Aqueous suspensions of nTiO₂ (5 nm and 10 nm) were used for all the treatments. Lettuce seeds were imbibed with water (control) or nTiO₂ suspensions (250 mg/L) for up to 12 hours and placed on moist paper towels in Petri dishes for germination. The nTiO₂ suspensions were freshly prepared and thoroughly agitated before all the treatment applications. Following the germination (after 10 days of sowing), the seedlings were transplanted into a peat-based mix growing medium, BM6 (Berger, Quebec, Canada) contained in 15 cm plastic pots (1 seedling/pot) (HC Companies, Twinsburg, OH, USA). The plants were grown in a greenhouse maintained approximately at 20˚C with solar radiation as the light source. Plants were watered every other day and fertigated approximately once a week with 100 mL of solution/plant containing 250 ppm N (Jack’s Peat-Lite, NPK: 20-10-20, JR Peters, Inc., Allentown, PA, USA). Plants were harvested after 4-5 weeks of transplanting. The experiment was conducted on a completely randomized design with 6 replications for each nanoparticle size.

In the first greenhouse study, the lettuce seeds were imbibed in water for up to 12 hours and directly sown in the moist peat-based growing medium (BM6) on July 17, 2023. After germination, the seedlings were thinned to retain one plant/ pot. Seedlings were watered every other day and fertigated once a week as described above. The 20-day-old lettuce seedlings were sprayed with freshly prepared nTiO₂ suspensions (5 nm and 10 nm) at 5, 10, 50, 100, 150, mg/L. Each plant was evenly sprayed with water (control) or nTiO₂ suspension ensuring its uniform coverage of the canopy and avoiding nTiO₂ contamination of the growing medium. The plants were irrigated before the foliar applications of nTiO₂ suspensions. The 5-week-old plants were harvested to measure their growth characteristics. The experiment was conducted on a completely randomized design with 6 replications.

A second greenhouse study was conducted to assess the impact of higher concentrations of nTiO₂ on the growth of lettuce plants. Lettuce seeds were imbibed in water for 12 hours and were germinated and then, transplanted into the peat-based growing medium as described above. After thirteen days of transplanting, seedlings were treated with a foliar spray of freshly prepared nTiO₂ (5 nm and 10 nm) suspensions at 500 mg/L, 2500 mg/L, and 4000 mg/L as described above. With the high concentration of TiO₂, agglomeration can be a problem, so, care was taken to apply well-dispersed nTiO₂ evenly over the foliage. The control plants were sprayed with water. Plants were irrigated every other day and fertigated once a week as described above. Plants were irrigated before the foliar applications of nTiO₂ suspensions. The experiment was conducted on a completely randomized design with 6 replications for each nanoparticle size.

All plant growth measurements were made on the plants grown from seeds imbibed with nTiO₂ and on those treated with nTiO₂ through foliar sprays before harvest.

As the lettuce variety has a loose-leaf canopy, plant height was measured from the base of the stem to the tallest leaf. The other growth characteristics measured were the number of fully opened leaves/plant and the leaf area of all the fully opened leaves using a leaf area meter (LI-3100, LI-COR, Inc., Lincoln, NE). To measure the plant biomass, plant shoots were separated from roots. The roots were washed free of the growing medium and were blotted dry with paper towels to remove the surface moisture. Shoot and root fresh weights from each plant were measured. They were then transferred to a drying oven at 70˚C and were dried for 4 days or until a constant weight was achieved before measuring the dry weights.

All the experiments were conducted on a completely randomized design with 6 replications with nTiO₂ nanoparticle sizes and their various concentrations as treatment variables. The Tukey HSD test was conducted at alpha 0.05 and p-values were obtained to perform multiple comparisons of the treatment means.

The response of lettuce plants grown from seeds treated with 5 nm and 10 nm nTiO₂ in relation to various plant growth characteristics including shoot fresh and dry biomass, root fresh and dry biomass, plant height, leaf area, and leaf number was studied at the time of harvest.

Lettuce plants grown from the seeds imbibed with nTiO₂ at 250 mg/L produced variable responses. The fresh shoot biomass of lettuce plants was significantly reduced by the seed treatment with 10 nm nTiO₂ by 18.1% compared to the control while, on the contrary, there was no significant impact on the fresh shoot biomass in plants receiving seed treatment with 5 nm nanoparticles (Figure 1(a)). This was partly due to a significant reduction in the number of leaves/ plant with 10 nm nTiO₂ seed treatment (Figure 1(b)). This shows that larger nanoparticle size had a significant negative impact on shoot biomass in lettuce compared to the smaller particle size. However, it should be noted that the size of nanoparticles can impact not only their absorption but more importantly their translocation leading to their differential accumulation in various plant parts [24] .

Many growth characteristics including plant height, dry shoot biomass, leaf area, and both fresh and dry root biomass were not affected by the seed treatment of either 5 nm or 10 nm nanoparticles (Table 1 and Figure 1(b)). Also, there was no significant impact on the seed germination in response to the different nanoparticle sizes (data not shown). Similar results were observed in kidney beans where nTiO₂ did not affect the germination but had a significant impact on early root growth [14] .

Although most of the growth characteristics were unaffected by nTiO₂ treatment, it is noteworthy that the negative impact of nTiO₂ (10 nm) on fresh shoot biomass has practical implications for the growers as it can notably affect the marketable yield. The reduction of shoot growth (biomass accumulation) when

nTiO₂ was applied to seeds may indicate its negative impact on the early growth of lettuce [14] . Similar observations on the negative effects of seed treatment of nTiO₂ have been made in other species including corn, cucumber, oats, onion, and tomato [15] [34] .

In the first greenhouse study, lettuce seedlings were treated with both 5 nm and 10 nm nTiO₂ nanoparticles at concentrations ranging from 5 mg/L to 150 mg/L as a foliar spray.

Foliar spray of 5 nm nTiO₂ did not have any significant impact on many of the growth characteristics of lettuce except the number of leaves per plant (Table 2). Plants treated with 10 mg/L and 150 mg/L of nTiO₂ (5 nm) produced more leaves/plant compared to the control plants, the highest leaf number/plant being at 150 mg/L of nTiO₂. The responses to other nTiO₂ concentrations were not

*FW and DW represent the fresh weight and dry weight, respectively.

*FW and DW represent the fresh weight and dry weight, respectively.

different from those in the control plants. Unlike the seed treatment of nTiO₂ (10 nm), the foliar sprays did not result in the reduction of shoot fresh biomass compared to the control plants (Figure 2(a)). Although there was an increase in shoot dry biomass in response to nTiO₂ (10 nm) at 10 mg/L, both shoot fresh and dry biomass accumulations at higher concentrations of nTiO₂ were similar to the those in the control.

Similarly, nTiO₂ concentrations did not have an impact on the leaf area compared to the control (Figure 2(b)). Overall, these observations suggest that the foliar application of nTiO₂ up to 150 mg/L did not significantly impact most of the growth characteristics in lettuce plants. Therefore in a separate study, we used higher concentrations of nTiO₂ as a foliar spray, to determine their impact on the growth of lettuce plants. Foliar sprays of both 5 nm and 10 nm nTiO₂ at concentrations ranging from 500 mg/L to 4000 mg/L were applied to the lettuce seedlings. Again, these treatments produced variable results on the shoot and root biomass with regard to the nanoparticle concentrations. The notable response of lettuce at higher concentrations of nTiO₂ was the reduction in fresh shoot biomass with 5 nm nTiO₂ and in dry shoot biomass with 10 nm nTiO₂ (Figure 3(a) and Figure 3(b)).

Fresh shoot biomass was reduced by approximately 20% compared to that in the control plants with the application of 5 nm nTiO₂ at 500 mg/L. However, 10 nm nTiO₂ at 500 mg/L reduced the shoot dry biomass by about 29% compared to that in the control plants (Figure 3(a) and Figure 3(b)).

However, these responses were variable at higher concentrations. Foliar applications of 5 nm or 10 nm nTiO₂ did not have any impact on other growth characteristics such as plant height, leaf area, root fresh and dry biomass (Table 3 and Table 4). Our results support the previous studies that show no impact of nTiO₂ on many aspects of plant growth [3] [14] . Furthermore, previous studies have also shown that nTiO₂ can have a negative impact on plant growth and even be toxic, especially at higher concentrations [17] . Hu et al. [25] showed that

*FW and DW represent the fresh weight and dry weight, respectively.

*FW and DW represent the fresh weight and dry weight, respectively.

the effects of nTiO₂ were dose-dependent in lettuce plants in that its higher concentrations suppressed chlorophyll content, biomass, and the nutritional quality of lettuce.

Considering the observation that the negative impact of plant growth (biomass accumulation) by nTiO₂ when it was applied to the seeds while foliar sprays produced little or no impact on overall growth may suggest that early stages of seedling growth including the germination process are more sensitive to nTiO₂ [14] . This is further supported by previous studies that find early growth in many plants including corn, oats, cabbage, and oilseed rape is sensitive to nTiO₂ [14] [15] .

Furthermore, foliar applications of nTiO₂ to lettuce seedlings, regardless of their size, showed some signs of toxicity. The foliar applications resulted in many brown necrotic spots or etiolated blotches on the leaves, which were more pronounced at higher concentrations of nTiO₂ (Figure 4). However, these symptoms were apparent with the foliar application of nTiO₂ even at concentrations as low as 50 mg/L. It is noteworthy that although the growth responses were variable with foliar application of nTiO₂, the higher concentrations of nTiO₂ produced signs of toxicity in lettuce. These observations support many previous studies involving the applications of high concentrations of nTiO₂ that result in injury caused by oxidative stress in several plant species [25] [28] [31] [35] . As nTiO₂ is a photocatalytic substance, it can generate reactive oxygen species in the presence of light, especially UV, which can be highly damaging to cells [23] . The primary effect of oxidative species induced by nTiO₂ is the damage to the chloroplast membranes resulting not only in reduced photosynthetic function but also in promoting autophagy, a process through which impaired cell organelles could be eliminated [31] [36] .

Similar findings of the negative impact of nTiO₂ on photosynthetic function and the overall growth in many plant species including lettuce have been reported [20] [25] [31] [32] . Our results on lettuce show that the application of both 5 nm and 10 nm nTiO₂ as a foliar spray can cause toxicity to the leaves in lettuce even at moderate concentrations of nTiO₂ (50 mg/L or higher).