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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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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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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Environmental Science & Technology
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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Environmental Science & Technology
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
5
crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone
6
and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux
7
rates of each metal individually and in the metal mixture over a three-week period, followed by
8
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
17
non-essential metals in the environment.
18 19
Keywords: Autoradiography, Bioaccumulation, Invertebrate, Metal, Organ distribution
20 21
INTRODUCTION
22 23
Metal bioaccumulation by aquatic invertebrates has received much attention over the past decades
24
due to the increasing concentrations of many potentially toxic metals in the environment and the
25
importance of understanding trophic transfer between organisms in aquatic food webs.1-5 The
26
nature of metal bioaccumulation is complex as exposure rarely occurs from a single source, rather
27
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
29
functions, organisms require certain metals, such as zinc, (e.g. for metallo-enzyme function7), while
30
other metals such as cadmium are not known have any metabolic function in aquatic invertebrates.
31
Interactions between such essential and non-essential metals upon bioaccumulation could
32
potentially affect the rates of uptake of each metal individually and the final organ location of metal
33
accumulation within the organisms.9, 10 Following accumulation, metals may remain in
34
metabolically available forms, which could result in toxicity to the organism, or be processed
35
internally and either removed from the body or stored in biologically inactive forms.9-11
36 37
Gamma spectrometry, involving the detection of gamma-emitting metal radioisotopes as tracers, is
38
a valuable tool for studying metal bioaccumulation in aquatic invertebrates, allowing the influx and
39
efflux of multiple metals to be analysed rapidly at multiple intervals during an exposure period
40
without sacrificing the organism.12-14 Autoradiography of cryosectioned organisms enables the
41
organ distribution of accumulated metals to be visualised and quantified.15-18 Organisms are snap-
42
frozen and cyrosectioned with thicknesses 10 MΩ·cm, Milli-RO, Millipore). All chemicals used were analytical
70
reagent grade or equivalent purity. Late juvenile (0.72±0.13 g wet weight; 10.1±1.1 mm post
71
orbital carapace length) M. australiense were obtained from a commercial prawn farm (Bingera
72
Weir Farm, Bundaberg, Queensland). The prawns were held in 43 L plastic storage containers
73
filled with synthetic river water (SRW: 1.92 g NaHCO3; 1.20 g CaSO4·2H2O; 2.46 g MgSO4·7H2O;
74
0.08 g KCl in 20 L de-ionised water) modified from a USEPA recipe.23 Prawns were fed twice
75
weekly with food pellets (Novo Rift JBL Sticks, JBL GmbH & Co., Germany: crude protein = 31%;
76
crude fat = 3%; crude fibre = 5.5%; crude ash = 11%), with uneaten food siphoned from the tanks
77
10 h after providing the food.
78 79
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
82
Australian Nuclear Science and Technology Organisation (ANSTO), Sydney, Australia. Both
83
isotopes were in their chloride form in 0.1 M HCl. The exposure of M. australiense was conducted
84
as previously described by Cresswell et al.1 Briefly, prawns were exposed to 32 kBq 109Cd/L and
85
19 kBq 65Zn/L in SRW contained in square 1.125 L polypropylene containers (Decor, Tellfresh;
86
hereafter referred to as exposure chambers). Analysis by inductively-coupled plasma mass
87
spectrometry (ICP-MS; Varian 820MS Quadropole; all samples run with internal standard
88
correction for matrix and drift correction) confirmed that these exposures were equivalent to 2.1 µg
89
Cd/L and 11.6 µg Zn/L respectively. In the absence of organic ligands in the exposure media, the
90
predominant species of both metals was likely Cd2+ (aq)24 and Zn2+ (aq)25. Each chamber contained
91
an internal polypropylene basket, which allowed the prawns to be removed from the chamber and
92
rinsed with ease prior to radioanalysis. Exposure solutions were introduced to the chambers for 24
93
h after which it was then discarded and replaced with fresh solution to condition each chamber prior
94
to the introduction of the prawns. Constant aeration was provided in all tests via a compressed air
95
line fed through a hole drilled in the lid of each chamber. All experiments were conducted at a
96
water temperature of 21±1°C on a 12 h:12 h light:dark regime in a temperature controlled room (set
97
at 21±1°C). Dissolved oxygen concentrations were maintained at 5.8±0.2 mg/L and 98±0.3%
98
saturation. Holding and exposure water physico-chemical parameters were as follows: pH 7.2±0.1;
99
conductivity 270± 40 µS/cm; hardness 85 mg/L as CaCO3 and alkalinity 30 mg CaCO3/L.
100 101
Prawns were exposed to 109Cd and 65Zn individually or as a mixture of both metals (i.e. a total of
102
three treatments with five replicates each) for 21 days with segregated feeding before being
103
transferred to clean exposure chambers with isotope-free SRW for 14 days to depurate. Animals
104
were radioanalysed (see below) every 24 h for the first seven days of exposure then three times per
105
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
107
activity and metal concentration respectively. Following the depuration period, prawns were
108
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
110
dish (35 mm diameter) and snap frozen in liquid nitrogen. Embedded prawn blocks were stored at -
111
80°C prior to cyrosectioning and autoradiography.
112 113
Gamma-spectrometry: Radioisotope detection and live animal radioanalysis
114 115
Gamma ray emissions from sources were determined using a 1.5×1.5” LaBr detector within a lead
116
chamber attached to a multi-channel spectrometer (Canberra InSpector 1000), connected to a PC
117
equipped with spectra analysis software (Genie 2000) using varying count times (from 1-10
118
minutes) to ensure propagated counting error was stomach > GI tract >
309
exoskeleton > abdominal tissue. Between subjects differences in organs uptake of Cd109 were
310
minor, as shown in Figure 2c. This strongly implies that the major route of bioaccumulation of
311
cadmium was via the gills rather than the stomach (through imbibing), as was expected. These
312
findings also suggest that a large proportion of cadmium assimilated via the gills remained
313
associated with this organ, even after two weeks of depuration in cadmium-free water. This may
314
indicate that only a fraction of the accumulated cadmium may be available for transfer to other
315
compartments within the organism, such as other internal organs. Potentially, metal that was
316
undergoing metabolic processes of detoxification (e.g. bound by soluble metalloproteins) may be
317
transported back to the gills for excretion during the two week depuration phase. As the organ
318
localisation was only determined at one time point, it was not possible to confirm which process
319
had taken place. Notably, the density of Cd109 within hepatopancreas was proportionally 35-70% of
320
that seen in the gills (Figure 2c).
321
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a)
b)
323 324 325 H
G AG
G
ng Cd/cm2
327
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
333
weeks followed by depuration in cadmium-free water for two weeks. a) & b): Autoradiographic imaging from 20 µm
334
sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and right side gill (b). Regions of
335
interest defining major organs: Exo = exoskeleton; GI = gastrointestinal tract (hindgut); H = hepatopancreas; S =
336
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
338
plotted represent mean ng 109Cd/cm2 for each organ ± SD (n=14).
339 340
Cadmium was present in the stomach and GI tract even though the prawns did not ingest any
341
radiolabelled food (all feeding was conducted in radioisotope- and metal-free water). This could
342
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
344
hepatopancreas, with subsequent extrusion from the cell followed by organismic excretory
345
mechanisms (in the stomach and GI tract) to return the metal to the environment.34 The majority of
346
cadmium in the hepatopancreas was likely present in soluble forms (e.g. associated with
347
metallothionein-like proteins) rather than as insoluble granules. Nunez-Nogueira et al.35 determined
348
that approximately 85% of the cadmium in the hepatopancreas of the marine decapod Penaeus
349
indicus was present in soluble forms following a 10-day exposure to 100 µg Cd/L. Furthermore,
350
radiolabelled cadmium present in the GI tract was approximately 5% of that found in the antennal
351
gland. This indicates that the cadmium was potentially being transported to the antennal gland for
352
excretion. However, this transport process in Macrobrachium is not known. Cadmium is believed
353
to be transferred between organs in the haemolymph by reversible binding to haemocyanin.36
354 355
It is therefore more likely that the presence of cadmium in the stomach and GI tract was from oral
356
or anal drinking. Fox37 conducted a series of microscopy examinations with freshwater and marine
357
prawns and confirmed that prawns (e.g. Atyaephyra desmaresti, Palaemon adspersus) imbibe the
358
surrounding water, a process that may improve food digestion in the gut. Similarly, Fox37 observed
359
that prawns would intake water anally, which when accompanied by intestinal antiperistalsis,
360
moved water forwards in the intestine towards the thorax. This water was then observed flowing
361
back towards the anus along with a fecal pellet, therefore acting as a natural enema to aid in
362
defecation. The ingestion of water containing a radioisotope potentially explains the presence of
363
cadmium radioisotope in the stomach and GI tract due to the adsorption of the metal ion to the
364
interior epithelial cells of these organs over three weeks of exposure.
365 366
All three replicates demonstrated cadmium activity in the antennal gland, which is believed to be
367
among the main organs for excretion of metals in decapod crustaceans2 and demonstrates a similar
368
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
370
14 days after ingesting 109Cd-radiolabelled food. Other non-essential trace metals have also been
371
found to exist in the antennal gland of decapods such as lead. Lead was found in the labyrinth cells
372
of the antennal gland of crayfish (Orconectes propinquus) exposed to lead and it was postulated that
373
phagocytotic haemocytes in the antennal gland were responsible for removing lead from the
374
haemolymph.39
375 376
These results suggest that while there was no significant reduction of whole body cadmium
377
concentrations during two weeks of depuration, the prawns were likely processing the
378
bioaccumulated cadmium in the hepatopancreas and beginning to transfer it to the antennal gland
379
for excretion.
380 381
Zinc
382 383
The spatial distribution of 65Zn from sagittal sections of prawn is shown in Figure 3 following three
384
weeks of exposure and two weeks of depuration. The profile of 65Zn accumulation into individual
385
organs also was assessed by relative density per unit area. The antennal gland of all three prawns
386
contained the greatest density of 65Zn per area by three orders of magnitude, with the profile of
387
uptake in remaining organs decreasing in activity per area as follows: hepatopancreas > eye > gill >
388
abdominal tissue = exoskeleton (Figures 3c and 3d). This stark difference between the antennal
389
gland and the other organs suggests that the prawns were processing the radiolabelled zinc and had
390
likely begun excretion. The excretion via the antennal gland is likely to be relatively slow as there
391
was no significant reduction in whole-body 65Zn during the two-week depuration period.
392
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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
408
sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and left side gill (b). Regions of
409
interest defining major organs: AG = antennal gland; H = hepatopancreas; AT = abdominal tissue; G = gill; Exo =
410
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.
416
This suggests that zinc was being processed differently to cadmium by being transferred internally
417
from the gill to the hepatopancreas for processing (e.g. detoxification and metabolism). Other
418
studies have found that most essential trace metals such as Fe, Cu and Zn accumulate in the cells of
419
the decapod hepatopancreas.39 Zinc accumulation within the eye has also been demonstrated for
420
marine decapods29, 40 and is thought to be due to high concentrations of zinc metalloenzymes known
421
to be associated with the visual process.29
422 423
Bryan41 measured concentrations of zinc in 18 species of decapods (freshwater and marine) and
424
suggested that the gills were the main site for the absorption of dissolved zinc and its subsequent
425
loss. While it is likely that the main site of zinc accumulation by M. australiense was the gills (as
426
little was found in the stomach or GI tract), the main site of transfer from the blood compartment
427
was undoubtedly via the antennal gland, presumably to be processed for excretion via urine. White
428
and Rainbow29 exposed the decapod P. elegans to 100 µg Zn/L in seawater with added 65Zn for 20
429
days, followed by a further 29 days in 100 µg Zn/L with no radiotracer. The major organs of the
430
shrimp were collected at different time points during the radiolabelled non-labelled exposure
431
periods and the total and radiolabelled zinc concentrations determined. The study found that total
432
zinc concentration did not appreciably change in any of the major organs over time apart from the
433
exoskeleton, which increased significantly (p
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Environmental Science & Technology
This document is confidential and is proprietary to the American Chemical Society and its authors. Do not copy or disclose without written permission. If you have received this item in error, notify the sender and delete all copies.
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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Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
5
crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone
6
and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux
7
rates of each metal individually and in the metal mixture over a three-week period, followed by
8
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
17
non-essential metals in the environment.
18 19
Keywords: Autoradiography, Bioaccumulation, Invertebrate, Metal, Organ distribution
20 21
INTRODUCTION
22 23
Metal bioaccumulation by aquatic invertebrates has received much attention over the past decades
24
due to the increasing concentrations of many potentially toxic metals in the environment and the
25
importance of understanding trophic transfer between organisms in aquatic food webs.1-5 The
26
nature of metal bioaccumulation is complex as exposure rarely occurs from a single source, rather
27
an organism is exposed to a cocktail of many different metals at the same time and in different ACS Paragon Plus Environment
Page 2 of 25
Page 3 of 25
Environmental Science & Technology
28
phases (i.e. dissolved and or associated with particles).1, 5-8 Furthermore, to regulate metabolic
29
functions, organisms require certain metals, such as zinc, (e.g. for metallo-enzyme function7), while
30
other metals such as cadmium are not known have any metabolic function in aquatic invertebrates.
31
Interactions between such essential and non-essential metals upon bioaccumulation could
32
potentially affect the rates of uptake of each metal individually and the final organ location of metal
33
accumulation within the organisms.9, 10 Following accumulation, metals may remain in
34
metabolically available forms, which could result in toxicity to the organism, or be processed
35
internally and either removed from the body or stored in biologically inactive forms.9-11
36 37
Gamma spectrometry, involving the detection of gamma-emitting metal radioisotopes as tracers, is
38
a valuable tool for studying metal bioaccumulation in aquatic invertebrates, allowing the influx and
39
efflux of multiple metals to be analysed rapidly at multiple intervals during an exposure period
40
without sacrificing the organism.12-14 Autoradiography of cryosectioned organisms enables the
41
organ distribution of accumulated metals to be visualised and quantified.15-18 Organisms are snap-
42
frozen and cyrosectioned with thicknesses 10 MΩ·cm, Milli-RO, Millipore). All chemicals used were analytical
70
reagent grade or equivalent purity. Late juvenile (0.72±0.13 g wet weight; 10.1±1.1 mm post
71
orbital carapace length) M. australiense were obtained from a commercial prawn farm (Bingera
72
Weir Farm, Bundaberg, Queensland). The prawns were held in 43 L plastic storage containers
73
filled with synthetic river water (SRW: 1.92 g NaHCO3; 1.20 g CaSO4·2H2O; 2.46 g MgSO4·7H2O;
74
0.08 g KCl in 20 L de-ionised water) modified from a USEPA recipe.23 Prawns were fed twice
75
weekly with food pellets (Novo Rift JBL Sticks, JBL GmbH & Co., Germany: crude protein = 31%;
76
crude fat = 3%; crude fibre = 5.5%; crude ash = 11%), with uneaten food siphoned from the tanks
77
10 h after providing the food.
78 79
109
Cd and 65Zn aqueous exposure
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80 81
109
Cd was obtained from Eckert & Ziegler Isotope Products Inc., Valencia, USA and 65Zn from the
82
Australian Nuclear Science and Technology Organisation (ANSTO), Sydney, Australia. Both
83
isotopes were in their chloride form in 0.1 M HCl. The exposure of M. australiense was conducted
84
as previously described by Cresswell et al.1 Briefly, prawns were exposed to 32 kBq 109Cd/L and
85
19 kBq 65Zn/L in SRW contained in square 1.125 L polypropylene containers (Decor, Tellfresh;
86
hereafter referred to as exposure chambers). Analysis by inductively-coupled plasma mass
87
spectrometry (ICP-MS; Varian 820MS Quadropole; all samples run with internal standard
88
correction for matrix and drift correction) confirmed that these exposures were equivalent to 2.1 µg
89
Cd/L and 11.6 µg Zn/L respectively. In the absence of organic ligands in the exposure media, the
90
predominant species of both metals was likely Cd2+ (aq)24 and Zn2+ (aq)25. Each chamber contained
91
an internal polypropylene basket, which allowed the prawns to be removed from the chamber and
92
rinsed with ease prior to radioanalysis. Exposure solutions were introduced to the chambers for 24
93
h after which it was then discarded and replaced with fresh solution to condition each chamber prior
94
to the introduction of the prawns. Constant aeration was provided in all tests via a compressed air
95
line fed through a hole drilled in the lid of each chamber. All experiments were conducted at a
96
water temperature of 21±1°C on a 12 h:12 h light:dark regime in a temperature controlled room (set
97
at 21±1°C). Dissolved oxygen concentrations were maintained at 5.8±0.2 mg/L and 98±0.3%
98
saturation. Holding and exposure water physico-chemical parameters were as follows: pH 7.2±0.1;
99
conductivity 270± 40 µS/cm; hardness 85 mg/L as CaCO3 and alkalinity 30 mg CaCO3/L.
100 101
Prawns were exposed to 109Cd and 65Zn individually or as a mixture of both metals (i.e. a total of
102
three treatments with five replicates each) for 21 days with segregated feeding before being
103
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
105
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
107
activity and metal concentration respectively. Following the depuration period, prawns were
108
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
110
dish (35 mm diameter) and snap frozen in liquid nitrogen. Embedded prawn blocks were stored at -
111
80°C prior to cyrosectioning and autoradiography.
112 113
Gamma-spectrometry: Radioisotope detection and live animal radioanalysis
114 115
Gamma ray emissions from sources were determined using a 1.5×1.5” LaBr detector within a lead
116
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
310
minor, as shown in Figure 2c. This strongly implies that the major route of bioaccumulation of
311
cadmium was via the gills rather than the stomach (through imbibing), as was expected. These
312
findings also suggest that a large proportion of cadmium assimilated via the gills remained
313
associated with this organ, even after two weeks of depuration in cadmium-free water. This may
314
indicate that only a fraction of the accumulated cadmium may be available for transfer to other
315
compartments within the organism, such as other internal organs. Potentially, metal that was
316
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
318
localisation was only determined at one time point, it was not possible to confirm which process
319
had taken place. Notably, the density of Cd109 within hepatopancreas was proportionally 35-70% of
320
that seen in the gills (Figure 2c).
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a)
b)
323 324 325 H
G AG
G
ng Cd/cm2
327
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
333
weeks followed by depuration in cadmium-free water for two weeks. a) & b): Autoradiographic imaging from 20 µm
334
sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and right side gill (b). Regions of
335
interest defining major organs: Exo = exoskeleton; GI = gastrointestinal tract (hindgut); H = hepatopancreas; S =
336
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
338
plotted represent mean ng 109Cd/cm2 for each organ ± SD (n=14).
339 340
Cadmium was present in the stomach and GI tract even though the prawns did not ingest any
341
radiolabelled food (all feeding was conducted in radioisotope- and metal-free water). This could
342
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
344
hepatopancreas, with subsequent extrusion from the cell followed by organismic excretory
345
mechanisms (in the stomach and GI tract) to return the metal to the environment.34 The majority of
346
cadmium in the hepatopancreas was likely present in soluble forms (e.g. associated with
347
metallothionein-like proteins) rather than as insoluble granules. Nunez-Nogueira et al.35 determined
348
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,
350
radiolabelled cadmium present in the GI tract was approximately 5% of that found in the antennal
351
gland. This indicates that the cadmium was potentially being transported to the antennal gland for
352
excretion. However, this transport process in Macrobrachium is not known. Cadmium is believed
353
to be transferred between organs in the haemolymph by reversible binding to haemocyanin.36
354 355
It is therefore more likely that the presence of cadmium in the stomach and GI tract was from oral
356
or anal drinking. Fox37 conducted a series of microscopy examinations with freshwater and marine
357
prawns and confirmed that prawns (e.g. Atyaephyra desmaresti, Palaemon adspersus) imbibe the
358
surrounding water, a process that may improve food digestion in the gut. Similarly, Fox37 observed
359
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
361
back towards the anus along with a fecal pellet, therefore acting as a natural enema to aid in
362
defecation. The ingestion of water containing a radioisotope potentially explains the presence of
363
cadmium radioisotope in the stomach and GI tract due to the adsorption of the metal ion to the
364
interior epithelial cells of these organs over three weeks of exposure.
365 366
All three replicates demonstrated cadmium activity in the antennal gland, which is believed to be
367
among the main organs for excretion of metals in decapod crustaceans2 and demonstrates a similar
368
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
371
found to exist in the antennal gland of decapods such as lead. Lead was found in the labyrinth cells
372
of the antennal gland of crayfish (Orconectes propinquus) exposed to lead and it was postulated that
373
phagocytotic haemocytes in the antennal gland were responsible for removing lead from the
374
haemolymph.39
375 376
These results suggest that while there was no significant reduction of whole body cadmium
377
concentrations during two weeks of depuration, the prawns were likely processing the
378
bioaccumulated cadmium in the hepatopancreas and beginning to transfer it to the antennal gland
379
for excretion.
380 381
Zinc
382 383
The spatial distribution of 65Zn from sagittal sections of prawn is shown in Figure 3 following three
384
weeks of exposure and two weeks of depuration. The profile of 65Zn accumulation into individual
385
organs also was assessed by relative density per unit area. The antennal gland of all three prawns
386
contained the greatest density of 65Zn per area by three orders of magnitude, with the profile of
387
uptake in remaining organs decreasing in activity per area as follows: hepatopancreas > eye > gill >
388
abdominal tissue = exoskeleton (Figures 3c and 3d). This stark difference between the antennal
389
gland and the other organs suggests that the prawns were processing the radiolabelled zinc and had
390
likely begun excretion. The excretion via the antennal gland is likely to be relatively slow as there
391
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
408
sagittal sections of prawns after exposure at two sagittal planes: center of prawn (a) and left side gill (b). Regions of
409
interest defining major organs: AG = antennal gland; H = hepatopancreas; AT = abdominal tissue; G = gill; Exo =
410
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
417
from the gill to the hepatopancreas for processing (e.g. detoxification and metabolism). Other
418
studies have found that most essential trace metals such as Fe, Cu and Zn accumulate in the cells of
419
the decapod hepatopancreas.39 Zinc accumulation within the eye has also been demonstrated for
420
marine decapods29, 40 and is thought to be due to high concentrations of zinc metalloenzymes known
421
to be associated with the visual process.29
422 423
Bryan41 measured concentrations of zinc in 18 species of decapods (freshwater and marine) and
424
suggested that the gills were the main site for the absorption of dissolved zinc and its subsequent
425
loss. While it is likely that the main site of zinc accumulation by M. australiense was the gills (as
426
little was found in the stomach or GI tract), the main site of transfer from the blood compartment
427
was undoubtedly via the antennal gland, presumably to be processed for excretion via urine. White
428
and Rainbow29 exposed the decapod P. elegans to 100 µg Zn/L in seawater with added 65Zn for 20
429
days, followed by a further 29 days in 100 µg Zn/L with no radiotracer. The major organs of the
430
shrimp were collected at different time points during the radiolabelled non-labelled exposure
431
periods and the total and radiolabelled zinc concentrations determined. The study found that total
432
zinc concentration did not appreciably change in any of the major organs over time apart from the
433
exoskeleton, which increased significantly (p
