Can fertilization with sewage sludge contaminate pineapple fruits with phthalates?

The aim of the present study was to evaluate the phthalate content in the soil and in the pineapple fruit fertilized with different types of sewage sludge and mineral fertilizer. The experiment was carried out in a greenhouse, in randomized block design, with seven treatments and three replications. The treatments were: control (C), mineral fertilization (MF), fertilizations with solarized sewage sludge (SS), sanitized sewage sludge with calcium oxide (CaS), dried sewage sludge (DS), composted sewage sludge (CS), and vermicomposted sewage sludge (VS). The contents of dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethyl hexyl phthalate (DEHP) in the soil and in the pineapple pulp were analyzed. Fertilization with sewage sludge sanitized with calcium oxide (0.012 mg kg), dried (0.017 mg kg), and solarized (0.031 mg kg) provided the lowest levels of DMP in the soil, while the sewage sludge sanitized with calcium oxide (0.046 mg kg), dried (0.054 mg kg), solarized (0.063 mg kg), and vermicompost (0.076 mg kg) provided the lowest levels of DEHP in pineapple pulp. Sludge fertilizations maintained the levels of DMP, DBP, and DEHP in soils, below the maximum limits for prevention, established by Brazilian environmental legislation. In addition, all phthalates levels in the pineapple pulp were below the critical safety limits for this food consumption.


Introduction
The basic sanitation sector, aiming to meet environmental requirements regarding the solid waste management, faces difficulties with the final destination of sewage sludge, since the waste must be correctly disposed, either in landfills, incinerated or even applied in agriculture (Song et al. 2013, Santos et al. 2017. Studies show that sewage sludge has been used in agriculture because it is a compound rich in organic matter (OM) and nutrients, mainly nitrogen and phosphorus (Melo et al. 2018, Florentino et al. 2019).
Among several crops produced in Brazil, pineapple production reached 1.6 million tons of 2019 (IBGE, 2019).
Pineapple is a fruit plant with high nutritional demand, mainly for nitrogen (Leonardo et al. 2019). Thus, sewage sludge and its derivatives can be used as nutrients source for this crop and as alternative in order to reduce its production costs (Mota et al. 2018).
However, one of the biggest obstacles to the sewage sludge application in agriculture is the contaminants presence in its composition, such as toxic organic compounds (Ignatowicz, 2017, Urbaniak et al. 2017). Among them, phthalate esters or just phthalates can be mentioned, which are chemical compounds used as plasticizers due to the benefits they provide to the final product such as flexibility and elasticity (Chang et al. 2021).
These substances may belong to the short alkyls group, such as dimethyl phthalate (DMP) and dibutyl phthalate (DBP), which are soluble in water, and the long alkyls group, such as diethyl hexyl phthalate (DEHP), which is less soluble in water and more recalcitrant. Some studies report that the exposure of these compounds to humans, especially pregnant women and fetuses, causes adverse health effects. However, they are prone to biodegradation, photodegradation and anaerobic degradation, which means that most of them do not persist for long in the environment (Chen et al. 2017a, Karačonji et al. 2017, Li et al. 2019, Podlecka et al. 2020.
Therefore, in order to reduce sewage sludge contaminants and make its use feasible in agricultural systems, it is necessary to sanitize (clean) it through physical, chemical, and/or biological processes, which cause changes in the nutrients bioavailability, in pathogens reduction and odor. There are some methods used for this purpose, as composting, churning, drying at high temperatures, solarization and vermicomposting, all of which promote changes in the physical and chemical characteristics of this residue (Nascimento et al. 2014, Martín-Díaz et al. 2020.
In this context, the aim of the present research was to evaluate the phthalates content in soil and in pineapple fruits pulp fertilized with sewage sludge derived from different hygiene processes.

Experiment location and climatic characteristics
The experiment was conducted from November 2014 to September 2016, in a greenhouse at the Institute of Agricultural Sciences (ICA) of the Federal University of Minas Gerais (UFMG), Montes Claros, Brazil, with latitude 16° 40' 58" S, longitude 43° 50' 25" W and "Aw" climatic classification (hot tropical with dry winter), according to Köppen (Alvares et al. 2013).

Cultivated variety and soil attributes
The pineapple seedlings of cultivar Pérola (Ananas comosus var. Comosus) were planted in soil samples from Cambissolo Háplico, belonging to the fruit cultivation area of the institution, whose chemical and physical attributes of the 0-20 cm deep layer are shown in Table 1.

Experiment installation
The experiment was arranged in randomized blocks with seven treatments and three replications, totaling 21 The sewage sludge used in the present research came from the Sewage Treatment Station of Montes Claros city, MG, called "ETE -Vieira". The ETE consists of preliminary treatment stages followed by an Upflow Anaerobic Sludge Blanket (UASB) and subsequent centrifugation of the sludge generated in the reactor (85% of moisture).
For the application of this sewage sludge in the soil, different sanitation processes were carried out to allow its safe use in the pineapple cultivation. Thus, the solarized sludge (SS) was obtained by drying the centrifuged sludge, in full sun, for 30 days, in a 30 cm layer. The sanitized sewage sludge with calcium oxide (CaS) originated by the addition of calcium oxide (CaO) in 50% proportion of sewage sludge dry mass, the dehydrated sludge was dried in a dryer at 350 ºC for 30 min. The Research, Society and Development, v. 10, n. 9, e9810917659, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17659 composted sludge resulted from the mixture of the centrifuged sludge to the grass pruning (Paspalum notatum) in the ratio of 1:5 m/m, to obtain C/N ratio of 30/1. The vermicomposted sludge was obtained using California Red earthworms (Eisenia foetida) in a pre-compound obtained from the mixture of the previous treatment, but with only one month of compost. The chemical attributes of the sludge after the cleaning processes are shown in Table 2.  (2006) Table 2).
The experimental units consisted of pots with 10 dm 3 capacity, filled with 8 dm 3 (10.4 kg) of air-dried soil, passed through a 4 mm sieve and mixed with the treatments mentioned above. The pineapple seedlings were the young type of cultivar Pérola, from tissue culture, and one seedling was planted per experimental unit. After planting and throughout the growing period, the pineapple seedlings were irrigated, keeping the soil moisture close to the field capacity.
Soil collection for phthalates quantification was carried out at the pineapple flowering beginning, at ten cultivation months. The collection was performed using a cylindrical metal collector and then the soil samples were air dried, passed through a 2 mm sieve and stored in paper bags.
The fruits were collected at the cultivation cycle end when they were approximately 50% of their yellowish area.
Afterwards, fruit pulp sample was taken, which was crushed in stainless blender and stored in glass container for further phthalates analysis.

Phthalates extraction in sludge, soil, and pineapple
The extraction of phthalates DMP, DBP, and DEHP in sewage sludge, soil and pineapple were carried out using solidliquid extraction with low temperature purification (SLE-LTP). The sewage sludge and soil samples were analyzed using methodologies proposed by Pereira et al. (2019).
On the other hand, the extraction of phthalates in pineapple was carried out by transferring 3.0 g of the sample to a glass vial (22 mL) and then 2 mL of distilled water and 8 mL of acetonitrile were added. The system was homogenized in vortex for 1 minute and subjected to freezing at -20 °C, for 3 hours. After that period, 2.5 mL of the organic phase was transferred to a glass test tube containing 350 mg of anhydrous sodium sulfate, homogenized for 1 minute and centrifuged at 4000 rpm for 10 minutes. Subsequently, 1 mL of the supernatant was transferred to the injection vial and analyzed by gas chromatography-mass spectrometry (GC-MS).

Chromatographic analysis
The quantification of the three phthalates (DMP, DBP, and DEHP) was performed in a gas chromatograph (Agilent The data were submitted to analysis of variance (ANOVA) and the treatment averages were compared by the Scott-Knott test at 0.05 of probability, by the software R version 3.6.3 (R Core Team, 2013).

Phthalates in Cambisolo Háplico samples
In the Cambisolo Háplico sample used in this study, the DMP (dimethyl phthalate) presence was not detected, however, contents of 0.015 and 0.021 mg kg -1 for DBP (dibutyl phthalate) and DEHP (diethyl hexyl phthalate) were found, respectively (Table 2). These values are lower than the maximum limits established for soil prevention, according to CONAMA Resolution N o 420 (Brasil, 2009), which is 0.25, 0.70, and 0.60 mg kg -1 , respectively, for DMP, DPB, and DEHP.

Phthalates in soil samples fertilized with sewage sludge
After pineapple cultivation, DMP (dimethyl phthalate) was quantified in soil samples from all treatments ( Figure 1A).
This substance quantification in control and mineral fertilization treatments allows inferring that there was DMP contribution by the better water used in irrigation, which may have been contaminated by the pesticides systematic use in the cultivation area (Sui et al. 2014, Ning et al. 2017. Anyway, treatments can be separated in relation to DMP levels in three main groups: the first formed by C treatment, with higher content of DMP, the second formed by MF, CS, and VS, with intermediate levels, and the third formed by SS, DS and CaS, with lower levels. The explanation for the fact mentioned above is that CaS and DS promoted less DMP input compared to the other sewage sludge (Table 2). In addition, the SS, as it is less decomposed, may have undergone more intense action by microorganisms, causing a greater DMP reduction.
The higher DMP levels in treatments CS and VS compared to SS and DS can also be explained by the decomposition degrees of these sewage sludges. In CS and VS, the organic matter was already much moisturized, promoting the diffusion of DMP in the humus porous matrix by adhesion and cohesion forces (van der Waals molecular forces). In this hydrophobic sorption, the moisturized soil organic matter acts as a solubilizing place for substances dissolved in water, analogous to octanol, with DMP partitioning into two phases (solution and soil organic matter) or by dissolution exclusively, that the less polar the substance, greater the tendency to be partitioned in the hydrophobic phase. Thus, DMP in CS and VS treatments were Research, Society andDevelopment, v. 10, n. 9, e9810917659, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17659 6 retained in the soil by hydrophobic partition, with low levels in the soil solution, and less availability for microbial degradation and plant absorption, when compared to SS and DS treatments (Silva, 2005, O'Connor, 1996. Anyway, when compared to the control (0.069 mg kg -1 ), the treatments with mineral fertilization (0.047 mg kg -1 ) and sewage sludge (0.031, 0.012, 0.017, 0.046 and 0.039 mg kg -1 for SS, CaS, LD, CS, and VS, respectively) promoted a greater reduction in soil DMP levels, which can be attributed to better fertility and, consequently, greater soil biological activity.
However, in the case of DBP, DEHP, and ∑DP, there were no differences between treatments (p> 0.05) (Figs. 1B, 1C and 1D), despite the differences in these substances contribution by sewage sludge treatments (Table 2). DEHP, followed by DBP, were the most abundant compounds in the present study (Figure 1). This fact corroborates with Roslev et al. (2007), who monitored a sewage treatment plant and found higher levels of DEHP and BBP congeners, followed by DBP and DMP in dehydrated sludge. Thus, these compounds abundance indicates greater population consumption of materials that have these phthalates in their composition. DEHP is widely used in the polymer industries and DBP is used in paints, adhesives, perfume fixers, among others (Meng et al. 2014, Vetta Química, 2015, Chen et al. 2017b).
Composting reduces phthalate content, mainly of DEHP, and its greatest reduction occurs in the thermophilic phase, which can be degraded by the bacteria Acinetobacter sp. and Micrococcus luteus, among others (Cheng et al. 2008, Pakou et al. 2009, Benjamin et al. 2015. Thus, the composting, solarization and thermal drying techniques are efficient in reducing of sewage sludge phthalates (Bagó et al. 2005, Gibson et al. 2007. DEHP was the phthalate most concentrated in the sewage sludge of the present research, with emphasis on the solarized sewage sludge (SS), which promoted the largest contribution of this substance (Table 2). Thus, considering the critical prevention level of 0.6 mg kg -1 of DEHP in the soil, required by CONAMA Resolution N o 420/2009(Brazil, 2009, and the content already existing in the soil (Table 1), it is estimated that pineapple fertilization with SS could be carried out a maximum of 6.6 times consecutively. In contrast, if composted sewage sludge (CS) were applied, with the lowest DEHP input, the number of times to reach the critical level would be 49.3. These values do not consider soil phthalates losses resulting from degradation processes or in other ways.

Phthalate in pineapple fruit treated with sewage sludge
DMP levels in the pineapple pulp were statistically similar between treatments (Figure 2), with values ranging from 0.02 to 0.04 mg kg -1 , while DBP levels were below the quantification limit by the analysis method used, which was 0.005 mg kg -1 .
In DEHP case, fruits from treatments with mineral fertilization (MF) and composted sludge (CS) had the DEHP highest levels (Figure 2), with averages of 0.15 and 0.11 mg kg -1 , respectively. However, for the other treatments, there were no significant differences between them, with values ranging from 0.02 to 0.08 mg kg -1 .
For ∑DP, the behavior was the same observed for the DEHP congener ( Figure 2), with highest values occurring in MF and CF treatments, with respective values of 0.19 and 0.14 mg kg -1 . In this case, there were also no significant differences between the other treatments, with a variation between the means of 0.05 to 0.10 mg kg -1 . Comparing the two congeners absorbed by the fruits, DEHP was absorbed in greater quantity than DMP (Figure 2), possibly due to its higher initial soil content and greater contribution through fertilization (Tables 1 and 2). However, DEHP has a higher molar mass, partition coefficient (Log Kow) and hydrophobicity, which would imply greater sorption by the soil's organic matter duo to its absorption by the plant (Cheng et al. 2008, Wang et al. 2015, Lin et al. 2017).
In Brazil, there is still no legislation that determines the maximum phthalate limit that can be ingested by humans without causing adverse health effects. However, according to Barros (2010), the acceptable daily intake of DEHP is 25 µg kg -1 of body weight per day for a man of 1.70 m height and 60 kg of body mass. In addition, the National Health Surveillance Agency (ANVISA) through the '' Technical regulation on the positive list of additives for plastic materials intended for the packaging and equipment preparation in contact with food '' determines that the acceptable migration limit of food packaging is 0.3 and 1.5 mg kg -1 of DBP and DEHP, respectively (Brasil, 2008).
Thus, considering the higher DEHP content observed in the pineapple fresh fruit (Figure 2), which was 0.15 mg kg -1 , and the daily intake of two pineapple slices with total fresh mass estimated at 180 g by one person of 60 kg of body mass, the rate of this substance ingestion would be only 0.45 µg kg -1 day -1 , that is, less than 2% of the maximum tolerable limit. In addition, the highest fruit DEHP content corresponded to only 10% of the maximum content allowed in food due to contact with plastics. Therefore, regardless of the fertilization type, the phthalate congener's levels in pineapple pulp were below the critical safety limits for this food consumption. It is worthy to mention that the present study evaluated the DMP, DBP, and DEHP congeners and did not consider pathogens and other contaminants that may be present in sewage sludge. And it is necessary to do more studies to understand the influence of those contaminants in pineapple cultivation treated with sewage sludge.

Conclusion
Fertilization with solarized, dried, sanitized with calcium oxide sewage sludge provides the dimethyl phthalate (DMP) lowest levels in soil cultivated with pineapple.
For pineapple pulp, fertilization with solarized, dried, sanitized with calcium oxide, and vermicomposted sewage sludge provides the lowest levels of diethyl hexyl phthalate (DEHP).
The fertilization with sewage sludge maintains the levels of dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethyl hexyl phthalate (DEHP) in soils, below the maximum limits for prevention, established by Brazilian environmental legislation.
Diethylexyl phthalate (DEHP) is the phthalate with the highest content in the soil and in the pineapple pulp, while dibutyl phthalate (DBP) is not quantified in the fruit.
The phthalate congener's contents in pineapple pulp were much below than the critical safety limits for this food consumption.
Finally, as a way to advance the scientific knowledge presented in this study, future investigations approaching analyzes of pathogens and other contaminants that may be present in sewage sludge.