doi:10.14720/aas.2020.116.1.1116 original research article / izvirni znanstveni članek Titanium dioxide nanoparticles alleviate cadmium toxicity in lentil (Lens culinaris Medic) seeds Hassan FEIZI 1 2, Nafiseh AGHELI 1, Hossein sAHABI 1 Received april 11, 2019; accepted May 29, 2020. Delo je prispelo 11. aprila 2019, sprejeto 29. maja 2020. Titanium dioxide nanoparticles alleviate cadmium toxicity in lentil (Lens culinaris Medic) seeds Abstract: This research was carried out with the aim of investigating the effects of different concentrations of titanium dioxide nanoparticles and cadmium on lentil seed germination and seedling early growth. The experiment was conducted as a factorial layout based on a completely randomized design with four replicates. Test plants were exposed to 0, 100, 250, and 500 ppm of cadmium, and then amended with 0, 100, 200, and 300 ppm of T^2 nanoparticles. Results demonstrated that the application of 300 ppm of titanium dioxide nanoparticles improved the mean germination time (MGT) and seed germination rate by 39 % and 62 % respectively. Most traits exhibited a decreasing trend as the concentration of cadmium increased. Furthermore, application of 500 ppm of cadmium concentration increased mean germination time compared to the control group (1.667 day). In conclusion, the application of titanium dioxide nanoparticles averted the effect of high cadmium stress on lentils, and maintained the length of the shoot compared to the control. However, further studies on plant life cycles should be performed to detect the inhibitory effects of titanium dioxide on heavy metal toxicity. Key words: heavy metals; cadmium; germination; nanoparticles Nanodelci titanovega dioksida zmanjšujejo strupenost kadmija pri kalečih semenih leče (Lens culinaris Medic) Izvleček: Namen raziskave je bil preučiti učinke različnih koncentracij nanodelcev titanovega dioksida in kadmija na kalitev semen leče in zgodnjo rast njenih sejank. Poskus je bil izveden kot popolni naključni faktorski poskus s štirimi ponovitvami. Testirane rastline so bile izpostavljene 0, 100, 250 in 500 ppm kadmija, s kasnejšim dodatkom 0, 100, 200 in 300 ppm nanodelcev Rezultati so pokazali, da je dodatek 300 ppm nanodelcev titanovega dioksida izboljšal poprečni čas ka-litve (MGT) za 39 % in hitrost kalitve za 62 %. Večina merjenih znakov je pokazala padajoč trend s povečevanjem koncentracije kadmija. Uporaba 500 ppm koncentracije kadmija je povečala poprečni čas kalitve za 1,667 dni v primerjavi s kontrolo. Zaključimo lahko, da je pri leči uporaba nanodelcev titanovega dioksida odpravila učinke stresa velikih koncentracij kadmija in ohranila dolžino poganjkov primerljivo s kontrolo. Vsekakor pa so potrebne še nadaljne raziskave življenskega kroga rastlin za ugotavljanje inhibitornih učinkov titanovega dioksida na toksičnost težkih kovin. Ključne besede: težke kovine; kadmij; kalitev; nanodelci 1 University of Torbat Heydarieh, Department of Plant Production, Torbat Heydarieh, Iran 2 Corresponding author, e-mail: h.feizi@torbath.ac.ir Acta agriculturae Slovenica, 116/1, 107-114, Ljubljana 2020 H. FEIZI et al. 1. introduction Nowadays, the need for agricultural production, along with the demand for plant raw materials for food production, has increased in the world. In this condition, the use of some branches of the industry in new research and the adoption of safe decisions are necessary to increase agricultural production. Anthropogenic activity degrades the soil, water, and atmosphere and this has led to the search for sustainable ways to improve food production (Feizi & Rezvani Moghadam, 2010). Soil contamination with heavy metals is one of the most important environmental problems worldwide. Upon gaining entry into agricultural lands, heavy metals can be absorbed by plants and transferred to humans through the food chain. The increase in the heavy metal content in the human body is due to the consumption of plant products grown in areas contaminated with these metals, inhaling soil particles and consuming contaminated water (Motesharezadeh et al., 2014). Among heavy metals, cadmium is one of the most toxic elements which has no biological role in humans. Cadmium (Cd) is an unnecessary element with high biological durability causing tubalization of leaves, chlorosis and reduced root and stem growth (Ahmadvand et al., 2012). In plants, Cd exposure frequently triggers negative effects on their growth and productivity. Recently, it has been proved that Cd can also increase root length and number, and consequently, root area and volume. In plant shoots, Cd can improve the content of photosynthetic pigments and enzymatic and non-enzymatic antioxidants are enhanced in both roots and shoots (Carvalhoa, et al. 2020). Although plants need some heavy metals such as zinc, iron, nickel, titanium etc. at very low concentrations, however, it leads to metabolic disorders and inhibitory growth of most plant species when the concentration of these metals is higher than the plant requirement. Recently, scientists and researchers have begun to develop efficient and inexpensive technologies, such as the use of microorganisms and living plants to remediate contaminated areas. (Ahmadvand et al., 2012). Titanium is one of the beneficial elements for plant, and can stimulate the absorption of other elements such as nitrogen, phosphorus, calcium, magnesium, iron, manganese and zinc (Pais, 1983). Ti02 nanoparticles have shown considerable advantages in absorbing and concentrating chemical pollutants owing to their exclusive properties such as high surface area-to-volume ratio, enhanced surface activity, and high affinity (Deng et al., 2017). In one study on fennel plants, it was observed that treatment with 60 ppm titanium dioxide nanoparticles significantly improved the seed germination percentage and germination rate (Feizi et al., 2013). In addition, the highest mean activity of ascorbate peroxidase, guaiacol peroxidase, and catalase enzymes on annual medic plant (Medicago scutellata L.) was found in foliar application of titanium nanoparticles in 0.06 % concentration (Dola-tabadi et al., 2015). Previous studies indicated that Ti02 nanoparticles had high efficiency and adsorption rate for Pb, Cd, Cu, Zn, and Ni (Engates & Shipley, 2011). The potential influence of Ti02 nanoparticles on the uptake and toxicity of heavy metals in biological systems has been documented. Ti02 nanoparticles significantly enhanced the bioaccumulation and toxicity of Pb, Cu, As(III), Zn, and Cd in zebra fish (Danio rerio Hamilton, 1882) larvae, Daphnia magna Straus, 1820, and carp (Cyprinus carpio Linnaeus, 1758) (Miao et al., 2015; Tan & Wang, 2014). However, Chen et al. (2015) reported that the bioavailability and toxicity of Cd and Cu are reduced in alga (Chlamydomo-nas reinhardtii P.A.Dang) and (Microcystis aeruginosa Kutzing ) and amphipod (Gammarusfossarum Linnaeus, 1758) by simultaneous exposure to Ti02 nanoparticles. Although both stimulatory and inhibitory impacts of nanoparticles on various crop species have been reported in the literature, there is limited information on the impact of Ti02 nanoparticles in heavy metal stress. This work investigated the impact of Ti02 nanoparticles in ameliorating the hazardous effects of Cd on seed germination and seedling growth of lentil. Furthermore, it assesses the co-effect of Ti02 nanoparticles and cadmium on lentil seedling. Therefore, our objectives were to (i) survey Cd-stressed lentil seed germination parameters using Ti02 nanoparticles, (ii) evaluate inhibitory effects of cadmium on lentil seedling and (iii) evaluate protective role of Tio2 nanoparticles under cadmium toxicity conditions. 2 materials and methods This research was carried out in the laboratory of Medicinal plants of Faculty of Agriculture, University of Torbat Heydarieh, Iran with the aim of investigating the co-effects of different concentrations of titanium dioxide nanoparticles and cadmium sulfate on lentil seed of Gachsaran cultivar (based on literature minimum concentration of Cd that could harm the lentil is unknown). This experiment was conducted as a factorial based on a completely randomized design with four replicates (in total 1600 seeds). We had a total 16 treatment combinations. We had four replicate for 16 treatments and considered 25 seeds in each treatment (25 seeds into each Petri dish). Consequently we calculated 4 x 4 x 4 x 25 = 1600 seeds. The first factor consisted of different levels of cadmium sulfate concentration of 0, 100, 250 and 500 60 Acta agriculturae Slovenica, 116/1 - 2020 Figure 1: image of nanosized Ti02 by Scanning Tunneling Microscope (STM) ppm, and the second factor included the concentrations of Ti02 nanoparticles of 0, 100, 200 and 300 ppm. Cadmium sulfate (CdS04) as a salt of cadmium was used for preparing cadmium stress treatment. First, healthy lentil seeds were selected and disinfected with sodium hypochlorite 5 % for 60 seconds, then washed with distilled water three times. The titanium dioxide nanoparticle powder was AER0XIDE® Ti02 P25 prepared from Evonik Degussa GmbH Company of Germany. The purity of nanoparticle powder was 99.8 % and the mean particle size was 21 nm. The required amount for each treatment level of titanium dioxide was weighted using a digital scale with a precision of 0.0001 g and by adding distilled water as the suspension. In order to obtain properly dispersed and stable Ti02 suspensions of each concentration, an ultra-sonica-tion treatment was applied to nanoparticles Ti02 powders dispersed in water for 15 min. After preparation, the nanoparticles were poured in different concentrations at separate Erlenmeyer flasks and the seeds were soaked in each concentration for 2 hours. The Erlenmeyer flasks were placed on a shaker to prevent the deposition of nanoparticles, so that the seeds had an identical contact surface with nanoparticles during the treatment. The size of Ti02 nanoparticles (Figure 1) were determined by Scanning Tunneling Microscope (STM) in Central Laboratory of Ferdowsi University of Mashhad, Iran. Then, they were placed in a 25-seed group with an appropriate distance from each other in each Petri dish. The experimental unit consisted of a sterile Petri dish with a culture medium of filter paper type (TP or paper culture method). Filter paper was packed in a double layer of aluminum foil for disinfection and placed in oven for 2 hours at a temperature of 120 °C, then, 5 ml of cadmium sulfate solution was added to each Petri dish. Distilled water was used for control treatment. Petri dishes were placed inside the germinator at 23 ± 1 ° C at 16/8 hours length in day / night (ISTA, 2009). The daily count of germinated seeds was performed every 12 hours in each experimental unit and seeds with seminal root length of more than two millimeters were counted as germinated seeds (ISTA, 2009). After a period of time necessary for planting seeds (10 days), ten seeding selected in each Petri dish. The length of seminal root and shoot was measured with the ruler and then, the roots and shoots were separated and placed them individually inside the pocket and placed in an oven at 70 °C for 48 hours. After drying the shoots and roots, they were weighed per treatment individually. In this experiment, traits such as the length of seminal root, and seedling, seedling mass, germination rate, germination percentage, seed vigor index I and II were evaluated. On the tenth day, the count of seminal root, shoot and whole seedling was measured and then the seedling dry weight was determined in the oven for a suitable time. Germination rate was calculated by the Maguire (1982) formula as equation (1): Germination Rate (GR) = (a \ 1) + (b - a / 2) + (c - b \ 3) + ... + (n - n - 1 / N) (1) MGT = Z (F.X) + ZF (2) In this equation, MGT: mean germination time (day), F: the number of new germinated seeds per day of the counting day X and X days of counting. For calculating the seed vigor index, equations (3) and (4) were used (Vashisth & Nagarajan, 2010). (3) Vigor index I = germination percentage x seedling length in cm (shoot + seminal root) Acta agriculturae Slovenica, 116/1 - 2020 139 H. FEIZI et al. (4) Vigor index II = germination percentage x seedling dry mass in mg (shoot + seminal root) Data from the experiment was first processed in Excel spreadsheet software, then statistical analysis of the data was done by MSTAT-C software. Means were compared by Duncan's Multiple range test. 3 results and discussion The results of analysis of variance showed that the application of different concentrations of titanium dioxide nanoparticles had a significant effect on all traits tested (Table 1). Also, different cadmium sulfate contents had a significant effect on other traits. The interaction between titanium dioxide nanoparticles and cadmium sulfate was significant on the rest of the studied traits except for the mean germination time. The results showed that all concentrations of titanium dioxide increased the seed germination percentage of lentil compared to control which increased 12.4 % relative to the control (Table 2). The application of 300 ppm of titanium dioxide nanoparticles also improved the mean germination time of lentil seeds by 39 % (Table 2). Seed germination rate was from 14.75 seeds per day in the control to 23.9 seeds per day was applied in the treatment of 300 ppm of titanium dioxide nanoparticles which was an increase of 62 % compared to the control. It seems that the nanoparticles have had a positive effect on the rate of water and oxygen exchange from the seed shell to different parts of the seed and ultimately significantly improved seed germination rate. According to the study conducted by Feizi et al. (2013) on fennel (Foeniculum vulgare L.), it was observed that, treatment of 60 mg l-1 titanium dioxide nanopar-ticles greatly improved the germination percentage and germination rate. But the treatment of 40 ppm of non-nanoparticles increased the mean germination time by 31.8 %. Also, other experimental results on the sage (Salvia officinalis L.) have shown that the concentration of 60 ppm of nanoparticles and non-nanoparticles of titanium dioxide improved germination percentage, and mean germination time compared to control. Furthermore, they declared that the germination rate in the treatment with 60 ppm nanoparticles of titanium dioxide was increased (Feizi et al., 2013). In the investigation of the effect of various concentrations of titanium dioxide nanoparticles on germination traits of silybum (Silybum marianum L.), Agheli et al. (2016) stated that the highest seed germination percentage was observed in the interaction of titanium dioxide nanoparticles treatment at a concentration of 100 ppm and magnetic field intensity of 30 mTesla (mT) and the application of titanium dioxide 60 Acta agriculturae Slovenica, 116/1 - 2020 nanoparticles with a concentration of 50 ppm and a magnetic field intensity of 60 mT. These treatments improved the germination percentage by 7.5 and 6.5 times the control treatment. The effect of different concentrations of titanium dioxide nanoparticles on the growth traits of seedling was different so that the shoot length was increased at lower concentrations, but the application of higher concentrations like 300 ppm resulted in increasing the shoot length. Nanoparticles at the concentration of 100 ppm resulted in a significant increase in shoot length from 2.273 cm to 2.623 cm. Nevertheless, application of 300 ppm concentration did not have affect its increase (Table 2). The different response of seminal root length was that only 300 ppm concentration caused a significant increase in seminal root length than in the control and increased its value by 26.2 %.The concentrations of titanium dioxide nanoparticles (100 and 300 ppm) significantly increased the seedling length compared to the control. The highest seedling length in the 100 ppm nanoparticle treatment was 12.9 % and increased its length from 4.04 cm to 4.56 cm (Table 2, Figure 2). Agheli et al. (2016) stated that the maximum seedling length of Silybum marianum was observed in the application of titanium dioxide nanoparticle treatment with a concentration of 100 ppm and a magnetic field intensity of 30 mT, and also the application of titanium dioxide nanoparticles with a concentration of 50 ppm and a magnetic field intensity of 60 mT. However, application of titanium dioxide nanoparticles had the highest significant effect on seminal root mass of lentil. so that application of concentration of 100 and 300 ppm caused a significant increase of 34 % and 43 % respectively. The lowest seminal root mass was obtained in the control and 200 ppm treatment. Titanium dioxide nanoparticles had no significant effect on shoot and seedling mass (Table 2). In the investigation of the effect of titanium dioxide particles on germination of wheat it was observed that, all treatments had significant effect on seminal root mass and the highest root mass was observed in treatments of two and 50 ppm of non-nanoparticles and 100 ppm of titanium dioxide nanoparticles (Feizi, 2012). The researchers showed that the effect of titanium dioxide nanoparticles on dry biomass was significant at 5 % probability level and reported the highest average (21481.4 kg ha-1) for biological yield for titanium dioxide nanoparticles and the lowest for control (17782.4 kg ha-1) (Moaveni et al., 2011a). seed vigor 1 was significantly affected by titanium dioxide nanoparticles and all concentrations significantly increased it compared to the control and increased its value by about 20 %. But seed vigor 2 was significant- Table 1: Analysis variance of effects of Ti02 nanopartcles and CdS04 on lentil seed and seedling traits Source of Shoot Root Seedling Shoot Root Seedling Germination Germination Vigor index Vigor index Variation df length Length Length mass mass mass MGT percent rate 1 2 Nano Ti02 (A) 3 0.392* 0.807* 1.314* 104.983* 3.473** 76.068 2.190* 384.766** 269.129** 20393.794** 1117827.3* Cd (B) 3 13.195** 106.481** 162.166** 573.845** 92.118** 783.060** 1.219* 1049.349** 243.520** 1635187.897** 15230258.6** AB 9 0.265* 7.412** 5.9780** 154.988* 4.228** 144.874* 0.158ns 196.571** 27.322* 62836.241** 1411315.1* Error 48 0.126 0.221 0.400 90.104 0.356 91.951 0.720 37.891 15.822 2932.194 684457.8 CV 14.67% 24.11% 14.46% 21.76% 23.18% 20.75% 65.05% 6.63% 19.15% 12.89% 19.11% V* denotes significant in 1 and 5 % probability level; ns denotes not si gnificant Table 2: Effects of Ti02 nanopartcles concentrations on lentil seed and seedling traits Seedling Germination TiO concentration Shoot Root Seedling Shoot mass Root mass mass MGT rate Germination Vigor index Vigor index (ppm) length length (cm) length (cm) (mg) (mg) (mg) (day) (seed/ day) (%) 1 2 0 2.273b 1.764b 4.036b 44.941 2.144b 47.09 1 1.388 ab 14.75b 85.63b 372.9 b 4131a 100 2.623 a 2.035 ab 4.658 a 40.66 1 2.875 a 43.541 1.065b 21.88 a 94.38 a 447.9 a 4141a 200 2.469 ab 1.768b 4.238 * 46.43 1 2.213 b 48.641 1.803 a 22.55 a 96.25 a 412.2 a 4696a 300 2.330b 2.226a 4.556a 42.491 3.069 a 45.56 1 0.9924b 23.90 a 95.31a 447.8 a 4351a * Means with the same letter are not significantly different at the 5 % probability. H. FEIZI et al. Figure 2: Root and shoot lengths of lentil seedling in Ti02 nanoparticles concentrations The left Petri dish shows control, Petri dish in center is 100 ppm T02 nanoparticles, The right petri dish is 200 ppm T02 nanoparticles in the medium. ly affected by the concentrations of titanium dioxide nanoparticles (Table 2). The results of exposing seeds to cadmium sulfate showed that, with increasing concentration of cadmium sulfate in growth medium, most traits showed a decreasing trend. The germination percentage of lentil seeds in control and 100 ppm concentration treatments was the highest (97.2 and 99.4 % respectively) and decreased significantly with increasing cadmium sulfate concentrations and, the lowest is obtained at the concentration of 500 ppm (81.3 %). Also, mean germination time in the control group showed the lowest amount and at the concentration of 500 ppm of cadmium sulfate reaches the highest value (1.667) (Table 3). According to Shafiq et al. (2008), decrease in germination of seeds could be due to the decomposition of food stored in the seeds as a result of application of cadmium. According to studies on the effect of heavy metals on maize germination, Parmon et al. (2014) opined that heavy metals had a significant effect on maize germination and seed strength. Low levels of metals increase the germination percentage (from 93 % to 98 % in lead nitrate, from 93 % to 97 % in bismuth nitrate and from 94 % to 96 % in cadmium) and seed strength (from 1200 to 1606 in bismuth nitrate and from 1568 to 1592 in lead nitrate), but high levels reduced both parameters. Mah-mood et al. (2005) also stated that different levels of copper had no significant effect on maize germination but reduced the early growth of maize. Application of cadmium sulfate at a concentration of 500 ppm compared to the control reduced the germination rate by 37 %. However, the effect of concentrations of 100 and 250 ppm on germination rate was not signifi- cant (Table 3). According to the results, it seems that lentil seeds can tolerate up to 100 ppm of cadmium sulfate in these conditions but higher concentrations cause toxicity for this plant. The results obtained by Vasilev (2003) about the trait of Hordeum vulgare L. on different levels of cadmium showed that, the maximum effect on reducing growth is related to treatment of 10 mg l-1 of cadmium. The maximum reduction in growth was about 20 percent compared to control treatment. The application of 100 ppm of cadmium sulfate increased the shoot length compared to the control from 2.876 cm to 3.264, and then at concentrations of 250 and 500 ppm significantly reduce it by 17.1 and 59.2 percent (Table 3, Figure 3). But seminal root length showed a significant decrease with increasing concentration of heavy metals. Seminal root length was decreased by 69, 92 and 100 %, respectively at the concentrations of 100, 250 and 500. In other words, the effect of cadmium sulfate toxicity on seminal root was much more than that of the shoot length, since the seminal root was directly exposed to stress, and it had the most contact with this. The concentration-response phenomenon that is characterized by low-dose stimulation and high-dose inhibition is called as hormesis or hormetic effect. An increased number of studies has described the improved performance of some plant species to Cd exposure, despite Cd accumulation in their roots and shoots. These results designate that plants have developed protective strategies to neutralize the side-effects from Cd toxicity or, more controversially, mechanisms that employ Cd as beneficial element (Carvalhoa et al. 2020). Carvalhoa et al. (2020) reported that carbonic anhy- 60 Acta agriculturae Slovenica, 116/1 - 2020 Table 3: Effects of CdS04 concentrations on lentil seed and seedling traits Cd concentration (ppm) Shoot length (cm) Root length ( cm ) Seedling length ( cm ) Shoot mass (mg) Root mass (mg) Seedling mass (mg) MGT (day) Germination rate (seed / day) Germination Vigor index (% ) 1 Vigor index 2 0 2.876b 5.659 a 8.535 1 41.29 bc 5.262 a 46.56 a 0.9996b 24.241 97.19 ab 831.4a 4531b 100 3.264a 1.716b 4.980 b 48.03 * 3.825 b 51.86 a 1.245 ab 22.271 99.38 a 494.4b 5155 1 250 2.382 c 0.4175 c 2.799 c 48.95 a 1.212 c 50.16 a 1.337ab 21.361 93.75b 261.9C 4712 ab 500 1.174 d 0.000 d 1.173 d 36.25 c 0.000 d 36.25 a 1.667a 15.20b 81.25 c 93.14d 2920 c * Means, in each column, followed by same letter are not significantly different at the 5 % probability level- using Duncan's Multiple Range Test. Table 4: Interaction effects of Ti02 nanoparticles and CdS04 concentrations on lentil seed and seedling traits Nano Ti02 (ppm) CdSO 4 (ppm) Root Shoot length length (cm) (cm) Seedling length (cm) Shoot Weight (mg) Root mass (mg) Seedling mass (mg) MGT (day) Germination rate (seed/day) Germination Vigor index (% ) 1 Vigor index 2 1 c 3 0 0 3.113 abc 3.128 d 6.240 c 41.60bc 3.600 cd 45.20 ^ 1.095 a 20.00 c g 93.75 ^ 582.7 d 4271bcd 1 100 2.895abc 3.928 c 6.823 c 49.05 ab 4.975b 54.03 * 1.405 a 16.13'« 98.75 a 673.4c 5343* 51 a 3 250 1.928 e 0.000 1 1.928 ef 49.30 ab 0.000 1 49.30 ^ 1.342 a 14.53« 91.25 ^ 176.1hi 4505 ^ S o 500 1.155 f 0.000 1 1.155 1 39.83bc 0.000 1 39.83 bcd 1.709 a 8.338 h 58.75 d 59.58' 2405 e P 100 0 3.173 ab 6.105 b 9.278 * 42.10bc 5.550b 47.65 ^ 0.8823 a 26.92 a 97.50 * 903.3 b 4641 ^ vf P 100 3.347a 0.9250 e 4.273 d 47.70 ab 3.250 cd 50.95 * 0.9313 a 20.29 b-g 100.0 a 427.3 ef 5095 ^ oT 250 2.722bcd 1.110 e 3.832 d 46.13 ab 2.700 de 48.83 ^ 0.8823 a 25.42 ad 93.75 ^ 354.7 f 4577abc