2.4. Determination of Plant Growth Characteristics

Plant growth characteristics, such as plant height and root length, were measured on day 35, day 50, and day 65 (at the end of the experiment). Plant height was measured from soil level to the terminal bud of a representative sample of plants and then averaged [25]. Similarly, root length was measured by collecting a representative sample of plants with minimum damage to the root systems and measuring the length from soil level to the tip of the root. At the conclusion of the column experiments on day 65, above-ground and below-ground dry biomass were measured by selecting sample plants from three areas (4 cm × 4 cm) of each column tested. To ensure the sample plants were representative of the population, the selection of these samples was performed in such a way as to include short, medium, and tall plants in each sample. The root systems were washed, cleaned, and separated from the rest of the plants. Both above-ground and below-ground plant tissue matter were separately dried in an oven at 60 °C for a minimum of 72 h and then weighed to calculate the plant above-ground and below-ground dry biomass density in g/cm² [26].

The chlorophyll contents of the leaves of plants were determined on day 65 by treating 0.5 g of fresh leaf samples in an 80% acetone solution [27]. The optical densities of the total chlorophyll (α and β) content were determined at specific wavelengths of 645 and 663 nm using a UV-VIS spectrophotometer (Model: UV-2600, Shimadzu Corporation, Kyoto, Japan). The total chlorophyll content was calculated using the formula in [28],

where, V is the volume (mL) of acetone used for chlorophyll extraction, d is the light path length (cm), D663 is the absorbance at 663 nm, D645 is the absorbance at 645 nm, and W is the weight of the leaf used and the total chlorophyll content (a and b) in 1 g of sample leaf. To evaluate the significant differences in plant characteristics, such as height and root length at different growth stages between gas-exposed and control groups within each vegetation type, a two-way ANOVA was conducted. Additionally, t-tests were used to assess the significant differences between the gas-exposed and control groups in terms of vegetation characteristics, including above-ground and below-ground dry biomass, as well as chlorophyll content. The statistical analyses were performed using SigmaPlot 14.0 software from Systat Software Inc., and statistical significance was indicated where p < 0.05.