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Dec 24, 2014 - This study used the radioisotopes 109Cd and 65Zn to explore the uptake, retention and organ distribution of these nonessential and esse...
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Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense Tom Cresswell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505254w • Publication Date (Web): 24 Dec 2014 Downloaded from http://pubs.acs.org on December 30, 2014

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Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Environmental Science & Technology es-2014-05254w.R1 Article 16-Dec-2014 Cresswell, Tom; Australian Nuclear Science and Technology Organisation, Institute for Environmental Research Simpson, Stuart; CSIRO Land and Water, Centre for Environmental Contaminants Research Mazumder, Debashish; ANSTO, Institute for Environmental Research Callaghan, Paul; ANSTO, LifeSciences Nguyen, An; ANSTO, LifeSciences

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Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense

Tom Cresswella*, Stuart L. Simpsonb, Debashish Mazumdera, Paul D. Callaghanc and An P. Nguyenc

a

Institute for Environmental Research, ANSTO, Locked Bag 2001 Kirrawee, NSW 2232, Australia

b

Centre for Environmental Contaminants Research, CSIRO Land and Water, New Illawarra Rd, Lucas Heights,

NSW 2234, Australia c

LifeSciences, ANSTO, Locked Bag 2001 Kirrawee, NSW 2232, Australia

ng Cd/cm2

TOC/Abstract Art

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ABSTRACT

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This study used the radioisotopes 109Cd and 65Zn to explore the uptake, retention and organ

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distribution of these non-essential and essential metals from solution by the freshwater decapod

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crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone

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and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux

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rates of each metal individually and in the metal mixture over a three-week period, followed by

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depuration for two weeks in metal-free water using live-animal gamma-spectrometry. Following

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exposure, prawns were cryosectioned and the spatial distribution of radionuclides visualized using

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autoradiography. Metal uptake and efflux rates were the same in the individual and mixed-metal

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exposures, and efflux rates were close to zero. The majority of cadmium uptake was localised

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within the gills and hepatopancreas, while zinc accumulated in the antennal gland at concentrations

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orders of magnitude greater than in other organs. This suggested that M. australiense may process

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zinc much faster than cadmium by internally transporting the accumulated zinc to the antennal

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gland. The combination of uptake studies and autoradiography greatly increases our understanding

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of how metal transport kinetics and internal processing may influence the toxicity of essential and

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non-essential metals in the environment.

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Keywords: Autoradiography, Bioaccumulation, Invertebrate, Metal, Organ distribution

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INTRODUCTION

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Metal bioaccumulation by aquatic invertebrates has received much attention over the past decades

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due to the increasing concentrations of many potentially toxic metals in the environment and the

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importance of understanding trophic transfer between organisms in aquatic food webs.1-5 The

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nature of metal bioaccumulation is complex as exposure rarely occurs from a single source, rather

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an organism is exposed to a cocktail of many different metals at the same time and in different ACS Paragon Plus Environment

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phases (i.e. dissolved and or associated with particles).1, 5-8 Furthermore, to regulate metabolic

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functions, organisms require certain metals, such as zinc, (e.g. for metallo-enzyme function7), while

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other metals such as cadmium are not known have any metabolic function in aquatic invertebrates.

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Interactions between such essential and non-essential metals upon bioaccumulation could

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potentially affect the rates of uptake of each metal individually and the final organ location of metal

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accumulation within the organisms.9, 10 Following accumulation, metals may remain in

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metabolically available forms, which could result in toxicity to the organism, or be processed

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internally and either removed from the body or stored in biologically inactive forms.9-11

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Gamma spectrometry, involving the detection of gamma-emitting metal radioisotopes as tracers, is

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a valuable tool for studying metal bioaccumulation in aquatic invertebrates, allowing the influx and

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efflux of multiple metals to be analysed rapidly at multiple intervals during an exposure period

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without sacrificing the organism.12-14 Autoradiography of cryosectioned organisms enables the

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organ distribution of accumulated metals to be visualised and quantified.15-18 Organisms are snap-

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frozen and cyrosectioned with thicknesses 10 MΩ·cm, Milli-RO, Millipore). All chemicals used were analytical

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reagent grade or equivalent purity. Late juvenile (0.72±0.13 g wet weight; 10.1±1.1 mm post

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orbital carapace length) M. australiense were obtained from a commercial prawn farm (Bingera

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Weir Farm, Bundaberg, Queensland). The prawns were held in 43 L plastic storage containers

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filled with synthetic river water (SRW: 1.92 g NaHCO3; 1.20 g CaSO4·2H2O; 2.46 g MgSO4·7H2O;

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0.08 g KCl in 20 L de-ionised water) modified from a USEPA recipe.23 Prawns were fed twice

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weekly with food pellets (Novo Rift JBL Sticks, JBL GmbH & Co., Germany: crude protein = 31%;

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crude fat = 3%; crude fibre = 5.5%; crude ash = 11%), with uneaten food siphoned from the tanks

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10 h after providing the food.

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109

Cd and 65Zn aqueous exposure

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Cd was obtained from Eckert & Ziegler Isotope Products Inc., Valencia, USA and 65Zn from the

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Australian Nuclear Science and Technology Organisation (ANSTO), Sydney, Australia. Both

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isotopes were in their chloride form in 0.1 M HCl. The exposure of M. australiense was conducted

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as previously described by Cresswell et al.1 Briefly, prawns were exposed to 32 kBq 109Cd/L and

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19 kBq 65Zn/L in SRW contained in square 1.125 L polypropylene containers (Decor, Tellfresh;

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hereafter referred to as exposure chambers). Analysis by inductively-coupled plasma mass

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spectrometry (ICP-MS; Varian 820MS Quadropole; all samples run with internal standard

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correction for matrix and drift correction) confirmed that these exposures were equivalent to 2.1 µg

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Cd/L and 11.6 µg Zn/L respectively. In the absence of organic ligands in the exposure media, the

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predominant species of both metals was likely Cd2+ (aq)24 and Zn2+ (aq)25. Each chamber contained

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an internal polypropylene basket, which allowed the prawns to be removed from the chamber and

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rinsed with ease prior to radioanalysis. Exposure solutions were introduced to the chambers for 24

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h after which it was then discarded and replaced with fresh solution to condition each chamber prior

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to the introduction of the prawns. Constant aeration was provided in all tests via a compressed air

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line fed through a hole drilled in the lid of each chamber. All experiments were conducted at a

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water temperature of 21±1°C on a 12 h:12 h light:dark regime in a temperature controlled room (set

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at 21±1°C). Dissolved oxygen concentrations were maintained at 5.8±0.2 mg/L and 98±0.3%

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saturation. Holding and exposure water physico-chemical parameters were as follows: pH 7.2±0.1;

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conductivity 270± 40 µS/cm; hardness 85 mg/L as CaCO3 and alkalinity 30 mg CaCO3/L.

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Prawns were exposed to 109Cd and 65Zn individually or as a mixture of both metals (i.e. a total of

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three treatments with five replicates each) for 21 days with segregated feeding before being

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transferred to clean exposure chambers with isotope-free SRW for 14 days to depurate. Animals

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were radioanalysed (see below) every 24 h for the first seven days of exposure then three times per

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week thereafter. Exposure solutions were renewed 100% at each prawn radioanalysis and sub-

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samples of exposure solutions were radioanalysed and analysed via ICP-MS to check exposure

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activity and metal concentration respectively. Following the depuration period, prawns were

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transferred to a -18°C freezer for 1 h to be euthanized but not frozen, before being embedded in an

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inert embedding resin (Cryomatrix, Thermo Fisher Scientific, Australia) within a small plastic Petri

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dish (35 mm diameter) and snap frozen in liquid nitrogen. Embedded prawn blocks were stored at -

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80°C prior to cyrosectioning and autoradiography.

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Gamma-spectrometry: Radioisotope detection and live animal radioanalysis

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Gamma ray emissions from sources were determined using a 1.5×1.5” LaBr detector within a lead

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chamber attached to a multi-channel spectrometer (Canberra InSpector 1000), connected to a PC

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equipped with spectra analysis software (Genie 2000) using varying count times (from 1-10

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minutes) to ensure propagated counting error was stomach > GI tract >

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exoskeleton > abdominal tissue. Between subjects differences in organs uptake of Cd109 were

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minor, as shown in Figure 2c. This strongly implies that the major route of bioaccumulation of

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cadmium was via the gills rather than the stomach (through imbibing), as was expected. These

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findings also suggest that a large proportion of cadmium assimilated via the gills remained

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associated with this organ, even after two weeks of depuration in cadmium-free water. This may

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indicate that only a fraction of the accumulated cadmium may be available for transfer to other

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compartments within the organism, such as other internal organs. Potentially, metal that was

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undergoing metabolic processes of detoxification (e.g. bound by soluble metalloproteins) may be

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transported back to the gills for excretion during the two week depuration phase. As the organ

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localisation was only determined at one time point, it was not possible to confirm which process

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had taken place. Notably, the density of Cd109 within hepatopancreas was proportionally 35-70% of

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that seen in the gills (Figure 2c).

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a)

b)

323 324 325 H

G AG

G

ng Cd/cm2

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S

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ng Cd/cm2

Exo GI

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329 330 80

c)

ng 109Cd/cm2

60

40

20

0

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Gill

Hepatopancreas Antennal gland Abdominal tissue Exoskeleton

Stomach

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Figure 2. Spatial distribution of 109Cd density in three individual M. australiense exposed to 2.1 µg Cd/L for three

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weeks followed by depuration in cadmium-free water for two weeks. a) & b): Autoradiographic imaging from 20 µm

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sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and right side gill (b). Regions of

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interest defining major organs: Exo = exoskeleton; GI = gastrointestinal tract (hindgut); H = hepatopancreas; S =

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stomach; G = gill; AG = antennal gland. Colour bars on autoradiographs represent calibration of images into ng Cd/cm2

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units. Each vertical bar of the same pattern on the bar graph (c) represents the organs of a single individual. Data

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plotted represent mean ng 109Cd/cm2 for each organ ± SD (n=14).

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Cadmium was present in the stomach and GI tract even though the prawns did not ingest any

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radiolabelled food (all feeding was conducted in radioisotope- and metal-free water). This could

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suggest the production of metal-containing insoluble granules (e.g. lysosomal residual bodies after

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the autolysis of cadmium-containing metalothioneins) within the epithelial cells of the

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hepatopancreas, with subsequent extrusion from the cell followed by organismic excretory

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mechanisms (in the stomach and GI tract) to return the metal to the environment.34 The majority of

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cadmium in the hepatopancreas was likely present in soluble forms (e.g. associated with

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metallothionein-like proteins) rather than as insoluble granules. Nunez-Nogueira et al.35 determined

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that approximately 85% of the cadmium in the hepatopancreas of the marine decapod Penaeus

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indicus was present in soluble forms following a 10-day exposure to 100 µg Cd/L. Furthermore,

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radiolabelled cadmium present in the GI tract was approximately 5% of that found in the antennal

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gland. This indicates that the cadmium was potentially being transported to the antennal gland for

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excretion. However, this transport process in Macrobrachium is not known. Cadmium is believed

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to be transferred between organs in the haemolymph by reversible binding to haemocyanin.36

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It is therefore more likely that the presence of cadmium in the stomach and GI tract was from oral

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or anal drinking. Fox37 conducted a series of microscopy examinations with freshwater and marine

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prawns and confirmed that prawns (e.g. Atyaephyra desmaresti, Palaemon adspersus) imbibe the

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surrounding water, a process that may improve food digestion in the gut. Similarly, Fox37 observed

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that prawns would intake water anally, which when accompanied by intestinal antiperistalsis,

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moved water forwards in the intestine towards the thorax. This water was then observed flowing

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back towards the anus along with a fecal pellet, therefore acting as a natural enema to aid in

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defecation. The ingestion of water containing a radioisotope potentially explains the presence of

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cadmium radioisotope in the stomach and GI tract due to the adsorption of the metal ion to the

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interior epithelial cells of these organs over three weeks of exposure.

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All three replicates demonstrated cadmium activity in the antennal gland, which is believed to be

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among the main organs for excretion of metals in decapod crustaceans2 and demonstrates a similar

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role to that of the kidney in fish and mammals, where cadmium has been shown to accumulate.38

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Rouleau et al.18 also observed cadmium in the antennal gland of the snow crab Chionoecetes opilio

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14 days after ingesting 109Cd-radiolabelled food. Other non-essential trace metals have also been

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found to exist in the antennal gland of decapods such as lead. Lead was found in the labyrinth cells

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of the antennal gland of crayfish (Orconectes propinquus) exposed to lead and it was postulated that

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phagocytotic haemocytes in the antennal gland were responsible for removing lead from the

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haemolymph.39

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These results suggest that while there was no significant reduction of whole body cadmium

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concentrations during two weeks of depuration, the prawns were likely processing the

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bioaccumulated cadmium in the hepatopancreas and beginning to transfer it to the antennal gland

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for excretion.

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Zinc

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The spatial distribution of 65Zn from sagittal sections of prawn is shown in Figure 3 following three

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weeks of exposure and two weeks of depuration. The profile of 65Zn accumulation into individual

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organs also was assessed by relative density per unit area. The antennal gland of all three prawns

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contained the greatest density of 65Zn per area by three orders of magnitude, with the profile of

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uptake in remaining organs decreasing in activity per area as follows: hepatopancreas > eye > gill >

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abdominal tissue = exoskeleton (Figures 3c and 3d). This stark difference between the antennal

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gland and the other organs suggests that the prawns were processing the radiolabelled zinc and had

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likely begun excretion. The excretion via the antennal gland is likely to be relatively slow as there

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was no significant reduction in whole-body 65Zn during the two-week depuration period.

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a)

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b)

394 395 396 397 AG

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H

AT

Eye

G

400 401

ng Zn/cm2

ng Zn/cm2

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402 403 404

15 ng 65Zn/cm2

d)

1E+4 ng Zn/cm2

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1E+5

off scale

c)

1E+3 1E+2 1E+1 1E+0 1E-1

10

G

H

AG

AT

Exo

Eye

5

0 Gill

Hepatopancreas Antennal gland Abdominal tissue

Exoskeleton

Eye

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Figure 3. Spatial distribution of 65Zn density in three individual M. australiense exposed to 11.6 µg Zn/L for three

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weeks followed by depuration in zinc-free water for two weeks. a) & b): Autoradiographic imaging from 20 µm

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sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and left side gill (b). Regions of

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interest defining major organs: AG = antennal gland; H = hepatopancreas; AT = abdominal tissue; G = gill; Exo =

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Exoskeleton. Colour bars on autoradiographs represent calibration of images into ng Zn/cm2. Each vertical bar of the

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same pattern on c) the bar graph represents the organs of a single individual. Data plotted represent mean ng 65Zn /cm2

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for each organ ± SD (n=14). Due to the antennal gland having the significant majority of zinc, the data are also plotted

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on a log scale (inset d).

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In contrast to cadmium, greater amounts of zinc were found in the hepatopancreas than the gill.

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This suggests that zinc was being processed differently to cadmium by being transferred internally

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from the gill to the hepatopancreas for processing (e.g. detoxification and metabolism). Other

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studies have found that most essential trace metals such as Fe, Cu and Zn accumulate in the cells of

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the decapod hepatopancreas.39 Zinc accumulation within the eye has also been demonstrated for

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marine decapods29, 40 and is thought to be due to high concentrations of zinc metalloenzymes known

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to be associated with the visual process.29

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Bryan41 measured concentrations of zinc in 18 species of decapods (freshwater and marine) and

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suggested that the gills were the main site for the absorption of dissolved zinc and its subsequent

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loss. While it is likely that the main site of zinc accumulation by M. australiense was the gills (as

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little was found in the stomach or GI tract), the main site of transfer from the blood compartment

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was undoubtedly via the antennal gland, presumably to be processed for excretion via urine. White

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and Rainbow29 exposed the decapod P. elegans to 100 µg Zn/L in seawater with added 65Zn for 20

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days, followed by a further 29 days in 100 µg Zn/L with no radiotracer. The major organs of the

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shrimp were collected at different time points during the radiolabelled non-labelled exposure

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periods and the total and radiolabelled zinc concentrations determined. The study found that total

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zinc concentration did not appreciably change in any of the major organs over time apart from the

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exoskeleton, which increased significantly (p