Acid–base and ionic fluxes in rainbow trout (Oncorhynchus mykiss) during exposure to chloramine-T

Aquatic Toxicology 43 (1998) 13 – 24

Acid–base and ionic fluxes in rainbow trout (Oncorhynchus mykiss) during exposure to chloramine-T Mark D. Powell *, Steve F. Perry Department of Biology, Uni6ersity of Ottawa, 30 Marie Curie, Ottawa, Ontario, K1N 6N5, Canada Received 7 August 1997; received in revised form 4 December 1997; accepted 15 December 1997

Abstract The effects of chloramine-T and its degradation products, sodium hypochlorite (NaOCl) and para-toluenesulphonamide (pTSA), on whole body acid–base and branchial and renal ion (Na + and Cl − ) fluxes were examined in rainbow trout (Oncorhynchus mykiss). Exposure to chloramine-T (3.5 h, 18 mg l − 1) resulted in increases in plasma total CO2 but no coincident rise in PaCO2 or reduction in blood pH. Exposure of fish to 2, 9 or 18 mg l − 1 chloramine-T (3.5 h duration) resulted in a reduction in net acid uptake suggesting the development of a metabolic alkalosis. Exposure to the chloramine-T breakdown product pTSA (dissolved in DMSO) resulted in increased net acid uptake (decreased acid excretion) suggesting a metabolic acidosis. Whole body ion fluxes demonstrated increases in the losses of both Na + and Cl − with chloramine-T, NaOCl and pTSA. However, the effect of DMSO alone could not be isolated. Confirmatory studies using fish in which the urinary bladder (to allow collection of urine) and dorsal aorta (to allow injection of [14C]polyethylene glycol 4000 ([14C]PEG), an extracellular fluid marker) were catheterised, revealed that changes in whole body ion fluxes during chloramine-T exposure could not be explained by increased renal efflux through urine flow, glomerular filtration or renal clearance. Branchial effluxes of [14C]PEG were not significantly affected by chloramine-T exposure suggesting that the changes in whole body ionic fluxes were caused by transcellular rather than paracellular processes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Metabolic acid–base fluxes; Ionic fluxes; Chloramine-T; Rainbow trout; Gill; Kidney

1. Introduction

* Corresponding author. Present address: School of Aquaculture, University of Tasmania, P.O. Box 1214 Launceston, Tasmania 7250, Australia. Tel.: + 61 3 63243813; fax: + 61 3 63243804; e-mail: [email protected]

Chloramine-T is a widely used disinfectant in freshwater aquaculture for the treatment of bacterial and parasitic diseases of gills (From, 1980; Bullock et al. 1991) and skin (Cross and Hursey, 1973). Such diseases are considered to be serious

0166-445X/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0166-445X(98)00040-X

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M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

limiting factors to freshwater aquaculture production (Daoust and Ferguson, 1983; Speare and Ferguson, 1989). Chloramine-T is used extensively as a prophylactic or disinfective treatment (Thorburn and Moccia, 1993). Therapeutically, it is used at a variety of doses, although treatments in the range of 5–20 mg l − 1 appear to be the most common (From, 1980; Bullock et al., 1991; Powell et al., 1994). The toxicity of chloramine-T, (in terms of LC50) was shown to decrease as stocking density increases (Bills et al., 1988a) and increase at lower pH, with elevated temperature and with reduced water hardness (Bills et al., 1988b). In all cases, however, the 96 h LC50 was higher than those concentrations used commercially. Chloramine-T degrades in solution due to nucleophilic substitution to release a hypochlorite ion (OCl − ) and para-toluenesulphonamide (pTSA). It is believed that the release of hypochlorite is the primary mechanism of both therapeutic action (antibacterial disinfection) (Booth and MacDonald, 1988) and toxicity (ultrastructural injury to epithelia; increased mucus secretion) (Powell et al., 1995; Powell and Perry, 1996). Hypochlorite is acutely toxic to fish (Heath, 1977; Seegert and Brooks, 1978; Zeitoun, 1978; Seegert et al., 1979; Brooks and Bartos, 1984) and was shown to cause ionic 0.1 – 0.6 mg l − 1(Block et al., 1978; Hose et al., 1983) and respiratory disturbances at 0.45 mg l − 1(Powell and Perry, 1996). Previous investigations have demonstrated that repeated intermittent exposure of rainbow trout to chloramine-T resulted in an apparent hyperplasia of branchial chloride cells and an increase in the chloride cell apical plasma membrane (Powell et al., 1995). These morphological changes coincided with reduced plasma ion concentrations (Powell et al., 1994) and were consistent with other work where changes in chloride cell fractional surface area of the gill was correlated with ionic uptake (Goss and Perry, 1993). Recently, it was shown that acute exposure of rainbow trout to therapeutic concentrations of chloramine-T (9 mg l − 1) caused transient respiratory and metabolic disturbances in the acid – base status of the fish which were rapidly corrected once the chloramine-T was removed (Powell and

Perry, 1996). Specifically, chloramine-T exposure induced a respiratory acidosis superimposed over a metabolic alkalosis, whereas exposure to the breakdown product pTSA caused a pure metabolic acidosis (Powell and Perry, 1996). The aim of this investigation was to test the hypothesis that blood acid–base disturbances caused by a single chloramine-T exposure correlated with changes in the acid–base fluxes across the gill. In addition, the effects of a single acute exposure on gill and renal ionic fluxes were examined in order to provide insight into potential ion losses which may occur during exposure, since reduced plasma ion concentrations have be en reported following repeated exposures to chloramine-T (Powell et al., 1994). 2. Materials and methods

2.1. Fish Rainbow trout (Oncorhynchus mykiss) were purchased from a commercial hatchery (Linwood Acres Trout Farm, Campbellcroft, Ontario) and acclimated to laboratory conditions for at least 3 weeks prior to use. During the acclimation period, fish were held in 300-l rectangular fibreglass tanks and maintained at 10oC in aerated dechlorinated city of Ottawa water (Na + , 118.9 9 2.4 mM; K + , 19.39 0.5 mM; Ca2 + , 365.8 9 8.2 mM; Cl − , 151.89 0.9 mM, pH 6.8). Residual chlorine levels were below those detectable using a N,N-ethyl-pphenylenediamine ferrous titrimetric method (Franson, 1978). Fish were fed on alternate days using a commercial pelleted diet. Food was withheld 24 h prior to experimental use.

2.2. Blood acid–base status and renal ionic fluxes In order to examine the potential contribution of renal ionic effluxes to the whole body ion fluxes (see below) while simultaneously assessing the blood acid–base staus during a static exposure to chloramine-T, fish were fitted with bladder and dorsal aortic catheters and exposed to 18 mg l − 1 chloramine-T. This concentration was chosen because it was sufficient to cause net losses of both sodium and chloride (see below).

M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

2.3. Surgical procedures Rainbow trout of mean weight ( 9 S.E.) 251.49 14.6 g (n=6) were anaesthetized with 80 mg l − 1 MS-222 and fitted with dorsal aortic catheters (PE50: Clay Adams) according to the method of Soivio et al. (1975). The bladder was then catheterised with a heat-flared PE60 polyethylene catheter (Clay Adams) according to Curtis and Wood (1991). Fish were allowed to recover for 24 h in individual black acrylic boxes (3.2-l volumes) with flowing aerated fresh water.

2.4. Experimantal procedures Following recovery from surgery, fish were injected with 0.5 ml 14C labelled polyethylene glycol ([14C]PEG, molecular weight=4000: Amersham) in Cortland’s saline (Wolf, 1963) to yield an activity of 0.3 mCi 100 g fish − 1. The catheter was then flushed with a further 0.25 ml of saline to ensure that no residual PEG was remaining in the catheter. Then, [14C]PEG was used as an extracellular marker to allow determination of glomerular filtration rate (GFR) as well as to determine the effects of chloramine-T exposure on branchial epithelial (paracellular) permeability (Wood and Pa¨rt, 1997). In a previous series of experiments, it was determined that a stable level of [14C]PEG was achieved in the plasma 12 h after the initial injection. All renal flux and acid – base experiments were therefore conducted 12 h post-injection with [14C]PEG. Water flow to the box was interrupted and the box sealed. Aeration and mixing of the water within the box was maintained by a curtain of bubbles provided from a perforated hose around the inner perimeter of the box. A 25-ml water sample was withdrawn to begin the flux period. After a period of 3.5 h a second water sample was removed and the water flow reinstated (pre-exposure flux). Urine was collected from the urinary catheters during the flux period. Following the pre-exposure flux the boxes were flushed for 1 h with fresh water. A second flux period was initiated with 18 mg l − 1 chloramine-T added to each box. Upon addition of the chloramine-T there was a 30-min mixing period prior to taking the

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first water sample (25 ml). After 3 h, a second 25-ml water sample was taken to end the flux period. 2.5. Analysis of water, plasma and urine A 500-ml blood sample was withdrawn from the arterial catheter at the beginning and end of the flux period and the blood pH determined using a Radiometer BMS Mk4 with a microcapillary G299A electrode. Arterial PO2 (PaO2) and O2 content (CaO2) were determined using a thermostatically controlled Radiometer E5046 electrode and an OxyconTM O2 content analyzer (Cameron Instruments, Port Aransas, TX), respectively. Haematocrit was determined from a sample of whole blood drawn into a microcapillary tube and centrifuged at 10000× g for 10 min. Haemoglobin content of the blood was determined with a commercial spectrophotometric assay kit (Sigma, St. Louis, MO.). Mean cellular haemoglobin content (MCHC) was calculated by the division of haemoglobin concentration by haematocrit. The remaining blood was centrifuged at 10 000g and the plasma total CO2 content (CaCO2) was determined with a Corning 965 total CO2 analyzer. The red cells were then resuspended in 500 ml of non-heparinised Cortland’s saline and re-injected into the fish. Arterial PCO2 (PaCO2) and plasma bicarbonate concentration ([HCO3− ]) were calculated using a rearrangement of the Henderson–Hasselbalch equation with constants from Boutilier et al., (1984). A 100-ml sample of plasma was dispersed in 10 ml of fluor (Amersham ACS II) for the determination of plasma (14C) activity. Plasma was then diluted 1000 × with deionised water and analyzed for Na + and Cl − as described below. A 5-ml sub-sample of water was mixed with 10 ml of fluor (Amersham ACS II) and the radioactivity (14C) of the water determined. Urine collected over the duration of the flux was diluted 100× with deionised water for determination of Na + and Cl − as described below. A 1-ml sample of urine was mixed with 10 ml of fluor (Amersham ACS II) for determination of urine (14C) activity. Activity of the water, plasma and urine samples were determined using a Canberra Packard TR1000 liquid scintillation counter.

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2.6. Whole body unidirectional ion fluxes 2.6.1. Experimental protocol Fish of mean weight (9 S.E.) 27.091.6 g were placed in individual 600 ml black acrylic boxes supplied with aerated flowing dechlorinated water 24 h prior to experimentation. To begin the flux, the water supply was stopped and the chamber was sealed. Some 22NaCl (Amersham) or H36Cl (Dupont) was added to each chamber to yield a final activity 0.2 mCi ml − 1. Mixing and aeration of the chamber were ensured using an air stone. Following a 30-min mixing period a 25-ml water sample was removed. After 3 h a second water sample was removed and the water flow was re-instated. This was called the pre-exposure (control) flux. After flushing the boxes for 1 h with flowing fresh water a second flux (called the exposure flux) was carried out. Chloramine-T (2, 9 or 18 mg l − 1 active ingredient, n =9), sodium hypochlorite (OCl − ) (0.2 mg l − 1 active ingredient, n=9) or para-toluenesulphonamide (pTSA) (9 mg l − 1 active ingredient in 0.017% dimethylsulphoxide (DMSO), n = 9) were added, where appropriate, to each box at the same time as the isotope. In a separate experiment, it was determined that 9 mg l − 1 chloramine-T resulted in 0.2 mg l − 1 free chlorine (according to the DPD method, Franson, 1978). This value is equivalent to other studies with chloramine-T (Bullock et al., 1991). A concentration of 9 mg l − 1 was chosen for pTSA since this concentration was the maximum which could be derived from full degradation of 9 mg l − 1 chloramine-T. All chemicals were first dissolved in 1 ml distilled water before adding to the flux chamber. The amount of isotope added varied between treatments in order to ensure a comparable specific activity of the water. A group of fish were also exposed to either 1 ml distilled water (0 mg l − 1, n =9) which was used to dissolve chloramine-T and dilute the sodium hypochlorite, or 0.017% DMSO (n =9) in distilled water.

(Amersham ACSII) and the radioactivity determined using an LKB Wallac 1215 Rackbeta liquid scintillation counter. A 5-ml sub-sample was then diluted 1:1 with deionised water and the Na + content was determined using a Varian Spectra AA plus atomic absorption spectrophotometer. Alternatively, a 1-ml sub-sample was analyzed for Cl − using a spectrophotometric assay (Zall et al., 1956). Total ammonium concentrations were determined using the salicylate-hypochlorite spectrophotometric method (Verdouw et al., 1978). Titratable alkalinity was determined according to the method of McDonald and Wood (1981). Water samples were gassed at room temperature with air for 15 min then titrated to pH 4.0 using 0.1M HCl. Titratable alkalinity measurements were made within 24 h of the experiment, water samples being stored refrigerated in sealed vials.

2.7. Calculations and statistical analysis Net and unidirectional ion fluxes were calculated according to the formulae: Jnet =

Where [X]0 is the concentration of ion X at the start of the flux (mmol l − 1), [X]T is the concentration of the ion at the end of the flux period (mmol l − 1), V is the volume of the flux chamber (l), T is the duration of the flux period (h) and W is the weight of the fish (g). Jin =

[(cpm0/s)− (cpmT /s)]V SA× WT

where cpm0 is the counts per minute at the start of the flux period, cpmT is the counts per minute at the end of the flux period, s is the sample volume (l), W is the weight of the fish (g), T is the duration of the flux period (h), V is the flux chamber volume (l) and SA is the average specific activity of the sample. SA=

2.6.2. Water analysis A 5-ml sub-sample of water was mixed with 10 ml aqueous liquid scintillation cocktail (fluor)

[X]0 − [X]TV TW

(cpm0 + cpmT )0.5 ([X]0 + [X]T )0.5

Unidirectional effluxes were thus calculated according to:

M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

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Table 1 The effect on respiratory variables at the beginning and end of a flux period, for trout exposed to 18 mg l−1 chloramine-T Variables

Pre-exposure

PaCO2 (kPa) pHa CaCO2 (mM) [HCO3−] (mM) PaO2 (kPa) CaO2 (mM) Hct (%) [Hb] (mM) O2:Hb (mlO2.g−1 Hb) MCHC

Exposure

Start

End

Start

End

0.2190.02 7.9590.07 6.3590.4 6.2590.39 11.9990.95 3.379 0.39 27.7592.09 1.359 0.10 1.1490.12 0.31 90.01

0.22 90.03 7.9290.05 6.16 90.52 6.05 90.51 11.28 9 0.59 2.829 0.25* 26.759 2.19 1.4490.12 0.9490.05 0.35 9 0.01

0.17 90.02 8.01 9 0.04 5.67 90.49 5.59 9 0.49 13.16 90.85 2.98 9 0.21 28.00 9 2.22 1.42 9 0.11 0.99 9 0.05 0.33 90.01

0.24 9B0.01 7.989 0.05 7.29 9 0.63* 7.17 9 0.62* 11.53 9 0.69* 2.35 90.29 26.25 92.27 1.36 90.12 0.82 90.05 0.33 9 0.01

Values are given as the mean9S.E. arterial carbon dioxide tension (PaCO2), arterial pH (pHa), total plasma carbon dioxide content (CaCO2) and plasma bicarbonate concentration ([HCO3−]), arterial oxygen tension (PaO2), arterial oxygen content (CaO2), haematocrit (Hct), haemoglobin concentration ([Hb]), O2 specifically bound to haemoglobin (O2:Hb) and mean cellular haemoglobin concentration (MCHC, [Hb] in g 100 ml−1/Hct). * Significant from start of flux.

Jout =Jnet − Jin Glomerular filtration rate (GFR) was calculated according to: GFR=

(dpmPEG)urine ×UFR (dpmPEG)plasma

where (dpmPEG)urine is disintegrations per minute recorded in the urine, (dpmPEG)plasma is disintegrations per minute in the plasma, UFR is the urine flow rate (ml g − 1 h − 1). The renal clearance ratio (RCR) for Na + and Cl − were calculated according to: RCRX =

UFR× [X]urine GFR ×[X]plasma

where [X]urine is the concentration of ion X in the urine (mmol l − 1) and [X]plasma is the concentration of ion X in the plasma (mmol l − 1). The concentration of metabolic protons added + or removed from the blood (dHm ) was estimated according to the formula of McDonald et al. (1980) with the non-bicarbonate buffering capacity b, estimated from haematocrit measurements (Wood et al., 1982): + =([HCO3− ]start −[HCO3− ]end) dHm

− b(pHstart − pHend)

where: [HCO3− ] is the plasma bicarbonate concentration at the start or end of the flux period and pH is the blood pH at the start or end of the flux period. Pre-exposure and exposure fluxes and measurements were compared using a paired t-test and each flux was tested for significance from zero using a one-tailed t-test. The most appropriate comparisons being made between pre-exposure and exposure fluxes for the same treatment. The fiducial limit was set at 0.05.

3. Results

3.1. Acid–base and respiratory data Arterial PCO2 levels did not increase significantly upon exposure to 18 mg l − 1 chloramine-T (P=0.085); neither was there a significant decrease in arterial pH. However, significant increases in both total CO2 content of the plasma and calculated bicarbonate concentrations were observed (Table 1). There was a significant decrease in PaO2 during the exposure to 18 mg l − 1 chloramine-T. In addition, there was a small but significant decrease in CaO2 during the pre-expo-

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M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

Fig. 1. The effects of exposure to either 0, 2, 9 or 18 mg l − 1 chloramine-T or its degradation products sodium hypochlorite (OCl − ), para-toluenesulphonamide (TSA) or dimethylsulphoxide (DMSO) on whole body flux rates of titratable alkalinity (J TA), total + ammonia (J Amm) and net acidic equivalents (J H net ). Positive values represent acidic equivalent uptake while negative values represent + TA and J Amm, signs considered. acidic equivalent excretion. Solid bars represent the net acidic equivalent flux (J H net ) as the sum of J Values are given as means 9S.E. (n= 9). * Significant difference from pre-exposure values.

sure flux period. No other significant effects, of either the flux period or the exposure to chloramine-T were demonstrated on any of the respiratory variables examined (Table 1). From the data presented it was possible to estimate a + metabolic acid load (dHm ) of approximately 0.51 mM whereas during exposure to chloramine-T, + there was a base excess (−dHm ) of approximately 1.27 mM.

3.2. Renal ion flux studies Exposure to 18 mg l − 1 chloramine-T had no significant effect on plasma or urine Na + and Cl − concentrations. Similarly, there was no significant effect of exposure on either urine flow rate or glomerular filtration rate. Renal efflux rates for Na + and Cl − were also not statistically significant between pre-exposure and during exposure to 18 mg l − 1 chloramine-T (Table 2). The renal clearance ratios for Na + and Cl − were unaffected by exposure to chloramine-T (Table 2). In addition, there was no significant increase in the branchial PEG efflux during exposure to 18 mg l − 1 chloramine-T.

3.3. Whole body acid–base and ion fluxes Significant decreases in net acid uptake (i.e. net H + flux less positive) occurred at all exposure concentrations of chloramine-T used except for the 0 mg l − 1, compared with the pre-exposure flux (Fig. 1). Similarly, there was a decrease in the net titratable alkalinity flux for fish exposed to 2 and 9 mg l − 1 chloramine-T, but this was not apparent at 18 mg l − 1 chloramine-T (Fig. 1). Fish exposed to NaOCl also showed a decrease in net acid uptake due, in part, to an increase in net ammonia excretion (Fig. 1). Fish exposed to pTSA showed an increase in net titratable alkalinity flux as well as a significant increase in net ammonium excretion (Fig. 1). The net effect was an increase in net acid uptake (a more positive net acid flux). However, there was a decrease in the net acid excretion in fish exposed to DMSO alone as well as significant decreases in net titratable alkalinity flux and net ammonium flux (Fig. 1). Net acid fluxes during the pre-exposure flux period were positive (fish were in a state of acid uptake) for all treatments at the start of the experiment, except for fish which were treated with DMSO.

[Na+]u (mM)

[Cl−]u (mM) UFR (ml g−1 h−1)

GFR (ml g−1 h−1)

J na out (mmol Kg−1 h−1)

J cl out

RCRNa

RCRCl

J PEG gill (mmol g–1 h−1)

134.199(4.81) 6.959(1.19) 18.019(2.52) 2.269(0.28) 4.639(1.00) 17.949(4.13) 39.389(6.58) 0.0229(0.025) 0.0889(0.030) −0.0219(0.011) 137.709(6.31) 8.799(1.78) 17.169(3.25) 2.279(0.04) 4.559(0.82) 23.369(6.83) 44.849(15.20) 0.0319(0.004) 0.0629(0.016) −0.0139(0.009)

[Cl−]pl (mM)

Values are given as the mean9S.D.

Pre-exposure 154.239(5.93) Exposure 156.909(7.63)

[Na+]pl (mM)

Table 2 Plasma ([X]pl) and urine ([X]u) concentrations, urine flow (UFR) and glomerular filtration (GFR) rates, and renal ion effluxes (J X out), renal clearance ratios RCRX and −1 branchial PEG efflux (J PEG chloramine-T gill ) of trout exposed to 18 mg l

M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24 19

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M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

Fig. 2. The effects of exposure to either 0, 2, 9 or 18 mg l − 1 chloramine-T or its degradation products sodium hypochlorite (OCl − ), para-toluenesulphonamide (TSA) or dimethylsulphoxide (DMSO) on unidirectional (open bars) and net (solid bars) whole body Na + flux. Positive values represent ionic uptake while negative values represent ionic losses. Values are given as means9 S.E. (n = 9). * Significant difference from pre-exposure values.

A significant increase in the net loss of Na + was measured in fish exposed to 18 mg l − 1 chloramine-T compared with pre-exposure values; however, there were no significant changes in the net Na + flux at lower concentrations (Fig. 2). Exposure to 9 mg l − 1 pTSA resulted in a significant increase in the Na + efflux which in turn resulted in a significant but negative net flux compared with the pre-exposure values (Fig. 2). There was also a significant increase in the negative net Na + flux in fish exposed to NaOCl (Fig. 2). Fish exposed to DMSO only showed a small but significant negative Na + net flux as compared with the small positive net flux during the pre-exposure period. No significant change in the Na + influx (as compared with the pre-exposure flux period) for any of the chemical treatments was observed (Fig. 2). Similarly, there were no significant changes in the net or unidirectional Na + fluxes of control (unexposed) fish during the exposure period (Fig. 2). Exposure to chloramine-T caused significant increases in the net loss of Cl − during exposure to 2 and 18 mg l − 1 concentrations (Fig. 3). However, a significant increase in the unidirectional efflux of Cl − was only seen in fish exposed to 18 mg l − 1 chloramine-T (Fig. 3). NaOCl exposure

also resulted in a net Cl − loss, but no change in the unidirectional efflux of Cl − (Fig. 3). There was a significant increase in the negative net Cl − flux upon exposure to pTSA (Fig. 3), but no significant changes in net Cl − flux after exposure to DMSO were measured (Fig. 3). In all treatments (chloramine-T, pTSA, NaOCl or DMSO), there was no effect of exposure on Cl − influx (Fig. 3). There were no significant effects of exposure on Cl − flux in the 0 mg l − 1 group.

4. Discussion Initially, it was essential to establish that a static exposure to chloramine-T resulted in arterial blood acid–base disturbances similar to those previously described by Powell and Perry (1996) when using a flow-through exposure system. The significant rise in plasma total CO2 content and calculated bicarbonate concentration were similar to that described previously (Powell and Perry, 1996). However, in the previous study, a significant increase in PaCO2 was also reported. Rebreathing of CO2, and subsequent hypercapnia, was evidently not a problem during the pre-exposure and exposure period, since there was no

M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

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Fig. 3. The effects of exposure to either 0, 2, 9 or 18 mg l − 1 chloramine-T or its degradation products sodium hypochlorite (OCl − ), para-toluenesulphonamide (TSA) or dimethylsulphoxide (DMSO) (B) on unidirectional (open bars) and net (solid bars) whole body Cl − flux. Positive values represent ionic uptake while negative values represent ionic losses. Values are given as means 9 S.E. (n =9). * Significant difference from pre-exposure values.

indication of a hypercapnic acidosis occurring in the blood. The rise in plasma bicarbonate was indicative of a metabolic alkalosis as demonstrated by the negative metabolic acid load (− + dHm ) when compared with the pre-exposure condition. The metabolic alkalosis that occurs upon exposure to chloramine-T can be explained in terms of the branchial exchange of acidic and basic equivalents. Clearly, our data shows that there was a marked reduction in the net acid uptake upon exposure to chloramine-T which was not evident in the 0 mg l − 1 group, but, an apparent increase in the net acid uptake upon exposure to pTSA and DMSO. Since pTSA had to be dissolved in DMSO prior to experimentation (due to the low solubility of pTSA) we cannot determine the effect of pTSA alone. In a previous study, we have demonstrated that a single chloramine-T exposure of 1 h duration causes a mixed respiratory/metabolic acid – base disturbance consisting of a mild respiratory acidosis superimposed over a metabolic alkalosis (Powell and Perry, 1996). The data presented in the present study from catheterised fish, in which significant increases in the measured plasma total CO2 and calculated plasma bicarbonate concentration without an accompanying decrease in pH, confirm

our suggestion of a metabolic alkalosis (as indicated by the estimation of base excess) being the predominant acid–base disturbance probably as a result of decreased branchial acid uptake. The significant increase in net acid uptake in pTSA exposed fish is suggestive of the development of a metabolic acidosis, which is also consistent with our previous study (Powell and Perry, 1996) but we cannot rule out a possible confounding effect of DMSO. Although we acknowledge that there was a large (and unexplainable) net uptake of acid in controls, the change in the net acid fluxes is consitent with the blood acid–base data (this study) and our previous findings (Powell and Perry, 1996). Differences in net ionic flux were not caused by the impairment of ionic uptake mechanisms since ionic influx rates were unaffected by chloramineT, NaOCl, pTSA or DMSO. Thus, the altered net ionic fluxes that were observed were probably caused by alterations in ionic permeability of the gill epithelium. Nevertheless, there were small but significant increases in the net losses of Na + and/or Cl − in fish exposed to chloramine-T, NaOCl or pTSA. Once again it is not possible to completely discount possible effects of DMSO which has been shown to cause ionic losses (at

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albeit lower concentrations) across the rat proximal tubule (Wilson et al., 1997). The recovery from exposure was not examined in this study because the correction of chloramine-T induced acid – base disturbances is rapid following the withdrawal of chloramine-T (Powell and Perry, 1996) and therefore probably of little pathological significance. In this study, we used a 3.5-h exposure period to assess the effects of chloramine-T on the acid – base/ ionoregulatory physiology of the gill, whereas shorter durations of exposure are frequently used commercially to treat gill diseases (From, 1980; Speare and Ferguson 1989; Bullock et al., 1991; Thorburn and Moccia, 1993). Even so, the ionic losses observed here upon exposure to chloramine-T were small and probably little pathological consequence, because a reduction in plasma ion concentrations was not seen in the blood from catheterised fish exposed to 18 mg l − 1: a concentration at which the greatest ionic losses were shown to occur, thus indicating a marked margin of safety for chloramine-T use. However, repeated exposure to chloramine-T may lead to progressive ionic losses which may result in the reduced plasma ion concentrations described previously (Powell et al., 1994). Exposure to hypochlorite, which is believed to be the primary disinfective component of chloramine-T resulted in increased net losses of both Na + and Cl − . As with chloramine-T, there was a significant decrease in the net acid uptake which supports the idea of a metabolic alkalosis. We have shown that hypochlorite exposure causes an acute respiratory acidosis (Powell and Perry, 1996). However, in the present study, NaOCl exposures were for a longer period and at a lower concentration of NaOCl (0.2 mg l − 1 active ingredient for 3 h compared with 0.45 mg l − 1 for 1 h, Powell and Perry (1996)). NaOCl has been demonstrated to cause acute ionic disturbances in marine fish (Block et al., 1978; Hose et al., 1983) probably because of branchial epithelial damage. Our results presented here are suggestive of increased branchial ionic losses. Chlorine (in the form of dissolved chlorine gas) has been shown to cause membrane lysis and the leakage of macromolecules from bacterial cells as

its primary mechanism of disinfection (Venkobacher et al., 1977). It is possible, therefore, that another chlorine derived oxidant, hypochlorite (from chloramine-T degradation) exerts a similar permeablising effect on the branchial epithelia of fish which may lead to acute branchial ion losses. Bladder catheterised fish were used to verify that the measured whole body fluxes primarily reflected branchial fluxes and to quantify the contribution from renal sources. The catheter method provided reliable estimates of urine flow rates and it was also possible to determine the ionic composition of the urine formed (Curtis and Wood, 1991). To determine glomerular filtration rate, it was necessary to use an extracellular fluid marker. [14C]PEG was the marker of choice since it has been used in other studies with success (Curtis and Wood, 1991, 1992). There was no effect of exposure to 18 mg l − 1 chloramine-T on the urine flow rate, glomerular filtration rate or on renal Na + and Cl − effluxes. Therefore, we eliminated the likelihood that differences in whole body ion fluxes were due to increased renal efflux. The use of [14C]PEG as an extracellular marker also allowed the estimation of branchial paracellular permeability (Wood and Pa¨rt, 1997). If whole body ion fluxes were not caused by increased renal efflux, then chloramine-T must be acting on the gill or the skin. There are two possible routes by which ions could be lost across epithelia, transcellularly (increased epithelial cell apical plasma membrane permeability) and paracellularly (direct loss from the extracellular space across the epithelial tight junction). Assuming that [14C]PEG would not be transported into epithelial cells, measurement of radioactivity in the water would be indicative of the paracellular permeability. There was no significant difference in the rates of [14C]PEG efflux across the gill with exposure to 18 mg l − 1 chloramine-T thus suggesting that altered ionic effluxes were probably not due to an increase in epithelial tight junctional permeability. Ionic effluxes (at least with 18 mg l − 1 chloramine-T) may therefore, have been due to transcellular processes.

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5. Conclusion In conclusion, we have demonstrated that a static exposure to chloramine-T results in acid – base disturbances similar to those seen with flowthrough treatments. Also, the metabolic alkalosis associated with chloramine-T exposure resulted in an increase in whole body acid excretion, whereas exposure to pTSA and DMSO resulted in increase in net acid uptake. This result was consistent with measured acid–base variables within the blood suggesting the development of a metabolic alkalosis with chloramine-T and compares favourably with previous studies indicating a metabolic acidosis occurs upon exposure to pTSA. This study has also demonstrated branchial ion losses with exposure to chloramine-T or its breakdown products, NaOCl or pTSA. These ion losses could not be correlated with increased branchial paracellular permeability to [14C]PEG. Although relatively minor in terms of pathological effects, this work suggests that caution should be used when using chloramine-T disinfection either prophylactically or therapeutically and suggests possible routes for branchial ion losses which may result in ionic disturbances in sub-lethal chlorine and chloramine-T exposed fish (Block et al., 1978; Hose et al., 1983; Powell et al., 1994).

Acknowledgements This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through an operating grant to SFP. MDP was the recipient of an NSERC postgraduate scholarship.

References Bills, T.D., Marking, L.L., Dawson, V.K., Howe, G.E., 1988a. Effects of organic matter and loading rates of fish on the toxicity of chloramine-T. US Dept. Int. Fish Wildlf. Serv. Invest. Fish Cont. 96, 1–4. Bills, T.D., Marking, L.L., Dawson, V.K., Rach, J.J., 1988b. Effects of environmental factors on the toxicity of chloramine-T to fish. US Dept. Int. Fish Wildlf. Serv. Invest. Fish Cont. 96, 1 – 6.

23

Block, R.M., Burton, D.T., Gullans, S.R., Richardson, L.B., 1978. Respiratory and osmoregulatory responses of white perch (Morone americana) exposed to chlorine and ozone in estuarine water. In: Jolley, R.L., Gorchev, H., Hamilton, D.H. (Eds.), Water Chlorination: Environmental and Health Effects, vol. 2. Ann Arbour Science Publishers, Ann Arbor, pp. 351 – 360. Booth, N.H., MacDonald, L.E., 1988. Veterinary Pharmacology and Therapeutics, 6th ed., Iowa State University Press, Ames, pp. 774 – 777. Boutilier, R.G., Heming, T.A., Iwama, G.K., 1984. Appendix: physicochemical parameters for use in fish respiratory physiology. In: Hoar, W.S., Randall, D.J. Jr (Eds.), Fish Physiology, vol. 10A. Academic Press, London, pp. 403 – 430. Brooks, A.S., Bartos, J.M., 1984. Effects of free and combined chlorine and duration on rainbow trout, channel catfish and emerald shiners. Trans. Am. Fish. Soc. 113, 786 – 793. Bullock, G.L., Herman, G.L., Waggy, C., 1991. Hatchery efficacy trials with chloramine-T for control of bacterial gill disease. J. Aquat. Anim. Health 3, 48 – 50. Cross, D.G., Hursey, P.A., 1973. Chloramine-T for the control of Ichthyophthirius multifilis (Fouquet). J. Fish Biol. 5, 789 – 798. Curtis, B.J., Wood, C.M., 1991. The function of the urinary bladder in vivo in the freshwater rainbow trout. J. Exp. Biol. 155, 567 – 583. Curtis, B.J., Wood, C.M., 1992. Kidney and urinary bladder responses of freshwater rainbow trout to isosmotic NaCl and NaHCO3 infusion. J. Exp. Biol. 173, 181 – 203. Daoust, P.Y., Ferguson, H.W., 1983. Gill diseases of cultured salmonids in Ontario. Can. J. Comp. Med. 47, 358 – 362. Franson, M.A., 1978. Standard methods for the examination of water and wastewater, 14th ed., American Public Health Association, Washington, DC, pp. 309 – 345. From, J., 1980. Chloramine-T for control of bacterial gill disease. Progr. Fish Cult. 42, 85 – 86. Goss, G.G., Perry, S.F., 1993. Physiological and morphological regulation of acid – base status during hypercapnia in rainbow trout (Oncorhynchus mykiss). Can. J. Zool. 71, 1673 – 1680. Heath, A.G., 1977. Toxicity of intermittent chlorination to freshwater fish: influence of temperature and chlorine form. Hydrobiology 56, 39 – 47. Hose, J.E., King, T.D., Zerba, K.E., Stoffel, R.J., Stephens, J.S. Jr, Dickinson, J.A., 1983. Does avoidance of chlorinated seawater protect fish against toxicity. Laboratory and field observations. In: Jolley, R.L., Brungs, W.A, Cotruvo, J.A., Cummings, B.B., Mattice, J.S., Jacobs, V.A. (Eds.), Water Chlorination: Environmental Impact and Health Effects, vol. 4. Ann Arbor Science Publishers, Ann Arbor, pp. 967 – 982. McDonald, D.G., Hobe, H., Wood, C.M., 1980. The influence of calcium on the physiological responses of the rainbow trout, Salmo gairdneri, to low environmental pH. J. Exp. Biol. 88, 109 – 113.

24

M.D. Powell, S.F. Perry / Aquatic Toxicology 43 (1998) 13–24

McDonald, D.G., Wood, C.M., 1981. Branchial and renal acid and ion fluxes in the rainbow trout, Salmo gairdneri. J. Exp. Biol. 98, 273 –287. Powell, M.D., Speare, D.J., MacNair, N., 1994. Effects of intermittent chloramine T exposure on growth, serum biochemistry and fin condition of juvenile rainbow trout (Oncorhynchus mykiss). Can. J. Fish. Aquat. Sci. 51, 1728– 1736. Powell, M.D., Wright, G.M., Speare, D.J., 1995. Morphological changes in rainbow trout (Oncorhynchus mykiss) gill epithelia following repeated intermittent exposure to chloramine-T. Can. J. Zool. 73, 154–165. Powell, M.D., Perry, S.F., 1996. Respiratory and acid–base disturbances in rainbow trout (Oncorhynchus mykiss) blood during exposure to chloramine-T, paratoluenesulphonamide and hypochlorite. Can. J. Fish. Aquat. Sci. 53, 701 – 708. Seegert, G.L., Brooks, A.S., 1978. The effects of intermittent chlorination on coho salmon, alewife, spottail shiner and rainbow smelt. Trans. Am. Fish. Soc. 107, 346–353. Seegert, G.L., Brooks, A.S., Vande Castle, J.R., Grandall, K., 1979. The effects of monochloramine on selected riverine fishes. Trans. Am. Fish. Soc. 108, 88–96. Speare, D.J., Ferguson, H.W., 1989. Clinical and pathological features of common gill diseases of cultured salmonids. Can. Vet. J. 30, 882 –887. Soivio, A., Nyholm, K., Westman, K., 1975. A technique for repeated sampling of blood of individual resting fish. J. Exp. Biol. 62, 207 – 217.

.

Thorburn, M.A., Moccia, R.D., 1993. Use of chemotherapeutics on trout farms in Ontario. J. Aquat. Anim. Health 5, 85 – 91. Venkobacher, C., Iyengar, L., Prabhakara Rao, A.V.S., 1977. Mechanism of disinfection: effect of chlorine on cell membrane functions. Water Res. 11, 727 – 729. Verdouw, H., van Etcheld, C.J.A., Dekkers, E.M., 1978. Ammonium determinations based on indophenol formation with sodium salicylate. Water Res. 12, 399 – 402. Wilson, R.W., Waring, M., Green, R., 1997. The role of active transport in potassium reabsorption in the proximal convoluted tubule of the anaesthetized rat. J. Physiol. 500, 155 – 164. Wolf, K., 1963. Physiological salines for freshwater fishes. Progr. Fish Cult. 25, 135 – 140. Wood, C.M., McDonald, D.G., McMahon, B.R., 1982. The influence of experimental anaemia on blood acid base regulation in vivo and in vitro in the starry flounder (Platichthys flesus) and the rainbow trout (Salmo gairdneri ). J. Exp. Biol. 96, 221 – 237. Wood, C.M., Pa¨rt, P., 1997. Cultured branchial epithelia from freshwater fish gills. J. Exp. Biol. 200, 1047 – 1059. Zall, D.M., Fisher, M.D., Garner, Q.M., 1956. Photometric determination of chloride in water. Anal. Chem. 28, 1665 – 1678. Zeitoun, I.H., 1978. Assessment of intermittently chlorinated heated effluents on survival of adult rainbow trout (Salmo gairdneri ) at power generating facilities. Environ. Sci. Tech. 12, 1173 – 1179.