COUPLING OF METHYLMERCURY UPTAKE WITH RESPIRATION AND WATER PUMPING INFRESHWATER TILAPIA OREOCHROMIS NILOTICUS

RUI WANG,y MING-HUNG WONG,z and WEN-XIONG WANG*yyThe Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

zCroucher Institute for Environmental Sciences, Hong Kong Baptist University, Hong Kong

(Submitted 10 April 2011; Returned for Revision 23 May 2011; Accepted 16 June 2011)

Abstract—The relationships among the uptake of toxic methylmercury (MeHg) and two important fish physiological processes—respiration and water pumping—in the Nile tilapia (Oreochromis niloticus) were explored in the present study. Coupled radiotracer andrespirometric techniques were applied to measure simultaneously the uptake rates of MeHg, water, and oxygen under variousenvironmental conditions (temperature, dissolved oxygen level, and water flow). A higher temperature enhanced MeHg influx and theoxygen consumption rate but had no effect on the water uptake, indicating the influence of metabolism onMeHg uptake. The fish showeda high tolerance to hypoxia, and the oxygen consumption rate was not affected until the dissolved oxygen concentration decreased toextremely low levels (below 1mg/L). The MeHg and water uptake rates increased simultaneously as the dissolved oxygen leveldecreased, suggesting the coupling of water flux and MeHg uptake. The influence of fish swimming performance on MeHg uptake wasalso investigated for the first time. Rapidly swimming fish showed significantly higher uptake rates of MeHg, water, and oxygen,confirming the coupling relationships among respiration, water pumping, and metal uptake. Moreover, these results support that MeHguptake is a rate-limiting process involving energy. Our study demonstrates the importance of physiological processes in understandingmercury bioaccumulation in fluctuating aquatic environments. Environ. Toxicol. Chem. 2011;30:2142–2147. # 2011 SETAC

Keywords—Methylmercury Bioaccumulation Oxygen consumption Water pumping

INTRODUCTION

Mercury is widely distributed in the natural environment. Itsconcentration has risen in aquatic systems because of anthro-pogenic deposition in recent decades [1]. In freshwater organ-isms, fish are recognized as the largest accumulator of toxicmethylmercury (MeHg), because they can accumulate MeHgrapidly and eliminate it slowly. Methylmercury constitutesmore than 80% of the overall accumulated Hg in fish [2]because of its high trophic transfer potential [3]. ConsumingHg-polluted fish is a severe health risk to humans (e.g., theMinamata disease), making understanding the bioaccumulationprocesses important.

Numerous studies have demonstrated the huge influence ofenvironmental conditions on Hg bioaccumulation. However,environmental changes can modify a series of complex bio-geochemical processes, making explaining quantitatively theirinfluences on Hg bioaccumulation difficult [3–5]. A variety offactors are responsible for the varied Hg concentrations in fish,including chemical (e.g., metal concentration and metal speci-ation) and ecological (e.g., abundance and distribution oforganisms) factors, by directly affecting the waterborne anddiet-borne Hg uptake [6–8]. Besides those factors, aquaticorganisms have developed a series of physiological regulationsthat allow them to adapt to fluctuating environments. Whethersuch physiological regulations could influence the metal bio-accumulation is an intriguing and seldom studied question.

In the present study, we investigated the influences of threeenvironmental factors—temperature, dissolved oxygen level,and water flow condition—on MeHg uptake in freshwater fish.These factors could not only influence the fish distribution and

habitat in aquatic systems [9] but also could affect the fishphysiological behavior [10]. For example, the respirationactivity (or metabolism, in terms of oxygen consumption rate)of a fish varies with its surrounding temperature and its life stage(i.e., a higher metabolic rate in warmer waters and in larger fish[11,12]). Gill ventilation, accompanied by simultaneouschanges of water-pumping activity, is always enhanced underhypoxic conditions [13]. We hypothesized that these twophysiological factors, respiration and water pumping activity,may be related to MeHg uptake, because the uptake of tracemetals, including MeHg, is generally depicted in the literatureas carrier-mediated transport or an energy-involved process[14,15] and thus may be affected by the metabolic rate.The fish gill is not only the respiratory organ but also the majorsite for ion and water exchanges; therefore, changes inwater-pumping activity may affect the accompanying ionuptake and excretion if MeHg uptake is a rate-limitingprocess.

Nile tilapia (Oreochromis niloticus) was chosen as the modelorganism in the present study because it can survive within awide range of temperature and dissolved oxygen levels, and itcan accumulate Hg from the environment [8]. Such hightolerance to environmental fluctuation is an important reasonfor its wide distribution around the world. In the present study,fish physiology was assumed to be affected by the changes indifferent environmental factors (temperature, oxygen level,water flow), and the uptake rates of MeHg, water, and oxygenwere then determined simultaneously under these conditions.Radiotracers (Me203Hg and 3H2O) were used to quantify MeHgand water influx. Oxygen consumption rates were determined inenclosed respirometric systems. For the first time, we coupleddirect and simultaneous measurements of water pumping,respiration activity, and MeHg uptake, emphasizing the impor-tance of fish physiology in understanding metal bioaccumula-tion in fluctuating environments.

Environmental Toxicology and Chemistry, Vol. 30, No. 9, pp. 2142–2147, 2011# 2011 SETAC

Printed in the USADOI: 10.1002/etc.604

* To whom correspondence may be addressed(wwang@ust.hk).

Published online 28 June 2011 in Wiley Online Library(wileyonlinelibrary.com).

2142

MATERIALS AND METHODS

Experimental organisms

Small individuals of freshwater tilapia (Oreochromisniloticus, 4 to 6 g in wet wt and 5–7 cm in total length) werecollected from a local fish farm in Yuen Long, Hong Kong,and acclimated in an ecologically balanced aquarium withcirculating dechlorinated tap water and continuous aeration.The fish were then acclimated in the aquarium for at least onemonth at 258C before use and fed with clean commercial fishfood (<3 ng/g MeHg).

Measurement of MeHg, water, oxygen uptake rates

The radiotracer technique was employed to measure non-invasively the MeHg aqueous uptake in tilapia in response tochanges in ambient temperature, dissolved oxygen (DO) level,and water flow [3]. The g-emitting radioisotope Me203Hg wasused to track the mercury influx. The isotope Me203Hg wassynthesized from 203Hg(II) (t1/2¼ 46.6 d, in 1M HCl, specificactivity¼ 82–131 GBq/g, purchased from Eckert and Ziegler)following a well-established method [16] and stored in a 0.005-M Na2CO3 solution. A standard synthetic freshwater [17] wasused as the uptake water medium in all experiments. Theradioactivity of Me203Hg in the fish body was determinedperiodically with a Wallac 1480 wizard 3’’ gamma counter.The gamma emission of 203Hg was determined at 232 keV.Counting times were adjusted to acceptable counting errors ofgenerally less than 5%. The MeHg uptake rates (VMeHg, ng/g/h)were then calculated using the data obtained during the expo-sure period.

The radioisotope 3H2O was used to track water influx in fishgills at different ambient temperatures, DO levels, and waterflow speeds to investigate the relationship between water influxand MeHg influx. For each experiment, specific amounts of3H2O (15ml/L) were spiked into the corresponding exposurecontainers, which held synthetic freshwater. After 1 h of expo-sure to the radioactive water, the fish were then removed, rinsed,and dissected. Fish gills from both sides were taken, and gilltissues were weighed and digested using 4ml tissue solubilizer(Soluene-350, PerkinElmer) at 558C overnight in 20-ml liquidscintillation vials. Digestion was completed when the solutionbecame clear.

The exposure water medium was also sampled at the begin-ning and end of the uptake period. The water and tissue solutionsamples (2ml) were mixed thoroughly with 18ml liquidscintillation cocktail (OptiPhase Hisafe 3, Perkin-Elmer LifeScience) by mechanical shaking. The radioactivity of all sam-ples was measured with a Beckman LS-6500 liquid scintillationcounter. A series of standard water samples were also measuredfollowing the same method and were used for quantification anddata correction. Each treatment was replicated five times.Counting times were adjusted to ensure that any propagatedcounting errors were kept to less than 5%. The water uptakerates (Vwater) in gills were then standardized to microliter ofwater per gram (wet wt) per h (ml/g/h).

The oxygen consumption rate (VO2) in fish is commonly usedto represent the metabolic rate of fish [12]. In our experiments,VO2 was determined individually in an enclosed respirationchamber (730ml, Strathkelvin Instruments) under variousconditions. The upper side of each chamber had three openings.One was connected to an oxygen meter to record continuouslythe dissolved O2 levels, and the other two were sealed withsilicon stoppers during exposure. The respiration chamberswere filled with synthetic freshwater. The water medium was

stirred with a magnetic stirrer. The fish were placed individuallyin each chamber and protected from the stirring by a perforatedPetri dish at the bottom of the chamber. Each treatment had fivereplicates. The required initial DO level in the water mediumwas obtained by adjusting the amount of N2 and O2 bubblinginto the continuously stirred water medium. The oxygenelectrode was calibrated before each experiment, and anybubbles in the chamber were removed before exposure. Fishwere exposed in the enclosed chambers for 1 h only to minimizetheir consumption of DO. The VO2 was then calculated from theamount of DO loss and standardized to micrograms of O2 pergram (wet wt) per hour (mg/g/h).

Influences of temperature

The physiology and metabolic rate of fish are greatlyinfluenced by water temperature [11,18], and thus the metaluptake rate of fish may be affected. In this experiment,O. niloticus were first acclimated in standard synthetic fresh-water under different temperatures (21, 27, and 328C) for 2 d toevacuate their guts and to avoid any stress from sudden changesof temperature in the following experiments. The fish were thenexposed individually in polypropylene beakers containing 1 Lsynthetic freshwater (open system with aeration) at correspond-ing water temperatures with equilibrated Me203Hg (100 ng/L).The exposure lasted for 8 h, during which the fish were taken outat 2, 4, 6, and 8 h and washed in a nonradioactive water mediumfor 1 to 2min to remove the weakly absorbed isotope. Fish werereturned to the chambers after radioassays. The radioactivity ofthe water medium was also determined at each time point andwas found to have decreased by less than 20% during the wholeexperiment. The VMeHg (ng/g/h) was then calculated as the slopeof the linear regression between newly accumulated MeHg inthe whole fish body and the exposure time. The VO2 at the testedtemperatures was determined in the enclosed respiration cham-bers after 1 h of exposure. The water mediumwas aerated beforeexposure and the initial (average) DO levels were determined as7.8mg/L at 218C, 7.2mg/L at 278C, and 6.5mg/L at 328C.Besides, the Vwater in fish gills was also measured at thosetemperatures after 1 h exposure in the 3H2O-spiked syntheticfreshwater medium as described previously.

Influences of ambient oxygen levels and water flow

The Nile tilapia (O. niloticus) is renowned for its hypoxiatolerance [19]. In this experiment, we explored quantitativelythe influence of DO levels on fish physiology (respiration andwater pumping) and MeHg uptake. First, we monitored con-tinuously the decrease in DO levels and changes in fish behaviorin the enclosed chamber. The water medium was stirred with amagnetic stirrer (water flow speed approximately 0.1m/s), sothat the fish would keep swimming to avoid being carried awayby the current. The exposure began at a high DO level and lasteduntil the fish could not swim normally against the water flowbecause of hypoxia. Then, the uptake rates of MeHg, water, andoxygen were all determined in the enclosed respiration cham-bers under a series of initial ambient DO levels (1, 3, 6, 8mg/L)at room temperature (218C). To avoid the fluctuation of DOlevels, the radioisotopes (Me203Hg and 3H2O) were quicklyspiked into the chambers through the small opening to obtainthe required initial concentrations (100 ng/L MeHg and 15ml/L3H2O) just before exposure. After 1 h exposure, the VMeHg

(whole body) and Vwater (in fish gills) were then quantified.In the calculation of VMeHg, we ignored the influence of MeHgabsorption because it was insignificant compared with MeHg

Influence of fish physiology on MeHg accumulation in tilapia Environ. Toxicol. Chem. 30, 2011 2143

influx in O. niloticus [3]. The VO2 was calculated from theamount of DO loss during the exposure.

The well-controlled respirometric systemwas further used toexplore the influences of fish swimming activity on oxygen,water, and MeHg uptake. Fish swimming activities werecontrolled by changing the water flow speed in the enclosedchambers, because we found the fish would always keepswimming to avoid being carried away by the current.Therefore, different swimming speeds could be induced bychanging the water stirring speed. The uptake rates of oxygen,water, and MeHg were determined simultaneously under threedifferent water flow speeds (0, 0.09m/s, and 0.17m/s). Theinitial oxygen levels were all adjusted to 8mg/L, and thespecific amounts of Me203Hg (100mg/L) and 3H2O (15ml/L)were added to each chamber just before the experiments. Theexposure lasted for 1 h, during which the changes in DO levelswere recorded for the VO2 determination. Similarly, at the end ofexposure, fish were taken out and radioassayed to quantify theVMeHg, and then the fish gills were digested and measured todetermine the Vwater.

Statistical analysis

Significant differences between treatments were determinedby one-way analysis of variance followed by Tukey’s post hoctest, where p¼ 0.05 in all cases. When the uptake of water oroxygen was significantly affected by the environmental factors(temperature, DO level, water flow), linear regressions wereperformed for the relationships between the MeHg uptake andwater or oxygen uptake. Statistical significance of the regres-sion was tested by analysis of variance, and the regression wasquantified by coefficient of determination r2.

RESULTS AND DISCUSSION

Influence of water temperature

During the 8-h aqueous exposure, theMeHg concentration inO. niloticus increased linearly with exposure time at all testedtemperatures. The calculated uptake rates (VMeHg) increasedwith the increase in water temperature. The lowest VMeHg wasfound at 218C (14.9� 1.3 ng/g/h) and the highest at 328C(25.3� 1.5 ng/g/h), exhibiting a 1.7-fold variation (Fig. 1A).Two possible mechanisms might explain the reduced MeHguptake rates at low temperatures. The first mechanism is relatedto the speed at which water flowed toward the fish gills. A lowwater pumping rate may result in a slow water flux and thuscause a slow delivery of MeHg into the epithelium cells whenthe MeHg uptake is a rate-limiting process. To test this hypoth-esis, the short-term Vwater in fish gills was determined at varioustemperatures using radioactive 3H2O. The freshwater Niletilapia showed no significant difference in water pumpingactivity among the tested temperatures (Fig. 2A).

We further tested the second hypothesis that the MeHguptake might be affected by temperature when the uptake isan energy-involved process, because a rise in temperature couldenhance the metabolism of organisms [12]. Mitochondria-richchloride cells, also the main location of the gill Naþ-Kþ-ATPase, might be a possible transportation site of mercury[13]. To test this hypothesis, we blocked the function of chloridecells by adding the metabolic blocker ouabain, which is knownas an NaþKþATPase inhibitor [20]. During the 4-h aqueousexposure to 100 ng/L spiked MeHg, neither the fish exposure inthe ouabain-containing (50mmol/L) water medium nor the fishpre-exposed for 0.5 h to ouabain (100mmol/L) showed anysignificant decrease in VMeHg compared with control (synthetic

freshwater), as shown in Figure 3, suggesting that the fish werenot sensitive to ouabain in vivo.

Similar to our results, previous perfusion experiments havealso reported high MeHg flux into fish gills and intestine at hightemperatures [15,21]. However, Andres et al. [15] showed thatHg accumulation in the gills and guts of blue crab decreased inthe presence of ouabain, but it was not fully inhibited, indicatinga partially energy-dependent Hg uptake mechanism. The fishmetabolic rates in terms of VO2 were further determined atthe tested temperatures. It was noticed that the fish had sig-nificantly higher VO2 at 328C (0.59 �0.05mg/g/h) than at 218C

Fig. 1. Methylmercury (MeHg) uptake inOreochromis niloticus influencedby various environmental factors. (A) Uptake under different temperatures(218C, 278C, and 328C), VMeHg were calculated during 8-h exposure; (B)uptake under different oxygen levels (7.5, 5.5, 2.5, and 0.7mg/L) during 1-hexposure in enclosed chambers; (C) uptake at different water flow conditions(0, 0.09, and 0.17m/s representing the water flow speeds). Data aremean� standard deviations (n¼ 5). DO¼ dissolved oxygen.

2144 Environ. Toxicol. Chem. 30, 2011 R. Wang et al.

(0.48� 0.05mg/g/h) and 278C (0.38� 0.01mg/g/h) as shownin Figure 2A. A positive correlation (r2> 0.99, Fig. 3A) wasfound between VO2 and VMeHg, indicating the coupling ofoxygen consumption and MeHg uptake. Therefore, our resultssuggest that the MeHg uptake could be enhanced at a highmetabolism (high water temperature), supporting the energy-involved uptake mechanism of MeHg.

In fact, enhanced Hg aqueous uptake at high temperaturewas found in various aquatic organisms, such as fish, crabs [15],and cladocerans [22]. According to the energetic-basedbiokinetic model, the overall metal bioaccumulation could bewell predicted by determining the metal uptake and eliminationrate in organisms [23]. In contrast to Hg uptake, the effect ofwater temperature on Hg elimination is conflicting. Previousstudies have shown that the elimination of Hg was not signifi-cantly affected by temperature in the cladoceran Daphniamagna [22], clam Corbicula fluminea [24], and freshwatermollusks [25], whereas Fowler et al. [26] noticed an enhancedMeHg elimination in mussels maintained in a warmer environ-ment. For fish, Trudel and Rasmussen [27] suggested that theMeHg elimination was positively correlated to water temper-ature in long-term experiments, based on numerous data fromearlier studies. Because the overall metal accumulation dependson both accumulation and elimination, the increased aqueousuptake and increased elimination ofMeHg at a high temperaturemay lead to an inconclusive accumulation result. Nevertheless,fish living in warmer aquatic systems always tended to havehigher MeHg levels [28]. Besides the influence of an enhancedfish metabolic rate [12], the increase in MeHg productionattributable to a higher methylation rate in bacteria at higherambient temperature might be another reason for the elevatedMeHg levels in fish in warmer seasons [29]. Moreover, a similarpositive correlation between accumulation and temperaturealso has been reported for other metals, such as Cd, Pb, andCu [30,31], indicating the influence of metabolism on metalaccumulation.

Influences of dissolved oxygen levels

After the fish were placed into the enclosed system, the DOlevels decreased linearly, and the fish could swim normallyuntil the DO level dropped below 1mg/L, at which point theyexperienced severe hypoxia (a typical curve is shown in Fig. 4).In our experiments, the Nile tilapia even managed to survive at aDO level of 0.3mg/L with depressed swimming activity, indi-cating its high tolerance to hypoxia. Similarly, in the short-termMeHg uptake experiments, the fish showed constant VO2 at highDO levels (7.5, 5.5, 2.5mg/L on average; Fig. 2B), and the VO2

decreased significantly under hypoxic conditions (0.7mg/L onaverage; Fig. 2B). The calculated VMeHg increased gradually(from 10.6� 3.6 ng/g/h to 34.1� 4.5 ng/g/h; Fig. 1B) with thedecrease in ambient DO level (from 7.5mg/L to 0.7mg/L;Fig. 1B), indicating a higher accumulation of MeHg in fishexposed to hypoxia. Consistent with our results, the accumu-lation of Hg in carp (Cyprinus carpio) exposed to hypoxia washigher than that in carp under normoxic condition [32]. Inaddition, finding that the respiration frequency of O. niloticuswas enhanced significantly in an environment with a lower DOwas intriguing. Therefore, we posited that the elevated MeHguptake at low DO levels might be related to the enhanced waterpumping activity.

A short-termwater uptake experiment at the tested DO levelswas then conducted to test this hypothesis. As expected, thecalculated Vwater showed a good positive relationship(r2¼ 0.980, p< 0.01) with VMeHg, indicating the coupling of

Control Ouabain A Ouabain B

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Fig. 3. The influence of ouabain on methylmercury (MeHg) uptake inOreochromis niloticus. Control: fish were exposed in ouabain-free syntheticfreshwater for 4 h; Ouabain (A) fish were exposed in an ouabain-containing(50mmol/L) water medium for 4 h; Ouabain (B) fish were preexposed in anouabain-containing (100mmol/L) water medium for half an hour, followedby 4 h exposure in the ouabain-free synthetic freshwater medium. Thespiked MeHg concentration was 100 ng/L in all treatments. Data aremean� standard deviations (n¼ 5).

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Fig. 2. The relationships among VMeHg, VO2, and Vwater under variousenvironmental conditions. (A) Under different temperatures; (B) underdifferent oxygen levels; (C) at different fish swimming speeds. Data aremean� standard deviations (n¼ 5).

Influence of fish physiology on MeHg accumulation in tilapia Environ. Toxicol. Chem. 30, 2011 2145

water pumping and MeHg uptake processes (Fig. 2B). In fact,such a coupling of ventilation and metal uptake was also foundin shrimp [33] and green mussels [34], even though the relation-ship between metal uptake and oxygen level was contradictoryfor these two species: hypoxia condition elevated the Cdaccumulation in shrimp but reduced the Cd and Zn accumu-lation in green mussels. This was attributable to species-specificphysiological responses (hypoxia condition enhanced the ven-tilation of shrimp but depressed that of green mussels).

Under hypoxic conditions, a series of physiologicalresponses would be evoked to increase the oxygen extractionefficiency or to decrease the energy expenditures of fish.These include depressing the swimming activity; increasingthe respiration frequency; and increasing the use of aquaticsurface respiration [11,35,36]. Such compensation mechanismsare species-specific, and thus result in species-specific toleranceto hypoxia. For example, the white sturgeon showed a signifi-cant reduction in oxygen consumption under moderatelyhypoxic conditions [35], whereas the reduction was not sig-nificant in rainbow trout [36], even in a more hypoxic condition.In contrast, the VO2 of Nile tilapia in our experiment did notdecrease until the DO dropped to an extremely low level (1mg/L), indicating its efficient compensation capability to hypoxia.The depressed swimming activity and increased respirationfrequency at low DO levels observed in our study might allcontribute to its high tolerance.

Influence of fish swimming performance

Fish swimming performance is related not only to fish healthbut also to the ambient environmental conditions. For example,fish living in rivers generally swimmore than fish living in lakesor ponds. Because swimming requires energy, the question ofwhether it influences the energy-involved metal uptake processis intriguing yet seldom studied. To satisfy such a high energyrequirement, fish increase their respiration when they areswimming [37]. We then hypothesized that the uptake of MeHgmight be affected by swimming performance because of theincreased water flux toward fish gills and the enhanced meta-bolism in those swimming fish. This hypothesis was furthertested using a combination of respirometric and radiotracertechniques.

In this experiment, the swimming speed of fish was con-trolled by adjusting the water flow conditions (no flow, mid

flow, and high flow), with corresponding water flow speeds of 0,0.09 and 0.17m/s. The VMeHg, VO2, and Vwater were determinedsimultaneously after 1 h of exposure to 3H2O- and Me203Hg-spiked water media with an initial DO level of 8mg/L. Asexpected, the calculated VMeHg was the highest for the fastswimming fish (32.3� 4.1 ng/g/h) and the lowest for the restingfish (10.9� 1.5 ng/g/h), exhibiting a threefold variation(Fig. 1C). In addition, the uptake of water and oxygen infast-swimming fish were also 2.2 times and 2.9 times higherthan those of resting fish, respectively. A positive correlationwas found between VMeHg and VO2 (r2¼ 1.000, p< 0.001,Fig. 2C) as well as between VMeHg and VWater (r2¼ 0.975,p< 0.05, Fig. 2C), suggesting the coupling of respiration, waterpumping, and MeHg uptake processes. The influence of waterflow has seldom been considered in previous studies. Yedilerand Jacobs [32] reported that the fish in a water flow systemaccumulated a higher Hg level than the fish in resting water, butthe underlying explanation was not clear. Our study for the firsttime quantitatively revealed the coupling relationship betweenphysiological processes (respiration and water pumping) andMeHg uptake.

Implications for MeHg bioaccumulation in fish

In aquatic systems, fish can highly accumulate the toxicMeHg because of its fast uptake rate and extremely lowelimination rate [3]. Previous studies suggested that the uptakeof MeHg in aquatic organisms involved a number of mecha-nisms, including both passive and active pathways [14,38]. Inour study, the coupling of respiration and MeHg uptake in fishdirectly supported that energy was at least partially involved inMeHg uptake. Some previous evidence pointed out that theMeHg uptake was likely through an anion transport system(especially via the chloride transporters [14,38]), whereasothers showed that MeHg transport was through the channelsfor amino acid transport [39]. Besides, a ligand exchangereaction with a rate-limiting step during uptake process wasalso reported [38]. In the present study, a positive relationshipwas found between water flux and MeHg uptake in fish,indicating that the uptake of MeHg appeared to be limitedby the rate of water flux toward the gill membrane surface,which was in agreement with the ligand exchange process.

Fish are known to accumulate Hg through both aqueous anddietary sources [3], and the specific bioaccumulation processwas highly related to the surrounding environment. One intri-guing question concerning the Hg bioaccumulation in fish is theinterspecies and inter-site differences of Hg levels in fish. Theinfluences of environmental factors on Hg bioaccumulationhave been well characterized over the past decades. For exam-ple, fish living in wetlands had high Hg concentrations becauseof the high in situ methylation rate [40]. The physical andchemical properties of water, such as pH, salinity, dissolvedorganic matter, and competitive ions, could influence the bio-accumulation by affecting the Hg speciation [5,6,38], and thefood conditions (quantity and quality) could greatly influencethe Hg level in predatory fish by affecting their dietary uptake[7]. In contrast, the potential internal factors such as thephysiology of organisms were seldom investigated. In fact,fish physiology may change with the surrounding environmentand further affect bioaccumulation. The present study clearlyshows that changes of respiration and water pumping activitiesunder varied environmental conditions (temperature, DO level,water flow speed) could contribute to varied Hg concentrationsin fish. However, the present study tested only the influences ofphysiological processes on aqueous Hg uptake, whereas their

Time (min)0 100 200 300 400

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Fig. 4. Typical change of dissolved oxygen (DO) levels in the 730-mlenclosed chamberswith time as a result of oxygen consumption by fish. Onlyone fish (approximately 5 g wet wt) was placed in each enclosed chamber.The water medium was not stirred.

2146 Environ. Toxicol. Chem. 30, 2011 R. Wang et al.

influences on diet-borne uptake need further study. Based onour results, some practices in fish farming (e.g., suitable temper-ature, sufficient aeration, and high-density fish culture) may bebeneficial to minimize the Hg level in fish.

In summary, our study has shown the influence of variousenvironmental factors on MeHg uptake in O. niloticus. TheVMeHg was elevated with high temperatures, low oxygen levels,and in fast water flow systems. The coupling relationshipsamong VO2, Vwater, and VMeHg were quantitatively revealed,suggesting the important role of physiological processes (suchas water pumping and respiration) in understanding Hg uptakein various aquatic systems. Besides MeHg, such couplingrelationships might exist for other metals as well if the uptakeis a metabolically controlled or rate-limiting process.

Acknowledgement—This study was supported by a Collaborative ResearchFund (HKBU1/07C), and a General Research Fund (663009) from the HongKongResearchGrants Council.We thank the anonymous reviewers for theirhelpful comments.

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