Microtox bioassay

Environ Sci Pollut Res DOI 10.1007/s11356-014-2715-0

RESEARCH ARTICLE

Acute toxicity of arsenic to Aliivibrio fischeri (Microtox® bioassay) as influenced by potential competitive–protective agents David A. Rubinos & Valeria Calvo & Luz Iglesias & María Teresa Barral

Received: 30 September 2013 / Accepted: 28 February 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract In this study, we investigated the effect of some potential alleviative compounds against the acute toxicity of arsenic (AsV, AsIII and DMAV) on Aliivibrio fischeri (formerly Vibrio fischeri), a bioluminescent model bacterium, through the Microtox® bioassay. The compounds studied differed in their mechanism of action, and they included the following: phosphate and glycerol, as chemical analogues (and potential competitors) of AsV or AsIII, respectively; citrate, a weak natural organic ligand; and the antioxidant ascorbic acid. Special attention was paid to phosphate effects, a widespread pollutant in natural environments. AsV was found to be more acutely toxic than AsIII to A. fischeri, in accordance with its higher interaction with the bacteria. Both AsV and AsIII were found to be much more acutely toxic than DMAV, which was essentially non-acutely toxic even at very high concentrations. Phosphate presence (at equimolar P/As ratios or higher) resulted in the almost total suppression of bioluminescence inhibition, suggesting it exerts an alleviative effect against AsV acute toxicity on A. fischeri. Interestingly, the uptake and the percentage of extracellular AsV were not affected by the addition of phosphate, suggesting that such protective effect does not result from the competition for their common transporters. In contrast, the acute toxicity of AsIII was essentially unaffected by phosphate. Glycerol did not decrease the acute toxicity or the uptake of AsIII by A. fischeri, denoting the

Responsible editor: Vera Slaveykova D. A. Rubinos (*) : V. Calvo : L. Iglesias : M. T. Barral Department of Soil Science and Agricultural Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain e-mail: [email protected]

likely occurrence of an additional mechanism for AsIII uptake in such bacteria. Similarly, citrate and ascorbic acid essentially did not caused alleviation of AsV or AsIII acute toxicity. As for environmental and operational implications, P could beneficially protect aquatic microorganisms against acute detrimental effects of AsV, whilst its presence could mask the toxicity due to AsV when assessed using the Microtox® bioassay, thus leading to seriously underestimate the actual ecological and health risks. Keywords Arsenic . Bacteria . Microtox . Phosphate . Alleviation . Toxic

Introduction Arsenic (As) is a highly toxic metalloid ubiquitously distributed in the environment. Although As has been traditionally linked to its poisonous nature, most concern nowadays on this element lays on the exposure to environmental As, mainly through the consumption of contaminated drinking water and food. Globally, As contamination in drinking water is a major public health issue and is exerting a devastating effect on human health in several parts of the world (Jain and Ali 2000), particularly in Asia, where chronic As poisoning has became epidemic with over 100 million people exposed to drinking water with high As concentrations (Wang et al. 2007). Arsenic is considered to pose the most significant potential threat to human health based on frequency of occurrence, toxicity and human exposure (Rosen and Liu 2009). Natural (weathering, erosion and dissolution of As-bearing rocks and minerals, volcanic emissions and thermal springs) and anthropogenic sources (smelter slag, coal combustion,

Environ Sci Pollut Res

runoff from mine tailings, hide tanning waste, pigment production for paints and dyes, the processing of pressure-treated wood, waste disposal, indiscriminate use of fertilizers, pesticides and herbicides, manufacturing and chemical spillage) contribute to the worldwide occurrence of As contamination (Duker et al. 2005; Oremland and Stolz 2003). The major current uses of As compounds are as pesticides, herbicides and fungicides (sodium and calcium salts of monomethylarsenate and dimethylarsenate), wood preservatives (chromated copper arsenate), as defoliant (cacodylic acid), as growth promoters for poultry and pigs (Roxarsone®, 4-hydroxy-3-nitrophenylarsonic acid) (O’Neill 1995; Hughes et al. 2011), and as chemotherapeutic drugs for the treatment of parasitic diseases (Melarsoprol® for human African trypanosomiasis) and cancer (Trisenox® for acute promyelocytic leukemia) (Rosen and Liu 2009; Mukhopadhyay and Beitz 2010). Arsenic exists in inorganic (iAs) and organic forms and in four oxidation states, +V (arsenate), +III (arsenite), 0 (arsenic) and−III (arsine) (Sharma and Sohn 2009), but when dealing with environmental exposure, toxicologists are primarily concerned with the As in the trivalent and pentavalent state (Hughes 2002), both widely present in natural waters. AsV species are more stable and predominate in oxidizing environmental conditions, whereas in reducing environmental conditions, As III species predominate (Duker et al. 2005). Biotransformed methylated derivatives of both As V (monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV) and trimethylarsine oxide (TMAO)) and AsIII (monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII)) are also compounds of environmental and toxicological relevance (Sharma and Sohn 2009). DMAV is the major human metabolite after exposure to iAs and it is a toxic compound widely used as herbicide (Kenyon and Hughes 2001). Other organoarsenic compounds that can be often found in food are arsenobetaine, arsenocholine, arsenosugars and arsenolipids (Hughes et al. 2011). Arsenic toxicity affects a wide variety or organisms, including humans (Cervantes et al. 1994). The toxicity of As to human health ranges from skin lesions to cancer, and it causes acute (from gastrointestinal distress to death) (Hughes et al. 2011) and chronic (damage of the liver, kidney, the respiratory, digestive, circulatory, neural, and renal systems, and the skin, and the occurrence of skin, brain, liver, lung, bladder, kidney and stomach cancers) adverse health effects (Ng et al. 2003; Sharma and Sohn 2009). Consequently, the current World Health Organization (WHO) recommended a guideline value for As in drinking water of 10 μg/L (WHO 2011). As for ecotoxicological effects, adverse effects of arsenicals on aquatic organisms have been reported even at low concentrations in water (19 to 48 μg/L) (Eisler 1994). There is a strong link between As toxicity and speciation. The chemical form and oxidation state of As influence its

acute and chronic toxicity and the mode of action of such toxicity. Both AsV and AsIII are toxic, but trivalent As is generally more toxicologically potent than pentavalent As (Hughes et al. 2011) and, usually, inorganic As species are more toxic than organic forms to living organisms (Sharma and Sohn 2009), excepting MMAIII and DMAIII (Petrick et al. 2000; Styblo et al. 2000). Despite the general assumption that AsIII is more toxic than AsV, the biouptake and toxicity of As species on both freshwater and marine microalgae and phytoplankton remains controversial (Karadjova et al. 2008), and several researchers observed equal of higher toxicity of inorganic AsV than AsIII to these organisms (Cullen et al. 1994; Karadjova et al. 2008; Knauer et al. 1999; Levy et al. 2005; Pawlik-Skowrońska et al. 2004). This different toxicity results from the different mode of entry and action of the chemical species of As. AsV enters the cell through phosphate transporters in both prokaryotes (through the Pit and Pst systems) and eukaryotes (Maciaszczyk-Dziubinska et al. 2012; Rosen and Liu 2009), and it replaces phosphate in many biochemical reactions. AsV competitively inhibits enzymes that use phosphate or have phosphorylated intermediates (Rosen and Tamás 2010), and it uncouples the formation of ATP (Hughes 2002), ultimately depleting the cell of its energy. On the other hand, AsIII is taken up in living cells mainly through aquaglyceroporins (the glycerol facilitator GlpF in Escherichia coli) (Bhattacharjee et al. 2008; Rosen and Tamás 2010), which are transmembrane proteins that allow the bidirectional transport of water, glycerol and other small uncharged solutes (Hachez and Chaumont 2010). AsIII toxicity is related to its high affinity for the sulfhydryl groups of biomolecules such as gluthathione (GSH) and lipoic acid, and the cysteinyl residues of many enzymes (Aposhian and Aposhian 2006; Sharma and Sohn 2009), inhibiting important biochemical reactions leading to cytotoxicity (Hughes 2002). AsIII also leads to the production of reactive oxygen species (ROS) by binding to reduced glutathione (Bhattacharjee et al. 2008). Diverse bioassays have been used to study the toxicity/ ecotoxicity of As, most of them based on animals, plants, algae or bacteria (Farre and Barcelo 2003). Even though the first three shows good sensitivity, they are expensive, time consuming and, often, require specialized equipment and skilled operators. In contrast, bacterial bioassays are reproducible, relatively cheap, rapid and simple (Parvez et al. 2006). Among the bacterial bioassays, the standardized Aliivibrio fischeri (formerly known as Vibrio fischeri) (Urbanczyk et al. 2007) bioassay (Microtox® test) has gained popularity, and it is used by many laboratories worldwide as routine and standard (ISO 11348–3 2007; ASTM Standard method D5560-96 2009) toxicity test for environmental toxicity monitoring, assessment and screening (Johnson 2005; Kaiser 1998; Parvez et al. 2006). The Microtox® acute toxicity test (Strategic Diagnostics Inc., Newark, DE, USA) is an acute


ecotoxicological test based on the inhibition of bioluminescence of the gram-negative marine bacterium A. fischeri when exposed to a toxic chemical. Despite its limitations, this bioassay has been referred as the most sensitive test across a wide range of chemicals compared with other bacterial assays (Parvez et al. 2006). Microtox® produced comparable results with other standard toxicity bioassays (Abbondanzi et al. 2003; DeZwart and Slooff 1983; Hsieh et al. 2004), and a significant strong correlation of A. fischeri median effective concentration (EC50) with median lethal concentrations (LC50) for many aquatic species has been reported (Kaiser 1998). Regardless of the various studies carried out using the Microtox® bioassay to evaluate the toxicity of As (Fulladosa et al. 2004, 2005, 2007a; Martín-Peinado et al. 2012; Tišler and Zagorc-Končan 2002), it is worth mentioning that the acute toxicity of As to A. fischeri in the presence of other chemicals and ligands with potential capacity to interfere such toxicity has been scarcely studied. This is particularly relevant for the case of P and As, which are chemically and structurally analogous elements and form species with comparable chemical properties; hence, some kind of effect of P on As uptake and toxicity is expected. Even though several studies have shown that P decreases the toxicity and uptake of AsV, and in some cases of AsIII as well (Karadjova et al. 2008), by aquatic microorganisms (Karadjova et al. 2008; Levy et al. 2005; Takahashi et al. 2001; Thiel 1988), its effect on As acute toxicity to A. fischeri has not been yet evaluated. Besides, As can form stable complexes with natural organic ligands in aqueous systems (Buschmann et al. 2006; Redman et al. 2002), which could influence the toxicity of As (Jones and Huang 2003). Therefore, the main objective of this article is to study the effect of diverse agents with potential competitive–protective action on the acute toxicity of different As species/forms— AsV, AsIII and DMAV—to A. fischeri (Microtox® bioassay), whilst gaining insight into the mechanisms by which As exerts toxicity on this model bacterium. The tested agents were the following: (1) phosphate, a widespread contaminant in natural waters and wastewaters, where it is usually present at higher concentrations than those of As (Sø et al. 2012) and which shares with AsV the same transporters to enter cells; (2) glycerol, a molecular mimic of As(OH)3 (Porquet and Filella 2007), which is taken up by living cells through the same channels as AsIII (Rosen and Liu 2009) and whose presence could competitively decrease AsIII uptake; (3) citrate, an example of a naturally occurring organic ligand expected to be found in natural waters as a consequence of biological activity (Klewicki and Morgan 1998) and which acts as an intermediate of ATP generation; and (4) ascorbic acid, an antioxidant which directly scavenges ROS, thus potentially protecting cells from As intoxication (Banerjee et al. 2009; Ramanathan et al. 2003; Singh and Rana 2007). An additional

objective is to test the suitability and potential pitfalls of the Microtox® bioassay when used to evaluate the acute toxicity of As-rich complex matrices where concur chemicals that could hide As toxicity.

Materials and methods Chemicals As V solutions were prepared by dilution of a 13.35 ± 0.03 mmol As/L (as As2O5) standard aqueous solution, whereas DMAV and AsIII solutions were prepared respectively by dissolution of cacodylic acid sodium salt 3-hydrate [C2H6AsNaO2 ·3H2O] and AsIII trioxide [As2O3] in deionized (MilliQ) water. Acidification (pH ~1) and mild heating was needed to achieve proper dissolution of As2O3 (Budavari et al. 1996). Phosphorous solutions were prepared by dissolution of NaH2PO4 ·H2O in water, as were glycerol [C3H5(OH)3], L(+)ascorbic acid [C6H8O6] and sodium citrate [Na3C6H5O7 · 2H2O] solutions. All the As solutions were prepared extemporaneously in order to minimize changes in As oxidation state. pH adjustment was made when necessary by addition of either 1 M/0.1 M HCl or NaOH solutions, prepared respectively by dilution of concentrated 35 % (Hiperpur-plus grade) HCl or by dissolution of solid NaOH. pH and Eh (for the Ascitrate solutions) were measured with a Thermo Scientific Orion Dual Star™ pH/ISE meter coupled to an Aquapro™ combination pH electrode or a Epoxy Sure-Flow™ combination redox electrode (Thermo Fisher Scientific Inc., Beverly, USA). All chemicals used were of ACS-ISO or higher grade and were purchased from Panreac (Barcelona, Spain), with the exception of As2O3, which is purchased from Sigma-Aldrich (St. Louis, MO, USA). All solutions were prepared in ultrapure water (18.2 MΩ/cm resistivity, MilliQ system (Millipore, Bedford, MA, USA)). All glassware was washed with diluted nitric acid overnight, followed by thoroughly rinse with ultrapure water. Microtox® bioassay protocol The toxicity tests were performed by means of the Microtox® acute bioassay, using a Microtox® Model 500 Toxicity Analyzer (Strategic Diagnostics Inc., Newark, USA). The apparatus includes a temperature-controlled incubator block for 30 tubes and a read well, both at 15±0.5 °C, a compartment at 5.5±1 °C to properly maintain the luminescent bacteria during the assays, and a digital display where the light levels are indicated. The bacteria A. fischeri (strain NRRL B− 11177) was used as test organisms, obtained as a freeze-dried bacterial form (Microtox® acute toxicity test reagent). This reagent was stored at −20 to −25 °C and reconstituted immediately before each analysis by addition of 1 mL of Microtox®


reconstitution solution (specially prepared non-toxic ultrapure water + 0.01 % NaCl) at 5.5±1 °C. The test cuvettes, lyophilized bacteria, reconstitution, diluent (2 % NaCl) and osmotic solutions (22 % NaCl) were purchased from Strategic Diagnostics Inc. A 90 mg/L phenol solution was used as a reference toxicant for quality control of the bacterial strain (Fulladosa et al. 2004). Determined EC 50 value for this toxicant was 30.81 mg/L (% effect 34.23 %, 95 % confidence range 28.31 to 41.38), which is in accordance with the Microtox® manufacturer’s recommended range (Azur Environmental 1998), and it is close to previously reported values for this compound (Fulladosa et al. 2004; Kaiser and Ribó 1988). In order to evaluate the toxicity of AsV, AsIII and DMAV, we developed dose–response curves by testing solutions of initial As concentration ranging from 0 to 3.0 mmol/L (2.1 mmol/L in the case of DMAV), following the extended (13-fold dilutions, 0.5 dilution factor) basic test of the Microtox® protocol (Azur Environmental 1998). Arsenic (testing samples) and diluent solutions were adjusted to pH 7±0.2 by addition of either 0.1 M HCl or NaOH just before performing the toxicity test. Light emission was recorded after 5 and 15 min of exposure to the toxicant, and the output data analyzed using the Microtox® Omni™ (version 1.18) software (Strategic Diagnostics Inc., Newark, USA). The results obtained were calculated as bioluminescence inhibition relative to the As-free control samples. EC50 and EC20—that represents a measurable threshold of As toxicity in the Microtox® bioassay (Tišler and Zagorc-Končan 2002; Fulladosa et al. 2005)—defined respectively as the concentration of chemical that produces a 50 and 20 % decrease in light emission of A. fischeri for a given time (5 or 15 min) of exposure (Fulladosa et al. 2007b), were calculated from the dose–response curve data by linear regression analysis of the relationship between the log of As concentration and the log of the gamma function (Γ). Γ is the ratio of lost/remaining light intensities (Fulladosa et al. 2007b), and it is computed in the Omni™ software as follows: I ¼ ðI c =I s Þ−1

ð1Þ

where Ic and Is are the intensity of bioluminescence of the control and a given sample concentration, respectively. EC50 and EC20 values were calculated from the intercept, providing a r2 >0.95 (Volpi Ghirardini et al. 2009). Each toxicity determination was carried out at least in triplicate. Competition–protection experiments The competitive–protective acute toxicity tests were performed by developing dose–response curves following the Microtox® protocol as described in the “Microtox® bioassay protocol” section and for the same As concentration range, but

in the presence of phosphate (for AsV, AsIII and DMAV), citrate or ascorbic acid (both for AsV and AsIII), or glycerol (only for AsIII). In all cases, As and the chemical were added at the same time, i.e. A. fischeri was simultaneously exposed to both substances. The experiments on phosphate effect were performed at 1:1, 1:10 and 1:100 AsV,III/P molar ratios, with the exception of DMAV, for which only 1:1 and 1:10 DMAV/P molar ratios were tested. Citrate and ascorbic acid were added well in excess (45 mM) of AsV and AsIII concentrations, whilst glycerol was tested at 1:1, 1:10 and 1:100 AsIII/glycerol molar ratios. The potential toxic effects of the competitive–protective chemicals on A. fischeri were checked by performing Microtox® bioassays for single solutions of each added chemical. The experiments were performed at pH 7.0±0.2, adjusted as described in the “Microtox® bioassay protocol” section and run at least in triplicate. Interaction of arsenic with A. fischeri In parallel with the toxicity experiments, the sorption/uptake of As by A. fischeri after 15 min of exposure was determined as a function of AsV, AsIII and DMAV concentration by filtering the bacteria suspensions through 0.45-μm syringe filters (Whatman Puradisc 25AS™), followed by analysis of the total As concentration in the filtrates. The potential competitive uptake of As by A. fischeri was studied for the binary arsenic (AsV, AsIII or DMAV)—phosphate and AsIII—glycerol systems as well. In view of the acute toxicity tests’ results, the uptake of AsV was determined as a function of added P concentration at 1:1, 1:10 and 1:100 As/P molar ratios, whilst for AsIII and DMAV, it was measured only at 1:1 molar ratios. In turn, the effect of glycerol on AsIII uptake was studied at a 1:1, 1:10 and 1:100 AsIII/glycerol molar ratios, as in the case of phosphate. Bacterial As (extra- plus intracellular) was calculated as the difference between added As concentration (determined experimentally from appropriate blanks, i.e. identical systems containing As solutions, but no bacteria) and dissolved As concentration in the filtrates. Loss of As due to adsorption to the test cuvette walls, precipitation, or retention when passing through the filter was checked, and measured As was ~106– 134 and ~84–101 % of theoretical added concentration for AsV and AsIII, respectively. Bacterial As concentrations were expressed on mole per cell basis, considering that each test cuvette contains roughly a million individual test organisms (Azur Environmental 1998). Extracellular (sorbed on the cell surface) As was distinguished in the systems AsV and AsV/P ratio (1:1) as a function of As concentration to test if competition between AsV and phosphate for sorption sites of cell surface occurred, as it has been described for other microorganisms (Levy et al. 2005). Extracellular As was determined after 15-min exposure period by filtering the cell suspensions and washing the cells on the membrane filter with 0.1 M


a 1.2

As(V)

Relative light emission (It/I0)

phosphate buffer (5 mL, 2 min of contact time), previous rinsed (2×5 mL) of the cells with arsenic-free Microtox® diluent solution to remove excess dissolved As remaining from the exposure stage (Karadjova et al 2008; Levy et al 2005). Arsenic was analyzed in all cases by inductively coupled plasma mass spectrometry (ICP-MS, Varian 820 MS) with collision reaction interface (CRI) technology to avoid the interference of chloride. Each ICP-MS measurement was made in triplicate, and the relative standard deviations were below 5 %. In the systems containing phosphate, P uptake was measured as well (ICP-MS).

1

As(III)

0.8

0.6

0.4

0.2

Theoretical aqueous speciation of arsenic

Data analysis Values were expressed as mean±standard deviation (n≥3). Statistical differences were evaluated by one-way ANOVA followed by post hoc analysis (Duncan test at p450b,c,d

5 min 71.4±3.1 95.5±15.4 >450b,c,d

a

15 min 2.0±0.6 9.9±2.7 >450b,c,d

15 min 37.2±10.7 59.7±2.3 >450b,c,d

Values are the mean±standard deviation (SD) of at least three replicates

b

Maximum tested concentration (450 mg/L)

c

Hormesis detected

d

Statistical calculations could not be performed on the 5- and 15-min data

This behaviour could be related with different concentrationdependent uptake kinetics of AsV and AsIII or with the mechanism through which these As species impair the bacteria. In the case of DMAV, there was no observable toxicity within the concentration range studied (figure not shown); besides, hormesis (i.e. a stimulatory effect on bacteria at low concentrations of chemical) was detected (up to ~5–8 % stimulatory effect) after exposure to DMAV, supporting the hypothesis that the acute toxicity of AsV decreases with methylation (Roy and Saha 2002). This agrees with the previously reported very low acute toxicity of this compound to A. fischeri (Fulladosa et al. 2007a, b) as well as on mammal cells (Dopp et al. 2005). Although the toxicological modes of action of DMAV are less well elucidated, its toxicity seems to be exerted mainly at the chronic effect level, being principally due to the peroxyl radical of DMAV and to the production of active oxygen species in vivo, inducing nucleic acid damage and cellular toxicity (Kenyon and Hughes 2001; Mandal et al. 2001). From the EC data obtained, the following sequence of decreasing acute toxicity of arsenicals to A. fischeri can be established: AsV >AsIII ≫DMAV. This agrees with the previously reported toxicity of As on A. fischeri (Fulladosa et al. 2005, 2007a; Planer-Friedrich et al. 2008) and on many freshwater (Knauer et al. 1999; Levy et al. 2005; PawlikSkowrońska et al. 2004) and marine microalgae (Cullen et al. 1994; Takimura et al. 1996). Some of the following reasons could contribute to the greater sensitivity of A. fischeri to AsV than to AsIII found: (i) the pH and ionic composition of the testing environment, which has been reported to greatly influence As speciation and solubility, and hence toxicity (Bradl et al. 2005; Fulladosa et al. 2004; Pawlik-Skowrońska et al. 2004); (ii) blockade of ATP and reduced flavin mononucleotide (FMNH2) generation in the presence of AsV by competitively substituting P, hence depleting the availability of the reduced form of nicotinamide

adenine dinucleotide phosphate (NADPH), which is essential for bioluminescence emission in A. Fischeri; and (iii) the preferential uptake of AsV compared with AsIII, as reported for other aquatic microorganisms (Levy et al. 2005; PawlikSkowrońska et al. 2004). In relation with the pH and ionic composition, the theoretical speciation calculations performed showed that in our experimental systems (pH 7.0±0.2), the main AsV and AsIII aqueous species were respectively the bivalent HAsO42− (82.34 % of total) and the neutral H3AsO3 (98.65 % of total), with only 1.35 % of AsIII occurring as the supposedly most toxic H2AsO3− species (Fulladosa et al. 2004). This species distribution could contribute to the observed higher toxicity of AsV, since Fulladosa et al. (2004) found lower EC50 values for AsIII at pH 9 (where H2AsO3− predominates) than for AsV at pH ≤7. Also, no precipitation of As was observed in the experimental systems, and calculated SI of AsV and AsIII solid phases were much lower than 0 even for the highest As concentration studied (SI= −24.137 for As2O5(s) and SI = −3.584 and −3.629 for arsenolite and claudetite, respectively), suggesting that solubility did not limit the availability and toxicity of both As species. The chloride-rich (2 % NaCl) ionic composition of the media should diminish the toxicity of AsV, thus Cl− seems not to be involved in the toxicity sequence found. Therefore, as we discuss below, the higher interaction of AsV with the bacteria seems to play a key role in the toxicity response observed.

Interaction of arsenic with A. fischeri The bacterial As concentration as a function of added As concentration in the medium, after 15 min of exposure, is shown in Fig. 2. Similar interaction patterns were observed for the three As forms studied, and the As concentration of the bacteria increased linearly (r2 =0.9988, r2 =0.9979 and r2 = 0.9813, for AsV, AsIII and DMAV, respectively) with the added As concentration within the experimental concentration range. A plateau in the plots was not observed in any case, indicating that saturation did not occur even at the highest tested As concentrations, corresponding to maximal concentrations on/ in the bacteria of [As]max (AsV) =(1.15±0.23)×10−12 mol/cell, [As]max (AsIII) =(3.67 ± 1.28)× 10−13 mol/cell and [As]max −12 mol/cell. (DMAV) =(1.94±0.22)×10 V Interestingly, the As retained by the bacteria after exposure was 2–5 times higher than that of AsIII within the same concentration range, which is in accordance with the observed differences in toxicity, hence suggesting that the higher toxicity of AsV than AsIII to A. fischeri could be related with its higher uptake. Higher availability and uptake of AsV than As I II have been also reported for the green algae Stichococcus bacillaris, and it has been suggested as the main


1E-11

1.2


[As]cell (mol/cell)

1E-12

a As(V) As(III) DMA(V)

1E-13

1E-14

1E-15

1E-16 1E-07

1E-06

0.00001 0.0001

0.001

III

1 0.8 0.6 0.4 As As:P (1:1) As:P (1:10) As:P (1:100) P

0.2

0.01

0

[As] (M) V

AsV

0

V

Fig. 2 Concentration (mol/cell) of As , As and DMA on/in Aliivibrio fischeri as a function of initial added concentration (mol/L) of the different As species in the tested media. pH 7.0±0.2, 15-min exposure time. Data represent mean±SD (error bars) of three replicate sets

0.5

1

1.5

2

2.5

3

AsV (mM)

b

factor responsible for its higher toxicity (Pawlik-Skowrońska et al. 2004). Regarding DMAV, its retention was also concentrationdependent and, in general, of the same order of that of AsV ([As](DMAV) =(7.86±0.16)×10−13 mol/cell) (Fig. 2). This behaviour, together with the lack of observable toxic effects of DMAV, shows that DMAV is much less acutely toxic than inorganic AsV or AsIII. Competitive–protective toxicity experiments


1.2

AsIII

1 0.8 0.6 0.4 As As:P (1:1) As:P (1:10) As:P (1:100) P

0.2 0

Effect of phosphate on toxicity of AsV, AsIII and DMAV

0

0.5

1

1.5

2

2.5

3

III

As (mM)

The effect of increasing phosphate concentrations on As toxicity to A. fischeri is displayed in Fig. 3 as dose–response curves. As can be seen, the presence of phosphate affects the As toxicity in a different way depending on the arsenical compound considered. In the case of AsV, no decay of bioluminescence (i.e. no toxicity) was observed in the presence of phosphate in the range of the tested AsV concentrations and for all the As/P molar ratios studied (Fig. 3a). The apparent absence of toxic effects caused that neither EC20 nor EC50 values could be precisely calculated (EC20 and EC50 >100 % of the highest AsV concentration). Furthermore, bioluminescence inhibition was below the threshold of 20 % effect even at the highest AsV concentration (Table 2), with the exception of the binary 1:100 AsV/P system at the highest AsV −P concentration, where a 30.4±1.8 highest % effect was observed, probably due to the extremely high concentration of phosphate causing some bioluminescence inhibition, as was observed in a parallel Microtox® test carried out with a 300 mM P solution without AsV (highest % effect

Fig. 3 Effect of phosphate on arsenic acute toxicity: dose–response curves for the toxicity of AsV (a) and AsIII (b) on Aliivibrio fischeri (Microtox® acute toxicity test) as a function of As species to P molar ratio (1:1, 1:10 and 1:100). Grey solid line shows the relative bioluminescence of a 300-mM P solution tested using the Microtox® bioassay. Toxicity curve in the absence of phosphate (dotted line) is also showed as reference. pH 7.0±0.2, 15-min exposure time. Data represent mean±SD (error bars) of at least three replicate sets

(15 min)=36.6±4.0 % at 300 mM P) (Fig. 3a). Besides, no enhancement in the bioluminescence was observed after exposure of A. fischeri neither to single phosphate nor to binary AsV/P solutions within the concentration range tested. The results obtained suggest that the simultaneous addition of P, even at equimolar ratios, exerts an alleviative effect against the acute toxicity of AsV to A. fischeri, at least up to aqueous concentrations as high as 3 mM AsV. The protective effect of phosphate against the toxicity of AsV has been generally ascribed to a decrease in the uptake

Environ Sci Pollut Res Table 2 Toxicity parameters (EC20, EC50 and the highest % effect) for arsenic compounds in the presence of simultaneously added phosphate as a function of As (DMAV)/P molar ratio, determined using the Microtox® acute toxicity bioassay System

EC20 ±SD (mg/L)a

EC50 ±SD (mg/L)a

AsV:P (1:1) AsV:P (1:10) AsV:P (1:100)

5 min >225c >225 –d

15 min >225 >225 –d

5 min >225 >225 >225

15 min >225 >225 >225

15 min 7.5±4.1 10.8±6.5 30.4±1.8

AsIII:P (1:1) AsIII:P (1:10) AsIII:P (1:100) DMAV:P (1:1)e DMAV:P (1:10)e

12.1±7.6 11.3±4.7 13.6±6.9 >450 >450

8.2±3.4 12.7±1.3 11.8±7.4 >450 >450

48.2±27.8 133.7±7.6 146.4±24.1 >450 >450

36.2±14.2 94.6±9.1 108.2±4.8 >450 >450

81.3±8.9 63.6±1.5 59.0±1.1 5.8±1.2 3.7±0.6

% effectmaxb

Reference EC values for As in the absence of phosphate are displayed in Table 1 Numbers in parentheses indicate As (or DMA) to P molar ratio a


b

Highest % effect

c

Maximum tested concentration (225 mg/L for AsV or AsIII ; 450 mg/L for DMAV )

d

Statistical calculations could not be performed on the 5- and 15-min data

e

Hormesis detected

and accumulation of AsV by the cells in the presence of phosphate (Karadjova et al. 2008; Levy et al. 2005; Takahashi et al. 2001), resulting from the competition between arsenate and phosphate for the common transporters used by both elements to enter the cell (Harold and Baarda 1966; Rosen and Liu 2009). Also, the presence of phosphate could decrease the amount of arsenate sorbed on the bacteria by hindering the approach of the negatively charged As oxyanions to the cell surface sites. To elucidate the mechanisms that are mainly responsible for the observed alleviative effect of phosphate, the retention of AsV by A. fischeri after exposure was measured as a function of added AsV and P concentrations and molar ratios. Besides, extracellular As was determined after exposure to equimolar AsV/P to assess the contribution of eventual AsV sorption on the cell surface to the overall process and to know how phosphate affects such sorption. Increasing P to AsV ratio did not resulted in a decrease in the amount of AsV retained by the bacteria within the concentration range studied. The As concentration on/in the bacteria increased linearly (r2 ≥0.99) with the added As concentration in the presence of P (Fig. 4a), leading to similar maximal bacterial As concentrations ([As]max =(1.23±0.07)×10−12, [As]max =(1.26± 0.14)×10−12 and [As]max =(0.94±0.23)×10−12 mol/cell, for 1:1, 1:10 and 1:100 AsV/P molar ratios, respectively) to those in absence of phosphate ([As]max =(1.15±0.23)×10−12 mol/cell). Interestingly, although the extracellular As concentration increased (up to [As]ext, max =(6.3±0.03)×10−15 and [As]ext, −15 mol/cell, in absence and in presence max =(5.9±0.02)×10 of P, respectively) with increasing concentrations of AsV added to the medium, the percentage of extracellular

(sorbed) AsV decreased as the concentration of added AsV increased (Fig. 5). Extracellular As (%) accounted for up to≈ 20 % of retained As at the lowest concentration (0.73 μM AsV), and it decreased progressively up to a nearly constant value of≈ 0.5 % at concentrations ≥50 μM AsV. The presence of equimolar P concentrations did not affect the percentages of extracellular As within the concentration range studied (Fig. 5), suggesting that sorption processes of AsV onto the cell surface do not mostly contribute to the effects observed on toxicity. In both cases, extracellular As concentration showed a strongly linear positive relationship with As concentration remaining in solution after the exposure period (i.e. displayed a high affinity linear isotherm). Thus, most As removed from solution must have been taken up by the bacteria, in accordance with the high toxicity observed at these high As concentrations, suggesting that soluble As is strongly and quickly bioavailable. Hence, the addition of phosphate did not seem to inhibit the interaction of AsV with A. fischeri within the concentration range studied, probably because a saturation of the arsenate– phosphate transporters was not reached even at the highest added AsV concentration, as suggested by the absence of a plateau in the As V retention patterns. Likewise, the Lineweaver–Burk plots used to depict the dependence of the short-term AsV uptake on P concentration, although linear (r2 =0.990–0.999) (Fig. 6), did not reveal the displacement to higher reciprocal values with increasing P to AsV molar ratio, as would be characteristic of a competitive relationship between phosphate and arsenate (Harold and Baarda 1966). Moreover, a shift to lower 1/Asuptake with increasing P to AsV molar ratio was observed, suggestive of an stimulatory effect

Environ Sci Pollut Res 25

a

As(V)

1E-11 V

As

Extracellular As (%)

[As]cell (mol/cell)

1E-13

1E-14 As

1E-15

15

10

5

As:P (1:1) As:P (1:10)

1E-16 1E-07

As(V):P

20

1E-12

As:P (1:100)

1E-06

0.00001 0.0001

[AsV]

0.001

0

0.01

1E-07

(M)

1E-06

0.00001

0.0001

0.001

0.01

[As] (M)

b

Fig. 5 Extracellular AsV (% of previous retained As on/in the cell) as a function of initial added As concentration (mol/L) in the absence and presence of equimolar As/P concentrations

1E-11

AsIII [As]cell (mol/cell)

1E-12

1E-13

1E-14

1E-15

1E-16 1E-07

As As:P (1:1)

1E-06

0.00001 0.0001

0.001

0.01

[AsIII] (M) Fig. 4 Effect of phosphate on arsenic retention by Aliivibrio fischeri: concentrations (mol/cell) of AsV (a) and AsIII (b) as a function of initial added bulk concentration (mol/L) of As in the tested media and as a function of As species to P molar ratio (1:1, 1:10 and 1:100 molar ratios). pH 7.0±0.2, 15-min exposure time. Data represent mean±SD (error bars) of three replicate sets

Interestingly, these maximal retained P concentrations were 0.16-, 3- and 36-fold the maximal cellular concentrations of As in these systems (in contrast with the 1-, 10- and 100-fold P/As in the bulk media). Such behaviour could be related either with a greater affinity of the phosphate transport system for AsV than that for phosphate (Harold and Baarda 1966) or with the inhibition of the rate and amount of P uptake in the presence of high concentrations of AsV, as it has been reported in other bacteria (Harold and Baarda 1966; Willsky and Malamy 1980). Despite the lower maximal retained P concentration than AsV in equimolar conditions, it has been recently reported that bacterial cells are capable of discriminating phosphate over arsenate at least 500-fold (Elias et al. 2012), and even much lower bulk concentrations (130–1300-fold) of

1/Asupt (mol/cell/15 min)

3E+15

of P on AsV uptake as more P is added. In relation to this, a tendency of the cells to increase the number and/or the efficiency of the transporters in response to additions of arsenate or phosphate has been reported (Hellweger et al. 2003; Karadjova et al. 2008). In fact, P retention increased as the P to AsV molar ratio increased and, within same molar ratio, as the bulk P concentration increased (Fig. 7). Increasing the P concentration by tenfold led to further, and almost proportional, increases in P retained up to [P]max =(1.96±0.48)×10−13, (3.74±2.00)×10−12 and (3.36±0.43)×10−11 mol/cell (for the 1:1, 1:10 and 1:100 AsV/P systems, respectively), suggesting improved capacity of A. fischeri uptake in response to an additional contaminant exposure, as it has been observed in other bacteria (Willsky and Malamy 1980).

As As:P (1:1) As:P (1:10) As:P (1:100)

2E+15

1E+15

0 0.0E+00

5.0E+05

1.0E+06

1.5E+06

1/[As] (M) Fig. 6 Lineweaver–Burk plots for AsV in the presence of different phosphate concentrations

Environ Sci Pollut Res 1E-10

against AsIII acute toxicity in the concentration range and for none of the AsIII/P molar ratios studied (Table 2). The lack of a clear protective effect of phosphate on A fischeri against AsIII acute toxicity agrees well with its different uptake pathway and toxicity mechanisms in cells, so transport competition between AsIII and P is unlikely. In fact, we found that the retention of AsIII by A. fischeri was not significantly altered in the presence of equimolar P concentrations (Fig. 4b), supporting the absence of protective effects of phosphate on A. fischeri upon acute exposure to AsIII. In a similar way, the presence of phosphate did not affect the bioluminescence and retention of DMAV (data not shown).

[P]cell (mol/cell)

1E-11

1E-12

1E-13

1E-14 As:P (1:1) As:P (1:10)

1E-15

As:P (1:100) 1E-16 1E-07

1E-06 0.00001 0.0001

0.001

0.01

0.1

1

[P] (M) Fig. 7 Concentration of phosphorous (mol/cell) on/in Aliivibrio fischeri as a function of initial bulk phosphorous concentration for the binary systems 1:1, 1:10 and 1:100 AsV/P. Data represent mean±SD (error bars) of three replicate sets

phosphate than arsenate exerted alleviation of the toxicity of arsenate in cyanobacterium (Takahashi et al. 2001). These results show that the capacity of A. fischeri for retaining phosphate and arsenate is enhanced as more anion is added to the system and that its actual maximal capacity is much higher than that found for the highest AsV concentration tested in the toxicity tests, which is in accordance with the observation of no competition between both elements within the concentration range studied. On the other hand, the SI calculations carried out using Visual MINTEQ did not predict precipitation of AsV in the systems containing phosphate (SI=−23.8 for As2O5(s)); hence, the solubility of AsV was not a limiting factor of availability and toxicity of As in the experimental systems. The obtained results suggest that the alleviative effect of phosphate on AsV toxicity cannot be explained merely by the competition between phosphate and arsenate for the cell surface or transport sites of A. fischeri, at least at the outermembrane level. Maybe such effect is due to the competition between both compounds at the metabolic pathways’ intracellular level, as it has been reported for the cyanobacterium Anabaena variabilis (Thiel 1988). Nonetheless, if a similar phosphate uptake system to that described for Vibrio cholerae (Mudrak and Tamayo 2012) exists in A. fischeri, then the competition between P and AsV at the periplasmic (for binding to the periplasmic P binding proteins: PstS and PstS2) and/or at the inner membrane level (for binding to the permease protein complex: PstCAB and PstCAB2) cannot be excluded. Further research is needed to definitely conclude at which cellular level P exerts its protective effect against AsV toxicity. In contrast to AsV, the dose–response relationships obtained for AsIII in the absence and presence of phosphate (Fig. 3b) did not show a significant alleviative effect of phosphate

Effect of glycerol The dose–response relationships obtained showed that glycerol did not alleviate the acute toxicity of AsIII on A. fischeri when both compounds were added simultaneously (Fig. 8a; Table 3). Similar bioluminescence inhibition patterns were observed in the absence and presence of glycerol, with maximal % effects of 70.3, 68.0, 73.3 and 76.9 % for the systems AsIII (no glycerol), 1:1, 1:10, and 1:100 AsIII/glycerol, respectively. Besides, although it has been shown that glycerol addition competitively decreases AsIII short-term uptake in a dose-dependent manner in rice roots (Meharg and Jardine 2003) and yeast (Wysocki et al. 2001), no significant differences in AsIII retention by the bacteria were observed between the systems containing or not different concentrations of glycerol (Fig. 8b). Considering that bacteria exposure to both AsIII and glycerol occurred simultaneously in our experiments, possible explanations for our observations could be that (a) AsIII has a higher affinity for the transport system than glycerol (Meharg and Jardine 2003) or (b) the smaller diameter and molecular volume of the As(OH)3 (2.563 to 2.807 Å, 59 cm3/ mol) than the retracted conformation of glycerol (2.684 to 3.071 Å, 71 cm3/mol) molecule offers to AsIII an additional advantage for its transit through the narrowest region constriction (pore diameter ~4 Å) (Rosen and Tamás 2010) of the GlpF channel (Porquet and Filella 2007), thus making less likely the uptake competition between these two compounds. Effect of citrate The addition of citrate, used as a model of low-molecular weight dissolved natural organic acids that affects As mobilization and speciation in natural media (Corsini et al. 2011), did not cause a clear alleviative effect against As acute toxicity (Table 4). Merely a slight bioluminescence enhancement was observed at high AsV concentrations (≥56 mg/L) in the presence of citrate. Such effect could result from a stimulatory effect of citrate on A. fischeri, as it has been recently reported for other bacteria (Bacillus, Pseudomonas, and Geobacter) (Corsini et al.


a

Table 3 EC20 and EC50 values for AsIII in the presence of simultaneously added glycerol (Gly) as a function of AsIII to glycerol molar ratio, determined using the Microtox® acute toxicity bioassay


1.2 1

System

EC20 ±SD (mg/L)a

EC50 ±SD (mg/L)a

AsIII alone AsIII:Gly (1:1) AsIII:Gly (1:10) AsIII:Gly (1:100)

5 min 14.9±8.6 11.3±2.2 7.3±2.0 13.4±2.1

5 min 106.7±11.8 123.9±16.0 95.7±21.2 65.8±7.9

0.8 0.6 0.4 As As:Gly (1:10) As:Gly (1:100)

0 0

0.5

1

15 min 58.9±3.0 66.2±13.3 58.1±1.9 37.1±2.6

Data obtained for a system containing AsIII alone is also showed for comparison

As:Gly (1:1)

0.2

15 min 10.4±5.7 7.2±1.0 11.5±5.1 7.4±1.0

Gly glycerol

1.5

2

2.5

3

a


III

As (mM)

Effect of ascorbic acid

b 1E-11

The presence of ascorbic acid at a concentration (45 mM) well in excess of As did not cause any ameliorative effect against AsV or AsIII acute toxicity on A. fischeri, and the calculated EC20 and EC50 values did not differ significantly upon addition of ascorbic acid (Table 4). This suggests that the toxicity of As to A. fischeri is mostly exerted through a direct mechanism, by reaction with cellular thiols (in the case of AsIII) and by substituting phosphate in biochemical reactions (in the case of AsV), at least in the short term.

[As]cell (mol/cell)

1E-12

1E-13

1E-14 As

1E-15

1E-16 1E-07

As:Gly (1:1) As:Gly (1:10) As:Gly (1:100)

1E-06

0.00001 0.0001

0.001

Conclusions

0.01

[AsIII] (M) III

Fig. 8 Effect of glycerol on the acute toxicity of As : dose–response curves (Microtox® acute toxicity test) (a) and retention (b) of AsIII by Aliivibrio fischeri as a function of AsIII to glycerol molar ratio (1:1, 1:10 and 1:100). Toxicity curve in the absence of glycerol (dotted line) is also shown as reference. pH 7.0±0.2, 15-min exposure time. Data represent mean±SD (error bars) of three replicate sets

Arsenic acute toxicity determined using the Microtox® bioassay depends on As speciation in the tested media. A. fischeri cells were much more sensitive to inorganic AsV and AsIII than to DMAV, which was essentially non-acutely toxic even at Table 4 EC20 and EC50 values for AsV and AsIII in the presence of simultaneously added Na citrate or ascorbic acid (45 mM initial concentration), determined using the Microtox® acute toxicity bioassay System

2011), or alternatively be due to the anionic nature of citrate (93 % of total amount present as CIT3−in our systems) which, when present near the cell surface, could electrostatically hinder the approach of arsenate to the transport system of the bacteria. In the case of AsIII, the dose–response curves obtained in the absence and presence of citrate overlapped each other, yielding similar EC20 and EC50 values (Table 4). This agrees with the fact that cells uptake AsIII as the neutral H3AsO3, thus electrostatic impediment for the approach to AsIII transporters is unlikely to occur in the presence of citrate. The results also show that an external citrate supplementation, aimed to stimulate the inhibited citric acid cycle by As, is unsuccessful in terms of alleviating As acute toxicity in prokaryotes.

V

As alone AsV–citrateb AsV–ascorbicb AsIII alone AsIII–citrate AsIII–ascorbic

EC20 ±SD (mg/L)a

EC50 ±SD (mg/L)a

5 min 3.2±2.5 9.6±5.4 6.0±3.3 10.2±2.4 13.2±5.3 14.2±3.3

5 min 82.5±32.9 151.7±3.2 63.9±10.4 95.5±15.4 135.3±21.9 81.3±8.5

15 min 4.7±1.3 4.7±0.8 3.6±0.7 9.9±2.7 7.9±4.1 10.2±2.2

15 min 31.8±13.9 70.8±14.6 25.1±2.7 59.7±2.3 68.2±8.5 50.7±5.9

Data obtained for a system containing AsV or AsIII alone are also showed for comparison a b


Initial concentration of citrate or ascorbic acid in the testing media, 45 mM


very high concentrations. Higher toxicity was found for AsV than for AsIII. However, in the presence of phosphate, this sequence of toxicity was changed, and AsIII was the form exhibiting the highest acute toxicity. The presence of phosphate—at equimolar AsV/P ratios or higher—exerts an alleviative effect of AsV acute toxicity to A. fischeri, visualized as the almost suppression of bioluminescence decay upon exposure to very high concentrations of As. Phosphate did not affect either the AsV retained by the bacteria or the percentage of extracellular AsV, suggesting that transport competition between both elements was not the main mechanism involved. In contrast, phosphate did not protect against AsIII acute toxicity. Glycerol does not decrease the acute toxicity of AsIII on A. fischeri even at concentrations exceeding (up to 1:100 AsIII/ P molar ratios) those of AsIII, suggestive of the likely occurrence of additional AsIII uptake routes in A. fischeri, but further research is needed on this aspect. Similarly, addition of citrate or ascorbic acid did not exert a clear protective effect against the acute toxicity of either AsV or AsIII. As for environmental and operational implications, the effect of phosphate on As toxicity is particularly relevant, since it suggests that P presence could protect aquatic microorganisms against AsV toxicity, maybe without limiting its availability, an aspect particularly valuable for bioremediation purposes. From an operational point of view, it means that the presence of phosphate could seriously mask the acute toxicity of AsV when Microtox® is used to screen leachates or extracts where both compounds concur (as usually happens in wastewaters, industrial leachates and soil extracts), hence seriously underestimating the actual risks. In contrast, neither P nor glycerol influences the acute toxicity of AsIII, and the results from Microtox® bioassay can be considered reliable in these cases. Solutions containing As and ascorbic acid, as for example soil extracts from sequential extraction procedures or solutions where ascorbic acid is added as antioxidant to preserve As species, can be satisfactorily tested using Microtox®. The results also indicate that AsV or AsIII acute toxicity, as the Microtox® results, would be not significantly affected by the presence of weak non-thiolic ligands in the aquatic media. Acknowledgments David A. Rubinos wishes to acknowledge the financial support of the Xunta de Galicia (Plan Galego de Investigaciónn, Innovación e Crecemento—I2C, Consellería de Educación e Ordenación Universitaria) and the European Social Fund.

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