**3. A single extraction study of a metal-polluted mine waste of Central Mexico**

### **3.1 Introduction**

In this study, we focused mainly on lowering the bioavailability and mobility of Cd, Cu, Pb, and Zn below official environmentally safe values and to warrant a biologically clean and sustainable ecosystem. To reach this goal, two schemes were visualized. First, we assayed the addition of widely used agronomic materials consisting of lime (Ca(OH)2); gypsum (CaSO42H2O); P-fertilizer (KH2PO4) and compost to "treat" a gradient of soil-fresh mine tailings mixtures to assess the treatment efficacy to abate the levels of the most toxic metal species available for plant growth; in solution, the free metal ion activity, (M2+)-value, for Cd2+, Cu2+, Pb2+, and Zn2+; and on the solid phase; the so-called DTPA-phytoavailable [17], and the acidsoluble fraction imposed by some international [22, 120] standards, and a national norm [23–28]; second, a bioassay was applied to find the conditions that allowed a sensitive indicator plant to grow in these "fertile" ameliorated media. Our studies proved to be useful in deriving soil-substrate quality criteria to establish specific strategies to verify the success of remediation processes. To evaluate the HM-toxicity abatement, both the bioavailable (acid-extractable) HM fraction and the chemical activity of the free metal ion, (M2+) were measured after incubation with the agrostabilizing treatments. Acid drainage was emulated using the standardized acetic acid extraction procedure required by norms [23–28] and standards [22, 121].

### **3.2 Water, DTPA, and acid-extractable heavy metal levels**

Extraction solutions to evaluate water-DTPA- and acid-extractable solutions consisted of 1) H2O-CO2 [24, 120] as saturation extract; 2) 1:2 ratio DTPA-extraction [17, 22], and 3) acetic acid (HAcO)-extraction [22], were used to obtain different species and fractions of metals from soil, mine waste, and mixtures. Extracts were analyzed for total dissolved Cd, Pb, Cu, and Zn by FAAS. Initial and in equilibrium (after incubation) extractable levels of metals, [M]HAcO, were determined at a 1:20 solid:liquid ratio in 0.2 M CH3COOH. Equilibrium-free HM ion activities, (M2+), were determined in the aqueous extracts of the treated mixtures by ASV [55, 56]. Calculations of (M2+) were carried out with MINEQL+ software [66, 67].

### **3.3 Experimental substrate mixtures and agrochemical treatments**

Enough total mass for speciation studies and bioassays of six different substrate systems were prepared by mixing soil and mine tailings at various ratios (w/w) to emulate different degrees of soil pollution as follows: A (100:0%), B (80:20%), C (60:40%), D (40:60%), E (20:80%), and F (0:100%) soil:mine waste material. The four agrochemical treatments tested consisted in adding lime [Ca(OH)2], gypsum [CaSO4�2H2O], P-fertilizer [KH2PO4], and compost [OM] at three different doses. Agronomic materials were stoichiometrically formulated according to the initial sum of the Cd, Cu, Pb, and Zn extractabilities in 0.2 N HAcO (highest dose), DTPA (medium dose), and H2O-CO2 (lowest dose). Compost dose was added to reach 5, 10, and 20% (w/w) of OM. Blank and treated mixture systems were incubated for three weeks to reach equilibrium, adding water to keep a 1:2.5 solid:liquid ratio. For Pfertilizer, the stoichiometric addition also considered the amount of exchangeable Ca2+ levels. This test helped to discriminate treatments that efficiently decreased the HM-extractable contents from those shown by the untreated blank mixtures.

### **3.4 Toxicity bioassays**

This bioassay was carried out only for the PO4 and OM treatments following international standard instructions (ISO 1993 [29], ISO 2005 [30]). At least seven barley (*Hordeum vulgare*) plants per experimental unit were grown in 100 mL black conical plastic pots (max/min radii of 3.7/1.9 cm and 5 cm height) to contain ca. 140 g of material of each of the six soil-mine waste mixtures, including the four ameliorating treatments and the three doses to give a total 72 pot systems. Root length was measured [32] and statistically analyzed with a 95–99% Fisher test of significance against the HM extracted from the mixture systems as an indicator to evaluate the efficacy of treatments and doses to lower the HM toxic effects and to assess the cleanness of the treated polluted mixtures.

### **3.5 Results**

The presence of high amounts of Pb and Zn is common in Zimapan and they are found in combination with As, mainly in minerals of arsenopyrite (AsFeS), scorodite (FeAsO4�2H2O), and in association with pyrrhotite (Fe1-xS), pyrite and marcasite (FeS2), sphalerite (ZnS) and galena (PbS), very common minerals in the area of Zimapan [3] such that As-levels are within the reported values for this element in soils which were 19–17,384 ppm in Mexico [121–124] and within 5200–40,853 ppm in mine tailings of Zimapan [3, 122, 125]. Regarding the four metals of interest levels found were within the reported values for soils 15–7200 ppm for Cu, 31–3400 ppm for Pb, and 26–6270 ppm for Zn [68, 122, 123], whereas for mine wastes in the country, our results were within the reported ranges of 186–2787 ppm, 910–9500 ppm, and 2218– 32,400 for the same metals, respectively [3, 121, 122]. Official Mexican regulations [23] established levels of Cd and Pb in soils of the order of 100–300 and 3–5 mg kg�<sup>1</sup> , respectively, as hazardous to crops. These limits were not exceeded in the soil sample extracts obtained with 0.2 M HAcO. Regarding Cu and Zn, levels higher than 0.2 mg kg�<sup>1</sup> and 1.0 mg kg�<sup>1</sup> , respectively, are reported as adequate for these micronutrients [23]. Accordingly, levels of Cd and Pb in mine tailing must not exceed 24 mg kg�<sup>1</sup> and 120 mg kg�<sup>1</sup> , respectively, in the aqueous and/or HAcO extracts, so that Pb is not within the allowable levels when extracted with HAcO 0.2 M [26].

Zn and Cu are not potentially toxic elements regulated by Mexican official norms. The efficacy of the agronomic treatments was evaluated by comparing the initial and final quantities of the studied metals, based on the acid-extractable fraction for each experimental mixture. **Figure 2** shows, in contrast with reported values, which found more than 87% decrease of the HCl-extractable concentrations of Cd, Cu, Pb, and Zn in polluted soils, after a combined CaCO3-CaHPO4 stabilizer was added [126], in our studies when agricultural lime and gypsum were applied, the [M]AcO-extracted did not show a significant HM level decrease, with respect to their initial concentrations, as compared with controls (see gypsum and lime graphs in **Figure 2**), moreover, redissolution process was observed for all metals except for Pb in the case of gypsum, and with some tendency to positive results for the E and F systems for Cu, Pb, and Zn. For Cd, gypsum worked well only when the soil fraction dominated (systems A, B, and C at lowest dose) but, in general, without significant differences between blank and treatments (F-test, 95%). In contrast with lime and gypsum, P-fertilizer showed excellent results (see **Figure 2** at lower left) when suppressing the acid-extractable levels of Cd, Pb and Zn at any dose getting for the latter diminutions of 92% of initial quantities. For Pb the lowest dose showed a biphasic behavior indicating there exist two distinct sites for sorption which agrees with results found elsewhere [127]. For Cu, a significant decrease of [M]AcO was observed only when the dose and mine

### **Figure 2.**

*Effect of adding ameliorating materials lime, Ca(OH)2 (upper left), gypsum, CaSO4*�*2H2O (upper right), Pfertilizer, KH2PO4 (lower left) and OM-compost (lower right), at low, medium, and high doses, over the 0.2 M acid-extractable levels (mg kg*�*<sup>1</sup> ) of Cd, Cu, Zn, and Pb (Y-axis), for six experimental mixtures soil:mine waste (s:mw): A: 100% soil, B: 80:20 s:mw, C: 60:40 s:mw, D: 40:60 s:mw, E: 20:80 s:mw, F: 100% mw. Curves show in yellow-orange the blank treatment (no ameliorating material); blue the low dose (ameliorating materials added based on the sum of the four concentrations of water-soluble metals); purple the medium dose (based on the sum of the four concentrations of the DTPA-extractable metals); and red the high dose (based on the sum of the four concentrations of the acid-extractable metals). For OM-compost, low, medium, and high doses were added to reach 5% (low dose), 10% (medium), and 20% (high dose) OM levels (w/w-basis), for the water-soluble, DTPAextractable, and acid-extractable metals, respectively.*

tailing contents were highest for systems C to F (F-test, 95%). **Figure 2** also reveals that compost showed the best results of all amending materials where HM level suppression was more homogeneous. For Cd and Zn this treatment showed a significance reduction of the extractable metal levels at the three doses tested, although results for Zn were much more pronounced. For Cu and Pb the decrease of the extractable metal where mine tailing material was higher (systems D to F) the abatement was significant with respect to the blank system A. However, where the soil was pure or slightly polluted (systems A to C) the effect was not significant, especially for Cu-lowest and medium doses where even the metal extractability increased. For Pb, only the highest and medium doses showed some efficacy in suppressing these values. Note again the biphasic sorption for Cu and Pb at all doses, but more pronounced at the lowest one. These results completely correspond with those obtained by other authors [128–130]; who added composts, biosolids, manure, and peat materials effectively reducing Cd, Pb, and Zn mobility. These results were also consistent with the aqueous free metal, [M2+] ac, levels determined by ASV (not shown). Increment in the doses produced an important drop in the activity of this toxic chemical species even in the pure tailing systems, obtaining, in the best case, a diminution of three to five orders of magnitude orders, for example, Cu and Cd system-E treated with OM, respect to control.

### **3.6 Biotoxicity assays**

Toxicity bioassay systems (A to F) and the P-fertilizer and compost treatments were tested at the medium doses, with the only intention of evaluating if there was a chance for a positive response when applying these amending materials and the indicator-sensitive plant could prove a fertile non-toxic media was created. Root length was the agronomic parameter measured [29, 30]. Fisher test was applied to root length (95% significance) to see differences among soil-mine waste mixture treatments (OM and P-fertilizer) and doses (low, medium, and high). **Figure 3** shows the results of these analyses. The effect of treatments including the null one was

### **Figure 3.**

*Histograms show the root length (cm) of barley (*Hordeum vulgare) *plants as affected by treatments. Upper row shows the results for the blank and compost added; lower row shows the results for blank and P-fertilizer, KH2PO4 added, both at low, medium, and high doses, respectively. Different lower-case letters mean significant differences among treatments, according to Fisher's test of 95% significance. Low, medium, and high PO4-doses were added based on the sum of the four concentrations of the water-soluble, DTPA-, and acid-extractable metals, respectively). For OM-compost, low, medium, and high doses corresponded to 5%, 10% and 20% (w/w-basis), respectively.*

investigated in the six soil:mine waste systems to evaluate the effect of mine waste incorporation and predominance in these emulated scenarios of polluted soils.

**Figure 3** shows that roots growing in the different mixture systems were considerably affected because of the increasing content of mine waste material added to the pristine soil, having a shortage of more than 75% of the length, when exposed to pure mine waste (blank-mixture F), respect to the pure soil (blank-mixture A). With the presence of P-fertilizer or OM treatments, a significant increment in root length with respect to control systems (PO4- and OM-mixture F) was observed, especially for the pure mine tailing mixtures (PO4-mixture F), where the highest doses improved the growth remarkably. The addition of P-fertilizer in medium and high doses was effective in providing a good media for the growth of the sensitive plant in pure mine tailing whereas compost as shown by the good response of root growth when the dose was highest. Based on these findings, it results clear the DTPA- and the acidextractable levels of Cd, Cu, Pb and Zn, gave a good indication of the phytoavailability-phytotoxicity levels being suppressed by the ameliorating material added. PO4-medium and high doses effectively corrected most of the growing problems shown on the blank (not amended) treatment and on the low doses of PO4 added which was based on the sum of all four water-extracted metals. For the OM-compost treatments, it is important to note that low, medium, and high OM levels were chosen based on what is recommended for optimum growth of plants, according to what FAO and other OM classifications suggest which consider 5–6% as the minimum good OMlevel to improve soil fertility [23].
