PHYSIOLOGICAL CHANGES IN TROUT (SALMO GAIRDNERZ ...

3 downloads 0 Views 469KB Size Report
Six rainbow trouts were exposed to a 2 mg/l concentration of resin acids for 24 h. The toxicants influenced respiration by causing a decrease in arterial Par, red ...
Toxicology Letters, 14 (1982) 103-l 10 Elsevier Biomedical Press

103

PHYSIOLOGICAL CHANGES IN TROUT (SALMO GAIRDNERZ) DURING A SHORT-TERM EXPOSURE TO RESIN ACIDS AND DURING RECOVERY (Pulp mill effluents; respiration;

liver function; ionoreg~ation;

fish)

MIKKO NIKINMAA and AIM0 O.J. OIKARI Division of Physiology,

Department

of Zoology,

University of Helsinki, Arkadiankatu

7, SF-00100

Helsinki IO (Finland)

(Received March 5th, 1982) (Revision received May 24th, 1982) (Accepted June 3rd, 1982)

SUMMARY Six rainbow trouts were exposed to a 2 mg/l concentration of resin acids for 24 h. The toxicants influenced respiration by causing a decrease in arterial Par, red cell volume and pH, and an increase in ATP/Hb ratio. These responses were reversible on recovery. A disturbance in hydromineral balance was shown by a decrease in plasma chloride concentration, which was reversible in all but one fish. This fish had additional metabolic difficulties as shown by increases in plasma lactate concentration and enzyme activity. An indication of liver dysfunction was the increase in plasma bilirubin concentration, continuing throughout recovery.

INTRODUCTION

The effluent discharges from pulp and paper mills, which contain resin acids as an important toxicant group, fall into two types: continuous discharge resulting in small increases in the toxicant concentration of water, and spill discharge resulting in sudden increases in toxicant concentrations to high levels and consecutive dilution after the end of the discharge. In the former case the physiological effects of the toxicants on fish are best studied by long-term exposure to low-toxicant levels [ 1,2]. The latter type of discharge can be simulated by short-term exposures to high toxicant levels [3-51, although in previous studies, little attention has been paid to the recovery phase and the time courses of the responses. The present study Abbreviations: dehydrogenase.

ASAT, aspartate

0378-4274/82/~-~/$02.75

aminotransferase;

LDH, lactate dehydrogenase;

0 Elsevier Biomedical Press

SDH, sorbitol

104

therefore

was carried

the respiratory

function

out to investigate of blood,

these questions.

but hydromineral

The main emphasis

parameters

and plasma

was on enzyme

levels were also determined. MATERIALS

AND METHODS

Rainbow trout (Salmo gairdneri, 8 Q 0, 871 k 71 g, 41 f 2 cm) were cannulated via the dorsal aorta [6] and acclimatized to dechlorinated Helsinki tap water for 2 weeks. The water pH was 7.2-7.4, PO2 115-140 mmHg, P,,, O-l mmHg and temperature 14.5-16.5”C. These water conditions were maintained during the resin acid exposure. 2 days prior to experimentation the fish were transferred to 300-I plastic tanks, and enclosed in individual restrainers [6] (2 fishes per tank). By using individual restrainers the activity of the animals was kept at a constant level at all stages of the experiment. Therefore, in the present experiment, the direct physiological effects of the toxicant were not masked by changes in activity. The resin-acid mixture used in the experiment (donated by Hercofinn Co., Finland) was a wood resin containing at least 95% resin acids with the following composition: pimaric 7.9%, sandaracopimaric 1.5%, levopimaric 4.7%, palustric 24.4%, isopimaric 4.1%, abietic 26.3%, dehydroabietic 10.4% and neoabietic acid 13.5% (analysed in Abe Akademi by Dr. Bjarne Holmbom according to Holmbom [7]). 6 fishes were exposed to resin acids. Blood samples (0.7 ml) were taken before the addition of resin acids, at 1, 3, 6, and 24 h during exposure, and at 3, 6, 24 and 48 h after the removal of resin acids. The samples were taken into hypodermic The cannulation technique used offers two syringes without anticoagulants. advantages for the present type of study. First, serial blood samples can be obtained from the same animals, and second, the pre-exposure values of each individual can be used as control values to which the experimental values can be compared. Therefore, the effects of the toxicant are not masked by variation between individual fishes. Several earlier reports [6, 8-121 have shown that, apart from the decrease in haemoglobin concentration, the blood values in rainbow trout are not affected by sampling via cannulae. This was ascertained for the present study by sampling two animals at the same time schedule as the experimental fish, but without subjecting them to the toxicants. Apart from a decrease in Hb concentration no changes were detected at different sampling times in these control fish (Table I). The resin acids (concentration in water 2 mg/l) were mixed into the tanks during a lo-min period. Thereafter they were added at such a rate that the outflowing resin acids were replaced by the inflow. At the end of the exposure the flow of resin acids was stopped and the water in the tanks changed at a rate of 300 l/h, giving 95% partial replacement of the water in 3 h. The following determinations were carried out on the blood samples: (a) The haematocrit value was measured in duplicate. (b) The modal volume of red cells was

105

TABLE

I

THE BLOOD

VALUES

The fish were sampled calculated

FOR TWO SAMPLING with the same

for all the measurements;

time interval

the material

P,, (mmHg) Med. vol. erythrocyte Lactate

(mg/l)

Plasma

pH

Erythrocyte ATP/Hb

@ms)

pH molar

ratio

as the exposed

fish. The mean

SEM

and

SEM were

n

96.5

3.2

18

245.1

2.1

18

118.0

15.4

18

7.71

0.015

18

7.29

0.025

18

0.82

0.06

18

0.2

18

133.7

(mM)

FISH

was homogeneous. x

Parameter

Cl-

CONTROL

determined with Coulter apparatus calibrated for rainbow trout blood (see [ 131). (c) The arterial Po2, plasma and red-cell pH were measured with Radiometer BMS3 Mk2, PHM 71 and 72 apparatus, the red-cell pH after freezing and thawing (see [12]). (d) The erythrocytic ATP concentration was determined with Boehringer Mannheim test kit No. 123 897, plasma lactate concentration with Boehringer Mannheim test kit No. 124 842, and plasma total bilirubin concentration with Boehringer Mannheim test kit No. 123 943. (e) Plasma Na+ and K+ concentrations were determined by flame spectrophotometry, and chloride concentration with a Radiometer CMT 10 chloride titrator. (f) Blood haemoglobin concentration was measured by the cyan-methaemoglobin method, and protein concentration by Lowry’s method [ 141. (g) The plasma LDH (EC 1.1.1.27) activity was determined by an optimized UV method according to Oikari et al. [15], plasma ASAT (EC 2.6.1 .l) activity with Boehringer Mannheim test kit No. 124 362, and plasma SDH (EC 1.1.1.14) activity with Boehringer Mannheim test kit No. 125 016, using 300 mM fructose concentration (see [ 161). The ATP/Hb molar ratio was calculated from the erythrocytic ATP concentration and Hb concentration. For statistical analyses either the paired t-test or the non-parametric Wilcoxon matched-pairs signed-ranks test was used. In all cases comparisons were made between samples taken from the same fish. RESULTS

AND DISCUSSION

The arterial P,,decreased towards the end of exposure (Fig. l), and the decrease continued during the first 3 h of recovery. Davis [3] suggested that such a decrease would be caused by a secretion of mucus on the gills and that this would lead to tissue hypoxia. However, in the present study the red-cell pH and volume decreased at the beginning of exposure (Fig. 2). These responses are exactly opposite to those observed in environmental hypoxia [12]. A decrease in the red-cell pH shifts the O2

106

p%

_

mmHg

110 *

100~ 90. 00. 70. 60.

mgll

. ?

600,

.q

2

..

&oo.

,.I ,y’

_...x

:’

200,

+@,&Q”

. . . ..v...’

,*,

+------0 P.. 01 3 6

Fig.

:

,.,p_._--.. -.-

.I’ .I’

-.

2.4 27 30

1. Blood PQ and plasma lactate concentration

(open bar) and during

recovery

(solid line). P-values

J 72 h

I.8

of rainbow

trout during

refer to paired

exposure

t-test carried

to 2 mg/l resin acids

out on the values from

each individual throughout the experiment. Bars indicate +SEM. The cross indicates the plasma concentration of the fish that died during the first 3 h of recovery. The triangles indicate values for the ‘def.’ fish (see text).

dissociation curve to the right via the Bohr effect [17] thus decreasing the amount of 0, loaded in the gills. Towards the end of the exposure the red-cell pH and volume returned to pre-exposure levels but, at the same time, the ATP/Hb ratio (Fig. 2) increased considerably thereby decreasing the blood O2 affinity [ 171. A disturbance in the red-cell volume also took place at the onset of recovery when the resin acids were removed from water. Thus the changes in red-cell volume seem to occur both when the plasma concentration of resin acids starts to increase and when the plasma concentration starts to decrease and in both cases the system is clearly in disequilibrium. When the resin acids were removed from water the ATP/Hb ratio also decreased rapidly. It thus seems that resin acids directly affect the function of red cells, but that these changes are reversible. The most noticeable effect of resin acids on the ion balance of the fish was the significant decrease in plasma Cl- concentration (Fig. 3) although the other ion concentrations studied (Na+ and K+) did not change. The change in plasma Cll was reversible in that after 24 h recovery Cl- concentrations returned to preexposure levels in all fish except one (later called ‘def.‘) in which case the level remained low throughout the recovery period.

107

PIO-1)