulbus and the incision closed with sutures. Finally, 1-cm2 brass impedance electrodes were sutured to the trailing edges of the operculae. Fish were then allowed to recover in flowing freshwater (15°C) overnight in a 12.5-l black acrylic respirometer box prior to experimentation. Following recovery, catheters were flushed with heparinized saline (25 IU ml-1 ammonium heparin: Sigma). Dorsal aortic (DAP) and ventral aortic (VAP) pressures were measured from the saline-filled dorsal aortic or bulbus arteriosus catheters connected to UFI model 1050BP (UFI, Morro Bay, Calif., USA) pressure transducers calibrated against a water column. Mean blood pressures were determined according to:
(Systolic pressure+Diastolic pressure)/2 Blood flow through the ventral aorta was measured using the implanted ultrasonic flow probe connected to a Transonics T106 small-animal blood-flow meter. The flow probe was pre-calibrated in the factory and verified in the laboratory by pumping (using a peristaltic pump) a known flow rate of saline (15°C) into the heart of a dead trout immersed in water. Inspired water PO2 was measured continuously by continuous siphon across a Radiometer E5046 oxy-
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Experimental Biology Online (EBO) 1999 Vol. 4:5 • ISSN: 1430-3418
gen electrode housed in a thermostated cuvette and measurement made with a Cameron Instruments blood gas monitor (Cameron Instruments, Port Aranas, Tex., USA). The opercular impedence electrodes were connected to a customized impedance converter and preamplifier. The outputs from the pressure transducers, blood flow meter, and impedance converter were relayed to a data acquisition system (Biopac Systems) and data recorded using Acknowledge 3.0 software. Experimental protocol Five hundred microliter arterial and venous blood samples were anerobically removed via the dorsal aortic and bulbus catheters respectively and replaced with twice the volume of saline so as not to affect the blood pressure recordings (Dr. Phil Byrne, University of Guelph, Canada, pers. comm). This was termed the pre-exposure sample. The hemoglobin concentration of duplicate 20 µl samples was determined using a commercial spectrophotometric kit (Sigma). Hematocrit was determined by drawing blood into microcapillary tubes and centrifuging at 10 000 g for 10 min. The oxygen content of the blood was determined from duplicate 40 µl samples using an Oxycon blood O2 content analyzer (Cameron Instrument). The remaining blood was centrifuged at 10 000 g for 30 s and duplicate 50 µl plasma samples analyzed for total CO2 content using a Corning 965 carbon dioxide analyzer.
Fig. 1. Water oxygen tension (PwO2) during a static exposure of a rainbow trout to chloramine-T (between vertical lines)
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The water flow to the respirometer box was stopped and the box aerated by means of an airstone to ensure that the inspired water PO2 (PwO2) did not fall below 135 mmHg (Fig. 1) and thus potentially cause an increase in ventilation volume (Smith and Jones, 1982). Chloramine-T (9 mg l-1 active ingredient, BDH, Toronto Ontario: analytical grade lot No. 106295/15998) that had been pre-dissolved in 25 ml of the same water supplying the respirometer was then added to the box. The fish were monitored for 45 min after which the water flow was reinstated and second 500 µl arterial and venous blood samples were taken and analyzed as above. Controls consisted of fish that were not exposed to chloramine-T although 25 ml of dechlorinated tap water was added. A 45min exposure was used because in previous studies (Powell and Perry, 1996) respiratory disturbances were observed within this time period. Calculations and statistical analysis Rates of oxygen uptake (MO2, in mmol kg-1 h-1) and carbon dioxide excretion (MCO2, in mmol kg-1 h-1) were calculated according to the formulae:
MO2 =
Q(CaO2 − CvO2 ) × 60 × 1000 W
where Q is mass-specific cardiac output blood flow (ml min-1 kg-1), CaO2 is arterial oxygen content (mM), CvO2 is venous oxygen content (mM), and W is the mass of the fish (kg).
MCO2 =
Q(Cv CO2 − Ca CO2 ) × 60 × 1000 W
Where Q is mass-specific cardiac output (ml min-1 kg-1), CaCO2 is arterial carbon dioxide content (mM), CvCO2 is venous carbon dioxide content (mM), W is the mass of the fish (kg). Branchial (Rg, cmH2O ml-1 min-1) and systemic (Rs, cmH2O ml-1 min-1) vascular resistance were calculated from the ventral and dorsal aortic pressure and mass-specific cardiac output data in the Acknowledge software according to the formulae:
Rg =
VAP − DAP Vb
Rg =
VAP − DAP Vb
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Experimental Biology Online (EBO) 1999 Vol. 4:5 • ISSN: 1430-3418
Where VAP is the mean ventral aortic pressure (cmH2O), DAP is the mean dorsal aortic pressure (cmH2O), and Vb is the cardiac output (ml min-1 kg-1). Mean cellular hemoglobin concentration was calculated by dividing the hematocrit by the total blood hemoglobin concentration. Oxygen specifically bound to hemoglobin was determined by dividing the measured CaO2 and CvO2 by the mean hemoglobin concentration of the blood sample. Data presented represent means ±1 standard error of the mean. Differences between pre-exposure and post-exposure samples (measured blood parameters, cardiac output, calculated O2 consumption, CO2 excretion, respiratory exchange ratio and perfusion conduction requirements) were compared using a paired t-test and comparisons between chloramine-T-exposed and control fish tested using a two-sample t-test (discrete data). Continuous data (VAP, DAP, Rg, Rs, ventilation frequency and amplitude) were compared using a twoway repeated-measures analysis of variance using a Bonferroni corrected ttest to isolate differences. P values
