- Email: [email protected]
The effect of copper on growth, food consumption and food conversion of perch Perca fluviatilis L. Offered maximal food rations
Aquatic Toxicology, 6 (1985 ) 105 - 113
105
Elsevier
A Q T 00145
T H E EFFECT OF C O P P E R ON G R O W T H , F O O D C O N S U M P T I O N A N D F O O D C O N V E R S I O N OF P E R C H PERCA FLUVIATILIS L. OFFERED MAXIMAL FOOD RATIONS
LARS C O L L V I N
Institute of Limnology, University of Lund, Box 3060, S-220 03 Lund, Sweden (Received 27 August 1984; revised version received and accepted 25 November 1984)
The effect of copper (1, 13, 22, 39 and 81 # g . 1 - ' ) on the maximal growth rate of perch (Perca fluviatilis) was followed for 30 days. After that, previously available food (Gammaruspulex) was removed and the starvation rates determined. Copper reduced the maximal growth rate at concentrations > 22 #g Cu • 1- ~. This reduction was mainly an effect of reduced food conversion efficiency, which was itself attributed to an increase in standard metabolism due to detoxication. Only at the highest concentration was food intake reduced. The reduction was immediate and food intake then remained at a constant and low level. In terms of growth and food conversion, the perch acclimated at the lowest effectconcentration, and started to acclimate at the highest concentration, but did not acclimate at all in terms of food consumption. Starved and acclimated perch lost weight at higher rates than starved and unexposed perch. This demonstrated that acclimated perch were still affected by copper due to increased energetic costs during conditions of starvation. Key words: copper; growth; food consumption; food conversion; acclimation; perch
INTRODUCTION
Copper exposure has a well documented negative effect on fish growth (e.g., O'Hara, 1971; L e t t e t al., 1976; Waiwood and Beamish, 1978; Collvin, 1984a). At sublethal exposure levels this effect lessens with the exposure time and eventually results in a return to the control rate, i.e., acclimation or compensation occurs (Lett et al., 1976; Waiwood and Beamish, 1978; Dixon and Sprague, 1981). Growth reductions in copper-exposed fish have so far only been observed for fish fed reduced rations (McKim and Benoit, 1971; Lett et al., 1976; Waiwood and Beamish, 1978; Collvin, 1984a). The reason for this is that up to a given food ration the growth of fish generally increases linearly with increasing food rations (Brett and Groves, 1979), but beyond this upper food ration excess energy is available which may be utilized, e.g., for detoxication without affecting growth (Lett et al., 1976; Farmer et al., 1979). In contrast to this general pattern, however, the perch (Perca fluviatilis) maintains a linear increase in growth rate even up to the maximal food 0166-445X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
106 ration, at least in a laboratory system with temperatures above 12°C (Lessmark, 1983). Fish need energy for metabolic processes and growth; energy not utilized is excreted (Warren and Davis, 1967; Brett and Groves, 1979). Metabolic energy is used for activity (locomotion), food processing (assimilation/excretion) and basic physiological processes (standard metabolism, Warren and Davis, 1967; Brett and Groves, 1979). This study aims to follow the effect of copper on the maximal growth rate of perch over 30 days and to evaluate this effect in terms of energy intake and energy expenditure. In addition, the study examines whether acclimation implies that copper no longer affects the perch energetically. MATERIALS AND METHODS The perch (n = 50) were randomly distributed a m o n g 5 100-1 glass aquaria. Tap water (Table I) continuously entered the aquaria via mixing vessels and was exchanged (95°-/0, theoretical) in 4.3 h (Sprague, 1973). To prevent the food f r o m drift: ing away a net was placed over the effluent pipe. The aquaria were covered with green plastic to minimize stress effects from outside the aquaria. The light-dark cycle was 15 h - 9 h. Stock solutions of copper (one per test concentration) were prepared f r o m a titrisol solution (CuC12, 1000 #g C u - 1 - 1). HNO3 (3 ml. 1- 1) was added to adjust p H to 1. The solutions were kept in 10-1 polyethylene bottles and were fed to the mixing vessels via a peristaltic p u m p (0.206 m l ' min-1). Alkalinity, p H , hardness, electrolytic conductivity and oxygen saturation of the test water were determined once weekly on duplicate samples (SIS, 1981). Temperature and flow rates were determined every second day. The effluent from the aquaria was sampled once weekly (40 ml, acidified with 5 drops of 7 M HNO3) and the copper concentration was determined using an atomic absorption spectrophotometer with a graphite furnace. The detection limit ( = 3 SD of 10 blanks) was 0.43/zg Cu.1-1 and the sensitivity (=0.0044 A U) was 0,65 #g Cu.1 - I
TABLE 1 Physical and chemical properties of the test water Variable
Mean
SD
n
Unit
Temperature Oxygen Electrolytic conductivity pH Alkalinity Total hardness (Ca+ Mg)
17.5 87 466 7.8 129 194
0.24 5.5 1.2 0.06 5.9 6.9
141 36 6 36 36 18
(°C) (°7o 02-saturation)
(#S.cm 1) (mg CaC03.1- t) (mg CaC03.1-~)
107 TABLE II Mean weights (SD, n=9-10) of the perch in the study Copper conc.
Perch weight (g wet weight)
~ g Cu.l 21)
Time of exposure (days)
1 13 22 39 81
3.8 3.8 4.3 3.9 3.9
(0.69) (0.38) (0.63) (0.41) (0.51)
7.6 7.4 8.4 6.4 3.7
(1.51) (1.04) , (1.78) (0.61) (1.14)
7.2 6.8 7.8 5.8 3.2
(1.42) (0.75) (1.63) (0.60) (1.01)
Perch (Table II) were caught by electro-fishing in the eutrophic lake S6vdeborgssj6n, sou'th Sweden (55°35'N, 13°42'E). They were individually marked with alcian-blue to follow the growth of each individual. They were kept under routine conditions and were acclimated to the test conditions for 4 weeks. The food organisms, Gammarus pulex, were kept in small volumes of tap water (5°C) until offered to the perch. The perch were given a maintenance ration of G. pulex for three out of four weeks before copper addition. During the fourth week the food rations were raised to unrestricted rations. Weight determinations during this period ensured that there was no difference ( P < 0.05, t-test; Sokal and Rohlf, 1969) in mean growth rate of perch between aquaria. The perch had continuous access to an unrestricted number of G. pulex for intervals of 5 days during copper exposure. Food additions were made twice daily to minimize the negative effect of copper on the G. pulex. Food items remaining after 5 days were sieved off and weighed to determine food consumption rates. Then, to obtain comparable growth data the perch were left without food for 24 h to allow for stomach evacuation (Persson, 1979). The growth rate, food consumption rate and food conversion efficiency of the perch were determined after each 6-day interval over the initial 30-day period of copper additions. After the 30-day growth period food was withdrawn for 9 days (days 30-39). The starvation rates were determined to examine if acclimated perch are still energetically influenced by copper and, if so, to determine the metabolic cost in terms of weight loss. The perch were tranquillized for 5 min in MS 222 (5 mg" l-1) before weighing. They were identified, blotted with a soft damp paper and weighed to the near~st 0.01 g. Specific growth rates and starvation rates of perch, G and Gs (°T0"day- 1), were determined using the equation (Brown, 1957):
G= 100 (lnW2-1nW1)/(T2- T1) where W1 and W2 are the wet weights (g ww) of a given perch at the start (T1) and
108
at the end (T2) of a given time interval. The food consumption rate, R (%. d a y - 1) and the food conversion efficiency, K (%) were determined as (Lessmark, 1983):
R = lOOC/(t/2(Wl + w2)) and, K = 100(w2
-
w1)/C
where C is the amount of food (g dw) consumed in the interval t (days). wl and wz are the ash-free dry weights (dw) of each group of perch at the start and at the end of that interval. Since individual differences in feeding were not determined, the effects of copper on R and K could not be evaluated statistically. Determinations of R and K were based on dw. dw was defined as the difference in weight determined at 65°C after 48 h and at 560°C after 24 h. The dw of perch was estimated from ww using the relation: dw = 0.206 ww + 0.0438 (r 2 = 0.938, n--57, 1-57 g ww perch from lake S6vdeborgssj6n, Lessmark op. cit.). The ww and dw of G. pulex were determined in a similar way as for perch. The mean ww of an individual G. p u l e x was 19.7 mg (SD 0.42, n = 5 groups of 350 G. pulex). The mean dw constituted 12.3% (SD 0.71, n = 5) of the mean ww. This proportion was used to convert the ww of the G. pulex into dw. The 'Friedman's two-way analysis of variance by ranks' was used to test for differences in growth rate over the 30-day period within a given concentration of copper. The 'Walsh test' was used to test for effects in growth rate between any two growth periods within a concentration of copper. The 'Approximate test of equality of means when the variances are unequal' was used to test for effects of copper on growth rate within a growth period. The 'Test of equality of the means of two samples whose variances are unequal' was used to test for differences between any two concentrations of copper within a growth period. Unless stated otherwise P < 0 . 0 5 was used as the level of statistical significance, and statistical tests were taken from Siegel (1956) and from Sokal and Rohlf (1969). RESULTS
There was no significant difference in the physical and chemical variables (except for copper) either between aquaria or within an aquarium over the test period (Table I). The mean concentrations of copper were: 1 (95o7o CL 1.2, control), 13 (2.0), 22 (1.0), 39 (1.8) and 81 (3.8) tzg Cu.1-1, respectively. The mean growth rate of the perch in the control group initially increased ( P < 0.001, Fig. la). Concurrently, the rate of food consumption increased from 8.9 to 11.8o7o •d a y - 1 (Fig. lb). Subsequently, the growth rate did not change ( P > 0.05) and was 3.0°7o .day -1 (SD 0.33, n = 36). Over this time, the perch fed at a mean rate o f ll.9oT0-day- x (SD 1.12, n = 4 ) and converted on average 24.807o (SD 1.71, n = 4 ) o f the food into growth (Fig. lb, c).
109
Copper addition up to 22 #g. 1- ~did not (P> 0.05) affect growth at any exposure time. Nor did the food consumption rates or the food conversion efficiencies appear to be affected. At 39 #g Cu. 1- t, however, copper depressed (P< 0.001) the growth rate for 18 days, after which the growth rate returned (P> 0.05) to control level (Fig. la). The food conversion efficiency but not the food consumption rate seemed to decrease initially (Fig. lb, c). At 81/~g Cu" I- 1, copper depressed the food consumption rate, the food conversion efficiency and, subsequently, the growth rate (P< 0.001, Fig. la-c). The mean food consumption rate was 5.7°T0-day-1 (SD 0.76, n = 5) over the 30-day period. The decrease in growth rate from -0.2070"day -~ over the initial 12 days to G (%.d "1) 4.
32-
10-1-2-
®
-3-
R (%'d "1) 15"
10-
5-
® K (%) 40-
20-
0
® -20Time of e x p o s u r e (days)
Fig. 1. The effect of copper on (a) growth rate (mean and SD, n = 9-10), (b) food consumption rate, and (c) food conversion efficiency of perch at 6-day intervals over 30 days. (O) 1, ( 0 ) 39, and (11) 81 #g Cu '1 - 1, respectively. (Effects at 13 and 22 #g Cu .1 - 1 did not differ from the control a n d are, for clarity, not shown.)
110
-0.8o70-day -1 over the next 12 days was not significant ( P = 0 . 0 5 6 ) . The final increase in g r o w t h rate to above maintenance growth, however, was significant ( P = 0 . 0 1 1 , Fig. la). The direction o f the effect o n f o o d conversion efficiency followed that on the growth rate. W i t h o u t feeding, the starvation rate o f the perch increased with increasing copper concentration ( P < 0.001, Table II, Fig. 2a). T h e increase in m e a n starvation rate, however, was significant ( P < 0.001) only above 22 pig Cu" 1- 1 (Fig. 2b). A regression (best-of-fit a m o n g linear, power, exponential and logarithmic) that relates the starvation rate, Gs (oT0"day-1) to the copper concentration, Ccu (ktg'l-1), was calculated to evaluate the energetic cost o f c o p p e r to perch: (Gs+2)=0.653
• e -0.010- ccu (1-2=0.53, n = 4 8 , P < 0 . 0 0 1 )
Thus, the starvation rate o f perch at 0 Ccu is estimated to be 0.65o70.day-1 (SD 0.95, n = 48). The rate constant, which states the increase in starvation rate with increasing copper concer~tration (up to 81 izg C u ' l - t ) is estimated to be - 0.010O7o " d a y - 1. (~g Cu. 1 - 1) - 1 (SD 0.0014, n = 48). Hence, an increase in copper c o n c e n t r a t i o n f r o m 20 to 30/~g- 1 - 1 will increase the starvation rate f r o m - 0.90 to - 1.00°7o" d a y - 1. A n equivalent increase f r o m 70 to 80/zg. 1 - 1 will result in an increase f r o m - 1.33 to - 1.40°70 • d a y - 1. DISCUSSION
F o o d c o n s u m p t i o n was reduced at a concentration o f 81/zg Cu" 1- 1 but appeared unaffected at and below 39/~g Cu" 1- 1. Thus, energy intake was not reduced below 81 #g Cu" 1- t. G r o w t h reduction at 39/xg (~u- 1- 1 is therefore a result o f an increased G pL.d-li
oi-
Copper conc.(iJg.I "1)
?
,
,
3,0
,
,
60
,
,
,
I
AG (%.d "~)
o.1o
'Z
,
.
|
J
- 0.4
-0.8
f~
Fig. 2. The effect of copper, from day 30 to day 39, on (a) the starvation rate of perch (mean and SD, n=9-10). (b) Difference in mean starvation rate between unexposed and copper-exposed perch.
111
energy expenditure (provided there is no decrease in energy utilization). At 81 /~g C u . l - 1 , however, the growth reduction could result either from the decreased energy intake alone or, more likely, from a decrease in energy intake in combination with an increase in energy expenditure. As perch have defined stomachs (like salmonids but in contrast to cyprinids), they can only fill them to fullness. In a food-rich environment the ingestion rate thus depends on the egestion rate. Egestion depends, among other things, on the assimilation efficiency and the excretion rate (F/~nge and Grove, 1979). Lett et al. (1976) showed that sublethal copper concentrations do not affect the assimilation rate of rainbow trout (Salmo gairdnerl). If the same holds true for perch then the excretion rate cannot be expected to increase since there was no increase in food consumption. The perch cannot, under this assumption, compensate for the reduction in growth rate by increasing the ingestion rate. What causes the reduction in growth rate in situations where food processing (here, energy intake) is unaffected? By definition (see above) either the activity a n d / o r the standard metabolism must increase. Except for an initial hyperactivity for 2-5 days the routine swimming activity of perch does not increase over a period o f 30 days at 55/zg Cu- l - 1 (Collvin, unpublished). The reduction in growth rate of copper-exposed perch is therefore mainly the result of an increase in standard metabolic rate. When perch acclimate to copper, the metal is continuously transported from the water via the gills to the liver (Collvin, 1984b). Furthermore, perch actively regulate the copper concentration in the gills and probably in the liver, which indicates that detoxicatory and excretory processes exist. Such physiological processes will require additional energy and cause the standard metabolic rate to increase. Since the food conversion efficiency reduced and since its variation followed that of the growth rate, I assume that the perch take this energy from the food. Copper interacts with the ligands of proteins and, therefore, affects the catalytic capacity of enzymes and the transport ability of membranes (Passow et al., 1961; Luoma, 1983). This leads to reduced efficiency of physiological reactions and may stimulate a higher production rate of, e.g., enzymes to generate the amount required to fulfill given physiological tasks. Thus, any copper-induced increase in synthesis o f substances will be expected to cause a higher rate of standard metabolism. Food conversion rate and growth rate of the perch returned to normal after 18 days at 39/~g Cu" 1- 1, while at 81 #g Cu" 1- 1 a return may have been initiated after 24 days. Unexposed perch that consume a food ration of 4-6%" d a y - 1 of their dw will, under the conditions of this study, grow at a rate of 0 . 8 - 1 . 5 % - d a y -1 (Lessmark, 1983; Collvin, 1984a). Growth rate and food conversion efficiency of the perch at 81 #g Cu. 1- 1 are therefore initially reduced more than can be accounted for by a mere reduction in food consumption rate. There are, in contrast to perch exposed at 39/~g C u - l - 1, indications (P = 0.056) of a further and trough-like reduction over 12 days. The mean growth rate after 30 days is 0.20/o . d a y - 1 and perch
112
at 81 ~tg C u ' 1 - 1 are therefore not, in terms of standard metabolism, fully acclimated. The time required before the onset of metabolic acclimation and full compensation thus appears to increase with increased copper concentration. Does acclimation (return of standard metabolism to control level) mean that copper no longer affects the perch energetically? If a higher starvation rate is found in the copper-exposed, acclimated and starved perch then the copper still influences the perch. Since this was the case I suggest that acclimated perch continue to take up and excrete copper. Why did the maintenance cost for detoxication eventually vanish for maximally feeding perch at 40 ttg Cu" 1- 1? Copper-exposed fish increase their concentration of metallothionein in the liver (Roch et al., 1982). This substance is ascribed the ability to detoxify, store and transport copper (Cherian and Goyer, 1978). Whatever substance(s) is/are responsible for detoxication of copper in perch, it/they require(s) ca. 20 days to reach full detoxicating strength at 40/~g Cu" 1- 1 (Collvin, 1984b; this study). The build up of detoxicating substances implies a mechanism for detoxication, energy for synthesis and elements for substance formation. The results show that at 40/tg C u ' 1- 1 the food supplied the perch with enough energy and elements for detoxication. Therefore, any effect appears to relate to the efficiency of the mechanism. When the detoxicating substance is synthesized at a rate sufficient tc detoxify all copper f r o m the inward flux of copper, it is then possible that vital physiological processes are left unaffected and that the perch may acclimate.
ACKNOWLEDGEMENTS
I thank Dr. A. S6dergren, Dr. P. Larsson and Dr. I.J. Winfield for constructive criticism of the manuscript.
REFERENCES Brett, J.R. and T.D.D. Groves, 1979. Physiological energetics. In: Fish physiology. Vol. VllI, Bioenergetics and growth, edited by W.S. Hoar, D.J. Randall and J.R. Brett, Academic Press, London, pp. 279-352. Brown, M.E., 1957. Experimental studies on growth. In: Physiology of fishes. Vol. I, edited by M.E. Brown, Academic Press, London, pp. 361-400. Cherian, M.G. and R.A. Goyer, 1978. Minireview. Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci. 23, 1-10. Collvin, L., 1984a. The effects of copper on maximum respiration rate and growth rate of perch, Perca fluviatilis L. Water Res. 18, 139-144. Collvin, L., 1984b. Uptake of copper in the gills and liver of perch, Percafluviatilis. Ecol. Bull. 36, in press. Dixon, D.G. and J.B. Sprague, 1981. Acclimation to copper by rainbow trout (Salmo gairdner~) - A modifying factor in toxicity. Can. J. Fish. Aquat. Sci. 38, 880-888. Farmer, G.J., D. Ashfield and H.S. Samant, 1979. Effects of zinc on juvenile atlantic salmon Salmo salar: Acute toxicity, food intake, growth and bioaccumulation. Environ. Pollut. 19, 103-117.
113 F~inge, R. and D. Grove, 1979. Digestion. In: Fish physiology. Vol. VIII, Bioenergetics and growth, edited by W.S. Hoar, D.J. Randall and J.R. Brett, Academic Press, London, pp. 161-260. Lessmark, O., 1983. Competition between perch (Perca fluviatilis) and roach (Rutilus rutilus) in south Swedish lakes. Ph.D. Thesis, Univ. of Lund, Lund, Sweden, 172 pp. Lett, P.F., G.J. Farmer and F.W.H. Beamish, 1976. Effect of copper on some aspects of the bioenergetics of rainbow trout (Salmo gairdnert). J. Fish. Res. Bd. Can. 33, 1335-1342. Luoma, S.N., 1983. Bioavailability of trace metals to aquatic organisms - A review. Sci. Total Environ. 28, 1-22. O'Hara, J., 1971. Alterations in oxygen consumption by bluegills exposed to sublethal treatment with copper. Water Res. 5, 321-327. McKim, J.M. and D.A. Benoit, 1971. Effects of long-term exposure to copper on survival, growth, and reproduction of brook trout (Salvelinus fontinalis). J. Fish. Res. Bd. Can. 28, 655-662. Passow, H., A. Rothstein and T.W. Clarkson, 1961. The general pharmacology of the heavy metals. Pharmacol. Rev. 13, 185-224. Persson, L., 1979. The effects of temperature and different food organisms on the rate of gastric evacuation in perch (Perca fluviatilis). Freshwater Biology 11, 131-138. Roch, M., J.A. McCarter, A.T. Matheson, M.J.R. Clark and R.W. Olafson, 1982. Hepatic metallothionein in rainbow trout (Salmo gairdnert) as an indicator of metal pollution in the Campbell River System. Can. J. Fish. Aquat. Sci. 39, 1596-1601. Siegel, S., 1956. Nonparametric statistics for the behavioral sciences, McGraw-Hill International Book Company, London, 312 pp. SIS, t981. Standardiseringskommissionen i Sverige, Box 3295, 103 66 Stockholm, Sweden. Sokal, R.R. and F.J. Rohlf, 1969. Biometry. The principles and practice of statistics in biological research. Freeman and Company, San Francisco, 757 pp. Sprague, J.B., 1973. The ABCs of pollutant bioassay using fish. In: Biological methods for the assessment of water quality, edited by J. Cairns Jr. and K.L. Dickson, ASTM STP 528, Philadelphia, pp. 6-30. Waiwood, K.G. and F.W.H. Beamish, 1978. The effect of copper, hardness and pH on the growth of rainbow trout, Salmo gairdneri. J. Fish. Biol. 13, 591-598. Warren, C.E. and G.E. Davis, 1967. Laboratory studies on the feeding, bioenergetics, and growth of fish. In: The biological basis of freshwater fish production, edited by S.D. Gerking, Blackwell, Oxford, pp. 175-214.
105
Elsevier
A Q T 00145
T H E EFFECT OF C O P P E R ON G R O W T H , F O O D C O N S U M P T I O N A N D F O O D C O N V E R S I O N OF P E R C H PERCA FLUVIATILIS L. OFFERED MAXIMAL FOOD RATIONS
LARS C O L L V I N
Institute of Limnology, University of Lund, Box 3060, S-220 03 Lund, Sweden (Received 27 August 1984; revised version received and accepted 25 November 1984)
The effect of copper (1, 13, 22, 39 and 81 # g . 1 - ' ) on the maximal growth rate of perch (Perca fluviatilis) was followed for 30 days. After that, previously available food (Gammaruspulex) was removed and the starvation rates determined. Copper reduced the maximal growth rate at concentrations > 22 #g Cu • 1- ~. This reduction was mainly an effect of reduced food conversion efficiency, which was itself attributed to an increase in standard metabolism due to detoxication. Only at the highest concentration was food intake reduced. The reduction was immediate and food intake then remained at a constant and low level. In terms of growth and food conversion, the perch acclimated at the lowest effectconcentration, and started to acclimate at the highest concentration, but did not acclimate at all in terms of food consumption. Starved and acclimated perch lost weight at higher rates than starved and unexposed perch. This demonstrated that acclimated perch were still affected by copper due to increased energetic costs during conditions of starvation. Key words: copper; growth; food consumption; food conversion; acclimation; perch
INTRODUCTION
Copper exposure has a well documented negative effect on fish growth (e.g., O'Hara, 1971; L e t t e t al., 1976; Waiwood and Beamish, 1978; Collvin, 1984a). At sublethal exposure levels this effect lessens with the exposure time and eventually results in a return to the control rate, i.e., acclimation or compensation occurs (Lett et al., 1976; Waiwood and Beamish, 1978; Dixon and Sprague, 1981). Growth reductions in copper-exposed fish have so far only been observed for fish fed reduced rations (McKim and Benoit, 1971; Lett et al., 1976; Waiwood and Beamish, 1978; Collvin, 1984a). The reason for this is that up to a given food ration the growth of fish generally increases linearly with increasing food rations (Brett and Groves, 1979), but beyond this upper food ration excess energy is available which may be utilized, e.g., for detoxication without affecting growth (Lett et al., 1976; Farmer et al., 1979). In contrast to this general pattern, however, the perch (Perca fluviatilis) maintains a linear increase in growth rate even up to the maximal food 0166-445X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
106 ration, at least in a laboratory system with temperatures above 12°C (Lessmark, 1983). Fish need energy for metabolic processes and growth; energy not utilized is excreted (Warren and Davis, 1967; Brett and Groves, 1979). Metabolic energy is used for activity (locomotion), food processing (assimilation/excretion) and basic physiological processes (standard metabolism, Warren and Davis, 1967; Brett and Groves, 1979). This study aims to follow the effect of copper on the maximal growth rate of perch over 30 days and to evaluate this effect in terms of energy intake and energy expenditure. In addition, the study examines whether acclimation implies that copper no longer affects the perch energetically. MATERIALS AND METHODS The perch (n = 50) were randomly distributed a m o n g 5 100-1 glass aquaria. Tap water (Table I) continuously entered the aquaria via mixing vessels and was exchanged (95°-/0, theoretical) in 4.3 h (Sprague, 1973). To prevent the food f r o m drift: ing away a net was placed over the effluent pipe. The aquaria were covered with green plastic to minimize stress effects from outside the aquaria. The light-dark cycle was 15 h - 9 h. Stock solutions of copper (one per test concentration) were prepared f r o m a titrisol solution (CuC12, 1000 #g C u - 1 - 1). HNO3 (3 ml. 1- 1) was added to adjust p H to 1. The solutions were kept in 10-1 polyethylene bottles and were fed to the mixing vessels via a peristaltic p u m p (0.206 m l ' min-1). Alkalinity, p H , hardness, electrolytic conductivity and oxygen saturation of the test water were determined once weekly on duplicate samples (SIS, 1981). Temperature and flow rates were determined every second day. The effluent from the aquaria was sampled once weekly (40 ml, acidified with 5 drops of 7 M HNO3) and the copper concentration was determined using an atomic absorption spectrophotometer with a graphite furnace. The detection limit ( = 3 SD of 10 blanks) was 0.43/zg Cu.1-1 and the sensitivity (=0.0044 A U) was 0,65 #g Cu.1 - I
TABLE 1 Physical and chemical properties of the test water Variable
Mean
SD
n
Unit
Temperature Oxygen Electrolytic conductivity pH Alkalinity Total hardness (Ca+ Mg)
17.5 87 466 7.8 129 194
0.24 5.5 1.2 0.06 5.9 6.9
141 36 6 36 36 18
(°C) (°7o 02-saturation)
(#S.cm 1) (mg CaC03.1- t) (mg CaC03.1-~)
107 TABLE II Mean weights (SD, n=9-10) of the perch in the study Copper conc.
Perch weight (g wet weight)
~ g Cu.l 21)
Time of exposure (days)
1 13 22 39 81
3.8 3.8 4.3 3.9 3.9
(0.69) (0.38) (0.63) (0.41) (0.51)
7.6 7.4 8.4 6.4 3.7
(1.51) (1.04) , (1.78) (0.61) (1.14)
7.2 6.8 7.8 5.8 3.2
(1.42) (0.75) (1.63) (0.60) (1.01)
Perch (Table II) were caught by electro-fishing in the eutrophic lake S6vdeborgssj6n, sou'th Sweden (55°35'N, 13°42'E). They were individually marked with alcian-blue to follow the growth of each individual. They were kept under routine conditions and were acclimated to the test conditions for 4 weeks. The food organisms, Gammarus pulex, were kept in small volumes of tap water (5°C) until offered to the perch. The perch were given a maintenance ration of G. pulex for three out of four weeks before copper addition. During the fourth week the food rations were raised to unrestricted rations. Weight determinations during this period ensured that there was no difference ( P < 0.05, t-test; Sokal and Rohlf, 1969) in mean growth rate of perch between aquaria. The perch had continuous access to an unrestricted number of G. pulex for intervals of 5 days during copper exposure. Food additions were made twice daily to minimize the negative effect of copper on the G. pulex. Food items remaining after 5 days were sieved off and weighed to determine food consumption rates. Then, to obtain comparable growth data the perch were left without food for 24 h to allow for stomach evacuation (Persson, 1979). The growth rate, food consumption rate and food conversion efficiency of the perch were determined after each 6-day interval over the initial 30-day period of copper additions. After the 30-day growth period food was withdrawn for 9 days (days 30-39). The starvation rates were determined to examine if acclimated perch are still energetically influenced by copper and, if so, to determine the metabolic cost in terms of weight loss. The perch were tranquillized for 5 min in MS 222 (5 mg" l-1) before weighing. They were identified, blotted with a soft damp paper and weighed to the near~st 0.01 g. Specific growth rates and starvation rates of perch, G and Gs (°T0"day- 1), were determined using the equation (Brown, 1957):
G= 100 (lnW2-1nW1)/(T2- T1) where W1 and W2 are the wet weights (g ww) of a given perch at the start (T1) and
108
at the end (T2) of a given time interval. The food consumption rate, R (%. d a y - 1) and the food conversion efficiency, K (%) were determined as (Lessmark, 1983):
R = lOOC/(t/2(Wl + w2)) and, K = 100(w2
-
w1)/C
where C is the amount of food (g dw) consumed in the interval t (days). wl and wz are the ash-free dry weights (dw) of each group of perch at the start and at the end of that interval. Since individual differences in feeding were not determined, the effects of copper on R and K could not be evaluated statistically. Determinations of R and K were based on dw. dw was defined as the difference in weight determined at 65°C after 48 h and at 560°C after 24 h. The dw of perch was estimated from ww using the relation: dw = 0.206 ww + 0.0438 (r 2 = 0.938, n--57, 1-57 g ww perch from lake S6vdeborgssj6n, Lessmark op. cit.). The ww and dw of G. pulex were determined in a similar way as for perch. The mean ww of an individual G. p u l e x was 19.7 mg (SD 0.42, n = 5 groups of 350 G. pulex). The mean dw constituted 12.3% (SD 0.71, n = 5) of the mean ww. This proportion was used to convert the ww of the G. pulex into dw. The 'Friedman's two-way analysis of variance by ranks' was used to test for differences in growth rate over the 30-day period within a given concentration of copper. The 'Walsh test' was used to test for effects in growth rate between any two growth periods within a concentration of copper. The 'Approximate test of equality of means when the variances are unequal' was used to test for effects of copper on growth rate within a growth period. The 'Test of equality of the means of two samples whose variances are unequal' was used to test for differences between any two concentrations of copper within a growth period. Unless stated otherwise P < 0 . 0 5 was used as the level of statistical significance, and statistical tests were taken from Siegel (1956) and from Sokal and Rohlf (1969). RESULTS
There was no significant difference in the physical and chemical variables (except for copper) either between aquaria or within an aquarium over the test period (Table I). The mean concentrations of copper were: 1 (95o7o CL 1.2, control), 13 (2.0), 22 (1.0), 39 (1.8) and 81 (3.8) tzg Cu.1-1, respectively. The mean growth rate of the perch in the control group initially increased ( P < 0.001, Fig. la). Concurrently, the rate of food consumption increased from 8.9 to 11.8o7o •d a y - 1 (Fig. lb). Subsequently, the growth rate did not change ( P > 0.05) and was 3.0°7o .day -1 (SD 0.33, n = 36). Over this time, the perch fed at a mean rate o f ll.9oT0-day- x (SD 1.12, n = 4 ) and converted on average 24.807o (SD 1.71, n = 4 ) o f the food into growth (Fig. lb, c).
109
Copper addition up to 22 #g. 1- ~did not (P> 0.05) affect growth at any exposure time. Nor did the food consumption rates or the food conversion efficiencies appear to be affected. At 39 #g Cu. 1- t, however, copper depressed (P< 0.001) the growth rate for 18 days, after which the growth rate returned (P> 0.05) to control level (Fig. la). The food conversion efficiency but not the food consumption rate seemed to decrease initially (Fig. lb, c). At 81/~g Cu" I- 1, copper depressed the food consumption rate, the food conversion efficiency and, subsequently, the growth rate (P< 0.001, Fig. la-c). The mean food consumption rate was 5.7°T0-day-1 (SD 0.76, n = 5) over the 30-day period. The decrease in growth rate from -0.2070"day -~ over the initial 12 days to G (%.d "1) 4.
32-
10-1-2-
®
-3-
R (%'d "1) 15"
10-
5-
® K (%) 40-
20-
0
® -20Time of e x p o s u r e (days)
Fig. 1. The effect of copper on (a) growth rate (mean and SD, n = 9-10), (b) food consumption rate, and (c) food conversion efficiency of perch at 6-day intervals over 30 days. (O) 1, ( 0 ) 39, and (11) 81 #g Cu '1 - 1, respectively. (Effects at 13 and 22 #g Cu .1 - 1 did not differ from the control a n d are, for clarity, not shown.)
110
-0.8o70-day -1 over the next 12 days was not significant ( P = 0 . 0 5 6 ) . The final increase in g r o w t h rate to above maintenance growth, however, was significant ( P = 0 . 0 1 1 , Fig. la). The direction o f the effect o n f o o d conversion efficiency followed that on the growth rate. W i t h o u t feeding, the starvation rate o f the perch increased with increasing copper concentration ( P < 0.001, Table II, Fig. 2a). T h e increase in m e a n starvation rate, however, was significant ( P < 0.001) only above 22 pig Cu" 1- 1 (Fig. 2b). A regression (best-of-fit a m o n g linear, power, exponential and logarithmic) that relates the starvation rate, Gs (oT0"day-1) to the copper concentration, Ccu (ktg'l-1), was calculated to evaluate the energetic cost o f c o p p e r to perch: (Gs+2)=0.653
• e -0.010- ccu (1-2=0.53, n = 4 8 , P < 0 . 0 0 1 )
Thus, the starvation rate o f perch at 0 Ccu is estimated to be 0.65o70.day-1 (SD 0.95, n = 48). The rate constant, which states the increase in starvation rate with increasing copper concer~tration (up to 81 izg C u ' l - t ) is estimated to be - 0.010O7o " d a y - 1. (~g Cu. 1 - 1) - 1 (SD 0.0014, n = 48). Hence, an increase in copper c o n c e n t r a t i o n f r o m 20 to 30/~g- 1 - 1 will increase the starvation rate f r o m - 0.90 to - 1.00°7o" d a y - 1. A n equivalent increase f r o m 70 to 80/zg. 1 - 1 will result in an increase f r o m - 1.33 to - 1.40°70 • d a y - 1. DISCUSSION
F o o d c o n s u m p t i o n was reduced at a concentration o f 81/zg Cu" 1- 1 but appeared unaffected at and below 39/~g Cu" 1- 1. Thus, energy intake was not reduced below 81 #g Cu" 1- t. G r o w t h reduction at 39/xg (~u- 1- 1 is therefore a result o f an increased G pL.d-li
oi-
Copper conc.(iJg.I "1)
?
,
,
3,0
,
,
60
,
,
,
I
AG (%.d "~)
o.1o
'Z
,
.
|
J
- 0.4
-0.8
f~
Fig. 2. The effect of copper, from day 30 to day 39, on (a) the starvation rate of perch (mean and SD, n=9-10). (b) Difference in mean starvation rate between unexposed and copper-exposed perch.
111
energy expenditure (provided there is no decrease in energy utilization). At 81 /~g C u . l - 1 , however, the growth reduction could result either from the decreased energy intake alone or, more likely, from a decrease in energy intake in combination with an increase in energy expenditure. As perch have defined stomachs (like salmonids but in contrast to cyprinids), they can only fill them to fullness. In a food-rich environment the ingestion rate thus depends on the egestion rate. Egestion depends, among other things, on the assimilation efficiency and the excretion rate (F/~nge and Grove, 1979). Lett et al. (1976) showed that sublethal copper concentrations do not affect the assimilation rate of rainbow trout (Salmo gairdnerl). If the same holds true for perch then the excretion rate cannot be expected to increase since there was no increase in food consumption. The perch cannot, under this assumption, compensate for the reduction in growth rate by increasing the ingestion rate. What causes the reduction in growth rate in situations where food processing (here, energy intake) is unaffected? By definition (see above) either the activity a n d / o r the standard metabolism must increase. Except for an initial hyperactivity for 2-5 days the routine swimming activity of perch does not increase over a period o f 30 days at 55/zg Cu- l - 1 (Collvin, unpublished). The reduction in growth rate of copper-exposed perch is therefore mainly the result of an increase in standard metabolic rate. When perch acclimate to copper, the metal is continuously transported from the water via the gills to the liver (Collvin, 1984b). Furthermore, perch actively regulate the copper concentration in the gills and probably in the liver, which indicates that detoxicatory and excretory processes exist. Such physiological processes will require additional energy and cause the standard metabolic rate to increase. Since the food conversion efficiency reduced and since its variation followed that of the growth rate, I assume that the perch take this energy from the food. Copper interacts with the ligands of proteins and, therefore, affects the catalytic capacity of enzymes and the transport ability of membranes (Passow et al., 1961; Luoma, 1983). This leads to reduced efficiency of physiological reactions and may stimulate a higher production rate of, e.g., enzymes to generate the amount required to fulfill given physiological tasks. Thus, any copper-induced increase in synthesis o f substances will be expected to cause a higher rate of standard metabolism. Food conversion rate and growth rate of the perch returned to normal after 18 days at 39/~g Cu" 1- 1, while at 81 #g Cu" 1- 1 a return may have been initiated after 24 days. Unexposed perch that consume a food ration of 4-6%" d a y - 1 of their dw will, under the conditions of this study, grow at a rate of 0 . 8 - 1 . 5 % - d a y -1 (Lessmark, 1983; Collvin, 1984a). Growth rate and food conversion efficiency of the perch at 81 #g Cu. 1- 1 are therefore initially reduced more than can be accounted for by a mere reduction in food consumption rate. There are, in contrast to perch exposed at 39/~g C u - l - 1, indications (P = 0.056) of a further and trough-like reduction over 12 days. The mean growth rate after 30 days is 0.20/o . d a y - 1 and perch
112
at 81 ~tg C u ' 1 - 1 are therefore not, in terms of standard metabolism, fully acclimated. The time required before the onset of metabolic acclimation and full compensation thus appears to increase with increased copper concentration. Does acclimation (return of standard metabolism to control level) mean that copper no longer affects the perch energetically? If a higher starvation rate is found in the copper-exposed, acclimated and starved perch then the copper still influences the perch. Since this was the case I suggest that acclimated perch continue to take up and excrete copper. Why did the maintenance cost for detoxication eventually vanish for maximally feeding perch at 40 ttg Cu" 1- 1? Copper-exposed fish increase their concentration of metallothionein in the liver (Roch et al., 1982). This substance is ascribed the ability to detoxify, store and transport copper (Cherian and Goyer, 1978). Whatever substance(s) is/are responsible for detoxication of copper in perch, it/they require(s) ca. 20 days to reach full detoxicating strength at 40/~g Cu" 1- 1 (Collvin, 1984b; this study). The build up of detoxicating substances implies a mechanism for detoxication, energy for synthesis and elements for substance formation. The results show that at 40/tg C u ' 1- 1 the food supplied the perch with enough energy and elements for detoxication. Therefore, any effect appears to relate to the efficiency of the mechanism. When the detoxicating substance is synthesized at a rate sufficient tc detoxify all copper f r o m the inward flux of copper, it is then possible that vital physiological processes are left unaffected and that the perch may acclimate.
ACKNOWLEDGEMENTS
I thank Dr. A. S6dergren, Dr. P. Larsson and Dr. I.J. Winfield for constructive criticism of the manuscript.
REFERENCES Brett, J.R. and T.D.D. Groves, 1979. Physiological energetics. In: Fish physiology. Vol. VllI, Bioenergetics and growth, edited by W.S. Hoar, D.J. Randall and J.R. Brett, Academic Press, London, pp. 279-352. Brown, M.E., 1957. Experimental studies on growth. In: Physiology of fishes. Vol. I, edited by M.E. Brown, Academic Press, London, pp. 361-400. Cherian, M.G. and R.A. Goyer, 1978. Minireview. Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci. 23, 1-10. Collvin, L., 1984a. The effects of copper on maximum respiration rate and growth rate of perch, Perca fluviatilis L. Water Res. 18, 139-144. Collvin, L., 1984b. Uptake of copper in the gills and liver of perch, Percafluviatilis. Ecol. Bull. 36, in press. Dixon, D.G. and J.B. Sprague, 1981. Acclimation to copper by rainbow trout (Salmo gairdner~) - A modifying factor in toxicity. Can. J. Fish. Aquat. Sci. 38, 880-888. Farmer, G.J., D. Ashfield and H.S. Samant, 1979. Effects of zinc on juvenile atlantic salmon Salmo salar: Acute toxicity, food intake, growth and bioaccumulation. Environ. Pollut. 19, 103-117.
113 F~inge, R. and D. Grove, 1979. Digestion. In: Fish physiology. Vol. VIII, Bioenergetics and growth, edited by W.S. Hoar, D.J. Randall and J.R. Brett, Academic Press, London, pp. 161-260. Lessmark, O., 1983. Competition between perch (Perca fluviatilis) and roach (Rutilus rutilus) in south Swedish lakes. Ph.D. Thesis, Univ. of Lund, Lund, Sweden, 172 pp. Lett, P.F., G.J. Farmer and F.W.H. Beamish, 1976. Effect of copper on some aspects of the bioenergetics of rainbow trout (Salmo gairdnert). J. Fish. Res. Bd. Can. 33, 1335-1342. Luoma, S.N., 1983. Bioavailability of trace metals to aquatic organisms - A review. Sci. Total Environ. 28, 1-22. O'Hara, J., 1971. Alterations in oxygen consumption by bluegills exposed to sublethal treatment with copper. Water Res. 5, 321-327. McKim, J.M. and D.A. Benoit, 1971. Effects of long-term exposure to copper on survival, growth, and reproduction of brook trout (Salvelinus fontinalis). J. Fish. Res. Bd. Can. 28, 655-662. Passow, H., A. Rothstein and T.W. Clarkson, 1961. The general pharmacology of the heavy metals. Pharmacol. Rev. 13, 185-224. Persson, L., 1979. The effects of temperature and different food organisms on the rate of gastric evacuation in perch (Perca fluviatilis). Freshwater Biology 11, 131-138. Roch, M., J.A. McCarter, A.T. Matheson, M.J.R. Clark and R.W. Olafson, 1982. Hepatic metallothionein in rainbow trout (Salmo gairdnert) as an indicator of metal pollution in the Campbell River System. Can. J. Fish. Aquat. Sci. 39, 1596-1601. Siegel, S., 1956. Nonparametric statistics for the behavioral sciences, McGraw-Hill International Book Company, London, 312 pp. SIS, t981. Standardiseringskommissionen i Sverige, Box 3295, 103 66 Stockholm, Sweden. Sokal, R.R. and F.J. Rohlf, 1969. Biometry. The principles and practice of statistics in biological research. Freeman and Company, San Francisco, 757 pp. Sprague, J.B., 1973. The ABCs of pollutant bioassay using fish. In: Biological methods for the assessment of water quality, edited by J. Cairns Jr. and K.L. Dickson, ASTM STP 528, Philadelphia, pp. 6-30. Waiwood, K.G. and F.W.H. Beamish, 1978. The effect of copper, hardness and pH on the growth of rainbow trout, Salmo gairdneri. J. Fish. Biol. 13, 591-598. Warren, C.E. and G.E. Davis, 1967. Laboratory studies on the feeding, bioenergetics, and growth of fish. In: The biological basis of freshwater fish production, edited by S.D. Gerking, Blackwell, Oxford, pp. 175-214.