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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

ACS Paragon Plus Environment

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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



1

ABSTRACT

2 3

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

4

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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.

78 79

109

Cd and 65Zn aqueous exposure


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109

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

109

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

328

ng Cd/cm2

Exo GI

326

329 330 80

c)

ng 109Cd/cm2

60

40

20

0

331

Gill

Hepatopancreas Antennal gland Abdominal tissue Exoskeleton

Stomach

332

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

337

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.

380 381

Zinc

382 383

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)

393

b)

394 395 396 397 AG

398

H

AT

Eye

G

400 401

ng Zn/cm2

ng Zn/cm2

399

402 403 404

15 ng 65Zn/cm2

d)

1E+4 ng Zn/cm2

20

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

405 406

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

407

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

411

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

412

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

413

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

422 423

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

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