The materials and reagents used in the experiment are biofertilizers, lettuce seeds, distilled water, zinc sulfate heptahydrate (ZnSO₄·7H₂O), 90 × 14 mm diameter sterile petri dishes, filter paper, 10 mL syringe, graduated cylinder, beakers, graduated ruler, tweezers, paper towels, aluminum foil, plastic cling wrap, record book, and laptop for encoding purposes.

A sample of the biofertilizer was taken to the Regional Soils Laboratory of the Department of Agriculture located in Parola, Iloilo City, Philippines. Soil test data (Table 1) from the laboratory analysis reflected that the biofertilizer produced from the bioreactor composting technology has pH level of 8.23, 1.51

percent nitrogen content, 30.2 percent total organic matter, 117 ppm phosphorus content, and 2311 ppm potassium content. The pH level of the biofertilizer (8.23) is notably higher than the optimum soil pH range recommended for lettuce growth. This conforms with the previous results observed by Hanapi et al. (2013) [16] which obtained a biofertilizer pH range of 8.20 - 8.50. Though higher than the neutral pH, the slightly alkaline pH is beneficial because it will contribute to neutralizing acidic agricultural soil [17] that is dominant in the locality. Chang and Yang (2009) concluded in their study that inoculated biofertilizers with tested microbes had a significantly higher ash, pH, total nitrogen, and soluble phosphorus content. Adding microbes can shorten the period of maturity, improve the quality, increase the soluble phosphorus content, and enhance the populations of phosphate-solubilizing and proteolytic bacteria in the biofertilizers [18] .

Notably, the NPK composition of the biofertilizer is very high compared to the NPK requirement for lettuce growth. Lettuce requires medium levels of fertilization, growing best with 150 to 200, which significantly increases plant height, number of leaves per plant, dry weight, and yield [19] [20] . Healthy P levels in soil range from 25 to 50 ppm [21] . When soil test P levels are above 40 ppm, only small amounts of P are required which are best applied as starter. With soil K test values above 150 ppm, K fertilization is not required as lettuce will likely not respond to K fertilization. A positive yield response is possible with an intermediate K availability (100 - 150 ppm) [22] [23] .

After ten days of observation, the radicle length of lettuce seedlings in each sample replicate was carefully measured using a graduated ruler. As observed (Table 2), the negative control plant produced the longest radicle length of lettuce with a mean of 44.30 (SE = 12.57). Treatment 5, which has the highest level of biofertilizer dilution (10%) yielded the lowest radicle length with a mean of 2.22 (SE = 1.47). The application of varying levels of biofertilizer dilution does not significantly affect the growth of lettuce seeds in terms of their radicle length H(3) = 10.567, p = 0.061 > 0.05.

The variations in radicle development may be attributed to the other phytotoxic component of the biofertilizer. Aside from the excessive amount of nutrient present in the materials, other components such as heavy metals and other

Values are mean ± standard error S.E. Values with same letters in the same column do not differ significantly at p < 0.05 using Dunn-Bonferroni test for hypocotyl length and Games-Howell for germination percentage.

organic pollutants are present as well. In the study of Zhou, Wieslander, & Wu (2016), heavy metals harm lettuce seeds which impede their germination and radicle growth. According to Zhou et al. (2016), leafy vegetables are highly able to uptake and accumulate heavy metals [24] . Heavy metals can be readily taken up by vegetable roots, and can be accumulated at high levels in the edible parts of vegetables, even heavy metal in the medium is at low levels [25] [26] .

The hypocotyl length of the lettuce showed significant differences in response to varying levels of biofertilizer dilution with Welch’s F(5, 5.163) = 8.175, p = 0.017 < 0.05. Lettuce seeds applied with 0.001% biofertilizer were the tallest with mean of 19.89 mm (SE = 1.03) which significantly differ to those applied with 10% biofertilizer which produced the lowest hypocotyl length with mean of 2.33 (SE = 2.33). Among the levels of biofertilizers, there are no significant mean differences compared to the negative control plant.

In most cases, lettuce prefers fertile soil with a pH ranging from 6 to 6.8. In a study conducted by Roosta (2011) magnesium (Mg) concentration in the leaves increased with elevating pH up to 7.0 and then decreased at 8.0, but iron (Fe), manganese (Mn), and zinc (Zn) concentrations decreased at higher solution pH levels [27] .

The level of pH in the growth media determines the availability and functions of nutrients in them. Excessive amounts of nutrients in the medium can cause toxicity in the growth of the plants. As noted in the nutrient analysis of the biofertilizer, high levels of P and K are present and are higher that the ideal amount required. Alkaline pH increases and decreases micronutrients affecting the early growth development of plants.

Poor hypocotyl development may also be attributed to the presence of other toxic substances like heavy metals. Studies suggest that heavy metals might cause an inhibition in the initial growth that alters water balance and nutrient absorption, thereby affecting their transportation to the aboveground plant parts and thus negatively affecting shoot growth and ultimately decreasing biomass accumulation [28] . Hence, it is non-germination of the lettuce seeds. This result, however, contradicts the observation of Spiassi et al. (2015) in their study for they found a relative relationship between the elongation of hypocotyl and increased levels of biofertilizer [29] .

Different levels of biofertilizer affected significantly the germination percentage of lettuce seeds F(5, 12) = 5.822, p = 0.006 < 0.05. Seeds subjected to negative control solution have the highest germination percentage with a mean of 76.67% (SE = 3.33) while those seeds subjected to 10% biofertilizer dilution have the lowest germination percentage with a mean of 10.00% (SE = 5.77). In contrast to the negative control plant, those group of seeds applied with 0.01% and 10% biofertilizer dilution differed significantly in terms of germination. Notably, all levels of biofertilizer dilution compared to 10% level of biofertilizer dilution have a significant mean difference in germination percentage.

Biofertilizers are substances containing variety of microbes having the capacity to enhance plant nutrient uptake by colonizing the rhizosphere and make the nutrients easily accessible to plant root hairs. In the study conducted by Bákonyi et al. (2013), they concluded that the usage of biofertilizers make germination more effective, thus significantly increasing the number of germinated seeds [30] .

The relative germination percentage is the determination of the viable seeds sown. It measures the total seeds germinated at the right conditions from the seed sown. Relative germination percentage (RGP) reflects germination behavior of seeds of a single batch subjected to different levels of treatment in contrast to the negative control plant. All levels of biofertilizer treatments indicated a decrease in relative germination percentage (Figure 1). Seeds subjected to 0.001% biofertilizer have the lowest decline of germination compared to the negative control plant, equivalent to only 4.17% (RGP = 95.83%). Those seeds applied with 10% biofertilizer have the highest reduction of germination percentage, equal to 86.9% (RGP = 13.10%).

As noted, biofertilizers can be good sources of nutrients such as Nitrogen, Phosphorous, Potassium, and total organic matter (TOM). During the early development of seedlings, a minimum amount of nutrients is required considering that seeds can initially utilize the nutrients within their system. Exposure of the seedlings to a higher concentration of nutrients at this stage can be detrimental to the development of the plant. Some studies showed that the increase in the dose of N in both the laboratory and field conditions decreased the germination rate of seedlings [31] . Statistical analysis proved that increasing the N dose

decreased the vigor value of wheat seeds, however, other authors found the opposite [32] , claiming that the vigor of the seeds improved by increasing the N dose. According to the study of Aydinalp & Marinova (2009), there was a reduction in seed germination as metal concentrations in the growing media increased in general [33] . The major effects of heavy metals on seeds are manifested by overall abnormalities and decrease in germination, reduced root and shoot elongation, dry weight, total soluble protein level [34] [35] , oxidative damage, membrane alteration, altered sugar and protein metabolisms, nutrient loss [24] - [36] all contributing to seed toxicity and productivity loss [34] .

Radicle growth is the primary root that emerges from the seeds upon germination. It grows downward in the soil and is used to seeds anchorage for a better crop stand. The relative radicle growth of seeds from different levels of biofertilizer versus the negative control plant reflects the viability of a population of seeds. All levels of biofertilizer dilutions showed increased radicle growth compared to the negative control solution except for the 10% biofertilizer dilution. Notably, the application of 0.1% biofertilizer dilution increased the efficiency and viability of the seeds by 48.44%, compared to the negative control which was the highest increase among the levels of biofertilizer. This was followed by seeds with 1% biofertilizer dilution with an increase of 37.20%. Seeds with 0.1% biofertilizer had a 28.95% increase and seeds applied with 0.01% dilution had an increase of 8.94%. Seeds that received the 10% biofertilizer showed an extremely high reduction in radicle growth, equivalent to 72.22% (RRG = 27.78%).

Though lettuce applied with 0.001% biofertilizer showed a slight decrease in germination percentage (4.17%), it produced substantially longer radicle length in contrast to the negative control plant by 48.44%. On the other hand, the highest decline in germination percentage (RRG = 13.10%) and radicle length (RRL = 27.78%) were both observed from the lettuce applied with 10% biofertilizer. These findings suggest that the low number of lettuce seeds that were able to germinate correspondingly produced significantly shorter radicle length when applied with a high concentration of biofertilizer in contrast to the negative control plant. One observed properties of biofertilizers that may contribute to the significantly lower relative radicle length is their high level of pH.

During seed germination, alkalinity/salinity results in many disorders and metabolic changes, such as solute leakage, K⁺ efflux, and α-amylase activity [37] . Firstly, it reduces moisture availability by inducing osmotic stress, creating nutrient imbalance and ionic toxicity [38] . Cell membranes are the hotspots for controlling solute’s active and passive transfer and regulating plant nutrient uptake [39] . Alkali salts, such as NaHCO₃ and Na₂CO₃, are the primary ion sources found in saline soils; Na⁺, K⁺, Ca²⁺, and Mg²⁺ are the main cations and C1⁻, NO 3 − , HCO 3 − , CO 3 2 − , and SO 4 2 − are the main anions. Indeed, studies have confirmed that alkaline salts damage plants more than neutral salts [40] [41] [42] [43] as cited by Wang et al. (2022).