Chemosphere 135 (2015) 335–340
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Specific anion effects in Artemia salina Pierandrea Lo Nostro a,b,⇑, Barry W. Ninham c, Emiliano Carretti a, Luigi Dei a, Piero Baglioni a a
Department of Chemistry ‘‘Ugo Schiff’’ and CSGI, University of Florence, 50019 Sesto Fiorentino, Firenze, Italy Enzo Ferroni Foundation, 50019 Sesto Fiorentino, Firenze, Italy c Department of Applied Mathematics, Research School of Physical Sciences and Engineering, Institute of Advanced Studies, Australian National University, Canberra 0200, Australia b
h i g h l i g h t s We studied the specific anion effect on the vitality of Artemia salina. The results are discussed in terms of the Hofmeister series. Strong kosmotropes and chaotropes have severe adverse effects on Artemia salina. The effect depends on the salt physico-chemical properties. The stability of oxygen bubbles and the ion adsorption partly explain the effects.
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 22 April 2015 Accepted 23 April 2015
Keywords: Artemia salina Brine shrimp Specific ion effects Hofmeister series
a b s t r a c t The specific anion effect on the vitality of Artemia salina was investigated by measuring the Lethal Time LT50 of the crustaceans in the presence of different sodium salts solutions at room temperature and at the same ionic strength as natural seawater. Fluoride, thiocyanate and perchlorate are the most toxic agents, while chloride, bromide and sulfate are well tolerated. The rates of oxygen consumption of brine shrimps were recorded in mixed NaCl + NaF or NaCl + NaSCN solutions as a function of time. The results are discussed in terms of the Hofmeister series, and suggest that, besides the biochemical processes that involve F, SCN and ClO 4 , the different physico-chemical properties of the strong kosmotropic and chaotropic anions may contribute in determining their strong toxicity for A. salina. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Artemia salina (brine shrimps) are branchiopod crustaceans that develop very well in hypersaline conditions, as in saltworks using solar evaporation for table salt and chemical industries, where they are essential for limiting algae bloom and favor the growth of halophilic bacteria (van Stappen, 1996). Adult Artemia have an elongated body, covered with a thin and flexible exoskeleton of chitin (Criel and Macrae, 2002). Brine shrimps possess a very efficient osmoregulatory system (Holliday et al., 1990), the capacity to synthesize efficient respiratory pigments to cope with the low O2 levels at high salinities, and the ability to produce dormant embryos (cysts) depending on the salt concentration in the environment. After 24-h incubation in seawater, the cysts release free-swimming larval nauplii.
⇑ Corresponding author at: Department of Chemistry ‘‘Ugo Schiff’’ and CSGI, University of Florence, 50019 Sesto Fiorentino, Firenze, Italy. E-mail address: [email protected]fi.it (P. Lo Nostro). URL: http://www.csgi.unifi.it/ (P. Lo Nostro). http://dx.doi.org/10.1016/j.chemosphere.2015.04.080 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
The best environment for Artemia are thalassohaline concentrated saltwaters where NaCl is the major component. However there are different species that are adapted to sulfate, carbonate, or potassium rich athalassohaline environments, spread almost all over the earth. For example, A. monica, a Californian subspecies of A. franciscana, has adapted very well to high concentrations of carbonate ions (van Stappen, 2002). In general, the composition of the aqueous medium in terms of nature and concentration of electrolytes is crucial for their survival and reproduction (Camargo et al., 2005). Trehalose serves in desiccation tolerance of cysts, acting as a stabilizer for the membrane structure (Criel and Macrae, 2002) through physical intercalation between the phospholipid headgroups. In Artemia, large amounts of the organic osmoregulating solutes (trehalose, glycerol and methylamines) may be involved in preventing macromolecular denaturation in hypersaline conditions or in the presence of toxic or foreign ions (Somero, 1986). Specific ion (Hofmeister) effects occur everywhere, in aqueous and non-aqueous solutions and at interfaces (Lo Nostro and Ninham, 2012). They reflect the different behavior of electrolytes
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in solution, and usually emerge when the salt concentration is larger than 0.1 M, but in some cases even in the micromolar range. The systematics of salt behavior over a range of different experimental conditions is typified by the so-called Hofmeister series. The observation, dating back to the 1870s, orders the behaviour of salts in a particular sequence, the so-called Hofmeister series, according to their effectiveness in precipitating the egg yolk albumin from a water dispersion (Kunz et al., 2004): For anions (with fixed cation) the sequence is (Lo Nostro and Ninham, 2012):
2 SO2 4 > HPO4 > F > CH3 COO > Cl > NO3 > Br > I > ClO4
> SCN : For cations (with fixed anion) it is: þ
NHþ4 > Kþ > Naþ > Li > Mg2þ > Ca2þ > CðNH2 Þþ3 : However there may be re-orderings along the series, depending on the hydrophobicity and surface charge of the colloid or substrate, on the solvent and on the concentration (Schwierz et al., 2010). In some cases a total inversion of the series is observed. The specificity of ions in biology and biochemistry is universal and crucial to life (Lo Nostro and Ninham, 2012). Life has adapted to conditions on Earth in a natural environment made up predominately of carbonates, sulfates, chlorides, phosphates and silicates for the anions, and sodium, potassium, magnesium, calcium and aluminum for the cations. What these ions have in common is an either strong or mild kosmotropic nature, i.e. a large q/r charge density, a strong hydration, a rather low ion polarizability, and a higher viscosity of their aqueous solutions compared to that of pure water at the same temperature. On the other hand, chaotropic salts contain large monovalent ions like cesium, iodide, thiocyanate or perchlorate. They possess a low free energy of hydration, a large polarizability and their solutions are less viscous than water at the same temperature (Ninham and Lo Nostro, 2010). The terminology and distinction between the words ‘‘kosmotropic’’ and ‘‘chaotropic’’ is accepted and useful but somewhat fuzzy, depending on the meaning of ‘‘water structure’’ (Ball and Hallsworth, 2015). This contribution addresses a class of Hofmeister phenomena as yet almost totally unexplored, the effect of salts on a living, multicelled organism. The effects reflect physical chemistry that takes place within the organism and not biochemistry alone. Hofmeister phenomena in bulk solutions and at interfaces are the subject of thousands of papers. But specific effects of different salts on living species are almost unexplored (Boas, 1926; Gellhorn, 1933). This is because living organisms are more difficult to handle – sometimes it is hard to define a standard system that can be representative of an entire population. Further, it is not easy to pin down how and where a specific salt affects the complex dynamic mechanisms that control life. Salts in general and trace amounts of certain ions determine the structure and functionality of the hierarchically lower structures that support life. For example, they participate in the osmotic regulation of cells and in the main living processes. Any living organism will suffer a serious stress when the concentrations of specific salt are varied or one is replaced with another. An example is the acidic rain-induced mobilization of Al3+ and Mn2+, which are toxic to plants and animals, in water basins (van Dama et al., 2008). Moreover, traces of other elements, like Mn, Ni, V, Cr, Zn, and Se, are necessary in plant and animal health for structure and function of enzymes (Hinojosa et al., 2010; Lindh, 2007). These have whole-animal effects, as is clear from the classic example of iodine deficiency (Dasgupta et al., 2008). Halophilic bacteria and other salt-loving species evolved in peculiar and
extreme environments where the concentration of salts is particularly high and prohibitive for most living species. The osmoregulating activity that these special forms of life have developed is indeed another case of Hofmeister specific ion effects, because all salts behave differently. In a previous paper we reported on the effect of different salts on the growth rate of Staphylococcus aureus and Pseudomonas aeruginosa (Lo Nostro et al., 2005). The present work is a further step up in an evolving systematics of phenomena that display Hofmeister effects. They range from activity coefficients and osmotic pressures of salt solutions (Parsons and Ninham, 2010), optical activity of amino acids (Rossi et al., 2007), absorption of water in natural fibres (Lo Nostro et al., 2002) and pH and buffers (Salis et al., 2006), to protein interactions and conformation (Dér et al., 2007), to enzyme activities (Kim et al., 2001; Voinescu et al., 2006; Salis et al., 2007) to single celled (bacteria) and now to multicelled organisms. The toxicity of some sodium and potassium salts, as well as of some alkali and alkaline-earth chlorides, on the membrane formation and on the nauplii of A. salina was investigated long time ago (Boone and Baas-Becking, 1931). In this contribution we discuss the effect of different anions at the same ionic strength of natural seawater on the vitality of brine shrimps (A. salina) at room temperature. The results show that the effect of the anion is (sodium salts):
2 Cl ; Br > NO3 > SO2 4 > CO3 > I > ClO4 > F ; SCN
This means, e.g., that in the presence of fluoride or thiocyanate the vitality of the brine shrimps was seriously reduced after a short period of time, while the individuals survived up to 25 h in the presence of pure chloride or bromide. Moreover, since the oxygen uptake is sensitive to temperature and salinity (Irwin et al., 2007), we measured the weight-specific rates of oxygen consumption of brine shrimps in mixed NaCl + NaF or NaCl + NaSCN solutions as a function of time. According to our results A. salina prefers salty waters that contain weakly kosmotropic/chaotropic species (such as chloride and bromide). These sit in the middle of the Hofmeister series. On the other hand the stronger kosmotropes (fluoride) and especially stronger chaotropes (thiocyanate, perchlorate and iodide) are severely toxic. Further studies on the effect of different cations and on the oxygen consumption of brine shrimps in the presence of electrolytes are currently in progress. 2. Materials and methods The cysts (from Premium SHG, Alessandria, Italy) were hydrated with artificial regenerated seawater (from Biotech Salt Oceanlife, Bologna, Italy). The breeding was fed with Hobby Liquizell (Dohse Aquaristik KG, Gelsdorf, Germany), thermostatted at 23 °C for 17 days, then at 27 °C for 11 days to promote the growth. Finally the tank was kept at 25 °C under continuous aeration. Sodium sulfate, carbonate, fluoride, chloride, bromide, iodide, thiocyanate, nitrate and perchlorate were purchased from Fluka (Milan, Italy) and used as received. 2.1. Vitality tests The individuals were kept fasting for 3 days before the vitality tests. 15 individual nauplii were transferred into the salt solution, and their vitality was assessed by visual inspection upon the addition of the salt, at the same ionic strength as seawater (507.2 mmol kg1 for the monovalent salts and 169.2 mmol kg1
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for the divalent salts). The salt solutions were prepared using Millipore water. The vitality tests were performed by evaluating the number of alive nauplii every 30 min for the first 10 h, then every hour for one day, and finally every 24 h for three days. In order to avoid the dilution of the receiving salt solution with the regenerate seawater where the animals were bred, the A. salina individuals were rapidly rinsed with Millipore water and then with the same receiving salt solution. 2.2. Oxygen solubility and respirometry An Ox-MR Unisense electrode was used for measuring the solubility of oxygen in the salt solutions at (25 ± 1) °C. The samples were magnetically stirred and the content of O2 was measured after 20 min of aeration of the solution and 10 min at rest. Each value is the average over three readings. The respirometry tests were carried out at (25 ± 1) °C with the same electrode and a special cage-holder where 5 individuals were located. The cage was transferred into the salt solutions and the oxygen consumption was recorded every hour. Each value is the average over 5 replicates. _ O ) were deterWeight-specific rates of oxygen consumption (M 2
mined based on the dry weight of the animals (Varó et al., 1998). The dry weight of the animals ranged from 0.1 to 0.7 mg. For the respirometry tests the salt solutions consisted in mixed NaCl/NaF or NaCl/NaSCN solutions with a mole fraction of NaF or NaSCN ranging between 0.05 and 0.20. 3. Results and discussion After the development of cysts and an appropriate lapse for their growth, the individuals of A. salina were transferred in the salt solutions at the same ionic strength of seawater, in order to investigate the specific effect of the anion, at the same temperature and growth time. 3.1. Vitality tests The vitality tests were performed in order to assess the macroscopic effect of each anion on the survival of the individuals. The vitality of the animals was assessed by inspecting their legs. We considered as dead an individual that did not swim and whose legs did not move. Fig. 1 shows the results of the vitality tests in the different salt solutions. The number of alive individuals (N) is plotted as a function of time (in log scale). The plot shows that in the case of NaF and NaSCN the animals slowly die. After 90 min in NaF or in NaSCN, 13 over 15 individuals
were in a deadly state, barely moving their legs. The remaining individuals were either swimming normally (in NaF) or slowly (in NaSCN). A similar situation occurred in the presence of NaClO4 but at higher contact times (less than 4 hours). In order to quantify the effect of the different salts we evaluated the Lethal Time 50 (LT50) as the time necessary for assessing the death of 50% of the individuals. Table 1 shows the value of LT50 for the different salt solutions and for the blank sample. NaF, NaClO4 and NaSCN are the most toxic salts for A. salina, while in NaCl and NaBr they survived better than in the blank solution. Actually A. salina performs normal ecdysis in NaCl and NaBr solutions (Boone and Baas-Becking, 1931). Our results are in line with those of Chrogan who showed that Artemia adults survive very well in NaCl and NaBr (0.5 M), while they are moribund or very slow after 24 h in Na2SO4 (0.25 M) and NaNO3 (1 M), and LiCl or NaHCO3, NaN3 and potassium salts are more toxic (Chrogan, 1958). The toxicity of fluoride has already been demonstrated in previous studies and is presumably related to the inhibition of different enzymes (including phosphatases, hexokinase, enolase, succinic dehydrogenase, pyruvic oxidase) and to the interruption of metabolic processes such as glycolysis and synthesis of proteins (Pankhurst et al., 1980; Camargo, 2003). On the other hand, the toxicity of thiocyanate in fish and crustaceans seems to be related to the substitution of chloride ions in the gill and to the perturbation of the ionic balance, resulting in respiratory problems (Epstein et al., 1973; Bhunia et al., 2000). Perchlorate is known to be toxic for fish (Crane et al., 2005; Park et al., 2006) as a hormone disruptor. In addition to these biochemical effects, we propose that other, physico-chemical mechanisms, can participate in determining the toxicity of these ions for A. salina. These have to do with (i) the interfacial adsorption of the anions at the chitin/water interface, and (ii) with their influence on the stabilization of air bubbles in solution. (i) Strong kosmotropes such as fluoride can significantly weaken the hydrogen bonds that involve water and amide bonds in chitin. On the other hand, strong chaotropes such as perchlorate, thiocyanate and iodide are able to deplete water from the hydrophobic hydration layer around chitin (Cho et al., 2008; Zhang and Cremer, 2010). This peculiar physico-chemical mechanism may operate synergistically with the chemical binding of these anions at the chitin/water interface that result in a potent toxic effect for Artemia. We recall that several anions adsorb on chitin and its derivatives with a selectivity trend that follows the Hofmeister sequence (Davila-Rodriguez et al., 2012):
SO2 4 > HCO3 > F > Cl > NO3 :
Table 1 LT50 (in min) and surface tension molar increment at the air/water interface (r, in lN m2 mol1) for the investigated sodium salt solutions. The concentration is 507.2 and 169.2 mmol kg1 for the 1:1 and 2:1 salts, respectively.
Fig. 1. Vitality tests. The number of alive individuals N is plotted versus time (in min) in the presence of different sodium salt solutions: NaF ( ), NaSCN ( ), NaClO4 ( ), NaI (d), Na2CO3 ( ), NaNO3 ( ), Na2SO4 ( ), NaBr ( ), and NaCl ( ).
Salt
LT50 (min)
r (lN m2 mol1)
NaF NaSCN NaClO4 Na2CO3 NaI NaNO3 Na2SO4 Blank NaBr NaCl
73 76 224 364 460 686 1120 1403 1463 1637
2.00 0.54 0.62 2.60 1.02 1.10 1.60 – 1.31 1.76
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capacity of the crustaceans. This hypothesis may explain the trend found in Fig. 2. In fact chaotropic ions such as SCN, ClO 4 and I , that have no effect on the coalescence of gas bubbles in solution, dramatically decrease the vitality of A. salina. 3.2. Respirometry We prepared mixed solutions of NaCl + NaF and of NaCl + NaSCN at different mole fractions of the two solutes and measured the oxygen consumption in order to detect the threshold value of toxicity for NaF and NaSCN (see Fig. 3a and b). In both cases we found an initial decrement (in absolute value) _ O , followed by a temporary increment, before death (zero in M 2
Fig. 2. LT50 versus the surface tension molar increment r for the different anions.
Since no data for the chitin/water interface tension in the presence of different electrolytes is reported in the literature, in Fig. 2 we plotted the variation of LT50 as a function of the surface tension molar increment r. This parameter is defined as r ¼ @ðDcÞ=@c, where c is the air/solution interface and c is the molar concentration of the electrolyte in solution. The values of r for the different salts are shown in Table 1. r reflects the adsorption/desorption of ions at the air/solution interface: kosmotropes such as fluoride possess a large value of r, while chaotropes like iodide show a rather low r (Ninham and Lo Nostro, 2010; Kunz et al., 2004; Lo Nostro and Ninham, 2012). (ii) The bubble–bubble coalescence phenomenon is a typical specific ion effect: some electrolytes stabilize the formation of air microbubbles in an aqueous solution and prevent their coalescence, while others have no effect with the formation of short-lived macrobubbles (Ninham and Yaminsky, 1997; Craig et al., 1993). For example, NaCl, NaBr and Na2SO4 prevent bubbles coalescence, while NaClO4 and NaSCN do not (Henry et al., 2007). This is why clean, non polluted seawater is foamy, while in fresh water air bubbles are not stable and collapse very quickly. We recall that the thorax of A. salina bears eleven pairs of phyllopods or swimming legs that are used for locomotion, osmoregulation, respiration and nutrition (Criel and Macrae, 2002). The osmoregulation mechanism developed by Artemia basically consists in drinking the external medium and transfer of the ionic solutes and fluid into the haemolymph. The outward ion transport is mainly carried out by specialized cells, the metepipodites that are located on the phyllopodia (Clegg and Trotman, 2002; Holliday et al., 1990). We argue that a change in the availability and size of oxygen microbubbles may modify and eventually hamper the breathing
value). The comparison of the two plots shows that thiocyanate is more or less as toxic as fluoride. The trends seem to suggest that initially all individuals suffer for the presence of the toxic anion (either fluoride or thiocyanate), depending on its concentration (each line in Fig. 3 refers to a different mole fraction of NaF or NaSCN compared to the background NaCl salt, as indicated in the figure legend). In fact the oxygen consumption decreases and the decrement is faster at higher concentrations of the toxic component. After about 5–7 h of regular decrease at minimal concentration of NaF or NaSCN (black lines) the individuals apparently start breathing more oxygen up to about 22 h, after which they quickly die. For higher concentration of NaF or NaSCN the respirometric curves are compressed and death is reached within the first 5– 10 h, depending on the toxic salt concentration. _ O (recovering step) Presumably the significant increment in M 2
before death is related to the intense production of osmoregulating molecules, such as trehalose and glucose, that the animals synthesize when experiencing a stressing condition (Hand et al., 2011; Glasheen and Hand, 1989). 3.3. Oxygen solubility In order to verify whether the different vitality of A. salina is related to the different oxygen solubility in the electrolyte solutions, we measured the concentration of saturated oxygen in the different solutions at 25 °C (see Table 2), and compared the data to those found in the literature (Millero et al., 2002). We recall that the oxygen solubility at 25 °C and 1 bar in fresh water and seawater (salinity 35‰) is 258 and 206 lM, respectively. The solubility of oxygen in an electrolyte solution depends on temperature, pressure, concentration and nature of the solute. At a given temperature, the solubility of oxygen a salt solution, [O2]s, depends on the salt concentration (m, in molal units) according to Eq. (1):
ln½O2 s ¼ ln½O2 w kS m
ð1Þ
_ O , in lmol g1 h1) as a function of time (in hours) for NaF + NaCl (left) and for NaSCN + NaCl (right) mixed solutions at different mole Fig. 3. Oxygen consumption (M 2 fractions: x = 0.05 (d), 0.10 ( ), 0.15 ( ), and 0.20 ( ).
P. Lo Nostro et al. / Chemosphere 135 (2015) 335–340 Table 2 Anion partial molal volume (ms, in cm3 mol1) for the investigated salts at 25 °C, and solubility of oxigen (in lmol kg1) at 25 °C for the investigated salts in water. The concentration is 507.2 and 169.2 mmol kg1 for the 1:1 and 2:1 salts, respectively. Salt
NaF NaCl NaBr NaI NaSCN NaNO3 NaClO4 Na2CO3 Na2SO4 a b
O2 solubility (lmol kg1) This work
From Millero et al. (2002)
218.0 ± 0.1 221.4 ± 0.1 225.0 ± 0.1 227.8 ± 0.1 229.8 ± 0.1 224.4 ± 0.1 231.1 ± 0.1 232.1 ± 0.1 219.8 ± 0.1
216 ± 7 220 ± 1 223 ± 1 230 ± 2 n/a 229 ± 1 n/a 271 ± 4 223 ± 2a
ms (cm3 mol1)b 1.2 17.8 24.7 36.2 35.7 29.0 44.1 – 14.0
From Millero et al. (2003). From Millero (1971).
Fig. 4. Oxygen solubility (in lmol kg1) as a function of the partial molal volume of the anion (ms, in cm3 mol1) at 25 °C and at atmospheric pressure. : data measured in this work and : taken from the literature (Millero et al., 2002, 2003). The line is a guide for the eye.
Here [O2]w is the solubility of oxygen in water at the same temperature and kS is the salting-out Setchenow constant, respectively (Millero et al., 2003). The scaled particle theory model describes the value of kS for 1:1 electrolytes as determined by the Gibbs free energy that is necessary to create the cavity for the dissolution of the oxygen microbubble in the solution, and the Gibbs free energy for the oxygen–water and oxygen–salt interactions (Millero et al., 2003). This explains why kS depends on the composition of the electrolyte. Fig. 4 illustrates the change in the oxygen solubility with the partial molal volume of the anion (ms). The values of ms were taken from the literature (Millero, 1971). At the same temperature and pressure, oxygen is more soluble in aqueous salt solutions where the anion is a chaotrope, with a larger partial molal volume, because it is easier to create the cavity to accommodate the dissolved gas. On the other hand, oxygen is less soluble in aqueous solutions of strong kosmotropes such as fluoride and sulfate. Comparing the Lethal Time LT50 to the oxygen solubility it is clear that there is no direct correlation between the salt-dependent solubility of oxygen and the effect of the anion on the vitality of A. salina, in fact low values of LT50 are found for salts that enhance the solubility of O2 (such as sodium perchlorate, thiocyanate and iodide), as well as for sodium fluoride that lowers to amount of dissolved oxygen. 4. Conclusions The effect of nine different sodium salts on the vitality and on the weight-specific rates of oxygen consumption of A. salina was
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investigated at room temperature. The evaluation of the Lethal Time LT50 shows that the crustaceans survive very well in sodium chloride and bromide, while the presence of foreign salt species such as sodium fluoride, thiocyanate, perchlorate, iodide and nitrate severely adverse their vitality and eventually lead to death. Besides the specific toxicity that has been reported in the literature for some anions such as fluoride and thiocyanate, which is mainly related to their biochemical activities, we propose that the adverse effect is also related to their ionic physico-chemical features. Fluoride and carbonate are strong kosmotropes, strongly hydrated and with large surface tension molar increment, that impair the strength of the exoskeleton’s chitin. On the other hand, iodide, thiocyanate and perchlorate are chaotropes, with weaker hydration and lower surface tension molar increments, that promote the anion adsorption at the chitin/water interface. The solubility of atmospheric oxygen in the presence of different electrolytes is not correlated with their effect on the vitality of the crustaceans. Instead, our results apparently suggest that the most toxic anions for A. salina are those that have no effect on bubble–bubble coalescence such as ClO (Henry 4 and SCN et al., 2007). In conclusion, A. salina has naturally adapted to even strong concentrations of chloride and bromide ions, that occupy the intermediate mild region of the Hofmeister sequence. The presence of strong kosmotropes (F or CO2 3 ) and especially of strong chao tropes (SCN, ClO 4 , I ) severely impair the vitality of the crustaceans, and suggest that the physico-chemical properties of these ions determine their toxic effect, besides the specific biochemical activities they perform in the respiratory or metabolic routes. Acknowledgements The Authors acknowledge the Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI, Florence), and the Enzo Ferroni Foundation (Florence) for partial financial support. References Bhunia, F., Saha, N.C., Kaviraj, A., 2000. Toxicity of thiocyanate to fish, plankton, worm, and aquatic ecosystem. Bull. Environ. Contam. Toxicol. 64, 197–204. Boas, F., 1926. Beiträge zur hylergographie. Uber die Wirkung von Salzen, namentlich Neutralsalzen, auf die Zelle (Hylergographic studies. The influence of neutral salts on the cell). Biochem. Z 176, 349–402. Boone, E., Baas-Becking, L.G.M., 1931. Salt effects on eggs and nauplii of Artemia Salina L. J. Gen. Physiol. 14 (6), 753–763. Camargo, J.A., 2003. Fluoride toxicity to aquatic organisms: a review. Chemosphere 50, 251–264. Camargo, W.N., Durán, G.C., Rada, O.C., Hernández, L.C., Linero, J.-C.G., Muelle, I.M., Sorgeloos, P., 2005. Determination of biological and physicochemical parameters of Artemia franciscana strains in hypersaline environments for aquaculture in the Colombian Caribbean. Saline Syst. 1, 9–19. Cho, Y., Zhang, Y., Christensen, T., Sagle, L.B., Chikoti, A., Cremer, P.S., 2008. Effects of hofmeister anions on the phase transition temperature of elastin-like polypeptides. J. Phys. Chem. B 112, 13765–13771. Chrogan, P.C., 1958. The survival of Artemia salina (L.) in various media. J. Exp. Biol. 35, 213–218. Clegg, J.S., Trotman, C.N.A., 2002. Physiological and biochemical aspects of artemia ecology. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 3. Craig, V.S.J., Ninham, B.W., Pashley, R.M., 1993. The effect of electrolytes on bubble coalescence in water. J. Phys. Chem. 97, 10192–10197. Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2005. Effects of ammonium perchlorate on thyroid function in developing fathead minnows, Pimephales promelas. Environ. Health Perspect. 113, 396–401. Criel, G.R.J., Macrae, T.H., 2002. Artemia morphology and structure. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 1. Dasgupta, P.K., Liu, Y., Dyke, J.V., 2008. Iodine nutrition: iodine content of iodized salt in the United States. Environ. Sci. Technol. 42, 1315–1323.
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Davila-Rodriguez, J.K., Escobar-Barrios, V.A., Rangel-Mendez, J.R., 2012. Removal of fluoride from drinking water by a chitin-based biocomposite in fixed-bed columns. J. Fluorine Chem. 140, 99–103. Dér, A., Kelemen, L., Fábián, L., Taneva, S.G., Fodor, E., Páli, T., Cupane, A., Cacace, M.G., Ramsden, J.J., 2007. Interfacial water structure controls protein conformation. J. Phys. Chem. B 111, 5344–5350. Epstein, F.H., Maetz, J., Renzis, G., 1973. Active transport of chloride by the teleost gill: inhibition by thiocyanate. Am. J. Physiol. 224, 1295–1299. Gellhorn, E., 1933. Experiments on the hofmeister series in heart muscle. Protoplasma 18, 411–419. Glasheen, J.S., Hand, S.C., 1989. Metabolic heat dissipation and internal solute levels of artemia embryos during changes in cell-associated water. J. Exp. Biol. 145, 263–282. Hand, S.C., Menze, M.A., Toner, M., Boswell, L., Moore, D., 2011. LEA proteins during water stress: not just for plants anymore. Annu. Rev. Physiol. 73, 115–134. Henry, C.L., Dalton, C.N., Scruton, L., Craig, V.S.J., 2007. Ion-specific coalescence of bubbles in mixed electrolyte solutions. J. Phys. Chem. C 111, 1015–1023. Hinojosa, M.B., Carreira, J.A., García-Ruíz, R., Rodríguez-Maroto, J.M., Daniell, T.J., Griffiths, B.S., 2010. Plant treatment, pollutant load, and soil type effects in rhizosphere ecology of trace element polluted soils. Ecotoxicol. Environ. Safety 73, 970–981. Holliday, C.W., Roye, D.B., Roer, R.D., 1990. Salinity-induced changes in branchial Na+/K+ ATPase activity and transepithelial potential difference in the brine shrimp Artemia Salina. J. Exp. Biol. 151, 279–296. Irwin, S., Wall, V., Davenport, J., 2007. Measurement of temperature and salinity effects on oxygen consumption of Artemia franciscana K., measured using fibreoptic oxygen microsensors. Hydrobiologia 575, 109–115. Kim, H.-K., Tuite, E., Nordén, B., Ninham, B.W., 2001. Co-ion dependence of DNA nuclease activity suggests hydrophobic cavitation as a potential source of activation energy. Eur. Phys. J. E 4, 411–417. Kunz, W., Lo Nostro, P., Ninham, B.W., 2004. The present state of affairs with Hofmeister effects. Curr. Op. Coll. Interface Sci. 9, 1–18. Lindh, U., 2007. Metal biology: aspects of beneficial effects. Ambio 36, 107–110. Lo Nostro, P., Ninham, B.W., 2012. Hofmeister phenomena: an update on ion specificity in biology. Chem. Rev. 112, 2286–2322. Lo Nostro, P., Fratoni, L., Ninham, B.W., Baglioni, P., 2002. Water absorbency by wool fibers: hofmeister effect. Biomacromolecules 3, 1217–1224. Lo Nostro, P., Ninham, B.W., Lo Nostro, A., Pesavento, G., Fratoni, L., Baglioni, P., 2005. Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa. Phys. Biol. 2, 1–7. Millero, F.J., 1971. The molal volumes of electrolytes. Chem. Rev. 71, 147–176. Millero, F.J., Huang, F., Laferiere, A.L., 2002. The solubility of oxygen in the major sea salts and their mixtures at 25 °C. Geochim. Cosmochim. Acta 66, 2349–2359. Millero, F.J., Huang, F., Graham, T.B., 2003. Solubility of oxygen in some 1–1, 2–1, 1– 2, and 2–2 electrolytes as a function of concentration at 25 °C. J. Solut. Chem. 32, 473–487.
Ninham, B.W., Lo Nostro, P., 2010. Molecular Forces and Self Assembly. In Colloid, Nano Sciences and Biology. Cambridge University Press, Cambridge. Ninham, B.W., Yaminsky, V.V., 1997. Ion binding and ion specificity: the hofmeister effect and onsager and lifshitz theories. Langmuir 13, 2097–2108. Ball, P., Hallsworth, J.E., 2015. Water structure and chaotropicity: their uses, abuses and biological implications. Phys Chem Chem Phys. http://dx.doi.org/10.1039/ c4cp04564e [Advance Article]. Pankhurst, N.V., Boyden, C.R., Wilson, J.B., 1980. The effect of a fluoride effluent on marine organisms. Environ. Pollut. 23, 299–312. Park, J.-W., Rinchard, J., Liu, F., Anderson, T.A., Kendall, R.J., Theodorakis, C.W., 2006. The thyroid endocrine disruptor perchlorate affects reproduction, growth, and survival of mosquitofish. Ecotoxicol. Environ. Safety 63, 343–352. Parsons, D.F., Ninham, B.W., 2010. Importance of accurate dynamic polarizabilities for the ionic dispersion interactions of alkali halides. Langmuir 26, 1816– 1823. Rossi, S., Lo Nostro, P., Lagi, M., Ninham, B.W., Baglioni, P., 2007. Specific anion effects on the optical rotation of a-amino acids. J. Phys. Chem. B 111, 10510– 10519. Salis, A., Pinna, M.C., Bilanicˇová, D., Monduzzi, M., Lo Nostro, P., Ninham, B.W., 2006. Specific anion effects on glass electrode pH measurements of buffer solutions: bulk and surface phenomena. J. Phys. Chem. B 110, 2949–2956. Salis, A., Bilanicˇová, D., Ninham, B.W., Monduzzi, M., 2007. Hofmeister effects in enzymatic activity: weak and strong electrolyte influences on the activity of candida rugosa lipase. J. Phys. Chem. B 111, 1149–1156. Schwierz, N., Horinek, D., Netz, R.R., 2010. Reversed anionic hofmeister series: the interplay of surface charge and surface polarity. Langmuir 26, 7370–7379. Somero, G.N., 1986. Protons, osmolytes, and fitness of internal milieu for protein function. Am. J. Physiol. 251, R197–R213. van Dama, R., Hogan, A., Harford, A., Markich, S., 2008. Toxicity and metal speciation characterisation of waste water from an abandoned gold mine in tropical northern Australia. Chemosphere 73, 305–313. van Stappen, G., 1996. Introduction, biology and ecology of Artemia. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture, Rome, FAO Fisheries Technical Paper. van Stappen, G., 2002. Zoogeography. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 4. Varó, I., Taylor, A.C., Amat, F., 1998. The effects of temperature and oxygen tension (P O2 ) on the oxygen consumption rates of adults of different Artemia strains. Comp. Biochem. Physiol. A: Physiol. 120, 385–390. Voinescu, A., Bauduin, P., Pinna, C., Touraud, D., Kunz, W., Ninham, B.W., 2006. Similarity of salt influences on the pH of buffers, polyelectrolytes, and proteins. J. Phys. Chem. B 110, 8870–8876. Zhang, Y., Cremer, P.S., 2010. Chemistry of hofmeister anions and osmolytes. Annu. Rev. Phys. Chem. 61, 63–83.
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Specific anion effects in Artemia salina Pierandrea Lo Nostro a,b,⇑, Barry W. Ninham c, Emiliano Carretti a, Luigi Dei a, Piero Baglioni a a
Department of Chemistry ‘‘Ugo Schiff’’ and CSGI, University of Florence, 50019 Sesto Fiorentino, Firenze, Italy Enzo Ferroni Foundation, 50019 Sesto Fiorentino, Firenze, Italy c Department of Applied Mathematics, Research School of Physical Sciences and Engineering, Institute of Advanced Studies, Australian National University, Canberra 0200, Australia b
h i g h l i g h t s We studied the specific anion effect on the vitality of Artemia salina. The results are discussed in terms of the Hofmeister series. Strong kosmotropes and chaotropes have severe adverse effects on Artemia salina. The effect depends on the salt physico-chemical properties. The stability of oxygen bubbles and the ion adsorption partly explain the effects.
a r t i c l e
i n f o
Article history: Received 6 March 2015 Received in revised form 22 April 2015 Accepted 23 April 2015
Keywords: Artemia salina Brine shrimp Specific ion effects Hofmeister series
a b s t r a c t The specific anion effect on the vitality of Artemia salina was investigated by measuring the Lethal Time LT50 of the crustaceans in the presence of different sodium salts solutions at room temperature and at the same ionic strength as natural seawater. Fluoride, thiocyanate and perchlorate are the most toxic agents, while chloride, bromide and sulfate are well tolerated. The rates of oxygen consumption of brine shrimps were recorded in mixed NaCl + NaF or NaCl + NaSCN solutions as a function of time. The results are discussed in terms of the Hofmeister series, and suggest that, besides the biochemical processes that involve F, SCN and ClO 4 , the different physico-chemical properties of the strong kosmotropic and chaotropic anions may contribute in determining their strong toxicity for A. salina. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Artemia salina (brine shrimps) are branchiopod crustaceans that develop very well in hypersaline conditions, as in saltworks using solar evaporation for table salt and chemical industries, where they are essential for limiting algae bloom and favor the growth of halophilic bacteria (van Stappen, 1996). Adult Artemia have an elongated body, covered with a thin and flexible exoskeleton of chitin (Criel and Macrae, 2002). Brine shrimps possess a very efficient osmoregulatory system (Holliday et al., 1990), the capacity to synthesize efficient respiratory pigments to cope with the low O2 levels at high salinities, and the ability to produce dormant embryos (cysts) depending on the salt concentration in the environment. After 24-h incubation in seawater, the cysts release free-swimming larval nauplii.
⇑ Corresponding author at: Department of Chemistry ‘‘Ugo Schiff’’ and CSGI, University of Florence, 50019 Sesto Fiorentino, Firenze, Italy. E-mail address: [email protected]fi.it (P. Lo Nostro). URL: http://www.csgi.unifi.it/ (P. Lo Nostro). http://dx.doi.org/10.1016/j.chemosphere.2015.04.080 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
The best environment for Artemia are thalassohaline concentrated saltwaters where NaCl is the major component. However there are different species that are adapted to sulfate, carbonate, or potassium rich athalassohaline environments, spread almost all over the earth. For example, A. monica, a Californian subspecies of A. franciscana, has adapted very well to high concentrations of carbonate ions (van Stappen, 2002). In general, the composition of the aqueous medium in terms of nature and concentration of electrolytes is crucial for their survival and reproduction (Camargo et al., 2005). Trehalose serves in desiccation tolerance of cysts, acting as a stabilizer for the membrane structure (Criel and Macrae, 2002) through physical intercalation between the phospholipid headgroups. In Artemia, large amounts of the organic osmoregulating solutes (trehalose, glycerol and methylamines) may be involved in preventing macromolecular denaturation in hypersaline conditions or in the presence of toxic or foreign ions (Somero, 1986). Specific ion (Hofmeister) effects occur everywhere, in aqueous and non-aqueous solutions and at interfaces (Lo Nostro and Ninham, 2012). They reflect the different behavior of electrolytes
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in solution, and usually emerge when the salt concentration is larger than 0.1 M, but in some cases even in the micromolar range. The systematics of salt behavior over a range of different experimental conditions is typified by the so-called Hofmeister series. The observation, dating back to the 1870s, orders the behaviour of salts in a particular sequence, the so-called Hofmeister series, according to their effectiveness in precipitating the egg yolk albumin from a water dispersion (Kunz et al., 2004): For anions (with fixed cation) the sequence is (Lo Nostro and Ninham, 2012):
2 SO2 4 > HPO4 > F > CH3 COO > Cl > NO3 > Br > I > ClO4
> SCN : For cations (with fixed anion) it is: þ
NHþ4 > Kþ > Naþ > Li > Mg2þ > Ca2þ > CðNH2 Þþ3 : However there may be re-orderings along the series, depending on the hydrophobicity and surface charge of the colloid or substrate, on the solvent and on the concentration (Schwierz et al., 2010). In some cases a total inversion of the series is observed. The specificity of ions in biology and biochemistry is universal and crucial to life (Lo Nostro and Ninham, 2012). Life has adapted to conditions on Earth in a natural environment made up predominately of carbonates, sulfates, chlorides, phosphates and silicates for the anions, and sodium, potassium, magnesium, calcium and aluminum for the cations. What these ions have in common is an either strong or mild kosmotropic nature, i.e. a large q/r charge density, a strong hydration, a rather low ion polarizability, and a higher viscosity of their aqueous solutions compared to that of pure water at the same temperature. On the other hand, chaotropic salts contain large monovalent ions like cesium, iodide, thiocyanate or perchlorate. They possess a low free energy of hydration, a large polarizability and their solutions are less viscous than water at the same temperature (Ninham and Lo Nostro, 2010). The terminology and distinction between the words ‘‘kosmotropic’’ and ‘‘chaotropic’’ is accepted and useful but somewhat fuzzy, depending on the meaning of ‘‘water structure’’ (Ball and Hallsworth, 2015). This contribution addresses a class of Hofmeister phenomena as yet almost totally unexplored, the effect of salts on a living, multicelled organism. The effects reflect physical chemistry that takes place within the organism and not biochemistry alone. Hofmeister phenomena in bulk solutions and at interfaces are the subject of thousands of papers. But specific effects of different salts on living species are almost unexplored (Boas, 1926; Gellhorn, 1933). This is because living organisms are more difficult to handle – sometimes it is hard to define a standard system that can be representative of an entire population. Further, it is not easy to pin down how and where a specific salt affects the complex dynamic mechanisms that control life. Salts in general and trace amounts of certain ions determine the structure and functionality of the hierarchically lower structures that support life. For example, they participate in the osmotic regulation of cells and in the main living processes. Any living organism will suffer a serious stress when the concentrations of specific salt are varied or one is replaced with another. An example is the acidic rain-induced mobilization of Al3+ and Mn2+, which are toxic to plants and animals, in water basins (van Dama et al., 2008). Moreover, traces of other elements, like Mn, Ni, V, Cr, Zn, and Se, are necessary in plant and animal health for structure and function of enzymes (Hinojosa et al., 2010; Lindh, 2007). These have whole-animal effects, as is clear from the classic example of iodine deficiency (Dasgupta et al., 2008). Halophilic bacteria and other salt-loving species evolved in peculiar and
extreme environments where the concentration of salts is particularly high and prohibitive for most living species. The osmoregulating activity that these special forms of life have developed is indeed another case of Hofmeister specific ion effects, because all salts behave differently. In a previous paper we reported on the effect of different salts on the growth rate of Staphylococcus aureus and Pseudomonas aeruginosa (Lo Nostro et al., 2005). The present work is a further step up in an evolving systematics of phenomena that display Hofmeister effects. They range from activity coefficients and osmotic pressures of salt solutions (Parsons and Ninham, 2010), optical activity of amino acids (Rossi et al., 2007), absorption of water in natural fibres (Lo Nostro et al., 2002) and pH and buffers (Salis et al., 2006), to protein interactions and conformation (Dér et al., 2007), to enzyme activities (Kim et al., 2001; Voinescu et al., 2006; Salis et al., 2007) to single celled (bacteria) and now to multicelled organisms. The toxicity of some sodium and potassium salts, as well as of some alkali and alkaline-earth chlorides, on the membrane formation and on the nauplii of A. salina was investigated long time ago (Boone and Baas-Becking, 1931). In this contribution we discuss the effect of different anions at the same ionic strength of natural seawater on the vitality of brine shrimps (A. salina) at room temperature. The results show that the effect of the anion is (sodium salts):
2 Cl ; Br > NO3 > SO2 4 > CO3 > I > ClO4 > F ; SCN
This means, e.g., that in the presence of fluoride or thiocyanate the vitality of the brine shrimps was seriously reduced after a short period of time, while the individuals survived up to 25 h in the presence of pure chloride or bromide. Moreover, since the oxygen uptake is sensitive to temperature and salinity (Irwin et al., 2007), we measured the weight-specific rates of oxygen consumption of brine shrimps in mixed NaCl + NaF or NaCl + NaSCN solutions as a function of time. According to our results A. salina prefers salty waters that contain weakly kosmotropic/chaotropic species (such as chloride and bromide). These sit in the middle of the Hofmeister series. On the other hand the stronger kosmotropes (fluoride) and especially stronger chaotropes (thiocyanate, perchlorate and iodide) are severely toxic. Further studies on the effect of different cations and on the oxygen consumption of brine shrimps in the presence of electrolytes are currently in progress. 2. Materials and methods The cysts (from Premium SHG, Alessandria, Italy) were hydrated with artificial regenerated seawater (from Biotech Salt Oceanlife, Bologna, Italy). The breeding was fed with Hobby Liquizell (Dohse Aquaristik KG, Gelsdorf, Germany), thermostatted at 23 °C for 17 days, then at 27 °C for 11 days to promote the growth. Finally the tank was kept at 25 °C under continuous aeration. Sodium sulfate, carbonate, fluoride, chloride, bromide, iodide, thiocyanate, nitrate and perchlorate were purchased from Fluka (Milan, Italy) and used as received. 2.1. Vitality tests The individuals were kept fasting for 3 days before the vitality tests. 15 individual nauplii were transferred into the salt solution, and their vitality was assessed by visual inspection upon the addition of the salt, at the same ionic strength as seawater (507.2 mmol kg1 for the monovalent salts and 169.2 mmol kg1
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for the divalent salts). The salt solutions were prepared using Millipore water. The vitality tests were performed by evaluating the number of alive nauplii every 30 min for the first 10 h, then every hour for one day, and finally every 24 h for three days. In order to avoid the dilution of the receiving salt solution with the regenerate seawater where the animals were bred, the A. salina individuals were rapidly rinsed with Millipore water and then with the same receiving salt solution. 2.2. Oxygen solubility and respirometry An Ox-MR Unisense electrode was used for measuring the solubility of oxygen in the salt solutions at (25 ± 1) °C. The samples were magnetically stirred and the content of O2 was measured after 20 min of aeration of the solution and 10 min at rest. Each value is the average over three readings. The respirometry tests were carried out at (25 ± 1) °C with the same electrode and a special cage-holder where 5 individuals were located. The cage was transferred into the salt solutions and the oxygen consumption was recorded every hour. Each value is the average over 5 replicates. _ O ) were deterWeight-specific rates of oxygen consumption (M 2
mined based on the dry weight of the animals (Varó et al., 1998). The dry weight of the animals ranged from 0.1 to 0.7 mg. For the respirometry tests the salt solutions consisted in mixed NaCl/NaF or NaCl/NaSCN solutions with a mole fraction of NaF or NaSCN ranging between 0.05 and 0.20. 3. Results and discussion After the development of cysts and an appropriate lapse for their growth, the individuals of A. salina were transferred in the salt solutions at the same ionic strength of seawater, in order to investigate the specific effect of the anion, at the same temperature and growth time. 3.1. Vitality tests The vitality tests were performed in order to assess the macroscopic effect of each anion on the survival of the individuals. The vitality of the animals was assessed by inspecting their legs. We considered as dead an individual that did not swim and whose legs did not move. Fig. 1 shows the results of the vitality tests in the different salt solutions. The number of alive individuals (N) is plotted as a function of time (in log scale). The plot shows that in the case of NaF and NaSCN the animals slowly die. After 90 min in NaF or in NaSCN, 13 over 15 individuals
were in a deadly state, barely moving their legs. The remaining individuals were either swimming normally (in NaF) or slowly (in NaSCN). A similar situation occurred in the presence of NaClO4 but at higher contact times (less than 4 hours). In order to quantify the effect of the different salts we evaluated the Lethal Time 50 (LT50) as the time necessary for assessing the death of 50% of the individuals. Table 1 shows the value of LT50 for the different salt solutions and for the blank sample. NaF, NaClO4 and NaSCN are the most toxic salts for A. salina, while in NaCl and NaBr they survived better than in the blank solution. Actually A. salina performs normal ecdysis in NaCl and NaBr solutions (Boone and Baas-Becking, 1931). Our results are in line with those of Chrogan who showed that Artemia adults survive very well in NaCl and NaBr (0.5 M), while they are moribund or very slow after 24 h in Na2SO4 (0.25 M) and NaNO3 (1 M), and LiCl or NaHCO3, NaN3 and potassium salts are more toxic (Chrogan, 1958). The toxicity of fluoride has already been demonstrated in previous studies and is presumably related to the inhibition of different enzymes (including phosphatases, hexokinase, enolase, succinic dehydrogenase, pyruvic oxidase) and to the interruption of metabolic processes such as glycolysis and synthesis of proteins (Pankhurst et al., 1980; Camargo, 2003). On the other hand, the toxicity of thiocyanate in fish and crustaceans seems to be related to the substitution of chloride ions in the gill and to the perturbation of the ionic balance, resulting in respiratory problems (Epstein et al., 1973; Bhunia et al., 2000). Perchlorate is known to be toxic for fish (Crane et al., 2005; Park et al., 2006) as a hormone disruptor. In addition to these biochemical effects, we propose that other, physico-chemical mechanisms, can participate in determining the toxicity of these ions for A. salina. These have to do with (i) the interfacial adsorption of the anions at the chitin/water interface, and (ii) with their influence on the stabilization of air bubbles in solution. (i) Strong kosmotropes such as fluoride can significantly weaken the hydrogen bonds that involve water and amide bonds in chitin. On the other hand, strong chaotropes such as perchlorate, thiocyanate and iodide are able to deplete water from the hydrophobic hydration layer around chitin (Cho et al., 2008; Zhang and Cremer, 2010). This peculiar physico-chemical mechanism may operate synergistically with the chemical binding of these anions at the chitin/water interface that result in a potent toxic effect for Artemia. We recall that several anions adsorb on chitin and its derivatives with a selectivity trend that follows the Hofmeister sequence (Davila-Rodriguez et al., 2012):
SO2 4 > HCO3 > F > Cl > NO3 :
Table 1 LT50 (in min) and surface tension molar increment at the air/water interface (r, in lN m2 mol1) for the investigated sodium salt solutions. The concentration is 507.2 and 169.2 mmol kg1 for the 1:1 and 2:1 salts, respectively.
Fig. 1. Vitality tests. The number of alive individuals N is plotted versus time (in min) in the presence of different sodium salt solutions: NaF ( ), NaSCN ( ), NaClO4 ( ), NaI (d), Na2CO3 ( ), NaNO3 ( ), Na2SO4 ( ), NaBr ( ), and NaCl ( ).
Salt
LT50 (min)
r (lN m2 mol1)
NaF NaSCN NaClO4 Na2CO3 NaI NaNO3 Na2SO4 Blank NaBr NaCl
73 76 224 364 460 686 1120 1403 1463 1637
2.00 0.54 0.62 2.60 1.02 1.10 1.60 – 1.31 1.76
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capacity of the crustaceans. This hypothesis may explain the trend found in Fig. 2. In fact chaotropic ions such as SCN, ClO 4 and I , that have no effect on the coalescence of gas bubbles in solution, dramatically decrease the vitality of A. salina. 3.2. Respirometry We prepared mixed solutions of NaCl + NaF and of NaCl + NaSCN at different mole fractions of the two solutes and measured the oxygen consumption in order to detect the threshold value of toxicity for NaF and NaSCN (see Fig. 3a and b). In both cases we found an initial decrement (in absolute value) _ O , followed by a temporary increment, before death (zero in M 2
Fig. 2. LT50 versus the surface tension molar increment r for the different anions.
Since no data for the chitin/water interface tension in the presence of different electrolytes is reported in the literature, in Fig. 2 we plotted the variation of LT50 as a function of the surface tension molar increment r. This parameter is defined as r ¼ @ðDcÞ=@c, where c is the air/solution interface and c is the molar concentration of the electrolyte in solution. The values of r for the different salts are shown in Table 1. r reflects the adsorption/desorption of ions at the air/solution interface: kosmotropes such as fluoride possess a large value of r, while chaotropes like iodide show a rather low r (Ninham and Lo Nostro, 2010; Kunz et al., 2004; Lo Nostro and Ninham, 2012). (ii) The bubble–bubble coalescence phenomenon is a typical specific ion effect: some electrolytes stabilize the formation of air microbubbles in an aqueous solution and prevent their coalescence, while others have no effect with the formation of short-lived macrobubbles (Ninham and Yaminsky, 1997; Craig et al., 1993). For example, NaCl, NaBr and Na2SO4 prevent bubbles coalescence, while NaClO4 and NaSCN do not (Henry et al., 2007). This is why clean, non polluted seawater is foamy, while in fresh water air bubbles are not stable and collapse very quickly. We recall that the thorax of A. salina bears eleven pairs of phyllopods or swimming legs that are used for locomotion, osmoregulation, respiration and nutrition (Criel and Macrae, 2002). The osmoregulation mechanism developed by Artemia basically consists in drinking the external medium and transfer of the ionic solutes and fluid into the haemolymph. The outward ion transport is mainly carried out by specialized cells, the metepipodites that are located on the phyllopodia (Clegg and Trotman, 2002; Holliday et al., 1990). We argue that a change in the availability and size of oxygen microbubbles may modify and eventually hamper the breathing
value). The comparison of the two plots shows that thiocyanate is more or less as toxic as fluoride. The trends seem to suggest that initially all individuals suffer for the presence of the toxic anion (either fluoride or thiocyanate), depending on its concentration (each line in Fig. 3 refers to a different mole fraction of NaF or NaSCN compared to the background NaCl salt, as indicated in the figure legend). In fact the oxygen consumption decreases and the decrement is faster at higher concentrations of the toxic component. After about 5–7 h of regular decrease at minimal concentration of NaF or NaSCN (black lines) the individuals apparently start breathing more oxygen up to about 22 h, after which they quickly die. For higher concentration of NaF or NaSCN the respirometric curves are compressed and death is reached within the first 5– 10 h, depending on the toxic salt concentration. _ O (recovering step) Presumably the significant increment in M 2
before death is related to the intense production of osmoregulating molecules, such as trehalose and glucose, that the animals synthesize when experiencing a stressing condition (Hand et al., 2011; Glasheen and Hand, 1989). 3.3. Oxygen solubility In order to verify whether the different vitality of A. salina is related to the different oxygen solubility in the electrolyte solutions, we measured the concentration of saturated oxygen in the different solutions at 25 °C (see Table 2), and compared the data to those found in the literature (Millero et al., 2002). We recall that the oxygen solubility at 25 °C and 1 bar in fresh water and seawater (salinity 35‰) is 258 and 206 lM, respectively. The solubility of oxygen in an electrolyte solution depends on temperature, pressure, concentration and nature of the solute. At a given temperature, the solubility of oxygen a salt solution, [O2]s, depends on the salt concentration (m, in molal units) according to Eq. (1):
ln½O2 s ¼ ln½O2 w kS m
ð1Þ
_ O , in lmol g1 h1) as a function of time (in hours) for NaF + NaCl (left) and for NaSCN + NaCl (right) mixed solutions at different mole Fig. 3. Oxygen consumption (M 2 fractions: x = 0.05 (d), 0.10 ( ), 0.15 ( ), and 0.20 ( ).
P. Lo Nostro et al. / Chemosphere 135 (2015) 335–340 Table 2 Anion partial molal volume (ms, in cm3 mol1) for the investigated salts at 25 °C, and solubility of oxigen (in lmol kg1) at 25 °C for the investigated salts in water. The concentration is 507.2 and 169.2 mmol kg1 for the 1:1 and 2:1 salts, respectively. Salt
NaF NaCl NaBr NaI NaSCN NaNO3 NaClO4 Na2CO3 Na2SO4 a b
O2 solubility (lmol kg1) This work
From Millero et al. (2002)
218.0 ± 0.1 221.4 ± 0.1 225.0 ± 0.1 227.8 ± 0.1 229.8 ± 0.1 224.4 ± 0.1 231.1 ± 0.1 232.1 ± 0.1 219.8 ± 0.1
216 ± 7 220 ± 1 223 ± 1 230 ± 2 n/a 229 ± 1 n/a 271 ± 4 223 ± 2a
ms (cm3 mol1)b 1.2 17.8 24.7 36.2 35.7 29.0 44.1 – 14.0
From Millero et al. (2003). From Millero (1971).
Fig. 4. Oxygen solubility (in lmol kg1) as a function of the partial molal volume of the anion (ms, in cm3 mol1) at 25 °C and at atmospheric pressure. : data measured in this work and : taken from the literature (Millero et al., 2002, 2003). The line is a guide for the eye.
Here [O2]w is the solubility of oxygen in water at the same temperature and kS is the salting-out Setchenow constant, respectively (Millero et al., 2003). The scaled particle theory model describes the value of kS for 1:1 electrolytes as determined by the Gibbs free energy that is necessary to create the cavity for the dissolution of the oxygen microbubble in the solution, and the Gibbs free energy for the oxygen–water and oxygen–salt interactions (Millero et al., 2003). This explains why kS depends on the composition of the electrolyte. Fig. 4 illustrates the change in the oxygen solubility with the partial molal volume of the anion (ms). The values of ms were taken from the literature (Millero, 1971). At the same temperature and pressure, oxygen is more soluble in aqueous salt solutions where the anion is a chaotrope, with a larger partial molal volume, because it is easier to create the cavity to accommodate the dissolved gas. On the other hand, oxygen is less soluble in aqueous solutions of strong kosmotropes such as fluoride and sulfate. Comparing the Lethal Time LT50 to the oxygen solubility it is clear that there is no direct correlation between the salt-dependent solubility of oxygen and the effect of the anion on the vitality of A. salina, in fact low values of LT50 are found for salts that enhance the solubility of O2 (such as sodium perchlorate, thiocyanate and iodide), as well as for sodium fluoride that lowers to amount of dissolved oxygen. 4. Conclusions The effect of nine different sodium salts on the vitality and on the weight-specific rates of oxygen consumption of A. salina was
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investigated at room temperature. The evaluation of the Lethal Time LT50 shows that the crustaceans survive very well in sodium chloride and bromide, while the presence of foreign salt species such as sodium fluoride, thiocyanate, perchlorate, iodide and nitrate severely adverse their vitality and eventually lead to death. Besides the specific toxicity that has been reported in the literature for some anions such as fluoride and thiocyanate, which is mainly related to their biochemical activities, we propose that the adverse effect is also related to their ionic physico-chemical features. Fluoride and carbonate are strong kosmotropes, strongly hydrated and with large surface tension molar increment, that impair the strength of the exoskeleton’s chitin. On the other hand, iodide, thiocyanate and perchlorate are chaotropes, with weaker hydration and lower surface tension molar increments, that promote the anion adsorption at the chitin/water interface. The solubility of atmospheric oxygen in the presence of different electrolytes is not correlated with their effect on the vitality of the crustaceans. Instead, our results apparently suggest that the most toxic anions for A. salina are those that have no effect on bubble–bubble coalescence such as ClO (Henry 4 and SCN et al., 2007). In conclusion, A. salina has naturally adapted to even strong concentrations of chloride and bromide ions, that occupy the intermediate mild region of the Hofmeister sequence. The presence of strong kosmotropes (F or CO2 3 ) and especially of strong chao tropes (SCN, ClO 4 , I ) severely impair the vitality of the crustaceans, and suggest that the physico-chemical properties of these ions determine their toxic effect, besides the specific biochemical activities they perform in the respiratory or metabolic routes. Acknowledgements The Authors acknowledge the Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGI, Florence), and the Enzo Ferroni Foundation (Florence) for partial financial support. References Bhunia, F., Saha, N.C., Kaviraj, A., 2000. Toxicity of thiocyanate to fish, plankton, worm, and aquatic ecosystem. Bull. Environ. Contam. Toxicol. 64, 197–204. Boas, F., 1926. Beiträge zur hylergographie. Uber die Wirkung von Salzen, namentlich Neutralsalzen, auf die Zelle (Hylergographic studies. The influence of neutral salts on the cell). Biochem. Z 176, 349–402. Boone, E., Baas-Becking, L.G.M., 1931. Salt effects on eggs and nauplii of Artemia Salina L. J. Gen. Physiol. 14 (6), 753–763. Camargo, J.A., 2003. Fluoride toxicity to aquatic organisms: a review. Chemosphere 50, 251–264. Camargo, W.N., Durán, G.C., Rada, O.C., Hernández, L.C., Linero, J.-C.G., Muelle, I.M., Sorgeloos, P., 2005. Determination of biological and physicochemical parameters of Artemia franciscana strains in hypersaline environments for aquaculture in the Colombian Caribbean. Saline Syst. 1, 9–19. Cho, Y., Zhang, Y., Christensen, T., Sagle, L.B., Chikoti, A., Cremer, P.S., 2008. Effects of hofmeister anions on the phase transition temperature of elastin-like polypeptides. J. Phys. Chem. B 112, 13765–13771. Chrogan, P.C., 1958. The survival of Artemia salina (L.) in various media. J. Exp. Biol. 35, 213–218. Clegg, J.S., Trotman, C.N.A., 2002. Physiological and biochemical aspects of artemia ecology. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 3. Craig, V.S.J., Ninham, B.W., Pashley, R.M., 1993. The effect of electrolytes on bubble coalescence in water. J. Phys. Chem. 97, 10192–10197. Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2005. Effects of ammonium perchlorate on thyroid function in developing fathead minnows, Pimephales promelas. Environ. Health Perspect. 113, 396–401. Criel, G.R.J., Macrae, T.H., 2002. Artemia morphology and structure. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 1. Dasgupta, P.K., Liu, Y., Dyke, J.V., 2008. Iodine nutrition: iodine content of iodized salt in the United States. Environ. Sci. Technol. 42, 1315–1323.
340
P. Lo Nostro et al. / Chemosphere 135 (2015) 335–340
Davila-Rodriguez, J.K., Escobar-Barrios, V.A., Rangel-Mendez, J.R., 2012. Removal of fluoride from drinking water by a chitin-based biocomposite in fixed-bed columns. J. Fluorine Chem. 140, 99–103. Dér, A., Kelemen, L., Fábián, L., Taneva, S.G., Fodor, E., Páli, T., Cupane, A., Cacace, M.G., Ramsden, J.J., 2007. Interfacial water structure controls protein conformation. J. Phys. Chem. B 111, 5344–5350. Epstein, F.H., Maetz, J., Renzis, G., 1973. Active transport of chloride by the teleost gill: inhibition by thiocyanate. Am. J. Physiol. 224, 1295–1299. Gellhorn, E., 1933. Experiments on the hofmeister series in heart muscle. Protoplasma 18, 411–419. Glasheen, J.S., Hand, S.C., 1989. Metabolic heat dissipation and internal solute levels of artemia embryos during changes in cell-associated water. J. Exp. Biol. 145, 263–282. Hand, S.C., Menze, M.A., Toner, M., Boswell, L., Moore, D., 2011. LEA proteins during water stress: not just for plants anymore. Annu. Rev. Physiol. 73, 115–134. Henry, C.L., Dalton, C.N., Scruton, L., Craig, V.S.J., 2007. Ion-specific coalescence of bubbles in mixed electrolyte solutions. J. Phys. Chem. C 111, 1015–1023. Hinojosa, M.B., Carreira, J.A., García-Ruíz, R., Rodríguez-Maroto, J.M., Daniell, T.J., Griffiths, B.S., 2010. Plant treatment, pollutant load, and soil type effects in rhizosphere ecology of trace element polluted soils. Ecotoxicol. Environ. Safety 73, 970–981. Holliday, C.W., Roye, D.B., Roer, R.D., 1990. Salinity-induced changes in branchial Na+/K+ ATPase activity and transepithelial potential difference in the brine shrimp Artemia Salina. J. Exp. Biol. 151, 279–296. Irwin, S., Wall, V., Davenport, J., 2007. Measurement of temperature and salinity effects on oxygen consumption of Artemia franciscana K., measured using fibreoptic oxygen microsensors. Hydrobiologia 575, 109–115. Kim, H.-K., Tuite, E., Nordén, B., Ninham, B.W., 2001. Co-ion dependence of DNA nuclease activity suggests hydrophobic cavitation as a potential source of activation energy. Eur. Phys. J. E 4, 411–417. Kunz, W., Lo Nostro, P., Ninham, B.W., 2004. The present state of affairs with Hofmeister effects. Curr. Op. Coll. Interface Sci. 9, 1–18. Lindh, U., 2007. Metal biology: aspects of beneficial effects. Ambio 36, 107–110. Lo Nostro, P., Ninham, B.W., 2012. Hofmeister phenomena: an update on ion specificity in biology. Chem. Rev. 112, 2286–2322. Lo Nostro, P., Fratoni, L., Ninham, B.W., Baglioni, P., 2002. Water absorbency by wool fibers: hofmeister effect. Biomacromolecules 3, 1217–1224. Lo Nostro, P., Ninham, B.W., Lo Nostro, A., Pesavento, G., Fratoni, L., Baglioni, P., 2005. Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa. Phys. Biol. 2, 1–7. Millero, F.J., 1971. The molal volumes of electrolytes. Chem. Rev. 71, 147–176. Millero, F.J., Huang, F., Laferiere, A.L., 2002. The solubility of oxygen in the major sea salts and their mixtures at 25 °C. Geochim. Cosmochim. Acta 66, 2349–2359. Millero, F.J., Huang, F., Graham, T.B., 2003. Solubility of oxygen in some 1–1, 2–1, 1– 2, and 2–2 electrolytes as a function of concentration at 25 °C. J. Solut. Chem. 32, 473–487.
Ninham, B.W., Lo Nostro, P., 2010. Molecular Forces and Self Assembly. In Colloid, Nano Sciences and Biology. Cambridge University Press, Cambridge. Ninham, B.W., Yaminsky, V.V., 1997. Ion binding and ion specificity: the hofmeister effect and onsager and lifshitz theories. Langmuir 13, 2097–2108. Ball, P., Hallsworth, J.E., 2015. Water structure and chaotropicity: their uses, abuses and biological implications. Phys Chem Chem Phys. http://dx.doi.org/10.1039/ c4cp04564e [Advance Article]. Pankhurst, N.V., Boyden, C.R., Wilson, J.B., 1980. The effect of a fluoride effluent on marine organisms. Environ. Pollut. 23, 299–312. Park, J.-W., Rinchard, J., Liu, F., Anderson, T.A., Kendall, R.J., Theodorakis, C.W., 2006. The thyroid endocrine disruptor perchlorate affects reproduction, growth, and survival of mosquitofish. Ecotoxicol. Environ. Safety 63, 343–352. Parsons, D.F., Ninham, B.W., 2010. Importance of accurate dynamic polarizabilities for the ionic dispersion interactions of alkali halides. Langmuir 26, 1816– 1823. Rossi, S., Lo Nostro, P., Lagi, M., Ninham, B.W., Baglioni, P., 2007. Specific anion effects on the optical rotation of a-amino acids. J. Phys. Chem. B 111, 10510– 10519. Salis, A., Pinna, M.C., Bilanicˇová, D., Monduzzi, M., Lo Nostro, P., Ninham, B.W., 2006. Specific anion effects on glass electrode pH measurements of buffer solutions: bulk and surface phenomena. J. Phys. Chem. B 110, 2949–2956. Salis, A., Bilanicˇová, D., Ninham, B.W., Monduzzi, M., 2007. Hofmeister effects in enzymatic activity: weak and strong electrolyte influences on the activity of candida rugosa lipase. J. Phys. Chem. B 111, 1149–1156. Schwierz, N., Horinek, D., Netz, R.R., 2010. Reversed anionic hofmeister series: the interplay of surface charge and surface polarity. Langmuir 26, 7370–7379. Somero, G.N., 1986. Protons, osmolytes, and fitness of internal milieu for protein function. Am. J. Physiol. 251, R197–R213. van Dama, R., Hogan, A., Harford, A., Markich, S., 2008. Toxicity and metal speciation characterisation of waste water from an abandoned gold mine in tropical northern Australia. Chemosphere 73, 305–313. van Stappen, G., 1996. Introduction, biology and ecology of Artemia. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the Production and Use of Live Food for Aquaculture, Rome, FAO Fisheries Technical Paper. van Stappen, G., 2002. Zoogeography. In: Abatzopoulos, Th.J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Springer Science+Business Media, Dordrecht. chapter 4. Varó, I., Taylor, A.C., Amat, F., 1998. The effects of temperature and oxygen tension (P O2 ) on the oxygen consumption rates of adults of different Artemia strains. Comp. Biochem. Physiol. A: Physiol. 120, 385–390. Voinescu, A., Bauduin, P., Pinna, C., Touraud, D., Kunz, W., Ninham, B.W., 2006. Similarity of salt influences on the pH of buffers, polyelectrolytes, and proteins. J. Phys. Chem. B 110, 8870–8876. Zhang, Y., Cremer, P.S., 2010. Chemistry of hofmeister anions and osmolytes. Annu. Rev. Phys. Chem. 61, 63–83.
