Bioaccumulation Kinetics and Organ Distribution of Cadmium and Zinc

Dec 24, 2014 - these nonessential and essential metals from solution by the freshwater decapod crustacean Macrobrachium australiense. Three treatments...
0 downloads 0 Views 676KB Size
Subscriber access provided by Johns Hopkins Libraries

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

Page 1 of 25

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

ACS Paragon Plus Environment

Page 4 of 25

Page 5 of 25

Environmental Science & Technology

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

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-

ACS Paragon Plus Environment

Environmental Science & Technology

106

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

ACS Paragon Plus Environment

Page 14 of 25

Page 15 of 25

Environmental Science & Technology

322

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

ACS Paragon Plus Environment

Environmental Science & Technology

343

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

ACS Paragon Plus Environment

Page 16 of 25

Page 17 of 25

Environmental Science & Technology

369

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

ACS Paragon Plus Environment

Environmental Science & Technology

Page 18 of 25

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).

ACS Paragon Plus Environment

Page 19 of 25

Environmental Science & Technology

414 415

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