Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

The High Salt Requirement of the Moderate Halophile Chromohalobacter salexigens DSM3043 Can Be Met Not Only by NaCl but by Other Ions

The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na⁺, K⁺, Rb⁺, or NH₄⁺, Cl⁻, Br⁻, NO₃⁻, or SO₄²⁻ ions. To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Calcium uptake by excised maize roots and interactions with alkali cations

Ca²⁺ uptake was studied in short-term experiments using 5-day-old excised maize roots. This tissue readily absorbs Ca²⁺, and inhibition by dinitrophenol and low temperature shows that the process is metabolically mediated. The uptake of Ca²⁺, like that of other cations, is influenced by the counter ion, the pH and concentration of the ambient solution, and the presence of other cations. The rate of uptake from various salts decreases in the following order: NO₃⁻ > Cl⁻ = Br⁻ > SO₄²⁻. K⁺ and H⁺ greatly interfere with Ca²⁺ absorption, while Li⁺ and Na⁺ have only slight effects.     

Sulfate Reduction in Freshwater Sediments Receiving Acid Mine Drainage

One arm of Lake Anna, Va., receives acid mine drainage (AMD) from Contrary Creek (SO₄²⁻ concentration = 2 to 20 mM, pH = 2.5 to 3.5). Acid-volatile sulfide concentrations, SO₄²⁻ reduction rates, and interstitial SO₄²⁻ concentrations were measured at various depths in the sediment at four stations in four seasons to assess the effects of the AMD-added SO₄²⁻ on bacterial SO₄²⁻ reduction. Acid-volatile sulfide concentrations were always an order of magnitude higher at the stations receiving AMD than at a control station in another arm of the lake that received no AMD. Summer SO₄²⁻ reduction rates were also an order of magnitude higher at stations that received AMD than at the control station (226 versus 13.5 mmol m⁻² day⁻¹), but winter values were inconclusive, probably due to low sediment temperature (6°C). Profiles of interstitial SO₄²⁻ concentrations at the AMD stations showed a rapid decrease with depth (from 1,270 to 6 μM in the top 6 cm) due to rapid SO₄²⁻ reduction. Bottom-water SO₄²⁻ concentrations in the AMD-receiving arm were highest in winter and lowest in summer. These data support the conclusion that there is a significant enhancement of SO₄²⁻ reduction in sediments receiving high SO₄²⁻ inputs from AMD.     

The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar

Net uptakes of K⁺ and NO₃⁻ were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K⁺ and NO₃⁻ uptake in the absence of NH₄⁺ or Cl⁻. Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH₄⁺, K⁺, Cl⁻, or NO₃⁻ affected the cultivars very differently: (a) the presence of NH₄⁺ as NH₄Cl stimulated net NO₃⁻ uptake in Prato barley but inhibited net NO₃⁻ uptake in Olli barley; (b) Cl⁻ inhibited net NO₃⁻ uptake in Prato but had little effect in Olli; and (c) NH₄⁺ as (NH₄)₂SO₄ inhibited net K⁺ uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl⁻ initially inhibited net K⁺ uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH₄⁺ and Cl⁻ uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption.     

Effects of Catchment and Riparian Landscape Setting on Water Chemistry and Seasonal Evolution of Water Quality in the Upper Han River Basin,China

Six-year (2005–2010) evolution of water chemistry (Cl⁻, NO₃ ⁻, SO₄ ²⁻, HCO₃ ⁻, Na⁺, K⁺, Ca²⁺ and Mg²⁺) and their interactions with morphological properties (i.e., slope and area), land cover, and hydrological seasonality were examined to identify controlling factors and processes governing patterns of stream water quality in the upper Han River, China. Correlation analysis and stepwise multiple regression models revealed significant correlations between ions (i.e., Cl⁻, SO₄ ²⁻, Na⁺ and K⁺) and land cover (i.e., vegetation and bare land) over the entire catchment in both high- and low-flow periods, and in the buffer zone the correlation was much more stronger in the low-flow period. Catchment with steeper slope (>15°) was negatively correlated with major ions, largely due to multicollinearity of basin characteristics. Land cover within the buffer zone explained slightly less of major elements than at catchment scale in the rainy season, whereas in the dry season, land cover along the river networks in particular this within 100 m riparian zone much better explained major elements rather than this over the entire catchment. Anthropogenic land uses (i.e., urban and agriculture) however could not explain water chemical variables, albeit EC, TDS, anthropogenic markers (Cl⁻, NO₃ ⁻, SO₄ ²), Na⁺, K⁺ and Ca²⁺ significantly increased during 2005–2010, which was corroborated by principal component analyses (PCA) that indicated anthropogenic inputs. Observations demonstrated much higher solute concentrations in the industrial-polluted river. Our results suggested that seasonal evolution of water quality in combined with spatial analysis at multiple scales should be a vital part of identifying the controls on spatio-temporal patterns of water quality.     

Ionic balance in different tissues of the tomato plant in relation to nitrate, urea, or ammonium nutrition

An investigation was carried out to study the cation-anion balance in different tissues of tomato plants supplied with nitrate, urea, or ammonium nitrogen in water culture.

Irrespective of the form of nutrition, a very close balance was found in the tissues investigated (leaves, petioles, stems, and roots) between total cations (Ca, Mg, K and Na), and total anions (NO₃⁻, H₂PO₄⁻, SO₄⁻⁻, Cl⁻) total non-volatile organic acids, oxalate, and uronic acids. In comparison with the tissues of the nitrate fed plants, the corresponding ammonium tissues contained lower concentrations of inorganic cations, and organic acids and a correspondingly higher proportion of inorganic anions. Tissues from the urea plants were intermediate between the other 2 treatments. These results were independent of concentration or dilution effects, caused by growth. In all tissues approximately equivalent amounts of diffusible cations (Ca⁺⁺, Mg⁺⁺, K⁺ and Na⁺), and diffusible anions (No₃⁻, SO₄⁻⁻, H₂PO₄⁻, Cl⁻) and non-volatile organic acids were found. An almost 1:1 ratio occurred between the levels of bound calcium and magnesium, and oxalate and uronic acids. This points to the fact that in the tomato plant the indiffusible anions are mainly oxalate and pectate. Approximately equivalent values were found for the alkalinity of the ash, and organic anions (total organic acids including oxalate, and uronic acids).

The influence of nitrate, urea, and ammonium nitrogen nutrition on the cation-anion balance and the organic acid content of the plant has been considered and the effects of these different nitrogen forms on both the pH of the plant and the nutrient medium and its consequences discussed.

     

Photosynthesis, growth, and the role of chloride

Previous studies with isolated chloroplasts have indicated that Cl⁻ is an essential cofactor for photosynthesis. Considerable support for the postulated Cl⁻ requirement in photosynthesis came from the observation that Cl⁻ is essential for growth. Data are presented which show that a 60% reduction in growth which occurred in Cl⁻ -deficient sugar beet (Beta vulgaris L.) was not due to an effect of Cl⁻ on the rate of photosynthesis in vivo (net CO₂ uptake per unit area of attached leaves). The principal effect of Cl⁻ deficiency was to lower cell multiplication rates in leaves, thus slowing down their growth and ultimately decreasing their area. The absence of an effect of Cl⁻ on photosynthesis in vivo was unlikely to have been due to Cl⁻ retention by the chloroplasts because their Cl⁻ concentration (measured after nonaqueous isolation) decreased progressively with decrease in leaf Cl⁻.     

Structural Effects of Cl and Other Anions on the Water Oxidizing Complex of Chloroplast Photosystem II

To further our understanding of the role of Cl⁻ and certain other monovalent anions in the oxygen evolving photosystem II of chloroplasts, dissociating and stabilizing anion effects on the extrinsic 17 and 23 kilodalton polypeptides of the photosynthetic water oxidizing complex were investigated. It was found that (a) the dissociation of the two polypeptides in Cl⁻ free media of pH ≈ 7 was enhanced by millimolar concentrations of the divalent anion SO₄²⁻ and also by divalent cations like Mg²⁺ and Ca²⁺; (b) the dissociation was opposed by relatively low concentrations of monovalent anions with an order of effectiveness Cl⁻ = Br⁻ > NO₃⁻ > F⁻ > ClO₄⁻; (c) at molar concentrations, SO₄²⁻ stabilized the binding of the 23 kilodalton polypeptide, while Cl⁻ and Br⁻ became dissociating agents, in agreement with studies by Blough and Sauer (1984 Biochim Biophys Acta 767: 377-381); (d) the binding of the polypeptides was strengthened at room temperature relative to 0°C, indicating an involvement of hydrophobic forces. It is suggested that a specific binding of Cl⁻, or certain substitutes, organizes the protein surfaces and/or the adjacent water layers in the water oxidizing complex in a way that not only stabilizes its assembly, but is essential for the catalytic mechanism as well. Binding of, or charge screening by, divalent ions interferes with this process. At high salt concentrations, all these effects are overridden by “lyotropic” actions of the solutes that affect the integrity of the water oxidizing protein complex by stabilizing or disrupting critical hydrophobic domains.     

Organic Acids and Ionic Balance in Xylem Exudate of Wheat during Nitrate or Sulfate Absorption

Experiments were designed to study the importance of organic acids as counterions for K⁺ translocation in the xylem during excess cation uptake. A comparison was made of xylem exudate from wheat seedlings treated 72 hours with either 1.0 millimolar KNO₃ or 0.5 millimolar K₂SO₄, both in the presence of 0.2 millimolar CaSO₄. Exudation from KNO₃ plants had twice the volume and twice the K⁺ and Ca²⁺ fluxes or rate of delivery to shoots, as K₂SO₄ plants. Malate flux was 25% higher in K₂SO₄ than in KNO₃ exudate. Malate was the principal anion accompanying K⁺ or Ca²⁺ in K₂SO₄ treatment, while in the KNO₃ treatment, NO₃⁻ was the principal anion. The contribution of SO₄²⁻ was negligible in both treatments. In a second experiment, exudate was collected every 4 hours during the daytime throughout a 72-hour treatment with KNO₃. Malate was the only anion present in exudate at first, just after the CaSO₄ pretreatment had ended. Malate concentration decreased and NO₃⁻ concentration increased with time and these concentrations were negatively correlated. By 62 hours, NO₃⁻ represented 80% of exudate anions. K⁺ and NO₃⁻ concentrations in exudate were strongly correlated with K⁺ and NO₃⁻ uptake, respectively. The first 36 hours of absorption from KNO₃ solution resembled the continuous absorption of K₂SO₄, in that malate was the principal counterion for translocation of K⁺.     

Influence of the level of nitrate nutrition on ion uptake and assimilation, organic Acid accumulation, and cation-anion balance in whole tomato plants

Tomato plants (Lycopersicon esculentum L. var. Ailsa Craig) were grown in water culture in nutrient solution in a series of 10 increasing levels of nitrate nutrition. Using whole plant data derived from analytical and yield data of individual plant parts, the fate of anion charge arising from increased NO₃ assimilation was followed in its distribution between organic anion accumulation in the plant and OH⁻ efflux into the nutrient solution as calculated by excess anion over cation uptake. With increasing NO₃ nutrition the bulk of the anion charge appeared as organic anion accumulation in the plants. OH⁻ efflux at a maximum accounted for only 20% of the anion charge shift. The major organic anion accumulated in response to nitrate assimilation was malate. The increase in organic anion accumulation was paralleled by an increase in cation concentration (K⁺, Ca²⁺, Mg²⁺, Na⁺). Total inorganic anion levels (NO₃⁻, SO₄²⁻, H₂PO₄⁻, Cl⁻) were relatively constant. The effect of increasing NO₃ nutrition in stimulating organic anion accumulation was much more pronounced in the tops than in the roots.     

The Influence of Sulphate Deposition on the Seasonal Variation of Peat Pore Water Methyl Hg in a Boreal Mire

In this paper we investigate the hypothesis that long-term sulphate (SO₄ ²⁻) deposition has made peatlands a larger source of methyl mercury (MeHg) to remote boreal lakes. This was done on experimental plots at a boreal, low sedge mire where the effect of long-term addition of SO₄ ²⁻ on peat pore water MeHg concentrations was observed weekly throughout the snow-free portion of 1999. The additions of SO₄ ²⁻ started in 1995. The seasonal mean of the pore water MeHg concentrations on the plots with 17 kg ha⁻¹ yr⁻¹ of sulphur (S) addition (1.3±0.08 ng L⁻¹, SE; n = 44) was significantly (p<0.0001) higher than the mean MeHg concentration on the plots with 3 kg ha⁻¹ yr⁻¹ of ambient S deposition (0.6±0.02 ng L⁻¹, SE; n = 44). The temporal variation in pore water MeHg concentrations during the snow free season was larger in the S-addition plots, with an amplitude of >2 ng L⁻¹ compared to +/−0.5 ng L⁻¹ in the ambient S deposition plots. The concentrations of pore water MeHg in the S-addition plots were positively correlated (r² = 0.21; p = 0.001) to the groundwater level, with the lowest concentrations of MeHg during the period with the lowest groundwater levels. The pore water MeHg concentrations were not correlated to total Hg, DOC concentration or pH. The results from this study indicate that the persistently higher pore water concentrations of MeHg in the S-addition plots are caused by the long-term additions of SO₄ ²⁻ to the mire surface. Since these waters are an important source of runoff, the results support the hypothesis that SO₄ ²⁻ deposition has increased the contribution of peatlands to MeHg in downstream aquatic systems. This would mean that the increased deposition of SO₄ ²⁻ in acid rain has contributed to the modern increase in the MeHg burdens of remote lakes hydrologically connected to peatlands.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

Effect of Ionic Composition of Suspending Solution on Virus Adsorption by a Soil Column

The effect of various electrolytes on the adsorption of poliovirus was measured in 250-cm-long soil columns with ceramic samplers at different depths. Viruses suspended in deionized water moved much farther through the soil than those suspended in tap water, whereas movement in sewage water was intermediate. The salt content of the tap water and sewage water promoted virus adsorption, but evidently the organic compounds in sewage retarded adsorption. When viruses were suspended in chloride solutions of K⁺, Na⁺, Ca⁺, and Mg²⁺, virus adsorption increased as the cation concentration and valence increased. The depth of virus penetration was related to the ionic strength of the solutions. Virus penetration data for NO₃⁻, SO₄²⁻, and H₂PO₄⁻ salts of K⁺, Na⁺, and Ca²⁺ indicated that other anions were more effective than Cl⁻ in promoting virus adsorption. Also, NH₄⁺ was more effective than other cations in limiting the penetration depth of viruses. It seems that ions composed of radicals are more effective than ions composed of single atoms in promoting virus adsorption. Al³⁺ was the most effective ion in limiting virus penetration, probably owing to flocculation of the viruses. Adding AlCl₃ concentrations to secondary sewage effluent to provide an Al³⁺ concentration of 0.1 mM reduced the virus penetration depth to 40 cm. These studies show that the ionic composition of the suspending solutions must be considered in predicting virus penetration depths, and it may be practical to add low concentrations of a flocculating agent such as AlCl₃ to sewage water to limit virus movement through very porous soils.     

Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H⁺. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl⁻, Br⁻, SCN⁻, and I⁻) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pH_e = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl⁻]_i under unlikely protonation conditions (pH_e = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H⁺ plays a minor role in dislodging the glutamate gate.     

Chemical Characteristics of Water-Soluble Ions in Particulate Matter in Three Metropolitan Areas in the North China Plain

PM_2.5 and PM₁₀ samples were collected simultaneously in each season in Beijing, Tianjin and Shijiazhuang to identify the characteristics of water-soluble ion compositions in the North China Plain. The water-soluble ions displayed significant seasonal variation. The dominant ions were NO₃ ⁻, SO₄ ²⁻, NH₄ ⁺ and Cl⁻, accounting for more than 90% and 86% to the mass of total water-soluble ions in PM_2.5 and PM₁₀, respectively. The anion/cation ratio indicated that the ion acidity of each city varied both between sites and seasonally. Over 50% of the ion species were enriched in small particles ≤1 µm in diameter. The [NO₃ ⁻]/[SO₄ ²⁻] ratio indicated that vehicles accounted for the majority of the particulate pollution in Beijing. Shijiazhuang, a city highly reliant on coal combustion, had a higher SO₄ ²⁻ concentration.