Effect of temperature on the uptake of waterborne strontium in the common carp, Cyprinus carpio (L.)

Aquatic Toxicology 54 (2001) 151– 160 www.elsevier.com/locate/aquatox

Effect of temperature on the uptake of waterborne strontium in the common carp, Cyprinus carpio (L.) Mohammed J. Chowdhury 1, Ronny Blust * Department of Biology, Uni6ersity of Antwerp (RUCA), Groenenborgerlaan 171, B-2020 Antwerp, Belgium Received 30 August 2000; received in revised form 17 February 2001; accepted 19 February 2001

Abstract The effect of temperature on the uptake kinetics of strontium (Sr) in the common carp (Cyprinus carpio) was studied in vivo, exposing pre-acclimated fish to a wide range of Sr concentrations in water (Srtotal = 0.2– 10 000 mM; Catotal =348 mM) at 10, 20, 25 and 30°C. Sr uptake rates were determined in the whole body, gills and blood of the fish after an exposure period of 3 h and were analyzed as a function of the free-ion activity of Sr and Ca in water. The uptake of Sr2 + by the whole body, gills and blood increased with temperature and showed saturation kinetics with the increase of Sr2 + activity. Analyzing the observed uptake rates with a Michaelis– Menten type model showed that the kinetic parameters (Jmax, Km and Ki) for both Sr2 + and its analogue Ca2 + are temperature dependent. Thermodynamic analysis of the temperature effects indicates that the Arrhenius activation energies (Ea) required for Sr2 + uptake (91.9 kJ mol − 1) and Ca2 + uptake (105.9 kJ mol − 1) in the whole body of carp were constant over the temperature range 10–25°C and showed a break in the Arrhenius plots above this temperature. The Arrhenius plot for the Sr2 + uptake in blood was similar to that for the whole body uptake with an Ea of 98.1 kJ mol − 1. However, the Ea for Sr2 + uptake in gills was much smaller and constant (58.1 kJ mol − 1) over the temperature range of 10–30°C. For a temperature change from 10 to 25°C, the Q10 for Sr2 + uptake in whole fish, gills and blood were 3.71, 2.29 and 4.05, respectively. Compared with Ca2 + uptake, Sr2 + uptake appears to require a lower activation energy for transport across the solution body interface in carp. The similar pattern of Arrhenius plots and magnitude of activation energies for Sr2 + uptake both in blood and whole fish suggest that the uptake into the blood across the basolateral membrane is the rate-limiting energy barrier and hence dictates the overall uptake rate of Sr2 + in whole fish. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Strontium; Calcium; Temperature; Metal; Uptake kinetics; Carp

1. Introduction * Corresponding author. Tel.: + 32-3-2180347; fax: + 32-32180497. E-mail address: [email protected] (R. Blust). 1 Present address: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1.

Strontium (Sr) is a calcium resembling element accumulated in the bone and other tissues of organisms. As a result radioactive Sr, which can contaminate water bodies via the controlled dis-

0166-445X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 1 7 6 - X

152

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

charge of nuclear installations and nuclear accidents, poses an environmental concern (Polikarpov, 1966; Coughtrey and Thorne, 1983; Kryshev and Sazykina, 1995). Limited information concerning the effect of temperature on Sr uptake by fish or other organisms exists. Only a few older reports indicate that the uptake of 89Sr and 90Sr by fish increases with the increase of temperature (Polikarpov, 1966; Kulikov, 1973). Temperature is an important environmental factor determining the uptake and toxicity of metals by aquatic organisms. It has influences on both the solution chemistry of metals, and physiology of organisms (Cairns et al., 1975; Cossins and Bowler, 1987; Schmidt-Nielsen, 1993). Within limits, a temperature increase accelerates many physiological processes, most notably the many catalytic systems involved in transport and metabolism (Hochachka and Somero, 1989). Temperature influences metal uptake by determining the rate of reactions at the solution body interfaces, altering equilibrium conditions, and causing organizational changes in membranes (Webb, 1963; Hazel, 1993). Metal uptake is considered to involve an initial reaction of the free metal ion with membrane-embedded systems such as channels, carriers and pumps to form a temporary complex that undergoes dissociation for the translocation of the metal across the membrane (Simkiss and Taylor, 1995). Most aquatic organisms are poikilothermic, that is, they are unable to control their body temperature within narrow limits. As a result, their physiology is highly dependent on ambient temperature. In a typical fish, the heat generated by metabolism is carried via the blood to gills, where it is dissipated to the environment (Hazel, 1993). Consequently, tissue temperatures in most fish attain at or within 1°C of the ambient water temperature (Carey et al., 1971; Reynolds et al., 1976). The reliable prediction of metal accumulation by an organism requires that the effects of environmental factors including temperature are well understood and rational models are constructed. It is well known that waterborne Ca is an important factor affecting the branchial uptake of Sr (Rosenthal, 1957; Brungs, 1965; Suzuki et al., 1972) as well as other divalent metals such as

cadmium, cobalt and zinc by fish (Spry and Wood, 1988, 1989; Verbost et al., 1989; Comhaire et al., 1994; Hogstrand et al., 1994, 1996; Hollis et al., 2000). These metals are reported to interact with Ca2 + during uptake via the transcellular calcium transport system in fish gills (Flik et al., 1995). We have shown that Sr2 + and Ca2 + inhibit each other in a completely competitive way during uptake by carp, and this phenomenon can be described by a Michaelis–Menten type model (Chowdhury et al., 2000). Recently we have developed a model that accounts for both the effect of waterborne Ca2 + and the effect of water pH on Sr2 + uptake by carp which reveals a partially non-competitive interaction between protons and Sr2 + during uptake (Chowdhury and Blust, 2001). In the present study, we report on the nature of the effect of temperature on the uptake kinetics of waterborne Sr2 + and Ca2 + in carp to further extend the model. It was investigated whether the effect of temperature on Sr2 + and Ca2 + uptake was primarily due to changes in the Jmax and/or the Km of the uptake kinetics. In addition, the energy of activation for Sr2 + and Ca2 + uptake was determined to evaluate the temperature sensitivity of the uptake process.

2. Materials and methods

2.1. Experimental animals One-month-old carp ( 1.5 g), Cyprinus carpio, were obtained from the fish hatchery of the Agricultural University of Wageningen, The Netherlands, and were grown for at least three months in the laboratory at 259 1°C and a pH of 7.6–8.0. A standard protocol, as outlined by Chowdhury et al. (2000) for water quality and feeding, was followed during this rearing period. Prior to the experiments, the fish were kept for an acclimation period of 16–18 d in plastic tanks containing 150 l moderately hard freshwater (acclimation water). They were divided in four groups according to the exposure temperatures 10, 20, 25 and 30°C. For the groups of 10, 20 and 30°C, the desired temperature was reached gradually starting from 25°C

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

(standard holding condition) over a 3-d period, and then the fish were kept at the final temperature for 15 d. The acclimation water, consisting of NaHCO3 (1143 mM), MgSO4 (500 mM), CaCl2 (348 mM), and KCl (54 mM), was prepared by dissolving the reagent-grade salts (Merck, Darmstadt, Germany) in deionized water according to standard methods (Clesceri et al., 1989). For each acclimation group, 35– 40 individuals were held in each tank. The pH of the acclimation water was maintained at 7.89 0.2. The water in the tank was continuously recirculated through a submerged filter containing lava stone and synthetic − foam. The water quality criteria (for NH+ 4 , NO2 − and NO3 ) were maintained as mentioned in Chowdhury et al. (2000). The fish were fed daily with frozen chironomid larvae at 8% of their body weight, but starved for 24 h before exposure.

2.2. Experimental procedures After acclimation the carp (4.9390.76 g) were exposed to different Sr concentrations (Srtotal = 0.2, 10, 100, 1000, 5000 and 10 000 mM; Catotal = 348 mM; pH 8.0) for 3 h at the temperature of acclimation (10, 20, 25 and 30°C). The uptake of Sr2 + and Ca2 + in the whole body, gills, and blood of the carp over a 3-h period was followed in this experiment. For this, the fish were individually transferred to polypropylene beakers containing 0.5 l of exposure water having the same basic composition as the water of acclimation. The beakers were placed in a thermostatic water bath (90.5°C). The exposure water with different Sr concentrations was prepared by using analytical-grade SrCl2 (Merck, Darmstadt, Germany). The solutions were spiked with radiotracers (85Sr and 45Ca; Amersham, Bucks, UK) of which the activities, as measured in the water, varied between 753 and 1488 kBq l − 1 for 85Sr, and 512 and 1201 kBq l − 1 for 45Ca depending on exposure conditions. Both stable and radioactive metals were added 24 h before the start of the exposure to achieve equilibrium. In addition, 10 mM of noncomplexing buffer (H)EPPS (Sigma, St. Louis, MO) was added to the exposure solutions to keep the pH at 8.0 9 0.1. The concentration of the stable Sr in the solutions was measured with a

153

graphite furnace atomic absorption spectrophotometer (SpectraAA. 800Z, Varian, Australia), and that of Ca and other elements with an inductively coupled plasma atomic emission spectrometer (ICP-AES, Liberty Series II, Varian, Australia). After exposure, fish were rinsed in 0.5 l tracerfree rinsing solution containing 1 mM of stable Sr for 10 min to remove the tracers adsorbed to the body surface, as described in Chowdhury et al. (2000). Three minutes before the end of rinsing, 400 mM of MS 222 (ACROS, USA) was added to the solution to anesthetize the fish. Then each fish was placed in a 18 ml scintillation vial (Maxi-vial, Canberra Packard, Meriden, CT) containing 10 ml of the rinsing solution and counted for 1 min in a Minaxi-g Auto-gamma 5530 counter (Canberra Packard) to determine the whole-body radioactivity of 85Sr. After counting, :100–120 ml of blood and the whole mass of gill soft tissue of each fish were collected. The blood and gill tissues were solubilized in Soluene-350 tissue solubilizer and mixed with Hionic-Fluor liquid scintillation cocktail (Canberra Packard) in scintillation vials. The vials were counted for 15 min for the radioactivity of 85Sr and 45Ca. The procedures for the preparation and counting of blood and gill samples are presented in detail in Chowdhury et al. (2000). To measure the 45Ca in whole fish, the carcass of each fish was transferred individually to scintillation glass vials, dried at 60°C for 3–5 d, and ashed at 600°C in a muffle furnace. The ash content in the vials of each fish was dissolved in 1 ml of 20% HNO3. The solution was added with 20 ml of the scintillation cocktail Ultima Gold-AB (Canberra Packard). Then the vials were stored in a dark place for 24 h for stabilization and counted for 15 min in a Packard Tri-carb Liquid Scintillation Analyzer (Model 1900 TR) to determine the radioactivity of 45Ca. The total counts of 45Ca in the whole fish were determined by adding the counts in the blood and gill samples to that of the carcass of each fish. The 45Ca counts were corrected according to the method described by Van Ginneken and Blust (1995) to eliminate the bcounts contributed by 85Sr and for color quenching in the solutions containing the ashes.

154

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

2.3. Calculation of uptake rates The uptake rates of Sr2 + and Ca2 + in the whole fish, gills, and blood ( jM in mmol kg − 1 h − 1) over 3 h were calculated using the following equation: jM = CM/(60EMWtSM)

(1)

where CM is the 85Sr or 45Ca activity (cpm) of the whole fish, gills, or blood after corrections for background radiation and radio-decay, EM, the counting efficiency of the tracers, W, the wet weight (kg) of the sample, t, the exposure time (h), and SM, the specific activity of the tracers in the exposure water (Bq 85Sr or 45Ca per mmol total Sr or Ca). The uptake rates of Sr2 + and Ca2 + for the gill samples were corrected for the blood trapped in the soft gill tissue according to the method described by Van Ginneken et al. (1996).

2.4. Kinetic modeling The data for the uptake of Sr2 + by whole fish, gills and blood were analyzed using a reversible reaction model for mediated transport (Weiss, 1996). Assuming equilibrium conditions, the relationship between the free metal ion activity and uptake rate is the Michaelis– Menten equation: jSr = Jmax(Sr2 + )/[Km +(Sr2 + )]

(2) −1

−1

where jSr is the uptake rate (mmol kg h ) of Sr2 + by carp, Jmax, the apparent maximum rate of Sr2 + uptake at pH 8.0, Sr2 + , the activity of free Sr ion in water (mM), and Km (mM), the apparent half-saturation constant for Sr2 + uptake in the presence of an inhibitor (Ca2 + ). Our previous study indicates that waterborne Ca2 + inhibits Sr2 + uptake and vice versa competitively by changing the apparent Km, but not Jmax (Chowdhury et al., 2000). To account for the inhibitory effect on metal uptake, a Michaelis– Menten type model for competitive inhibition was used (Weiss, 1996): jM = JmaxM(M 2 + )/[Km{1 + (I 2 + )/(Ki) +(M 2 + )}] (3) where JmaxM is the apparent maximum rate of Sr2 + or Ca2 + uptake by carp at pH 8.0, M 2 +

(mM), the activity of the free metal ion in question, Km (mM), is the true half-saturation constant for the metal in the uninhibited process and Ki (mM), the inhibitor constant for the activity of the inhibiting metal ion (I 2 + = Ca2 + or Sr2 + ).

2.5. Chemical speciation The equilibrium concentrations and activities of free Sr2 + and Ca2 + in the exposure solutions were calculated using MinteqA2, a geochemical assessment model for environmental systems (Allison et al., 1991). The database of stability constants in MinteqA2 was used for the equilibrium reactions of most common elements. The database was extended with stability constants of reactions not included in the original one, such as the Sr species. The necessary stability constants and enthalpies were taken from the National Institute of Standards and Technology database (Martell and Smith, 1997).

2.6. Statistics The kinetic parameters (Jmax, Km, Ki) were estimated by iterative fitting of the Michaelis– Menten type equations using the nonlinear regression methods included in the computer software Statistica (StatSoft, Tulsa, OK). The fitting of the models was evaluated with the coefficients of determination (r 2) and significance levels (p) of the parameter estimates. One-way analysis of variance was used to find significant differences among uptake rates and parameter estimates for different temperatures. Specific differences were detected using the Duncan new multiple range test.

3. Results

3.1. Chemical speciation Chemical speciation modeling showed that changes in temperature had a small effect on the speciation of Sr and Ca at constant hydrogen ion activity. Over the entire range of experimental temperatures (10–30°C), the concentration of free

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

Sr2 + and Ca2 + ions varied between 95% and 98% of the total metal concentrations in the exposure water (Srtotal =0.2– 10 000 mM; Catotal =348 mM; pH: 8.0). However, the speciation of the metal ions changed considerably with the changes in the concentration of Sr in the exposure water. The most important effect was the change in the ionic strength of the medium that ranged from 0.008 M at 0.2 mM Sr (10°C) to 0.037 M at 9761 mM Sr (30°C). The activity coefficients of the metal species depend on the charge of the ion, the ionic strength and temperature of the medium. For the present experimental conditions, the activity coefficients calculated using the Davies equation decreased from 0.692 to 0.484 for both Sr2 + and Ca2 + . Due to this effect the free Sr2 + activity varied between 0.133 and 4545 mM, and the free Ca2 + activity varied between 230 and 165 mM with the increase of the nominal concentrations of Sr ranging from 0.2 to 10 000 mM (actual 0.2– 9761 mM) in the exposure water. At any given total concentration of the metals, the activity of free Sr2 + and Ca2 + did not vary by more than 3% with the change in temperature from 10 to 30°C.

155

3.3. Modeling the uptake of strontium The uptake rates of Sr2 + in whole fish at different temperatures were analyzed using the Michaelis–Menten type model (Eq. (2)), assuming that only the free ionic form of the metal in the exposure water is available to the fish. The model explains 96–98% of the variation in Sr2 + uptake at Sr concentrations ranging from 0.2 to 10 000 + = 0.13–4669 mM) and at experimenmM (Sr2activity tal temperatures ranging from 10 to 30°C. The estimated values, as presented in Table 1, show that the apparent Jmax increased significantly (PB 0.001) with the increase of temperature up to 25°C and then decreased at 30°C. With the change of temperature, the values for the apparent Km increased showing a minimum at 25°C (860.3937.7 mM). However, the difference between the apparent Km for 25 and 30°C was not statistically significant (P\ 0.05). The uptake rates of Sr2 + in the gills and blood were also modeled by fitting the same equation (Eq. (2)) to the observed data. The model explains 87–95% of variations in Sr2 + uptake of gills, and

3.2. Uptake of strontium and calcium by carp At all temperatures, Sr2 + uptake rates showed saturation kinetics with the increase of Sr2 + activity in the water, indicating that a facilitated process is involved (Fig. 1(A) and Fig. 2(A)). With increasing temperatures of exposure, the uptake rates of Sr2 + in whole fish and blood increased up to 25°C, and decreased significantly (PB 0.001) at 30°C. The uptake rates in gills also increased up to 25°C, but those at 25 and 30°C were not significantly different (P \0.25). The uptake of Ca2 + decreased with increasing 2+ Sr activity in the water (Fig. 1(B) and Fig. 2(B)), revealing the competitive nature of the interaction between these two ions. For a given concentration of Sr2 + in water, the uptake of Ca2 + was also observed to increase with increasing temperature of exposure up to 25°C, as found for Sr2 + . At 30°C, the uptake rates of Ca2 + were significantly (PB0.05) lower than those at 25°C in whole fish, but this was not observed for gills and blood (Fig. 1(B) and Fig. 2(B)).

Fig. 1. Effect of Sr2 + activity in the exposure water on the kinetics of Sr2 + (A) and Ca2 + (B) uptake in the whole body of carp at 10 ("), 20 (), 25 ( ), and 30 ( )°C. Data points represent mean uptake rates with standard deviations over a period of 3 h (n = 5 –7; Srtotal =0.2 – 10 000 mM; Catotal =348 mM; pH: 8.0). The solid lines are predictions by a competitive inhibition model of the Michaelis – Menten type (Eq. (3)).

156

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160 Table 2 Uptake of Sr2+ in gills and blood of carp at different temperatures (pH: 8.0): estimates of the apparent Jmax and Km with standard error ( 9 SE) and coefficients of determination (r 2)a for the nonlinear fitting curves Temperature (°C) Gills 10 20 25 30 Blood 10 20 25 30

Fig. 2. Effect of Sr2 + activity in the exposure water on the kinetics of Sr2 + (A) and Ca2 + (B) uptake in the gills and blood of carp at 10 ("), 20 (), 25 ( ), and 30 ( )°C. Data points represent mean uptake rates with standard deviations over a period of 3 h (n= 5–6; Srtotal = 0.2–10 000 mM; Catotal =348 mM; pH: 8.0). The solid lines are predictions by a Michaelis – Menten type model (Eq. (2)).

92 –96% of variations in Sr2 + uptake of blood with the same ranges of Sr concentrations and experimental temperatures. The estimated values of the kinetic parameters (Table 2) show that the apparent Jmax for gills increased with the increase of temperature from 10 to 30°C. The apparent Jmax for blood increased up to 25°C, then decreased significantly at 30°C, as found for whole fish. For both the gills and blood, the values of the apparent Km did not show any significant difference over the temperature range tested.

Jmax

Km

r2

n

206.1 926.0b 504.9 940.4b 872.1 927.1 968.6 963.7

1545.1 9524.4 1105.3 9 441.9 877.5 996.4 811.4 9197.5

0.95 0.87 0.91 0.95

32 36 32 36

39.5 94.9b 102.2 910.0b 365.6 916.2 177.0 916.5b

1850.8 9575.6 1555.3 9421.0 1687.6 9203.0 1617.2 9 410.7

0.95 0.94 0.92 0.96

32 36 32 36

The parameters (Jmax in mmol kg−1 h−1 in tissue and Km in mM free-ion activity in water) are derived from a Michaelis– Menten type model (Eq. (2)) for Sr2+ uptake. All estimates are statistically significant (PB0.01). b Significantly different from the value at 25°C for the same tissue (PB0.001). a

4. Discussion The uptake of Sr2 + in the present study showed saturation kinetics at all experimental temperatures (10–30°C), indicating that the uptake is a facilitated process (Stein, 1990). The observed saturation kinetics of Sr2 + uptake are consistent with our previous results on Sr2 + and Ca2 + uptake under variable environmental conditions such as Ca-hardness (Chowdhury et al., 2000) and pH (Chowdhury and Blust, 2001). The change in

Table 1 Uptake of Sr2+ by whole fish at different temperatures (pH: 8.0): estimates of the kinetic parameters with standard error ( 9 SE)a and coefficients of determination (r 2) for the nonlinear fitting curves Temperature (°C)

Apparent Jmax (mmol kg−1 h−1)

Apparent Km (mM)

KmSr (mM)

KiCa (mM)

r2

na

10 20 25 30

40.69 0.7b 155.49 4.0b 296.49 3.5 219.7911.9b

1479.3 977.9b 1517.5 9109.6b 860.3 937.7 1026.1 986.3

226.6 98.8b 298.0 916.5b 113.6 93.1 366.1 922.7b

35.7 91.7 47.0 9 3.1 27.5 90.9 107.3 9 9.8b

0.98 0.96 0.97 0.97

36 36 36 36

(69) (71) (71) (71)

a The apparent Jmax and Km are derived from Eq. (2) (n =36) and represent the values at pH 8.0 and Catotal =348 mM, respectively. KmSr and KiCa are the true parameter values for the uninhibited process, and are derived from Eq. (3) for the whole body uptake of Sr2+ (n in parentheses). b Significantly different from the value for 25°C in the same column (PB0.001).

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

the apparent Jmax of Sr2 + with temperature (Jmax(10 – 25°C) =40.6– 296.4 mmol kg − 1 h − 1; Jmax(30°C) = 219.7 mmol kg − 1 h − 1) reveals that the uptake process is temperature dependent. In the present study, the carp were acclimated to the temperatures of exposure (10– 30°C) for 16 –18 d, and this range of temperatures is well within the tolerance limits (5– 30°C) for carp (Martinez et al., 1988). However, the uptake of Sr2 + and Ca2 + at 30°C was significantly lower than that at 25°C (Fig. 1 and Fig. 2). The decreased uptake indicates that the characteristics of the biological interface are altered between 25 and 30°C resulting in a slower uptake rate despite a higher exposure temperature. The Km, which is inversely proportional to the affinity of the substrate for the binding site, is often affected by changes in ambient temperature. The effect of temperature on the weak bonding forces that stabilize proteins and other biochemical structures, is considered the thermodynamic basis for temperature induced changes in Km (Hazel and Prosser, 1974; Hazel, 1993). The kinetic data for most of the enzymatic or metabolic processes in fish exhibit a minimum Km at temperatures corresponding to the acclimation or optimum temperature, resulting in a U-shaped Km-temperature response (Hazel and Prosser, 1974; Somero and Hochachka, 1976; Hochachka and Somero, 1989). The lowest Km for the whole body uptake of Sr2 + at 25°C agrees with this observation. However, the apparent Km for gills and blood did not change significantly with temperature (Table 2). This may be attributed to the fact that Sr2 + is continuously transferred from gills and blood to other tissues, and as a result, the uptake rates of Sr2 + for gills and blood do not represent total uptake over the exposure period of 3 h. Temperature influences reaction rates by changing the proportion of reactive molecules in a given population that possesses the energy of activation (Ea) to react (Hazel and Prosser, 1974; Cossins and Bowler, 1987; Horton et al., 1993). The temperature dependence of a reaction can be described by the Arrhenius equation: 6 = A exp[ − Ea/RT], where 6 represents the observed velocity of a reaction, A the Arrhenius pre-expo-

157

nential constant related to the collision frequency of molecules, R the universal gas constant, and T the absolute temperature. The natural logarithm of the reaction rate plotted against reciprocal temperature yields a straight line with a slope of − Ea/R (Cossins and Bowler, 1987). The activation energies for Sr2 + uptake (EaSr) in whole fish (Fig. 3(A)) and blood (Fig. 3(B)) calculated from the linear relationship between the reciprocal temperatures ranging from 10 to 25°C and the natural logarithm of the apparent Jmax for Sr are 91.9 and 98.1 kJ mol − 1, respectively. For whole fish and blood the apparent Jmax for Sr is considerably lower at 30°C than at 25°C, resulting in a break in the curve. However, the EaSr for gills was considerably smaller and constant (58.1 kJ mol − 1) showing no break in the Arrhenius plot over the temperature range of 10–30°C (Fig. 3(B)). The activity of the basolateral Ca2 + -ATPase which is, together with apical Ca2 + channels, responsible

Fig. 3. (A) Arrhenius plot for the effect of temperature on the uptake rates of Sr2 + ( ) and Ca2 + () in the whole body of carp. (B) Arrhenius plot for the effect of temperature on the uptake rates of Sr2 + in the gills ( ) and blood (") of carp. Data points represent the apparent maximum uptake rates (Jmax) of Sr2 + and Ca2 + at pH 8.0. The Arrhenius activation energies (Ea in kJ mol − 1), calculated from the slopes of the regression lines, are shown.

158

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

for the transepithelial transport of Ca2 + (Flik et al., 1995, 1996) and probably of Sr2 + in fish, is highly temperature sensitive, exhibiting a break mostly at about 20°C in the Arrhenius plot (Lee et al., 1994; Thomas and Karon, 1994). As a result, the basolateral uptake of Sr2 + in blood probably showed a distinct break in the Arrhenius plot, while apical uptake of Sr2 + in gills did not over a temperature range of 10– 30°C. Moreover, the high energy of activation for uptake in whole body and blood compared to uptake in gills indicates that the transport of the metal across the basolateral membrane of the gill epithelium is the rate limiting step rather than the transport across the apical membrane. Since the uptake of Sr2 + in carp is found to be inhibited by waterborne Ca2 + by changing the apparent Km (Chowdhury et al., 2000), it is of interest to determine the true Km for Sr2 + uptake (KmSr) and its Ki for the inhibition of Ca2 + (KiCa) at different temperatures. For this, the uptake rates of Sr2 + under variable Ca concentrations (Catotal = 10–10 000 mM; Srtotal =0.27 – 2.0 mM) were calculated. If the energy of activation is known, the velocity of a reaction for one temperature can be calculated from the reaction rate at another temperature from the equation: loge61 = Ea/R[1/T2 –1/T1]+loge62 (Cossins and Bowler, 1987). The uptake rates of Sr2 + at 10 and 20°C were calculated from this equation, using the energy of activation for Sr (EaSr =91.9 kJ mol − 1), and the known uptake rates in the whole body of carp at 25°C (pH: 8.0) for the same concentrations of Sr and Ca (Chowdhury et al., 2000). The Michaelis–Menten type model for competitive inhibition (Eq. (3)) was fitted to the pooled data of the calculated and observed Sr2 + uptake rates in whole fish for different temperatures, using the previously estimated values (Table 1) for the apparent Jmax of Sr. The estimated values of KmSr and KiCa for the whole body uptake of Sr2 + are presented in Table 1. The activation energy for the whole body uptake of Sr2 + at 30°C was not determined since only two data sets (25 and 30°C) were available, and thus, the KmSr and KiCa for this temperature were estimated by fitting the same model (Eq. (3)) only to the data obtained in this study. The curves as shown in Fig. 1(A),

explain most of the observed variations in Sr2 + uptake by whole fish for the tested temperatures. As found for the apparent Km of Sr2 + , the values for KmSr and KiCa increased with the increase or decrease of temperature from 25°C, indicating that the kinetic parameters for both Sr2 + and Ca2 + are temperature sensitive. However, the relative sensitivity appears to be higher for KmSr than KiCa at low temperatures; with a temperature decrease of 15°C (25–10°C), the KmSr increases by a factor of 2.4–3.1, while KiCa by a factor of 1.3– 1.6. Since Sr2 + and Ca2 + interact competitively for the same uptake system (Chowdhury et al., 2000), the values of KmSr and KiCa for Sr uptake should be equal or almost equal to those of KmCa (true Km for Ca) and KiSr for Ca2 + uptake, i.e., KmSr : KiSr and KmCa : KiCa. Therefore, we can estimate the apparent Jmax of Ca (JmaxCa) by fitting the competitive inhibition model (Eq. (3)) to the observed data for Ca2 + uptake at different temperatures. As shown in Fig. 1(B), the model yielded a good fit (r 2 = 0.74–0.88; n= 36) to the Ca2 + uptake data over the Sr concentrations of 0.2– + 10 000 mM (Sr2activity = 0.13–4669 mM). The estimated values for JmaxCa are 15.69 0.8, 61.69 2.7, 154.59 3.2 and 149.89 4.7 mmol kg − 1 h − 1 at 10, 20, 25 and 30°C, respectively. The Arrhenius plot (Fig. 3(A)) for the natural logarithm of JmaxCa also shows a similar trend with a break at 25°C, and an activation energy (EaCa) of 105.9 kJ mol − 1 for the temperature range 10–25°C. The higher activation energy of Ca2 + than Sr2 + indicates that Ca2 + faces a higher energy barrier for transport across the solution body interfaces in carp. The Arrhenius activation energy is an enthalpy-derived parameter and can be expressed as Ea = DH + RT, where DH is the enthalpy of activation for the reaction (Webb, 1963; Cossins and Bowler, 1987). The calculated enthalpy of Ca2 + uptake (103.5 kJ mol − 1) for the temperature range 10–25°C is :1.15 times higher than that of Sr uptake (89.5 kJ mol − 1). This is consistent with the order of difference (1.10) for the hydration enthalpy of these metal ions (Ca: 1592.4 kJ mol − 1, Sr: 1444.7 kJ mol − 1) (Burgess, 1978). The movement of an ion through a channel often involves a dehydration step. The higher the energy

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

of hydration the more energy is required to strip off the water of hydration surrounding the central ion and the higher the enthalpy of uptake. When the energy of activation (Ea) for a reaction rate is known, the temperature coefficient (Q10) for any temperature change for constant Ea can be calculated from the thermodynamic expression: Q10 = exp[Ea/R{10/(T1T2)}], where T1 and T2 are absolute temperatures (SchmidtNielsen, 1993). The Ea for Sr2 + uptake in the whole body (91.9 kJ mol − 1), blood (98.1 kJ mol − 1) and gills (58.1 kJ mol − 1) of carp were constant over the temperature range 10– 25°C, and the calculated Q10 of Sr2 + uptake for this temperature range were 3.71, 4.05 and 2.29, respectively. The lower Q10 for gills than blood indicates that the process of Sr2 + uptake across the apical membrane is less sensitive to temperature than that across the basolateral membrane of the epithelium. In conclusion, the uptake of Sr2 + by the whole body, gills and blood of carp increased with temperature and showed saturation kinetics with the increase of Sr2 + activity in water. Analyzing the observed uptake rates with a Michaelis–Menten type model has shown that the kinetic parameters (Jmax, Km and Ki) for both Sr2 + and Ca2 + are temperature sensitive. Thermodynamic analysis of the temperature effect indicates that the free energy required for Sr2 + uptake in carp is constant over the temperature range 10– 25°C. Compared with Ca2 + uptake, Sr2 + uptake appears to require a lower activation energy for transport across the solution body interfaces in carp. The similar pattern of Arrhenius plots and magnitude of activation energies for Sr2 + uptake both in blood and whole fish suggest that the uptake across the basolateral membrane into the blood is the rate-limiting energy barrier and hence dictates the overall uptake in the whole body of carp.

Acknowledgements We thank the European Commission for financial support provided for this work under the Nuclear Fission Safety RTD program (Radiation protection contract c F14P-CT95-0018). M.J.C.

159

is thankful to the University of Chittagong, Bangladesh for granting study leave. References Allison, J.D., Brown, D.S., Novo-Gradac, K.J., 1991. MinteqA2/ProdefA2, a geochemical assessment model for environmental systems, EPA/600/3-91/021, USEPA Environmental Research Lab, Athens, GA, USA. Brungs, W.A., 1965. Experimental uptake of strontium-85 by freshwater organisms. Health Phys. 11, 41 – 46. Burgess, J., 1978. Metal Ions in Solution. Ellis Horwood, Chichester. Cairns, J. Jr., Heath, A.G., Parker, B.C., 1975. The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologia 47, 135 – 171. Carey, F.G., Teal, J.M., Kanwisher, J.W., Lawson, K.D., Beckett, J.S., 1971. Warm-bodied fish. Am. Zool. 11, 137 – 145. Chowdhury, M.J., Blust, R., 2001. A mechanistic model for the uptake of waterborne strontium in the common carp (Cyprinus carpio L.). Environ. Sci. Technol., 35, 669 – 675. Chowdhury, M.J., Van Ginneken, L., Blust, R., 2000. Kinetics of waterborne strontium uptake in the common carp, Cyprinus carpio, at different calcium levels. Environ. Toxicol. Chem. 19, 622 – 630. Clesceri, L.S., Greenberg, A.E., Trussell, R.R., 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, Washington, DC. Comhaire, S., Blust, R., Van Ginneken, L., Vanderborght, O.L.J., 1994. Cobalt uptake across the gills of the common carp, Cyprinus carpio, as a function of calcium concentration in the water of acclimation and exposure. Comp. Biochem. Phys. 109C, 63 – 76. Cossins, A.R., Bowler, K., 1987. Temperature Biology of Animals. Chapman & Hall, London. Coughtrey, P.J., Thorne, M.C., 1983. Radionuclide Distribution and Transport in Terrestrial and Aquatic Ecosystems: A Critical Review of Data, vol. 1. A.A. Balkema, Rotterdam. Flik, G., Verbost, P.M., Wendelaar Bonga, S.E., 1995. Calcium transport processes in fishes. In: Wood, C.M., Shuttleworth, T.J. (Eds.), Fish Physiology. Cellular and Molecular Approaches to Fish Ionic Regulation, vol. 14. Academic Press, New York, pp. 317 – 343. Flik, G., Klaren, P.H.M., Schoenmakers, T.J.M., Bijvelds, M.J.C., Verbost, P.M., Wendelaar Bonga, S.E., 1996. Cellular calcium transport in fish: unique and universal mechanisms. Physiol. Zool. 69, 403 – 417. Hazel, J.R., 1993. Thermal biology. In: Evans, D.H. (Ed.), The Physiology of Fishes. CRC Press, Boca Raton, FL, pp. 427 – 467. Hazel, J.R., Prosser, C.L., 1974. Molecular mechanisms of temperature compensation in poikilotherms. Physiol. Rev. 54, 620 – 677.

160

M.J. Chowdhury, R. Blust / Aquatic Toxicology 54 (2001) 151–160

Hochachka, P.W., Somero, G.N., 1989. Biochemical Adaptation. Princeton University Press, Princeton. Hogstrand, C., Wilson, R.W., Polgar, D., Wood, C.M., 1994. Effects of zinc on the kinetics of branchial calcium uptake in freshwater rainbow trout during adaptation to waterborne zinc. J. Exp. Biol. 186, 55 –73. Hogstrand, C., Verbost, P.M., Wendelaar Bonga, S.E., Wood, C.M., 1996. Mechanism of zinc uptake in gills of freshwater rainbow trout: interplay with calcium transport. Am. J. Physiol. (Regulatory Integrative Comp. Physiol.) 270, R1141 – R1147. Hollis, L., McGeer, J.C., McDonald, D.G., Wood, C.M., 2000. Effects of long term sublethal Cd exposure in rainbow trout during soft water exposure: implications for biotic ligand modeling. Aquat. Toxicol. 51, 93 –105. Horton, H.R., Moran, L.A., Ochs, R.S., Rawn, J.D., Scrimgeour, K.G., 1993. Principles of Biochemistry. Prentice Hall, New Jersey. Kryshev, I.I., Sazykina, T.G., 1995. Radiological consequences of radioactive contamination of the Kara and Barents Seas. J. Environ. Radioactivity 29, 213 – 223. Kulikov, N.V., 1973. Radioecology of freshwater plants and animals. In: Klechkovskii, V.M., Polikarpov, G.G., Aleksakhin, R.M. (Eds.), Radioecology. Wiley, New York, pp. 323 – 337. Lee, A.G., East, J.M., Henderson, I.M.H., Starling, A.P., Warmerdam, J.Q.M., 1994. Phospholipid requirements of the Ca2 + -ATPase. In: Cossins, A.R. (Ed.), Temperature Adaptation of Biological Membranes. Portland Press, London, pp. 13– 29. Martell, A.E., Smith, R.M., 1997. Critical stability constants of metal complexes database, version 3.0. National Institute of Standards and Technology (NIST), Gaithersburg, MD. Martinez, I., Viscor, G., Palomeque, J., 1988. Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes. J. Fish Biol. 32, 247 – 252. Polikarpov, G.G., 1966. Radioecology of Aquatic Organisms: The Accumulation, and Biological Effect of Radioactive Substances. Reinhold, New York. Reynolds, W.W., McCauley, R.W., Casterlin, M.F., Crawsha, L.I., 1976. Body temperatures of behaviorally thermoregulating largemouth black bass (Micropterus salmonides). Comp. Biochem. Physiol. A 54, 461 –472. Rosenthal, H.L., 1957. The metabolism of strontium-90 and calcium-45 by Lebistes. Biol. Bull. 113, 442 –450.

Schmidt-Nielsen, K., 1993. Animal Physiology: Adaptation and Environment. Cambridge University Press, Cambridge. Simkiss, K., Taylor, M.G., 1995. Transport of metals across membranes. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. Wiley, New York, pp. 1– 44. Spry, D.J., Wood, C.M., 1988. Zinc influx across the isolated, perfused head preparation of the rainbow trout (Salmo gairdneri ) in hard and soft water. Can. J. Fish Aquat. Sci. 45, 2206 – 2215. Spry, D.J., Wood, C.M., 1989. A kinetic method for the measurement of zinc influx in vivo in the rainbow trout, and the effects of waterborne calcium on flux rates. J. Exp. Biol. 142, 425 – 446. Somero, G.N., Hochachka, P.W., 1976. Biochemical adaptations to temperature. In: Newell, R.C. (Ed.), Adaptation to Environment: Essays on the Physiology of Marine Animals. Butterworths, London, pp. 125 – 190. Stein, W.D., 1990. Channels, Carriers and Pumps: An Introduction to Membrane Transport. Academic Press, San Diego. Suzuki, Y., Nakamura, R., Ueda, T., 1972. Accumulation of strontium and calcium in freshwater fishes of Japan. J. Radiat. Res. 13, 199 – 207. Thomas, D.D., Karon, B.S., 1994. Temperature dependence of molecular dynamics and calcium-ATPase activity in sarcoplasmic reticulum. In: Cossins, A.R. (Ed.), Temperature Adaptation of Biological Membranes. Portland Press, London, pp. 1 – 12. Van Ginneken, L., Blust, R., 1995. Sequential determination of a combined g/b and a pure g-emitter by gamma and liquid scintillation counting: application to the transport of metals across fish gills. Anal. Biochem. 224, 92 – 99. Van Ginneken, L., Comhaire, S., Blust, R., 1996. Determination of blood content in teleost gills using a liquid scintillation analyser: Procedure for the correction of branchial metal accumulation rates. Comp. Biochem. Phys. C 115, 33 – 40. Verbost, P.M., Van Rooij, J., Flik, G., Wenderlaar Bonga, S.E., 1989. The movement of cadmium through freshwater branchial epithelium and its interference with calcium transport. J. Exp. Biol. 145, 185 – 197. Webb, J.L., 1963. Enzyme and Metabolic Inhibitors. General Principles of Inhibition, vol. 1. Academic Press, New York. Weiss, T.F., 1996. Cellular Biophysics. The MIT Press, Massachusetts.

.