Germination and phytotoxicity test on sunflower seeds

The sunflower seed germination test was conducted at the Agro-environmental Management Laboratory belonging to the Faculty of Agronomy of the National University of San Agustin de Arequipa - Peru (LGA-UNSA). To determine their viability, 10 seeds were deposited on a plastic tray with absorbent paper (^{Dos-Santos et al., 2017}). The test was performed in triplicate, and each tray was irrigated with water. The number of germinated seeds was recorded after 10 days, and the results were expressed as a percentage of germinated seeds.

The phytotoxicity test on sunflower seeds was carried out in the same laboratory to verify their viability when exposed to Pb solutions at concentrations of 35, 70 and 105 ppm applied by irrigation. A Pb(NO₃)₂ solution was used as a Pb source to define the most appropriate concentration for the treatments. In this test, Pb levels were related to the environmental quality standards (EQSs) for agricultural soils established in the Peruvian regulations (^{Ministerio del Ambiente del Perú [MINAM], 2017}). For the test, 10 sunflower seeds were deposited in a plastic tray with absorbent paper (^{Dos-Santos et al., 2017}) and the corresponding Pb dilution. The test was carried out in triplicate for each concentration.

Initial soil and vermicompost analysis

The agricultural soil exposed to remediation was collected on land adjacent to an area of high vehicular traffic called the Arequipa-Peru bypass road (UTM WGS-84 North and East coordinates of 8188920 and 223859, respectively). The Pb concentration in the initial soil sample was obtained at the UNSA Research and Services Laboratory (LABINVSERV) with an atomic absorption spectrophotometer (Analyst™ 300, Perkin Elmer®). Complementary properties of the soil sample and vermicompost were determined at the Soil, Water and Seed Analysis Laboratory of the Agricultural Experimental Station (LAS-EEA-INIA, Arequipa) using the methodologies proposed by ^{Bazán-Tapia (2017)}: Walkley-Black (organic matter), potentiometer (pH), conductometer (electrical conductivity [EC]), saturation with ammonium acetate (cation exchange capacity [CEC]), micro Kjeldahl (total N), modified Olsen (available P) and spectrophotometry (exchangeable K). The sand, silt and clay particles in the soil were determined with a hydrometer, texture was obtained with the textural triangle, and bulk density was determined by relating dry mass and soil volume ^{(Bazán-Tapia, 2017}).

Research development

The study was carried out in the plant propagation greenhouse belonging to the UNSA’s Faculty of Agronomy. For this, 12 pots were used, each containing 5 kg of agricultural soil contaminated with 105 ppm Pb in the form of Pb(NO₃)₂. This concentration was determined from the phytotoxicity test. The Pb solution was applied by localized irrigation, with an incubation period of 15 days. The incorporation of vermicompost was 0.25 kg for each treatment (5 % based on the weight of the agricultural soil). Subsequently, three sunflower seeds were placed per treatment. During the development of the sunflower plant, directed irrigation was applied and a container was installed to collect the leachate from the drainage.

The treatments studied were: T1 (soil with Pb, vermicompost and sunflower), T2 (soil with Pb and vermicompost), T3 (soil with Pb and sunflower) and T4 (soil with Pb).

Evaluations

Statistical analysis

To study the effect of sunflower and vermicompost on Pb recovery in an artificially contaminated agricultural soil, a completely randomized experimental design with four treatments and three replicates was established, which generated twelve experimental units. The experimental unit was a pot with contaminated soil. The values obtained were subjected to an analysis of variance and a Tukey's mean comparison test (P ≤ 0.05). Statistical analyses were performed with the SPSS version 22 software (^{International Business Machines [IBM], 2013}).

Sunflower seed germination

After 10 days, 96.7 ± 0.47 % germination was obtained in sunflower seeds, which demonstrated their high viability to continue their development. A germinated seed was considered when there was a radicle present with a minimum length of 2 mm. According to ^{Dos-Santos et al. (2017)}, sunflower seed germination starts 4 days after sowing and ends at 13 days, as happened in this research. At 15 days, all seeds had germinated.

Phytotoxicity in sunflower seeds

The phytotoxicity test was carried out to determine seed tolerance to Pb (35, 70 and 105 ppm). Pb tolerance was determined by the ability of the seeds to germinate. Results showed a high germination percentage (90 %) at 10 days, even at the highest Pb concentration (105 ppm) (Table 1). At all Pb levels, 100 % germination was achieved at 15 days. These results allowed choosing the 105 ppm Pb dose for application to the soil as part of the treatments.

According to the phytotoxicity results, 105 ppm did not affect sunflower seed germination. This agrees with what was reported by ^{Gutiérrez-Espinoza et al. (2011)}, who evaluated the effect of different concentrations of Pb(NO₂)₃ (from 25 to 400 mg·L^-1) on sunflower seed germination and found that seeds germinated in all treatments. These authors argue that sunflower can be employed for Pb phytoremediation as indicated in other research (^{Ahmadreza et al., 2020}; ^{Gómez et al., 2018}; ^{Moslehi et al., 2019}; ^{Munive et al., 2020}; ^{Nehnevajova et al., 2005}; ^{Ortiz-Cano et al., 2009}; ^{Zhao et al., 2019}).

Initial characterization of soil and vermicompost

The soil presented an initial Pb concentration of 16.05 ppm (Table 2), which was contaminated with 105 ppm Pb, resulting in a concentration of 121.05 ppm Pb (initial Pb level in the treatments). This value exceeds the EQS established for soils in agricultural areas, which is 70 ppm (^{MINAM, 2017}).

The soil had a sandy loam texture, which facilitates sunflower root development. Organic matter content was moderate, so it requires the incorporation of a source of organic matter, such as vermicompost, to improve the Pb remediation process (^{Ortiz-Bernad et al., 2007}; ^{Volke-Sepúlveda & Velasco-Trejo, 2002}). The soil had an initial pH close to neutrality, and did not present salinity problems. Although its CEC was limited, it should increase with the incorporation of vermicompost (^{Munive et al., 2020}). The initial nitrogen level was deficient, but mineralization of the organic matter incorporated in the form of vermicompost increases its content (^{Vázquez & Loli, 2018}). The applied vermicompost composition offers a good N level, and P and K values were sufficient to ensure sunflower growth.

Regarding the characterization of the vermicompost, good organic matter content was detected (Table 2). The carbon-nitrogen ratio was low, which facilitated the subsequent humification process of the medium. The CEC was high in the material, with good nutrient (N, P, K) inputs for the remedial plant. Organic matter and CEC favor the exchange of heavy elements, and make the bioremediation process of soils contaminated with heavy elements viable (^{Ortiz-Bernad et al., 2007}; ^{Vázquez & Loli, 2018}).

Lead in soil after the remediation process

T2 (soil with Pb and vermicompost) had the highest level of recovered Pb (Table 3), which resulted in the lowest remaining Pb content in the soil, with a significant statistical difference with respect to the other treatments.

In soil, Pb is generally found in ionic form, oxides and hydroxides (^{Jiménez et al., 2017}). Its mobility in the soil is limited, so it accumulates on the surface. This mobility depends on the organic matter content and CEC, among other soil characteristics (^{Karande et al., 2019}). When Pb reacts, it can form insoluble compounds in the form of lead phosphates, carbonates and hydroxides (^{Hettiarachchi & Pierzynski, 2004}).

In this work, it was evident that the use of vermicompost facilitated Pb recovery. This behavior is mainly associated with the high organic matter content (20.63 %) and the good CEC (54.738 cmol·kg^-1). The organic matter contained in vermicompost is transformed into humic substances, especially humic acids, during the decomposition and mineralization process through resynthesis and polymerization reactions. Soil recovery from heavy elements, such as Pb, has been attributed to the action of humic acids (^{Rubenacker et al., 2011}). According to ^{Zhang et al. (2019)}, vermicompost has the ability to form organo-metal complexes between Pb and humic substances. Therefore, it is believed that the formation of functional groups (-COOH) acted as an active center to immobilize Pb. The results obtained are similar to those published by ^{Carrillo-González, Maldonado-Torres, González-Chávez, and Cruz-Díaz (2014)}, who found that Pb availability in a contaminated soil decreased with the addition of vermicompost, being inversely proportional to the applied dose; this was associated with the formation and precipitation of Pb oxide.

The high CEC of the vermicompost improved the formation of organo-metal complexes of Pb and functional groups. According to ^{Yuvaraj et al. (2021)}, this effect is due to the fact that the humic substances originated by the vermicompost make the process of cation exchange of fractionated heavy metals in contaminated soils viable.

Regarding recovery efficiency, Table 4 shows that T2 had the best performance in the recovery of Pb from the soil, although all treatments removed Pb below the EQS for agricultural soils (70 ppm). Therefore, it was deduced that all treatments performed well in Pb removal.

The best recovery efficiency, evidenced in T2, allows inferring that Pb removal assisted with amendments, such as vermicompost, is an effective method to reduce the bioavailability of this toxic soil element (^{Branzini & Zubillaga, 2010}); this is due to its high stability, bacterial fiber content and assimilable nutrients (^{Manaf et al., 2009}). The incorporation of vermicompost favors the immobilization of contaminants by improving the formation of colloidal complexes and the biological activity of the soil (^{Carrillo-González et al., 2014}). ^{Obaji et al. (2017)} point out that vermicompost, used as a soil amendment, has high potential in Pb removal due to its persistent nature and formation of active sites, which favor the sorption of heavy metals.

The performance of T1 revealed that sunflower, supplemented with vermicompost, is also efficient in Pb recovery, removing it below the EQS. ^{Jun et al. (2020)} note that Pb can enter the plant through the roots and concentrate in the foliar part; therefore, plant growth and development should be favored during the phytoremediation process (^{Rostami & Azhdarpoor, 2019}). ^{Zhang et al. (2019)} argue that phytoremediation should be assisted with soil amendments, such as vermicompost, to facilitate the bioaccumulation of metals in plant tissues. The incorporation of vermicompost into the soil facilitates the establishment of sunflower plants to remove Pb because it favors organic matter, CEC and soil biological activity (^{Branzini & Zubillaga, 2010}).

Behavior of sunflower plants in the remediation of contaminated soil

Regarding the BF of the aerial part and roots of sunflower plants, Table 5 indicates that both treatments (T1 and T3) showed values < 1, behaving as an exclusive plant species (^{Audet & Charest, 2007}). However, T1 registered lower values than T3 due to the use of vermicompost, which favors Pb bioconcentration in the aerial part and roots of sunflower. This behavior agrees with the observations of ^{Malkowski, Kurtyka, Kita, and Karcz (2005)}, who indicate that Pb accumulates in the root system in the form of Pb phosphate, which stimulates cell wall thickening.

Some plant species can extract and accumulate Pb in the root, stem and leaves to stabilize it. These species form organic compounds and enclose Pb in the cell wall and vacuole (^{Gutiérrez-Espinoza et al., 2011}). Sunflower has high Pb extraction capacity and tolerance to Pb because it develops mechanisms to enhance antioxidant enzyme activity, Pb deposition in non-active parts of the plant and stimulation of plant osmolytes (^{Moslehi et al., 2019}).

^{Marmiroli, Antonioli, Maestri, and Marmiroli (2005)} argue that part of the flux of heavy metals, such as Pb, can be retained in the cell wall by the ligno-cellulosic structure. Plant species can reduce the toxicity of metals in their environment by adopting different abilities to resist, such as the exclusion of the metal, which limits its transfer to the aerial part, or the accumulation of the metal in the leaf part in non-toxic forms (^{Alderete-Suarez, Valles-Aragón, Canales-Reyes, Peralta-Pérez, & Orrantia-Borunda, 2019}).

The TF of T1 was < 1 (Table 6), revealing that the roots of the sunflower plant, in the presence of vermicompost, promote the Pb phytostabilization mechanism (^{Audet & Charest, 2007}; ^{Deng et al., 2004}). In this way, Pb is retained in the sunflower root system with the help of vermicompost, whose function is to capture Pb in the clay-humic complex of the soil (^{Branzini & Zubillaga, 2010}; ^{Carrillo-González et al., 2014}). Pb in an ionic state enters the plant through the roots by passive diffusion, and once absorbed by the plant, most of it is retained in the roots by ion exchange bonds in the cell wall (^{Alvarado, Dasgupta-Schubert, Ambriz, Sánchez-Yañez, & Villegas, 2011}).

The T3 results registered a TF > 1, which indicates that the sunflower plant in the absence of vermicompost enhances the translocation of Pb from the roots to the aerial part of the plant. In this case, as there is no vermicompost, the Pb phytoextraction mechanism of the sunflower plant is boosted (^{Audet & Charest, 2007}; ^{Deng et al., 2004}; ^{Ortiz-Cano et al., 2009}), and concentrates the Pb in the aerial part of the plant (^{Nehnevajova et al., 2005}).

^{Munive et al. (2020)} obtained similar results (BF of 0.08 and TF of 1.2) when using vermicompost and sunflower as a Pb phytoremediation plant. ^{Alaboudi, Ahmed, and Brodie (2018)} demonstrated that the sunflower, by increasing its biomass, was able to remove toxic substances from contaminated soil because of its ability to adapt to adverse environments.

Finally, a soil analysis was performed in T2 (soil with Pb and vermicompost) at the end of the remediation process, since this treatment achieved the highest efficiency in Pb recovery. The analysis found a sandy loam texture, without variation with respect to the initial soil texture. Organic matter increased from 3.91 to 4.12 %, which is associated with the incorporation of organic matter through vermicompost. The pH changed slightly from 6.78 to 6.93, and salinity increased to 3.11 dS·m^-1, although this value does not imply salinity problems. CEC increased from 9.72 to 17.872 cmol·kg^-1 due to the increase in organic matter. Total N, available P and exchangeable K presented values of 0.47 %, 16.250 ppm and 218.733 ppm, respectively, which were higher than those identified at the beginning of the remediation process and are associated with decomposition processes of the soil organic matter and with the vermicompost, which through its mineralization released these nutrients. Consequently, T2 was not only the most efficient in Pb recovery, but also favored soil properties.