2.1 Soil, sewage sludge, and biochar sampling and their properties
An agricultural soil (within depth 0–30 cm) was sampled from the Azandarian region located in the Hamedan province, Iran. The SS sample was collected from the municipal wastewater treatment plants in the Sanandaj province, Iran. Soil and SS samples were collected in 2018. The biochar sample was prepared from SS through slow pyrolysis at a temperature of 450 ºC for 7 h.
The soil, biochar and SS samples were analyzed by methods suggested by Rowell [20]. Electrical conductivity (EC) and pH were determined in deionized water with a soil-solution ratio of 1:5. The content of OM was measured by the dichromate oxidation procedure. Calcium carbonates equivalent (CCE) was determined by the acid neutralization method. Determination of cation exchange capacity (CEC) was done by a neutral 1.0 M NH4OAc saturation method. Dithionite-citrate-bicarbonate Fe (Fed) was measured by the method given by Mehra and Jackson [21]. The surface structure of SS and biochar was analyzed by JSM-840A scanning electron microscopy (SEM). Fourier transform infrared (FTIR) spectrum of SS and biochar was used to determine the chemical functional groups by a Perkin-Elmer FTIR spectrometer 17,259 using KBr disks.
2.2 Greenhouse experiment
The pot experiment was designed for untreated, control (C) and spiked soils (S). For preparing the spiked soils, the PTE amounts equivalent to 10, 250, and 400 mg kg-1 of cadmium (Cd), copper (Cu), and zinc (Zn), respectively, were added to the soil by using chloride salts. These contents were selected based on the average values of regulatory standards of PTEs in agricultural soil reported by He et al. [22]. The non-spiked (control soil) and spiked soils were incubated at field moisture for three weeks. Finally, the SS or biochar was mixed with the soils at rate of 0%, 2%, 5% and 10% on a dry weight basis. After the incubation period, cherry tomato (Lycopersicon esculentum L.) seeds were sown in peat moss and perlite combination placed in multiple-cell containers. After emergence about 14 days after sowing, one cherry tomato was planted in each of the pot (2 kg of soil each). The experiment was conducted in three replicates in a glass house at temperatures of 20–30 ºC. The period of experiment was three months (from December 2018 to March 2019). The pots were irrigated with deionized water approximately up to field capacity.
Rhizosphere and bulk soil samples were separated by pulling plants out of the soil and gently shaking them by hand at the end of the greenhouse experiment. The soil that remained on the roots was regarded as the rhizosphere soil, and the soil that separated from the roots was considered as the bulk soil [23]. Then rhizosphere or bulk samples were air-dried and thoroughly mixed for analysis.
2.3 Plant analysis
The fresh weight of each part (root, shoot, and fruit) was measured. Then, samples were placed in an oven at 70 ºC for 48 h and the dry weight of each portion was taken. The PTE content in plants was measured via the HNO3/H2O2 wet digestion method given by Cao et al. [24]. Firstly, 0.3 g of the plant samples was placed in a Teflon container. Then, 5 mL of concentrated nitric was added and the samples were left overnight. The next day, 2 ml H2O2 was added and samples were placed in a microwave digester until the mixture became clear. The aliquots collected in the bottle diluted to a final volume of 25 ml with deionized water. At the end of the digestion period, PTE content in roots, shoots, and fruits was analyzed by atomic absorption spectrophotometry (AAS).
2.4 Bioconcentration factor
The bioconcentration factor (BCF) was calculated as the ratio of PTE content in plant tissues (mg kg-1) at harvest and the content of total PTEs in soil (mg kg-1) [25]. This factor was determined for the root (BCFroot), shoot (BCFshoot), and fruit (BCFfruit) of the plant.
2.5 Human health risk assessment
Estimated daily intake (EDI) (mg kg-1 day-1) and health risk index (HRI) of PTEs via cherry tomato consumption was calculated as follows [26, 27]:
where FIR is the daily vegetable consumption of 109.0 g per person and day [28], C is the PTE content in the fresh weight of cherry tomato fruits (mg kg−1), W is the average body weight of 70.7 kg adult and 32.9 kg children [27], ORD is the oral reference dose and the values of 0.001, 0.04 and 0.30 mg-1 kg-1 day-1 were considered for Cd, Cu, and Zn, respectively [29].
If the EDI of PTEs exceeds the ORD, the HRI becomes ≥ 1.0, and human is exposed to a level of contamination with potential adverse health effects [30].
2.6 Determination of total and DTPA-extractable PTEs
Total PTEs contents in the soil, SS, and biochar were determined via the procedure proposed by Pietrzak and McPhail [31]. To determine the total PTEs content, 2 g of each sample was weighed into 50 ml tubes and 2.5 ml of 70 wt.% of HNO3 was added. The samples were left overnight to digest at room temperature. The next day, 7.5 ml of 70 wt.% HNO3 was added to each tube and the mixtures were placed in a boiling water bath. Samples were digested at 100 ºC for 8 h. After this time, samples were centrifuged for 3 min at 3500 rpm, and the supernatant was filtered into 50 ml volumetric flasks. The residual wet solid was rinsed with 10 ml and then with 5 ml of deionized water, centrifuged and the aliquots added into the 50 ml volumetric flask. Finally, PTE concentrations were analyzed by AAS.
Analysis of DTPA-extractable PTEs was carried out by the method suggested by Lindsay and Norvell [32].
2.7 Sequential extraction
The sequential extraction of PTEs was performed using the procedure suggested by Tessier et al. [33] which was modified by Jaradat et al. [34]. In this procedure, PTEs were classified into solution and exchangeable (F1; EXCH), bound to carbonate phase (F2; CARB), bound to iron and manganese oxides (F3; Fe-Mn OX), bound to organic matter and sulfides (F4; OM), and residual (F5; RES).
The mobility factor (MF) of PTEs was determined for the samples by equation 3 [35]. The units of MF and F1 to F5 are % and mg kg-1, respectively.
As a check on the values from the sequential method, the sum of the five fractions (F1+F2+F3+F4+F5 in mg kg-1) and the total content of PTEs (TC in mg kg-1) determined by concentrated nitric acid was compared in the recovery rate (R in %) equation:
2.8 Quality control of data and statistical analysis
To validate the analytical method, certified reference salts (Merck, Darmstadt, Germany) for each PTE were used to spike the soil. All experiments were carried out in triplicate and a blank sample was applied for each measurement. The instruments were calibrated by standard solutions before each measurement. The detection limits of AAS for Cd, Cu, and Zn were 0.02, 0.03, and 0.01 mg l-1. After the incubation period of soil spiking with Cd, Cu, and Zn salts, the average total PTEs recovery percentages of Cd, Cu, and Zn for spiked soils were 92.8, 95.0, and 93.7, respectively.
Statistical analyses using a significant level of p < 0.05 were carried out using SAS [36]. One-way ANOVAs followed by Duncan’s multiple range post-hoc test were applied to identify significant differences among treatments for data on bioavailability, fractionations, and uptake of PTEs. The experimental design was completely randomized with three replicates. Correlations between PTE contents in plants and extraction from soil were determined using Pearson’s correlation analysis.