Bioavailability and Solubility of Heavy Metals and Trace Elements during Composting of Cow Manure and Tree Litter

Objectives. To characterize the total content of heavymetals (Cd, Cr, Cu, Hg,Mo, Ni, Pb, and Zn) and trace elements (As and Se) of interest, their bioavailability and solubility during the composting of cow manure and tree litter in piles inoculated with beneficial microorganism (IBM), or not inoculated (NBM), on the university campus of the Universidad Nacional Agraria La Molina (UNALM).Methods.)e investigation evaluated composting in six piles, three IBM piles and three NBM piles, for 120 days. Every 30 days, a composite sample was taken from each pile. )e raw materials used were cattle manure and tree litter. )e variables analyzed were the total concentrations (content) of metals and trace elements of interest, extracted with diethylenetriaminepentaacetic acid/pentatetic acid (DTPA) (for the bioavailability study) and extracted with deionized water (for the solubility study). Results. )e average values (n� 3) of the total content (mg·kg) on day 120 in IBM were in the following order: Zn (404.33)>Cu (86.33)>Pb (71.2)>Cr (34.33)>As (28.0)>Ni (13.83)>Mo (2.86)> Se (1.38)>Cd (1.32)>Hg (0.39) and in NBM: Zn (466.0)>Cu (112.23)>Pb (73.23)>Cr (35.33)>As (29.67)>Ni (14.37)>Mo (3.23)> Se (1.55)>Cd (1.38)>Hg (0.38). )e values complied with the Austrian Compost Standard for Landscaping and Land Rehabilitation and the Peruvian Standard for Nonorganic Agriculture. Significant differences were observed in some elements (p< 0.05) between 0 and 120 days in both types of pile (IBM and NBM). Conclusions. For nonorganic agriculture in Peru, urban compost is recommended, whereby raw organic materials are inoculated with beneficial microorganisms during the composting process to reduce the bioavailability of Cu and Zn, as these elements showed less bioavailability than noninoculated piles. )e composting process for both piles (IBM and NBM) reduced the solubility in Cu, Pb, and Zn.


Introduction
e heavy metals present in plants and their bioavailability are a concern because of the multiple adverse effects on human health that these chemical elements have [1][2][3] as well as carcinogenic and neurological effects [4,5]. e heavy metals present in plants in urban areas come mainly from air pollution generated by burning fuel and emissions from the manufacturing industry [6][7][8][9]. Other forms of contamination include irrigation water [10], inorganic fertilizer [11], and organic fertilization with compost [12,13]. e world's population has migrated mainly from rural areas to urban areas [14], thus increasing the demand for food in the latter. Due to this need, the practice of urban agriculture is recommended [15] to produce plant food. However, research has shown that there is contamination in crops in these areas [8,16].
Compost is an amendment that is used in urban agriculture [15], especially in poorer areas, because it is a lowcost technique for recycling organic matter and is easy to handle [17]. Compost has the virtue of being a soil improver and also provides nutrients to plants [18,19]. However, due to the raw materials used, it can contain significant contents of heavy metals [18,[20][21][22][23][24]. It is known that green spaces with arboreal vegetation in an urban environment retain heavy metals in the foliage due to the precipitation of atmospheric dust produced mainly by the use of fossil fuels by vehicular traffic [7,[25][26][27][28][29][30][31]. It is also known that cattle manure contains significant amounts of some heavy metals [32][33][34][35][36] which is due to the fact that cattle feed has nutrients fortified with these elements and that, in some cases, the quantities supplied are greater than the absorption in the digestive process, whereby these are excreted [34,35].
Humic and fulvic acids are generated during the composting process, which can influence the mobility of metals and trace elements, thus reducing their bioavailability for plants [24,[37][38][39]. e total content of heavy metals and trace elements analyzed during the composting process is not an adequate indicator of their bioavailability [18,21]. erefore, it is important to use an extractant such as pentatetic acid (DTPA) to determine their bioavailability [21,40] or deionized water to determine their solubility [24,37,41,42].
Inoculation with beneficial microorganisms (BMs) is a bioremediation technique used in areas with soil contaminated by heavy metals whereby their bioavailability and solubility are reduced [43,44]. Due to the presence of these polluting elements in the raw materials used in composting in urban areas, it could be a useful technique to reduce their availability in the compost produced [45]. It is known that BM can improve the composting process, reducing the required time and increasing the contents of macroelements such as N, P, and K [46,47]. One of the metabolic properties of these organisms is that they can promote changes in the speciation of metals towards less polluting forms, and they can adsorb them on their own structures [48]. BMs are considered to be microbial consortia made up of yeasts and bacteria that increase the metabolism of the degradation of the raw organic components of the composting piles, especially the lignocellulose elements, towards more assimilable intermediates in the food chain of the microbiological ecosystem [49]. e objective of this investigation was the characterization of the total content of heavy metals and trace elements of interest, as well as their bioavailability and solubility, during the composting of cow manure and tree litter in piles inoculated, or not inoculated, with beneficial microorganisms on the university campus of the Universidad Nacional Agraria La Molina (UNALM).

Location.
e university campus of UNALM is located in the district of La Molina, in the province of Lima, department of Lima, in Peru. It is at an altitude of 246 meters above sea level, and the average temperature is between 15 and 26°C ( Figure 1).

Raw Materials for the Investigation Stages.
e research had two distinct stages. e characterization stage analyzed the total contents of heavy metals, Cd, Cr, Cu, Hg, Mo, Ni, Pb, and Zn, and of trace elements, As and Se, of interest (HM&TE) in the products from co-composting cow manure (COW-M) and grass clipping (GRASS-C) and from composting of tree litter (TREE-L), two types of compost that are produced on the UNALM campus. e evaluation stage investigated the total contents, bioavailability, and solubility of the HM&TE during the composting of COW-M and TREE-L in piles inoculated with beneficial microorganisms (IBMs) and in piles which were not inoculated with microorganisms (NBM). e piles were of pyramidal form, 0.8 m high with a 3 m base for the co-composting (manure and vegetal material) and 1.5 m high with a 5 m base in the case of the composting (only tree litter). Table 1 shows the raw materials used, their weights, proportion, and composting days.

Characterization of the Raw Materials of the Evaluation Stage.
For the evaluation stage, physicochemical variables and the total contents of heavy metals and trace elements of interest (HM tot ) were recorded in the raw materials prior to the beginning of the composting process, as shown in Table 2. Most of the physicochemical values for COW-M were higher than for TREE-L, except for humidity (H%) and ammoniacal nitrogen (NH4 + -N). e organic matter (OM) content was 26.6% higher in COW-M than in TREE-L. e electrical conductivity (EC) in the COW-M was higher, due to the type of cattle feed. Similar results were found in Cáceres and Marfa [50]. TREE-L concentrations for most HM tot exceeded COW-M, except for Cu, Se, and Zn. e total Pb content in the TREE-L was more than five times the value in COW-M. Ahmed et al. [51] also found an important difference between Pb in poultry manure (1.3 mg·kg −1 ) and in sawdust (16.0 mg·kg −1 ), more than twelve times the value. Soobhany et al. [52] obtained average total concentrations of Cu and Zn lower values in leaves and dry branches (84.3 mg·kg −1 and 65.3 mg·kg −1 respectively) than in cattle manure (137.1 mg·kg −1 and 596.0 mg·kg −1 respectively) which is corroborated in the present results where the TREE-L had lower values than the COW-M in Cu and Zn (see Table 3), which, as mentioned above, may be due to the feed received by cattle and especially those used for milk production.

Generation, Inoculation, and Characterization of Beneficial Microorganisms in the Evaluation Stage.
e generation of inoculum with BM was performed in two stages: the first was the generation of the stock solution (SS) and the second was the activation and enrichment of the SS for inoculation in the field. e SS or "efficient EM1 microorganisms" as Dr. Teruo Higa calls it [53] was prepared from cabbage leaf extract (Brassica oleracea L. var. capitata). In the second stage, nutrients were added and a bundle of plants. ese plants were wrapped in silk textile to produce a filtrate that was decomposing and possibly contributing in the generation of BM in the SS. e plants were "rue" (Ruta graveolens L.), "horsetail" (Equisetum arvense L.), and "rosemary" (Rosmarinus officinalis L.). e plants selected  Figure 1: Satellite image of the university campus with the facilities which contributed to the investigation.  [54][55][56][57][58]. e activation procedure is as suggested by Dr. Teruo Higa [53] prior to definitive field application. e first inoculation was carried out with a 20% dilution. In other words, 4 L of the activated solution was taken and diluted in 20 L of water for the irrigation of piles P1, P2, and P3. Next, in each turning over, which was performed weekly, during the first month inoculation it was with a 1% dilution (200 mL in 20 L). at is to say, four inoculations were made at 1% (Figure 2). is inoculation process followed the guidelines mentioned by APROLAB [59] and OISCA-BID [53]. On days 0 (T0) and 30 (T30), samples were taken in triplicate from the activated stock solution (ASS) for microbiological analysis. On both occasions, the pH was measured, and an average of 3.21 and 3.19, respectively, was obtained (Figure 2). Figure "a" of supporting information shows an image of the triplicate samples of the activated stock solution.
e average results are shown in Table 4 where it is observed that the presence of BM is in the order of millions, whereas the presence (Table 5) of pathogenic microorganisms (PM) is almost nil or minimal. On day 0 (T0) and day 30 (T30), the molds and yeasts were, respectively, independently counted. Alvarez [56] found on average for the yeasts in rue 5.5 × 10 4 UFC·mL −1 and in rosemary 5.6 × 10 5 UFC·mL −1 , for Bacillus spp. 1.46 × 10 7 UFC·mL −1 in rue and 6.67 × 10 5 UFC mL −1 in rosemary, for Lactobacillus spp. 6.43 × 10 12 UFC·mL −1 in rue and 3.47 × 10 12 UFC·mL −1 in rosemary, and for actinomycetes 8.67 × 10 3 UFC·mL −1 in rue and 1.6 × 10 5 UFC·mL −1 in rosemary. e results for the ASS of cabbage enriched with rue, rosemary, and horsetail are slightly higher in yeasts, slightly lower in Bacillus spp. and actinomycetes and significantly lower in Lactobacillus spp. than analyzed by Alvarez [56] for rue and rosemary but even so representing important values.

Methods.
e variables analyzed and the methods of analysis are given below, followed by the chronology of the samples taken in each type of compost (Table 6). e results were obtained for homogenized samples composed of five subsamples (four vertices and crown) from the COW-M cocomposting piles (Figure "b" of supporting information shows an image of the 120 day sampling), and for TREE-L alone, samples were composed of three subsamples taken from the base of the pile. e 500 g samples were collected in Ziplock TM polypropylene bags which were sent to a laboratory for analysis in triplicate. In the case of microbiological variables, they were also analyzed in triplicate. e viability time was 24 hours, and all were delivered within 12 hours of collection.

Analyzed Secondary Variables.
Twenty-three secondary variables were analyzed, eleven physicochemical and twelve microbiological ones.
e compost extract was stored in plastic vials at 4°C for further analysis [21].

(3) Concentration of Heavy Metals and Trace Elements of
Interest Extracted with Deionized Water (HM H2O ). 1 g of compost sample was taken. e sample was extracted with 15 mL of deionized water (by reverse osmosis and ion exchange with EC < 1 μS·m −1 ) by mechanical agitation at 120 rpm for two hours and then centrifuged at 4000 rpm for 5 minutes. It was filtered with a 0.45 μm nylon membrane. e compost extract was stored in plastic vials at 4°C for further analysis [21].
Analyses of the digestates of the primary variables were performed on a Perkin Elmer spectrometer, model Elan DRC-e. Table 6 shows the stages of the investigation, primary variables, secondary variables, and measurement dates.

Statistical Analysis (Evaluation Stage).
e experimental design of the investigation in its evaluation stage was completely randomized (completely randomized design). e investigation considered the IBM and NBM factors, five treatments (composting days: 0, 30, 60, 90, and 120), and three repetitions (composting piles). Fifty-three variables have been evaluated, divided into thirty main variables and twenty-three (23) secondary variables. Table "a" of supporting information shows factors, treatments, and their respective coding.

Secondary Variables.
Twelve physicochemical variables (H%, pH, EC, TOC, TN, C/N ratio, HA, FA, HA/FA ratio, NH4 + -N, OM, and Da) were registered. T°was discarded due to the nonexistence in the literature of information concerning its influence on the bioavailability of the heavy metals and trace elements of interest, and the C/ N and HA/FA rates used to determine the maturation of compost were incorporated [17]. Eleven microbiological variables include mesophilic aerobes, thermophilic aerobes, Lactobacillus spp., molds and yeasts, Pseudomonas spp., Bacillus spp., actinomycetes, total coliforms, fecal coliforms, Escherichia coli, and helminths. e variable Salmonella spp. was withdrawn from the statistical analysis as its results are qualitative.

Primary
Variables. Primary variables are thirty principal variables considering the three analysis groups (HM tot , HM DTPA and HM H20 ) and the ten elements under analysis (As, Cd, Cr, Cu, Hg, Mo, Ni, Pb, Se, and Zn).

Analysis of the Variables.
e analysis of the (primary and secondary) variables was carried out using the decomposition technique of the variability of the data called analysis of variance (ANOVA). is test allowed the comparison of the mean concentration or units of each variable at different composting days (treatments) on the basis of the completely randomized design, or factor design, used in this investigation. For correct application, the ANOVA of a factor requires compliance with the assumption of normal distribution and homogeneity of variances [71]. e AndersonDarling test was used or the verification of normality and the Bartlett test for homogeneity. For the compliance with both assumptions, the p value associated with each of these tests should be greater than the level of significance, 0.05. e ANOVA analysis was performed when it was verified that the two assumptions, variance homogeneity and normal distribution, were met. When these assumptions were not met, it was decided to use the KruskalWallis Test or nonparametric method or free distribution [71]. ese analyses were carried out using the statistical software R ™ Version 3.3.2.
In order to analyze how primary and secondary physicochemical variables vary with respect to the application factors of beneficial microorganisms and treatments, general linear and mixed models were made to consider the heterogeneity of variance during the analyzed period. en, the most appropriate model was selected using the values of Akaike (AIC), Schwarz (BIC), and the maximum likelihood test (LRT) [72]. For all measured variables, normality was analyzed using the ShapiroFrance test [73]. In the case of microbiological secondary variables, generalized linear binomial negative models were used [72]. In order to study the degree of association between primary and secondary variables, the Pearson linear correlation coefficient was used in those variables that did not show influence by the inoculation factor. is comparative value is reinforced with the p value (if it is <0.05, it is significant and if it is >0.05, it is not significant). is correlation was obtained by processing the information with the InfoStat ™ software, version 2017.1.2.
Correlations were evaluated between the 53 variables, all against all. For the present article, the primary variables were selected that exceeded the correlation of 0.5, positive or negative, in relation to the fraction of humic acids (AHs).
Using the InfoStat ™ software, version 2017.1.2, an unrestricted sorting analysis was made of the principal components. Fifty-three variables were used for this (primary and secondary, excepting Sallmonella spp. as it has qualitative values) initially through a first run, where abundant values greater than 1 were obtained. From this, a reduction of variables was made, through the ordering of the variables and their weights in the first components, working with the absolute values and observing which variables have greater weight in the first two components. From this new arrangement, two autovalues with values greater than 1 were obtained (the variables that had less weight in the first two axes were eliminated). Finally, the variables obtained for components 1 and 2 explain 78% of the variance.

Secondary Variables (Physicochemical).
e results of the recorded physicochemical variables (pH, EC, TOC, TN, and C/N ratio) of the compost products (T111/T240) of the characterization stage are shown in Figure 3. It is observed that, in all mean values, manure compost exceeds tree litter. Compared with the Chilean standard [74], we have the pH complying for both types of compost and both classes (A and B); for the EC, only tree litter complies for class B compost; TN is complied with for both types of compost and for both classes, and the C/N ratio is met in both types of compost and for both classes. It is important to emphasize the value of CE, which can be associated with the type of cattle feed [75].
is presence of EC in this type of manure can generate the increase of this value in the soils where it is applied, in the medium or long term, which can generate processes of salinization and infertility [76,77].

Primary Variables (Total Content of Heavy Metal and Trace Elements of Interest).
e HM tot of the TREE-L composting product (T120) showed the following order in its mean values (mg·kg In the cases of Cr and Pb for TREE-L compost, it exceeds by more than three and two times the mean value of the COW-M and GRASS-C compost, respectively (Figure 4). e higher HM tot value in the compost generated from the TREE-L of the Botanical Garden of the UNALM shows what was pointed out by several authors [7,[25][26][27][28][29][30] on the retention of these elements in the foliage due to the precipitation of atmospheric dust. e botanical garden, due to its proximity to La Universidad Avenue, acts as a "biofilter" for the university due to the retention of these pollutants. On the other hand, the compost generated from COW-M and GRASS-C, not being exposed at the same intensity and frequency to air pollution, shows lower levels of heavy metals, but Cu and Zn do show a significant presence due to the feeding of dairy cattle [32][33][34][35][36].
is stage was carried out 24 months before the evaluation stage, giving knowledge of the concentration of heavy  Applied and Environmental Soil Science 7 metals and trace elements of interest in significant amounts in the TREE-L compost, so it was decided to use this raw material for the next (evaluation) stage mixed with COW-M. Two figures of microbiological variables with the physicochemical variables HA, pH, ratio C/N, and T°have been added in order to be able to see the behavior during the composting process of the average values in the IBM and NBM piles. Figure 5 groups the microbiological variables that were counted as colony-forming units per gram (CFU·g −1 ) and Figure 6 as most probable number per gram (MPN·g −1 ). ese grouped figures do not consider the variable Salmonella spp. as it has qualitative values nor the helminths as they have a differentiated accounting to the groupings. e first group figure, considered also as BM, is made up of mesophilic aerobes and thermophilic aerobes, Bacillus spp., Lactobacillus spp., molds and yeasts, and actinomycetes, considering the order of descending population magnitude, and the second figure, considered also as PM, of Pseudomonas spp., total coliforms, fecal coliforms, and Escherichia coli, considering the order of descending population magnitude. As can be seen at the beginning of the process (Day 0), the population of microorganisms is higher than that observed at the end of the composting process (Day 120). In the cases of the second grouping, it is important to observe the reduction of pathogens such as fecal coliforms, which are the only microbiological variables that showed a statistically significant (p < 0.05) reduction in its population in the IBM and NBM piles. ese population changes with significant values at the beginning and decreasing during the composting process have also been observed by Ahmed et al. [80] and Haroun et al. [81]. e sterilization of pathogens in composting piles is related to the maximum temperatures reached and the retention time [17,41,51]. In both types of piles, the temperature reached 56°C (Figure 2), but in IBM, the thermophilic phase lasted 20 days, while in NBM, it lasted 18 days and in no case was the temperature in the range of 60 to 70°C, which is recommended for the sterilization of the majority of pathogens [17,51]. Despite this, the maximum values recommended by the Peruvian regulations [82,83] were met in the IBM piles, except for helminths, and the NBM piles also did not reach the value for eliminating total coliforms in the composting product (Day 120).

Primary Variables.
e results of the 10 metals and trace elements of interest, the main variables of this investigation, are presented graphically and described in grouped and differentiated form in HM tot , HM DTPA , and HM H2O for the IBM and NBM piles and the physicochemical variables (pH, T°, C/N ratio, and HA).  (Figure 7). Cd, Pb, and Zn exceeded the values of the Austrian Standard [84] for compost with organic agriculture in IBM and NBM, just as it as found with Cu in NBM. In addition, As also exceeded the Brazilian Standard [85] for compost with organic agriculture in IBM and NBM. Nevertheless, the compost complied with it for landscaping and land rehabilitation of the Austrian Standard, and also with Peru's regulations [82,83] for compost for nonorganic agriculture. e hierarchical distribution of the elements differs from the one observed in the products of composting in the characterization stage, as although for the four composts Zn is the highest, Pb is the second, displacing Cu to third place, followed by As in fourth place (Section 3.1.2). Ciavatta et al. [40] found distributions of the average concentration (mg·kg −1 ) of heavy metals, in compost from municipal household waste in Italy during 55 days of composting in an aerated static pile, in summer: Zn (1 037) > Pb (981) > Cu > (737) > Cr (162) > Cd (6, 7) and in winter: Zn (981) > Cu (399) > Pb (287) > Cr (69) > Ni (47) > Cd (3.9). Hanc et al. [24] recorded at the end of 84 days of composting household waste with garden waste in a reactor with medium aeration that the average concentration values (mg·kg −1 ) were Zn (167.1) > Cu (29.1) > Pb > (16.9) > Cd (0.27). In both investigations, Zn leads the total concentration values, and the other three are Cu, Pb, and Cr, similar to those recorded in this investigation. e sums of the means of the ten elements of interest on day 0 for IBM and NBM (mg·kg −1 ) were 501.03 ± 65.77 and 520. 53  ere is no trend in the variations indicating any influence of inoculation with BM, but there was found differences among 120 days samples of Cu. e increase in concentration could be due to the loss of mass resulting from mineralization and volatilization of carbon and nitrogen [17,21,52,86,87]. e reduction in Mo may be associated with the effect of the pH on its mobility and possible leaching [88]. Ciavatta et al. [40] showed an increase in the summer in the sequence: Ni (752%) > Pb (554%) > Cd (379%) > Cu (303%) > Zn (66%); in winter: Cd (550%) > Ni (104%) > Zn (77%) > Cr (47%) > Cu (19%) > Pb (6%). Hanc et al. [24] observed that the order of increases was Zn (84%) > Cu (49%) > Pb (22%); in the case of Cd, there was a reduction of 4%. Similar to this investigation, most of the average total concentrations of heavy metals increased at the end of the composting process, although with differences in the incremental values, by type of compost and season.   Figure 8 shows the evolution of the average values of the heavy metals and trace elements of interest extracted with DTPA during the composting process in IBM and NBM piles and physicochemical variables (pH, T°, C/N ratio, and HA). e sums of the means of the ten elements of interest on day 0 for IBM and NBM (mg·kg −1 ) were 119.69 ± 7.50 and 123.13 ± 8.14, and on day 120, they presented values of 131. 76 [40] observed in 60-day compost increases in the concentration of Cu, Pb, Ni, and Cd, but for Zn and Cr, it remained almost constant. e opposite was found by Huang et al. [21] as they showed a 63-day composting reduction in Cu and Zn extracted with DTPA. e results are dissimilar to what was mentioned by the authors and the investigation. However, they do agree in the case of Cu and that mentioned by Ciavatta et al. [40].  Figure 9 shows the evolution of the average values of the heavy metals and trace elements of interest extracted with deionized water during the composting process in IBM and NBM piles and physicochemical variables (pH, T°, C/N ratio, and HA). e sums of the means of the ten elements of interest on day 0 for IBM and NBM (mg·kg −1 ) were 13.09 ± 2.53 and 11.64 ± 1.94, and on day 120, they presented values of 7.10 ± 0.62 and 7.38 ± 0.44, respectively. is meant an average reduction of 45. ese results indicate that the composting process reduced the solubility and leaching of the metals other than Mo. Statistical significance (p < 0.05) was observed in the reduction in Cu, Ni, Pb, and Zn for both types of piles, from which it can also be stated, without statistical significance, that in absolute terms, there are higher solubility reduction values for these elements in IBM piles than in NBM piles. Ciavatta et al. [40], Huang et al. [21], Castaldi et al. [37], Hargreave et al. [41], and Ramdani et al. [89] showed a reduction in the concentration of Cd, Cr, Cu, Ni, Pb, and Zn extracted with deionized water in composting between 60 and 100 days, which resembles the results found in the present investigation.

Statistical Analysis of the Primary and Secondary Variables of the Evaluation Stage
(1) Analysis of the Primary Variables. Zn, Cu, and Pb are the three elements with the highest total concentration, and they also exceeded the maximum values for organic agriculture [84,85]. e Cu showed a significant difference, in both types of piles (IBM and NMB), between day 0 and 120 in the concentration extracted with DTPA and deonized water. For Pb, the difference was shown in the extraction with deionized water. For Zn, there were differences in total concentration and in extraction with deionized water (Figure 10).

Correlation of Variables.
On the basis of those variables that were significant (p value >0.05) in the Pearson correlation with a coefficient value greater than 0.5 and did not show any difference between the factors (inoculation and noninoculation), it was observed for the elements Cu, Ni, Zn, Cd, Pb, Se, and Hg extracted with water that they have a significant negative correlation with the humic acid fraction (Table 7). e same happens with Se, Ni, and Hg extracted e opposite occurs with Mo extracted with pentatetic acid which shows a positive correlation with AH and in the total concentration of Cd, Cr, Ni, Pb, Hg, Zn, and Se (Table 8). Castaldi et al. [37] found a correlation of −0.95; −0.95; −0.92, and −0.90 (p < 0.01), respectively between the Pb, Cd, Zn, and Cu extracted with deionized water and the AH after 100 days of composting of solid municipal waste in Italy, values higher than those found in this investigation, but which confirm the affinity between these two variables for these elements.

Unrestricted Sorting Analysis with Examination of
Principal Components. Figure 11 shows that the variables C/ N and OM (%) were highly correlated, as well as Da (g·cm −3 ) with [Cr] tot (mg·kg −1 ) and that those variables with a negative correlation were [Zn] DTPA (mg·kg) and FA (%). In the majority     ere is variation in bioavailability between day 0 and 120 with a statistical significance (p < 0.05) in Cu, Cr, Mo, Ni, and Se for both pile types (IBM and NBM) in Cu, with 62.7 % increase (from 6.89 to 11.21 mg·kg −1 ) in IBM and 162.2 % increase (from 6.16 to 16.15 mg·kg −1 ) in NBM.; in Mo, with 353.7% increase (from 0.082 to 0.372 mg·kg −1 ) in IBM and 182.1% increase (from 0.145 to 0.409 mg·kg −1 ) in NBM; in Cr, with 32.7% reduction (from 0.52 to 0.35 mg·kg −1 ) for IBM and 43.9 % reduction (from 0.57 to 0.32 mg·kg −1 ) in NBM; in Ni, with 68.0% reduction (from 1.03 to 0.33 mg·kg −1 ) in IBM and 38.3% reduction (from 0.60 to 0.37 mg·kg −1 ) in NBM; in Se, with 45.7% reduction (from 0.13 to 0.07 mg·kg −1 ) for IBM and 42.6% reduction (from 0.12 to 0.07 mg·kg −1 ) in NBM.
ere is variation in solubility between day 0 and 120 with a statistical significance (p < 0.05) in Cu, Ni, Pb, and Zn for both pile types (IBM and NBM) in Cu, with 76.3% reduction (from 2.62 to 0.62 mg·kg −1 ) in IBM and 68.9% reduction (from 2.25 to 0.70 mg·kg −1 ) in NBM; in Ni, with 66.4% reduction (from 0.268 to 0.09 mg·kg −1 ) in IBM and 55.0 % reduction (from 0.2 mg kg −1 to 0.09 mg·kg −1 ) in NBM; in Pb, with 63.8% reduction (from 0.719 to 0.26 mg·kg −1 ) in IBM and 47.9% reduction (from 0.547 to 0.285 mg·kg −1 ) in NBM; in Zn, with 64.5% reduction (from 4.764 to 1.689 mg·kg −1 ) in IBM and 52.9 % reduction (from 3.933 to 1.852 mg·kg −1 ) in NBM. e increase in the total content of Hg, Se, and Zn is due to the loss of mass resulting from mineralization and volatilization of carbon and nitrogen [24]. e reduction in the total Mo content may be due to mobility resulting from the increase in pH [1]. e increased bioavailability of Cu and Mo is linked to mineralization and chelation processes that in some cases increase availability [1] and also to higher pH (>5.5) which influences availability in Mo [3]. e composting process reduced the solubility and leaching of Cu, Ni, Pb, and Zn, and there were higher solubility reduction values in the inoculated piles than in the noninoculated piles. It is understood that the oxidation process and the formation of organometallic complexes influenced the reduction of the soluble content of the mentioned elements [21,37,42,90,91]. [Se]DTPA (mg kg -1 ) Figure 11: Unrestricted sorting analysis (with examination of principal components). the compost matrix of the mentioned elements, which would reduce their mobility and increase their concentration (bioaugmentation) while the organic matter mineralizes [3,24,[92][93][94]. is affinity is confirmed by comparing the results of variation between days 0 and 120 for IBM and NBM. It should be noted that the As, which had the smallest increase in total concentration (<10%) for both piles, confirming the nonaffinity with the AH [1,3].

On the Influence of Humic and Fulvic Acids on
ere is a negative correlation between the concentration of the humic acid fraction and the concentration extracted with pentatetic acid for Se, Ni, and Hg in the following order: Se (−0.84) > Ni (−0.77) > Hg (−0.66). A chelating or sequestering effect in the megastructure of the humic acid fraction can be understood, which generated a phenomenon of "immobility" in the compost matrix [90]. is is confirmed by the analysis of the variation between days 0 and 120 for inoculated and noninoculated piles because these three elements present the greatest reductions in their extracted concentrations. It is highlighted that the As, which had the lowest reduction extracted with pentatetic acid (<6%) for both piles, confirming the nonaffinity with the AH [1,3].
ere is a positive correlation (+0.62) between the concentration of the humic acid fraction and the concentration of Mo extracted with pentatetic acid. is can infer a possible affinity in the disposition of this element for plants in the presence of humic acids [3,88]. is is confirmed by the analysis of the variation between days 0 and 120 for inoculated and noninoculated piles because this element presents the greatest increase of its extracted concentration.
ere is a negative correlation between the concentration of the humic acid fraction and the concentration from the extraction with deionized water of Cu, Ni, Zn, Cd, Pb, Se, and Hg in the following order: Cu (−0. It is possible that the increased concentration of the humic acid fraction during the composting process influenced the reduction of the solubility of these elements, which infers a redistribution of the water-soluble fractions to more stable forms strongly bound to the humic acid fractions [12,37,90]. is is confirmed by the analysis of the variation between days 0 and 120 for IBM and NBM, due to the fact that these elements present the greatest reductions of their extracted concentrations. It should be noted that the As, which had the smallest reduction in the concentration extracted with deionized water (<6%) for both piles, confirms the nonaffinity with the HA [1,3]. e concentration of the fulvic acid fraction showed no significant correlation with any of the analyzed primary variables. Humic and fulvic acids are organic substances that can have a high capacity for generating bonds with heavy metals, due to the large number of functional groups. In this investigation, this was only indicated with humic acid for some heavy metals, which may be due to the raw materials and the types of extractants which were used [21,95] Comparing day 0 (T0) and day 120 (T120), the total Cu content was reduced by 16.45% (from 103.33 to 86.33 mg·kg −1 ) in IBM and increased by 48.32% (from 75.67 to 112.23 mg on the influence of inoculation with beneficial microorganisms on total concentration, bioavailability, and solubility of the heavy metals and trace elements of interest kg −1 ) in NBM. Comparing the concentration in the final product (T120) of IBM and NBM, it was observed that the former registered 23.08% less concentration of total Cu. Based on what has been recorded, it can be concluded that the inoculation of beneficial microorganisms reduced the total Cu concentration.
Comparing day 0 (T0) and day 120 (T120), the bioavailability of Cu increased 62.7% (from 6.89 to 11.21 mg·kg −1 ) in IBM and 162.2% (from 6.16 to 16.15 mg·kg −1 ) in NBM. Comparing the bioavailability of the Cu extracted in the final product (T120), we have 30.6% less concentration in IBM. From the above, it is concluded that the inoculation of the beneficial microorganisms reduced the bioavailability of Cu.
Comparing day 0 (T0) and day 120 (T120), the bioavailability of Zn increased by 9.94% (from 83.32 to 91.60 mg·kg −1 ) in IBM and by 39.71% (from 85.14 to 118.95 mg·kg −1 ) in NBM. Comparing the bioavailable concentration of Zn in the final product (T120), we have 23% less in IBM. From the above, it is concluded that the inoculation of the beneficial microorganisms reduced the bioavailability of Zn. e inoculation of beneficial microorganisms did not influence, with statistical significance (p < 0.05), the extraction with deionized water of the heavy metals and trace elements of interest or the solubility of these elements. e lower bioavailability of Cu and Zn in inoculated piles than in noninoculated piles may be due to the fact that inoculation increased the bioaugmentation of these elements in the cells of microorganisms, or the chelation with organic functional groups, or the sequestration in dead cells [45]. While the reduction in the concentration of the total Cu content remains unknown because of its low probability of volatilization, one possibility is that refractory compounds are generated that prevent its quantification [3].

Conclusions
e composting product made with tree litter and cattle manure from an urban area cannot be used in organic agriculture because As, Cd, Cu, Pb, and Zn do not comply with the maximum allowable values of Brazil and Austria. However, it is possible to use it in land rehabilitation and landscaping considering the Austrian standard and also in nonorganic agriculture in Peru. e presence of Cu and Zn in the composting product is due to the type of cattle feed and to atmospheric contamination, while for As, Cd, and Pb, it is mainly due to the atmospheric contamination. Inoculation of beneficial microorganisms reduces the bioavailability of Cu and Zn in comparison with the compost from piles which were not inoculated. e composting process reduced in both pile types (IBM and NBM) and the solubility of Cu, Pb and Zn. Fulvic acids did not show a significant correlation with any of the elements; however, humic acids did show affinity with Cd, Cu, Pb, and Zn but not with As.
is affinity confirms that the soils of green spaces can be sinks for heavy metals. Likewise, we can infer that the compost of the cattle manure can be recommended as an amendment to soils to reduce the leaching of Cu and Zn. e responses of the ten elements under analysis are different, as we have observed. It is therefore unlikely that their bioavailability and solubility can be reduced with a single treatment technique. Specifically, in the case of As, its behavior associated with an increase in pH must be taken into account, for which other techniques such as the aggregation of biochar, zeolites, or vermicomposting could be considered. Regarding the presence of helminth oocysts, consideration should be given to increasing the frequency of turning and to inoculation with a greater activated stock solution of beneficial microorganisms in order to reach temperatures between 60 and 70°C and so eliminate them.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.