Introduction

The biosolids are stabilized sewage sludge derived from urban and industrial wastewater treatment processes (SEMARNAT, 2002). Their final disposal has become an environmental problem that has been increasing since the last decade of the 20th century (^{Gavalda et al., 2005}). The options to dispose of biosolids include incineration, landfill and recycling as organic fertilizers for agricultural use (^{Alves et al., 2006}). The use of biosolids as agricultural soil improvers may be environmentally and economically viable and is considered a sustainable option for final disposal. The biosolids can be used as an economical source of nutrients in agriculture because they have characteristics that improve soil fertility and can increase yields and generate economic gains for producers (O’Connor et al., 2005).

The benefits of the agricultural use of biosolids include, the positive impact on the physical and chemical properties of the soil, the reduction of the soil density and the increase of the porosity, which improves the structural stability (^{Roig et al., 2012}). These changes generally result in an increase in water retention capacity, especially in coarse textured soils and in the long term improve water mobility and resistance to soil erosion (^{Samaras et al., 2008}). The biosolids have high contents of organic matter, nitrogen and potassium.

The amounts of soluble nutrients are initially small and nutrient uptake by the plant must await the mineralization of the organic constituents of the biosolids (^{Guo et al., 2012}), which makes possible the availability of nutrients in the medium and long term. The main restrictions for the agricultural use of biosolids are the heavy metal content and the presence of pathogenic microorganisms. The heavy metals can be toxic to plants, animals or humans, due to possible transfer and bioaccumulation through the trophic networks. The risk depends on the concentration present in the soil, on the biosolids and their availability and mobility (^{Castro et al., 2009}). The strategies for the agricultural use of biosolids are generally not based on scientific studies that support the doses applied to specific crops.

Therefore, it is necessary to carry out research prior to the application of biosolids in the field for a given crop, because the effects that will occur will depend on factors such as the climate, the type of soil, the quality of the seed, the quality of the biosolids and agricultural management. The city of Puebla (19º 02’ 37’’ north latitude, 98º 11’ 53’’ longitude west), capital of the state of the same name and located in the central region of Mexico, has a population of two million inhabitants and counts with five wastewater treatment plants generating 200 t day^-1 of sewage sludge.

These residues are stabilized in the same treatment plants by means of anaerobic digestion, thereby eliminating a large number of pathogenic microorganisms (^{Pepper et al., 2006}); they are then dehydrated to give them the appropriate quality for use in agricultural soils (SEMARNAT, 2002). The biosolids produced in the city of Puebla are used as organic amendments in agricultural soils, with low fertility and low content of organic matter, of rural communities located south of the city, where maize (Zea mays L.). The objective of this study was to evaluate, under greenhouse conditions, the effects that different doses of biosolids produce on the quality of the maize plant, the heavy metal content in it and the chemical characteristics of the soil of the southern zone of the municipality of Puebla.

Materials and methods

The project was developed at the Technological Institute of Puebla (19º 02’ 37’’ north latitude, 98º 11’ 53’’ west longitude) under controlled greenhouse conditions (except temperature). The average relative humidity was 48.5%; the irrigation was manual, three times a week. The experiment was carried out from September to December 2013. The duration was 120 days. In this experimental crop, soil of the community called La Paz Tlaxcolpan was used in the municipality of Puebla, Mexico (18° 54’ 21’’ north latitude, 98° 13’ 16’’ longitude west), which is one of the places where the biosolids are applied to the corn crop. This soil is classified as a cambisol eutrico (FAO, 2014). The selected seed from local maize was used by producers.

The biosolids used were of urban origin. In the wastewater treatment plant are stabilized by means of anaerobic digestion and subsequently dehydrated. Samples were taken in the month of august 2013. The biosolids were dried at room temperature for 10 days. Once dried they were placed in polyethylene bags, for later use in the formation of different mixtures with the soil. The determination of heavy metals in biosolids was performed according to the established standard (SEMARNAT, 2002); for the determination of texture, pH, CE, MO and CIC in biosolids were used the methods applied to soil samples (^{SEMARNAT, 2000}).

The doses used were determined on the basis of the calculation of the maximum amount of biosolids applied in the soils of the community of La Paz Tlaxcolpan, 400 t ha^-1. To determine the doses were considered, an apparent density of 1.2 t m^-3 and a depth of the arable layer of 20 cm for the soil; for biosolids the density used in the calculations was 1 t m^-3. With these data it was determined that 2.5 million kg of soil (corresponding to one hectare) apply 400 000 kg of biosolids. The ratio would be 160 g of biosolids per kilogram of soil at a dose of 400 t ha^-1. From these data the soil-biosolid relationships were calculated for the different doses.

The experimental units were pots (polyethylene bags 40 cm x 40 cm). In each pot were placed 10 kg of soil and its corresponding amount of biosolids for the different doses (on a dry basis). The soil and biosolids were mixed five days prior to planting. In each pot three seeds were planted. The germination occurred between six and 10 days after sowing. The percentage of germination was 94%. 15 days after sowing the weeding was done in each pot with the purpose of leaving a single plant in each pot.

A completely randomized experimental design with six treatments and four replicates was used for a total of 24 experimental units. The treatments corresponded to the different doses of biosolids as follows: treatment 1 (T1)= 100 t ha^-1, treatment 2 (T2)= 200 t ha^-1, treatment 3 (T3)= 300 t ha^-1, treatment 4 (T4)= 400 t ha^-1, treatment 5 (T5)= 500 t ha^-1, control treatment (TC)= soil without biosolids. The vegetative variables and physico-chemical characteristics of the soil-biosolids mixture were measured in each experimental unit.

The vegetative variables measured in the maize plant were as follows (^{Avendaño-Arrazate et al., 2008}): plant height (AP) was recorded by measuring the total length from the base of the stem to the base of the spike; basal diameter of the stem (DBT), measured with a vernier base of the stem; number of leaves (NH); leaf area per plant (AFP), the area of one leaf per plant was determined by the equation: long leaf x width leaf x 0.75 (^{Montgomery, 1971}) and multiplied by the number of leaves per plant.

The yield variable, total biomass (BT), was measured by weighing the dry weight of the plants harvested in each experimental unit. The variables identified in the mixture soil-biosolids and biosolids were: texture, pH, organic matter (MO), electrical conductivity (CE), cation exchange capacity (CIC) and heavy metals removable (Cd, Cu, Fe Mn, Pb, Zn) with diethylene triaminepentaacetic acid (DTPA). For these variables only one determination was made. The methods used were those established in the corresponding standard (^{SEMARNAT, 2000}).

For the analysis of plant tissue samples, the wet digestion method was used (^{López-Ritas and López-Melida, 1978}). The concentrations of Cd, Cu, Pb and Zn were determined in root, stem and leaf of the plant. The quantification of the metal concentrations in all extracts obtained was performed with an equipment (Varian Liberty Series II) of inductively coupled plasma atomic emission spectroscopy (ICP-AES). A statistical analysis was performed, applying an analysis of variance to the variables measured in the plant, using the Tukey test to perform the comparison of means between treatments, with a level of significance of 95% and thus to determine possible effects of the different doses of Biosolids. The SPSS program was used (Statistical Package for Social Sciences) 15.0.

Results and discussion

The physicochemical characteristics of the soil and the biosolids used are shown in Table 1. The soil and biosolids had a slightly acidic pH. By the value obtained from CE, the soil had negligible effects of salinity; in contrast, biosolids showed an CE that corresponds to the conditions of a saline soil. The MO content was low for soil and high for biosolids. The CIC was high for both, which is mainly due to the clay content that both present in its texture (^{Porta et al., 2003}), the high content of organic matter in the biosolids is the cause of its greater CIC due to the high surface area that it possesses and that results in a greater capacity of adsorption to retain metallic cations (^{Basta et al., 2005}).

Table 1 Physical and chemical properties of soil and biosolids used.  

The values of Fe, Mn, Cu and Zn (DTPA extractable) are adequate in the soil; the concentrations of Cd and Pb were found to be below what was considered normal (^{SEMARNAT, 2000}). The concentrations of the heavy metals determined in the biosolids (Table 1), are below the established in the official Mexican norm in “permissible maximum limits for heavy metals in biosolids”, and according to the same, the biosolids used can be classified in the category of “excellent”, for use as soil improvers.

The chemical variables measured in the soil-biosolids mixture (Table 2), presented some important trends with respect to the increase of applied biosolids dose. It was observed that the pH of the TC was slightly acidic and when the biosolids were applied to the soil, the pH rose to be slightly alkaline for T1 and T2; for T3, T4 and T5, the pH dropped to a value considered neutral (SEMARNAT, 2002). The highest values of pH were from treatments with lower doses of biosolids (100 t ha^-1 and 200 t ha^-1); as the dose increased the pH dropped.

Table 2 Chemical properties of the different treatments (soil-biosolids mixture). 

The electrical conductivity (CE) showed an inverse behavior to that of pH; increased directly with the dose of biosolids. The lowest value was TC and the highest value was T5. ^{Schroeder et al. (2008)}, in a field study with wheat (Triticum aestivum) and ^{Bañuelos et al. (2007)}, in a field study with apricot (Prunus armaniaca), found the same tendency in the CE regarding the application of biosolids. This situation could imply a risk of salinity for the soil. This phenomenon can be explained by two effects related to the addition of biosolids to the soil; the first involves the MO content contributed.

The conduction of electricity in soils is mainly done through continuous macro and micro pores that are filled with water. The MO promotes the formation and stabilization of aggregates in the soil, generating continuous pores, which increases the conductivity of electricity in the soil; the MO also produces a greater moisture retention in the soils, which also implies a higher CE (^{Zhang and Wienhold, 2002}).

The other factor that has an impact on the CE increase is the texture of the soil-biosolids mixture. The loamy clay texture of the soil and the clayey texture of the biosolids imply a high content of fine particles, which have a much closer particle-particle contact, which produces a high microporosity, able to retain water with more force and consequently greater electrical conduction is generated (^{Sudduth et al., 2003}).

Regarding the extractable metals content with DTPA (Table 2), the Cd presented low concentrations in all treatments. The control soil had the lowest concentration and in treatments with biosolids increased slightly with increasing biosolids dose. This means that the Cd is supplied by the biosolids. This can be attributed to the formation of complexes between organic matter and metal cations. The Cd has a high affinity with organic matter, specifically with the thiol functional group (-SH), present in the humid organic matter (^{Li et al., 2001}); ^{Hettiarachchi et al., 2003}; ^{Kukier et al., 2010}).

The Cu, Fe, Pb and Zn metals increased their concentrations and their availability in direct relation with the increase of the dose of biosolids. The affinity of Cu, Fe, Pb and Zn with MO has been extensively documented (^{Kabata-Pendias and Pendias, 2001}; ^{Basta et al., 2005}). The Cu and Pb form strong inner-surface surface complexes with organic matter and are therefore contributed by biosolids to the soil (^{Adriano, 2001}). In the soil, Pb is relatively immobile because it is strongly adsorbed by solid soil fractions such as mineral clays, Fe and Mn oxides and hydroxides and soil organic matter and biosolids (^{González-Flores et al., 2011}). The Pb showed low availability, which increased in direct ratio with the dose of biosolids.

The results of the response of the vegetative variables of the maize plant to the different doses of biosolids are shown in Table 3. Compared with the TC, for which there is a notable difference, all treatments with biosolids increased the values of all the measured variables. In general, higher plants with a larger number of leaves were obtained with a thicker stem and, consequently, a higher production of dry biomass, but higher values corresponded to treatments with higher doses of biosolids.

Table 3 Vegetative variables as a function of applied biosolid dose (mean ± S, n = 4). 

The value of the variables tends to increase to a higher dose of biosolids. These results can be attributed to the great contribution of biosolids to the soil of organic matter and nitrogen, which favors a better development of the plant and a higher production of biomass (^{Haynes, 2005}; ^{Van Wieringen et al., 2005}). The increase in biomass production in direct function with the dose of biosolids was also observed by ^{Hernández et al. (2005)} in a study under greenhouse conditions on the production of sorghum (Sorghum vulgare). Similar results were found by ^{Wang et al. (2008)} in a field study on grassland production (Zoysia japonica) when applying biosolids. ^{Jurado et al. (2007)} and ^{Delibacak et al. (2009)} correlated the increase of total nitrogen with the increase of the dose of biosolids in the production of semi-arid grasslands and peanut (Arachis hypogaea), respectively.

The T3 produced higher plant height; in T3 and in T5 the plants had a greater number of leaves; the basal diameter of the stem was higher in T5; the largest leaf area and the highest biomass production occurred in T4 (Table 3). However, the analysis of variance and Tukey’s test for mean comparison indicated that there is no significant statistical difference (p≤ 0.05) between biosolids treatments. That is, they showed a similar behavior for all measured variables.

In spite of the Cd that contained the soil and the one contributed by the biosolids (Table 1), this metal was not detected anywhere in the plant in any treatment. ^{Torri and Lavado (2009)}, in a study at greenhouse level with english grass (Lolium perenne L.), obtained similar results; did not detect Cd in plant tissue when they applied different doses of biosolids. ^{Kukier et al. (2010)} found a very low availability of Cd in the soil when applying biosolids in a study with lettuce (Lactuca sativa L.).

This suggests that the Cd is strongly retained in the solid fractions of the soil-biosolids mixture; as mentioned above Cd can form very stable complexes with organic matter. Another fraction of the soil-biosolids mixture that participates in the retention of Cd is the Fe and Mn oxides, where Cd is strongly adsorbed (^{Hettiarachchi et al., 2003}). The slightly alkaline pH also influences the low availability of Cd.

In the Figure 1 shows the results found for the content of Cu in the root, stem and leaves of the maize plant in the different treatments. The highest concentration of Cu was found in the root, in all treatments; lower concentrations of this metal were obtained in the aerial parts of the plant. The trend was upward in direct function with the dose of biosolids. This result agrees with that obtained by ^{Hernandez-Herrera et al. (2005)}, in their study with forage sorghum. According to ^{Kabata-Pendias and Pendias (2001)}; ^{Baker (1990)} the root tissues possess a powerful capacity to retain the Cu and avoid its transport to the aerial parts, therefore the mobility of this element is limited.

Figure 1 Concentration of copper in the different parts of the plant as a function of the biosolid dose.  

^{Bañuelos and Ajwa (1999)} report that copper is strongly chelated by the organic acids present in the radical system. ^{Mehra and Farago (1994)} indicate a concentration of 13 mg kg^-1 of Cu in the maize plant as adequate for the normal development of the plant. The concentrations in stem and leaf (Figure 1) were below 10 mg kg^-1 and were lower than those found in the root; were almost constant in all treatments with biosolids. This could represent a nutritional deficiency of the plant. The slightly alkaline pH of all biosolids treatments also appears to influence the lower absorption of Cu by the plant.

The concentrations of Zn in the different parts of the plant (Figure 2), followed the same trend as those of copper; higher in the root and smaller in the stem. Although they were, in general, higher. In similar studies with maize, ^{De las Heras et al. (2005)} at the greenhouse level and ^{Kidd et al. (2007)} in a field work, obtained results that agree with those obtained in this research, regarding the concentrations of Zn in the plant. The Zn presented high availability in the soil-biosolids mixture, in all treatments (Table 2).

Figure 2 Concentration of zinc in the different parts of the plant as a function of the biosolid dose.  

According to ^{Mehra and Farago (1994)}, Zn is an element that is easily absorbed by the plant in different chemical forms (Zn hydrated, Zn²⁺, and in organic chelates). ^{Bañuelos and Ajwa (1999)} establish that unlike copper, zinc is weakly chelated by the organic acids present in the root, which causes it to be considered a moving element and therefore its translocation to the aerial parts of the plant occurs relatively easily. This would explain the high concentrations found in the leaves, especially in the treatments with higher doses of biosolids, T4 and T5. The concentrations of Zn found in stem and leaves are adequate to avoid the deficiency, which would present with levels below 20 mg kg^-1. On the other hand the same concentrations are below to cause a possible toxicity in the plant, which would be reached with levels of 300 mg kg^-1 (^{Kabata-Pendias and Mukherjee, 2007}).

The concentrations of Pb in the different parts of the corn plant had a downward trend when the biosolid dose increased (Figure 3). In general, the concentrations were low relative to the available Pb in the soil-biosolids mixture (Table 2); the behavior of Pb in the plant tissue was similar to that of Cu and Zn; that is, higher root content and lower stem content. ^{Kidd et al. (2007)} in a study of maize cultivation in greenhouse, applying several types of biosolids reported not having detected Pb in any part of the plant; however, ^{Intawongse and Dean (2006)} in applying biosolids to lettuce (Latuca sativa L.) and spinach (Spinacia oleracea L.), under greenhouse conditions, reported low concentrations of Pb in general, but the same trend found in this study for the maize: higher root content and lower leaf content.

Figure 3 Concentration of plumbum in the different parts of the plant as a function of the dosage of biosolids.  

The plumbum is considered one of the least mobile elements in the soil and therefore of low availability (^{Davies, 1990}; ^{Mehra and Farago, 1994}; ^{Kabata-Pendias and Pendias, 2001}). The data of Table 2 confirm this behavior, which can be attributed to the high affinity of Pb with organic matter, with which it can form specific adsorption complexes (^{Basta et al., 2005}). Therefore, the higher the amount of biosolids and higher MO content, the higher the retention of Pb in the soil. In this way, the absorption and subsequent translocation of Pb to the aerial parts of the maize plant is limited (^{Zimdahl and Hassett, 1977}; ^{Castro et al., 2009}). This would explain why the treatments with lower doses of biosolids (lower content of organic matter) present less retention of Pb in the mixture soil-biosolids and, therefore, cause that their presence in the different parts of the plant is greater. The organic matter has a primordial role in the retention of Pb in the soil.

The concentrations of the three extractable metals are positively correlated with the doses of biosolids applied (Table 4). That is to say, at a higher dose of biosolids, the higher the concentration available in the soil biosolids mixture; This positive correlation between doses of biosolids and extractable concentration of Cu and Zn is similar to that reported by ^{Lavado et al. (2007)} in their research with maize and wheat at the field level.

Table 4 Correlations between the concentration of extractable metals of the soil and the content of the plant.  

The correlation between the metal content in the different parts of the plant and the extractable concentration in the soil-biosolids mixture, Cu showed a positive correlation between roots and leaves (Table 4). This would suggest that if DTPA-extractable Cu content increases in the soil-biosolids mixture, its concentration can be expected to increase in the roots and leaves of the maize plant. For Zn no correlations were found, the Pb only showed a negative correlation in the leaves. This result could mean that if the availability of Pb in the soil-biosolids mixture increased, this would not be reflected in an increase in its content in any part of the maize plant.

Conclusions

The vegetative variables had the highest values with doses of 300 t ha^-1 to 500 t ha^-1; therefore, with the application of these doses of biosolids a better quality plant is obtained. According to the analysis of variance, statistically there is no difference between the treatments, which means that with any of the applied doses the same plant quality is obtained. The pH, MO and CIC of the soil-biosolids mixture showed the most appropriate values for the development of the plant with doses of 400 t ha^-1 and 500 t ha^-1. The CE is suitable in all doses of applied biosolids. The concentrations of essential micronutrients in the soil (Fe, Mn, Cu and Zn), present adequate values in all treatments.

The concentrations of metals in the plant tissue (Cu, Zn and Pb) indicate that there may be copper deficiency in the maize plant, because in all treatments were obtained values below 10 mg kg^-1. The Zinc showed satisfactory values for plant development in all treatments. The plumbum had very low values in all treatments and the risk of toxicity to the plant is low. The organic matter contained in the biosolids seems to be the main factor influencing the behavior of the other variables.

By the results found in all variables measured in this research, and considering the quality of the plant obtained, the improvement of the chemical properties of the soil and the minimum risk of toxicity due to the presence of heavy metals in the plant, the dose of biosolids more suitable for the soil cambisol eutrico, located in the southern part of the municipality of Puebla, is 400 t ha^-1.