environments

Article Lead Complexation by Humic and Their Analogs: A Voltammetric Study

Spencer Steinberg * and Vernon Hodge Department of Chemistry, University of Nevada, Las Vegas, NV 89154, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-702-895-3599

 Received: 28 September 2020; Accepted: 23 October 2020; Published: 26 October 2020 

Abstract: Differential pulse polarography (DPP) was used to assess the interaction of Pb2+ with various humic analogs and several humic acids. DPP analysis demonstrated that the reduction 2+ peak maximum (Ep) for Pb shifted to more negative values in the presence of humic acids and 2+ humic acid analogs. The observed Ep for Pb in the presence of humic acids and humic acid analogs 2+ 2+ is influenced by ligand concentration, solution pH and Pb concentration. Shifts in the Ep for Pb are related to the reduction potential and can be rationalized using the Lingane equation.

Keywords: lead; differential pulse polarography; ; humic substance

1. Introduction When metal cations interact with natural organic matter, metal ligand complexes of various strengths can form. Natural organic matter in and can therefore have a significant influence on chemical speciation of various metals in the environment. For example, Grosell et al. [1] demonstrated that humic acid can reduce the toxicity of lead for fathead minnows during laboratory tank exposure. García-Mina et al. [2] reviewed the role that metal-humic complexes can play in plant micronutrient uptake. Senesi and Loffredo [3] reviewed the role of humic substances on the speciation of metals in . Senesi and Lofferedo [4] reviewed the various spectroscopic methods utilized for studying the influence of humic acid metal complexes on metal speciation in soil. Some metal humate complexes have been demonstrated to be electrochemically inert [5–7], while other metals that form complexes are still available for electrochemical reduction [8]. These latter complexes may, for example, be reduced to their zero-valance forms by a cathodic scan on a mercury electrode. This reduction reaction, of the complexed metal, occurs at a more negative potential than for the simple metal ion. The shift in reduction potential is a function of the metal ligand stability constant and the ligand concentration in solution, and this shift can be rationalized by reference to the Lingane equation [9]. When the ligand concentration is sufficiently higher than the metal concentration, and the metal ion equilibration with the ligand is sufficiently rapid, the shift in reduction potential is a linear function of the log of the ligand concentration, as predicted by the Lingane [9] equation. The shift in the reduction potential will also be a function of the pH of the solution if the speciation of the ligand and its affinity for the metal is influenced by pH. This phenomenon was also observed by Greter et al. [10]. These investigators noted the shift in apparent reduction potential in the presence of humic material and provided compelling evidence for the adsorption of the Pb2+ humate complex onto the mercury electrode. Similar observations were made by Turner et al. (1987), where the differential pulse polarography (DPP) measurement in the presence of fulvic acids yielded peak maxima at a significantly more negative value than fulvic acid-free solutions [11]. Wallman et al. [12] also observed that Pb2+ fulvic acid complexes adsorbed onto a static mercury electrode and were detectable by cyclic voltammetry (CV). The adsorbed Pb2+ was reduced during the cathodic scan, and the peak current

Environments 2020, 7, 94; doi:10.3390/environments7110094 www.mdpi.com/journal/environments Environments 2020, 7, 94 2 of 18 progressively shifted to more negative peak voltages with increased adsorption time. These authors noted that the Pb2+ fulvic acid complexes were labile on the time scale of CV. In a previous publication, using anodic stripping voltammetry (ASV), we demonstrated that copper is strongly bound by water-extractable and by humic acids [13]. A significant fraction of the bound copper was electrochemically inert, and the formation of this inert fraction was time-dependent. The presence of this electrochemically inert fraction is manifested by a nonlinear Copper ASV response when organic matter is titrated with Cu2+. Similar observations have been made by other investigators. Our investigations with with Pb2+ using ASV indicate that the concentration of the inert Pb2+ complexes is at least an order of magnitude lower than for Cu2+ (results not shown). Thus, ASV results for Pb2+ do not show the pronounced nonlinearity of response vs. concentration that was observed for Cu2+. However, during our investigations, we noticed that there was a notable shift 2+ in the DPP peak maximum (Ep) potential for Pb in the presence of humic acids when compared to the 2+ Ep of Pb in humic-free buffer solutions. In the current investigation, we sought to further elucidate the interaction of Pb2+ with various humic acids by examining the shift in the reduction potential of Pb2+ in the presence of humic acids. Our results demonstrated that Pb2+ interacts with humic material primarily by the formation of an electrochemically active (reducible) complex. This Pb2+ humate complex, although in rapid equilibrium with the solution species, is reduced at a more negative reduction potential (100 to 200 mV) than uncomplexed Pb2+.

2. Materials and Methods

2.1. Chemicals and Soil Samples Deionized water was redistilled in an all-glass still before use. All reagents used were of ACS grade or better and employed without further purification. Humic acid samples were purchased from the International Humic Substances Society (IHSS) (leonardite humic acid standard, Pahokee humic acid standard, Suwannee River Natural Organic Matter (NOM) and Elliot soil humic acid standard V). In addition, a commercial leonardite humic acid was obtained from TeraVita (Lancaster, PA, USA). This commercially available humic material is extracted from leonardite and is marketed as a soil amendment (TeraVita product information). Dimethyldichlorosilane was obtained from Alpha Aesar (Haverhill, MA, USA). Buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), MES (2-(N-morpholino) ethanesulfonic acid) and PIPES (piperazine-N,N0-bis (2-ethanesulfonic acid)) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Acetic acid and were obtained from Baker (Phillipsburg, NJ, USA). Buffer concentrations were 0.1 M, with 1-M NaNO3 added as a background electrolyte. For example, the HEPES pH 8 buffer was prepared by weighing out HEPES sodium salt to make a 0.100-M solution upon dilution. Similarly, enough, NaNO3 was weighed out so that the final solution was 1.00 M in NaNO3. The buffer salt was titrated to pH 8 with 6-M HCl and then diluted to volume. MOPs pH 7, MES pH 6.15 and acetate (sodium acetate) were prepared the same way. The pH of the solution was verified be within 0.02 pH units of the buffer, with a pH meter calibrated with National Institute of Standards and Technology (NIST) traceable standard buffers. Pb2+ standards were prepared by dilution of a commercial lead atomic adsorption standard purchased from VWR (Visalia, CA, USA). Thus, Pb2+ was added from an acidic standard solution, and we were concerned that the addition of these acidic aliquots would result in a significant change in pH. The potential for pH change was most significant for samples containing 15 mL of humic solution and only 1.0 mL of the 0.1-M buffer. Therefore, for some experiments, the humic acid was dissolved in 0.1-M pH 8 HEPES buffer solution or in 0.1-M HEPES buffer with 1.0-M NaNO3 added to increase the ionic strength of the solution. The increased concentration of the buffer increased the buffer capacity of the test solution and prevented any significant excursions in pH. Environments 2020, 7, 94 3 of 18

2.2. Instrumentation A CHI (Austin, TX, USA) model 660 A potentiostat was used in conjunction with a BASi MF-9058 controlled growth mercury electrode cell stand with magnetic stirrer. The electrochemical cell was purged with nitrogen before all measurements, and the cell head space was blanketed with nitrogen during all measurements. The controlled growth mercury electrode was equipped with a 150-µm glass capillary and was used in the static mercury electrode mode. A platinum wire served as the auxiliary electrode, and an Ag/AgCl reference electrode was employed for all measurements. The test solutions were stirred using a Teflon™-coated stir bar. All measurements were carried out in an MF-1084 glass cell manufactured by BASi. In order to minimize the adsorption of Pb2+ to the cell walls, the following cleaning and deactivating procedure was developed. The glass cell was washed first with Alkonox™ and then exhaustively rinsed with distilled water. The vessels were then soaked in concentrated nitric acid and again rinsed with distilled water and oven-dried at 110 ◦C. In order to minimize the adsorption of metals, the vessel was salinized by immersion in 5% dichlorodimethylsilane in toluene for approximately one hour. The vessel was then rinsed with methanol and dried at 40 ◦C. The cleaning and deactivation of the glass cell was repeated before each titration.

2.3. Voltammetry A 15.0-mL aliquot of sample was placed into the cleaned and salinized glass titration vessel. A 1.0-mL aliquot of 0.1-M buffer was added, and the sample was placed on the “cell stand” and purged for 15 min before starting the titration experiment. Pb2+ stock solutions used for titrations were in the concentration range of 10 ppm to 1000 ppm. Alternatively, humic acid solutions were prepared in 100-mM buffer solutions. A 15-mL aliquot of this solution was placed in the cell and purged with nitrogen before measurements were initiated. Most of the measurements for this study were performed using DPP. With the DPP, fixed voltage pulses were superimposed on a linear potential ramp [14]. The resulting current at the mercury drop electrode was sampled before the pulse and after the pulse. The difference in these two currents was plotted against the applied potential, resulting in an experimental voltammogram. The voltammogram consists of a current peak that is proportional to the concentration of the metal cation or the metal complex being reduced. The DPP peak position (Ep) is related to the reduction potential of the metal cation or metal complex. Due to various experimental conditions, and possibly kinetic contributions, Ep will only present an estimate of the thermodynamic reduction potential. In these DPP experiments, the initial voltage was set to 200 mV and the final voltage to 800 mV. The BASi instrument was − − set to the static dropping mode. A signal from the CHI potentiostat was used to initiate and control the mercury drop formation. The mercury electrode voltage was decreased by 5 mV with each mercury drop. For most measurements, the differential pulse amplitude was set to 50 mV, and the − pulse width was 50 ms, while the sampling time was 10 ms and the pulse period was set to 1 s. The mercury drop size was set to 14 on the BASi console.

2.4. Size Exclusion Chromatography Size exclusion chromatography (SEC) was performed on an SRI Model 210D HPLC equipped with a Model 401 UV/VIS detector. Separations were performed using a 7.5 mm 30 cm TSK × G2000SW column. The column was protected using a 7.5 mm 7.5 cm TSK G2000SW guard column. × The column eluent was 0.05-M pH 7 phosphate buffer containing 20% methanol. The column flow rate was maintained at 0.500 mL/min. The column was calibrated using polystyrene sulfonate standards (1.2 K, 4.4 K, 14.3 K and 35 K) purchased from American Polymer Standard (Mentor, OH, USA), and 4-hydroxybenzoic acid was used as a low molecular weight marker standard. Chromatograms were recorded at 250 nm and 280 nm. Little difference in the apparent elution times for the humic materials was observed at these two different wavelengths. Environments 2020, 7, 94 4 of 18

3. Results and Discussion Differential pulse polarography provides estimates of the reduction potentials for metals cations such as Pb2+, Cd2+, Zn2+ and Cu2+ [15]. Our observations indicated that the presence of various ligating small molecules (3,4-dihyroxybenzoic acid and salicylic acid) and humic acids results in a shift 2+ in the Ep for Pb to a more negative voltage. The magnitude of these potential shifts increases with increasing concentrations of complex forming ligands. The shift in the voltammogram peak maximum (Ep) can be understood utilizing an equation originally presented by Lingane [9]. The principle assumption is that the metal cation (M) is complexed by a ligand (L) with the formation of MLp and that this reaction is in rapid equilibrium with a stability constant KL. The equilibrium expression (Equation (1)) is given by: h i MLp fc K f = p p (1) [M][L] fm fL

In Equation (1), fc, fm and fL represent activity coefficients for the complex, the metal ion and the ligand, respectively, and p is the stoichiometric coefficient for the complex. The Lingane equation for apparent reduction potential of the metal complex is given by Equation (2).

p p 0.0592 KL[L] fa fL kc E00 = E0 log (2) − n fcka

The kc and the ka terms are kinetic terms related to the diffusion of the complex in solution and the diffusion of the metal in the mercury electrode, respectively. The term fa represents the activity coefficient of the metal on the mercury electrode. Expanding the Log term (Equation (3)):

0.0592 fakc 0.0592 P p E00 = E0 log + logKL[L] fL (3) − n fcka n

For a simple metal ion, the equation reduces to the following (Equation (4)):

0.0592 fakm E00 = E0 log (4) − n fmka

The km term is a kinetic term related to the diffusion of the uncomplexed metal in solution. The shift in the peak maximum is the difference in E00 between the complex metal ion and the simple metal ion in the buffer solution and is given by f, Equation (5).

p 0.0592 kc fm fL 0.0592 p ∆E = log logKL[L] (5) − n km fc − n

For low molecular weight complexing agents, the ratio kc/km is likely to be close to one, and the contribution of the activity coefficients to ∆E is also small or, at least, constant. Therefore, the shift in the reduction potential measured by the differential pulse is primarily a function of the stability constant and the ligand concentration (Equation (6)).

0.0592 p ∆E logKL[L] (6) ≈ − n

2+ The kc/km term may be important for humic substances, as diffusion rates for humic-bound Pb is likely to be slower than for lower molecular weight Pb2+ species [15]. The measurements of Ep made during this investigation can be used to estimate a value for ∆E. If the concentration of the ligand (L) and p are available, it is then possible to estimate the value of KL. If the natural abundance and stoichiometry of the complexing groups on the humic material were Environments 2020, 7, 94 5 of 18 well-characterized, it would be possible to estimate the magnitude of the stability constant for a complex formation for Pb2+ with the humic material. It is tempting to use the dissociable proton content of humic materials as an estimate of the abundance of complexing groups; however, past research has indicated that not all of these groups are available to complex metals [3,16,17]. Furthermore, many of the humic substance-binding sites are likely to be multidentate, and some binding may involve the nonspecific accumulation of counter ions in the vicinity of the negatively charged humic substance [3,18–20]. Using the stability constants imbedded in Visual Minteq [21] and the conditions of these experiments, we calculated the speciation of Pb2+. At pH 8, and in the absence of humic matter, 2+ 2+ + Pb would exist primarily as Pb , PbOH and Pb(OH)2 (44.7%, 53.9% and 1.31%), respectively. The HEPES, MOPS and PIPES buffering agents utilized were selected because of their minimal interactions with metal species and should not impact this distribution [22]. The shift in Ep that we measured represents a change in Pb2+ speciation, where there is some hydrolysis to a ligated form of Pb2+. The ligated species may be mono- or multidentate and could involve mixed ligand complexes as well. The differential DPP method was tested with two low molecular weight humic acid analogs (3,4-dihydroxybenzoic acid and salicylic acid) and a polyacrylic acid sample of 5100 g/mol. The and functionalities on these compounds are similar to the ligating functionalities on humic materials [23–25]. 3,4-dihydroxybenzoic acid was utilized in this study as a proxy for humic materials. The effect of this on the reduction of Pb2+ (2–10 uM) was examined at three different pH values, and the results are presented in Figure1. The concentration of 3,4-dihydroxybenzoic acid was 1.25 mM for all of these measurements. This ligand has been reported to react with various metals through the ring hydroxyl groups [26]. The pH of the solution was maintained with 1 mL of buffer in 15 mL of sample. The impact of this ligand and pH on the reduction of Pb2+ is clear. The pH 5 sample 2+ showed minimal shift from the reduction potential of uncomplexed Pb , and the observed Ep was very close to a ligand-free buffer solution. Measurements at pH 6.15 and 7.84 showed a shift toward a more negative voltage. The greater shift for the pH 7.84 measurements was consistent with the increased ionization of the ring hydroxyl groups, making a complex formation more favorable. On the other hand, Khayat et al. [27] examined the interaction of 3,4-dihydroxybenzoic acid and Pb2+ using a potentiometric approach at much higher Pb2+ and ligand concentrations. In their study, a precipitate was formed at pH 3 to 4.5 that appeared to be consistent with the formation of a complex through the ionized carboxylic acid group. Khayat et al. [27] did note that the precipitate appeared to dissolve at a higher pH, with the possible formation of a strong complexation through the phenolic groups. Salicylic acid has been used by other investigators as a low molecular weight humic acid analog [23–25]. The effect of salicylic acid on the reduction of lead was, therefore, investigated. Since the sodium salicylate is fairly soluble, it was possible to vary the concentration over a fairly large concentration range and, therefore, test the prediction of the Lingane equation. Measurements at two different pH values are summarized in Figure2 and illustrate that the salicylate 2+ complexation can shift the reduction potential for Pb to more negative values. It is interesting that the Ep at pH 6.15 and 7.84 are very close. This observation possibly indicates that the phenolic hydroxyl group of 2+ salicylic acid does not ionize when the Pb salicylate (Sal) complex forms. The shift in the Ep increased with the logarithmic concentration of salicylic acid in the solution, as predicted by the Lingane equation. The slopes of the two lines are 34.4 and 36.8 mV/log (Sal), respectively. This differs from the slope predicted by the Nernst equation (29.6 at 25 ◦C) but is still reasonably close. There are very few measurements of the stability constants between salicylate and Pb2+ in the literature. Furia and Porto [28] investigated the interaction of Pb2+ with hydrogen salicylate using potentiometric titration. These investigators measured the stability constants at a rather high ionic strength (3-M KClO4) and extrapolated the constant to 0 ionic 2 strength. Their results indicated that, at pH 7.84, PbSal and Pb(Sal)2 − should be the dominant species. Using the Furia and Porto [28] stability constants and a variant of the DeFord Hume equation [29–31], which is similar to the Lingane equation, we calculated that the shift in reduction potential should be Environments 2020, 7, x FOR PEER REVIEW 5 of 18

binding may involve the nonspecific accumulation of counter ions in the vicinity of the negatively charged humic substance [3,18–20]. Using the stability constants imbedded in Visual Minteq [21] and the conditions of these experiments, we calculated the speciation of Pb2+. At pH 8, and in the absence of humic matter, Pb2+ would exist primarily as Pb2+, PbOH+ and Pb(OH)2 (44.7%, 53.9% and 1.31%), respectively. The HEPES, MOPS and PIPES buffering agents utilized were selected because of their minimal interactions with metal species and should not impact this distribution [22]. The shift in Ep that we measured represents a change in Pb2+ speciation, where there is some hydrolysis to a ligated form of Pb2+. The ligated species may be mono- or multidentate and could involve mixed ligand complexes as well. The differential DPP method was tested with two low molecular weight humic acid analogs (3,4- dihydroxybenzoic acid and salicylic acid) and a polyacrylic acid sample of 5100 g/mol. The carboxylic acid and phenol functionalities on these compounds are similar to the ligating functionalities on humic materials [23–25]. 3,4-dihydroxybenzoic acid was utilized in this study as a proxy for humic materials. The effect of this organic acid on the reduction of Pb2+ (2–10 uM) was examined at three different pH values, and the results are presented in Figure 1. The concentration of 3,4-dihydroxybenzoic acid was 1.25 mM for all of these measurements. This ligand has been reported to react with various metals through the ring hydroxyl groups [26]. The pH of the solution was maintained with 1 mL of buffer in 15 mL of sample. The impact of this ligand and pH on the reduction of Pb2+ is clear. The pH 5 sample showed minimal shift from the reduction potential of uncomplexed Pb2+, and the observed Ep was very close to a ligand-free buffer solution. Measurements at pH 6.15 and 7.84 showed a shift toward a more Environments 2020, 7, 94 6 of 18 negative voltage. The greater shift for the pH 7.84 measurements was consistent with the increased ionization of the ring hydroxyl groups, making a complex formation more favorable. On the other 2+ closerhand, to a factorKhayat of et two al. times[27] examined greater atthe pH interaction 7.84 than of what 3,4-dihydroxybenzoic we observed. Furthermore, acid and Pb there using should a potentiometric approach at much higher Pb2+ and ligand concentrations. In their study, a precipitate be a significant difference between pH 6.15 and pH 7.84. The calculation indicated that the shift for pH Environmentswas formed 2020 at, 7pH, x FOR 3 to PEER 4.5 REVIEWthat appeared to be consistent with the formation of a complex through6 of 18 7.84 should be about four times greater than for pH 6.15. Clearly, the salicylate ligation of Pb2+ requires the ionized carboxylic acid group. Khayat et al. [27] did note that the precipitate appeared to dissolve additionalat a higherFigure investigation. 1.pH, Differential with the pulsepossible polarography formation (D ofPP) a strong results complexationfor the reduction through of Pb2+ thein the phenolic presence groups. of 1.25 mM 3,4-dihydroxybenzoic acid at three different pH values. The peak maximum (Ep) becomes increasingly negative-0.32 as the pH increases.

Salicylic acid has-0.34 been used by other investigators as a low molecular weight humic acid analog [23–25]. The effect of salicylic acid on the reduction of lead was, therefore, investigated. Since the

sodium salicylate is-0.36 fairly soluble, it was possible to vary the concentration over a fairly large concentration range and, therefore, test the prediction of the Lingane equation. Measurements at two different pH values are summarized in Figure 2 and illustrate that the -0.38 salicylate complexation can shift the reduction potential for Pb2+ to more negative values. It is Ep interesting that the Ep at pH 6.15 and 7.84 are very close. This observation possibly indicates that the -0.40 phenolic hydroxyl group of salicylic acid does not ionize when the Pb2+ salicylate (Sal) complex forms. The shift in the Ep increased with the logarithmic concentration of salicylic acid in the solution, as predicted by the Lingane-0.42 equation. The slopes of the two lines are 34.4 and 36.8 mV/log (Sal), respectively. This differs from the slope predicted by the Nernst equation (29.6 at 25 °C) but is still reasonably close. There-0.44 are very few measurements of the stability constants between salicylate and Pb2+ in the literature. Furia and Porto [28] investigated the interaction of Pb2+ with hydrogen salicylate using potentiometric-0.46 titration. These investigators measured the stability constants at a rather high 0246810 ionic strength (3-M KClO4) and extrapolated the constant to 0 ionic strength. Their results indicated Pb(uM) that, at pH 7.84, PbSal and Pb(Sal)22− should be the dominant species. Using the Furia and Porto [28] stability constants and a variant of thepH DeFord 7.84 Hume equation [29–31], which is similar to the Lingane pH 6.15 equation, we calculated that the shiftpH in 5 reduction potential should be closer to a factor of two times

greater at pH 7.84 than what we observed. Furthermore, there should be a significant difference Figurebetween 1. pHDiff 6.15erential and pulsepH 7.84. polarography The calculation (DPP) indicated results for that the the reduction shift for ofpH Pb 7.842+ in should the presence be about of 2+ 1.25four mMtimes 3,4-dihydroxybenzoic greater than for pH acid 6.15. at threeClearly, diff erentthe salicylate pH values. ligation The peak of Pb maximum requires (E padditional) becomes increasinglyinvestigation. negative as the pH increases.

FigureFigure 2. DPP 2. DPP results results for for Pb 2Pb+ 2+in in the the presence presence ofof a range of of sali salicyliccylic acid acid concentrations. concentrations. There There was was little difference in the results for pH 6.15 and pH 7.85. The linear relationship between Ep and log little difference in the results for pH 6.15 and pH 7.85. The linear relationship between Ep and log (salicylic(salicylic acid) acid) is consistent is consistent with with the the Lingane Lingane equation. equation.

Environments 20202020,, 77,, 94x FOR PEER REVIEW 7 of 18

Previous workers have considered polycarboxylic acids to be viable models for humic Previous workers have considered polycarboxylic acids to be viable models for humic substances substances and have examined the interaction of metal cations with polycarboxylic acids of various and have examined the interaction of metal cations with polycarboxylic acids of various molecular molecular weights [32]. In recent years, this polymer view of humic substances has been challenged weights [32]. In recent years, this polymer view of humic substances has been challenged by several by several groups [33,34]. Nevertheless, we examined the interaction of Pb2+ with 100-mg/L sodium groups [33,34]. Nevertheless, we examined the interaction of Pb2+ with 100-mg/L sodium polyacrylate polyacrylate of 5100 g/mol at both pH 6.12 and pH 8. The results presented in Figure 3 show little of 5100 g/mol at both pH 6.12 and pH 8. The results presented in Figure3 show little di fference between difference between the two pH values. In addition, the presence of the 1-M NaNO3 background the two pH values. In addition, the presence of the 1-M NaNO background electrolyte made little electrolyte made little difference in the negative shift of the Pb3 2+ reduction peak (results are not difference in the negative shift of the Pb2+ reduction peak (results are not shown). shown).

-0.30

-0.32

-0.34

-0.36

-0.38 p

E -0.40

-0.42

-0.44

-0.46

-0.48

-0.50 024681012 Pb2+ (uM) V pH 8 V pH 6.12 V (buffer only)

2+ Figure 3. DPP results for Pb 2+ inin the the presence presence of of 100-mg/L 100-mg/L polyacry polyacrylate.late. There There is is little little difference difference in the results for pH 6.126.12 andand pHpH 8.8.

The small didifferencefference in results for the twotwo pHpH valuesvalues may indicate that there is little change in the ionizationionization of thethe carboxylatecarboxylate groupsgroups betweenbetween pH 66 andand 8.8. The carboxylatecarboxylate concentration of the polyacrylate shouldshould bebe about 10.6 mMmM/g./g. This is somewhat larger than the carboxylate concentrations of the humic acids examined in this study but notnot largelylargely so.so. The lack of pH dependencedependence probably results from thethe absenceabsence ofof phenolicphenolic groupsgroups inin polyacrylate.polyacrylate. The Pahokee peat peat humic humic acid acid and and Pb Pb2+ 2interaction+ interaction was was examined examined at four at four different different pH values pH values and andat different at different Pb2+ concentrations. Pb2+ concentrations. The Pahokee The Pahokee peat humic peat acid humic concentration acid concentration was 100 wasmg/L, 100 and mg the/L, 2+ 2+ andresults the are results presented are presented in Figure in 4. Figure As expe4.cted, As expected, the impact the of impact humic of acid humic on the acid Ep on for the Pb E pincreasedfor Pb increasedwith the withpH, thewhich pH, increased which increased the ionization the ionization of the of humic the humic acid acid and and the the availability availability of of metal 22++ complexing ligands. It was also notednoted thatthat thethe shiftshift inin EEpp decreaseddecreased asas thethe PbPb concentration in the solution was increased. This concentration dependence for E p indicatesindicates that that there there is a spectrum of binding interactions of Pb22++ withwith the the humic humic material, material, with with the the more stable complexes formed at low 22++ Pb concentrations.concentrations. We We also also observed observed that that the the shift shift in in the E p waswas a function of the concentration of humic acid (Figure5 5).).

Environments 2020, 7, 94 8 of 18 Environments 2020, 7, x FOR PEER REVIEW 8 of 18 Environments 2020, 7, x FOR PEER REVIEW 8 of 18

Figure 4. DPP results for Pb2+in the presence of 100-mg/L Pahokee peat humic acid at pH 5, 6.15, 6.76 Figure 4. DPPDPP results for Pb 22++inin the the presence presence of of 100-mg/L 100-mg/L Pahokee Pahokee peat peat humic humic acid acid at at pH pH 5, 5, 6.15, 6.76 and 7.84. and 7.84.

2+ FigureFigure 5. 5. DPPDPP results results for for Pb Pb2+ atat pH pH 88 in in the the presence presence of of varyin varyingg concentrations concentrations of of the the Pahokee Pahokee peat peat Figure 5. DPP results for Pb2+ at pH 8 in the presence of varying concentrations of the Pahokee peat humichumic acid. acid. humic acid. These experiments with the Pahokee peat humic acid were conducted at pH 8 in a 0.100-M These experiments with the Pahokee peat humic acid were conducted at pH 8 in a 0.100-M HEPESThese buff experimentser with 1.0-M with NaNO the3 Pahokeeas a background peat humic electrolyte. acid were These conducted results illustrateat pH 8 in that a the0.100-M shift HEPES buffer with 1.0-M NaNO3 as a background electrolyte. These results illustrate that the shift in HEPES buffer with 1.0-M NaNO3 as a background electrolyte. These results illustrate that the shift in reduction potential for the Pb2+ humic acid complex is a function of the humic acid concentration. reduction potential for the Pb2+ humic acid complex is a function of the humic acid concentration.

Environments 2020, 7, 94 9 of 18

Environments 2020, 7, x FOR PEER REVIEW 9 of 18 in reductionEnvironments potential 2020, 7, x FOR for PEER the PbREVIEW2+ humic acid complex is a function of the humic acid concentration.9 of 18 These results are plotted in Figure 6 as a function of the Log of the humic acid concentration in mg/L These results are plotted in Figure6 as a function of the Log of the humic acid concentration in mg /L forThese three resultsdifferent are Pb plotted2+ concentrations. in Figure 6 as The a function linearity of ofthe these Log ofplots the conformshumic acid to concentration the general form in mg/L of the 2+ forLingane threefor three di equation.fferent different Pb Pbconcentrations.2+ concentrations.The The linearitylinearity of of these these plots plots conforms conforms to the to thegeneral general form form of the of the LinganeLingane equation. equation.

2+2+ FigureFigureFigure 6. 6.DPP 6.DPP DPP results results results for for for Pb Pb Pb2+ atat at pH pH 88 plottedplotted as as a a functionfunction function of of of th th theee log log log of of ofthe the the Pahokee Pahokee Pahokee peat peat peathumic humic humic acid acid acid 2+2+ concentrationconcentration (mg (mg/L)/L) at threeat three di differentfferent concentrations concentrations of of Pb Pb2+(( 1.611.61 uM, uM,  N4.824.82 uM uM and and  9.63H 9.63 uM). uM). concentration (mg/L) at three different concentrations of Pb (• 1.61 uM,  4.82 uM and  9.63 uM). A similar observation is illustrated in Figure 7 for Pb2+2 +in the presence of a commercially available A similarA similar observation observation is is illustrated illustrated in in Figure Figure7 7 for for Pb Pb2+ inin the the presence presence of of a commercially a commercially available available TeraVita leonardite humic acid. These experiments were conducted at pH 8 in 0.100-M HEPES buffer TeraVitaTeraVita leonardite leonardite humic humic acid. acid. These These experimentsexperiments were conducted conducted at at pH pH 8 8in in 0.100-M 0.100-M HEPES HEPES buffer buff er with 1.0-M NaNO3 as a background electrolyte. It is clear that the shift of the reduction potential for withwith 1.0-M 1.0-M NaNO NaNO3 as3 as a a background background electrolyte.electrolyte. It is is clear clear that that the the shift shift of of the the reduction reduction potential potential for for 2+ Pb2+ is a function of the humic acid concentration. PbPb2+is is a a function function of of the the humic humic acidacid concentration.concentration.

Figure 7. DPP results for Pb2+ at pH 8 in the presence of TeraVita leonardite humic acid. The Ep value

shifts toward a more negative voltage with an increase in the humic acid concentration. 2+2+ FigureFigure 7. 7.DPP DPP results results for for Pb Pb atat pH 8 in the presence presence of of TeraVita TeraVita leonardite leonardite humic humic acid. acid. The The Ep E valuep value shifts shifts toward toward a morea more negative negative voltage voltage with with anan increaseincrease in the humic humic acid acid concentration. concentration.

Environments 2020, 7, x FOR PEER REVIEW 10 of 18

In Figure 8, the Ep values for Pb2+ in the presence of various humic acids, at a concentration of 100 mg/L, at pH 8 are compared. The HEPES buffer was 0.100 M, and the NaNO3 was 1.00 M. The 2+ resultsEnvironments indicate2020, 7the, 94 greatest shift in the Pb reduction potential was observed for the Elliot soil humic10 of 18 acid samples, and the lowest shift was observed for the Suwannee River natural organic matter. The two leonardite samples displayed roughly the same behavior. For all of the humic acids, the Ep shifts 2+ In Figure8, the E values for Pb2+ in the presence of various humic acids, at a concentration a more negative voltagesp at lower Pb concentrations. The Ep for the various humic acids did not correlateof 100 mg with/L, atthe pH average 8 are compared.molecular weight The HEPES or IHSS bu carboxylicffer was 0.100 acid and M, and phenolic the NaNO hydroxyl3 was contents. 1.00 M. 2+ ForThe example, results indicate according the greatestto results shift published in the Pb by thereduction IHSS, the potential Suwanee was River observed NOM for had the the Elliot highest soil phenolichumic acid content samples, yet andexhibited the lowest the least shift negative was observed Pb2+ reduction for the Suwannee potentials River [35]. natural organic matter. The twoWe leonarditefurther examined samples the displayed impact roughly of the the1.0-M same NaNO behavior.3 background For all of electrolyte the humic acids,on the the DPP Ep 2+ shifts a more negative voltages at lower Pb concentrations. The Ep for the various humic acids did not reduction Environmentspotentials. 2020 ,We 7, x FOR compared PEER REVIEW the results for the Elliot soil humic acid, the Suwannee10 of 18 River NOMcorrelate and with the theIHSS average leonardite molecular with and weight without or IHSS 1.0-M carboxylic NaNO3 acidin Figures and phenolic 9–11. The hydroxyl presence contents. of the 2+ backgroundFor example, electrolyte accordingIn Figure 8, thedid to E results p notvalues induce publishedfor Pb a in major the by presence the difference IHSS, of various the or Suwanee humicconsistent acids, River at impact a concentration NOM on had the of the reduction highest phenolic content100 mg/L, yet at pH exhibited 8 are compared. the least The negative HEPES buffer Pb2+ wasreduction 0.100 M, and potentials the NaNO [353 was]. 1.00 M. The potentials.results indicate the greatest shift in the Pb2+ reduction potential was observed for the Elliot soil humic acid samples, and the lowest shift was observed for the Suwannee River natural organic matter. The two leonardite samples displayed roughly the same behavior. For all of the humic acids, the Ep shifts a more negative voltages at lower Pb2+ concentrations. The Ep for the various humic acids did not correlate with the average molecular weight or IHSS carboxylic acid and phenolic hydroxyl contents. For example, according to results published by the IHSS, the Suwanee River NOM had the highest phenolic content yet exhibited the least negative Pb2+ reduction potentials [35]. We further examined the impact of the 1.0-M NaNO3 background electrolyte on the DPP reduction potentials. We compared the results for the Elliot soil humic acid, the Suwannee River NOM and the IHSS leonardite with and without 1.0-M NaNO3 in Figures 9–11. The presence of the background electrolyte did not induce a major difference or consistent impact on the reduction potentials.

Figure 8. 8. ComparisonComparison of of DPP DPP results results for for Pb2+ Pb at2+ pHat 8 pH in the 8 in presense the presense of 100 of mg/L 100 of mg the/L various of the various humic acids.humic acids.

We further examined the impact of the 1.0-M NaNO3 background electrolyte on the DPP reduction potentials. We compared the results for the Elliot soil humic acid, the Suwannee River

NOM and the IHSS leonardite with and without 1.0-M NaNO3 in Figures9–11. The presence of the background electrolyteFigure 8. Comparison did not of induce DPP results a major for Pb difference2+ at pH 8 in the or presense consistent of 100 impactmg/L of the on various the reduction humic potentials. acids.

Figure 9. Comparison of DPP results for Pb2+ at pH 8 in the presence of 100 mg/L Elliot soil humic

acid with and without 1.00-M NaNO3. 2+ 2+ Figure 9. ComparisonFigure 9. Comparison of DPP of results DPP results for Pbfor Pbat at pH pH 8 8 in inthe the presencepresence of of 100 100 mg/L mg Elliot/L Elliot soil humic soil humic acid acid with and without 1.00-M NaNO3. with and without 1.00-M NaNO . 3 Environments 2020, 7, 94 11 of 18 EnvironmentsEnvironments 2020 2020, ,7 7, ,x x FOR FOR PEER PEER REVIEW REVIEW 1111 of of 18 18

FigureFigureFigure 10. 10.10.Comparison ComparisonComparison of ofof DPP DPPDPP results resultsresults for forfor PbPbPb22+2++ atat pHpH 88 8 inin in thethe the presencepresence presence ofof of 100-mg/L100-mg/L 100-mg/ LSuwanneeSuwannee Suwannee RiverRiver River NOMNOMNOM with withwith and andand without withoutwithout 1.00-M 1.00-M1.00-M NaNO NaNONaNO333. .

2+ FigureFigureFigure 11. 11.11.Comparison ComparisonComparison of of DPPof DPPDPP results resultsresults for for Pbfor PbPbat2+2+ pHatat pHpH 8 in 88 the inin presencethethe presencepresence of 100-mg ofof 100-mg/L100-mg/L/L International InternationalInternational Humic SubstancesHumicHumic SubstancesSubstances Society (IHSS) SocietySociety leonardite (IHSS)(IHSS) leonarditeleonardite humicacid humic humic with acidacid and withwith without andand withoutwithout 1.00-M 1.00-M 1.00-M NaNO NaNO NaNO3. 33. .

Environments 2020, 7, 94 12 of 18 Environments 2020, 7, x FOR PEER REVIEW 12 of 18

OurOur previous work investigated the the impact impact of of soluble soluble organic organic matter matter in in arid soil extracts on 2+ coppercopper speciation [13]. [13]. DPP DPP results for Pb 2+ inin a a water water extract extract from from a a soil soil sampled sampled from from the the root root zone zone ofof a a creosote bush is shown in Figure 12.12. The The location location of of this this soil soil was was from from a a roadside roadside site site located located in in HalloranHalloran Springs, Springs, California, California, USA. USA. This This soil soil was discussed in Steinberg and Hodge [[13].13]. The The total organicorganic carbon carbon content content of of this soil sample water extract was was measured at at 35 mg/L. mg/L. Assuming that humichumic material is is approximately 50% 50% carbon carbon indicates indicates that that this extract would have approximately 70-mg/L70-mg/L humic material. This This creosote creosote soil soil extrac extractt was examined using the DPP method. The The results results 2+ areare similar to the humic materials examined in this study. The reduction potentialpotential ofof PbPb2+ inin these these soil soil extractsextracts shows similarsimilar pHpH dependencedependence to to that that observed observed in in the the humic humic acid acid solution. solution. In In addition, addition, the the Ep 2+ Epshift shift was was dependent dependent on the on Pb the concentration,Pb2+ concentration, indicating indicating a heterogeneous a heterogeneous distribution distribution of lead bindingof lead bindinggroups ingroups the extract. in the extract.

2+ FigureFigure 12. ComparisonComparison ofof DPP DPP results results for for Pb Pbin2+ thein presencethe presence of a creosoteof a creo soilsote extract soil atextract three diat ffthreeerent differentpH values. pH values.

TheThe results results from from size size exclusion exclusion chromatography chromatography of of the the various various humic humic materials materials are are shown shown in in Figure 13. The number-averaged (Mn) and weight-averaged (Mw) molecular weights derived Figure 13. The number-averaged (Mn) and weight-averaged (Mw) molecular weights derived from thefrom SEC the are SEC tabulated are tabulated in Table in 1. Table The1 .most The striki mostng strikingresults are results the large are the differences large di ffinerences number- in averagednumber-averaged vs. weight-averaged vs. weight-averaged molecular molecular weights and weights that andall of that the allsamples of the had samples rather had low rather average low molecularaverage molecular weights weightsand an andoverall an overallvery broad very broadmolecular molecular weight weight distribution. distribution. Furthermore, Furthermore, all of all the of humicthe humic materials materials examined examined contained contained a large a large contribution contribution from from low low molecular molecular weight weight components. components. These observations are consistent with the results of other studies [36–38]. The Ep results did not show These observations are consistent with the results of other studies [36–38]. The Ep results did not show any apparent correlation with Mn or Mw of the humic substance. any apparent correlation with Mn or Mw of the humic substance.

Table 1. The weight average (Mw) and number average (Mn) molecular weights for the humic Table 1. The weight average (Mw) and number average (Mn) molecular weights for the humic substances as determined by SEC (Size exclusion chromatography) with detection at 280 nm are substances as determined by SEC (Size exclusion chromatography) with detection at 280 nm are reported. Natural Organic Matter (NOM), TV: TeraVita, IHSS: International Humic Substances Society reported. Natural Organic Matter (NOM), TV: TeraVita, IHSS: International Humic Substances and HA: humic acid. Society and HA: humic acid.

Sample Mw Mn Sample Mw Mn LeonarditeLeonardite TeraVita TeraVita 1020 1020 175 175 Leonardite IHSS 2360 537 Leonardite IHSS 2360 537 Pahokee HA 2020 191 SuwanneePahokee NOM HA 1150 2020 191 648 Elliot SoilSuwannee HA NOM 9490 1150 648 1410 Elliot Soil HA 9490 1410

Environments 2020, 7, 94 13 of 18

Environments 2020, 7, x FOR PEER REVIEW 13 of 18

Environments 2020, 7, x FOR PEER REVIEW 13 of 18

FigureFigure 13. SEC 13. SEC results results for for humic humic materials materials utilized utilized in this investigations. investigations. All All of the of thehumic humic materials materials had a broad distribution of molecular weights with a significant contribution of materials that are less had a broad distribution of molecular weights with a significant contribution of materials that are less than 500 mW. than 500Figure mW. 13. SEC results for humic materials utilized in this investigations. All of the humic materials Thehad a observedbroad distribution Ep may of molecularbe partially weights a function with a significant of unknown contribution electrode of materials kinetic that factors are less and The observed Ep may be partially a function of unknown electrode kinetic factors instrumentalthan 500 mW.settings, as well as the reduction potential of the complexed species. Therefore, we andexamined instrumental the influence settings, of the as various well DPP as parameters the reduction on the position potential of the of observed the complexed reduction peak species. Therefore,for PahokeeThe we observed examined peat humic Ep the acidmay influence in be order partially ofto thegauge variousa thefunction magnitude DPP of parametersunknown of the influence electrode on the of position variouskinetic instrumental offactors the observedand reductionparametersinstrumental peak on for settings, the Pahokee observations. as well peat as humicAll the of reductionthese acid experi in potential orderments to were of gauge the done complexed the with magnitude 15 mL species. of the of Therefore, Pahokee the influence peat we of variousHAexamined instrumental 100 mg/L the influencein 100-mM parameters of HEPES the various on pH the 8DPP observations.and parameters 1.00-M NaNO Allon the3 of and theseposition with experiments 4.8-uM of the observedPb2+. wereFigure reduction done 14 indicates with peak 15 mL for Pahokee peat humic acid in order to gauge the magnitude of the influence of various instrumental of thethat Pahokee increasing peat the HA voltage 100 increment mg/L in 100-mM(ΔV) between HEPES mercury pH drops 8 and tends 1.00-M to yield NaNO a more3 and negative with E 4.8-uMp, parameters on the observations. All of these experiments were done with 15 mL of the Pahokee peat Pb2+.although Figure 14 there indicates was little that change increasing in the peak the voltage position incrementbetween the (∆ 2-V) and between 4-mV increments. mercury drops tends to HA 100 mg/L in 100-mM HEPES pH 8 and 1.00-M NaNO3 and with 4.8-uM Pb2+. Figure 14 indicates yield a more negative Ep, although there was little change in the peak position between the 2- and that increasing-0.470 the voltage increment (ΔV) between mercury drops tends to yield a more negative Ep, 4-mValthough increments. there was little change in the peak position between the 2- and 4-mV increments.

-0.470 -0.475

-0.475 -0.480 Ep

-0.480 Ep -0.485

-0.485 -0.490 02468101214 ΔV (mV) -0.490 02468101214 Figure 14. The influence of the DPP voltage increment (ΔΔVV) (mV)on the Ep value. The results are shown for Pahokee peat humic acid at 100 mg/L at a pH of 8 and in the presence of 1.0-M NaNO3. The Pb2+

concentration was 4.8 uM. Figure 14. The influence of the DPP voltage increment (ΔV) on the Ep value. The results are shown Figure 14. The influence of the DPP voltage increment (∆V) on the Ep value. The results are shown for Pahokee peat humic acid at 100 mg/L at a pH of 8 and in the presence of 1.0-M NaNO3. The Pb2+ for Pahokee peat humic acid at 100 mg/L at a pH of 8 and in the presence of 1.0-M NaNO . The Pb2+ concentration was 4.8 uM. 3 concentration was 4.8 uM.

Environments 2020, 7, 94 14 of 18 Environments 2020, 7, x FOR PEER REVIEW 14 of 18 The results presented in Figure 15 indicate that increasing the pulse amplitude leads to a less The results presented in Figure 15 indicate that increasing the pulse amplitude leads to a less negative Ep value. negative Ep value. Environments 2020, 7, x FOR PEER REVIEW 14 of 18

The results presented in Figure 15 indicate that increasing the pulse amplitude leads to a less negative Ep value.-0.450 -0.455 -0.460 -0.465-0.450 -0.470-0.455 -0.460 -0.475 Ep -0.465 -0.480 -0.470 -0.485-0.475 Ep -0.490-0.480 -0.495-0.485 -0.500-0.490 -0.4950 20406080100120 -0.500 Pulse Amplitude (mV) 0 20406080100120 Pulse Amplitude (mV) Figure 15. TheThe influence influence of the DPP pulse amplitude on the Ep value. The The results results are shown for Pahokee peat humic acid at 100 mg/L and a pH of 8 and in the presence of 1.0-M NaNO3. The Pb22++ PahokeeFigure peat 15. humic The influence acid at 100of the mg DPP/L and pulse a pHamplitude of 8 and onin the the Ep presence value. The of results 1.0-M NaNOare shown3. The for Pb concentration was 4.8 uM. concentrationPahokee peat was humic 4.8 uM. acid at 100 mg/L and a pH of 8 and in the presence of 1.0-M NaNO3. The Pb2+ concentration was 4.8 uM. The influence influence of the pulse period (drop time) is shown in FigureFigure 1616.. The shortest pulse period examinedThe had influence a less negativenegative of the pulse EEpp valueperiod than (drop the time) procedure is shown usedin Figure forfor sample16.sample The shortest analysis.analysis. pulse Pulse period periods examined had a less negative Ep value than the procedure used for sample analysis. Pulse periods from 1 to 3 s showed little change in the Ep.. from 1 to 3 s showed little change in the Ep. Pahokee IHSS HA 100 mg/L pH 8 100 mM Hepes 1M Pahokee IHSS HA 100 mg/L pH 8 100 mM Hepes 1M NaNO3NaNO3 4.84.8 uM PbPb -0.450-0.450

-0.455-0.455

-0.460-0.460

-0.465-0.465

-0.470 -0.470Ep Ep -0.475 -0.475 -0.480 -0.480 -0.485 -0.485 -0.490 -0.490 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 0.00 0.50 1.00 1.50Pulse Period 2.00(s) 2.50 3.00 3.50 Pulse Period (s) Figure 16. The influence of the DPP pulse period on the Ep value. The results are shown for Pahokee Figure 16. The influence of the DPP pulse period on the Ep value. The results are shown for Pahokee peat humic acid at 100 mg/L and a pH of 8 and in the presence of 1.0-M NaNO3. The Pb2+ concentration peat humic acid at 100 mg/L and a pH of 8 and in the presence of 1.0-M NaNO . The Pb2+ concentration Figurewas 16. 4.8 The uM. influence of the DPP pulse period on the Ep value. The results3 are shown for Pahokee waspeat 4.8humic uM. acid at 100 mg/L and a pH of 8 and in the presence of 1.0-M NaNO3. The Pb2+ concentration was 4.8 uM.

Environments 2020, 7, 94 15 of 18

It is clear that the observed Ep values are a function of the DPP conditions selected. Thus, caution in using a strictly thermodynamic interpretation of DPP observations is in order. Nevertheless, we used these results to estimate the magnitude of the Pb2+ humic acid stability constants. To form this estimate, we utilized Equation (6), along with the observed Ep from humic-free solutions, to estimate 2+ the value for a conditional logKL for the Pb binding by the various humic substances examined in this study. For the purposes of this calculation, we assumed that the shift in Ep from a humic-free to a humic-containing solution was equivalent to ∆E in Equation (6). We also assumed that p in Equation (6) was equal to 1. We estimated the molar concentration of the humic substance (L) by dividing the mass concentration by either Mn or Mw. Using these highly simplified assumptions, we rearranged Equation (6) into Equation (7) to produce an estimate for log KL. These estimates were performed with the results from lowest concentration of Pb2+ used in the study (1.6 uM). ! ∆Ep logKL 2 + plog[L] (7) ≈ − 0.0592

The results of this very preliminary calculation of conditional stability constants are reported in Table2. These calculations assumed a 1:1 stoichiometry for the Pb 2+ humic acid complex. The calculation ignores the heterogeneity of humic acids and the potential contribution of electrostatic effects to metal binding. It is quite possible that the binding capacity of these humic acids is much higher than this calculated molarity. If so, the estimated log KL value could be considerably reduced. The metal-binding capacity for humic substances has not been well-established. For example, Logan et al. [39] and Sahu and Banjeree [40] utilized ion-selective electrodes with a graphical analysis to estimate both the metal-binding capacities and metal humic stability constants. Both parameters were found to be pH-dependent.

Table 2. Estimated conditional logK for humic substances at pH 8.

Sample logK (Mw) logK (Mn) Elliot Soil 11.8 11.0 Leonardite IHSS 10.9 10.3 Pahokee Peat 10.8 9.8 Leonardite 9.5 8.7 TeraVita Suwannee NOM 8.1 7.8

Schnitzer and Skinner [41] and Schnitzer and Hansen [42] examined metal binding by a soil fulvic acid at low pH (5 and lower) using Schubert’s ion exchange method. The molecular weight (Mn) of the fulvic acid was estimated using vapor pressure osmometry. Using the estimated molecular weight and the method of continuous variation, Schnitzer and Hansen [42] determined that the complex involved a metal-to-fulvic acid ratio of 1:1 for Pb2+ and other divalent metals at an ionic strength of 0.1, although, at lower ionic strength, this ratio decreased. These authors indicated that, at a lower ionic strength, mixed or polynuclear complexes were likely forming. The Log KL (~6) values for Pb2+ estimated in these studies was much lower than those reported here; however, the pH of these measurements was considerably lower as well. The assumptions used for our estimates of the Log KL are consistent with Schnitzer and Hansen’s observation of 1:1 stoichiometry.

4. Conclusions

2+ Differential pulse polarography (DPP) measurements demonstrate a shift in Ep for Pb as a function of the organic matter content and pH. This shift, which is also a function of the Pb2+ concentration, is consistent with the formation of humate complexes that are reduced at a more negative potential than the free metal ion. The correlation of the shift with the concentration of humic acid also conforms to the predictions of the Lingane equation and likely indicates a rapid Environments 2020, 7, 94 16 of 18 equilibrium between complexed and uncomplexed Pb2+ species. The observed increased complexation with an increase in pH is consistent with the observation of other investigators. As noted, the shift in the Ep is dependent on the concentration of humic acid and decreases as the concentration of Pb2+ increases. This observation is consistent with the existence of a heterogeneous distribution of binding sites for Pb2+. It is clear from these results that the interpretation of DPP reduction potential shifts is complicated by the absorption of the Pb2+-humate complex on the electrode. This Pb2+–humate adsorption probably explains the negative shift of the Ep with the increase in pulse period (drop lifetime). In addition, we observed that the reduction current also increased with the pulse period (results not shown). We did some cyclic voltammetry (CV) studies (not shown) that showed that the signal increased with the deposition time, or the time elapsed between the mercury drop formation and the initiation of the CV scan. It is clear that the adsorption of the complex to the drop is a complication in the interpretation of the data. The humic acids selected for these experiments (with the exception of the TeraVita product) were obtained from the IHSS. The Ep observed for the various samples did not show any correlation with the reported carboxylic acid or phenolic acid contents of the samples. The molecular weights of these humic substances were examined using SEC with a method that was calibrated using polystyrene sulfonate samples. The observed number-averaged molecular weights (Mn) of the humic samples were all below 1000, with the exception of the Elliot soil. The Ep measurements did not show any clear relationship to the molecular weight. Furthermore, all of these substances showed a very broad distribution of molecular, with a large contribution from low molecular weight components. Our goal is to utilize the results from this analytical method, along with other measurements of complexation, to calibrate and possibly refine a humic acid model so that improved predictions for the fate and transport of Pb2+ in aquatic and soil environments will be possible.

Author Contributions: S.S. conceptualization, methodology, experimental measurements and writing—original draft preparation. V.H. writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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