Carbonate Disequilibrium in the External Boundary Layer of

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Ecotoxicology and Human Environmental Health

Carbonate disequilibrium in the boundary layer of freshwater chrysophytes: Implications for contaminant uptake Michel Lavoie, Jerome F.L. Duval, J. A. Raven, Frédéric Maps, Béchir Béjaoui, David John Kieber, and Warwick Vincent Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00843 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Environmental Science & Technology

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Carbonate disequilibrium in the boundary layer of freshwater chrysophytes:

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Implications for contaminant uptake

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Michel Lavoie1*, Jérôme F. L. Duval2,3, John A. Raven4,5, Frédéric Maps1, Béchir Béjaoui6,

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David J. Kieber7, Warwick F. Vincent1

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Département de biologie, Université Laval, Québec, Québec, Canada G1V 0A6

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UMR7360, Vandoeuvre-lès-Nancy F-54501, France.

Québec-Océan, Takuvik Unité Mixte Internationale (Université Laval-CNRS) and

CNRS, LIEC (Laboratoire Interdisciplinaire des Environnements Continentaux),

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Université de Lorraine, LIEC, UMR7360, Vandoeuvre-lès-Nancy, F-54501, France

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Division of Plant Science, University of Dundee at the James Hutton Institute, Invergowrie,

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Dundee DD2 5DA, United Kingdom (permanent address of JAR)

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Sydney, Ultimo, NSW 2007, Australia

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rue du 2 mars 1934, 2025, Salammbô, Tunisie.

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and Forestry, Syracuse, New York, USA 13210

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*

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Word count: 5633 words (from the Abstract to the end of the conclusion including

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acknowledgments and supporting information abstract) + 2 figures x 600 words + 1 figure x

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300 words = 7133 words-equivalent

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Running head: Phytoplankton boundary layer and metal speciation

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Keywords: pH, carbonate, boundary layer, metals, bioavailability, phytoplankton

Functional Plant Biology and Climate Change Cluster (C3), University of Technology

Laboratoire du Milieu Marin, Institut National des Sciences et Technologies de la Mer, 28,

Department of Chemistry, State University of New York, College of Environmental Science

corresponding author: [email protected]

ACS Paragon Plus Environment

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Abstract

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phycosphere and the resulting modulations of contaminant speciation and uptake is poorly

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characterized. Here we modeled the effect of algal C and N uptake on carbonate cycling and

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speciation of selected contaminants in the phycosphere (external boundary layer) of

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chrysophytes, a key phytoplankton group in oligotrophic systems. We calculated enrichments

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in H+ concentration relative to that in the bulk solution (pH 7.0) of approximately 40% for

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NH4+-grown cells or depletions of approximately 500% for NO3--grown cells at the algal

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membrane surface of a 5-µm or 30-µm radius cell or colony, respectively. Such changes are

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mainly due to direct H+ uptake or release at the plasmalemma if NO3- or NH4+ is the N source,

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respectively. Due to these pH changes in the boundary layer, competition between H+ and

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metals for uptake is enhanced, which contributes to a decrease in potential metal uptake. Our

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model suggests that the uptake of protonated weakly acidic organic acids (HA) is greater in

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NH4+-grown cells compared to NO3--grown cells. The account of chemical reactions in the

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algal boundary layer could improve ecological risk assessments for a wide range of

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contaminants.

The interplay between biological and chemical reactions in the freshwater phytoplankton

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Abstract Art efflux

HA

uptake

NO3- OHMez+

efflux

HA

uptake

Reactive boundary layer Mez+

NH4+ H+

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1 Introduction

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Chrysophyte species in the Chrysophyceae and the closely-related class Synurophyceae

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constitute a dominant or sub-dominant portion of phytoplankton biomass in lakes, with

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moderate to low productivity.1 With diverse carbon acquisition mechanisms, ranging from

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photoautotrophy in all Synurophyceae to phagomixotrophy in many Chrysophyceae, these

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algal classes play an important role in determining the carbon flow through the food web,

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including in Canadian Shield water bodies that represent ~50% of the world’s freshwater

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lakes.2 Several freshwater chrysophyte habitats are impacted by metals released from mining

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industries3,4 and organic contaminants discharged from urban areas.5,6 Anthropogenic

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eutrophication of freshwater systems as well as global change related-CO2 increases and

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concomitant acidification7 can also affect the physiology and diversity of chrysophytes.8 Such

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pH changes may further impact the uptake and toxicity of several inorganic and hydrophobic

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contaminants in phytoplankton.9-11 For freshwater phytoplankton, chemical reactions in the

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phycosphere (external boundary layer) surrounding cells rather than those in the well-mixed

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bulk phase dictate the overall interactions between algae and their external aquatic medium.

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However, due to practical and technical limitations, in-depth microscale analysis of these

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chemical interactions has been proven difficult and has received little attention.12,13


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A major uncertainty is how photosynthesis affects carbonate chemistry in the immediate

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vicinity of freshwater algal cells, and what the consequences are in terms of speciation and

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uptake of nutrients and contaminants. This issue remain difficult to evaluate for cyanobacteria

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and algae because of their carbon concentrating mechanisms (CCM), which increases algal

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photosynthesis at low inorganic carbon concentrations.14 In contrast, chrysophytes lack such a

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CCM and hence soley rely on diffusive CO2 transport for photosynthesis.15-18 They are

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therefore an attractive model for the analysis of carbonate system and associated contaminant

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disequilibria in the critical reactive layer adjacent to the algal cell surface.

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Whereas modeling work and experiments have shown that marine phytoplankton can

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substantially deplete carbon dioxide at the cell surface, which is particularly true for large

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phytoplankton species,19-21 little information is available for phytoplankton species growing in

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freshwater environments, many of which are poorly buffered.22,23 A better knowledge of the

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ecophysiology of CCM-lacking algae is needed to assess the impacts of global climate change

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and contaminant pollution on organic carbon production by freshwater algae. Such a coupling

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between algal cell physiology and ecotoxicology is of increasing interest especially given the

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ongoing development of metal toxicity models that account for physiological regulation of

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metal transport systems,24-26 and given the increasing recognition of the importance of algal

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physiological state on pesticide toxicity.27,28

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The aim of the present study is to model and evaluate the effect of photosynthesis on

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carbonate chemistry within the boundary layer of chrysophyte cells or colonies of different

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sizes. The proposed model simulates reactive carbon transfers with an explicit account of

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species diffusion and intertwined acid/base disequilibria as coupled to C and N nutrient

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uptake, and the associated effect on the uptake of inorganic and organic contaminants.


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2. Theory

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2.1. Modeling the influence of algal metabolism on the carbonate system in the boundary

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layer

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For strictly phototrophic cells grown under continuous light conditions as experienced at high

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latitudes in summer, adopting a cell size scaling derived for algal growth from laboratory

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experiments, the net carbon fixation rate (d[CO2] /dt) can be written as the product of the

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carbon cell quota (Qc, pgC/cell) and the specific growth rate (µ):

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d[CO2]/dt = Qc µ

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The cell carbon quota in freshwater phytoplankton grown under continuous light scales

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linearly with cell volume with a proportionality coefficient of 0.279 pg C µm-3 according to

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the following equation:29

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Qc = 0.279 Vc

2

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where Vc is the cell volume (µm3). This relationship is valid for cells with biovolumes

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between ~2 and 100,000 µm3, i.e., a cell radius in the range 1.3 to 30 µm assuming spherical

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geometry for chrysophytes such as Dinobryon and Mallomonas.30 However, Dinobryon

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generally occur as loose colonies of cells unlike the chrysophyte Synura, which forms more

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compact colonies.

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The nutritional mode of several freshwater phytoplankton species lacking CCM varies widely

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from strictly phototrophic to phagomixotrophic so that cells may acquire carbon not only

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from photosynthesis but also by the phagotrophy variant of heterotrophy. The current study


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considers spherical cells without CCM growing photoautotrophically without or with (see

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below) phagocytosis, which is important in several protist species. Photosynthesis has been

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shown to provide more than 75% of cell carbon in the mixotroph Dinobryon31 whereas

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several other chrysophytes are strict (e.g., Mallomonas and Synura) or possibly facultative

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(Hydrurus) photoautotrophs.15

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Based on eq. 2, the mean cellular organic carbon concentration in a range of freshwater algae

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growing in nutrient-sufficient conditions is 2.3 × 104 mol C per m3 of cell biovolume. This is

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a good approximation of the cellular carbon concentration of 2.7 × 104 mol C m-3 in

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Dinobryon divergens 32 and of 2.6 × 104 mol C m-3 in Dinobryon cylindrum .31

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Adopting a theoretical power law relating cell volume to growth rate (µ = constant Vc-0.25) (33

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and references therein), which is at the lower end of the range of values of the exponent for

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nano- and micro-phytoplankton,34 and a growth rate of 0.4 d-1 as measured for Dinobryon

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(equivalent spherical radius = 4 µm) in an axenic culture,31 the specific growth rate (µ in d-1)

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as a function of cell radius (rc in m) can be evaluated from:

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µ = (2.90 x 10-5)rc-0.75

3

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It should be noted that this theoretical cell size-scaling relationship minimizes growth rates

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(C and N nutrient uptake rates) and hence boundary layer perturbations in the carbonate

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system and contaminant speciation. This choice adds robustness to our modeling approach.

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Combining eq.1 for the net CO2 uptake flux (mol CO2 m-2 d-1) by an alga with eq. 3, and

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assuming a spherical cell with a mean intracellular C concentration per cell surface area of 2.3

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× 104 mol C per m3 (see calculation above), with rc in m, it comes:


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d[CO2]/dt = (3.16 x 10-6) rc0.25

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The carbon uptake rate (d[CO2]/dt) is assumed to be independent of bulk-solution pH from 5

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to 8, even though photosynthesis in Mallomonas sp. has been shown to vary by ~30%

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between pH 5 and 7 and by 3- to 4-fold between pH 6 and 8.18 The effect of pH on carbon

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uptake is specifically considered in the discussion section.

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Calculations were also performed considering mixotrophic growth based on data from

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Dinobryon cultures with added bacteria. This chrysophyte can grow at 0.7 d-1 and acquire

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75% or more of its internal cell C through photosynthesis, with the remaining carbon

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provided by ingestion of bacteria.31,35 Further assuming an identical C:N ratio in bacteria and

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algae of 116:16, it follows that ≤ 25% of algal cell N can be provided by ingestion of bacteria.

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Since the cellular fate of bacterial N and its associated coupling with H+/OH- exchange at the

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plasmalemma is unknown to our knowledge, no model corrections were made regarding the

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impact of bacterial N uptake on H+/OH- exchange at the plasmalemma. We further considered

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the amount of bacterial carbon respired as CO2 by assuming a 1.05 mean molar ratio of CO2

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production to organic C taken up through heterotrophy in the mixotrophic chrysophyte

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Ochromonas malhamensis.32 Therefore, in the case of mixotrophic growth, the net CO2

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uptake rate for a given cell radius decreased by 25% due to a decrease in photosynthesis, and

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by 25% due to bacterial organic carbon respiration, but increased by 75% due to an increase

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in growth rate relative to the phototrophic case. As a result, the net CO2 uptake rate (mol m-2

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d-1) for mixotrophic growth was only 14% lower than that for a strict phototroph. This uptake

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can thus be expressed as a function of rc according to:

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d[CO2]/dt = (2.77 x 10-6)rc0.25

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2.2 Modeling the carbonate distribution in the algal boundary layer

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We modeled the effect of pH (and carbonate speciation) in the bulk solution on the pH and

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carbonate speciation prevailing in the algal boundary layer. To that end and for simplicity, we

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considered that the bulk-solution dissolved CO2 concentration was in equilibrium with the

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atmosphere, as an approximation of carbonate system for several oligotrophic freshwater

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lakes. We varied the bulk-solution pH from 5 to 8, encompassing the range of pH values

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typically measured in freshwater systems from acidic Canadian Shield lakes to lakes with

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limestone in the catchment basin. We assumed that the bulk-solution concentrations of all

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inorganic carbon species and the bulk-solution pH did not change over time as expected under

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non-bloom, low algal cell density conditions. The steady-state CO2, HCO3-, CO32-, OH- and

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H+ concentration gradients in the external boundary layer were evaluated as a function of

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cell/colony size. All calculations were performed at 1 mol m-3 ionic strength (I), and model

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outcomes were evaluated to verify that the model changed by less than 30% percent over a

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range of I applicable in freshwater systems (from 0.5 to 5 mol m-3). The rate of change in

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concentration of each chemical species in the boundary layer (dc/dt) was further given by the

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following diffusion-reaction equation:20,36

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+ =

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is the diffusion Laplacian operator in spherical geometry and R is the sum of all where

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reaction rates involving the chemical species of interest. The diffusion rate of each inorganic

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carbon species is defined by:

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c = D

( )

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where D and [c] are the diffusion coefficient and concentration of a given chemical species,

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respectively, and r is the radial distance from the center of the spherical cell. A common value

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for D of 1.18 x 10-9 m2 s-1 was adopted for all carbonate species to maintain electroneutrality

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(SI section 1). The extent of disequilibrium under steady-state diffusion conditions in the

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carbonate system due to algal nutrient C uptake was estimated taking into account the rate

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constants of the relevant chemical reactions involving CO2, HCO3-, CO32- and H+/OH- in the

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external boundary layer. Direct H+ release or uptake at the plasmalemma due to NH4+ or NO3-

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uptake, respectively, was further considered (0.15 mol H+ per mol C taken up, assuming a 1:1

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N:H+ co-transport and a Redfield C:N ratio).37 At steady-state, the time rate of change in the

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concentration of inorganic carbon species and H+/OH- are zero, and eq. 6 can be solved for

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the concentration of CO2, HCO3- and CO32-, H+ and OH- as a function of the distance from the

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cell surface using two boundary conditions: 1) the equilibrium concentration of chemical

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species in the bulk solution beyond the external boundary layer, which is determined from the

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atmospheric CO2 pressure and bulk-solution pH, and 2) the uptake flux of CO2, which is the

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only inorganic carbon species taken up by the cell. Note that CO2 uptake is not coupled to net

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H+ transport at the cell membrane, since CO2 assimilation produces neutral carbohydrates. A

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proton is taken up or released, however, at the cell membrane due to NO3- and NH4+

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assimilation, respectively (see section 2.1). All chemical equations and modeling procedures

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used to determine R in eq. 6, are detailed in the SI, section 2.

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2.3 Modeling metal speciation in the boundary layer as a function of pH


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To test the effect of external boundary-layer pH on metal uptake, we focussed on cadmium

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Cd(II), which has been extensively studied in ecotoxicology laboratory experiments using

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well-defined inorganic media. Cd(II) was chosen for its high toxicity10 as well as its relatively

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simple speciation, with a large fraction of Cd(II) present as the free, hydrated ion or as

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inorganic complexes at pHs greater than 7.5; typically less than 25% of total Cd is complexed

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to dissolved organic matter (DOM) in low DOM freshwater environments that are often

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associated with oligotrophic conditions.4,38 Cadmium speciation was evaluated in the external

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algal boundary layer considering a total Cd concentration of 1 × 10-6 mol m-3 and

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complexation with OH- and CO32- (SI, section 3). It was further assumed that DOM was not

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present in the external boundary layer for simplification in order to mimic experimental media

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routinely used in short-term metal uptake experiments. Cadmium uptake rates by freshwater

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phytoplankton are generally much slower (ca. by one order of magnitude) than metal

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diffusion from the bulk solution to the algal membrane surface. Therefore, Cd uptake is not

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expected to be limited by diffusion, but rather by the internalisation step particularly at

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nanomolar Cd concentrations, provided that Cd uptake kinetics in green algae is comparable

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to that in chrysophytes.10,24 Furthermore, since metal internalisation kinetics are much slower

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than that for other chemical species considered in this work (e.g., CO2), algal Cd uptake was

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ignored by setting the corresponding flux at the algal surface to zero as a boundary condition;

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this is detailed in the SI section 3. As a second boundary condition, it was assumed that Cd

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speciation was at equilibrium in the bulk solution.

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2.4 Modeling the speciation of ionisable weak acid/base organic contaminants in the

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boundary layer


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Protonation/deprotonation of weak acid/base organic contaminants is important from an

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ecotoxicological viewpoint, since ionic species do not freely pass through the cell membrane

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and therefore are generally much less bioavailable (or toxic) for living cells than neutral

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species that are able to cross algal membranes via simple diffusion.23 Here we considered two

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examples of ionisable organic contaminants whose uptake may not be limited by diffusion in

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the external boundary layer but rather may be controlled by changes in their speciation.

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Contaminants were selected in such a way that their modeled membrane permeability (Pm)

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was much smaller than the modeled boundary layer permeability (Pbl). The Pm term was

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calculated using the Xiang and Anderson empirical equations39 and Pbl was determined by

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dividing the diffusion coefficient of the organic contaminant by the external boundary layer

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thickness (see SI section 4.1).

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As a first example, we calculated the boundary layer speciation of the polar herbicide maleic

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hydrazide (MA) (Kow = 0.72, pKa = 5.64), which contaminates freshwater systems and may

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affect phytoplankton.40 Secondly, we estimated the speciation of a mildly hydrophobic

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antifungal pharmaceutical clindamycin (Clind) (pKa = 7.79, Kow = 101.83), which is released in

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freshwater and is of potential concern for phytoplankton.41 For each case, the calculated

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exchange rate constant of the neutral and charged species with H+ were considered. It should

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be noted that the proportion of the neutral species relative to the charged contaminant

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concentration dominates at pH < pKa or pH > pKa, for MA or Clind, respectively. Two

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boundary conditions were considered: 1) organic contaminant uptake was set to 0 since the

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uptake is limited by the membrane transport process; and 2) organic contamination speciation

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in the bulk solution is at equilibrium. The modeling procedure for these contaminants is

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further detailed in SI section 4.


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2.5 Model assumptions

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Since the thickness of the cell wall in unicellular algae is less than 10% of the thickness of the

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effective external boundary layer in small to giant-celled algae, diffusion of inorganic carbon

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species and H+ were assumed to be controlled by the external boundary layer,23 and thus the

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potential effect of the cell wall on diffusive fluxes was not considered in the model.

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Therefore, throughout this study, enrichment at the cell or colony surface refers to the nearest

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point of the external boundary layer at the surface of the algal plasmalemma. In all

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calculations, it was assumed that dissolved CO2 and N concentrations sustained maximal algal

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growth. We also assumed that C and N uptake rates were not significantly affected by the

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presence of contaminants as expected at low contaminant concentrations or under short-term

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exposure. Algal cells or colonies were assumed spherical and interstitial spaces within loose

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colony of cells were not considered for simplicity. The influence of turbulence was neglected

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at the micrometer scale (see SI section 5)42 as was the effect of cell swimming and sinking

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rates on diffusion in the boundary layer; potential influences of turbulence, swimming and

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gravitational sinking are discussed in SI section 6. The role of heterotrophic bacteria in CO2

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production within the algal boundary layer was also not considered in the model based on

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calculations performed in SI section 7. The role of other processes that may regulate cellular

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acid/base reactions (e.g., production of cell intermediary metabolites, nucleic acids and

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proteins as well as organic acid excretion) are neglected, since these processes are not

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constrained for chrysophytes. The role of cell-surface charge on the enrichment of ions was

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also not considered because of the insignificant or small differences in ionic bio-partitioning

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reported for Chlorophyceae with increasing pH (see SI section 8 and discussions for further


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details).43,44 Reactions involving reactive oxygen species were not considered due to

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insufficient knowledge regarding these reactions in the external boundary layer. Finally, since

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the steady-state concentration gradient and chemical equilibria for CO2 and other chemical

273

species are achieved within milliseconds to minutes, much shorter than algal doubling times,

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any effect of cell-number changes on model results due to algal growth were not included (see

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SI section 9).

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2.6 Model implementation

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All simulations were run and all figures plotted using R software (v 1.0.153).45 All scripts

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including functions are freely available on github (DOI: 10.5281/zenodo.1252222). The R-

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package “Reactran” (v 1.4.3.1) was used to solve the differential diffusion-reaction equation

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(eq. 6) in spherical coordinates using the cell radius and bulk-solution pH as model input

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variables, and a set of model parameters (C and N uptake rates, equilibrium and rate

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constants, ionic strength and bulk contaminant speciation).46 Steady-state concentrations of

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carbonate species or contaminants were computed in the external boundary layer using

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unidimensional model grids over a distance from the algal cell membrane of 60 µm (for cell

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radius of 5 µm) or 100 µm (for cell or colony radius of 30-um) with a model grid resolution

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of 10,000 elements.

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3. RESULTS AND DISCUSSION

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3.1 Carbonate system disequilibrium and associated pH gradients in the boundary layer

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as a function of nitrogen source


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Photosynthesis in model chrysophyte cells or colonies of 5 or 30-µm radius led to significant

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gradients in CO2, H+/OH-, HCO3-, CO32- in the external boundary layer (Fig. 1 and 2). Slow

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CO2 hydration or dehydration kinetics relative to diffusion generated a CO2 drawdown in the

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boundary layer; N uptake altered H+/OH- concentrations at the membrane surface (Fig. 1 and

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2 and SI section 9). At pH 7, for a 5 µm-radius chrysophyte cell (representative of small

297

Chromulina or Mallomonas), algal metabolism resulted in an increase or decrease of cell-

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surface [H+] by around 30-40% for cells grown on NH4+ or NO3-, respectively (Fig 1 and 2).

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This change was almost entirely accounted for by direct H+/OH- exchange at the

300

plasmalemma due to N uptake and not a decrease in carbonate system buffering due to CO2

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uptake (SI section 10).

302

The proton gradient was even more pronounced for larger cells. The H+ concentration at the

303

surface of a 30-µm radius cell/colony was depleted by approximately 5-fold if NO3- was the N

304

source, whereas a 3.5-fold cell-surface enrichment in H+ was obtained for the same

305

cell/colony size when NH4+ was the N source (Fig. 1 and 2). However, cell surface depletion

306

in H+ due to C uptake (assuming no inorganic N uptake) only reached ~25% for a 30 µm-

307

radius cell/colony despite a decrease in cell surface CO2 concentrations of more than 3.5-fold,

308

indicating that HCO3- buffering remained high (SI section 10). Indeed, over the range of cell

309

radii tested between 5 and 30 µm, and at pH 7, HCO3- concentrations changed by less than

310

1%, and therefore, as for the 5-µm radius cells, the proton gradient was mostly due to nitrogen

311

uptake (and direct H+ exchange at the cell membrane) rather than to a change in carbonate

312

buffering. Uptake of N influences more the cell surface pH than CO2 uptake since N uptake

313

and assimilation, unlike CO2 uptake, is coupled to net H+ transport at the cell membrane,

314

which directly affects cell surface pH, and this effect may be significant depending on


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boundary layer pH buffering, algal cell size and N uptake rate. By contrast, CO2 uptake

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increases the cell surface pH indirectly through H+ consumption via thereaction (H+ + HCO3-

317

-> CO2 + H2O, see SI section 2), a reaction that is slow relative to diffusion leading to kinetic

318

limitation of CO2 replenishment at the cell surface and minimizing the associated pH rise. As

319

stated previously, whereas bicarbonate concentrations did not change, carbonate

320

concentrations did increase, but concentrations of this species were quite low and it therefore

321

only played a minor role (ca.

Carbonate Disequilibrium in the External Boundary Layer of

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