Cadmium accumulation and toxicity in the unicellular alga Pseudokirchneriella subcapitata: Influence of metal-binding exudates and exposure time

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Environmental Toxicology

CADMIUM ACCUMULATION AND TOXICITY IN THE UNICELLULAR ALGA

PSEUDOKIRCHNERIELLA SUBCAPITATA: INFLUENCE OF METAL-BINDING

EXUDATES AND EXPOSURE TIME

NATHALIE PAQUET, MICHEL LAVOIE, FRÉDÉRIC MALONEY, JÉRÔME F.L. DUVAL, PETER G.C.

CAMPBELL, and CLAUDE FORTIN

Environ Toxicol Chem., Accepted Article • DOI: 10.1002/etc.2927

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Environmental Toxicology Environmental Toxicology and Chemistry DOI 10.1002/etc.2927

CADMIUM ACCUMULATION AND TOXICITY IN THE UNICELLULAR ALGA

PSEUDOKIRCHNERIELLA SUBCAPITATA: INFLUENCE OF METAL-BINDING EXUDATES AND EXPOSURE TIME

Running title: Evolution of cadmium accumulation and toxicity over time

NATHALIE PAQUET, †‡ MICHEL LAVOIE, †§ FRÉDÉRIC MALONEY, †|| JÉRÔME F.L. DUVAL, #

PETER G.C. CAMPBELL, † and CLAUDE FORTIN†

† Institut national de la recherche scientifique, Centre Eau Terre Environnement (INRS-ETE), Québec, Québec, Canada

‡ Centre d’expertise en analyse environnementale du Québec, Ministère du développement

durable, de l’Environnement et de la Lutte contre les changements climatiques, Québec, Québec,

Canada

§ Québec-Océan and Takuvik Joint Université Laval/CNRS Research Units, Département de Biologie, Université Laval, Québec, Québec, Canada

|| Administration portuaire de Québec, Québec, Québec, Canada

# Laboratoire interdisciplinaire des Environnements Continentaux (LIEC), CNRS-Université de Lorraine, Vandoeuvre- lès-Nancy cedex, France

*Address correspondence to [email protected]

Additional Supporting Information may be found in the online version of this article.

This article is protected by copyright. All rights reserved

Submitted 14 October 2014; Returned for Revision 31 January 2015; Accepted 2 February 2015

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Abstract: Predicting metal availability and toxicity for chronic (several hours or days) metal

exposure scenarios, even for unicellular algae, is a major challenge to existing toxicity models.

This is because several factors affecting metal uptake/toxicity, such as the release of metal-

binding exudates, changes in the kinetics of metal uptake/toxicity over time and algal

physiological acclimation to internalized metals, are still poorly understood. The present study

assessed the influence of these factors on Cd uptake and toxicity in laboratory batch cultures of

the freshwater alga, Pseudokircheneriella subcapitata. To do so, changes in the free Cd2+

concentrations due to the release of metal-binding algal exudates were monitored, 109Cd

accumulation in algal cells was measured and Cd-induced inhibition of algal growth as a

function of exposure time (from 12 to 96 h) was followed. Our results indicate that metal-binding

exudates may decrease the proportion of the free Cd2+ ion in solution up to twofold, a decrease

that affect Cd uptake and toxicity. Pseudokirchneriella subcapitata has the capacity to decrease

net Cd uptake rate on short-time scales (< 24 h), but this reduction in the Cd uptake rate

disappeared after 24 h and Cd toxicity occurred at relatively high Cd concentrations in solution.

These data illustrate some of the pitfalls of standard algal toxicity assays, which were designed

for short-term exposures, and suggest how robust chronic bioassays might be developed.

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Keywords: Phytoplankton, Ecological risk assessment, Metal uptake, Toxicity testing, Green

alga

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INTRODUCTION

The biotic ligand model (BLM) has been shown to accurately predict short-term uptake

(< 1 h) of cationic metals and their acute toxicity in algae, although some exceptions exist [1, 2].

However, predicting metal accumulation and toxicity in algae under chronic metal exposure

conditions (several hours or days) is still challenging and cannot be easily achieved [3-6]. In

chronic metal exposure, poorly understood complicating factors need to be taken into account in

order to model metal uptake and toxicity. Such factors include growth biodilution of internalized

metal [7], acclimation of the targeted organisms to the presence of the toxic metal [8], metal

efflux [9], possible depletion of metals from bulk solution due to the necessarily finite size of the

metal-supplying medium [10, 11], and production of metal-binding exudates [12]. The latter

situation can be particularly problematic in weakly buffered culture media without strong ligands

(e.g. NTA, EDTA) in which metal-binding exudates can significantly affect metal speciation,

uptake and toxicity [13-16]. Toxicity assays, which often need to be performed on natural waters

without addition of strong metal chelators in order to evaluate metal toxicity in situ [17, 18],

might then be biased by the release of metal-binding exudates by the algae.

Until now, studies about the effect of metal-binding exudates on metal uptake and

toxicity have focused mainly on marine algae [13-15, 19] with fewer reports dealing with

freshwater algal species [16, 20, 21]. In many of these studies, the free metal ion concentration

was not measured and the effect of metal-binding exudates on metal speciation thus remains

poorly documented, particularly in freshwater algal cultures. Other complications in chronic

exposures such as changes in the kinetics of metal uptake in algae and the time scale over which

Cd exerts its toxicity have not received much attention in the literature. Therefore, improvement

in our fundamental knowledge of metal uptake/toxicity in algae over chronic exposure periods is

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critically needed to refine current chronic toxicity models as well as to evaluate potential

weaknesses in toxicity assay procedures.

In the present study, the freshwater alga Pseudokirchneriella subcapitata was used as the

test organism. This species is one of the most commonly used bioassay algae around the world

[17, 18, 22, 23] and is widely distributed across nearly all types of inland waters [24, 25]. The

objectives of this research were to: 1) evaluate the effect of metal-binding exudates produced by

the freshwater alga P. subcapitata on Cd speciation; 2) evaluate Cd accumulation kinetics in P.

subcapitata in chronic (96 h) exposures to Cd and; 3) investigate the link between Cd speciation,

Cd accumulation and toxicity in P. subcapitata as a function of exposure time.

MATERIALS AND METHODS

Test organisms and culture conditions

Pure agar cultures of the unicellular green alga Pseudokirchneriella subcapitata

(Korshikov) Hindak (CPCC 37) were purchased from the University of Toronto Culture

Collection (UTCC), now known as the Canadian Phycological Culture Center (CPCC).

Asynchronous batch cultures were grown axenically (periodically checked by culture plating) in

100 mL of CEAEQ (Centre d’expertise en analyse environnemental du Québec) culture medium

with the 3-(N-morpholino)-propanesulfonic acid (MOPS) pH buffer but without any metal

chelating agent (Table 1). Stock algae cultures were maintained in 250 mL Erlenmeyer glass

flasks under constant rotary agitation (60 rpm), illumination (100 μmol m-2 s-1) and temperature

(20 ± 1 °C). A 2-mL sub-sample of algal culture was transferred to a sterile culture medium

every week using a sterile serological pipette. Culture media (without trace metals) as well as

material (Erlenmeyer flasks and filtration apparatus) destined for algal cultures were autoclaved

at 121 °C for 15 min. Culture media were then supplemented with a filter-sterilized trace metal

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mix (0.2 µm polycarbonate membrane, 47 mm). All plasticware and glassware were soaked for

at least 24 h in 10% (v/v) HNO3 and rinsed seven times with ultrapure water (18 MΩ cm;

Nanopure grade) and then dried under a class 100 laminar flow hood where all manipulations

that required precautions against possible contamination by airborne particulates were

performed.

Fe oxyhydroxides and trace metal speciation in the CEAEQ medium

Since the CEAEQ medium did not contain a trace metal buffer to stabilize iron in

solution, the predicted amount of trace metals (Cu, Co, Zn, Cd) adsorbed on amorphous

ferrihydrite (Feox) was calculated with the two- layer surface complexation model [26]. The

MINEQL+ chemical speciation programme version 4.6 [27] with updated formation constants for

aqueous inorganic complexes and intrinsic surface complexation constants for cations on Feox

[28] was used. Even when it is assumed that virtually all the added Fe precipitated in the

medium, the modeled adsorption of essential (Co, Zn, Cu) and non-essential (Cd) metal ions on

Feox was quantitatively unimportant (<10 %; except for Cu, for which up to 50% of Cu may be

adsorbed to Fe colloids). Moreover, at the highest [Cd] used in the present study, MINEQL+

simulations show that competition between Cd and Cu remains low, such that Cu adsorption on

Feox decreases by only 17%. Our calculations thus suggest that Fe colloids will not appreciably

affect Cd accumulation and toxicity in our experiments at constant [Cu2+].

Preparation of exposure media

A stock of acidified 109Cd radioisotope solution (3.45 mCi mg-1 Cd, Amersham) was

prepared in fluorinated ethylene propylene copolymer (Teflon®) flasks. The exposure media

consisting of CEAEQ medium spiked with 109Cd were left to equilibrate 24 h after addition of

109Cd and pH adjustment with small additions of NaOH or 0.1 M HCl. In all exposure media, the

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Cd addition step (targeted initial total Cd concentrations from 8 to 270 nM) did not increase the

ionic strength significantly (i.e., it remained constant at 7.5 meq L-1). Each exposure

concentration was tested in triplicate in 70 mL of CEAEQ medium using polycarbonate 250 mL

Erlenmeyer flasks. The pH was buffered at 7.0 ± 0.1 by the addition of MOPS (10 mM; the pH

fluctuated by less than 0.08 units after the 96-hour exposure). Cadmium speciation in the

exposure solutions was calculated with the chemical speciation model MINEQL+ (version 4.6;

[29]) with updated equilibrium constants [28], using total measured metal concentrations (at t = 0

or 96 h) as input data. All calculations were constrained to a fixed input pH (7.0) at equilibrium

with the atmosphere (pCO2 = 10-3.5 atm).. MINEQL+ simulations indicated that around 98% of

the total initial Cd in the CEAEQ exposure solutions is present as free Cd2+ ions.

Measurements of cadmium accumulation, cell density and cell size

Exponentially growing cells from the stock cultures were gently harvested on 2 µm

polycarbonate filters (Poretics, Fisher Scientific) using a vacuum pressure of <13 kPa. Harvested

cells were rinsed five times with CEAEQ-R medium (simplified culture medium; Table 1) and

resuspended in 20 mL of CEAEQ medium. Cell diameter, surface and volume (assuming

spherical cell shape) were determined in the algal concentrate resuspension with a Multisizer 3

Coulter Counter (70-µm aperture, Beckman). Afterwards, algal cells were inoculated into Cd

exposure media under a laminar flow hood. We decreased the initial cell density from 10,000 to

2,500 cells mL-1 to potentially minimize metal depletion over time and mimic more closely the

cell densities generally encountered in oligotrophic freshwater ecosystems [21]. Immediately

after inoculation, algal cultures were placed in the environmental chamber. Subsamples of the

algal cultures inoculated at an initial cell density of 2,500 cells mL-1 were taken after 0, 12, 24,

48, 72 and 96 h in order to measure cell sizes with the Coulter Counter and cell densities with a

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FacsCalibur flow cytometer (see details of measurement using flow cytometry in the next

section). Algal cultures inoculated at an initial cell density of 10,000 cells mL-1 were only

subsampled at 96 h.

At each time step where cell density and size were analyzed, 10-mL aliquots of the algal

cultures were gently filtered through two superimposed 2-µm polycarbonate membranes. The

filtrates were recovered to analyse the dissolved cadmium concentration remaining in the culture

medium. Afterwards, the filters were soaked for 10 min with 5 mL of 2 × 10-4 M EDTA solution

(in simplified CEAEQ-R medium); the EDTA solutions were then gently filtered and another 5

mL of EDTA was rapidly passed through the filters. The EDTA rinse removes the 109Cd bound

to the surface of the algal cells and the Cd remaining on the filter membrane is thus operationally

defined as intracellular [30, 31]. Filter radioactivity (intracellular algal Cd content) was measured

with a scintillation counter (Wallac 1414; Perkin Elmer Life Sciences) by placing the filters in

borosilicate vials containing 20 mL of Ecolume scintillation cocktail (ICN). The activity of the

lower filter was subtracted from that measured on the upper filter to correct for passive retention

of 109Cd by the polycarbonate filters. 109Cadmium activities in the filtrates of exposure media

(dissolved Cd in the exposure media) or after the EDTA rinsing step (cadmium adsorbed on the

algae) were also measured by scintillation counting. Mass balance calculations indicate that

losses of Cd on the filtration apparatus, pipette tips and due to handling of filters were 11 ± (SD)

6%.

Ion-exchange technique

Free [Cd2+] in initial exposure media (before inoculation of algal cells) as well as in

filtered samples from algal cultures at the end of the exposure (96 h; 0.2 µm, polycarbonate

filters) was measured using an ion-exchange technique (IET) [32] in algal cultures inoculated at

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2,500 cells mL-1. For the cultures inoculated at 10,000 cells mL-1, the free Cd2+ concentrations

were measured at the end of the exposure but not at the beginning. Briefly, the technique consists

of bringing a calibrated ion-exchange resin to equilibrium with the test solution. The metal

bound to the resin is then eluted and the original free metal ion concentration is back calculated

from a previous metal- resin calibration (See the SI.1 section of Supplemental Data,for details

about the IET technique).

Flow cytometry

Algal cell density was analyzed using a FACSCalibur (BD BioSciences) flow cytometer.

Algae subsamples (1 mL) were first vortexed for 5 minutes and then injected into the flow

cytometer. The algal cells were then hydrodynamically focused into a fluid stream so that the

cells passed individually through the light source. The pigments of the algal cells were excited by

a laser emitted by a 488 nm xenon- ion excitation lamp and the autofluorescence of the algae was

detected as red fluorescence (660-700 nm) by a FL3 fluorescence detector equipped with

fluorescence emission filters. Nonalgal particles and dead cells were excluded from the analysis

by setting an acquisition threshold on FL3 (positioned below the distribution of healthy control

cells). All parameters were collected as logarithmic signals and analyses were performed at a

high flow rate (60 µL/min), with acquisition over 120 s. The stability of the rate of aspiration

was measured after each 10 samples using TRUCount tubes (BioScience, # 340334).

Concentrations of algal cells (Cpop in cell/mL) were determined using the following equation 6

[33]:

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where “occurrence number” represents the number of fluorescence signals detected, “Vtot” is the

total sample volume comprising the fixing solution (mL), “Valgae” is the volume of culture

medium add in the cytometer (mL), “Raspiration” is the aspiration rate of the flow cytometer

(mL/min ) and “Treading” is the time for analysing one sample (min).

Growth rate calculation

Cell densities of algal cultures exposed to various total [Cd] and monitored at each time

points by flow cytometry were multiplied by the mean biovolume per cell (µm3 cell-1) measured

with the Coulter counter yielding total cell volume concentrations per mL (µm3 of alga mL-1).

The total cell volume concentrations per mL were then used to calculate the specific growth rate

(µ) of algal cultures exposed to various total [Cd] with the following equation (7):

where t0 and t represent the beginning and end of the experiment and Nt and Nt0 are total cell

volume concentrations (µm3 of alga mL-1) at time t and t0 respectively.

Cadmium toxicity

The toxicity of Cd was evaluated by measuring the culture yield (cell number or total cell

volume) at each exposure time step (12 to 96 h). The culture yield was then plotted as a function

of the total dissolved [Cd] (initial and mean exposure concentration), free final measured [Cd2+],

intracellular Cd concentrations or Cd cell quotas. The data were fitted with the macro REGTOX

(http://www.normalesup.org/~vindimian/en_index.html) to calculate the EC50 with the Hill

approach. The 95% confidence intervals (95%CI) around the mean EC50 values were estimated

by a bootstrap non parametric simulation.

Statistics

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Preliminary conditions of parametric statistical tests were evaluated and, if these

conditions were violated, parametric tests were performed on Box-Cox transformed data.

Statistical analyses were performed using Systat software (Version 10.0) and results were

considered to be significant if p < 0.05. Unless otherwise indicated, errors are given as ± one

standard deviation. Differences among effective Cd concentrations inhibiting algal growth by

50%, as determined with the cell yield or the cell volume yield, were conservatively evaluated by

comparing the 95%CI of the EC50.

RESULTS

Cadmium speciation: complexation by metal-binding exudates

The slope of the theoretical regression representing the expected initial Cd2+

concentrations in solution modeled with MINEQL+ (I = 200 meq L-1, CEAEQ medium, ionic

strength adjusted with NaNO3) as a function of the total initial Cd concentration was of 0.83

(dotted line, Figure 1). At mean measured total initial [Cd] < 62 nM, the slope of the regression

of the free measured [Cd2+] as a function of total measured initial [Cd] was close to the dotted

theoretical line (slope = 0.64 ± (SE) 0.11, regression not shown) as expected. However, when

including all the measured Cd concentrations at t=0, the slope of the regression of the free

measured [Cd2+] as a function of the measured total [Cd] was of 0.44 ± (SE) 0.06 (Figure 1, open

circles, regression not shown), which is almost two times less than the expected slope based on

MINEQL+ calculations. At t = 96 h, the slope of the regression of measured Cd2+ as a function

of total Cd (including all the Cd concentrations) further decreased to 0.26 ± (SE) 0.01 (Figure 1,

close circles, regression not shown). At total measured [Cd] < 20 nM, the mean measured free

[Cd2+] was up to an order of magnitude lower after the 96 h exposure compared to the initial

exposure conditions, although this difference was not statistically significant because of the high

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variability in the measured free [Cd2+] at t = 96 h. However, for total measured [Cd] > 40 nM,

the mean measured free [Cd2+] decreased significantly by about 50% at the end of the exposure.

Total dissolved cadmium concentrations

Over the first 12 h, the total dissolved [Cd] in solution showed an apparent decrease of 15

to 40% compared to the initial total [Cd] (t = 0), for which there was only one replicate (n = 1).

In the interval between 12 and 96 h, the total dissolved [Cd] decreased further but by no more

than 20%. For the experiments performed at an initial cell density of 2,500 cells mL-1, we thus

decided to average all measured total dissolved [Cd] between 12 and 96 h (denoted “mean

exposure total [Cd]”) when studying the relationship between Cd accumulation or toxicity over

time and the total [Cd] in solution. For the 96-h Cd exposure experiment performed at an initial

cell density of 2,500 cells mL-1 and 10,000 cells mL-1; the total dissolved [Cd] decreased by 54 ±

(SD) 9% and 18 ± (SD) 3% respectively.

Cadmium accumulation as a function of time and dissolved [Cd]

Intracellular cadmium concentration in P. subcapitata cells exposed to relatively low

mean exposure total [Cd] (< 50 nM) remained stable (p > 0.05) after 24 h of exposure (see insert

in Figure 2A). However, at relatively high mean exposure total [Cd] (> 56 nM), intracellular Cd

concentrations diverged appreciably as the exposure time was extended. Generally, Cd

accumulation increased as mean exposure total [Cd] increased, except for t = 12 h, where

intracellular Cd concentrations were surprisingly constant across a wide range of mean exposure

total [Cd] (Figure 2B). This constancy in intracellular Cd concentrations was confirmed

statistically since the slope of the regression relating the intracellular Cd concentration to the

mean exposure total [Cd] between 89 and 202 nM was not significantly different from 0 (t-test

analysis).

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Cadmium toxicity

Algal cells inoculated at 2,500 cells mL-1 grew exponentially (µ = 0.058 ± SE 0.003 h-1)

over the 96 h exposure in the control medium (no Cd added; full circles, Figure 3). At a mean

exposure total [Cd] of 18 nM, P. subcapitata cells also grew exponentially over the 96 h

exposure, but the specific cellular growth rate decreased by approximately 26% (µ = 0.046 ± SE

0.0009 h-1) with respect to control cells. For mean exposure total [Cd] 56 nM, the specific

growth rate changed over the exposure period; the algal cells grew at the maximal rate for the

first 24 h and then continued to grow at a much lower rate (5.3 times less than the control at 56

nM mean exposure total [Cd]) from 48 to 96 h (mean µ = 0.011 ± SE 0.001 h-1; solid line in

Figure 3). Note that the algal cells inoculated at 10,000 cells mL-1 in control medium with no

added Cd grew exponentially over the 96 h exposure at a specific growth rate of 0.035 ± SE 0.1

h-1, which was significantly slower than the specific growth rate of algal cells inoculated at 2,500

mL-1.

Figure 4 and Table SI.1 show the EC50 values calculated for the cell yield (panels A to

D) or the total cellular volume yield (panels E to H) as a function of the exposure time; the EC50

values were calculated in terms of total initial [Cd], final free [Cd2+], mean exposure total [Cd],

intracellular [Cd] or Cd quotas. No effect on cell growth was observed after 12 or 24 h across the

whole range of Cd concentrations and thus no EC50s could be computed at these time steps. The

EC50s expressed as initial total [Cd], mean exposure total [Cd] or final free [Cd2+] all tended to

decrease over the exposure time, but this decrease was less clear when total cellular volume yield

was chosen as an endpoint instead of the cell number yield. When considering the cell number

yield as an endpoint, this decrease in EC50s over time was statistically significant as

conservatively evaluated on the basis of the absence of overlapping 95%CI for the EC50 values

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at t = 48, 72 and 96 h. However, for the EC50s at 48, 72 and 96 h calculated on the basis of the

total cellular volume yield instead of the cell number yield, the 95%CI overlapped and the

statistical significance of the decrease in EC50 values over time could not be demonstrated.

For the toxicity assays performed at very low initial cell densities (2,500 cells mL-1), the

EC50 values at 96 h calculated from the effect of total initial [Cd] or Cd accumulation on cell

number yield or total cell volume yield tended to be lower than the EC50s measured at higher

initial cell density (10,000 cells mL-1). The differences in these EC50s were statistically

significant (except when considering the intracellular Cd concentrations) on the basis of the

absence of overlap between the 95%CI at high or low initial cell density. The intracellular Cd

concentrations inhibiting by 50% the total cell volume yield at 96 h of exposure for the cultures

inoculated at a low initial cell density were particularly variable (1.09 × 10-19 mol Cd µm-3;

95%CI = 6.0 × 10-20 to 6.8 × 10-19 mol Cd µm-3) and the 95%CI of this EC50 overlapped with

the 95%CI of the EC50 at high initial cell density; hence, the significance of this difference

could not be demonstrated conservatively.

DISCUSS ION

Cadmium speciation and metal-binding exudates

The free measured initial Cd2+ concentrations at total measured [Cd] < 62 nM was close

(although the data variability was high) to the modeled free Cd2+ concentrations suggesting that

no Cd complexation occurred initially in the culture medium before algal inoculation (Figure 1,

open circles). However, when taking into account all total measured [Cd] at t = 0 Figure, the

slope of the regression of the free measured Cd2+ concentration as a function of the total

measured [Cd] (Figure 1, open circles) was almost two times less than the expected slope based

on MINEQL+ calculations (Figure1, dotted line). This discrepancy could be due to the low

number of replicates (n = 1) used for the measurements of total [Cd] at t = 0 or to an unexpected

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Cd complexation at t = 0. Although there are some uncertainties on the degree of Cd

complexation at t = 0, the data clearly show that Cd complexation was more pronounced at t = 96

h (Figure1, solid line) than at t = 0 h (Figure1, dashed line), which strongly suggests that metal-

binding exudates produced by the algae complexed a significant portion of total Cd in solution.

In a natural freshwater environment, metals such as Cd, Co and Zn are typically found in

large proportions as the free ion species [34-36] due to their relatively low affinity for dissolved

organic matter, which poorly buffers the free metal ion concentrations. Hence, one might expect

that metal-binding exudates produced by phytoplankton in oligotrophic but particularly in

eutrophic systems would sometimes affect metal speciation, as suggested by several free metal

ion measurements in ecosystems in different trophic states [37-41]. Koukal et al. [16] also

suggested that metal-binding exudates released by P. subcapitata could decrease Cd, Cu, Pb and

Zn uptake and toxicity due to metal complexation in solution and/or to an indirect effect of

organic exudates on algae. The present study affords additional information about the interaction

between metal-binding ligands of algal origin and Cd speciation. For laboratory experiments in

synthetic media, the results show that algal exudates secreted by the freshwater algae P.

subcapitata may significantly decrease the free Cd2+ concentration, particularly at Cd

concentrations > 40 nM for which Cd complexation significantly increased at t = 96 h relative to

t = 0 (Figure1). Such a decrease would be expected to affect Cd accumulation and toxicity in

algae as well as in other aquatic organisms. The results also suggest that toxicity assays

performed in natural waters without adding strong metal chelators might be biased by the release

of metal-binding exudates by the algae and subsequent Cd complexation. The results thus

demonstrate the importance of keeping cellular density as low as possible in toxicity assays using

P. subcapitata to better reflect the metal sensitivity of the cells.

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Effect of exposure time on steady-state Cd accumulation

Contrary to acute (<1 h) metal exposure experiments, chronic algal experiments (i.e.,

several hours or a few days) bring into play several poorly understood factors such as growth

kinetics (and metal biodilution), algae acclimation to varying metal concentrations, release of

metal-binding exudates, and metal efflux. All of these physiological responses of algae to metals

may potentially affect metal accumulation and toxicity in phytoplankton. When algae are

acclimated to the exposure medium, a steady-state can be attained or approximated in which

metal uptake is balanced by metal loss. In the present study, chronic Cd accumulation in P.

subcapitata cells exposed to relatively low [Cd] (i.e. mean exposure [Cd] < 60 nM) tended to

reach a steady-state in Cd accumulation after an exposure time of 24 h (Figure 2A). In batch

cultures of P. subcapitata where depletion in dissolved [Cd] (and free Cd2+) is negligible and

specific growth rate is constant, Cd uptake is expected to be balanced only by growth biodilution

[7, 8, 42] since Cd efflux in P. subcapitata was shown to be negligible in an earlier study [42].

The specific growth rate of P. subcapitata cells exposed to a low mean exposure total [Cd] of 18

nM was constant over the 96-hour exposure (Figure 4). However, at higher mean exposure total

[Cd] (56 nM and above), specific algal growth rates decreased after 24 h of exposure (Figure 3).

Such a decrease in growth rate after 24 h suggests that Cd slowly exerts its toxic effect within the

algal cells (see the last section of the discussion about Cd toxicity for details). This delay in the

expression of Cd toxicity on growth rate will tend to postpone reaching a steady-state in

intracellular Cd contents. Figure 2A effectively demonstrates that at the mean exposure Cd

concentrations of 56 nM and 89 nM (i.e. highly toxic concentrations, Figure 4), intracellular Cd

concentrations level off only after 72 h whereas at mean exposure Cd concentrations of 108 nM

or greater, intracellular Cd concentrations did not reach a steady-state over the 96-h exposure.

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Note that an increase in Cd complexation by algal exudates over time (Figure 1) will tend to

decrease Cd uptake and should also affect whether or not a steady-state in Cd accumulation may

be achieved over the 96-h Cd exposure experiment.

Cadmium uptake as a function of mean exposure total [Cd] and exposure time

Cadmium accumulation as a function of the mean measured exposure total [Cd] shows an

upward curvature as toxic Cd concentrations are reached (Figure 2B). Positive Cd feedback on

Cd accumulation (i.e., Cd uptake inhibits growth rate, which decreases growth biodilution) is a

plausible hypothesis that could explain the non-linear relationship between Cd accumulation and

[Cd] observed in Figure 2. If true, one might expect that the extent of this possible positive

feedback on Cd accumulation, as well as the sensitivity of Cd uptake to variations in [Cd2+], will

depend on the number, affinity and capacity of Cd transport system(s) involved.

Another interesting result is the capacity of P. subcapitata to maintain intracellular Cd

concentrations at nearly stable levels after an exposure time of 12 h across a wide range of mean

exposure total [Cd] (from 89 to 202 nM; Figure 2, full circles). These results suggest that algal

cells can reduce net Cd uptake rates on a short-time scale. Such a response of P. subcapitata was

also reported by Errécalde and Campbell [42], who demonstrated that Cd uptake in the presence

of high [Cd2+] (280 and 600 nM) induced a decrease in Cd uptake rates after an exposure of just

10 min.

Cadmium toxicity (EC50) as a function of the initial cell density

Standard toxicity assays such as those performed by the CEAEQ, Environment Canada

and the USEPA measure metal toxicity using 96 h exposures of P. subcapitata cells grown in

batch cultures with initial algal inoculums of 10,000 cells mL-1 [17, 18, 43]. In the present study,

Cd toxicity based on cell yields (expressed as total initial Cd) using the standardized initial cell

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density used by several environmental agency (10,000 cells mL-1) was around three times higher

(EC50 three times lower) when a low initial cell density (2,500 cells mL-1) was used. The present

study thus demonstrates that the sensitivity of standardized toxicity assays may be improved by

decreasing the initial algal density. Higher algal sensitivity to total initial Cd as the initial cell

density decreased cannot be explained by lower metal depletion at lower initial cell density

because metal depletion at t = 96 h increased, not decreased, as the initial cell density decreased,

i.e. 54 ± (SD) 9% and 18 ± (SD) 3% of total dissolved Cd was lost at 2,500 cells mL-1 and

10,000 cells mL-1 respectively. Higher metal depletion observed at lower initial cell density

might be explained by inter-experimental differences in specific growth rates. For instance,

control algal cells inoculated at 10,000 cells mL-1 grew more slowly than control algal cells

inoculated at 2,500 cells mL-1, and this variation in growth rate, presumably due to slight

changes in the physiological state of algal cells, may lead to large changes in the amount of Cd

taken up by the cells (and depleted from the culture medium) after a long-term Cd exposure of

four days. Instead, we propose that the higher reported algal sensitivity to total initial Cd as the

initial cell density decreased (Figure 4) might be at least partly explained by a lower release of

metal-binding exudates (explained by the four times lower initial cell density) and, thus a higher

proportion of free Cd2+ at a given total [Cd]. Such effects of the initial cell density on metal-

binding exudate production, metal complexation and toxicity have also been suggested by

Franklin et al. [21] to explain the inverse relationship that they observed between initial cell

density (from 103 cells mL-1 to 105 cells mL-1) and Cu toxicity in P. subcapitata. However, the

effect of initial cell density on Cd toxicity was not a simple function of the final measured Cd2+

concentration since the EC50 values (expressed in terms of the final measured Cd2+

concentration) calculated on the basis of total cell volume and cell number yield were

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approximately 20 and 15 times higher when the cultures were inoculated at 10,000 cells mL-1

than at 2,500 cells mL-1. We hypothesize that variations in algal physiological states (related to

differences in the algal density and/or inter-experimental variability) could disproportionately

affect the sensitivity of P. subcapitata to the free Cd2+ ions. However, other experiments are

needed to confirm or infirm the above hypothesis.

Cadmium toxicity (EC50) as a function of time and endpoint

The present study showed that EC50 values calculated on the basis of total cell volume

yields of P. subcapitata and expressed in terms of initial total [Cd] or mean exposure total [Cd]

tended to decrease (toxicity increased) with time. For exposure periods of 48, 72 and 96 h, the

decrease in EC50 with time was mainly due to an increasing divergence between cellular volume

yields of control and Cd-exposed cells as the exposure time increased. Such a result occurred

with a constant growth rate over time (at mean exposure total [Cd] ≤ 18 nM; Figure 3) or with a

decreasing growth rate over time (at mean exposure total [Cd] 56 nM; Figure 3). This

increasing trend in Cd toxicity over time was more pronounced when the cell number yield was

used as the endpoint rather than the total cell volume yield. The difference in the decreasing

trend in the EC50s over time between both endpoints can be explained by the increase in

biovolume per algal cell at toxic Cd concentrations; this increase in biovolume per cell

compensated in part for the decrease in cell yields. For similar reasons, toxicity expressed in

terms of total Cd or final free Cd2+ also were consistently greater when the cell number yield was

used as the endpoint. Cellular volume yield as an endpoint for Cd2+ toxicity was also shown to be

less sensitive than the cell number yield in another recent study investigating Cd toxicity in a

different green algal species, Chlamydomonas reinhardtii [44].

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Cadmium effects on cell number or total cell volume yield (Figure 4) as well as on

growth rate (Figure 3) remained undetectable after exposure times of 12 to 24 h, even in the very

highly contaminated cultures (i.e., for high mean exposure total [Cd] reaching up to 202 nM and

leading to high intracellular Cd concentrations; Figure 2). These results strongly suggest that Cd

exerts its toxicity slowly within the cells, leading to little growth inhibition over the first 24 h of

exposure. Small inhibitions of algal growth rate (e.g. by < 20%) will not lead to a large decrease

in cell yield after 12 or 24 h; such small decreases in cell yield will remain difficult to measure

accurately. For the two above reasons (i.e., toxicity delay and difficulty of measuring growth

yield inhibition after 12 or 24 h in the presence of a relatively small growth rate inhibition),

EC5024 hours values based on growth rates or cell yields should not be considered as reliable

toxicity endpoints in Cd toxicity assays using P. subcapitata.

CONCLUSION

Cadmium accumulation and toxicity in the freshwater alga P. subcapitata can be

influenced not only by the free Cd2+ ion, but also by the Cd exposure time (i.e., expression of

toxicity may be delayed after initial Cd accumulation). While P. subcapitata may actively

decrease Cd uptake on short-time scales (< 24 h), Cd toxicity inevitably occurred over chronic (>

24 h) exposures to relatively high Cd concentrations in solution. The present study also

demonstrates that metal-binding exudates released by P. subcapitata may significantly decrease

the proportion of total Cd present as the free Cd2+ ion. Such a decrease in Cd2+ concentration is

expected to decrease Cd uptake and toxicity in freshwater organisms. The use of flow cytometry

to follow algal growth at very low cellular densities can be helpful to minimize such exudation

and also possibly metal depletion over time. The present results afford insights about chronic Cd

toxicity mechanisms in a freshwater alga species commonly used in standard toxicity assays.

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Such information should be helpful for the elaboration of robust algal bioassay as well as for the

derivation of water quality guidelines.

SUPPLEMENTAL DATA

Table S1. (52 KB. PDF).

Acknowledgement—We gratefully thank L. Martel, R. Cardin and C. Bastien from the Centre

d’expertise en analyse environnementale du Québec (CEAEQ) for their collaboration. We also

acknowledge the technical assistance provided by BD Bioscience, L. Rancourt, M. Bordeleau,

and S. Duval. ML held a scholarship from the Fonds Québecois de recherche sur la nature et les

technologies (FQRNT). CF and PGCC are supported by the Canada Research Chair programme.

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Figure 1. Free Cd2+ ion concentrations as measured by the ion-exchange technique at t = 0 (open

circles) or t = 96 h (full circles) as a function of total measured Cd concentrations. The dotted

line represents the expected initial Cd2+ concentrations modeled with MINEQL+ (I = 200 meq L-

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1, CEAEQ medium, ionic strength adjusted with NaNO3) (slope of 0.83). The short dashed line

and the solid line are least-square best fits of linear regressions for the data at t = 0 and t = 96 h

respectively. Insert shows data on a finer scale. The initial cell density was 2,500 cells mL-1.

Error bars represent the standard deviation of two to four replicates. In the insert, only the upper

error bars are shown for the data at t = 96 h.

Figure 2. Intracellular Cd concentrations (mol Cd µm-3) as a function of exposure time for

different mean exposure total [Cd] (Panel A) and as a function of the measured mean exposure

total [Cd], for different exposure times (Panel B). Insert shows results on a smaller scale. Error

bars represent the standard deviation of three replicates.

Figure 3. Natural logarithm of the total cellular volume per mL (µm3 of alga mL-1) as a function

of exposure time to various measured mean exposure total Cd concentrations (between t = 12 h

and t = 96 h). The dotted, small dash and solid lines represent linear regressions for the control

(R2 = 0.97), the 18 nM (R2 = 0.99) and the 56 nM (from 48 to 96 h) (R2 = 0.98) Cd

concentrations. The initial cell density was 2,500 cells mL-1. Error bars represent the standard

deviation of three replicates.

Figure 4. Effective Cd concentrations (A, E: initial measured total [Cd]; B, F: measured mean

exposure total [Cd] between 12 and 96 h; C, G: final measured free [Cd2+]; D, H: intracellular

Cd) inhibiting by 50% the cell yield (cell number mL-1) (A to D) or the total cell volume (µm-3 of

alga mL-1) (E to H) measured after 48, 72 and 96 h of exposure to Cd. The EC50s (except EC50s

expressed as measured mean exposure total [Cd]) at 96 h were calculated for cultures inoculated

at two different initial cell density (2,500 cells mL-1 or 10,000 cells mL-1); all other time points

are at an initial cell density of 2,500 cells mL-1. No significant Cd toxicity was measured after 12

or 24 h of exposure. Error bars represent the confidence intervals at 95% (95%CI).

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1

Supporting information for

Cadmium accumulation and toxicity in Pseudokirchneriella subcapitata: influence of

metal-binding exudates and exposure time

Authors: Nathalie Paquet†‡, Michel Lavoie†§, Frédéric Maloney†||, Jérôme F.L. Duval#,

Peter G.C. Campbell†, Claude Fortin†1

† Institut national de la recherche scientifique, Centre Eau Terre Environnement (INRS-

ETE), 490 de la Couronne, Québec (Québec), Canada G1K 9A9.

‡ Present address: Centre d’expertise en analyse environnementale du Québec, Ministère

du développement durable, de l’Environnement et de la Lutte contre les changements

climatiques, 2700 rue Einstein, Québec (Québec), Canada G1P 3W8.

§ Present address: Québec-Océan and Takuvik Joint Université Laval/CNRS Research

Units, Département de Biologie, Université Laval, Québec (Québec), Canada G1K 7P4.

|| Present address: Administration portuaire de Québec, P.O. Box 80, Haute-Ville,

Québec (Québec), Canada G1R 4M8.

# Laboratoire interdisciplinaire des Environnements Continentaux (LIEC), CNRS-

Université de Lorraine, B.P. 40, Vandoeuvre-lès-Nancy cedex, France

1 Corresponding author: [email protected]

2

1. Description of the ion-exchange technique

In this technique, the free Cd2+ ion concentrations equilibrate with a sulfonic acid-type resin that has been pre-calibrated with Cd standard solutions.

↔ . 1 This reaction is described by a conditional equilibrium constant :

. 2

where R = resin, = resin-binding sites occupied mainly by Na, the predominant major cations (mol L-1), and = resin-binding sites occupied by metal M (mol L-1). In the presence of a sufficiently high concentration of counter-ion (Na+), the Na+ concentration in solution and adsorbed on the resin is negligibly affected by the free metal ion which is present at trace levels (i.e. [RNa+] >> [RMz+]). Then, eq. 2 can be rearranged yielding a distribution coefficient , , valid at fixed ionic strength (200 meq

L-1 adjusted with 1 M NaNO3) and pH (7.0).

, , . 3

Before analysing samples of culture media, the resin is firstly calibrated with solutions of known [Cd2+] (e.g. without significant complexation by ligands or in the presence of significant metal-ligand complexes with well-known stability constants) in order to compute , , for Cd. This coefficient can be calculated by measuring the amount of

metal bound to the resin , which is obtained after elution of the resin with a volume V (4 mL) of strong acid (1.5 M HNO3). According to the quantity of resin used (mr = 7 mg; weighed on a micro-balance; Sartorius, Edgewood, NY, USA) and the concentration of metal measured in the standard eluate ( ) (mol L-1), can be calculated with Eq.4.

. 4

Analyses of CEAEQ culture media adjusted at an ionic strength of 200 meq L-1 with NaNO3 and spiked with 100 nM total Cd (MINEQL+ simulation predict 83 nM Cd2+ due to Cd complexation by high nitrate concentrations) yielded a , , of 0.41 ± 0.05 L g-1

(n = 9).

3

After calibration of the resin, the ionic strength of sample solutions was adjusted to that of the standard IET matrix (200 meq L-1) with NaNO3 1 M (dilution did not exceed 1% of the sample volume). Then, samples were passed through the resin for 4 min (V = 20 mL), after which equilibration between the ion-exchange resin and the solution is achieved (i.e. the metal concentration entering and exiting the resin column are equal). Combining Eq. 3 and 4 yields Eq. 5:

, ,

. 5

To determine free Cd2+ concentrations in culture medium samples, cadmium bound to the resin is first eluted with 2 ml of 1.5 M HNO3 in pre-weighted borosilicate vials (Opticlear, Fisher Scientific, Ottawa, ON, Canada). Thereafter, the eluates collected in the vials were weighted again and the exact volume of elution was computed using the density of the nitric acid eluent solution (1.055 g mL-1). Cadmium concentrations in

sample eluates ( ) were then measured by counting the 109Cd radioisotope

with a scintillation counter (Wallac 1414; Perkin Elmer Life Sciences, Turku, Finland) using Ecolume scintillation cocktail (ICN, Costa Mesa, CA, USA). Finally, the free [Cd2+] of the samples was computed with eq. 5. Analytical blanks were also run in parallel of each sample to evaluate background contamination of samples from the nitric acid used for elution of the resin column and were subtracted from the sample values.

4

2. Tabulated EC50 values obtained in the present study The EC50 values that are plotted in Fig. 4 of the main text body are shown in the Table SI.1 below.

Cd toxicity based on the cell yield (cell number mL-1) EC5048h-2,500 EC5072h-2,500 EC5096h-2,500 EC5096h-10,000 Initial measured total [Cd] (nM) 74 (59-89) 44 (39-50) 23 (16-32) 82 (75-89) Mean measured exposure [Cd] (nM) 44 (35-55) 25 (21-29) 11 (7.1-16) - Final measured free [Cd2+] (nM) - - 1.3 (0.21-2.3) 35 (33-37) Intracellular Cd quotas (× 10-17 mol Cd cell-1) 2.8 (1.9-3.8) 0.85 (0.67-1.1) 0.35 (0.025-0.79) 4.9 (4.4-5.3)

Cd toxicity based on the total cell volume (µm-3 of alga mL-1)

EC5048h-2,500 EC5072h-2,500 EC5096h-2,500 EC5096h-10,000 Initial measured total [Cd] (nM) 99 (73-128) 73 (59-88) 66 (52-80) 112 (100-119) Mean measured exposure [Cd] (nM) 59 (45-76) 46 (36-56) 47 (36-58) - Final measured free [Cd2+] (nM) - - 1.4 (0.42-2.9) 50 (42-59) Intracellular Cd quotas (× 10-19 mol Cd µm-3) 4.3 (2.6-6.6) 2.4 (0.84-5.4) 1.1 (0.60-6.8) 7.5 (6.2-8.5) Table SI.1: Effective Cd concentrations (expressed as initial measured total [Cd], measured mean exposure total [Cd] between 12 and 96 h, final measured free [Cd2+], and intracellular Cd) inhibiting by 50% the cell yield (cell number mL-1) or the total cell volume (µm-3 of alga mL-1) measured after 48, 72 and 96 h of exposure to Cd. The EC50s at 96 h were calculated for cultures inoculated at two different initial cell density (2,500 cells mL-1 or 10,000 cells mL-1) except for EC50s expressed as a function of measured mean total exposure [Cd]; all other time points are at an initial cell density of 2,500 cells mL-1. No significant Cd toxicity was observed after 12 or 24 h of exposure. Data are presented as mean EC50time-initial cell density ± 95%CI.