Article pubs.acs.org/est
Metal Transfer among Organs Following Short- and Long-Term Exposures Using Autoradiography: Cadmium Bioaccumulation by the Freshwater Prawn Macrobrachium australiense Tom Cresswell,*,† Debashish Mazumder,† Paul D. Callaghan,† An Nguyen,† Michael Corry,† and Stuart L. Simpson‡ †
Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, Sydney, New South Wales 2232, Australia ‡ CSIRO Land and Water, Centre for Environmental Contaminants Research, Sydney, New South Wales 2232, Australia S Supporting Information *
ABSTRACT: The uptake, depuration, and organ distribution of the radioisotope 109Cd were used to explore the internal kinetics of this nonessential metal following accumulation from waterborne cadmium by the freshwater decapod crustacean Macrobrachium australiense. Short- (6 h) and long-term (7 to 14 days) exposures to the radioisotope in solutions of 0.56 μg Cd/L were followed by depuration in metal- and isotope-free water for up to 21 days. The anatomical distribution of the radionuclide was visualized using autoradiography at predefined time points. The gills did not become saturated with cadmium after 14 days of exposure and demonstrated a greater rate of cadmium uptake relative to the hepatopancreas. Cadmium concentrations decreased rapidly during depuration from both gills and hepatopancreas after short exposures but slowly following long-term exposures. This suggests that the duration of cadmium exposure influences the depuration rate for this organism. The study demonstrates the complex behavior of cadmium accumulated by M. australiense and improves our understanding of how exposure duration will influence the internal location and potential toxicity of metals.
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INTRODUCTION Metals and metalloids present in the Earth’s crust that are liberated during mineral extraction can often find their way into the aquatic environment and interact with organisms inhabiting those areas. It is well-known that metals such as cadmium accumulate in aquatic invertebrates through water and dietary exposures.1−4 In decapod crustaceans, the mechanism for cadmium accumulation from water has been documented as occurring through the binding to a membrane transporter (possibly zinc transporter) and in ionic form via membrane channels for the uptake of calcium, occurring mainly at the gills and other poorly characterized pathways via the gastrointestinal system.5−7 Once within the body, cadmium tends to accumulate within the hepatopancreas,3 where detoxification and storage takes place.1 What is less well-known is the kinetics of metal transfer between the organ of entry into the body and the hepatopancreas during differing periods of exposure or depuration post-exposure. The internal location and the rates of influx and efflux of metals will influence the toxicity metals exhibit when they exceed tissue-specific local toxicity thresholds for metal that is not detoxified (e.g., exceeds a threshold for metal present in a metabolically active form).8,9 Greater understanding of metal uptake and biochemical modification/incorporation within the organism also has © XXXX American Chemical Society
relevance to the development of water quality criteria (WQC) or guideline values (WQGVs) for metals and how these are applied for management of risks posed to aquatic decapod crustaceans exposed intermittently to contaminated waters. 9,10 Previous studies on changes in the organ biodistribution of cadmium in invertebrates have been reported by Soegianto et al.,11 Pedersen et al.,6 and White and Rainbow.12 Such studies frequently utilize controlled exposures to radio- or stable-metal isotopes, following which the organism is euthanized and dissected to enable examination and metal analyses on specific tissues. The majority of these studies have used room-temperature dissections, which, during the investigation of organ distribution of labile metal species, can potentially lead to the loss of organ integrity and leakage of labile elements. The use of sensitive radiotracer techniques, coupled with whole-body autoradiography, allows for a detailed examination of organ biodistribution of the metal of interest while ensuring maximal organ integrity.3,13−16 Received: Revised: Accepted: Published: A
December 21, 2016 March 5, 2017 March 16, 2017 March 16, 2017 DOI: 10.1021/acs.est.6b06471 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
5.5%; and crude ash, 11%) every third day, as this frequency of feeding was determined to provide sufficient nutrition while reducing excess nitrogen build-up.3,7,20 A total of 6 h after feeding, uneaten food and fecal material was removed from the individual tanks by siphoning, and a 10 L water replacement was made. To determine background concentrations of cadmium within the whole prawns, seven holding prawns from the supplier were weighed wet, freeze-dried, weighed dry, and predigested in Teflon tubes with concentrated HNO3 (Merck, Tracepur) overnight. Total tissue digestion was achieved via a pressurized microwave (MARS Xpress). Following digestion, samples were diluted with deionized water (18 MΩ·cm, Milli-Q, Millipore) and analyzed by inductively coupled plasma-mass spectrometry (ICP-MS; Varian 820MS Quadropole; all samples run with internal standard correction for matrix drift and drift correction). Samples of a certified reference material (CRM; DORM-3, fish protein; Environment Canada) were digested and analyzed in the same manner. CRM recoveries for cadmium were within 6% of the certified value. The mean whole body background concentrations of prawns was 0.17 ± 0.01 μg Cd/g wet weight (n = 7). Preparation of Dissolved 109Cd Exposure Conditions. A nominal exposure solution of 40 kBq 109Cd/L (0.6 μg Cd/L; 5.3 nM Cd) was prepared in SRW from a carrier-free parent stock (Eckert and Ziegler, California; 0.1 M HCl). The addition of the stock to the SRW did not affect the physicochemical parameters described above. Individual polypropylene containers (1.125 L) with an internal polypropylene basket and lid were used as exposure tanks (one prawn per tank). A compressed air line connected to each container, via a hole in the lid, provided constant aeration. Prior to exposure, all tanks were conditioned for 24 h with the 109Cd exposure solution. The conditioning cadmium solution was discarded and refreshed with fresh 109Cd solution prior to introducing the prawns. Gamma analysis of radioisotopes (described below) of 10 mL aliquots taken daily from three random exposure tanks showed that the mean exposure throughout the study was 37 ± 9.4 kBq 109Cd/L (0.56 ± 0.14 μg Cd/L; n = 31 aliquots). These data are shown in Figure S1. The environmentally relevant exposure concentration of 0.6 μg/L is between the 80% and 90% species protection concentrations that are applied for soft freshwaters in Australia, WQGVs of 0.8 and 0.4 μg Cd/L, respectively.21 The exposure of prawns to the 109Cd-radiolabeled solution was undertaken in two experiments, as described in the following subsections. Experiment 1: Continuous Exposure. In the first experiment, 24 prawns were exposed in individual containers for 1, 3, 6, 24, 48, 96, 168, and 336 h to identical 109Cd solutions (as prepared above) to investigate the kinetics of organ transfer of cadmium during continuous exposure. At each time point, three prawns were removed from the exposures for whole body radioanalysis and autoradiography. Exposure solutions were replaced every 48 h. Experiment 2: Short- and Long-Term Exposures Followed by Depuration. The second experiment consisted of exposing prawns for short-term (6 h; 15 prawns) and long-term (7 days; 15 prawns) periods to identical 109Cd solutions (as prepared above). Each individual prawn was exposed in its own exposure chamber. Exposure solutions were replaced every 48 h. Following exposure, three prawns from each exposure period were removed for whole-body radioanalysis and autoradiography. The remaining prawns were rinsed (see the Supporting
The use of radioisotope tracers in an ecotoxicological context also allows for individual organisms to be tracked over time regarding their metal accumulation kinetics3,7 and can be conducted at exposure concentrations relevant to most field conditions (e.g., typically stomach. The gills and hepatopancreas were the only organs that showed any consistency of 109Cd presence among organisms in both treatment phases. There were several prawns that did show the presence of 109Cd in the antennal gland and in the stomach (likely from imbibing).3 The antennal gland is believed to be one of the main organs for excretion of metals in decapod crustaceans22 and has previously been shown to concentrate cadmium and zinc in M. australiense.3 However, 109Cd was only found in the antennal gland of some prawns (c. 25%) from the current study, suggesting that the excretion and storage of cadmium via this organ is possibly intermittent. Rouleau et al.14 found 109Cd within the antennal gland of the snow crab Chionoecetes opilio 3 days after feeding on radiolabeled food, which increased in that organ 14 days after ingestion, suggesting a net transport of bioaccumulated cadmium to the antennal gland post-ingestion. Further work with M. australiense is required to better understand the role of the antennal gland in cadmium regulation. Whole-Body and Organ Accumulation Kinetics of 109 Cd. The whole-body accumulation of 109Cd indicated linear accumulation and did not reach saturation over the 14 days of dissolved cadmium exposure (Figure 1a). This observation was in agreement with previous studies of M. australiense and other Macrobrachium species3,7 and for marine prawns of the Penaeid family (e.g., Penaeus stylirostris23). On an individual organ scale, 109 Cd was present in the gills within 1 h, while there was an apparent lag of up to 2 days in detecting substantial 109Cd in the hepatopancreas (Figure 1b). While there was detectable 109 Cd present in the hepatopancreas after exposure for 6 h (Figure 3a), the low accumulation rate of 109Cd within the hepatopancreas after this period suggests that some level of acute saturation of gill-binding sites may be required before the transfer of cadmium from the gills to the hepatopancreas via the hemolymph.24 Furthermore, the accumulation profile of 109Cd in both the gills and hepatopancreas was mostly linear over the 14 day exposure period, and the gills accumulated at a rate four times greater than the hepatopancreas (based on the slopes of the linear regressions presented in Figure 1b). Such a difference in accumulation rate between the gills and the hepatopancreas was not observed for the euryhaline palaemonid Palaemon elegans, which demonstrated comparable uptake rates between the gills and hepatopancreas when exposed to dissolved cadmium over 20 days.12 In that study, the hepatopancreas likely accumulated cadmium more rapidly than the gills. However, the P. elegans were exposed to much-greater concentrations of dissolved cadmium (100 μg/L) compared to this study, which will have likely increased the transfer rate of cadmium from the gills to the hepatopancreas in P. elegans. Whole-Body and Organ Depuration Kinetics of 109Cd. The whole-body 109Cd concentrations were 0.025 ± 0.007 and 0.105 ± 0.001 μg Cd/g wet weight (w-w) (mean ± SE; n = 3) for prawns after the short-term (6 h) and long-term (7 days) exposures to 0.56 μg Cd/L, respectively. These concentrations were comparable to the equivalent time points for whole-body 109 Cd concentrations during the 14 day accumulation study reported in Figure 1a (0.028 ± 0.013 and 0.139 ± 0.025 μg Cd/g w-w for 6 h and 7 days of exposure, respectively). The whole-body depuration of 109Cd was markedly different following short-term exposure compared to those following the long-term exposure (Figure 2). There was a sharp decrease in
Figure 2. Whole-body depuration profiles following short-term (6 h) and long-term (7 days) exposure to 0.56 μg Cd/L. Best fit of logarithmic curves are presented as R2. Data represent mean ± SE; n = 3.
whole-body 109Cd concentration by approximately 60% within the first 24 h of depuration following the short-term exposure and then a gradual decrease in whole-body concentrations between 1 and 20 days of depuration to approximately 20% of the starting value. In contrast, following the long-term exposure, no decreases in whole-body concentrations were detected in the first 24 h, a decrease of approximately 50% was detected by 7 days, and then no further decreases to day 20 of the depuration were detected. These results suggest that M. australiense accumulates cadmium from the dissolved phase rapidly and may excrete cadmium quite rapidly following a short-term “pulse” of exposure but less rapidly following a longer exposure and uptake period, e.g., as was demonstrated for this species by Cresswell et al.7 following a long-term exposure. By the end of the short-term and long-term exposure periods, the 109Cd organ activities within the gills and hepatopancreas were comparable to the equivalent time points for organ 109Cd activities during the 14 day accumulation study reported in Figure 1b (Table S1). The organ distribution of cadmium within the gills during depuration was different following the short-term (6 h) exposure compared with the long-term exposure (7 days), as shown in Figure 3. Following the long-term (7 days) exposure, the depuration of cadmium from the gills was relatively slow, with no detected loss in 109Cd activity during the first 24 h of depuration and a loss of 25% after 21 days of depuration. In contrast, following the short-term (6 h) exposure, there was an average of 64% loss in cadmium within the gills in the first 24 h of depuration and a further 10% loss by 20 days of the depuration. This may be explained by the relatively labile binding of cadmium to the gills during the short-term exposure and then the rapid desorption of this labile cadmium to the surrounding clean water during the first 24 h of depuration. The increased rate of transfer of cadmium from the gills following the short-term exposure may also have been a function of the lower absolute concentration in the gills following this exposure (2.4 ± 1.2 ng Cd/cm2) compared with that of the gills following the long-term exposure (7.3 ± 0.6 ng Cd/cm2; Table S1). Conversely to the short-term exposure, cadmium associated with the gills following 7 days of continuous exposure was observed to be considerably less labile. The depuration of 25% of gill 109Cd activity in 21 days of depuration following the long-term exposure likely represents the intra- and extracellular D
DOI: 10.1021/acs.est.6b06471 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 3. Depuration of 109Cd from the gills of M. australiense following either short-term (6 h) or long-term (7 days) exposure to 0.56 μg Cd/L. Depuration kinetics of gills following each exposure period are shown in (a) with data represented by mean ± SE (n = 3) starting with 100% remaining at t = 0 days of depuration; false-color autoradiograph of 109Cd in (b) the gills of a prawn immediately following 6 h of exposure and (c) 1 day into the depuration phase of a separate prawn following a 6 h exposure. Best fit of log curves in depuration profiles (see panel a) are provided. Note: data point for 1 day of depuration for the 7 days of exposure data set was excluded for the log curve fit. Scale bars on autoradiographs represent 1 cm.
Figure 4. Depuration of 109Cd from the hepatopancreas of M. australiense following either short-term (6 h) or long-term (7 days) exposure to 0.56 μg Cd/L. Depuration kinetics of hepatopancreas following each exposure period are shown in (a) with data represented by mean ± SE (n = 3) starting with 100% remaining at t = 0 days of depuration; false-color autoradiograph of 109Cd in (b) the hepatopancreas of prawn immediately following 6 h of exposure, (c) whole prawn 1 day into depuration following 6 h of exposure, and (d) whole prawn 20 days into depuration following 6 h of exposure. Scale bars on autoradiographs represent 1 cm. Multiphasic depuration and subsequent influx for 6 h of exposure shown in (a) with a straight line connecting each point; linear regression and associated R2 is shown in (a) for 7 days of exposure.
decapod crustaceans, such as the shore crab Carcinus maenas25 and the freshwater amphipod Gammarus pulex, as reviewed by Henry et al.26 The depuration of 109Cd activity from the hepatopancreas was also markedly different following the short-term (6 h) exposure compared with the long-term exposure (7 days), as shown in Figure 4. Approximately 88 ± 6% of cadmium
depuration kinetics of cadmium removal from the gill apical cells to the surrounding uncontaminated media. Depuration of cadmium from the gills following both short-term and longterm exposures suggests that the gills are a site of influx and efflux, as opposed to an organ of net metal storage for M. australiense. Cadmium influx and efflux across the gills, rather than net storage in that organ, has been demonstrated for other E
DOI: 10.1021/acs.est.6b06471 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
a short industrial effluent release or rainfall flushing contaminated water into a river system) is likely able to lower its organ and whole-body cadmium concentrations, thereby reducing the possibility of toxicity occurring. Conversely, if a prawn were chronically exposed to cadmium (e.g., residing downstream of a more continuous industrial effluent) the efflux rates for individual organs and the whole organism will be low, resulting in a continuing net influx and uptake of cadmium and potential toxic effects. These findings have important implications for how WQGVs (or WQC) are applied for assessing ecotoxicity risks from short-term exposures (e.g.,
