## 2 Materials and Methods  
### Production, Sampling and Status of Biosolids  
The biosolids were sampled monthly during 2018 at a UWWTP in Guanajuato, Mexico, located at geographic coordinates 100\({}^{\circ}\)51\({}^{\prime}\)64\({}^{\prime\prime}\) W and 20\({}^{\circ}\)30\({}^{\prime}\)78\({}^{\prime\prime}\) N (Figure 1). The biosolids were produced by biological treatments and aerobic digestion processes. After the aerobic digestion process, the produced solids were dehydrated by means of pressed band filtration systems. The biosolids were taken six times over a four-hour period on the conveyor belt at the outlet of the filter press. Approximately 30 kg in total of the sample on a wet basis (85%) were collected during each sampling performed [21]. The samples were taken at random in triplicate from day 7 to day 23 of each month, for 1 year, with a total of 36 samples. The samples were taken to the laboratory, dried in the air, sieved through a 2 mm diameter mesh and stored at 4 \({}^{\circ}\)C for further analysis. The analysis of their physicochemical and biological properties did not exceed the storage time of 1 week [45; 46].  
As an antecedent, the biosolids produced in this study have been evaluated from August 2014 to December 2018 (three times a year randomly), complying with the physicochemical standards established in current national and international regulations [21; 22; 23; 41] for their correct final disposal. These standards only include the content of pathogens, parasites, and heavy metals in biosolids produced in a UWWTP. The biosolids have been below the permissible limits both nationally and internationally for their use (Table 1). Therefore, biosolids before this study were applied to agricultural soils but without characterisations under other parameters that are potentially applicable as biological and molecular indicators of agronomic interest or fertility for soils.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Parameter**} & \multirow{2}{*}{**Value \({}^{*}\)**} & \multicolumn{3}{c}{**MPL of Contaminants in Biosolids**} \\ \cline{3-5}  & & **Mexico** & **EE. UU.** & **E.U.** \\ \hline Fecal coliforms (MPN g\({}^{-1}\)) & 210,000 & \textless{}2,000,000 & \textless{}2,000,000 & N.R. \\ _Salmonella sp._ (MPN g\({}^{-1}\)) & \textless{}3 & \textless{}300 & \textless{}4 & N.R. \\ Helmith Eggs (eggs g\({}^{-1}\)) & \textless{}1 & \textless{}35 & \textless{}1 & N.R. \\ As (mg kg\({}^{-1}\)) & 1.81 & 75 & 41 & N.R. \\ Cd (mg kg\({}^{-1}\)) & 7.41 & 85 & 39 & 20–40 \\ Cu (mg kg\({}^{-1}\)) & 107.28 & 4300 & 1500 & 1000–1750 \\ Cr (mg kg\({}^{-1}\)) & 28.46 & 3000 & N.R. & N.R. \\ Hg (mg kg\({}^{-1}\)) & 1.03 & 840 & 17 & 16–25 \\ Ni (mg kg\({}^{-1}\)) & 30.19 & 57 & 420 & 300–400 \\ Pb (mg kg\({}^{-1}\)) & 38.19 & 420 & 300 & 750–1200 \\ Zn (mg kg\({}^{-1}\)) & 707.95 & 7500 & 2800 & 2500–4000 \\ \hline \hline \end{tabular} \({}^{*}\) The results are reported on a dry basis and are the average of the data obtained in the half-yearly analyses carried out by UWTP of Celaya from 2014 to December 2018. MPL = Maximum permissible limit; EE. UU. = United States; E.U. = European Union; N.R. = Not regulated. MPL data were obtained for Mexico from NOM-004-SEMARNAT-2002, for the United States from EPA Standard 40 CFR- 503 and for the European Union from 86/278/EEC [21; 22; 23].  
\end{table}
Table 1: Regulatory standards for the application of biosolids in soils established in national and international standards.