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Uptake kinetics and subcellular compartmentalization explain lethal but not sublethal effects of cadmium in two closely related amphipod species Lena Jakob, Daria S. Bedulina, Denis V. Axenov-Gribanov, Michael Ginzburg, Zhanna M. Shatilina, Yulia A. Lubyaga, Ekaterina V. Madyarova, Anton N. Gurkov, Maxim A. Timofeyev, Hans-O. Portner, Franz J. Sartoris, Rolf Altenburger, and Till Luckenbach Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

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Page 1 of 36

Environmental Science & Technology

1

Uptake kinetics and subcellular compartmentalization explain lethal

2

but not sublethal effects of cadmium in two closely related amphipod

3

species

4 5

Lena Jakob* ab, Daria S. Bedulina c, Denis V. Axenov-Gribanov c, Michael

6

Ginzburg a, Zhanna M. Shatilina c, Yulia A. Lubyaga c, Ekaterina V.

7

Madyarova c, Anton N. Gurkov c, Maxim A. Timofeyev c, Hans-O. Pörtner ab,

8

Franz J. Sartoris a, Rolf Altenburger d and Till Luckenbach* d

9 10

a

11

for Polar and Marine Research, Am Handelshafen 12, 27570, Bremerhaven, Germany

12

b

13

Bremen, Germany

14

c

Institute of Biology, Irkutsk State University, Karl Marx str.1, 664003 Irkutsk, Russia

15

d

Department of Bioanalytical Ecotoxicology, UFZ – Helmholtz Centre for Environmental

16

Research, Permoserstr. 15, 04318 Leipzig, Germany

Department of Integrative Ecophysiology, Alfred Wegener Institute Helmholtz Centre

Faculty of Biology and Chemistry, University of Bremen, Leobener Straße, 28359

17 18

*Lena

19

Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570,

20

Bremerhaven, Germany, +49 471 4831 1331, [email protected]

Jakob, Department of Integrative Ecophysiology, Alfred Wegener Institute

21 22

*Till

23

Environmental Research, Permoserstr. 15, 04318 Leipzig, Germany, +49 341 235 1514,

24

[email protected]

Luckenbach, Department of Bioanalytical Ecotoxicology, UFZ – Helmholtz Centre for

ACS Paragon Plus Environment

1


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25

Abstract

26

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod

27

species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower

28

fresh weights of E. cyaneus) and cellular stress response (CSR) capacity potentially

29

causing species-related differences in uptake, internal sequestration and toxic

30

sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd

31

uptake rates, related to a given exposure concentration, were higher and lethal

32

concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6°C).

33

Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E.

34

cyaneus: 18 nM (2.0 µg L-1); E. verrucosus: 115 nM (12.9 µg L-1); 4 weeks exposure; 6°C),

35

Cd amounts in metal sensitive tissue fractions (MSF) related to fresh weight were

36

similar in both species (E. cyaneus: 0.25±0.06 μg g-1; E. verrucosus: 0.26±0.07 μg g-1),

37

whereas relative Cd amounts in the biologically detoxified heat stable protein fraction

38

were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size

39

appears to mainly explain species-related differences in Cd uptake and sensitivities.

40

Only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates

41

when exposed to Cd at LC1 over 4 weeks indicating metabolic depression and pointing

42

to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

43 44

Key words: Crustacea, heavy metal, metabolic rate, ventilation, sublethal effects,

45

metabolic depression, metal sensitive fraction, aquatic toxicology, Baikal, amphipods


2

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46

Introduction

47

Susceptibility of aquatic organisms to dissolved metals varies largely due to differences

48

in bioavailability, bioaccumulation kinetics and internal distribution of metals

49

Dissolved cadmium (Cd) enters the body of aquatic organisms mainly through

50

permeable body surface such as gill epithelia 2. According to the biotic ligand model

51

(BLM) only the free ion is available for uptake

52

permeability of epithelia to dissolved hydrophilic trace metal ions; uptake is thus

53

thought to be facilitated by diffusion through calcium (Ca) channels as Cd2+ and Ca2+

54

have the same charge and similar radii 6. Moreover, uptake is mediated by specific Ca2+

55

transport mechanisms such as Ca2+-ATPases and Na+/Ca2+-exchangers 7,8 and the highly

56

variable Ca2+ (Cd2+) uptake rates across aquatic organisms can be related to species-

57

specific Ca2+ demands 9. Thus, both Cd speciation in the exposure medium and

58

physiological regulation of transport systems are important factors for Cd toxicity

59

Consequently, Cd uptake was explained by the phylogenetic relationships of species

60

when accounting for body size 10, because (permeable) body surface increases with body

61

size to a lesser degree than body volume when comparing organisms of the same species

62

or very closely related species. Gill surface area in amphipods, for instance, was shown

63

to be relatively large in small amphipods compared to larger ones 11. Besides uptake of

64

dissolved Cd via epithelia uptake of metals via food has also been shown to be an

65

important exposure route 12,13.

3–5.

1.

This is due to the intrinsically low

3–5.

66

There is no known critical threshold of total body concentration of accumulated metal

67

for a given species determining the onset of toxic effects 1,14. Rather, toxicity is related to

68

a critical concentration of metabolically available accumulated metal that builds up

69

when the rate of metal influx exceeds the combined rates of excretion and detoxification

70

1.

Bioaccumulation kinetics can be deduced from the biodynamic metal bioaccumulation


3


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71

model, which combines site-specific, geochemical data from a site of interest with

72

parameterization of key physiological constants for a species from that site

73

Metabolically available accumulated metal can operationally be determined by

74

subcellular compartmentalization 16,17. The underlying principle of this approach is that

75

metal toxicity is predominantly explained by metal associated with enzymes and cell

76

organelles contained within the “metal sensitive fraction” (MSF) post tissue

77

fractionation. By contrast, metal that is bound to heat stable proteins (including

78

metallothionein-like proteins (MTLP)) or metal rich granules (MRG) is considered as

79

biologically detoxified

80

Moreover, behavioral and sublethal effects of aquatic organisms to the same

81

concentrations of metabolically available metal have not been studied yet across a set of

82

different species and effects may vary depending on the stress response strategy.

16–18.

15.

However, the fractions are often not well characterized.

83

Animals adopt different strategies to cope with environmental stress such as elevated

84

trace metal concentrations in their environment eliciting various cellular, physiological

85

and behavioral responses

86

of resistance against adverse conditions such as exposure to chemical or thermal

87

stressors

88

equipped with is activated as a reaction to damage of biological macromolecules

89

regardless of the type of stress

90

mitigate or repair the damage of macromolecules and mediate transformation or

91

sequestration of stress causing agents 20. Behavioral and physiological strategies can be

92

directed to reduce stress impact and may include down-scaling of metabolic activity 21.

20.

19.

Cellular stress response systems are important mediators

The universal stress response system that cells of animals are generally

20.

Components of the cellular stress response system

93

Crustaceans are particularly sensitive to toxic metals like Cd 22 and are therefore used

94

as experimental organisms for chemical risk assessments. Amphipods (Amphipoda,

95

Crustacea) endemic to Lake Baikal constitute key components of benthic communities


4

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23.

96

from littoral to deepwater zones of the lake.

97

site, is located in south-east Siberia and by volume is the largest freshwater body on

98

earth

99

concentrations of 0.0042 µg L-1 were measured in the Selenga shallow and were below

100

the detection limit (0.002 µg L-1) in the bay of Listvyanka. Concentrations in biota

101

(phytoplankton and invertebrates) were 0.0723 – 0.732 µg g dry weight-1 in the Selenga

102

shallow and 0.0375 – 1.42 µg g dry weight-1 in the bay of Listvyanka

103

industrialization and further sources of pollution (e.g., thawing of permafrost,

104

agriculture and tourism) of the region have increased significantly and progressively

105

during the last decades

106

millions of years 28; thus, it is unclear whether Lake Baikal endemics are able to adapt to

107

a rapidly changing environment. Pollutants entering the lake nowadays could persist for

108

centuries because water exchange in Lake Baikal proceeds over 377 - 400 years 29. The

109

extremely low total mineralization of 120 mg L-1 30 and low calcium ion concentration in

110

Lake Baikal water (Ca2+ = 15.2-16.1 mg L-1)30,31. Along these lines, increased uptake of

111

heavy metals were observed under reduced salinity conditions

112

concentrations of dissolved organic carbon (DOC), which may reduce bioavailability of

113

Cd, are low in Lake Baikal (90-110 µM) 34.

114

24.

Lake Baikal, a UNESCO world heritage

Cd concentrations of Lake Baikal water and biota are presently low; water

26,27.

25.

However,

The abiotic conditions of Lake Baikal were stable for

32,33.

Moreover,

The objectives of this study were to study uptake of waterborne Cd, subcellular

115

compartmentalization

and

sublethal

physiological

116

metabolic rate) of amphipods endemic to Lake Baikal in a comparative approach. The

117

investigated species are closely related but differ with regard to parameters determining

118

chemical uptake and toxic sensitivity. The body size of Eulimnogammarus cyaneus

119

(Dybowski, 1874) is considerably smaller than that of Eulimnogammarus verrucosus

120

(Gerstfeld, 1858) and the body surface in relation to the body volume (and, by extension,


effects

(ventilation

and

5


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121

the permeable body surface) is therefore larger. In consequence, higher Cd uptake rates

122

can be assumed for E. cyaneus than for E. verrucosus. Calcium requirements are likely

123

similar in the two species as they are closely related, live in the same habitat and have

124

similarly shaped exoskeletons. E. cyaneus, however, may possess a higher potential to

125

mitigate the effects of internal Cd than E. verrucosus, owing to its higher constitutive

126

levels of cellular stress response related proteins

127

higher upper thermal limit of E. cyaneus compared to E. verrucosus, which in contrast to

128

E. cyaneus migrates from the upper littoral zone to deeper waters when water

129

temperatures close to the shoreline may rise 36.

35.

This is in accordance with the

130

Concentration-mortality relationships were determined to derive low biologically

131

effective doses, which were then applied to investigate physiological responses on an

132

effect-scaled basis. Uptake kinetics (increase of internal metal over time) and metal

133

compartmentalization were studied to discriminate between the influences of these two

134

factors on effect propagation. The extent to which sublethal concentrations elicit

135

adverse functional effects was investigated through the quantification of physiological

136

parameters. Experimental exposures were performed over four weeks to simulate long-

137

term exposure conditions in situ.

138 139

Material and Methods

140

ANIMAL SAMPLING

141

Adult (identified by size

142

kick-sampling 38 in the littoral zone of Lake Baikal at depths of 0 - 1.2 m in proximity to

143

the Bolshie Koty settlement area (51°9137” N, 105°0691” E), which represents a pristine

144

field site in the area of the Pribaikalsky National Park. For the location of Lake Baikal in

145

Eastern Siberia/Russia and for the location of the sampling sites refer to Protopopova et

37)

E. cyaneus and E. verrucosus specimens were collected by


6

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39.

146

al.

Water taken in about 12 km distance to this site contained no detectable

147

concentrations of Cd (detection limit: 0.002 µg L-1)

148

individuals with visible parasites (i.e., leeches) were excluded. Immediately after

149

sampling, amphipods were transported to the laboratory in insulated boxes filled with

150

Lake Baikal water. Specimens of E. cyaneus (per tank: n = 150 - 200, fresh weight: 20 - 44

151

mg per individual) and E. verrucosus (per tank: n = 25, fresh weight: 418 - 942 mg per

152

individual) were quickly transferred into separate 2 L polypropylene (PP) tanks (high

153

density PP of food grade quality) containing aerated Lake Baikal water acclimated to 6°C

154

corresponding to the reported annual mean water temperature for the littoral of Lake

155

Baikal

156

from the littoral, which were previously cleaned with boiling Lake Baikal water. Animals

157

were kept in acclimation tanks for at least two days to assure that all animals were

158

intact. Experiments were performed in 2013 (July - October) at the field station of

159

Irkutsk State University in Bolshie Koty.

31,34,40.

25.

Egg-carrying females and

About two thirds of each tank bottom were covered with small pebbles

160 161

LONG-TERM CONCENTRATION-MORTALITY RELATIONSHIPS

162

Prior to experiments, tanks were prepared by keeping them filled with Lake Baikal

163

water for two weeks exchanging the water once every other day. Preliminary

164

experiments revealed that Lake Baikal water stored in uncleaned plastic containers for

165

four weeks contained 0.059 nM Cd (6.6 ng L-1), which is far below the experimental

166

concentrations (see below). Other contaminants were not determined. Tanks were then

167

pre-soaked with water containing CdCl2 at the respective exposure concentrations for

168

two days. CdCl2 was chosen due to its high solubility in water (120g in 100 g water;

169

25°C)

170

had no visible effects on the studied species at concentrations 22 times higher than the

41.

Further, preliminary experiments revealed that chloride in the form of NaCl


7


171

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highest experimental chloride concentrations used in this study.

172

Individuals of E. cyaneus (n = 15) and E. verrucosus (n = 10) were placed in each plastic

173

container with 0.4 and 1.2 L of well-aerated CdCl2-contaminated 6°C Lake Baikal water,

174

respectively. As only 10-15 individuals were added to each tank, no pebbles were used

175

in this setup because animals could attach to the aeration tube or tank corners. In

176

parallel to a water control without Cd, treatments were set up with nominal

177

concentrations of 8.7, 35, 105, 210, 419, 559, 1117 and 2235 nM Cd (0.98, 3.9, 11.8, 23.6,

178

47.1, 62.8, 125.7 and 251.3 µg L-1); three tank replicates per concentration) following

179

the OECD guideline for the testing of chemicals that requires at least five geometric

180

concentrations

181

concentrations were based on previously conducted range-finding tests. Actual Cd water

182

concentrations were measured at the start and after four days as this was the rate of

183

water changes in all experimental concentrations (as described below). Tanks were

184

randomly distributed in a laboratory refrigerator. During the 4-weeks exposure, the

185

omnivorous animals were fed ad libitum with a mix of amphipods, algae and water

186

plants collected in the littoral of Lake Baikal (frozen, air-dried at ≈30°C and roughly

187

mortared). The food that was applied in this study had a Cd content of 0.26 ± 0.04 μg g

188

dry weight-1, which is in line with reported data as low background concentrations of Cd

189

were measured in amphipods and phytoplankton of the sampling area (range: 0.0375 –

190

0.804 µg g dry weight-1) 25. In this study the route of Cd uptake was not considered. Cd

191

uptake was related to the concentration in water and physiological effects are based on

192

Cd concentrations determined in different subcellular fractions (see below). Mortality

193

was monitored daily and dead animals were removed.

42.

Information on Cd speciation is given below. The applied

194 195

PHYSIOLOGICAL EXPERIMENTS


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Experimental design and animal maintenance

197

Animals were exposed for four weeks at 6.0 ± 0.8°C to their species-specific nominal LC1

198

(lethal concentrations for 1% of the test groups after 4 weeks exposure to dissolved Cd

199

at 6°C; LC1 for E. verrucosus = 115 nM (12.9 µg L-1), LC1 for E. cyaneus = 18 nM (2.0 µg L-

200

1)

201

consumption and ventilation rates were determined weekly as well as hemolymph pH

202

(measured only in E. verrucosus; small size of individuals precluded measurements in E.

203

cyaneus). Hemolymph samples for analysis of major cations (Na+, K+, Mg2+ and Ca2+) and

204

whole animal samples for Cd analysis (directly taken from tanks) were taken at the same

205

time points, frozen in liquid nitrogen and stored at -80°C. All parameters were analyzed

206

in parallel also in controls.

as sublethal concentration. Exposure water was changed every fourth day. Oxygen

207 208

Routine metabolic rate and resting ventilation

209

Experimental set up and measurement procedures are described in detail elsewhere 36.

210

In brief, routine metabolic rate was measured as oxygen consumption in flow-through

211

respiration chambers equipped with optical sensors Microx TX3 and OXY4 and Oxygen

212

Micro-Optodes

213

Germany). Teflon® chambers were closed with plexi-glass lids and submerged in 2 L

214

temperature-controlled water tanks (6.0 ± 0.2°C). For analysis of ventilation rates (=

215

pleopod beat rates) animals were placed in Teflon® chambers with perforated

216

depressions to allow unhindered water circulation and pleopod movements were video-

217

recorded. Video sequences (if necessary in slow-motion) were analyzed by eye at the

218

computer screen to quantify pleopod beat rates (Hz). Chamber for oxygen and

219

ventilation measurements are depicted in Fig. S1 (supporting information).

(NTH-PSt1-L5-TF-NS*35**x1,20-PC3,1-YOP)

(Presens,

Regensburg,

220


9


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221

Hemolymph ion regulation and pH

222

Concentrations of the major hemolymph cations Na+, K+, Mg2+ and Ca2+ were determined

223

in diluted hemolymph samples. For hemolymph extraction, amphipods were carefully

224

patted dry with lint-free paper and glass capillaries with ultrafine tips were introduced

225

dorsally without damaging the intestines. Samples were stored at -20 to -25°C prior to

226

analysis. Ion concentrations were determined chromatographically with a Dionex ICS

227

1500 equipped with an Ion Pac CS 16 column operated at 40°C. Methane sulfonic acid

228

(30 mM) was used as eluent at a flow rate of 0.36 mL min-1. Three point calibrations of

229

the Dionex™ Combined Six Cation Standard-I (Dionex GmbH, Idstein, Germany) were

230

used for peak identification and peak area based quantification.

231

Hemolymph pH was measured with a microelectrode (InLab® Ultra-Micro, Mettler

232

Toledo, Giesen, Germany) in hemolymph immediately after extraction. Measurements

233

were performed at 6.0 ± 0.2°C in a water bath.

234 235

CADMIUM ANALYSIS

236

Subcellular cadmium compartmentalization

237

Cd was quantified in five subcellular compartments: (1) heat stable proteins (HSP;

238

fraction contains MTLPs), (2) metal rich granules and undigested exoskeleton

239

(MRG+exo), (3) heat denaturable proteins (HDP), (4) organelles and (5) cell debris. For

240

quantification of Cd in these compartments, amphipods were subjected to fractionation

241

with differential centrifugation

242

pulex was adapted

243

each sample; E. verrucosus samples consisted either of the anterior or the posterior half

244

of an individual that were separated with a scalpel. Prior to tissue extraction, weakly

245

bound external Cd was removed by washing the animals for about 20 sec in 10 mM

43.

16.

A protocol on subcellular fractionation in Gammarus

Due to the small size of E. cyaneus four animals were pooled for


10

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246

EDTA solution with a physiological content of NaCl and KCl to prevent cellular water

247

loss by osmosis and subsequent quick rinsing with deionized water. This cleaning

248

procedure was repeated once. Tissues were then homogenized with a laboratory

249

blender (Ultra-Turrax, IKA, Staufen, Germany) for 20 - 30 sec at 17,000 rpm in 1.5 mL

250

ice-cold Tris-HCl buffer (pH 8.0). Homogenates were centrifuged at 1,800 x g (15 min,

251

4°C). The supernatant was ultracentrifuged at 100,000 x g (1 h, 4°C) in order to produce

252

a pellet containing mitochondria, lysosomes and microsomes (= organelle fraction). The

253

ultracentrifuge supernatant was incubated at 80°C for 10 min and kept on ice for 1 h to

254

precipitate HDP. HSP and HDP fractions were separated by ultracentrifugation at 50,000

255

x g (12 min). The pellet of the first centrifugation step was resuspended in 1 mL

256

deionized water and heated to 100°C for 2 min. Subsequently, it was digested with 1.5

257

mL 1M NaOH (1h, 70°C) and centrifuged at 10,000 x g (10 min) for separating the MRG +

258

exo fraction which was pelleted; the supernatant contained digested exoskeleton, nuclei

259

and cell debris (= debris fraction). All fractions were transferred to microwave Teflon®

260

tubes; 5 mL of 65% HNO3 (p.a. degree; Merck, Darmstadt, Germany) were added and

261

incubated for 4 h. Later, 0.5 mL of 30% H2O2 (p.a. degree; Merck Darmstadt, Germany)

262

were added and the samples were subjected to complete digestion in a microwave-

263

system (CEM MARSXpress, Matthews, USA). Power was gradually increased from 100 to

264

500 W within 5 min and held at 800 W for 15 min. After cooling, the samples were

265

diluted with deionized water in volumetric flasks (25 mL). Rhodium Standard for ICP

266

(TraceCERT®, Sigma-Aldrich, Darmstadt, Germany) was added to each sample at a

267

concentration of 10 μg L-1 and served to ensure measuring quality of the ICP-MS device.

268

Blanks were randomly included and treated like samples.

269 270

Cadmium quantification and speciation


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271

Cd was determined by inductively coupled plasma mass spectrometry (ICP-MS; Elan

272

6000 (PerkinElmer, Waltham, USA)) using Argon as nebulizer gas at a flow rate of 0.9 L

273

min-1 (plasma power: 1000 W) in unfiltered water samples (one sample of each

274

concentration of the concentration-mortality experiment and three samples of each LC1

275

exposure) stored in Falcon tubes taken at day 1 (day of water change) and 4 (shortly

276

prior to water change) from aquaria as well as in tissue samples (10 samples of each

277

species per time point). Samples were kept frozen prior to analysis. Each sample was

278

analyzed in triplicate with ICP-MS. Cd concentrations of external standard solutions

279

were 0.1, 1 and 10 μg L-1 (Multi-element Calibration Standard 3, PerkinElmer, Waltham,

280

USA). Nominal and measured water concentrations of Cd in concentration-mortality

281

experiments and LC1 exposures are summarized in the supporting information in Table

282

S1 and S2, respectively.

283

The software PHREEQC

284

(supporting information; Table S3). It was revealed that Cd is mainly available in the

285

free ionic form (>95%). The pH is ranging from 7.7 to 8.3 in the surface water of Lake

286

Baikal

287

the calculations. Data on the chemistry of Lake Baikal water, which we used to model

288

speciation is available in the supporting information (Table S4).

23

44

was used to model Cd speciation in Lake Baikal water

due to its low buffering capacity; the broad range of pH was accounted for in

289 290

DATA ANALYSIS AND STATISTICS

291

The modified non-linear HILL model (Equation 1, supporting information) was fitted to

292

concentration-mortality data in order to derive effective lethal concentrations for

293

certain percentages of the test groups (LCx%).

294

To analyze whether exposure to the species-specific LC1 of Cd and exposure time had an

295

significant effect (p > 0.05) on ventilation and oxygen consumption a two-way ANOVA


12

Page 13 of 36


296

was performed using the package “car” 45 for R. The Shapiro-Wilk test was used to test

297

for normality and the Fligner-Killeen test was used to test for equal variances.

298

Heteroscedastic data was “White-corrected” for covariances

299

Sidak’s multiple comparison test was applied to reveal whether Cd exposed groups were

300

different from their parallel control groups and to analyze the time effect using

301

GraphPad Prism version 7 (GraphPad Software, La Jolla, California, USA). Means of two

302

groups were compared by the t-test or the Mann-Whitney-U test depending on the

303

assumptions. Data are presented as means ± standard error of the mean (SE) unless

304

otherwise indicated.

46

with the “hc3” model

47.

305 306

Results

307

CADMIUM WATER CONCENTRATIONS

308

Measured concentrations deviated from nominal concentrations by 24 and 11% at the

309

lowest Cd concentration applied (8.7 nM; 0.98 µg L-1) in E. verrucosus and E. cyaneus,

310

respectively. Deviations from nominal concentrations increased progressively with

311

increasing test concentrations reaching 40% (E. verrucosus) and 38% (E. cyaneus) at the

312

highest exposure concentration (2235 nM; 251.3 µg L-1). Between water changes (every

313

fourth day) Cd concentrations decreased by 26 and 56% in the lowest exposure

314

concentration and by 36 and 26% in the highest test concentration in treatments with E.

315

verrucosus and E. cyaneus, respectively (Table S1, supporting information).

316

In LC1-exposures, measured concentrations deviated from nominal ones by 0 - 17%

317

and decreased by 27 - 54% within four days (Table S2, supporting information). All

318

nominal concentrations are based on the measured concentration of a Cd stock solution

319

(4.4 mM; 0.5 g L-1) applied in all experiments presented here.

320


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Page 14 of 36

321

MORTALITY

322

Time periods until lethality (lethal times; LT) were significantly shorter for E. cyaneus

323

than for E. verrucosus in treatments with identical CdCl2 concentrations as shown for the

324

1117 nM Cd (125.7 µg L-1) treatments in Fig. 1B (LT50 E. verrucosus = 7.6 d (95% CI =

325

6.8, 8.4) and LT50 E. cyaneus 3.1 d (95% CI = 2.8, 3.4)). Higher Cd sensitivity of E.

326

cyaneus compared to E. verrucosus with respect to mortality is also reflected by nominal

327

LC50 values derived from nonlinear regressions for the concentration-mortality

328

relationships, which were significantly lower for E. cyaneus than for E. verrucosus (159

329

nM (95% CI = 141, 179); (18 µg L-1; 95% CI = 16, 20) vs. 366 nM (95% CI = 316, 406);

330

(41 µg L-1; 95% CI = 36, 46) (Fig. 1A). No mortality was observed in controls.

331 332

In LC1 experiments, mortality in treatments and controls was < 1.5% during the entire experimental period.

333 334

ROUTINE METABOLIC RATE AND RESTING VENTILATION

335

Exposure to species-specific nominal LC1 of dissolved Cd (4 weeks, 6°C) caused a highly

336

significant reduction of oxygen consumption and ventilation in E. verrucosus but not in E.

337

cyaneus (ANOVA; p < 0.0001 and p ≥ 0.6641, respectively) (Fig. 2). Oxygen consumption

338

and ventilation rates of E. verrucosus were reduced by 15 - 36% and 18 - 38%,

339

respectively, in comparison to parallel controls. Exposure time had also a weak effect in

340

E. verrucosus but not in E. cyaneus (p ≤ 0.0332 and p ≥ 0.2581, respectively). However,

341

the post-hoc test revealed that the time effect is only due to oxygen consumption data in

342

both the control group and the Cd-exposed group that differed at week 1 and 3. For

343

ventilation, differences occurred between week 2 and 3 only in the Cd-treated animals,

344

but overall, there was no progressive increase or decrease of physiological rates over

345

exposure time.


14

Page 15 of 36


346

When both species were exposed to the same Cd concentration (35 nM; 3.9 µg L-1) for

347

four weeks, E. verrucosus showed a decrease in oxygen consumption by 26% compared

348

to controls (control: 2.1±0.2 vs. treatment: 1.6±0.2; p = 0.045), whereas no differences

349

from controls were found for E. cyaneus (control: 6.0±0.4 vs. treatment: 5.7±0.4; p =

350

0.507) (data not displayed).

351 352

HEMOLYMPH ION CONCENTRATIONS AND pH

353

In E. verrucosus and E. cyaneus specimens exposed to Cd at the respective LC1 (4 weeks,

354

waterborne Cd LC1; 6°C) for four weeks, hemolymph concentrations of the major

355

cations Ca2+, Na+, K+ and Mg2+ were not significantly altered from control values.

356

For hemolymph pH (only determined in E. verrucosus) no significant differences were

357

seen between Cd treatments and controls after one week of exposure, but from week

358

two on, the pH of the hemolymph of treated animals was slightly but statistically

359

significantly decreased in comparison to control samples (mean difference in pH: 0.062;

360

p = 0.037) (Fig. S2, supporting information).

361 362

CADMIUM BODY BURDENS AND COMPARTMENTALIZATION

363

Total Cd body burdens (i.e., sums of Cd of all different tissue fractions, which are

364

displayed in Fig. 3) progressively increased in E. verrucosus and did not reach steady-

365

state within the entire exposure time (Fig. 4). In E. cyaneus, total Cd tissue

366

concentrations increased until week 2 of exposure and remained more or less

367

unchanged thereafter (Fig. 4). After four weeks, bioconcentration factors (BCF) of total

368

accumulated Cd were 79 and 500 for E. verrucosus and E. cyaneus, respectively. Major

369

increases of Cd concentrations of the different fractions were seen between weeks 1 and

370

2 in E. cyaneus and between weeks 2 and 3 in E. verrucosus (Fig. S3, supporting


15


Page 16 of 36

371

information). Cd concentrations of the MSF, representing the accumulated bioavailable

372

Cd, were more than two-fold higher in E. cyaneus compared to E. verrucosus in week 3

373

but almost equal in weeks 2 and 4 (Fig. 4). Biologically detoxified fraction of Cd (BDF-

374

Cd) was generally higher in E. cyaneus than in E. verrucosus, however, differences were

375

only significant after two weeks of exposure (p = 0.048). The toxicological role of Cd that

376

is associated with cell debris (debris fractions) is not clearly understood and thus

377

neither included in the MSF nor in the BDF as in other studies

378

makes a contribution of 9 and 14% to the total Cd body burden after four weeks in E.

379

verrucosus and E. cyaneus, respectively. After 4 weeks, the MSF-Cd accounted for 24 and

380

20% and the BDF-Cd for 63 and 70% of the total Cd in E. verrucosus and E. cyaneus,

381

respectively. The distribution of Cd among the different cellular compartments

382

(percentages of the fractions in relation to total body concentration) was similar to that

383

found in Gammarus pulex 43.

48.

The debris fraction

384

When exposed to the same external Cd concentration (105 nM; 11.8 µgL-1) for four

385

weeks, the BDF-Cd and MSF-Cd determined in E. cyaneus exceeded that found in E.

386

verrucosus by 3.7 and 7.2-fold, respectively (Fig. S4, supporting information).

387 388

Discussion

389

In the present study we examined the toxic impact of Cd on two phylogenetically closely

390

related amphipod species, E. verrucosus and E. cyaneus, that differ with regard to

391

parameters that may determine metal uptake and toxic sensitivity, namely body size and

392

titer of cellular stress-response related heat shock proteins 70 (Hsp70). The

393

physiological effects of exposure to the same lethal concentration of Cd (1117 nM CdCl2)

394

were observed at shorter incubation times in E. cyaneus than in E. verrucosus. LC50 (4

395

weeks waterborne Cd LC50; 6°C) values (based on nominal concentrations) were 366


16

Page 17 of 36


396

and 159 nM for E. verrucosus and E. cyaneus, respectively. The lower sensitivity of E.

397

verrucosus to Cd is likely associated with lower uptake rates and may be due to its larger

398

body size. Whereas during the 4-week exposure to species-specific LC1 (4 weeks

399

waterborne Cd LC1; 6°C) accumulation showed saturation in E. cyaneus this was not the

400

case for E. verrucosus. The BDF of Cd was higher in E. cyaneus than in E. verrucosus,

401

which shows that biological detoxification of Cd is more effective in E. cyaneus and may

402

mitigate toxic Cd effects to a higher degree. E. verrucosus, by contrast, displayed reduced

403

metabolism in response to sublethal Cd concentrations, which may have added to the

404

relatively low Cd uptake in specimens of this species. Metabolic depression following Cd

405

exposure was only determined in E. verrucosus and occurred at Cd concentrations that

406

were substantially below lethal levels. Further, the degree of metabolic depression did

407

not show concentration or time dependency and paralleled decreases in ventilation.

408

Thus, the decreases may not be directly related to metabolically available Cd and may

409

rather be the result of specific molecular cascades stimulated by Cd exposure.

410 411

UPTAKE KINETICS AND COMPARTMENTALIZATION OF TOTAL CD BODY BURDEN

412

Cd uptake proceeded slower in the larger E. verrucosus than in E. cyaneus, which is likely

413

associated with differences in the body surface to body volume ratios of the two species

414

(Fig. 4 and Fig. S3) as observed in previous studies on aquatic insects 10. Dependencies of

415

Cd accumulation kinetics in relation to body size have been earlier reported for other

416

crustaceans (mysid shrimps) 49 and aquatic insects 50. Although those studies focused on

417

surface bound metal while we studied internal metal here, similar scaling coefficients

418

can be expected across studies as they are due to the same body surface to body volume

419

dependencies

420

clean Lake Baikal water) further support the uptake/body size dependency described

10.

Observed total body concentrations of Cd in control animals (kept in


17


Page 18 of 36

421

above as concentrations of 0.03 (range: 0.03 - 0.04) and 0.09 (range: 0.08 – 0.10) μg g

422

FW-1 were determined in E. verrucosus and E. cyaneus, respectively (n = 4) (data not

423

shown). The contribution of particle-bound Cd, which was not investigated in this study

424

needs to be researched in future studies as the applied food could have adsorbed Cd in

425

case it was not immediately consumed by the animals. Other studies found that Cd

426

uptake via food is a major uptake route 13,48.

427

Whether bioaccumulation of Cd reaches steady state within the experimental

428

exposure period depends on external concentration, lifetime, uptake and elimination

429

rates, as well as on internal storage capacity, which is often related to body size.

430

Amphipods are net accumulators of trace metals and their steady state body

431

concentrations typically increase with local metal availability

432

amphipod Melita plumulosa has recently been described as a very weak net

433

bioaccumulator of copper

434

equilibrium is approached after approximately two weeks. A similar time period until

435

reaching equilibrium has been observed in the small freshwater amphipod Hyalella

436

azteca when exposed to 0.8 nM of Cd

437

increased progressively over the entire experimental period and did not reach a steady

438

state. This comparison indicates that it is important to consider uptake kinetics when

439

estimating metal toxicity as otherwise the sensitivity of species with slow Cd

440

accumulation (e.g. E. verrucosus) might be misleadingly interpreted to be comparatively

441

low and field effects may consequently be underestimated.

48.

1,51.

However, the

In E. cyaneus exposed to its LC1, our data indicate that

52.

However, Cd concentrations in E. verrucosus

442

Previously, it has been stated that Cd toxicity occurs when excretion and

443

detoxification rates are exceeded by Cd uptake leading to metabolically available excess

444

Cd 1. Our results emphasize that compartmentalization of the total accumulated metal

445

contributes to explain differences in toxicity because only a portion of the total body


18

Page 19 of 36


17,48.

446

burden exerts toxic effects

447

similar accumulation into potentially sensitive compartments has been previously

448

shown for aquatic insects

449

derivation of species-specific effect-scaled concentrations (4 weeks waterborne Cd LC1;

450

6°C) resulted in the same MSF of Cd in the species studied here (E. verrucosus = 0.26 ±

451

0.07 μg g fresh weight-1; E. cyaneus = 0.25 ± 0.06 μg g fresh weight-1 (6°C, 4 wk); Fig. 3).

452

Based on these observations, we found that MSF-Cd is a good predictor for lethal effects

453

in E. verrucosus and E. cyaneus. By contrast, the total Cd body burden was higher in E.

454

cyaneus mainly due to a higher Cd content in the HSP fraction containing proteins

455

related to the universal CSR. Although induction of heat shock proteins (also part of the

456

CSR) by Cd was found in both species in a 24 h-study 53, E. cyaneus started from a 5-fold

457

higher constitutive absolute concentration of Hsp70 than E. verrucosus under normal

458

physiological conditions 35,39.

10.

Similar susceptibility of closely related species due to

In accordance with the results of Buchwalter et al.

10,

459

Animals exposed to high bioavailable metal concentrations may show higher amounts

460

of metal detoxified in lysosomes contained in the organelle fraction 13,48,54. Pre-exposure

461

to high concentrations of bioavailable Cd can be excluded as a confounding factor in our

462

study since the Cd concentration at our sampling site was low

463

applicability, lysosomes/microsomes can be isolated upon subcellular metal partitioning

464

55.

25.

However, for general

465

In conclusion, differences in uptake kinetics and Cd compartmentalization likely

466

explain the observed differences in mortality. Metal accumulation and sequestration are

467

species-specific and only explicit consideration of the uptake kinetics as well as

468

determining the MSF of Cd is important because this is likely key in determining

469

effective lethal concentrations. Therefore, considering both uptake kinetics and


19


Page 20 of 36

470

compartmentalization of metals will likely improve the quality of models aiming to

471

predict biologically effective lethal effects.

472

The large deviations of measured from nominal Cd water concentrations are likely

473

due to adsorption of Cd to tank walls, food particles and stones needed to provide

474

shelter for the amphipods as it is known that the free Cd ion can be removed from

475

aqueous solutions by chelation, electrostatic interaction, such as ion exchange or the

476

formation of ionic pairs 56. Storage capacities for contaminated water are very limited in

477

the field station at Lake Baikal where the experiments were carried out; more frequent

478

water changes in the semi-static system or use of a flow-through system were therefore

479

not possible. However, as Cd concentrations decreased similarly in the experimental

480

setups for both examined species, the deviations of measured from nominal

481

concentrations do not affect the overall conclusions regarding species-specific

482

differences in Cd uptake and sequestration and Cd effects found in this study.

483 484

MECHANISMS OF TOXIC ACTION AND STRESS RESPONSE

485

Uptake kinetics and compartmentalization of Cd provide explanations for the

486

different species-specific concentration-mortality relationships but not for the different

487

physiological responses (i.e., reduction of ventilation and metabolic rates) to low Cd

488

doses. In contrast to E. cyaneus, E. verrucosus showed metabolic depression upon

489

exposures to its LC1 (115 nM) as well as to 35 nM for four weeks (6°C). The degree of

490

metabolic depression was comparable both Cd concentrations, i.e., it showed no

491

concentration dependence. In animals from the LC1 treatment metabolic rate and

492

ventilation were measured weekly; there was no time dependence although the internal

493

metabolically available Cd increased over time during exposure (Fig. 4). Metabolic

494

depression is a strategy that has been recorded in all major animal phyla and is reported


20

Page 21 of 36


495

to maximize survival time under adverse environmental conditions such as

496

hyperthermia, hypoxia, desiccation, hypersalinity and food deprivation

497

strategy of decreasing metabolic rate involves a reduction in energy utilization and

498

implies extended use of stored energy reserves, and usually involves reduced activity

499

and physiological rates (e.g., heart rate and ventilation) 21,57.

21,57.

The

500

Studies on the estuarine crab Chasmagnathus granulata or the white shrimp

501

Litopenaeus vannamei showed that Cd causes gill impairment (e.g. necrotic tissue, gill

502

thickening) and consequently hinders oxygen diffusion across the gill surface at higher

503

Cd concentrations than applied here

504

concentrations can likely be excluded as reason for decreased oxygen consumption in E.

505

verrucosus since we neither found a progressive decrease of these rates over time nor

506

dose-dependence of the effect as seen in other studies 33,60. Moreover, ventilation rates

507

in E. verrucosus decreased in concert with lowered oxygen consumption rates and it

508

seems obvious that both observations are mechanistically linked. The finding of only a

509

minor effect on the pH of the hemolymph in E. verrucosus and the unchanged

510

concentrations of major hemolymph cations in E. verrucosus and E. cyaneus in LC1

511

exposures support the assumption that hemolymph homeostasis was only negligibly

512

affected and, by extension, that severe toxic effects were absent. The slight changes in

513

pH of hemolymph may be explained by binding of Cd by hemocyanin leading to a

514

reduced buffering capacity as Cd2+ replaces Ca2+ at its binding sites

515

transporting hemocyanin is the major hemolymph protein in crustaceans (90 - 95%) 62

516

acting as pH buffer. Further, the altered metabolism may have caused the slightly

517

changed hemolymph pH.

58,59.

However, gill impairment at sublethal Cd

61.

The oxygen

518 519

PERSPECTIVES


21


Page 22 of 36

520

The ecophysiology of the two amphipod species studied here may contribute to explain

521

their different reactions to low effect-scaled concentrations of Cd. E. verrucosus is a cold

522

stenothermal species, which reproduces in winter, and has a comparably low upper

523

thermal limit in comparison to the summer-reproducing species E. cyaneus

524

Hypoxemia, which develops in the animal toward both ends of the thermal window,

525

likely contributes to the pattern of oxidative stress

526

oxidative stress

527

hypoxia

528

accordance with previous propositions 66. E. verrucosus exhibits behavioral plasticity to

529

cope with adverse environmental conditions. In summer, it escapes rising temperatures

530

in the upper littoral by migration to deeper and cooler waters 36,67 and in experimental

531

setups it was shown to avoid reduced oxygen concentrations

532

Lake Baikal is generally densely populated by a species-rich amphipod community; e.g.,

533

93 species are found at the sampling site in Bolshie Koty.

534

body size of adult E. verrucosus individuals is advantageous for withstanding the

535

competitive pressure of different species communities in different zones enabling them

536

to migrate to different habitats with different species compositions. However, escaping

537

from environmental pollution may not be possible.

65,

64

63.

36.

As Cd was also shown to induce

and causes depletion of oxygen availability in cells, which causes

we suggest an at least partly similar mode of action of the two stressors in

68.

69.

The littoral benthos of

The comparatively large

538

The finding of metabolic depression in E. verrucosus continuously occurring at Cd

539

levels in the low effect range (4 weeks waterborne Cd LC1; 6°C) over the entire exposure

540

time was striking and points to potentially severe effects even of low pollution levels on

541

biota of Lake Baikal and maybe also other water bodies. Metabolic depression is

542

typically paralleled by reduced activity, food uptake and reproduction 21,57. In the short

543

term, this strategy may help to avoid adverse impacts of chemicals. However, in the long

544

run, metabolic depression may have negative consequences on the population level. Less


22

Page 23 of 36


545

active organisms are more vulnerable to predation, reduced food uptake may result in

546

depletion of energy reserves and reduced reproduction and growth will lead to lower

547

recruitment. Thus, even concentrations of chemicals far below lethal ones may have

548

dramatic consequences on E. verrucosus populations in the wild. Amphipod diversity in

549

the littoral of Lake Baikal is extremely high but E. verrucosus as also E. cyaneus are

550

among species with large population sizes and impacts on their population structure is

551

likely to have dramatic consequences on the ecosystem.

552

In contrast to E. verrucosus, E. cyaneus is a sedentary species of the upper littoral and

553

does not migrate to escape elevated water temperatures. The species is exposed to large

554

changes in environmental conditions, such as seasonal temperature fluctuations from

555

close to freezing in winter to up to 20°C in summer 70. E. cyaneus did not show metabolic

556

depression in our experiments. It might rather respond to adverse conditions by

557

increasing cellular stress defense mechanisms, e.g., by increasing catalase activity at low

558

Cd concentrations as shown in other freshwater crustaceans

559

increased energy demand to support these processes and might alter the energy budget

560

of E. cyaneus. Shifts in energy budget at unchanged respiration rates have been observed

561

e.g. in Porcelio scaber when exposed to Cd 72.

71.

This may lead to

562

In order to identify possible further processes that could ultimately lead to reduced

563

Darwinian fitness of a species, further sublethal effects, such as on somatic growth,

564

reproduction, enzyme activities and energy state caused by low doses of Cd remain to be

565

studied in the here investigated species. Moreover, recent studies show that different

566

heavy metals may be handled individually by the organism considering metal

567

compartmentalization

568

Baikal endemic amphipod to low concentrations of a chemical stressor underscores the

13,48,73,74.

However, the extreme sensitivity of an important Lake


23


Page 24 of 36

569

necessity of water management strategies required to strictly avoid the chemical

570

contamination of Lake Baikal waters.

571 572

Acknowledgements

573

We highly acknowledge Ilsetraut Stölting for performing ICP-MS analyses and Birgit

574

Daus for technical advice. Moreover, our thanks go to Sabine Kasten and Ingrid Stimac

575

who provided their laboratory and microwave equipment. The students of Irkutsk State

576

University and Michael Meyer are acknowledged for laboratory assistance. This study

577

was performed within the LabEglo project HRJRG-221, financed by the bilateral funding

578

program “Helmholtz-Russia Joint Research Groups” (HRJRG) from the Helmholtz

579

Association and the Russian Foundation for Basic Research (RFBR). Russian participants

580

received further funding by the Russian Science Foundation (grant: 17-14-01063), the

581

RFBR (grants: 16-34-60060, 16-34-00687 and 15-04-06685) and a project part of

582

“Goszadanie” funded by the Ministry of Education and Science of the Russian Federation

583

(6.1387.2017PCH).

584 585

Supporting information

586

Hill model fitted to mortality data (Equation 1); nominal and measured Cd water

587

concentrations (Table S1 and S2); data on Cd speciation modeled with PHREEQC (Table

588

S3); data on Lake Baikal water chemistry used for modeling Cd speciation (Table S4);

589

oxygen and ventilation chambers (Fig. S1); hemolymph pH of E. verrucosus during 4 wk

590

LC1 (4 weeks waterborne Cd LC1; 6°C) exposures (Fig. S2); Cd concentrations in

591

different subcellular compartments of E. verrucosus and E. cyaneus during 4 wk LC1 (4

592

weeks waterborne Cd LC1; 6°C) exposures (Fig. S3) and after 4 wk exposure to 105 nM

593

CdCl2 (Fig. S4).


24

Page 25 of 36


594

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803

Figure captions

804

Fig. 1

805

(closed circles, black curve) exposed to different nominal concentrations of CdCl2 (0, 8.7,

806

35, 105, 210, 419, 559, 1117 and 2235 nM) (A) and to a nominal concentration of 1117

807

nM CdCl2 (B) for 4 weeks (6°C) (n = 3). Vertical lines mark the LC50 (A) and the LT50

808

values (B), respectively.

Cumulative mortality of E. verrucosus (open circles, grey curve) and E. cyaneus

809 810 811

Fig. 2

Oxygen consumption and resting ventilation (pleopod beating) of E. verrucosus

812

(A, C) and E. cyaneus (B, D) exposed to their species-specific LC1 (4 weeks waterborne

813

cadmium LC1; 6°C); LC1 E. verrucosus = 115 nM, LC1 E. cyaneus = 18 nM for 4 weeks at

814

6°C. Circles represent LC1 treatments and rhombs parallel controls. Data are displayed

815

as means ± SE (n = 7 - 13). Statistically significant differences between treatments and

816

parallel controls are indicated by asterisks (*p < 0.05; ****p < 0.0001).

817 818 819

Fig. 3

Cadmium concentration in different subcellular fractions of E. verrucosus (A)

820

and E. cyaneus (B) exposed to their species-specific LC1 (4 weeks waterborne cadmium

821

LC1; 6°C), LC1 E. verrucosus = 115 nM, LC1 E. cyaneus = 18 nM for 4 weeks at 6°C. Metal

822

sensitive fraction (MSF) = Organelle (mitochondria, lysosomes and microsomes) + HDP

823

(heat denaturable proteins); E. verrucosus = 0.26 ± 0.07 and E. cyaneus = 0.25 ± 0.06,

824

Biologically detoxified fraction (BDF) = MRG+exo (metal rich granules+exoskeleton) +

825

HSP + MTLPs (heat stable proteins including metallothionein-like proteins); E.

826

verrucosus = 0.66 ± 0.11 and E. cyaneus = 0.83 ± 0.13 and the toxicologically not well


30

Page 31 of 36


827

characterized debris fraction = Debris (cell debris). Data are presented as means ± SE in

828

μg g (fresh weight)-1 (n = 5-10).

829 830

Fig. 4

Total, biologically detoxified (BDF) and metal sensitive (MSF) Cd in E.

831

verrucosus (A) and E. cyaneus (B) upon exposure to their species-specific LC1 (4 weeks

832

waterborne cadmium LC1; 6°C; LC1 of E. verrucosus = 115 nM, LC1 of E. cyaneus = 18

833

nM) for up to four weeks. The MSF represents the biologically available Cd fraction

834

associated with toxic effects; the BDF comprises sequestered Cd. Linear models were fit

835

to data of E. verrucosus and a ligand binding (+ nonspecific binding) model was fitted to

836

the data sets of E. cyaneus y =

Bmax x + N s x . Data are presented as means ± SE (n = 5-10). Kd + x


31


Graphical abstract 84x34mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36


Fig. 1 Cumulative mortality of E. verrucosus (open circles, grey curve) and E. cyaneus (closed circles, black curve) exposed to different nominal concentrations of CdCl2 (0, 8.7, 35, 105, 210, 419, 559, 1117 and 2235 nM) (A) and to a nominal concentration of 1117 nM CdCl2 (B) for 4 weeks (6°C) (n = 3). Vertical lines mark the LC50 (A) and the LT50 values (B), respectively. 143x76mm (300 x 300 DPI)



Fig. 2 Oxygen consumption and resting ventilation (pleopod beating) of E. verrucosus (A, C) and E. cyaneus (B, D) exposed to their species-specific LC1 (4 weeks waterborne cadmium LC1; 6°C); LC1 E. verrucosus = 115 nM, LC1 E. cyaneus = 18 nM for 4 weeks at 6°C. Circles represent LC1 treatments and rhombs parallel controls. Data are displayed as means ± SE (n = 7 - 13). Statistically significant differences between treatments and parallel controls are indicated by asterisks (*p < 0.05; ****p < 0.0001). 137x112mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36


Fig. 3 Cadmium concentration in different subcellular fractions of E. verrucosus (A) and E. cyaneus (B) exposed to their species-specific LC1 (4 weeks waterborne cadmium LC1; 6°C), LC1 E. verrucosus = 115 nM, LC1 E. cyaneus = 18 nM for 4 weeks at 6°C. Metal sensitive fraction (MSF) = Organelle (mitochondria, lysosomes and microsomes) + HDP (heat denaturable proteins); E. verrucosus = 0.26 ± 0.07 and E. cyaneus = 0.25 ± 0.06, Biologically detoxified fraction (BDF) = MRG+exo (metal rich granules+exoskeleton) + HSP + MTLPs (heat stable proteins including metallothionein-like proteins); E. verrucosus = 0.66 ± 0.11 and E. cyaneus = 0.83 ± 0.13 and the toxicologically not well characterized debris fraction = Debris (cell debris). Data are presented as means ± SE in µg g fresh weight-1 (n = 5-10). 140x166mm (300 x 300 DPI)



Page 36 of 36

Fig. 4 Total, biologically detoxified (BDF) and metal sensitive (MSF) Cd in E. verrucosus (A) and E. cyaneus (B) upon exposure to their species-specific LC1 (4 weeks waterborne cadmium LC1; 6°C; LC1 of E. verrucosus = 115 nM, LC1 of E. cyaneus = 18 nM) for up to four weeks. The MSF represents the biologically available Cd fraction associated with toxic effects; the BDF comprises sequestered Cd. Linear models were fit to data of E. verrucosus and a ligand binding (+ nonspecific binding) model was fitted to the data sets of E. cyaneus. Data are presented as means ± SE (n = 5-10). 138x88mm (300 x 300 DPI)


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