Respiration and Osmoregulation in Rainbow Trout with Gills Damaged by Zinc Sulphate

Damage to the gill epithelium occurs when hatched fish are killed within a few hours by solutions of a variety of substances, including detergents (Schmid & Mann, 1962), phenols (Christie & Battle, 1963) and salts of heavy metals (Schweiger, 1957; Haider, 1964). When fish are exposed to solutions of the above poisons until the gill opercula have ceased to move, the epithelium covering the secondary lamellae is sloughed off from the underlying pillar-cell system and forms a detached but usually continuous layer of tissue lying above and between the desquamated lamellae. The blood spaces outlined by the pillar cell flanges may be contracted. Mucus is secreted and precipitated over the body surfaces generally, but is not seen to affect the gills. There is no haemorrhage from the gills. Gill changes before the cessation of oper-cular movements have not been studied.

It has not been shown that the destruction of the gill epithelium causes death through the breakdown of any vital gill function. Gas exchange, extra-renal excretion and ionic exchange could be obstructed or facilitated as a result of these changes, since the lifting of the epithelial tissue would generally increase the diffusion distance from water to blood, whereas any breaks in the epithelium would locally reduce the diffusion distance. Death might result primarily from asphyxia, or from osmotic stress. The present paper describes the rate of oxygen uptake and the blood osmotic concentration of rainbow trout exposed to toxic concentrations of zinc sulphate, in an attempt to identify the physiological cause of death.

Rainbow trout (Salmo gairdneri Richardson) from the Surrey Trout Farm, Nails-worth, Wilts., were held at 15±1°C. in a mixture of Bristol tap water (Table 1) and deionized water. The total hardness was adjusted by dilution to 50 ± 5 p.p.m. as CaCO₃. The pH was 7·1 to 7·5. The dissolved oxygen tension exceeded 130 mm. Hg. (O₂ > 8 p.p.m.). These conditions were maintained in all experiments unless otherwise stated. Fish were held for at least 7 days and were fed with fresh liver twice weekly. They were starved for 3 days prior to use in experiments.

Eleven trout, weight 210 ± 60 g. (mean ± standard deviation), total length 29 ± 2 cm., were anaesthetized singly in 100 p.p.m. tricaine methanesulphonate (MS 222) and placed on a portable operating table (Smith & Bell, 1964), modified and built by G. F. Holeton. The gills were irrigated with water containing 70 p.p.m. MS 222. The buccal cavity was cannulated through the snout, posterior to the buccal valve, usine Portex PP 160 polythene tubing, internal diameter 1·14 mm., with a heat-flared end (Saunders, 1961). One opercular cavity was cannulated through the centre of the operculum, at the junction of the opercular and preopercular bones, using PP 90 tubing, i.d. 0·86 mm., with a heat-flared end (Saunders, 1961). Pairs of electrodes made from Johnson and Matthey diamel-coated stainless-steel wire, SWG 45, were implanted under the skin below and anterior to the heart in ten of the eleven fish. The dorsal aorta of four of the fish was also cannulated using PP 60 tubing tipped with a number 21, short-bevel hypodermic needle (Smith & Bell, 1964). Approximately 100 international units of heparin in 0·5 ml. Cortland teleost salt solution (Wolf, 1963) were injected into the aorta, and the cannula was plugged. Each fish was then revived in water free of anaesthetic.

While still partially sedated, the fish was transferred, head downstream, to a Perspex chamber, 33 cm. by 6 cm. by 7 cm. high (Fig. 1). Water entered the chamber through a nozzle which directed the current evenly in all directions. The two (or three) cannulas left the chamber through a small side tube sealed with a rubber cap. The end of the chamber was plugged with a rubber bung. The electrode wires left the chamber between the bung and its Perspex seating, ending in terminals made of flexible springs attached to the outside of the fish chamber. The water inside the chamber was earthed by a stainless-steel wire grid.

Water was pumped by an Eheim No. 383 pump through a flow meter to the fish chamber at a maximum rate of 800 ml./min. From the chamber it passed through a three-way tap to a fibre-glass filter. Water drained through the filter by gravity to a 6o 1. reservoir. The reservoir was connected to the pump by a second three-way tap. A second 60 1. reservoir containing zinc sulphate solution (Zn 40 p.p.m.) was also connected to the pump through the second tap. The circulation was completed by the first tap, which could direct water or zinc solution through a second filter to the second reservoir. Both reservoirs were vigorously aerated ( > 150 mm. Hg, O₂ > 9·5 p.p.m.). The water upstream and downstream of the fish chamber, and from the buccal and opercular cavities, could be sampled through cannulas a, b, c and d, respectively. All four cannulas were clamped to regulate water flow at 2 ml./min. The blood could be sampled through cannula e, being returned to the fish after test.

The cannulas were led in turn to a glass cuvette (dead space 0·1 ml.) surrounded by a water jacket maintained at the same temperature as the fish. The cuvette contained a Beckman oxygen macro-electrode connected to a Beckman physiological gas analyser, model 160. The oxygen tension was measured in the water entering and leaving the fish chamber, in the water entering the buccal cavity of the fish and leaving its opercular cavity and in the dorsal aortic blood. The readings were recorded on a Devices two-channel pen recorder after amplification by a Devices DC2C pre-amplifier.

The heart electrodes were connected to the second channel of the pen recorder after amplification by a Devicés AG7C pre-amplifier, to record heart rate. In six cases the opercular and coughing rates could also be distinguished on the same trace. In the other fish, opercular and coughing rates were timed direct, using a stop watch.

Water flow through the fish chamber was controlled to allow a reduction in of 30 mm. Hg (2 p.p.m. O₂). Thus the fish inspired water at 120 mm. Hg. Each fish was maintained under these conditions overnight. The following day the water flow was first reduced so that the inspired was 40 mm. Hg for 1 hr., then increased to its former rate for 1 hr. Observations were made after 30 and 60 min. at each flow rate. Zinc_ sulphate solution was then circulated to the fish, the of the inspired water being 120 mm. Hg. Observations were continued at 15 to 30 min. intervals until the gill opérenla ceased moving and the heart stopped beating, survival time being defined as time to immobilization of the gill opérenla. Because most fish struggled when the toxic solution was introduced, fish were exposed in later experiments to water containing 40 p.p.m. Zn plus 20 p.p.m. MS 222. This concentration of anaesthetic in zinc-free water was sufficient to quieten the fish without depressing their rate of oxygen consumption, or affecting balance or changing their reaction to visual stimuli.

The following calculations were made on the observations with each fish, the symbols being as far as possible those used in mammalian respiratory physiology.

Terms: ⁠, Routine^* rate of oxygen uptake (mg./kg. fish per hr.); ⁠, ventilation volume of water through gills (l./kg. fish per min.) ; U, utilization of oxygen, or dissolved oxygen removed by fish (%); P, partial pressure of oxygen (mm. Hg); F, flow of water through fish chamber (ml./min.) ; W, wet weight of fish (g.).

Subscripts: I, Water inspired through buccal cavity; E, water expired through opercular cavity; in, water entering fish chamber; out, water leaving fish chamber.

Eleven trout weighing 145±16 g. were anaesthetized as before. Approximately 0·75 ml. blood (15% of the estimated blood volume) was taken from the heart by cardiac puncture. The fish were returned to their holding tanks for 7 days to recuperate. Seven of the fish were then transferred to 2001. of zinc sulphate solution (40 p.p.m. Zn, > 130 mm. Hg) and four fish to a 100l, tank of zinc-free water. A second blood sample was obtained from the unanaesthetized zinc-treated fish at gill opérenla immobilization and from the control fish (under anaesthetic) after a similar time interval. The osmotic concentration of the blood was measured in a Mechano-Lab vapour-pressure osmometer.

Three trout weighing 250 + 50 g. were exposed to 200 1. of zinc sulphate solution (Zn 40 p.p.m., >130 mm. Hg) until gill immobilization, and 1 ml. blood samples were collected by cardiac puncture. Blood samples were similarly collected from five anaesthetized control trout of similar size. Some of the blood was centrifuged, and all samples were diluted 1:50 in N/200 hydrochloric acid. Concentrations of sodium, potassium, calcium, magnesium and zinc were measured in a Unicam atomic-absorption spectrophotometer.

Salt solutions containing 0·13−6·2 mg. Zn plus 100 i.u. heparin per ml. were prepared by mixing Cortland teleost salt solution with an approximately isotonic solution of zinc sulphate (5 % ZnSO_4.7, H₂O) followed by filtering, adding heparin and assaying for zinc by spectrophotometry. Eight trout (250 ± 50 g.) were anaesthetized individually and mounted on the operating table with the gills irrigated with water containing 70 p.p.m. MS 222. The dorsal aorta was cannulated. One millilitre of blood was withdrawn and 1 ml. of solution injected. After initial dilution in the blood, these treatments were estimated to have produced concentrations of zinc ranging from 13 to 620 p.p.m. Zn plus 10 i.u./ml. heparin. Approximately 0·75 ml. blood was collected after 3−60 min. Survivors were returned to their holding tanks. Blood collected before treatments was analysed by spectrophotometry for sodium, potassium, calcium, magnesium and zinc; blood collected after treatments was assayed for zinc only.

The mean routine rate of oxygen uptake by eleven resting trout was about roo mg./kg. fish per hr. in zinc-free water at a of 120 mm. Hg and slightly more at a of 40 mm. Hg (Fig. 2). At a of 120 mm. Hg in zinc sulphate solution (Zn 40 p.p.m.) the followed one of two patterns, depending on the fishes’ activity. In five unanaesthetized fish which struggled when first immersed in zinc, the trebled briefly before declining to the resting level after 40% of the survival time had elapsed. Five lightly sedated fish and one unanaesthetized fish remained quiet when zinc was introduced. Their oxygen consumption remained steady at the resting level until 80% of the survival time. Collapse of oxygen uptake by both groups was then rapid.

Ventilation volume was generally low in high and high in low in zinc-free water (Fig. 3). In five of the six quiet fish the increased steadily on exposure to zinc. After 65−95 % of the survival time it reached six times the rate at high in zinc-free water. In four out of five struggling fish the VG fluctuated between four and ten times the resting level in high throughout the first 95 % of the survival time. In the remaining two fish the opercular cannulas were blocked. In all eleven fish the ventilation rate collapsed rapidly in the final 5 % of survival time.

Oxygen utilization (U) was inversely correlated with ⁠, being high in high and low in low ⁠, in zinc-free water (Fig. 4). In five quiet fish and four struggling fish it declined from 40 to 4% upon progressive exposure to zinc; but there was wide individual variation largely depending on the fishes’ activity.

The mean opercular rate of the eleven fish in zinc-free water was about 70 beats/ min. in high ⁠, and 100 beats/min. in low (Fig. 5). It rose steadily to 120 beats/ min. after 65−85 % of the survival time in zinc, and declined rapidly after 95 % survival time. The graphs for individual fish were similar in form, and the large standard deviations were due to consistent differences in the opercular rates of individual fish.

The mean coughing rate of the eleven fish was 1·1 coughs/100 opercular beats in high and almost nil in low (Fig. 6). It rose steadily on exposure of fish to zinc, reached a maximum of 20 coughs/100 beats after 85 % survival time, and then declined rapidly.

The mean heart rate of ten of the eleven fish was 60 beats/min. at high and 46 beats/min. at low (Fig. 7). The rate remained above do beats/min. on exposure of the fish to zinc until 50 % of the survival time had elapsed. It then declined to 29 beats/min. after 95% survival time, but accelerated to 38 beats/min. after the oper-cula became immobilized. Synchronization of opercular movements with heart beat occurred in five fish out of six examined under low in zinc-free water and in four fish out of six exposed for 65−85 % survival time in zinc solution.

The height of the QRS wave from five of the eleven fish was low in high and slightly greater in low (Fig. 8). The QRS wave rose steadily on exposure of the fish to zinc, reaching a maximum of three-fold after 75−95% survival time. Collapse was then rapid.

The oxygen tension in the dorsal aortic blood (Fig. 9) was high in high water and low in low ⁠. Blood declined steadily on exposure of fish to zinc sulphate solution.

The osmotic concentration of the blood of seven fish (from a second batch) decreased by 77 % from 323 to 298 milliosmoles upon their immobilization in a toxic solution of zinc sulphate (Table 2). The drop was highly significant (P = 0·01) but is within the normal biological range of the species (325 ±16 milliosmoles, n = 11). The weight of the seven fish rose by 1·9 % (144·6−147·4 g.) during the same treatment, but the rise was not statistically significant (P = 0·2).

The concentrations of sodium, potassium, calcium, magnesium and zinc in the blood of three fish (from a third batch) immobilized in zinc sulphate solution were1 generally similar to concentrations of these ions in thirteen control fish (Table 3). In particular, there was no rise in the zinc concentration, and there was a fall in the sodium concentration, the latter being consistent with the observed decrease in osmotic concentration (Table 2).

The measured concentrations of zinc in the blood of four trout (from a fourth batch) which survived injections of zinc sulphate solution were 27,35, 51 and 96 p.p.m. Zn. The concentrations in four trout which succumbed were 130, 140, 250 and 680 p.p.m. Zn, compared with control values of 24±15 p.p.m. Zn, ( n = 13).

Loss of osmotic control may. be ruled out as the cause of death in trout exposed to a rapidly lethal solution of zinc sulphate because the changes in blood osmotic concentration, recorded centrally, were too slight to exceed the normal range. Similarly, the small changes recorded centrally in the blood concentrations of sodium, potassium, calcium, magnesium and zinc were unlikely to cause general physiological imbalance, although local changes in gill tissue concentrations may have produced structural gill damage and loss of vital function.

When zinc sulphate solution was injected through the dorsal aorta, fish survived a four-fold increase in the normal zinc concentration in blood. Mortality in fish injected with higher concentrations of zinc was probably caused by mechanical obstruction of the blood vessels, since similar concentrations of zinc in vitro produced dense precipitation when added to trout serum and to zinc-free Cortland teleost salt solution.

In contrast, drastic changes were noted in all observed respiratory parameters when trout were exposed to a rapidly lethal solution of zinc sulphate rich in oxygen. In quiet fish the routine rate of oxygen uptake remained unchanged until 80 % of the survival time had elapsed, and then declined rapidly. Ventilation volume, opercular rate, coughing rate and QRS wave height rose steadily to maximum values at 80−90% survival time. They then collapsed. Oxygen utilization, heart rate and the of dorsal aortic blood fell steadily throughout the period of exposure. Extreme bradycardia sometimes led to synchrony of the breathing and cardiac rhythms. In struggling fish survival time was shorter, oxygen uptake and ventilation volume were higher and oxygen utilization was lower than in quiet fish, but values of the other respiratory parameters were similar in both groups.

Generally, fish exposed to zinc had to work increasingly hard to maintain an adequate rate of oxygen uptake. Failure to do so resulted fairly quickly in respiratory failure. Except for the large increases in coughing rate and in QRS wave height the observed changes were similar to those observed when the same fish had been previously exposed to hypoxia. The similarities suggest that gas exchange became increasingly difficult during exposure to zinc. The increased coughing rate during exposure to zinc (occupying up to 20% of the total opercular movements) probably reduced gill aeration even further since studies with ink showed that flow over the gills was usually reversed during coughs. The significance of the increase in QRS wave height is unexplained, though it suggests changes in the physiology of the heart.

Partial support for the view that gas exchange becomes difficult in zinc-poisoned fish is supplied by Jones (1938), who observed that the opercular rate of sticklebacks doubled in toxic solutions of zinc sulphate and later demonstrated (1947) that an increase in opercular rate coincided with a decrease in rate of oxygen uptake in sticklebacks exposed to salts of lead, copper and mercury.

Although there is little literature on the physiological action of toxic substances on aquatic animals, there is more on the normal parameters measured in the present study, under a variety of natural environmental conditions. These will now be discussed in as far as they shed fight on the problem of toxic action.

The routine rate of oxygen uptake in zinc-free water (Fig. 2) is similar to standard^* and routine rates reported in several recent studies using salmonids (Table 4). This supports the view that spontaneous activity in the present study was low and that the rate of oxygen uptake of quiet fish was not initially affected by exposure to zinc. The oxygen uptake of struggling fish rose in zinc sulphate solution to three times the routine rate, i.e. approximately to the activeFig. 2) is similar to standard^† rate, but the fish were unable to maintain this level of activity after 50% of their survival time had elapsed. Possibly their inability to extract sufficient oxygen from the water was already the limiting factor.

Increases in ventilation volume in trout of up to thirteen times the minimum rate have been measured by Holeton & Randall (1967) and up to five times by Stevens & Randall (1967). Present results gave a maximum ventilation volume of about eleven times the minimum rate. This was associated with an increase of the opercular rate from 74 to 120 beats/min. and an increase in respiratory stroke-volume from 6·7 to 48 ml. Hughes & Shelton (1962,Fig. 17) estimated that active fish spend such a high proportion of their total energy resources on respiration that the theoretical limit of ventilation volume is about ten times that of the resting rate.

Coughing has been studied by Schaumburg, Howard & Walden (1967) in salmon exposed to kraft pulp-mill effluent and DDT. They found a correlation between concentration of pollutant and cough frequency. Their suggestion that the cough response may be related to the environmental at the gill surface was not confirmed in the present study, because the trout rarely coughed under hypoxia.

Bradycardia has been observed in diving mammals, birds, reptiles and in frogs (Andersen, 1966) as a response to low oxygen tensions in the blood. Holeton & Randall (1967) observed bradycardia in trout exposed to hypoxia, but G. F. Holeton (personal communication) did not observe it in trout exposed to carbon monoxide. The bradycardia in zinc-poisoned fish (Fig. 7) is believed to be the first report of the phenomenum in a fish uninfluenced by drugs and with a high concentration of accessible oxygen in the surrounding environment.

The main route of oxygen uptake in fish is believed to be from the water through the gills to the blood, passing through the epithelium of the secondary lamellae, the basement membrane and the flanges of the pillar cells (Hughes & Shelton, 1962). The low oxygen tension observed in blood from the dorsal aorta of zinc-poisoned fish suggests that a block to this pathway occurs in the gills.

Histological examination of the gills of 50 g. trout, after exposure of the fish to zinc sulphate solution (40 p.p.m. Zn), will be described in a later paper. In that study damage to the gills was severe after at least 90 % of the survival time had elapsed, but only slight after 75% of the survival time. This suggests that most gill damage occurred between 75 % and 90 % of the survival time. Thus gill damage in small trout occurred after a proportion of the survival time similar to that relating to respiratory changes in large trout.

It seems probable that the most extreme changes in respiration physiology were the result of gill damage modifying gas exchange and thereby imposing an internal hypoxia on the fish. However, less extreme respiratory changes probably preceded structural gill changes. A reduction in the rate of gas transfer through the gills could result principally from an increase in diffusion distance from water to blood, a decrease in effective respiratory area, a drop in permeability and changed flow patterns in water and blood. None of these possibilities has been investigated, and this is considered to be a suitable area for future work.

I wish to thank Professor H. Heller for the use of the vapour-pressure osmometer and Professor G. M. Hughes for facilities and financial support through a grant from the Natural Environment Research Council.